International Review of Neurobiology Volume 36

International Review of

NEUROBIOLOGY VOLUME 36

Editorial Board W. Ross ADEY

PAULJANSSEN

J U L ~ U SAXELROD

SEYMOUR KETY

Ross BALDESSARINI

KEITH KILLAM

SIRROGERBANNISTER

CONANKORNETSKY

FLOYD BLOOM

ABELLAJTHA

DANIELBOVET

BORISLEBEDEV

PHILLIPBRADLEY

PAULMANDEL

YURIBUROV

HUMPHRY OSMOND

Jost DELGADO

RODOLFOPAOLETTI

SIRJOHN ECCLES

SOLOMON SNYDER

JOEL

ELKES

STEPHEN SZARA

H. J. EYSENCK

MARATVARTANIAN

KJELL FUXE

STEPHENWAXMAN

Bo HOLMSTEDT

RICHARDWYATT

International Review of

Edifedby RONALD J. BRADLEY Department of Psychiatry LSU Medical Center Shreveport, Louisiana

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

VOLUME 36

ACADEMIC PRESS Son Diego New York

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This book is printed on acid-free paper.

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Copyright 0 1994 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- 12-366836-0

PFUNTFD IN THE UNITED STATES OF AMERICA 94 95 9 6 9 7 98 9 9 B B 9 8 7 6

5

4

3 2 1

CONTENTS

Ca2+,N-Methybaspartate Receptors, and AIDS-Related Neuronal Injury

STUARTA. LIPTON ..................... I. Introduction . , . . . . . . . . . . . . . . . . . . . . . . 11. Neuronal Loss in the CNS of AIDS Pa 111. gpl20-Induced Neuronal Injury Is Ameliorated by Calci .............. Channel Antagonists . . . , . . . . . . . , . . . . . . . . . . 1V. Involvement of the NMDA Receptor in gpl20-Induced ..__....,......... Neuronal Injury . . . . . . . , . . . . . . . . . . V . Indirect Neuronal Injury Mediated by .................... Stimulated Monocytic Cells , . . . . . . . . 1 VI. Possible Involvement of Astrocytes, 0 HIV-1 Proteins in Neuronal Injury . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Overstimulation of NMDA Receptors, a Final Common Pathway . . . . VIII. Development of Clinically Tolerated NMDA Antagonists for HIVRelated Neuronal Injury . . . . . . . . . , , . . . . . . . ....’.’...’....’.’ IX. Excitatory Amino Acid Antagonist Treatments on the Horizon . . . . . . ......................... X. Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I

1 3 4

6 8

12 15

17 21 23 24

Processing of Alzheimer Ap-Amyloid Precursor Protein: Cell Biology, Regulation, and Role in Alzheimer Disease

SAMGANDYAND PAULGREENCARD Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alzheimer Disease Is Associated with an Intracranial Amyloidosis . . . . APP Structure Gives Clues to Some of Its Functions . . . . . . . . . . . . . . . APP Is Processed via Several Distinct Enzymatic and Subcellular Pathways . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . V. “Alternative” Pathways of APP Metabolism Provide Clues to the Source of A@-Amyloid . , . . , . . , . . . . . . . , . , . . . . , . . . . . . . . . . . . . . . . . . VI. AP-Amyloid Is a Normal Constituent of Body Fluids and the Conditioned Medium of Cultured Cells . . . . , . . . . . . . . . . . . . . . . . . . . . VII. Evidence Suggests the Existence of an Enzyme, P-Secretase, That Cleaves APP at the Amino Terminus of the AP-Amyloid Domain . . . . 1. 11. Ill. IV.

V

29 30 31

32 34 36

37

vi

CONTENTS

VIII. APP Mutations in Familial Cerebral Amyloidoses Occur within or near the AP-Amyloid Domain, Segregate with Disease in Affected Kindreds, and Yield APP Molecules That Display Some Proamyloidogenic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Signal Transduction via Protein Phosphorylation Regulates the Relative Utilization of APP Processing Pathways . . . . . . . . . . . . . . . . . . . X. Beyond Ap-Amyloid: Other Molecular Factors in Amyloidogenesis and Factors Differentiating Aging-Related Cerebral Amyloidosis from Alzheimer Disease ............................................ References ...................................................

37 39

42 44

Molecular Neurobiology of the GABAA Receptor

SUSANM. J. DUNN,ALANN. BATESON,AND IAN L. MARTIN I. Introduction ....................

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

11. Pharmacology of the GABAARece ............. 111. Biochemistry . . . . . . . . . . . . . . . . . . . . . .............. IV. Molecular Cloning of Receptor Subunits . . . . ...............

V. Characterization of the Receptor Family . . . . . . . . . . . . . . V1. The Future ................... .................

51 52 59 71 74 87 88

The Pharmacology and Function of Central GABAs Receptors

DAVIDD. MOTT AND DARRELL V. LEWIS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacology of GABAB Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 I. Properties of GABABReceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Function of GABA, Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ................................................... 11.

97 98 104 126 209 210

The Role of the Amygdala in Emotional Learning

MICHAEL DAVIS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Electrophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Anatomical Connections between the Amygdala and Brain Areas

V.

Involved in Fear and Anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elicitation of Fear by Electrical or Chemical Stimulation of the Amygdala . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225 226 227

228 23 1

CONTENTS

VI. Effects of Amygdala Lesions on Conditioned Fear ........ VII. Effects of Amygdala Lesions on Unconditioned Fear . . . . . . . . . . . . . . . VIII. Effects of Local Infusion of Drugs into the Amygdala o n Measures of Fear and Anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. T h e Role of the Amygdala in Attention . . . . . . . . . . . . . . . . . . . . . . . . . . X. T h e Amygdala Is Critical for the Fear-Potentiated Startle Effect XI. Are Aversive Memories Actually Stored in the Amygdala? . . . . . XI!. Is the Amygdala Absolutely Essential for Fear-Potentiated Startle? . . . XIII. Can Initial Fear Conditioning Occur without the Amygdala? . . . . . . . . XIV. T h e Role of Excitatory Amino Acid Receptors in the Amygdala in Fear Conditioning . . . . . . . . . . . . . .. xv. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . .. .. ..

vii 234 235 236 238 240 24 1 244 245 250 258 259

Excitotoxicity and Neurological Disorders: Involvement of Membrane Phospholipids

AKHLAQA. FAROOQUI AND LLOYD A. HORROCKS I. 11. 111. IV. V.

VI. VII. VIII. IX.

Introduction ....................... .. Classification of Excitatory Amino Acid Receptors . . . . . . . . . . . . . . . . . Excitatory Amino Acid Receptors and Neural Membrane ........ Phospholipid Metabolism . . . Role of Enhanced Excitatory Am lipi Metabolism in Developing Brain .. .. Possible Mechanism of Cell Injur Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Excitatory Amino Acid Receptors, Phospholipid Metabolism, and Neurological Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Excitatory Amino Acid Receptor Antagonists and the Treatment of Neurological Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . ........ Summary . . . . . . . . . ........ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

267 269 280 287 288 29 1 307 310 313 313

Injury-Related Behavior and Neuronal Plasticity: An Evolutionary Perspective on Sensitization, Hyperalgesia, and Analgesia

EDGART. WALTERS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolutionary Considerations .................................... Adaptive Behavioral Reactions to Injury . . . . . . . . . . . . . . . . . . . . . . . . . IV. Classes of Injury-Related Behavioral Modifiability . . . . . . . . . . . . . . . . . V. Injury Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.

11. 111.

325 327 330 342 36 1

...

Vlll

CONTENTS

VI. Mechanisms of Rapid Nociceptive Sensitization .................... VII. Mechanisms of Long-Term Nociceptive Sensitization . . . . . . . . . . . . . . . VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

367 386 407 412

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

429 44 1

Ca2+, N-METHYL-D-ASPARTATE RECEPTORS, A N D AIDS-RELATED NEURONAL INJURY

Stuart A. Lipton Laboratory of Cellular and Molecular Neuroscience, Deportment of Neurology, Children’s Hospital, Beth Israel Hospital, Brigham and Women’s Hospital, Massachusetts General Hospital, and Program in Neuroscience, Howard Medical School, Boston, Massachusetts 021 15

I. Introduction 11. Neuronal Loss in the CNS of AIDS Patients 111. gpl20-Induced Neuronal Injury Is Ameliorated by Calcium Channel Antagonists

IV. Involvement of the NMDA Receptor in gpl20-Induced Neuronal Injury V. Indirect Neuronal Injury Mediated by HIV-Infected or gp 120-Stimulated Monocytic Cells VI. Possible Involvement of Astrocytes, Oligodendrocytes, and Other HIV- 1 Proteins in Neuronal Injury A. Astrocytes and HIV-Related Neuronal Damage B. gp120 Binding to the Oligodendrocyte Surface Molecule GalC C. gp120 Binding to Sulfatide and Myelin-Associated Clycoprotein D. gp120 Attenuates p-Adrenergic Stimulation of Astrocytes and Microglia E. Neurotoxicity of Other HIV- 1 Proteins F. Direct Effects of HIV-Infected Macrophages on Neurons VII. Overstimulation of NMDA Receptors, a Final Common Pathway VIII. Development of Clinically Tolerated NMDA Antagonists for HIV-Related Neuronal Injury A. Sites of Action of Potential Clinically Tolerated NMDA Antagonists B. NMDA Open-Channel Blockers C. NMDA Receptor Redox Modulatory Site X. Excitatory Amino Acid Antagonist ‘Treatments on the Horizon XI. Conclusion References

1. Introduction

A large number of adult patients and children with acquired immunodeficiency syndrome (AIDS) eventually suffer from neurological manifestations, including dysfunction of cognition, movement, and sensation. How can human immunodeficiency virus type 1 (HIV-1) result in neuronal damage if neurons themselves are not infected by the virus? This article reviews a series of experiments leading to a hypothesis that accounts at least in part for the neurotoxicity observed in the brains of INTERNATlONAL REVlEW OF NEUROBIOLOGY, VOL. 36

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Copyright 0 1994 bv Academic Press, lnc. All rights of reproduction In any form reserved.

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STUART A. LIPTON

AIDS patients. There is growing support for the idea of the existence of HIV- o r immune-related toxins that lead indirectly to the injury or demise of neurons via a potentially complex web of interactions among macrophages (or microglia), astrocytes, and neurons. HIV-infected macrophages/microglia or macrophages activated by HIV- 1 envelope protein gp120 appear to secrete excitants/neurotoxins including plateletactivating factor, arachidonate, and its metabolites, and possibly cysteine, nitric oxide, and superoxide anion. In addition, interferon-? (IFN-7) stimulation of macrophages induces release of the glutamate-like agonist quinolinate. Furthermore, HIV-infected macrophage production of cytokines, including TNF-a and ILl-p, contributes to astrogliosis. A final common pathway for neuronal susceptibility appears to be operative, similar to that observed in stroke, trauma, epilepsy, and several neurodegenerative diseases, possibly including Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. This mechanism involves the activation of voltage-dependent Ca2+channels and N-methyl-D-aspartate (NMDA) receptor-operated channels, and therefore offers hope for future pharmacological intervention. This review focuses on clinically tolerated calcium channel antagonists and NMDA antagonists with the potential for trials in humans with AIDS dementia in the near future. Our laboratory has a long-standing interest in the relationship of neuronal viability/outgrowth to intracellular Ca'+ levels (Lipton and Kater, 1989). Glutamate, o r a related excitatory amino acid (EAA), is the major excitatory neurotransmitter that controls the level of intracellular neuronal Ca2+ ([Ca2+Ii).Escalating concentrations of glutamate have been measured in vim following focal stroke and head injury (Choi, 1988; Meldrum and Garthwaite, 1990). As a result, there is an immediate elevation in [Ca2+Ii that precedes neurotoxicity by -24 h. Although the rise in [Ca2+Iimay not account by itself for the ensuing neuronal injury, several laboratories have now reported that prevention of the increase in [Ca2+Iileads to the amelioration of anticipated neuronal cell death (Choi, 1988; Meldrum and Garthwaite, 1990). Excessive intracellular Ca2+is thought to contribute to the triggering of a series of potentially neurotoxic events leading to cellular necrosis or apoptosis; these events include overactivation of the enzymes protein kinase C, Ca2+/ calmodulin-dependent protein kinase 11, phospholipases, proteases, protein phosphatases, xanthine oxidase, nitric oxide synthase, and endonucleases (Lipton and Rosenberg, 1994). There are many mechanisms involved in intracellular calcium homeostasis, and this subject is beyond the scope of this article (but see Miller, 1991). Here we will consider modes of Ca2+ entry into neurons during these pathological processes. Two major routes of entry of Ca2+ occur

Ca2+ CHANNELS, NMDA, A N D AIDS-RELATED NEURONAL INJURY

3

via ion channels that are permeable to Ca2+ and can be summarized as follows: (a) glutamate o r related EAAs trigger voltage-dependent calcium channels (VDCC) by depolarizing the cell membrane; the major VDCC subtype that is chronically activated by prolonged depolarizations is the L-type calcium channel (Lipton, 1991b). (b) glutamate or related EAAs activate ligand-gated ion channels directly; the major glutamate receptor-operated channel that is permeable to Ca2+ under these conditions is the NMDA subtype, but other types may also contribute (Choi, 1988; Lipton and Rosenberg, 1994). We have shown that activation of these channel types can control neuronal plasticity during normal development, but, in excessive amounts, our laboratory and others have shown that this stimulation can lead to neuronal death, e.g., after a stroke (Lipton and Kater, 1989). Similar mechanisms may obtain in various neurodegenerative conditions. In fact, this mechanism may represent a final common pathway of neuronal injury, although not involved in the primary pathophysiology of a neurologic disorder. Most importantly, this pathway makes the disease process amenable to pharmacotherapy. This line of reasoning led us to think that this mechanism might be involved in AIDS-related neuronal injury.

II. Neuronal Loss in the CNS of AIDS Patients

A significant number of adults and children with AIDS eventually develop neurological manifestations including dementia, myelopathy, and peripheral neuropathy; as many as 50% of infected children have neurological deficits presenting as delayed developmental milestones. These deficits occur even in the absence of superinfection with opportunistic organisms or associated malignancies (Price et al., 1988). Among the several neuropathological manifestations of AIDS in the brain is neuronal loss. In selected brains from AIDS patients, several groups (Ketzler et al., 1990; Everall et al., 1991; Wiley et al., 1991; Tenhula et al., 1992) have demonstrated loss of 18-50% of cortical neurons and retinal ganglion cell neurons. In addition, in the neocortex there is a loss in the complexity of dendritic arborization as well as presynaptic area (Masliah et al., 1992). The question remains, however, in at least this subset of patients with neurological manifestations, how can neurons be injured and yet not be infected?

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STUART A. LIPTON

111. gpl20-Induced Neuronal Injury Is Ameliorated by Calcium Channel Antagonists

The major cell type infected with HIV- 1 in the CNS is the macrophage or microglial cell. These cells act as a reservoir for the virus and quite possibly release virus or viral proteins o r protein fragments. Possibly accounting at least in part for the injury to neurons is the observation first made in uitro by Brenneman and colleagues that picomolar concentrations of the envelope protein of HIV-1, gp120, can induce neuronal loss in rodent hippocampal cultures (Brenneman et al., 1988). Subsequently, our group demonstrated that in mixed cultures of neurons and glia, picomolar gp120 could increase [Ca2+liin rodent hippocampal neurons and retinal ganglion cells within a few minutes of application (Dreyer et al., 1990). Recently, similar findings were reported by Thayer's group (Lo et al., 1992), who were also able to resolve the increase in [Ca' +Ii into discrete oscillations by monitoring the calcium signal on a faster time scale. Within the next 24 h, neuronal injury ensues (Dreyer et al., 1990). Several groups have now confirmed that picomolar concentrations of gp120 can cause injury in a variety of neuronal preparations, including rat cortical neurons (Miiller et al., 1992; Dawson et al., 1993) and cerebellar granule cells (Savio and Levi, 1993). Both the early rise in [Ca2+Iiand the delayed neuronal injury can be largely prevented by antagonists of the L-type VDCC, including nimodipine (100 nM in 5% serum o r approximately 4 nM free drug) (Dreyer et al., 1990; Dawson et al., 1993; Savio and Levi, 1993). Other antagonists of the L-type VDCC are also effective to some degree (Lipton, 1991a) (Table I). Not only are rat retinal ganglion cells and cortical neurons in vitro partially protected by nimodipine and other voltage-dependent Ca2 channel antagonists, but also in a rat pup animal model, stereotactic injection of gp120 into the cortex produces a lesion consisting of cellular infiltrates of foamy macrophages and putative neuronal injury that is prevented by concomitant intraperitoneal administration of nimodipine (Lipton and Jensen, 1992). Additional in vivo evidence that low concentrations of gp120 are associated with neuronal injury has come from experiments of Brenneman, Hill, Ruff, Pert, and co-workers, who have found that intraventricular injections of gp120 into rats result in dystrophic neurites in hippocampal pyramidal cells as weil as behavioral deficits; moreover, cerebrospinal fluid of HIV-infected patients has gp120-like neurotoxic activity (Mervis et al., 1990; Buzy et al., 1992; Glowa et al., 1992). This evidence points to a potential role of gp120 in a neurodegenerative process. Because of these developments, the AIDS Clinical Trials Groups (ACTG) of the +

5

Ca2+ CHANNELS, NMIIA, A N D AIDS-RELATED NEURONAL INJURY

TABLE I VOLTAGE-DEPENDENT CALCIUM CHANNEL ANTAGONISTS ATTENUATEgp 120-MEDIATED NEURONAL INJURYI N VITRO _____

_____~

________

_________~

____

Amelioration"of gp 120-induced neuronal injury by voltagedependent calcium channel antagonists of the class

Dihydropyridine*

Dipheny lalkylamine piperazine derivative'

Phenylalkylamined

++++

++

+

Benzothiazepine' -

Note. Adapted from Lipton, 1992d. With permission from Hogrefe & Huber Publishers, Seattle. ' An increasing number of pluses indicates greater potency. Nimodipine and nifedipine (10-100 nM in 5% serum; -4-40 nM free drug). Flunarizine (10 p M in 5% serum). Verapamil (100 p M in 5% serum). Diltiazem (1 p M in 5% serum).

NIH Division of AIDS has asked us to begin a multicenter, randomized double-blind, placebo-controlled clinical trial to test the effects of nimodipine in adult patients with HIV-associated cognitive/motor complex (a subset of which has the more debilitating AIDS dementia complex), and this study is currently ongoing. Nevertheless, these developments do not tell us the mechanism of action of gp120 on neurons, which more recent evidence has led us to believe is an indirect pathway via macrophages/microglia (see below). For example, we noted that only neurons clustered in groups, presumably with synaptic contacts, were vulnerable to gp120, and this fact suggested that cellular interactions were necessary to produce injury. Moreover, the HIV envelope protein does not appear to act directly on calcium channels; in whole-cell and single-channel patch-clamp recordings, picomolar gp120 does not increase calcium current per se (H. S.-V. Chen, M. Plummer, P. Hess, S . A. Lipton, unpublished findings, 1990). It is possible that calcium channel antagonists ameliorate gp 120-induced neuronal injury by reducing the overall intracellular Ca" burden of the neurons. After all, Ca2+can accumulate in neurons during normal activity with each action potential fired, and nimodipine may be only indirectly beneficial by helping offset an increased calcium load due to another mechanism.

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STUART A. LIPTON

IV. Involvement of the NMDA Receptor in gpl20-Induced Neuronal Injury

As outlined above, there is another prominent mode of Ca2+entry via channels directly coupled to EAAIglutamate receptors. T h e type of glutamate receptor subtype that is primarily (but not exclusively)involved in this regard is named after NMDA, a glutamate analog that is a selective agonist of this receptor (however, NMDA does not occur naturally in the body). We reasoned that since gp120 causes an early rise in [Ca2+Ii and delayed neurotoxicity, similar to glutamate acting at the NMDA receptor, perhaps glutamate or a closely related molecule was involved in HIV-related neuronal injury. Furthermore, it was well known that VDCC antagonists such as nimodipine could block some forms of glutamate neurotoxicity (Abele et al., 1990; Weiss et al., 1990; Sucher et al., 1991). Therefore, it was certainly possible that glutamate or a related NMDA agonist was somehow involved in gp120-induced neuronal damage. In addition, Heyes and colleagues (Heyes et al., 1989, 1991) had found that cerebrospinal fluid (CSF) levels of quinolinate, a naturally occurring (albeit weak) NMDA agonist, was correlated with the degree of dementia in AIDS patients. To test the possibility that EAAs were involved, the following experiments were undertaken. NMDA antagonists were assessed for their ability to prevent gp 120-induced neuronal injury. We found that MK-80 1 (dizocilpine), an open-channel blocker of NMDA receptor-coupled ion channels, prevented gp 120-induced neuronal injury (Lipton et al., 1990, 1991). D-2-Amino-5-phosphonovalerate (APV), a competitive antagonist at the glutamate binding site of the NMDA receptor, was partially effective in ameliorating this form of neuronal injury. Recently, other groups have obtained similar results using NMDA antagonists or inhibitors of nitric oxide synthase (nitric oxide, or NO., is believed to be involved in one of the toxic pathways activated by NMDA receptor stimulation) (Muller et al., 1992; Dawson et al., 1993; Savio and Levi, 1993). In contrast, CNQX, a non-NMDA antagonist, did not protect from gpl20-induced neuronal damage, at least to retinal ganglion cells (Lipton et al., 1990, 1991). Another possible link between the effects of gp120 and NMDA receptor activation arises from the observation that one form of neuronal injury in both the brains of AIDS patients (Masliah et al., 1992) and the brains of rats injected with gp120 (Mervis et al., 1990) or the brains of transgenic gpl20 mice (Toggas et al., 1994) involves dystrophic neurites. These neurites are excessively tortuous and display a paucity of branches. Some of these neurites may be retracting, giving them a “bald” appearance. We and others have found a similar pattern of dystrophic neurites,

Ca2+ CHANNELS, NMDA, AND AlDS-RELATED NEURONAL INJURY

7

including retraction of growth cones, in response to sublethal concentrations of NMDA or glutamate in cultured rat retinal ganglion cells and hippocampal neurons (Mattson et al., 1988; Lipton and Kater, 1989; Offermann et al., 1991). Furthermore, these effects are dependent on influx of Ca2+ into the neurons. These findings indicate that the endpoints for neuronal injury related to gp120 or excitotoxicity should include more subtle changes than death, and these alterations in neuronal cytoarchitecture could have important consequences for neuronal function and plasticity (Lipton and Kater, 1989). T h e simplest potential explanation for all of these findings is that gp120 might simulate an NMDA-evoked current, or somehow augment such currents. T o examine this idea, we used the patch-clamp technique to determine whether gp 120 affected membrane currents. However, in whole-cell recordings, using both conventional and perforated-patch techniques, no effect of picomolar gp120 was observed, even in recordings lasting tens of minutes. Similarly, no enhancement of glutamateor NMDA-evoked currents was encountered (Lipton et al., 1991). Interestingly, nanomolar concentrations of g p 120 (a 1000-fold excess over the levels used in the aforementioned experiments) have been reported to block NMDA receptor-operated ion channels, preventing NMDAevoked increases in Ca2+ influx (Sweetnam et al., 1993). This finding may account, at least in part, for the dose-response curve of gp120induced neuronal injury, which has an inverted “U” shape (Brenneman et al., 1988); that is, at high nanomolar concentrations in contrast to picomolar concentrations, gp 120 no longer induces neuronal cell injury. Nevertheless, during HIV-1 infection in the brain it appears unlikely that nanomolar concentrations of gp 120 actually occur because conventional ELISA and Western blots are sensitive to these concentrations but have failed to detect their presence. The next possible explanation that we considered is that endogenous levels of glutamate become toxic in the presence of picomolar gp120. To test this hypothesis, the enzyme glutamate-pyruvate transaminase (GPT) was used to degrade the endogenous glutamate in our retinal cultures. High-pressure liquid chromatography (HPLC) analysis of amino acids was used to verify glutamate degradation (-25 p M decreased to less than 5 F M ) . Under these conditions, the catabolism of endogenous glutamate in uitro protected neurons from gp 120-induced injury to rat retinal ganglion cells (Lipton et al., 1990, 1991). Recently, Dawson et al. (1993) have also found that 25 ,uM glutamate was necessary in order for them to observe neurotoxicity in rat cortical cultures in the presence of 100 pM gp120. Taken together, these data argue that concurrent activation of NMDA receptors is needed for neuronal injury

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STUART A. LIPTON

by gp120 in AIDS. These experiments d o not tell us, however, whether the action of gp120 is mediated directly on neurons or indirectly via an intervening cell type, such as astrocytes or macrophages/microglia.

V. Indirect Neuronal Injury Mediated by HIV-Infected or gpl20-Stimulated Monocytic Cells

It is still not known definitively whether the adverse effects of gp120 are mediated directly on neurons, via glial cells such as microglia and astrocytes, or by a combination of these mechanisms. To determine at least some of the cell types involved in neurotoxicity, we performed the following experiment. L-Leucine methyl ester was used to deplete monocytoid cells from cultures of mixed glia and neurons. Under these conditions, gp120 no longer injured neurons, suggesting that at least under our culture conditions, macrophages/microglial were necessary to mediate the neurotoxic effects of gp120 (Lipton, 1992~).It is well known that gp120 binds to CD4 on human monocytic as well as lymphocytic cells, and, in fact, this appears to be the major (but probably not exclusive) route of entry of the virus into these cells. Human macrophages, monocytes, and microglia, but apparently not rat or mouse cells, possess the proper CD4 molecule to bind gp120; however, lack of known receptors does not, of course, rule out alternative mechanisms of binding or toxicity. In several laboratories, for instance, it has not been necessary to have human macrophages present to observe gp 120-induced neuronal injury in cultures. Along these same lines, in our laboratory’s cultures of rat retinal cells, anti-rat CD4 antibodies do not block the neuronal injury to retinal ganglion cells engendered by gp 120 which follows an EAA pathway, whereas anti-gp120 antiserum completely blocks this toxic effect (Kaiser et al., 1990). On the other hand, gp120 incubation with the human monocyte cell line THP-1 also produces the release of neurotoxins that follow an EAA pathway to cell injury; antibodies directed against the CD4-binding region of gp120, but not against the V 3 loop of gp120, block this toxic effect (Giulian, 1993). Thus, there appear to be both CD4- and non-CD4-mediated mechanisms of gp 120-induced neuronal injury, in a sense paralleling a similar situation concerning CD4- and non-CD4-mediated mechanisms for viral entry into cells. In conjunction with the data of other laboratories, the aforementioned results suggest the following model of HIV-related neuronal injury (Fig. 1). HIV-infected macrophages (Giulian et al., 1990; Pulliam et al., 1991) or gpl20-stimulated macrophages (Lipton, 1992c; Giulian, 1993) release neurotoxic products. These neurotoxins include relatively

Ca2+ CHANNELS, NMDA, AND AIDS-RELATED NEURONAL INJURY

9

small, heat stable compounds, which have recently begun to be characterized by Gendelman and colleagues (Genis et al., 1992). They found that the products released by HIV-infected macrophages include a product of phospholipase A, activity, platelet-activating factor (PAF), and arachidonic acid and its metabolites. Under their conditions, these substances are released only in the presence of astrocytes, implying some positive feedback loop between astrocytes and macrophages. HIV-infected macrophages also release the cytokines TNF-a and IL-lP, which have been shown to stimulate astrocyte proliferation (Chung and Benveniste, 1990; Selmaj et al., 1990), another feature of HIV encephalitis. In addition, the cytokines present in conditioned medium from lipopolysaccharide (LPS)-treated astrocytes can stimulate HIV- 1 gene expression in monocytic cells (Vitkovic et al., 1990). Under certain in vztro conditions, TNF-a and IL-1 can be associated with the death of oligodendrocytes and by implication, demyelination (see below) (Robbins et al., 1987). Moreover, there are multiple, complex interactions and feedback loops affecting cytokine and arachidonic acid metabolite production by macrophages and astrocytes. For example, TNF-a enhances IL- 1 production in macrophages (Morganati-Kossmann et al., 1992). Arachidonic acid metabolites can influence the production of TNF-a and IL-1P in macrophages, and, in turn, TNF can amplify arachidonic acid metabolism, in response to IL-1. PAF can enhance TNF and IL-1 production, and, in turn, PAF synthesis can be stimulated with TNF, IL-lP, or interferon-y (IFN-.)I)in human monocytes (Pignol el al., 1987; Valone and Epstein, 1988; Valone et al., 1988; Conti et al., 1989; Dubois et al., 1989; Poubelle et al., 1991).Finally, the same arachidonic acid metabolites and cytokines released by HIV-infected macrophages appear to be produced by gpl20-stimulated monocytic cells. For example, this HIV glycoprotein induces the release of arachidonate, its metabolites, TNF-a, and IL-1P from human monocytes (Merrill et al., 1989; Wahl et al., 1989; Merrill and Chen, 1991). It remains to be shown definitively however, that gp 120-stimulated macrophages also release PAF, although these experiments are currently in progress (H. Nottet, H. E. Gendelman, and S. A. Lipton, unpublished observations, 1994). T h e cytokines TNF-a and IL-IP in the amounts produced by HIVinfected or gp120-stimulated macrophages do not appear to be neurotoxic in and of themselves (Genis et al., 1992). Could, however, the arachidonic acid metabolites emanating from HIV-infected or gp 120stimulated macrophages be involved in neurotoxicity? Several arachidonic acid metabolites as well as of PAF have excitatory effects on neurons (Palmer et al., 1981; Kornecki and Ehrlich, 1988, 1991; Lindsberg et al., 1991; Manzini and Meini, 1991a,b; Meini et al., 1992), and this may

10

NOLdI7 ’V LXVnLS

STUART A. LIPTON

1-AIH

FIG. 1. Current models of HIV-related neuronal injury. Previous work has shown that HIV-infected macrophages/microglia release factors that lead to neurotoxicity. These factors include platelet-activating factor (PAF), arachidonic acid and its metabolites, as well as cytokines and other as yet unidentified substances. Macrophages and astrocytes have mutual feedback loops (signified by the reciprocal arrows). The excitatory action of the macrophage factor may lead to an increase in neuronal Ca2+ and the consequent release of glutamate. In turn glutamate overexcites neighboring neurons leading to an increase in intracellular Ca2+,neuronal injury, and subsequent further release of glutamate. This final common pathway of neurotoxic action can be blocked by NMDA antagonists. For certain neurons, this form of damage can also be ameliorated to some degree by calcium channel antagonists or non-NMDA receptor antagonists. The major pathway of entry of HIV-1 into monocytoid cells is via gp120 binding, and therefore it is not surprising that gp120 (or a fragment thereof) is capable of activating uninfected macrophages to release similar factors to those secreted in response to frank HIV infection. Cytokines participate in this cellular network in several ways. For example, HIV infection or gp120 stimulation of macrophages enhances their production of TNFa and IL-lp (solid arrow). The TNF-a and IL-lp produced by macrophages stimulate astrocytosis. Astrocytes appear to feedback (dashed arrow) onto monocytic cells by an as yet unknown mechanism to increase the macrophage production of these cytokines. TNFa may also increase voltage-dependent calcium currents in neurons. Interferon-? (IFNy), known to be elevated in the CNS of patients with AIDS, can induce macrophage/ microgliosis and macrophage production of quinolinate (an NMDA-like agonist) and PAF; in conjunction with IL-Ip, IFN-y can induce nitric oxide synthase (NOS) expression with consequent NO. production in cultured astrocytes and in this manner may potentiate NMDA receptor-mediated neurotoxicity in mixed neuronal-glial cultures. NO. has recently been shown to react with superoxide anion (02.-) to yield a neurotoxic substance, probably in the form on ONOO- (peroxynitrite). It is conceivable that such cytokine stimulation of the inducible form of NOS in macrophages or astrocytes may thereby contribute to HIVrelated neurotoxicity. In addition, the constitutive form of NOS (cNOS)has been implicated

Ca'+ CHANNELS, N M D A , A N D AIDS-RELATED NEURONAL INJURY

11

represent at least one pathway whereby PAF and these arachidonic acid metabolites evoke EAA-induced neurotoxicity. In particular, PAF has recently been shown to increase intracellular neuronal Ca2+and lead to enhanced neurotransmission, apparently by increasing the release of presynaptic glutamate (Bito et al., 1992; Clark et al., 1992). Although the elevated levels of arachidonate and metabolites released from HIVinfected macrophages may cycle up and down, the increased concentration of PAF is a persistent and potentially most important factor (H. E. Gendelman, personal communication, 1994). In collaboration with Gendelman's group, our laboratory has begun a series of experiments which suggest that under specific conditions, the elevated levels of PAF that have been measured in cultures of HlV-infected monocytic cells as well as in the cerebrospinal fluid of patients with the AIDS dementia complex (Genis et al., 1992; H. E. Gendelman, personal communication, 1994) can be toxic to neurons in vitro. It is possible that TNF-a also contributes to this process by increasing voltage-dependent Ca2+ currents (Soliven and Albert, 1992). Additional glutamate receptor activation may occur as a consequence of these events, as neurons are excited or injured and release their stores of glutamate onto neighboring neurons (Lipton et al., 1990b, 1991; Lipton, 1992; Lo et al., 1992). One line of evidence for this supposition lies in the finding, as detailed above, that NMDA antagonists or enzymatic degradation of glutamate ameliorates g p 120induced neuronal injury in mixed neuronal-glial cultures (Lipton et al., 1990, 1991). Moreover, PAF-related neuronal injury also appears to be ameliorated by NMDA antagonists. Intensive investigation in several laboratories is currently under way to study this potential pathway for neuronal injury that is triggered by HIV-infected or gpl20-stimulated macrophages. Also, as alluded to above, another possible link between HIV-1 infection and EAA-induced neurotoxicity involves quinolinate, an endogenous NMDA agonist that is increased in the cerebrospinal fluid of patients in gp120 neurotoxicity; the neuronal form of the enzyme (cNOS) is activated by a rise in intracellular Ca2+after stimulation of the NMDA receptor, and inhibitors of this enzyme have been reported to prevent gp120 neurotoxicity. The coat protein gp120 may have additional direct or indirect effects on astrocytes, e.g., to decrease growth factor production or to inhibit glutamate reuptake, for example, via arachidonic acid. Arachidonate has also been recently reported to enhance NMDA-evoked currents and therefore could contribute to neurotoxicity not only by enhancing net glutamate efflux but also by increasing its effectiveness at the NMDA receptor. Also, we have recently shown that gp120 enhances cysteine secretion from macrophages Cysteine is a known NMDA agonist and could therefore represent at least one of the neurotoxic substances released from stimulated macrophages.

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STUART A. LIPTON

with the AIDS dementia complex (Heyes et al., 1991). Quinolinate levels are known to be influenced by cytokines that are increased after HIV1 infection. For example, it is known that IFN-y is present in the brains of patients with AIDS (Tyor et al., 1992), and human macrophages activated by IFN-y release substantial amounts of quinolinate (Heyes et al., 1992). In addition, under some conditions, e.g., following neuronal loss, quinolate can also be produced by astrocytes (Speciale et al., 1987; Kohler et al., 1988). Quinolinate, therefore, may also contribute to neuronal injury by activating NMDA receptors during HIV infection, although this scenario appears to also be true for a variety of CNS infections. Recently, we have found that another NMDA agonist, cysteine, is released from gp120-stimulated human macrophages, at least in nitro (M. Yeh and S. A. Lipton, in preparation).

VI. Possible Involvement of Astrocytes, Oligodendrocytes, and Other HIV-1 Proteins in Neuronal Injury

A. ASTROCYTES AND HIV-RELATED NEURONAL DAMAGE In at least some model systems, the presence of astrocytes is necessary for HIV-infected macrophages to release substantial amounts of their neurotoxic factors (Genis et al., 1992). In addition, astrocytes may be important in mediating HIV-related neuronal injury in other ways. For example, in murine hippocampal cultures Brenneman et al. (1988) found that gp- 120-induced neurotoxicity can be prevented by the presence of vasoactive intestinal polypeptide (VIP) or by a five amino acid substance with sequence homology t.0 VIP, peptide T. These workers also found that VIP acts on astrocytes to increase oscillations in intracellular Ca2+ and to release factors necessary for normal neuronal outgrowth and survival (Brenneman et al., 1990). Thus, these results raise the possibility that gp120 may compete with endogenous VIP for a receptor, most likely on astrocytes, that is critical to normal neuronal function. The receptor may bear some resemblance to mouse CD4 because mouse anti-CD4 antibodies blocked the toxic effects of gp120 in this system (Brenneman et al., 1988). This effect of gp120 is hypothesized to prevent the release of such astrocyte factors that are necessary to prevent neuronal injury and suggests that one pathway for neuronal damage is an indirect one that is mediated via astrocytes. Although not specifically illustrated in Fig. 1, gp120 might therefore interact with a receptor on

Ca2+ CHANNELS, NMDA, AND AIDS-RELATED NEURONAL INJURY

13

astrocytes. Hence, neurotoxicity may in part be realized by interfering with the normal function of astrocytes and their release of a neuronal growth factor(s) (Giulian et al., 1993) (see Fig. 1). It is also possible that gp120 might affect astrocytes in some other manner, e.g., to inhibit their ability to take up glutamate or even to reverse the reuptake process, resulting in a net efflux, and thus contributing to EAA-induced neurotoxicity. Such an effect would contribute to the apparent increase in sensitivity of neurons to glutaniate toxicity in the presence of gp 120 and would also help explain the requirement for some glutamate (-25 pLM) to be present in the culture medium in order to observe gpl20-induced neurotoxicity (Lipton et al., 1990, 1991; Dawson et al., 1993). Future experiments will have to be designed to explore these possibilities.

B.

gp120 BINDING TO

THE

OLIGODENDROCYTE SURFACE MOLECULEGalC

T h e envelope protein g p 120 has also been shown to bind to galactosyl ceramide (GalC), a molecule on the surface of the oligodendrocyte, which represents the cell type responsible for myelination in the CNS (Bhat et al., 1991; Harouse et al., 1991). Relatively high concentrations of gp120 (nanomolar) were necessary to observe this binding compared to the low (picomolar) concentrations of the coat protein that have been found to lead to neurotoxicity. Nonetheless, the findings concerning binding to GalC raise the possibility of participation of gp120 in myelin disruption and, therefore, a further indirect influence on the welfare of neurons. Future studies will be necessary to determine the significance of this potential pathway for cellular injury. C. gp120 BINDING TO SULFATIDE A N D MYELIN-ASSOCIATED GLYCOPROTEIN Recently, in addition to GalC, gp120 has been shown to bind to sulfatide (Gals), a sulfated glycoprotein implicated in sensory neuritis, and to myelin-associated glycoprotein ( M A G ) , an autoantigen in demyeh a t i n g neuropathy (van den Berg et al., 1992). Similar to GalC, binding became significant in the nanomolar range of gp120. The authors speculate that this binding could have implications for the peripheral nervous system, e.g., in an acute or chronic demyelinating neuropathy or a painful sensory axonal neuropathy such as that frequently observed during HIV infection. However, as alluded to above, the significance of binding to

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nanomolar gp120 levels in the nervous system remains uncertain. The same group of workers has recently published a preliminary report that gp 120 may bind to neurons in immunofluorescence assays (Latov et al., 1993), but it is not clear whether the concentration of gp120 required to see this effect is attained in the CNS during HIV-1 infection.

D. gp 120 ATTENUATES /I-ADRENERGIC STIMULATION OF ASTROCYTES AND MICROCLIA Other effects of gp120 have also been reported. Levi's group have found that picomolar gp 120 can attenuate /I-adrenergic stimulation of CAMPin astrocytes and microglia (Levi et al., 1993). When added alone, gp120 modestly enhanced the basal levels of CAMP. These effects of gp120 could also interfere with P-adrenergic modulation of cytokine production, e.g., of TNF-a. Thus, gp120 may have other, complex effects on glial cells in the CNS.

E. NEUROTOXICITY OF OTHERHIV-1 PROTEINS In addition to gp120, two other HIV-1 proteins have been reported to affect neurons o r neuronal-like cells, raising the possibility of their involvement in HIV-related neuronal injury. T h e nuclear protein tat was shown to be toxic to glioma and neuroblastoma cell lines in uitro and to mice in uiuo (Gourdou et al., 1990; Sabatier et al., 1991). The basic region of the peptide (amino acid residues 49-57) appears to act nonspecifically to increase the leakage conductance of the membrane, thus altering cell permeability. Moreover, neurotoxicity of the related Maedi-Visna virus peptide was ameliorated by NMDA antagonists or by inhibitors of nitric oxide synthase (Hayman et al., 1993),reminiscent of the pharmacology of antagonism of the neurotoxic effects of gp120 and HIV-infected macrophages. Further work will be necessary to attempt to relate these findings with the tat peptide to the neuropathology encountered in the brains of patients with HIV-l-associated cognitive/motor complex. Another HIV-1 protein, Nef, has also been shown to affect neuronal cell function. Nef shares sequence and structural features with scorpion toxin peptides; both recombinant Nef protein and a synthetic portion of scorpion peptide increase total K f current in chick dorsal root ganglion cells (Werner et al,, 199I).

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15

F. DIRECTEFFECTS OF HIV-INFECTED MACROPHAGES ON NEURONS Finally, it is possible that HIV-infected monocytoid cells may have a cytopathic effect on neurons by direct contact (Tardieu et al., 1992). This mechanism does not preclude, however, an additional mechanism of neuronal injury mediated by soluble factors leading to excessive stimulation of NMDA receptors (Lipton, 1993).

WI. Overstimulation of NMDA Receptors, a Final Common Pathway

From the foregoing, there appear to be at least two sites of potential interaction of HIV-related neurotoxins with NMDA receptors (Fig. 1). First, quinolinate emanating from macrophages may directly stimulate neurons. Second, after excitation by quinolinate, PAF, and possibly arachidonic acid metabolites, or after injury due to other toxic pathways, neurons would release glutamate onto second-order neurons and astrocytes might fail to take up the glutamate. This “bad neighbor hypothesis” is in some ways similar to the damage thought to occur in the penumbra of a stroke-glutamate released by injured neurons contributes to further injury to neighboring neurons. Moreover, NMDA antagonists ameliorate HIV-related neuronal injury induced by either HIV-infected macrophages (Giulian et al., 1990; H. E. Gendelman, personal communication, 1993) or, as mentioned earlier, gpl20-activated macrophages (Lipton et al., 1990,199 1). Furthermore, in some cases calcium channel antagonists can attenuate this form of damage [(Dreyer et al., 1990; Lipton, 1991b; L. Pulliam, personal communication, 1991) (Table 11). In general, the pharmacology of neuroprotection from noxious agents depends on the repertoire and diversity of ion channel types in a particular class of neurons (Lipton, 1991b). For example, neurons lacking NMDA receptors will obviously not be protected by NMDA antagonists. Conversely, if NMDA receptorassociated channels are the predominant channel in a specific neuronal cell type whereby Ca2+enters the cell, then the lethal effects of excessive stimulation by glutamate may be ameliorated with NMDA antagonists. Some non-NMDA receptor-associated channels are directly permeable to Ca2+,but most appear not to be (e.g., those containing the GluR2 receptor subunit). However, depolarization of neurons by stimulation of non-NMDA receptors will trigger VDCCs. If sufficient L-type calcium channels exist on a particular neuronal cell type, then the excessive influx

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S T U A R T A. LIPTON

TABLE I1 PROTECTIVE EFFECTS OF CALCIUM CHANNEL ANTAGONISTSAND NMDA ANTAGONISTS AGAINST gp12O-INDUCED A N D HIV-INFECTED MACROPHAGE-MEDIATED NEURONAL INJURY rrv VITRO Attenuation of neuronal iniury by Insult gp120 glycoprotein or fragment HIV-infected macrophage toxin(s)

Ca2+channel anlagonists"

NMDA antagonist

non-NMDA antagonist

+

+

-

+b

+

-

Note. Adapted from Lipton, 1992d. With permission from Hogrefe & Huber Publishers, Seattle. Nimodipine or nifedipine (10-100 nh4 in 5% serum or -4-40 nM free dihydropyridine). L. Pulliam (personal communication, 199I)-results in human brain cell aggregates [but see also earlier work (Ciulian et al., 1990) that found no protective effective of high concentrations of calcium channel antagonists in chick ciliary and rat spinal cord neurons: nonetheless, it is possible that in these latter experiments the high concentration of calcium channel antagonist used was deleterious in and of itself or that these antagonists were not effective in the neuronal cell types tested].

of Ca'+ via these channels could lead to toxic consequences. Hence, in some cell types such as hippocampal pyramidal cells, cortical neurons, and retinal ganglion cells, there is evidence that calcium channel antagonists may attenuate damage due to activation of either NMDA or nonNMDA receptors (Abele et al., 1990; Weiss et al., 1990; Sucher et al., 1991). Along similar lines of reasoning, the release or action of glutamate may be involved in the final common pathway of neuronal injury by HIV-infected macrophages or by gpl20-stimulated macrophages. Thus, either NMDA or non-NMDA receptor activation may play a role in this form of toxicity depending on the exact repertoire of ion channels in a particular cell type. In fact, it has been suggested that non-NMDA receptors could also be important in contributing in the neurotoxic events triggered by gp120 (V. L. Dawson et al., 1992, 1993). Nevertheless, the majority of findings to date suggest that NMDA receptor-mediated neuronal injury plays a predominant role in the pathogenesis of the neurological manifestations of AIDS in the CNS (Lipton, 1992b; Lipton and Jensen, 1992).

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VIII. Development of Clinically Tolerated N M D A Antagonists for HIV-Related Neuronal lniury

NMDA receptors may be involved in HIV-related neurotoxicity at two separate sites, located (a)on the primary neuron injured by factors released from glial cells and (b) on neurons that are secondarily affected (see above and Fig. 1).This fact has provided an impetus for our laboratory to begin a drug development program for clinically tolerated NMDA antagonists, as described below.

A. SITESOF ACTIONOF POTENTIAL CLINICALLY TOLERATED NMDA ANTAGONISTS Despite concerns about the potential complexity of EAA receptor pharmacology, we can consider currently available agents that appear to work on broad classes of these receptors. For the purposes of this review, we will concentrate mainly on NMDA antagonists that appear to be clinically tolerated and therefore can be considered for human trials. There are several modulatory sites on the NMDA receptor-channel complex that could potentially be used to modify the activity of the receptor-operated ion channels and thus to prevent the excessive influx of Ca2+ (Fig. 2). The first site is the glutamate or NMDA binding site. An antagonist acting here would be competitive in nature, i.e., competing for the site with an EAA. For both theoretical and practical reasons, a competitive inhibitor might not be as desirable an antagonist as one that is not competitive for the glutamate binding site. A competitive antagonist would perforce eliminate the normal, physiological activity of the NMDA receptor even before it would affect potentially excessive levels of glutamate. Thus, cognition and memory, thought to be related to long-term potentiation (LTP), might be compromised as well as other important functions mediated by excitatory transmission in the brain. In any event, as part of the disease process, escalating levels of glutamate might be able to overcome o r “out-compete” such an antagonist.

B. NMDA OPEN-CHANNEL BLOCKERS I n contrast, other modulatory sites should be able to inhibit the effects of high levels of glutamate in compromised areas of the brain while

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STUART A. LIPTON

NMDA or giu glycine

J.

FIG.2. Sites of potential antagonist action on the NMDA receptor-channel complex. Competitive antagonists can compete with NMDA or glutamate (glu) for binding to the agonist site. Several antagonists to the glycine coagonist site have been described that are chlorinated and sulfated derivatives of kynurenic acid. These inhibitors do not compete with glutamate and hence are designated noncompetitive. It is not yet known whether any will prove to be tolerated clinically. H + effects are transmitted through another noncompetitive site; decreasing pH acts to downregulate channel activity. Other sites for polyamines and Zn2+ can also be used to affect receptor-channel function. Sites that inhibit channel activity by binding Mg2+or drugs such as MK-801, phencyclidine, and memantine (Mem.) are within the electric field of the channel and are only exposed when the channel is previously opened by agonist (termed uncompetitive antagonism). Finally, a redox modulatory site [probably a disulfide bond, or at least a long-lasting covalent modification of a thiol group, that can be converted to free sulfhydryl groups (S-S + 2-SH)] is affected by chemical reducing and oxidizing agents. Oxidation can favor the disulfide conformation (S-S) over free thiol (-SH) groups and thus downregulate channel activity. Several nitroso compounds can transfer the NO group to the thiol(s) of the NMDA receptor's redox site, producing RS-NO (NO' equivalents), and thus also leading to downregulation of channel activity.

leaving relatively spared the effects of normal neurotransmission in other regions of the brain (Karschin et al., 1988; Levy and Lipton, 1990; Chen et al., 1992). For example, one site that appears to have this advantageous effect is located in the channel itself. There are drugs that only block the channel when it is open; i.e., the antagonist can only gain access to the channel in the open state. On average, escalating levels of glutamate result in the channels remaining open for a greater fraction of time. Under these conditions, there is a better chance for an open-channel blocking drug to enter the channel and block it. The result of such a mechanism of action is that the untoward effects of greater (pathological) concentrations of glutamate are inhibited to a greater extent than those of lower (physiological)concentrations (Chen et al., 1992). Unfortunately, some of these open-channel blockers, which include phencyclidine (angel

Ca*+ CHANNELS, NMDA, A N D AIDS-RELATED NEURONAL INJURY

19

dust) and MK-801 (dizocilpine), have neuropsychiatric side effects and probably cannot be safely administered (Koek et al., 1988). Another concern with NMDA antagonists, such as phencyclidine and MK-801, is the development of reversible neuronal vacuolization (Olney et al., 1989). A problem with MK-801 is that once it enters an open channel, it leaves the channel only very slowly (half time >1 h). In practical terms this means that the degree of blockade builds up after MK-801 administration because each molecule of the antagonist entering a channel effectively does not leave. Several members of this open-channel blocking class of agents, however, are tolerated, such as ketamine and dextromethorphan o r the related molecule dextrorphan (Choi, 1987; Choi et al., 1987; MacDonald et al., 1987; Davies et al., 1988; O’Shaughnessy and Lodge, 1988). Unfortunately, it is not clear whether these particular drugs are sufficiently potent NMDA antagonists at clinically tolerated doses. Nevertheless, the fact that certain members of this open-channel blocker family are clinically tolerated appears to be associated with their rapid kinetics of interaction with the channel (the kinetic parameters are composed of the onrate and off-rate for channel blockade) (Rogawski and Porter, 1990; Chen et al., 1992). Most importantly, the safe drugs, such as memantine (see Bormann, 1989, and the discussion below), leave the channel promptly, with an off-rate -5 s at micromolar concentrations (Chen et al., 1992). Mg2+also blocks open NMDA channels, and this may be the basis for its antiepileptic and neuroprotective effects (Goldman and Finkbeiner, 1988; Wolf et al., 1990,1991). These beneficial effects, however, may not be robust, probably because Mg2+ leaves the channel so quickly that it may not act effectively to offset toxic levels of glutamate. In addition, these charged channel-blocking drugs act to a lesser degree when neurons are depolarized (become more positively charged), e.g., under conditions of energy compromise (Zeevalk and Nicklas, 1992). In summary, an agent that remains in the channel for at least some period of time is necessary to block the effects of glutamate overstimulation. Of the known NMDA open-channel blockers, memantine is one candidate for clinical trials to combat neurological disorders, such as HIV-associated cognitive/motor complex, with a component of NMDA receptor-mediated neurotoxicity because memantine has been used clinically in Germany for over a dozen years in the treatment of Parkinson’s disease and spasticity. Memantine is a congener of amantadine, the wellknown antiviral and antiparkinsonian drug used in the United States. Amantadine, however, is considerably less potent on NMDA receptoroperated ion channels at clinically tolerated doses (Chen et al., 1992),

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probably precluding its use for these other neurological diseases. It may be no accident that memantine both inhibits NMDA receptor responses and alleviates parkinsonian symptoms; one theory of Parkinson’s disease is that neurons die, at least in part, due to a form of NMDA receptormediated toxicity. C. NMDA RECEPTOR REDOXMODULATORY SITE

Another modulatory site on the NMDA receptor-channel complex of possible clinical utility in the near future was discovered several years ago in our laboratory and has been termed the redox modulatory sites (Aizenman et al., 1989). This site consists of one or more sulfhydryl groups; these sulfhydryl groups may possibly be in close approximation and form a disulfide bond under oxidizing conditions. Under chemical reducing conditions that favor the formation of free thiol (-SH) groups over a disulfide, the opening frequency of NMDA receptor-associated channels increases (Aizenman et al., 1989; Tang and Aizenman, 1993), and thus there is a net increase in Ca2+ influx through the channels (Reynolds et al., 1990; Sucher et al., 1990) and an increase in the extent of NMDA receptor-mediated neurotoxicity (Levy et al., 1990; Aizenman and Hartnett, 1992). Conversely, redox reagents that mildly oxidize the NMDA receptor, for example, to reform disulfide bonds o r form ligands on the free thiol groups, might prove useful in combating the myriad of neurological maladies following a final common pathway of NMDA receptor-mediated neuronal damage (Aizenman et al., 1990). Indeed, several such redox reagents have recently been reported, including quite surprisingly the common nitroso compound nitroglycerin (Lei et al., 1992). One mechanism of nitroglycerin’s action in this regard is mediated by a substance related to nitric oxide (NO.), but in a different redox state, for example, in the form of RS-NO (nitrosonium ion equivalents, N O + , with on less electron than NO.) (Stamler et at., 1992). Nitric oxide (NO-) itself can participate in reactions to form products that are toxic to nerve cells, such as peroxynitrite (ONOO-) and its breakdown products including hydroxyl radical (H0.)-like compounds (Beckman el al., 1990; Dawson et al., 1991; Radi et al., 1991; T . M. Dawson et al., 1992; Lei et al., 1992; Lipton et al., 1993). In other redox states, however, monoxides of nitrogen can interact with thiol groups, such as those constituting the redox modulatory sites of the NMDA receptor, by a nitrosylation reaction, resulting in transfer of the NO group to a thiol (Stamler et al., 1992; Lipton et al., 1993). This action results in downregulation of NMDA receptor activity and protects neurons from excessive stimulation of the receptor (Lei et al., 1992). Patients can be made tolerant

Ca2+ CHANNELS, NMDA, AND AIDS-RELATED NEURONAL INJURY

21

to the cardiovascular effects of nitroglycerin within hours of continuous therapy. Under these conditions, our laboratory has shown in animal models that the extent of NMDA receptor-mediated neurotoxicity can be markedly attenuated in the absence of behavioral or systemic side effects of the drug (Manchester et al., 1993). Nevertheless, the exact dosing regimen must be carefully worked out before this technique is applied to humans. Other promising reagents that appear to act either directly or indirectly on the NMDA redox modulatory site include oxidized glutathione (Gilbert et al., 1991; Levy et al., 1991; Sucher and Lipton, 1991) and the putative essential nutrient and redox cofactor pyrroloquinoline quinone (PQQ) (Aizenman et al., 1992). In addition, there are other important modulatory sites of the NMDA receptor, several of which are illustrated in Fig. 2. Antagonists of each of these sites could possibly be useful in the treatment or prevention of NMDA receptor-mediated neurotoxicity. For the purposes of this review, I have chosen to highlight only two of these, the ion channel and redox modulatory sites. The other modulatory sites may become therapeutically relevant, however, if clinically tolerated antagonists can be developed to interact with them. Intensive research efforts along these lines are now under way in both academic institutions and the pharmaceutical industry, which are exploring, for example, antagonists of the glycine coagonist site of the NMDA receptor. Since NMDA and non-NMDA receptor stimulation alike lead to neuronal depolarization and consequent activation of VDCCs, blockade of VDCCs might also ameliorate neurotoxicity, as discussed above. It has become apparent that different subpopulations of neurons have different repertoires of VDCCs, so it might be anticipated that an antagonist specific for a particular type of calcium channel may be effective only in certain regions of the brain o r for certain cell types (vide supra) (Lipton, 1991a,b; Regan et al., 1991). Therefore, it will be important to develop antagonists specific for these various types of calcium channels, and many investigators are working in this area. Currently available in the clinics are CNS-permeable antagonists of the L-type calcium channel, such as nimodipine. Other calcium channel antagonists that are permeable to the blood-brain barrier are also being tested in multicenter trials for entities other than the AIDS dementia complex (Lipton, 1991b).

IX. Excitatory Amino Acid Antagonist Treatments on the Horizon

Among the aforementioned classes of NMDA antagonists, the pharmaceutical industry is currently sponsoring in humans Phase 1/11 studies

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for stroke using the putative open-channel blockers CNS 1102 (Cambridge Neuroscience, Inc.) and dextrorphan (Roche); there is interest in testing the related compound dextromethorphan for amyotrophic lateral sclerosis (ALS). There is some evidence that the dextrorphan-like compounds also antagonize VDCCs as well as NMDA receptor-operated channels, which might be a helpful dual property (Carpenter etal., 1988). Other current clinical trials include a Phase I1 study (for stroke) using the NMDA competitive antagonist CGS 19755 (Ciba-Geigy). All of these trials to date have involved dose escalation and safety. Other companies are currently investigating both NMDA and non-NMDA antagonists, but for proprietary reasons information is scanty, and the indications d o not yet include the AIDS dementia complex. Based on animal testing, it is quite possible that for various forms of glutamate-related neurotoxicity, a combination of agents may be the most effective, e.g., combining calcium channel antagonists with NMDA antagonists (Uematsu et al., 1991; Hewitt and Corbett, 1992; Rod and Auer, 1992). Human clinical studies for indications other than the AIDS dementia complex are also in progress using agents that work downstream from EAA receptors. These include gangliosides (GMl),which are being tested for improvement of outcome after stroke (Rocca et al., 1992), and the 2 l-aminosteroid, tirilazad mesylate. However, recent reports that gangliosides can result in a polyneuropathy resembling Guillain-Barre syndrome have caused several authorities to conclude that clinical studies with ganglioside in humans should be suspended pending further assessment of this problem (Figueras et al., 1992; Raschetti et al., 1992). Finally, a case can be made that the proven NMDA open-channel blocker memantine (as well as its less potent cogener, amantadine) has already been in clinical use for years because it is known to ameliorate some of the symptoms of Parkinson’s disease. Furthermore, it is now known that the level of memantine (2-12 p M ) achieved in the human brain during this form of treatment (Wesemann et al., 1980) can afford protection from NMDA receptor-mediated neurotoxicity both in vitro and in vim (Seif el Nasr et al., 1990; Erdo and Schafer, 1991; Chen et al., 1992; Keilhoff and Wolf, 1992; Osborne and Quack, 1992). Recently, our laboratory and another independent group have reported that low micromolar levels of memantine can also protect neurons from damage induced by gp120 in uitro (Lipton, 1992a; Muller et al., 1992) and in vivo in an animal model (Lipton and Jensen, 1992). These preliminary findings raise the possibility that a clinically tolerated NMDA antagonist, memantine, might be useful in the treatment or prevention of the AIDS dementia complex. Therefore, it has been proposed to study the use of memantine as an adjunctive therapy with anti-retroviral drugs such as

Ca2+ CHANNELS, NMD.4, AND AIDS-RELATED NEURONAL INJURY

23

zidovudine o r didanosine, and the AIDS Clinical Trials Group of the NIH is currently considering this option.

X. Conclusion

Although it is likely that a complex web of cell interactions leads to neuronal loss in AIDS, HIV-infected macrophages or gpl20-stimulated macrophages release toxins whose action appears to be mediated by a final common pathway involving excessive stimulation of neurons by EAAs, such as glutamate and quinolinate. This represents at least one complete pathway to neuronal injury that is amenable to pharmacotherapy, although other pathways may also exist. A strong body of scientific evidence supports the premise that the mechanism for this form of HIV-related neuronal injury is similar to that currently thought to be responsible for a wide variety of acute and chronic neurological diseases (Choi, 1988; Meldrum and Garthwaite, 1990; Lipton, 1992b). EAAs apparently exert this excitotoxic effect by engendering an excessive influx of Ca2+ into neurons. Currently, there is intensive investigation to discover clinically tolerated drugs to combat the neurotoxic effects associated with the excessive stimulation of glutamate receptors or the events triggered downstream to receptor activation. One therapeutic approach has been to use glutamate receptor antagonists, and although several promising drugs are already in hand, additional agents are needed. With the possibility of a final common pathophysiology involving EAA receptors for many disorders of the central nervous system, including at least in part the AIDS dementia complex, the future development and testing of safe and effective EAA antagonists should become a high priority.

Acknowledgments

I thank my co-workers, Drs. E. B. Dreyer, N. J. Sucher, V. H . 4 . Chen, P. K. Kaiser, M. Oyola, S. Lei, J. Pellegrini, D. Zhang, and Y.-B. Choi for insightful discussions, and Dr. D. Leifer for comments on an earlier version of the manuscript. This work was supported by NIH Grants HD29587, EY05477, EY09024, NS07264; the American Foundation for AIDS Research; and an Established Investigator Award from the American Heart Association.

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PROCESSING OF ALZHEIMER AP-AMYLOID PRECURSOR PROTEIN: CELL BIOLOGY, REGULATION, AND ROLE IN ALZHEIMER DISEASE

Sam Gandy* and Paul Greengardt *Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021 and tLaboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021

I. 11. 111. 1V. V.

VI. VII.

VIII.

IX. X.

Introduction Alzheimer Disease Is Associated with an lntracranial Amyloidosis APP Structure Gives Clues to Some of Its Functions APP Is Processed via Several Distinct Enzymatic and Subcellular Pathways “Alternative” Pathways of APP Metabolism Provide Clues to the Source of AP-Am yloid A@-Amyloid Is a Normal Constituent of Body Fluids and the Conditioned Medium of Cultured Cells Evidence Suggests the Existence of an Enzyme, p-Secretase, That Cleaves APP at the Amino Terminus of the AP-Amyloid Domain APP Mutations in Familial Cerebral Amyloidoses Occur within or near the AP-Amyloid Domain, Segregate with Disease in Affected Kindreds, and Yield APP Molecules That Display Some Proamyloidogenic Properties Signal Transduction via Protein Phosphorylation Regulates the Relative Utilization of APP Processing Pathways Beyond AP-Amyloid: Other Molecular Factors in Amyloidogenesis and Factors Differentiating Aging-Related Cerebral Amyloidosis from Alzheimer Disease References

1. Introduction

Alzheimer disease (AD) is characterized by an intracranial amyloidosis that develops in an age-dependent manner, and that appears to be dependent on the production of AP-amyloid by proteolysis of its integral membrane precursor, the Alzheimer A@-amyloidprecursor protein (APP). Evidence causally linking APP to Alzheimer disease has been provided by the discovery of mutations within the APP coding sequence that segregate with disease phenotypes in autosomal dominant familial cerebral amyloidoses, including some types of familial Alzheimer disease (FAD). Although FAD is rare ((10% of all AD), the characteristic clinicopathologiINTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 96

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Copyright B 1994 by Academic Press. Inc. All rights of reproduction in any form reserved.

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SAM GANDY A N D PAUL GREENGARD

cal features-amyloid plaques, neurofibrillary tangles, synaptic and neuronal loss, neurotransmitter deficits, dementia-are apparently indistinguishable when FAD is compared with typical, common, “nonfamilial,” or sporadic AD (SAD). The nature and regulation of pathways for the cellular processing of APP have been extensively characterized and recent data demonstrate that soluble AP-amyloid is released from various cells and tissues in the course of normal cellular metabolism. To date, studies of APP catabolic intermediates and soluble AP-amyloid in SAD tissues and fluids have not provided specific SAD-associated changes in APP metabolism. However, studies of some clinically relevant mutant APP molecules from FAD families have yielded evidence that APP mutations can lead to enhanced generation or aggregability of AP-amyloid, consistent with a pathogenic role in AD. In addition, genetic loci for FAD have been discovered which are distinct from the immediate regulatory and coding regions of the APP gene, indicating that defects in molecules other than APP can also specify cerebral amyloidogenesis and FAD. It remains to be elucidated which, if any, of these rare genetic causes of AD is most relevant to our understanding of typical, comtnon SAD.

II. Alzheimer Disease Is Associated with an lntracranial Amyloidosis

Amyloid is a generic description applied to a heterogeneous class of tissue protein precipitates that have the common feature of @pleated sheet secondary structure, a characteristic that confers affinity for the histochemical dye Congo red (Tomlinson and Corsellis, 1984). Amyloids may be deposited in a general manner throughout the body (systemic amyloids) or confined to a particular organ (e.g., cerebral amyloid). AD is characterized by clinical evidence of cognitive failure in association with cerebral amyloidosis, cerebral intraneuronal neurofibrillary pathology, neuronal and synaptic loss, and neurotransmitter deficits (Tomlinson and Corsellis, 1984). T h e cerebral amyloid of AD is deposited around meningeal and cerebral vessels, as well as in gray matter. In gray matter, the deposits coalesce into structures known as plaques. Parenchymal amyloid plaques are distributed in brain in a characteristic fashion, differentially affecting the various cerebral and cerebellar lobes and cortical laminae. The main constituent of cerebrovascular amyloid was purified and sequenced by Glenner and Wong in 1984. This 40- to 42-amino acid

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polypeptide, designated “P protein” (Glenner and Wong 1984a,b) [or, according to Masters and colleagues (1985), “A4”; now standardized as AP by Husby et al., 19931, is derived from a 695- to 770-amino acid precursor, termed the AP-amyloid precursor protein (APP; Fig. l ) , which was discovered by molecular cloning (Goldgaber et al., 1987; Tanzi et al., 1987, 1988; Kang et al., 1987; Robakis et al., 1987; Ponte et al., 1988; Kitaguchi et al., 1988).

111. APP Structure Gives Clues to Some of Its Functions

T h e deduced amino acid sequence of APP predicts a protein with a single transmembrane domain (Goldgaber et al., 1987; Tanzi et al., 1987, 1988; Kang et al., 1987; Robakis et al., 1987; Ponte et al., 1988; Kitaguchi et al., 1988). Isoform diversity is generated by alternative mRNA splicing, and isoforms of 751 and 770 amino acids include a protease inhibitor domain [“Kunitz-type protease inhibitor” domain (KPI) (Ponte et al., 1988; Tanzi et al., 1988; Kitaguchi e l a l . , 1988)l in the extracellular region

KPI

ox-2

OlA4

n

H

coated pn targeting

Potential phosphorylation sites (lhr-654, Ser-655)

FIG. 1. Structure of the Alzheimer A@-amyloidprecursor protein. (Courtesy of Dr. Gregg Caporaso; numbering according to APP695, Kang et al., 1987. pIA4 domain = Ap domain; see text or Husby et al., 1993.)

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of the APP molecule. The ectodomains of the protease inhibitor-bearing isoforms of APP are identical to molecules that had been identified previously based on their tight association with proteases, and thus were designated “protease nexin 11” (PN-11; Van Nostrand and Cunningham, 1987; Oltersdorf el al., 1989; Van Nostrand et al., 1989). Identical molecules are also present in the platelet a-granules, where they were described under the name “factor XIa inhibitor” (XIaI; Smith et al., 1990; Bush et al., 1990). On degranulation of the platelet, factor XIaI/PN-II/ APP exerts an antiproteolytic effect on activated factor XIa at late steps of the coagulation cascade. Recent evidence suggests that KPIlacking isoforms may also act as regulators of proteolysis (Miyazaki et al., 1993). Another physiological role(s) for APP is yet unknown, although evidence from several independent lines of inquiry suggests that APP may play a role in transmembrane signal transduction (Nishimoto et al., 1993) and/or calcium metabolism (Mattson et al., 1993; Arispe et al., 1993). In addition, potential functional motifs in APP have been recognized by the presence of consensus sequences or by experimental implication. Some of these motifs suggest a role in metal ion binding (Bush et al., 1992),heparin binding (Schubert etal., 1989),cell-cell interaction (Konig et al., 1992), and/or functioning as a receptor for a currently unrecognized ligand (Kang et al., 1987; Chen et al., 1990). In some investigations, Saitoh and colleagues have accumulated evidence that APP may play a role in regulating cell growth (Saitoh et al., 1989). Recently, novel APPlike proteins (APLPs) have been discovered (Wasco et al., 1992,1993; Slunt et al., 1994), suggesting that APP may be a member of a larger family of related molecules. APLPs are highly homologous to APP and to each other, but APLPs lack the A@-amyloiddomain and therefore cannot serve as precursors to AP-amyloid.

IV. APP Is Processed via Several Distinct Enzymatic and Subcellular Pathways

APP is initially synthesized and cotranslationally inserted into membranes in the endoplasmic reticulum (ER). Studies of APP metabolism in the presence of either brefeldin A or monensin have, to date, failed to implicate the ER as an important site for discrete proteolytic processing of APP (Caporaso et al., 1992a). Following its exit from the ER, APP traverses the Golgi apparatus, where it is subjected to N- and O-glycosylation, tyrosyl sulfation, and

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sialylation (Weidmann et al., 1989; Oltersdorf et al., 1990). APP is also phosphorylated in both the extracellular and the cytoplasmic domains and preliminary evidence implies that some of these events may occur in an early compartment of the central vacuolar pathway (Knops et al., 1993; Suzuki et al., 1994).In addition, some APP molecules are chondroitin-sulfated in their ectodomains (Shioi et al., 1992). T h e proteolytic processing steps for APP have been a subject of intense interest, in part due to early evidence that excluded the possibility that AD was frequently associated with APP gene mutations or with disordered APP transcription (Koo et al., 1990). One attractive possibility, then, was that AD might be a disorder of APP processing. This possibility was strengthened by early evidence for an APP processing pathway that precluded AP-amyloid generation (Sisodia et al., 1990; Esch et al., 1990), implying that a defect in this pathway might underlie AD. Several proteolytic cleavage products of APP processing have now been definitively identified by purification and sequencing. The first to be identified (Weidemann etal., 1989)was a fragment that results primarily from the cleavage that occurs within the AP-amyloid domain. A large amino-terminal fragment of the APP extracellular domain [protease nexin-I1 (Van Nostrand and Cunningham, 1987; Oltersdorf et al., 1989; Van Nostrand et al., 1989) or s-APP or APPs, for soluble APP (Citron et al., 1992)], is released into the medium of cultured cells and into the cerebrospinal fluid (Weidemann et al., 1989; Palmert et al., 1989; Oltersdorf et al., 1990), leaving associated with the cell, a small nonamyloidogenic carboxyl-terminal fragment. This pathway is currently designated the a-secretory cleavage/release processing pathway for APP, so-called because the (yet undiscovered) enzyme that performs this nonamyloidogenic cleavage/release has been designated “a-secretase” (Esch el al., 1990; Seubert et al., 1993). Thus, one important processing event in the biology of APP acts to preclude amyloidogenesis by proteolyzing APP within the AP-amyloid domain. Few details are available concerning the molecular nature of asecretase, although it is very likely to be a member of a class of enzymes that regulates the “shedding” of ectodomains from a wide variety of transmembrane molecules, including growth factor precursors, cell adhesion molecules, receptors, and ectoenzymes (Ehlers and Riordan, 1991). Surprisingly, these enzymes appear to act primarily at or near the cell surface and to specify cleavage of substrates at a certain distance from the plasma membrane, largely without regard for the primary sequence surrounding the cleavage site (Sisodia, 1992; also Maruyama et al., 1991; Sahasrabudhe et al., 1992). Based on studies of proteolytic

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processing of the TGF-a precursor and the c-kit ligand precursors [which also appear to be cleaved by similar, cell-surface proteinase activities (Pandiella and Massague, 1991; Pandiella et al., 1992)], it appears that “secretase-like” activities may be heterogeneous at the molecular level (i.e., several individual proteinase species probably exist). This conclusion is based on the observation that, depending on the substrate assayed, slightly different protease inhibitor sensitivity profiles were obtained in studies of TGF-a “secretase” in side-by-side comparison with c-kit ligand family “secretases.” Intracellular signal transduction, especially via protein kinase C, is commonly an important regulatory mechanism for processing of molecules via “secretase-like” pathways (see below). The possibility currently exists that the activities of some secretases are regulated by the phosphorylation state of the enzymes themselves; if true, this would provide the first known examples of proteases whose activities are regulated by their states of phosphorylation.

V. “Alternotive“ Pathways of APP Metabolism ProvideClues to the Source of Ap-Amyloid

Due to issues of peptide conformation, peptide aggregation, and antibody reagent insensitivity, the AP-amyloid molecule was not initially detected as a normal metabolite of APP, neither in brain, nor in cerebrospinal fluid, nor in a cell culture system. In fact, until mid-1992, APamyloid was generally, described as being an “abnormal” metabolite of APP. Instead, early clues into AP-amyloidogenesis were provided by the observation of electrophoretic microheterogeneity of carboxyl-terminal fragments of APP. Such microheterogeneity was detected in association with high-level overexpression of human APP using recombinant vaccinia viruses (Wolf et al., 1990), baculoviruses (Gandy et al., 1992b), or stable transfection (Golde et al., 1992),in association with supraphysiological levels of protein phosphorylation (Buxbaum et al., 1990), and in human cerebral vessels (Tamaoka et al., 1992) and cortex (Estus et al., 1992). Antigenic characterization of carboxyl-terminal fragments of APP in cerebral vessels (Tamaoka et al., 1992) and cortex (Estus et al., 1992),in transfected cells (Golde et nl., 1992),and in the baculoviral overexpression system (Gandy et al., 1992b) provided the evidence that supported the possibility of “alternative” cleavage of APP molecules, giving rise to carboxyl-terminal fragments containing the complete AP-amyloid sequence, which in turn might give rise to AP-amyloid. Protein sequencing of the various putative amyloidogenic carboxyl-terminal species (candi-

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date intermediates in the pathway to AP-amyloid deposition) has recently provided for their definitive identification (Cheung et al., 1994). The “alternative” (i.e.. non-intra-AP) cleavage suggested by this microheterogeneity prompted a search for additional intracellular routes for APP trafficking and cleavage. The existence of trafficking routes other than the a-secretory cleavage/release processing pathway was also suggested by the estimation that only about 20% of immature molecules are recovered as released molecules (in PC-12 cells; Caporaso et al., 1992b). Since evidence failed to suggest the existence of an important degradative pathway for APP in the ER (Caporaso et al., 1992a), several groups undertook experiments to determine whether acidic (endosomal/ lysosomal or trans-Golgi network) compartments of the cell were important in APP metabolism (Cole et al., 1989; Caporaso et al., 1992a; Golde et al., 1992; Haass et al., 1992a; Knops et al., 1992). The possibility of endosomal metabolism of APP was bolstered by the discovery of a clathrin-coated vesicle (CCV) targeting motif in the LDL receptor (Chen et ul., 1990). This motif, NPXY, was required for proper internalization of the LDL receptor and was also present in the sequence of the cytoplasmic tail of APP (Fig. 1). The copurification of APP with CCVs was subsequently demonstrated directly (Nordstedt et al., 1993). T h e fact that APP contains an NPXY motif associates APP with a host of cellsurface receptors and suggests the possibility that APP may be a receptor for a yet undiscovered or unrecognized ligand. I n other efforts to dissect the process of AP-amyloidogenesis, vesicleneutralizing agents (such as chloroquine and ammonium chloride) were applied to cultured cells, and these compounds were associated with greatly enhanced recovery of full-length APP and an array of carboxylterminal fragments, including nonamyloidogenic and potentially amyloidogenic fragments (Caporaso e l ul., 1992a; Estus et al., 1992; Golde et al., 1992; Haass et al., 1992a; Knops et al., 1992). A similar array of fragments was recovered from purified lysosomes (Haass et al., 1992a). This led to the formulation that both the potentially amyloidogenic carboxyl-terminal fragments and A@-amyloidmight be generated primarily in lysosomes. However, no AP-amyloid could be recovered from lysosomes (Haass et al., 1993), making this a less likely (but not impossible) scenario. T h e likelihood that AP-amyloid is generated in lysosomes was further diminished by the observation that vesicular neutralization fails to diminish consistently AP-amyloid production in certain cell types (Busciglio et al., 1993; see also below), although neutralization-induced stabilization of the standard array of potentially amyloidogenic carboxylterminal fragments is consistently apparent.

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VI. Ap-Amyloid Is a Normal Constituent of Body Fluids and the Conditioned Medium of Cultured Cells

Until mid-1992, the prevailing notion of A@-aniyloidwas that of an abnormal, potentially toxic species, the production of which was perhaps relatively restricted to the brain in humans (and perhaps a few other species), and which occurred primarily in association with aging and AD. This concept became obsolete with the discovery by several groups that a soluble AP-amyloid species (presumably a forerunner of the aggregated fibrillar species that is deposited in senile plaque cores) is detectable in body fluids from various species and in the conditioned medium of cultured cells (Haass et al., 1992b; Seubert et al., 1992; Shoji et al., 1992), but is not detectable in the lysates of cultured cells. This so-called “soluble AP-amyloid” is apparently generated in a cellular compartment distal to the ER since brefeldin abolishes its generation and does not result in its accumulation inside cells (Haass et al., 1993). Vesicular neutralization compounds are effective in inhibiting AP-amyloid release from some cell types (Shoji et al., 1992), but this is not true for all cell types studied (Busciglio et al., 1993). The precise cellular locus (loci) involved in the amino- and carboxyl-terminal cleavages responsible for AP-amyloid generation has(have) not yet been unequivocally established. T h e consistent inability to recover AP-amyloid from cell lysates or from purified vesicles has led to a shift in focus away from the terminal degradative compartments of the cell (i.e., lysosomes) as possible sources for the generation of AP-amyloid. One plausible scenario for AP-amyloid production is that cleavage at the AD-amyloid amino-terminus is catalyzed by p-secretase (see below) in the precell surface limb of the constitutive secretory pathway, perhaps beginning in the trans-Golgi network (TGN). Cell-type-dependent variations in sensitivity of the TGN to neutralizing compounds may explain the observed dissociability of APamyloid generation from the apparent stabilization by these compounds of potentially amyloidogenic carboxyl-terminal fragments. Still unexplained is the cellular mechanism by which the carboxylterminus of AP-amyloid is generated, since this region of the APP molecule resides within an intramembranous domain. A plausible and conventional scenario for this step might involve the trafficking of APP or a potentially amyloidogenic fragment into a multivesicular body where vesiculated APP or an APP fragment may be liberated from the bilayer (Candy et al., 1992b). This is supported by ultrastructural evidence that multivesicular bodies are immunoreactive for APP epitopes (Caporaso et al., 1994). A multivesicular body containing wholly intraluminal

ALZHEIMER AP-AMYLOID PRECURSOR PROTEIN

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AP-amyloid could effect release of A@-amyloid into the extracellular space.

VII. Evidence Suggests the Existence of an Enzyme, p-Secretase, That Cleaves APP at the Amino Terminus of the Ap-Amyloid Domain

T h e possibility of heterogeneous cleavage along the constitutive secretory pathway (i.e., cleavage in the precell surface pathway or at the cellsurface) was initially discounted (Golde et al., 1992). However, Seubert and colleagues (1993) extended this line of investigation and succeeded in preparing an antibody that was specific for the free methionyl residue that would reside at the predicted carboxyl-terminus of such an alternatively cleaved and released attenuated PN-II-like (or APPs-like) molecule. This species was successfully detected as a component of the PN-II/APPs pool of cleaved and released APP ectodomains. The importance of this activity, designated “p-secretase,” was subsequently established by the discovery that a pathogenic FAD mutation in APP results in dramatic increases in AP-amyloid generation, which is probably attributable to an increase in P-secretase-type cleavage of APP, because the mutation enhances the substrate properties for cleavage by P-secretase (Felsenstein et al., 1994).

VIII. APP Mutations in Familic Cerebral Amyloidoses Occur within or near the A@Amyloid Domain, Segregate with Disease in Affected Kindreds, and Yield APP Molecules That Display Some Proamyloidogenic Properties

Certain mutations associated with familial cerebral amyloidoses have been identified within or near the AP-amyloid region of the coding sequence of the APP gene. These mutations segregate with the clinical phenotypes of either hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWAD or FAD-Dutch; Fig. 1 ; van Duinen e t a ) . , 1987; Levy et al., 1990; Van Broeckhoven et al., 1990) or more typical familial Alzheimer disease (FAD; Fig. 1 ; Goate et al., 1991; Naruse et al., 1991; Murrell et al., 1991; Chartier-Harlin et al., 1991; Mullan et al., 1992a), and provide support for the notion that aberrant APP metabolism is a key feature of AD.

38

SAM CANDY AND PAUL CREENGARD

In FAD-Dutch, an uncharged glutamine residue is substituted for a charged glutamate residue at position 693 of APP770.This mutated residue is located in the extracellular region of APP, within the AP-amyloid domain, where it apparently exerts its proamyloidogenic effect by generating AP-amyloid molecules that bear enhanced aggregation properties (Wisniewski et al., 1991). Mutations in APP that are apparently pathogenic for more typical FAD have also been discovered. In the first discovered FAD mutation (Goate et al., 199l),an isoleucine residue is substituted for a valine residue at position 717 of APP,,,, within the transmembrane domain (Fig. l), at a position just downstream from the carboxyl-terminus of the APamyloid domain. Although a conservative substitution, the mutation segregates with FAD in pedigrees of American, European, and Asian origins, arguing against the possibility that the mutations represent irrelevant polymorphisms. Other pedigrees have been discovered in which affected members have either phenylalanyl (Murrell et al., 1991)or glycyl (Chartier-Harlin et al., 1991) residues at position 7 17. Neuropathological examination has verified the similarity of these individuals to typical SAD neuropathology (reviewed by Rossor, 1992; see also Lantos et al., 1992; Mann et al., 1992; Ghetti et al., 1992; Cairns et al., 1993; Kennedy et al., 1993). Although the 717 mutant APPs are the most common of the FADcausing APP mutations, the mechanism by which the 717-mutant APPs exert their effects remains to be clarified. The location of the missense substitution raises the possibility either that the mutation may directly affect proteolytic cleavage (e.g., by leading to the production of extended, perhaps more hydrophobic and thus hyperaggregable AP-amyloid molecules; Cai et al., 1993) or that the mutation may otherwise influence the function, trafficking, or biology of the APP molecule. Missense mutations in other integral molecules are associated with alterations in their biological activities (e.g., the oncogene neu, Bargmann et al., 1986), their trafficking and proteolysis (e.g., T-cell receptor, Bonifacino et al., 1990), or their ability to effect functional physiological changes in response to phosphorylation of their cytoplasmic domains (e.g., CFTR, Schoumacher el al., 1987; Li et al., 1988, 1989; Hwang et al., 1989; Wagner et al., 1991). It has also been hypothesized that the FAD mutation may lead to abnormal APP translation as a result of a disturbance in the secondary structure of APP mRNA (Tanzi and Hyman, 1991; D. Goldgaber, personal communication, 1991). Which, if any, of these models accounts for the pathogenesis of APP-717 mutant FAD remains a mystery. Another FAD pedigree has been discovered and has proven to be substantially more informative in elucidating the cell biological conse-

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quences of the pathogenic mutation. In a large Swedish kindred, tandem missense mutations occur at the amino terminus of the AP-amyloid domain (Mullan et al., 1992a). Transfection of cultured cells with APP molecules containing the “Swedish” missense mutations results in the production of six- to eightfold excess soluble A@-amyloidabove that generated from wild-type APP (Citron et al., 1992; Cai et al., 1993). This is the first (and, to date, only) example of Alzheimer disease apparently caused by excessive AP-amyloid production. Based on the models of FAD-Dutch and FAD-Swedish, an important issue for clarification in sporadic Alzheimer disease will be to establish whether hyperaggregation or hyperproduction of AP-amyloid (or neither) is an important predisposing factor(s) to this much more commonly encountered clinical entity.

IX. Signal Transduction via Protein Phosphorylation Regulates the Relative Utilization of APP Processing Pathways

As noted, the protease that cleaves APP within the A@-amyloiddomain, as part of the nonamyloidogenic cleavage/release pathway (asecretase), and the proteases that cleave APP at other sites within the molecule to generate AP-am yloid (P-secretase and perhaps others) have not yet been identified. Nevertheless, some progress has been made toward understanding the regulation of APP cleavage. For example, the relative utilization of the various alternative APP processing pathways appears to be at least partially cell-type determined, with transfected AtT20 cells secreting virtually all APP molecules (Overly et al., 1991) whereas glia release little or none (Haass et al., 1991). In neuronal-like cells, the state of differentiation also plays a role in determining the relative utilization of the pathways (Baskin et al., 1992; Hung et al., 1992), with the differentiated neuronal phenotype being associated with relatively diminished basal utilization of the nonamyloidogenic asecretase cleavage/release pathway (Hung et al., 1992). Certain signal transduction systems that involve protein phosphorylation are potent regulators of APP cleavage, acting in some cases, perhaps, by altering the relative activity of nonamyloidogenic cleavage by asecretase. The role of protein kinase C (PKC) in this process has received the most attention. In many types of cultured cells, activation of PKC by phorbol esters dramatically stimulates APP proteolysis (Buxbaum et al., 1990) and cleavage/release (Caporaso et al., 1992b; Gillespie et al., 1992; Sinha and Lieberburg, 1992) via the a-secretase pathway. PKC-

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SAM GANDY AND PAUL GREENGARD

stimulated a-secretory cleavage of APP may also be induced by the application of neurotransmitters and other first messenger compounds whose receptors are linked to PKC (Nitsch et al., 1992; Buxbaum et al., 1992; Lahiri et al., 1992). Okadaic acid, an inhibitor of protein phosphatases 1 and 2A (Cohen et al., 1990), also increases APP proteolysis and release via thea-secretase pathway (Buxbaum etal., 1990; Caporasoetal., 1992b). Thus, either stimulation of PKC or inhibition of protein phosphatases 1 and 2A is sufficient to produce a dramatic acceleration of nonamyloidogenic APP degradation. Furthermore, this PKC-activated processing can be demonstrated to occur at the expense of amyloidogenic APP degradation, resulting in diminished generation of AP-amyloid ( S . Sinha, 1992; Buxbaum et al., 1993; Hung et al., 1993). These results suggest that defects in signal-dependent regulation of APP cleavage may contribute to the pathogenesis of AD, a possibility supported by evidence that deficits in cholinergic neurotransmission (Davies and Maloney, 19’76) and in protein kinase C activity (Cole et al., 1988; Van Huynh et al., 1989; Masliah et al., 1991) accompany AD. By extension, then, the possibility exists that pharmacological modulation of APP metabolism via signal transduction might be therapeutically beneficial in individuals with AD (Candy et al., 1991, 199237; Gandy and Greengard, 1992). Complicating these notions, however, is the observation that PKC is also a potent regulator of APP expression (Goldgaber et al., 1989), although these pleiotropic effects of PKC may be dissociable at the level of the PKC isoenzyme involved (Hata et al., 1993). In addition to the attention to regulation of the nonamyloidogenic a-secretase pathway as a source of candidate etiologic defects and therapeutic opportunities, it may also be fruitful to study the potentially amyloidogenic psecretase pathway in an analogous fashion. Further work will be required to elucidate the importance of signal transduction systems as important candidate defects or therapeutic targets in AD. The enormous pharmacological experience with compounds that affect signal transduction makes such an approach particularly attractive for targeting therapy. The probable causal relationship between aberrant protein phosphorylation and neurofibrillary tangle formation (another component of Alzheimer structural pathology) adds to the attractiveness of protein phosphorylation pathways as potential therapeutic targets in AD. The mechanism by which stimulation or inhibition of intracellular protein phosphorylation regulates the processing of APP (including evaluation of the effect of changing the phosphorylation state of APP per se) remains to be fully elucidated. Protein kinase C rapidly phosphorylates a seryl residue in the cytoplasmic domain of APP (Fig. I), using either a synthetic peptide (Gandy et al., 1988; Suzuki et al., 1992)or APP holopro-

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tein (Suzuki et al., 1992) as substrate. Moreover, APP species are phosphorylated on this and other seryl and threonyl residues in intact cells and in brain (Suzuki et al., 1994; M. Oishi, T. Suzuki, A. Czernik, A. C. Nairn, and P. Greengard, personal communication). Characterization of the various APP residues phosphorylated in intact cells is under way to determine which sites of phosphorylation are utilized and to determine the possible existence of novel APP phosphorylation sites and APP kinases (Knops et d.,1993; Hung and Selkoe, 1994; Suzuki et d.,1994; Oishi et al., personal communication). Once the sites for APP phosphorylation in intact cells are established, analysis of the processing of phosphorylation-site mutant APP molecules can be used to elucidate the role of direct phosphorylation of APP. This approach has already been applied to certain cytoplasmic phosphorylation sites in APP (da Cruz e Silva et al., 1993; Hung and Selkoe, 1994; Vitek et al., personal communication). These experiments have demonstrated that changes in the phosphorylation state of the APP cytoplasmic domain are not necessary for the phenomenon of phosphorylation-regulated a-secretory cleavage of APP to occur. These observations have led to the proposal that proteins of the processing/cleavage/release pathway may be phosphoprotein mediators of “regulated-” o r “activatedprocessing” (da Cruz e Silva et al., 1993; Hung and Selkoe, 1994). Activation of proteolysis by phosphorylation has been demonstrated for a number of integral membrane proteins, including the polyimmunoglobulin receptor (pIgR; Casanova et al., 1990), the transforming growth factor-a (TGF-a) precursor (Pandiella and Massague, 199l ) , and the receptor for colony-stimulating factor- 1 (CSFl R; Downing et al., 1989). Direct phosphorylation of pIgR appears to be crucial to the activation of its trafficking and processing; phosphorylation of the TGF-a precursor has not been demonstrated; CSFlR is known to be a phosphoprotein, but the relationship between its phosphorylation and its proteolysis is not yet established. In general terms, the possible mechanisms for activated processing of integral molecules can be conceptualized as involving either activation or redistribution of either the substrate (i.e., APP) or the enzyme (i.e., secretase). Based on the APP cytoplasmic tail mutational analyses described above (da Cruz e Silva et al., 1993; Hung and Selkoe, 1994), the “substrate activation” model (Gandy et al., 1988, 1991, 1992a,b) is inadequate to explain activated processing of APP. Furthermore, in recent immunofluorescent studies of APP in cultured cells that were incubated in the absence or presence of PKC-activating phorbol esters (Caporaso et al., 1994),no obvious phorbol-dependent redistribution of APP immunoreactivity was apparent at steady state. A more detailed

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analysis of APP distribution following PKC activation is underway, as suggested by the model of Luini and De Matteis (1993). Along a related line of investigation, Bosenberg and colleagues (1993) have succeeded in demonstrating apparently faithful activated processing of TGF-a precursor in porated cells in the virtual absence of cytosol, and in the presence of N-ethylmaleimide or 2.5 M NaCI. The preservation of activated processing under such conditions suggests that extensive vesicular trafficking is probably not required for activated processing of TGF-a and is consistent with a model of enzyme activation by direct phosphorylation. Studies are under way to determine whether activated APP processing has similar features.

X. Beyond Ap-Amyloid: Other Molecular Factors in Amyloidogenesis and Factors Differentiating Aging-Related Cerebral Amyloidosis from Alsheimer Disease

Since APP can be metabolized along several nonamyloidogenic or potentially amyloidogenic pathways, AD might be a clinicopathological phenotype that is due to a metabolic imbalance of the relative utilization of a nonamyloidogenic pathway(s) versus a potentially amyloidogenic pathway(s). To examine a possible correlation between APP metabolic pathway utilization and AD, some investigators have sought to identify AD-related changes in APP metabolism. Diminished levels of the large amino-terminal fragment of APP have been reported in the cerebrospinal fluid from patients with AD and from patients with the cerebrovascular AP-amyloidosis HCHWAD or FAD-Dutch (van Nostrand et al., 1992a,b). According to these reports, decreased levels of the released APP aminoterminal fragment were characteristic of the CSF from AD and FADDutch patients, but not that from age-matched controls, although there was some overlap between AD patients and patients with non-Alzheimertype dementia. To date, however, AD-specific changes in the levels of potentially amyloidogenic carboxyl-terminal fragments have not been observed in AD cortex (Nordstedt et al., 1991; Estus et al., 1992). Further, as noted in a preceding section, the metabolism of some mutant APP molecules and their carboxyl-terminal fragments in transfected cells appears to proceed in standard fashion (Cai et al., 1993; Felsenstein and Lewis-Higgins, 1993) (including apparently "normal" secretory cleavage), unperturbed by the presence of either the APP7"-"' FAD mutation o r the APP693-"1"FAD-Dutch mutation (numbering according to APP,7, isoform).

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CSF levels of soluble AP-amyloid in normal aging and AD have been investigated to determine whether a correlation exists between CSF soluble AP-amyloid levels and the predisposition to AD. An initial study failed to detect an obvious relationship (Shoji et al., 1992), and that observation has been recently confirmed (Wisniewski et al., 1993). Thus, there appear to be other important factors-perhaps downstream events in the metabolism of APP fragments or soluble AP-amyloid-that play key roles in AP-fibrillogenesis. In support of this latter possibility is the evidence that an important effect of the FAD-Dutch mutation is to accelerate A@-amyloidfibril formation (Wisniewski et al., 1991). Other factors contributing to A@-amyloiddeposition and fibril formation may include the processing of soluble AP-amyloid into an aggregated form (Burdick et al., 1992; Dyrks et al., 1992) and/or the association of APamyloid with other molecules, such as a,-ACT (Abraham et al., 1988), heparan sulfate proteoglycan (Snow et al., 1992), apolipoprotein E (Wisniewski and Frangione, 1992; Strittmatter et al., 1993), and P component (Wisniewski and Frangione. 1992). In addition, deposited AP-amyloid plaques may serve as nucleation foci and act to recruit additional APamyloid deposition (Maggio et al., 1992). Events beyond AP-amyloid deposition may also be crucial in determining the eventual toxicity of AP-amyloid plaques. Although aggregation of A@-amyloidis important for in vitro models of neurotoxicity (Mattson and Rydel, 1992; Pike et al., 1993), the relevance of these phenomena for the pathogenesis of AD is unclear, since AP-amyloid deposits may occur in normal aging, in the absence of any evident proximate neuronal injury (Crystal et al., 1988; Masliah et al., 1990; Berg et al., 1993; Delaere et al., 1993). This suggests that other events must distinguish “simple“ cerebral amyloidosis from “full-blown” AD. One intriguing possible contributing factor is the association of complement components with AD-amyloid (Rogers et al., 1992). In cerebellum, where AP-amyloid deposits appear to cause no injury, plaques are apparently free of associated complement, whereas in the forebrain, complement associates with plaques, perhaps becoming activated and injuring the surrounding cells (Lue and Rogers, 1992). Other, yet undiscovered, plaque-associated molecules may also play important roles. It is also possible that Alzheimer neuropathology may be a final end product that can develop through a host of independent initiating molecular abnormalities, analogous to the manner in which disorders of either oxygen radical metabolism (Rosen et al., 1993) or cytoskeletal protein expression (Brady, 1993; Cote et al., 1993; Xu et al., 1993) can lead to a clinicopathological picture of motor neuron disease. Similarly, in the

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case of Alzheimer disease, it is unknown whether, for example, in some situations, cytoskeletal phosphorylation abnormalities could be initiating events, and AP-amyloid deposits could occur secondarily. In support of this possibility is the recent demonstration that toxin- or lesion-induced nerve terminal degeneration can be associated with altered, potentially amyloidogenic APP metabolism (Iverfeldt et al., 1993). Further, A@amyloid deposition may “decorate” the periphery of amyloid plaques primarily composed of prion protein (Ikeda et al., 1992). The most promising leads for furthering our understanding of the molecular pathology of AD beyond APP metabolism lie in elucidating the role of apolipoprotein E allelic variation in determining predisposition to SAD (Saunders et al., 1993) and in the eventual discovery of the gene that causes the most common form of FAD, a form caused by a gene that resides on chromosome 14 (Schellenberg et al., 1992; St. GeorgeHyslop et al., 1992; Van Broeckhoven et al., 1992; Mullan et al., 199213). T h e identity of this gene is entirely unknown: it may represent a molecule that regulates APP expression or degradation, analogous to the lysozyme protease enzyme defect that was recently discovered to underlie hereditary systemic amyloidosis (Pepys et al., 1993). Alternatively, the chromosome 14 mutant molecule may implicate neurofibrillary components or may point in an entirely unexpected direction. In any event, discovery of the chromosome 14 FAD gene may prove to be an important step toward the eventual unravelling of the molecular basis of typical, common SAD, and it is this information that offers the most promise for ultimately providing us with a full understanding of Alzheimer disease and enabling its rational treatment. Acknowledgments

This work was supported by USPHS grants AG-11508 (to S.G.), and AG-09464 and AG-10491 (to P.G.). S.G. is the recipient of a Cornell Scholar Award in the Biomedical Sciences. The authors thank Drs. S. Sisodia and D. Selkoe for their critical review of the manuscript.

References

Abraham, C. R., Selkoe, D. J., and Potter, H. (1988). Cell (Cambridge, Mass.) 52,487-501. Arispe, N., Rojas, E., and Pollard, H. B. (1993). Proc. Null. Acad. Scz. U.S.A. 90, 567-571.

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Suzuki, T., Nairn, A. C., Candy, S. E., and Greengard, P. (19Y2).Neuroscience (Oxford)48, 755-76 1. Suzuki, T., Oishi, M., Marshak, D. R., Czernik, A. J., Nairn, A. C., and Greengard, P. (1994). E M B O J . 13, 1114-1122. 1-amaoka, A., Kalaria, R. N . , Lieberburg, I., and Selkoe, D. (1992). Proc. Natl. Acad. Sci. U.S.A. 89, 1345-1349. Tanzi, R., and Hyman, B. (1991). Nature (London) 350, 564. Tanzi, R. E., Gusella, J. F., Watkins, P. C . , Bruns, C . A. P., St. George-Hyslop, P., Van Keuren, M. L., Patterson, D., Pagan, S., Kurnit, D. M., and Neve, R. L. (1987). Science 235, 880-884. Tanzi, R. E., McClatchey, A. I., Lamperti, E. D., Villa-Kornaroff, L., Gusella, J. F., and Neve, R. L. (1988). Nature (London) 331, 528-530. Tomlinson, B. E., and Corsellis, J. A. N. (1984). In “Greenfield’s Neuropathology” Fourth edition. (J. H. Adams, J. A. N . Corsellis, and L. W. Duchen, eds.), 4th Ed. pp. 95- 1025. New York. Van Broeckhoven. C., Haan, I . , Bakker, E., Hardy, J. A,, Van Hul, W., Wehnert, A., Vegter-Van der Vlis, M., and Roos, R. A. C. (1990). Science 248, 1120-1 122. Van Broeckhoven, C., Backhovens, H., Cruts, M., De Winter, G., Bruyland, M., Cras. P., and Martin, J.-J. (1992). Nut. Genet. 2, 335-339. van Duinen, S. G., Castaiio, E. M., Prelli, F.. Bots, G. T h . A. M., Luyendijk, W., and Frangione, B. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 5991-5994. Van Huynh, T., Cole, G., Katzman, R., Huang, K.-P., and Saitoh, T. (1989). Arch. Neurol. 46, 1195-1 199. Van Nostrand, W. E., and Cunningham, D. D. (1987).J. Biol. Chem. 262, 8508-8514. Van Nostrand, W. E., Wagner, S. L., Suzuki, M., Choi, B. H., Farrow, J. S., Geddes, J. W., Cotman, C. W., and Cunningham, D. D. (1989). Nature (London)341, 546-549. Van Nostrand, W., Wagner, S.. Shankle, W. R., Farrow, J. S., Dick, M., Rozemuller, J. M., Kuiper, M. A., Wolters, E. C., Zimmerman, J., Cotman, C. W., and Cunningham, D. D. (1992a). Proc. Natl. Acad. Sci. U.S.A. 89, 2551-2555. Van Nostrand, W. E., Wagner, S. L., Haan, J . , Bakker, E., and Roos, R. A. (1992b).Ann. Neurol. 32, 215-218. Wagner, J., Cozens, A,, Schulman, H., Gruenert, D., Stryer, L., and Gardner, P. (1991). Nature (London) 349, 793-796. Wasco, W., Bupp, K., Magendantz, M., Gusella,,j. F., Tanzi, R. E., and Solomon, F. (1992). Proc. Natl. Acad. Scz. U.S.A. 89, 10758-10762. Wasco, W., Gurubhagavatula, S., Paradis, M. D., Romano, D. M., Sisodia, S. S., Hyrnan, R. T., Neve, R. L., and Tanzi, R. E. (1993). Proc. Natl. Acad. Scz. U.S.A. 5, 95-100. Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., and Beyreuther, K. (1989). Cell (Cambridge, Mass.)57, 115-126. Wisniewski, T., and Frangione, B. (1992). Neurosci. Lett. 135, 235-238. Wisniewski, T., Ghiso, J., and Frangione, B. (1991). Biochem. Biophys. Res. Commun. 179, 1247-1254. Wisniewski, T., Wegiel, J., Wisniewski, H. M., and Frangione, B. (1993). Neurology 43, A422. Wolf, D.. Quon, D., Wang, Y., and Cordell, B. (1990). EMBO J. 9, 2079-2084. Xu, Z., Cork, L. C., Griffin, J. W., and Cleveland, D. W. (1993). Cell (Cambrzdge, Mass.) 73, 23-33.

MOLECULAR NEUROBIOLOGY OF THE GABAA RECEPTOR

Susan M. J. Dunn, Alan N. Bateson, and Ian L. Martin Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

I. Introduction 11. Pharmacology of the GABA,, Receptor A. T h e GABA Site B. T h e Benzodiazepine Site C. T h e Barbiturate Site D. T h e Steroid Site 111. Biochemistry A. Receptor Isolation B. Ligand Binding Sites C. Protein Modification D. Immunological Characterization IV. Molecular Cloning of Receptor Subunits A. Initial Isolation of Receptor Subunit cDNAs B. lsolation of a cDNA Encoding a y2Subunit C. Receptor Heterogeneity Revealed hy Multiple Subunits D. Further Heterogeneity Arises from Alternate Splicing V. Characterization of the Receptor Family A. Heterologous Expression Reveals Different Functional Attributes of GABA,A Receptor Subunits B. Gene Expression C. Immunocytochernical Localization D. Assembly of Subunits VI. T h e Future References

I. Introduction

The first indication that y-aminobutyric acid (GABA) played an important role in the mammalian central nervous system dates back to the late 1940s when high concentrations of this amino acid were identified in mouse brain (Roberts and Frankel, 1949, 1950). However, the initial evidence that GABA functioned as a neurotransmitter, and was not merely an essential amino acid, came from studies with invertebrates. Application of GABA to isolated muscle fibers from lobster and crayfish was found to produce an attenuation of the stretch-receptor discharge (Bazemore et al., 1957). Later, stimulation of the inhibitory nerves inINTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 36

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nervating lobster skeletal muscle was found to result in the release of GABA into the extracellular space, whereas similar stimulation of the excitatory neurons in this preparation did not (Kravitz et al., 1962). In mammals, GABA is essentially localized to the central nervous system where it exhibits a differential topographical distribution (lversen and Bloom, 1972; Ottersen and Storm-Mathison, 1984). It is located in nerve terminals (Neal and Iversen, 1969) from which it can be released by depolarizing stimulii in a calcium-dependent manner (Bradford, 1970). Subsequent to release it is removed from the synaptic cleft by high affinity uptake systems (Iversen and Neal, 1968). These transporters are present in both neurons and glia, although the substrate specificity in the two cell types appears to be different (Iversen and Kelly, 1975). In the mammalian CNS, iontophoretic application of GABA usually results in an inhibitory hyperpolarizing response (Krnjevic and Schwartz, 1967; Obata et al., 1970), which is blocked by the alkaloid bicuculline (Curtis et al., 1970). The hyperpolarizing response is due to an increase in the chloride conductance of neuronal membranes (Curtis et al., 1968; Kelly et al., 1969) resulting in the passage of chloride ions into the cell. However, depolarizing responses have also been observed in superior cervical ganglion cells (Adams and Brown, 1975; Gallagher et al., 1978) and synaptic terminals (Schmidt, 1971). The receptor that responds to GABA application with an increase in chloride conductance of the neuronal membrane and is blocked by bicuculline is now termed the GABAA receptor. Until the late 1970s this was the only recognized response to the application of GABA. Subsequently bicuculline-insensitive GABA responses that could be mimicked by baclofen (P-p-chlorophenyl-GABA) were found, suggesting the existence of a novel type of GABA receptor (Bowery et al., 1980),now commonly known as the GABABreceptor. This receptor appears to be G-protein coupled giving rise to both decreases in calcium conductance and increases in potassium conductance ( Wojcik et al., 1989). Although the GABAA receptor of the invertebrate exhibits many properties in common with those found in vertebrates, only the GABAA receptor of the vertebrate CNS will be discussed in the remainder of this review.

II. Pharmacology of the GABAA Receptor

The GABAA receptor i s responsible for the majority of neuronal inhibition in the vertebrate CNS. It is widely distributed in the CNS and

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it has received considerable attention as the site of action of a number of important centrally acting drugs, probably the most visible of which are the benzodiazepines. Recent electron microscope images of the receptor, purified from pig brain, show that it is structurally similar to the nicotinic acetylcholine receptor, It comprises five distinct protein subunits arranged in a pentameric array around a central pit that presumably forms the ion channel (Nayeem et al., 1994). The precise identity of the five proteins that form this structure are unknown for any GABA, receptor in vivo. Molecular cloning studies have now revealed the existence of a large family of gene products that may assemble to produce functional GABA, receptors (Section IV). Hence the GABA, receptor in the mammalian brain is not a single entity, but a family of closely related receptor oligomers with properties that are similar but distinct. Much of our understanding of the pharmacology of the GABAA receptor has been obtained from the study of neurones of the mammalian CNS. This will be reviewed briefly here to provide a backgound for the detailed discussion that will appear later in this review; for convenience we will address the subject in terms of the recognition sites on the receptor complex that have been the most extensively studied.

A. THEGABA SITE T h e ubiquitous distribution of the GABA, receptor in the mammalian CNS has been revealed by the use of [3H]GABAradioligand binding techniques (Enna and Snyder, 1975), whereas autoradiographic studies have demonstrated their distinct topographical localization (Zukin et al., 1974; Palacios etal., 1981). Subsequent use of immunocytochemical methods has allowed rather more detailed anatomical information to be obtained (Richards et al., 1987). I n the cerebellum, where the greatest detail is available, all five major cell types exhibit varying degrees of receptor immunoreactivity, with the granule cells showing the highest density. Interestingly, there is evidence that the receptor is not only expressed in regions of the cell that receive GABAergic input, but also, for example, on Golgi cell somata where no synaptic contacts are found. With the two monoclonal antibodies used in this study, no immunoreactivity was detected on axons, nerve terminals, or glial cells (Somogyi et al., 1989). The distribution of the GABAA receptor at both synaptic and nonsynaptic sites is quite distinct from that of the closely associated glycine receptor, which appears to be mainly associated with synapses (Triller et al., 1985). Initial electrophysiological studies of the GABAA receptor demonstrated that its activation by GABA resulted in an increased chloride conductance of the supporting neuronal membrane (Curtis et al., 1968;

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Kelly et al., 1969). The agonist response exhibits positive cooperativity, consistent with the presence of two agonist binding sites on the receptor complex (Dichter, 1980; Sakmann et al., 1983; Bormann and Clapham, 1985; Akaike et al., 1985). The agonist induced current decreases on continued exposure to high agonist concentrations as a result of receptor desensitization (Adams and Brown, 1975; Akaike et al., 1987; Mathers, 1987). It is now clear that the agonist-induced conductance increase is due to the opening of the integral ion channel of the GABA, receptor. This channel is anion selective, potassium being about 20 times less permeant than chloride, and studies of anion specificity suggest that the minimum open pore size of the channel is about 0.56 nm (Bormann et al., 1987). T h e channel properties were first investigated using fluctuation noise analysis, which indicated that the channel had a conductance o f 16 pS and an average open time of about 24.5 ms (Barker et al., 1982); the mean open time, but not the channel conductance, was dependent on the activating agonist (Barker and Mathers, 1981). However, the introduction of single-channel current recording techniques (Hamill et al., 1981) has shown that the gating characteristics of the channel are much more complex. The GABA-activated channel of mouse spinal cord neurons in primary culture exhibits four conductance states of 44, 30, 19, and 12 pS, although their relative frequencies of occurrence vary; the 30-pS channel is most commonly observed, accounting for 83% of the openings, whereas the lowest and highest conductance states occur very infrequently (Bormann et al., 1987). Channel activity is a stochastic process that has been subjected to detailed statistical analysis. Single-channel currents, recorded from mouse spinal cord neurons in culture, show that the main conductance state of the GABA, receptor opens either singly or in bursts of several openings. Increasing the concentration of the agonist, GABA, results in an increase in the frequency with which the channels open and an increase in the average time for which the channels remain open, together with an increased frequency of occurrence of bursts of channel openings and a lengthening of the durations of the bursts of channel openings. It has no effect on the single-channel conductance (Macdonald et al., 1989a; Twyman et al., 1990). Although the detailed electrophysiological studies discussed above were carried out with the natural agonist GABA, the properties of the recognition site and the agonist recognition process itself have been investigated in some detail using a number of GABA analogues. Studies with [3H]GABAshow a heterogeneity in binding at equilibrium and this observation is common to all the other agonist ligands used (Wang et al., 1979; O l s e n e t a l . , 1981; Browner etal., 1981; Olsen and Snowman, 1982,

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1983; Jordan et al., 1982; Falch and Krogsgaard-Larsen, 1982; Burch et al., 1983). T h e details of these interactions are discussed in Section III,B, 1. The recognition properties of the binding site for GABA have been explored by the systematic modification of the structure of this amino acid (see Krogsgaard-Larsen et al., 1984, 1986) and certain general criteria have been established; GABA agonists are zwitterionic with a charge separation of about 0.48 nm. A number of GABA isosteres, in which the carboxyl moiety has been conformationally restricted, such as muscimol, are more potent than the natural agonist (Krogsgaard-Larsen et al., 1979). Substitution of the amino terminal invariably leads to a decrease in the potency of the compound; N-methyl-GABA is substantially less potent than the parent amino acid and the N,N-dimethyl derivative is inactive (Krogsgaard-Larsen and Johnston, 1978). However, the conformation of the basic nitrogen may be restricted, as in, for example, imidazole-4-acetic acid, without significant loss of potency (Johnston et al., 1979). Rationalizations for these structural characteristics continue to appear in the literature (Lipkowitz et al., 1989). Bicuculline acts as a competitive antagonist at the GABAA receptor (Curtis et al., 1970; Simmonds, 1978, 1980; Barker et al., 1983), as do its methochloride and methiodide quaternary salts, which are more stable in aqueous solution (Pong and Graham, 1972; Johnston et al., 1972). The stereochemistry of the active enantiomer is known to be erythro(1S,9R) (Hill et al., 1974; Collins and Hill, 1974), and structure-activity studies have been carried out with a series of 45 bicuculline-related phthalideisoquinolines (Kardos et al., 1984). [3H]Bicuculline has been shown to bind to brain membranes in a saturable manner, and the interaction appears to take place with a homogeneous population of sites (Mohler and Okada, 1977a, 1978a). The topographical distribution of these binding sites, as determined by autoradiography, is similar, but not identical, to that found for [3H]muscimol (Olsen et al., 1990). T h e phenylaminopyridazine SR9553 I also exhibits competitive inhibition of GABAA receptor-mediated responses and is about 20 times more potent than bicuculline (Heaulme et al., 1987; Mienville and Vicini, 1987).Two additional compounds display competitive antagonism at the GABA, receptor, pitrazepin (Gahwiler et al., 1984; Kemp et al., 1985) and R5135 (Hunt and Clements-Jewery, 198 I ) , although both lack specificity for this receptor. SITE B. THEBENZODIAZEPINE T h e benzodiazepines were introduced into clinical practice in 1960. They remain the most frequently prescribed psychoactive drugs and are

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used largely for the treatment of anxiety and insomnia although they are also potent anticonvulsants and muscle relaxants (see for example Martin, 1990).Their mechanism of action remained unknown until some 15 years after their introduction. The first clues were obtained when intravenous diazepam was found to cause an increase in presynaptic inhibition in the spinal cord (Schmidt et al., 1967), although it was not until 1972 that this inhibition was satisfactorily shown to be mediated by GABA (Barker and Nicoll, 1972; Davidoff, 1972). Pharmacological studies demonstrated that the action of the benzodiazepines was to facilitate, in some ill-defined manner, GABA, receptor-mediated transmission (Polc et al., 1974). Subsequent studies have shown that the phenomenon is general, the benzodiazepines produce a facilitation of GABA, receptor-mediated transmission at both pre- and post-synaptic sites in the mammalian CNS (Haefely and Polc, 1983). Chlordiazepoxide was shown to produce a leftward shift of the GABA dose-response curve in chick spinal cord and sensory neurons with no increase in the maximum response (Choi et al., 1981). Subsequent fluctuation analysis indicated that this was due to changes in the gating characteristics of the GABA-activated channel; the benzodiazepines appeared to increase the frequency of channel opening with little effect on the channel open time or channel conductance (Study and Barker, 1981).These studies have now been extended by single-channel analysis, which indicates that the reason for the observed increase in channel opening frequency is due not to single-channel events but to an increased occurrence of bursting activity, although the average duration of the bursts did not increase in the presence of the benzodiazepines (Twyman et al., 1989). In 1977 saturable, specific, high-affinity binding sites for ['Hldiazepam were found differentially distributed in the mammalian brain with identical properties in all brain regions studied. (Mohler and Okada, 197713; Squires and Braestrup, 1977). The observations that the affinity of other benzodiazepines for this binding site was highly correlated with their efficiency in a variety of pharmacological tests suggested that this binding site was the pharmacological receptor through which these drugs produced their overt effects (Mohler and Okada, 1977b; Braestrup et at., 1977). The binding sites are restricted largely to the mammalian CNS where the highest densities, of around 1 pmollmg protein, can be found in cortical areas (Squires and Braestrup, 1977; Mohler and Okada, 1978b). Outside the CNS, benzodiazepine recognition sites, identical to those in the brain, have been found in chromaffin granule cells (Kataoka et a/., 1984) and the pituitary (Brown and Martin, 1984). A binding site with similar, but quite distinct, recognition properties is found in the

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periphery (Braestrup and Squires, 1977), where it is associated with the mitochondria1 outer membrane (Anholt et al., 1986) and is thought to be involved in steroidogenesis (Baulieu, 1991). T h e distribution of the benzodiazepine binding sites in the mammalian CNS has been mapped in considerable detail (Richards et al., 1983) and, although they exhibit a similar distribution to the GABA, receptor binding sites, the match is not precise, suggesting that perhaps not all GABA, receptors have an associated benzodiazepine recognition site. However, observations that the affinity of [3H]diazepam is increased in the presence of high concentrations of GABA, an effect that is blocked by the competitive GABA,\ receptor antagonist bicuculline, provided strong evidence that there is a close association between the two recognition sites. Subsequent studies have shown that the purification of the benzodiazepine recognition site results in the copurification of the GABA, receptor, indicating that the two recognition sites are located on a common protein structure, a hypothesis that has received conclusive support from recent molecular biological studies (see below). It is now clear that several structurally disparate compounds recognize the benzodiazepine site with high affinity. T h e first of these compounds was the triazolopyridazine CL2 18872. This compound displaced diazepam o r flunitrazepam binding from the cerebellum with an apparently higher affinity than in the hippocampus (Squires et al., 1979); it was argued that these t w o brain regions contained subtly different subtypes of benzodiazepine binding sites that could be distinguished by CL2 18872, but not by diazepam or flunitrazepam. T h e site in the cerebellum with a higher affinity for CL218872 was termed the Bzl site and that with a lower affinity and present in the hippocampus, the Bz2 site. Later studies showed ethyl P-carboline-3-carboxylateto exhibit the same properties (Nielsen and Braestrup, 1980; see Martin et al., 1983); however, there was little evidence then of the remarkable complexity that this receptor would reveal later. T h e classical benzodiazepines possess a clearly defined profile of action as anticonvulsants, sedative/hypnotics, anxiolytics, and muscle relaxants. However, studies with ethyl @-carboline-3-carboxylaterevealed that the compound was a proconvulsant, having properties diametrically opposed to those of the classical benzodiazepines (Tenen and Hirsch, 1980; Cowan et al., 198 1);compounds of this type were termed inverse agonists. Subsequently compounds have been found that exhibit no overt behavioral actions but are able to block the effects of both agonists and inverse agonists (Nutt et al., 1982; Polc et al., 1982). T h e benzodiazepine recognition site is thus a modulatory site on the GABA, receptor. The inverse agonists exhibit precisely the characteristics expected: they shift the

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GABA dose-response curve to the right whereas the agonist benzodiazepines shift it to the left (Kemp et al., 1987). Thus the agonists display positive efficacy at the benzodiazepine recognition site of the GABA, receptor whereas the inverse agonists exhibit negative efficacy. The inverse agonists appear to produce effects on channel gating opposite to those of the agonist benzodiazepines; they decrease the apparent channel opening frequency, and produce no change in the channel open time or channel conductance (Barker et al., 1984). Both partial agonists and partial inverse agonists exist (Chan and Farb, 1985). There is evidence that the partial agonists may not produce the physical dependence apparent with the full agonists (Moreau et al., 1990), and thus be suitable targets for the development of more acceptable anxiolytics (Haefely et al., 1990). C. THEBARBITURATE SITE Anticonvulsant and anesthetic barbiturates potentiate the electrophysiological response to GABA but their mechanism of action is distinct from that of the benzodiazepines. Phenobarbital shifts the GABA dose-response curve to the left but, at higher concentrations, also increases the maximum response (Gallagher et al., 1981). High concentrations of the barbiturates are able to directly activate GABAA receptors (Nicoll et al., 1975; Simmonds, 1981; Higashi and Nishi, 1982), but at lower concentrations they appear to facilitate GABA-mediated transmission by increasing the channel open time, while having no effect on channel conductance or opening frequency (Study and Barker, 1981). Studies of channel gating characteristics have revealed that the barbiturates increase the channel burst duration but have no effect on bursting frequency (Twyman et al., 1989; Macdonald et al., 1989b). Unlike the benzodiazepines, no specific high-affinity binding sites for the barbiturates have been identified. However, in vitro the barbiturates enhance GABA, and benzodiazepine receptor agonist binding (Asano and Ogasawara, 1981; Olsen and Snowman, 1982),presumably by some allosteric interaction within the receptor complex. Although studies of the channel gating characteristics suggest that the convulsant picrotoxin acts in a manner reciprocal to that found for the barbiturates, i.e., it decreases channel burst duration (Twyman et al., 1989), it would appear that the barbiturates interact with a different site than picrotoxin. T h e evidence for this is somewhat indirect: picrotoxin competitively displaces the cage convulsant ligand tert-butylbicyclophosphorothionate (TBPS) from its specific high-affinity binding sites on brain membranes in a competitive fashion (Squires et al., 1983), whereas the interaction of the barbiturates with the TBPS binding sites is complex (Trifiletti et al., 1984).

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D. THESTEROID SITE It has been known for some time that steroid derivatives are effective and potent anesthetics (Selye, 1942; Figdor et al., 1957). Many of the effects of steroids result from their intracellular actions on the regulation of gene expression, but their ability to induce rapidly anesthesia suggested alternative mechanisms of action. Both electrophysiological and radioligand binding experiments suggest that the action of the steroid derivative alphaxalone is the result of potentiation of GABA, receptormediated transmission (Scholfield, 1980; Harrison and Simmonds, 1984). Subsequent studies with dissociated adrenal chromaffin cells, in culture, have shown that concentrations of alphaxalone in excess of 30 nM dosedependently potentiate GABA,-mediated responses and, further, that the response is additive with that of phenobarbitone, suggesting that the effects are not mediated via the same mechanism (Cottrell et al., 1987). Indeed, studies at the single-channel level seem to suggest that the effects of both androsterone and progesterone on channel gating in mouse spinal cord neurons in primary cell culture are distinct from those of the barbiturates; the steroids appear to increase both the channel opening frequency and the channel open lifetime (Twyman and Macdonald, 1992). I n addition to the major recognition sites described above, the GABAA receptor also appears to carry binding sites for a number of other modulators including Zn2+,avermectin, and, possibly, alcohol (reviewed by Sieghart, 1992). T h e GABAA receptor of the mammalian CNS is thus, functionally, a complex protein displaying a variety of allosteric interactions that may lead to increases or decreases in GABA-mediated transmission. Biochemical studies of the GABA, receptor in its natural membrane environment and after its purification have proved invaluable in contributing to our present understanding of the structure and function of this receptor.

111. Biochemistry

A. RECEPTOR ISOLATION 1. Purification T h e density of the GABA, receptor in mammalian brain is around 1 pmol/mg protein and initial biochemical characterization of the receptor indicated that it had a molecular weight of approximately 250,000 (see below). Assuming that there is one binding site per receptor oligo-

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mer, this suggested that a purification factor of about 4000 would be required to isolate the protein in a pure state. At that time, the common belief was that all GABAA receptors carried a benzodiazepine binding site and this site was, therefore, selected as the basis for purification of the receptor by affinity chromatography. In these purification attempts, the ability of the isolated receptor preparations to bind radiolabeled drugs from each of the pharmacological classes described above, in addition to retention of the expected allosteric interactions between different binding sites, has been widely used to assess purification success. In this respect, the GABAergic agonist [3H]muscimol, the benzodiazepine [3H]flunitrazepam, and t.he channel blocker [35]TBPShave been particularly important in following the fate of isolated GABAA receptors. GABA, receptors have been solubilized from brain membrane preparations using a variety of nondenaturing detergents. As discussed in detail previously (Stephenson and Barnard, 1986), the use of sodium deoxycholate in the presence of a cocktail of protease inhibitors gives the greatest efficiency of solubilization as assessed by the number of high-affinity binding sites for [3H]muscimol and [3H]flunitrazepam in the extract. However, in sodium deoxycholate, not only is the receptor unstable but binding sites for picrotoxinin and related channel blockers are lost and barbiturates lose their ability to potentiate benzodiazepine binding. These receptor characteristics were, however, shown to be preserved when solubilization was carried out using the zwitterionic detergent CHAPS (Sigel and Barnard, 1984) or the nonionic detergent @octylglucoside (Hammond and Martin, 1986; Bristow and Martin, 1987) in the presence of protease inhibitors and exogenous lipid. Following solubilization, several different immobilized benzodiazepines have been used to purify GABAA receptors by affinity chromatography. After absorption of GABAAreceptors to the affinity resin, specific elution has usually been achieved using the water-soluble benzodiazepines chlorazepate or flurazepam (reviewed in Stephenson and Barnard, 1986). Using these procedures, purification factors of 2000-5000 may be achieved, although the yield of benzodiazepine binding sites is low with only 2-5% of the activity in the original membranes being recovered. In most reports, the purified preparations have been demonstrated to carry a single population of binding sites for [3H]flunitrazepam (Kdvalues of 4-28 nM) and a single high-affinity binding site for [3H]muscimol, although lower affinity muscimol binding sites have also been observed (see below). The ratio of muscimol : flunitrazepam binding sites have been reported to range from 0.35 to 3.8 (reviewed by Stephenson, 1988). Several allosteric interactions have also been demonstrated to be preserved in purified receptors including the stimulation of benzodiazepine binding by GABA (Schoch and Mohler, 1983; Sigel and Barnard, 1984)

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and barbiturates (Sigel and Barnard, 1984; Olsen et al., 1984). Binding sites for [35S]TBPShave also been measured in the purified protein (Sigel and Barnard, 1984; Trifiletti et al., 1984; Stephenson et al., 1986; Olsen et al., 1984) but these rapidly inactivate on storage (Sigel and Barnard, 1984; Olsen et al., 1984). It has been shown that retention of the ability of isolated receptors to bind [35S]TBPSis critically dependent on the presence of exogenous phospholipids during purification (Stephenson et al., 1986). This lipid dependence is consistent with the results of a study in which it was shown that treatment of rat brain membranes with phospholipase A, decreased the binding of [35S]TBPSand also inhibited the barbiturate stimulation of benzodiazepine binding (Havoundjian et al., 1986). In a detailed study of binding site stability, Bristow and Martin ( 1987) have shown that binding sites in CHAPS-solubilized preparations can be protected from inactivation by inclusion of a natural brain lipid extract supplemented with cholesterol hemisuccinate. Examination of purified GABA, receptors by polyacrylamide gel electrophoresis in the presence of SDS revealed a multisubunit structure. In most cases two major subunits, a (53 kDa) and p (57 kDa), were observed (reviewed by Stephenson, 1988; Sieghart, 1991). Based on an overall complex molecular weight of 220,000-240,000 determined by radiation inactivation (Chang and Barnard, 1982) or by gel filtration chromatography (Sigel et al., 1983; Martini et al., 1982), it was proposed that the purified GABA, receptor complex was a heterotetramer with a stoichiometry of a@, (Casalotti et al., 1986; Mamalaki et al., 1987). Molecular cloning has since revealed the presence of many more subunits and this has prompted more careful scrutiny of gel patterns, resulting in the observation of microheterogeneity within the bands and additional bands being observed by protein staining, photoaffinity labeling, and immunological techniques (see below). Early attempts to obtain amino acid sequences of isolated, intact subunits failed to provide sequence information, presumably due to their blocked N-termini. However, Schofield et al. (1987) obtained pxtial sequences of proteolytic fragments and used these to design oligonucleotide probes that were used in the initial cloning of an a and a p subunit from bovine brain. When coexpressed in X e n o p w oocytes, these subunits were shown to form GABA-gated chloride channels with many (although not all) of the pharmacological properties of the native receptor (Section IV,A).

2. Reconstitution Although many groups have reported the successful solubilization and purification of GABA, receptors, it was only recently that such preparations were shown to form functional GABA-gated chloride chan-

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nels after reconstitution into lipid vesicles. In early studies, solubilized (Hammond and Martin, 1986)and affinity-purified (Schoch et al., 1984; Sigel et al., 1985)receptors were reconstituted in liposomes and, although these preparations retained the ability to bind various radiolabeled ligands, no chloride flux responses were reported. In the first report of successful functional reconstitution, Hirouchi et ul. (1987) demonstrated GABA-stimulated %I- influx in vesicles reconstituted with solubilized and/or purified GABA, receptors and this response was blocked by bicuculline and stimulated by flunitrazepam. Bristow and Martin (1990) reconstituted purified GABAA receptors using a mixture of natural brain lipids and cholesterol hemisuccinate and showed that [3H]flunitrazepam binding to these preparations was potentiated by GABA, pentobarbital, and the pyrazolopyridine cartazolate. GABA-mediated ( 100 p M ) 36Clflux responses were also potentiated by flunitrazepam, pentobarbital, and cartazolate. Using an alternative fluorescence technique to measure chloride flux, GABA, receptors were solubilized in 0-octylglucoside and reconstituted using a mixture of asolectin and native brain membranes (Dunn et al., 1989a,b). By following changes in the fluorescence of a chloride-sensitive dye, 6-methoxy-N-(3-sulfopropyl)quinolinium (MSQ), trapped within the vesicles, muscimol was shown to stimulate chloride influx in a dose-dependent manner with half-maximal response occurring at 0.3 p M (Dunn et al., 1989b). This flux was inhibited by prior desensitization of the receptor and by bicuculline and picrotoxin but was stimulated by diazepam (Dunn et al., 1989a). These preparations did not, however, display any sensitivity to barbiturates perhaps due to the lability of their binding sites (Section III,A, 1). Similar techniques have since been extended to studies of the affinity-purified protein(s) that displays similar functional properties (Thuynsma and Dunn, 1991). Although the receptor preparations that have been used in reconstitution procedures are likely to contain more than a single receptor subtype, within the limitations of the techniques used, no functional heterogeneity has been detected. It is likely that identification of functional heterogeneity will require more sophisticated analysis, e.g., biophysical analysis of single channels after reconstitution of purified protein in lipid bilayers. In this respect, the ability to purify native receptor subtypes by, for example, immunoaffinity chromatography (Section III,D) will be important. SITES B. LIGANDBINDING

1. Radiolabeled Ligund Binding Studies There have been many studies of the binding of radiolabeled agonists, benzodiazepines, and other channel modulators to GABAA receptors in

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purified brain membranes and these have been extensively reviewed before (Olsen et al., 1981; Olsen and Venter, 1986; Stephenson, 1988). At equilibrium, the binding of GABAergic agonists is heterogeneous, with the binding of r3H]GABA being characterized by a high-affinity component ( K d values of 10-20 nM) with one or more low-affinity sites with dissociation constants in the range 100 nM to 1 p M (Fischer and Olsen, 1986). The antagonist bicuculline inhibits high-affinity GABA binding with an IC,, of about 6 p M , and it appears to inhibit more potently the lower affinity sites with an IC,o of 0.8 p M (Olsen and Snowman, 1983). Since micromolar concentrations of agonists are required to induce chloride flux and to modulate the binding of benzodiazepines (reviewed by Fischer and Olsen, 1986), the lower affinity sites have been correlated with physiological function. N o function has been ascribed to the high-affinity sites and it is usually assumed that these reflect an inactivated, desensitized state of the receptor. It has not, however, been unambiguously determined whether high- and low-affinity sites represent binding to distinct binding sites or to interconverting states of the receptor (see Agey and Dunn, 1989). In studies using affinitypurified receptor preparations, both high- and low-affinity sites have been shown to exist (Sigel and Barnard, 1984; Schoch et al., 1984) but, since GABA, receptors are now known to be heterogeneous, this may reflect the presence of multiple receptor subtypes rather than multiple sites on the one receptor. The latter possibility is, however, supported by the results of 36Cl- flux experiments using cell-free preparations from rat brain, from which it has been suggested that the GABA binding sites involved in channel activation are distinct from those that induce receptor desensitization (Cash and Subbarao, 1987). A similar model proposing multiple sites and parallel pathways for activation and inactivation has previously been proposed for the nicotinic acetylcholine receptor (Dunn and Raftery, 1982). In contrast to the heterogeneity of [3H]GABAor [3H]muscimol binding to brain membranes or purified GABA, receptors, the binding of a classical benzodiazepine agonist such as [3H]diazepam is to an apparently single population of receptors, although the number of receptors shows a brain regional distribution (reviewed by Martin, 1987). As described above, benzodiazepine receptors can be pharmacologically subdivided into two major groups, on the basis of their sensitivity to the triazolopyridazine CL2 18872 and P-carboline derivatives (Martin, 1987). As noted above, micromolar concentrations of GABA or muscimol potentiate the binding of agonist benzodiazepines, whereas they inhibit that of inverse agonists and have no effect on the affinity for benzodiazepine antagonists (reviewed by Martin, 1987). The manner by which the agonist benzodiazepines modulate the characteristics of GABA-activated channels (Section

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11,A) is compatible with the suggestion that these compounds increase the affinity of the receptor for GABAergic agonists. Although such modulation of low-affinity GABA binding has been reported (Skerritt et al., 1982), this effect has not been observed consistently and thus remains somewhat controversial (see Haefely, 1990). The binding of barbiturates has not been directly measured but, in micromolar concentrations, they interact allosterically to increase the number of binding sites for GABA and to increase the affinity for benzodiazepines, responses that are blocked by picrotoxin (reviewed by Fischer and Olsen, 1986). Both [3H]picrotoxinin and the cage convulsant [35S]TBPShave been used in direct binding studies; these ligands appear to compete for the same binding site. This site may, however, be distinct from the barbiturate site (see Fischer and Olsen, 1986). 2. Thermodynamics Thermodynamic analysis of drug-receptor interactions can provide valuable information about the driving forces involved in complex formation and can sometimes be used to distinguish the modes of binding of different types of drugs. In the case of the p-adrenergic receptor, for example, it has been shown that agonist binding, but not antagonist binding, is temperature-dependent and whereas antagonist binding is largely entropy-driven, the binding of agonists occurs with a decrease in enthalpy to overcome an infavorable change in entropy (Wieland et al., 1979). With respect to GABA, receptors, the major goal in the application of thermodynamic analysis has been to identify possible differences in the modes of binding of benzodiazepine agonists, antagonists, and inverse agonists. In early studies of the binding of [3H]diazepam to rat brain membranes it was shown that binding decreased dramatically with increasing temperature (Mohler and Okada, 1977b; Braestrup and Squires, 1977). Speth et al. (1979) showed that for [3H]flunitrazepam, the rate constant for dissociation increased more than the association rate with increasing temperature, resulting in overall decrease in affinity. The van t’Hoff plot deviated from the classical relationship and was biphasic with an inflection point at 16°C. Analysis of the temperature dependence of binding of the agonist [’H]clonazepam also gave a biphasic van t’Hoff plot with an inflection point at 21”C, whereas the van t’Hoff plot for [“H]Ro15-1788 binding was linear (Mohler and Richards, 1981), suggesting differences in the modes of binding of the two ligands. To explain these results, a multistep model of binding was proposed, in which it was suggested that benzodiazepine agonists and antagonists bind to the receptor with similar thermodynamics: above 2 1”C, agonists, but not

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antagonists, can induce a further conformational change of the receptor-ligand complex. This was an attractive suggestion and evidence for such an agonist-induced conformational change was soon obtained in kinetic studies of radiolabeled ligand binding (Quast and Mahlmann, 1982; Chiu et al., 1983). However, subsequent more detailed thermodynamic studies appeared to contradict this suggestion (Kochman and Hirsch, 1982; Quast et al.. 1982) and it has also been pointed out that such a sequential two-step model would not predict curvilinearity in van t'Hoff plots (Doble, 1983). In a parallel study of the thermodynamics of ['H]flunitrazepam and the convulsant ['HH]ethyl P-carboline3-carboxylate binding to rat brain membranes, Doble (1983) found that the temperature dependence of the affinity constants of both ligands deviated from the simple relationship suggesting in each case an increase in enthalpic drive over the temperature range studied. After the effects of a series of different benzodiazepines and P-carbolines were studied, it was concluded that there was no simple correlation between biological effects and temperature dependence of binding (Doble, 1983). In the early studies described above, the degree of potential heterogeneity in GABA, receptors was unknown and the curvilinearity of the van t'Hoff plots that has been observed is possibly explained by the presence of multiple receptor subtypes having different temperature sensitivities. T o address this issue, Maguire et al. (1992) have recently studied the thermodynamics of [SH]Ro15-1788 binding to rat cerebellar membranes, in which the receptor population is apparently more homogenous with the drug binding to B z l receptors only (Massotti et al., 1991). The affinity for [3H]Ro15-1788was again found to decrease with temperature but the van t'Hoff plots for a series of benzodiazepine agonists, antagonists, and P-carbolines were linear and, with the exception of methyl-6,7-dimethoxy-~-carboline-3-carboxylate (DMCM) whose binding was entropy driven, the binding of all other ligands was driven by enthalpy. There, thus, appears to be no relationship between the different types of ligands and their thermodynamic properties, leading to the conclusion that binding and receptor modulation are distinct steps (Maguire et al., 1992). I n contrast to earlier results, Prince and Simmonds (1992) recently found that [3H]flunitrazepam binding to crude rat brain membranes was characterized by a linear van t'Hoff plot. Since these preparations are likely to contain multiple receptor subtypes, the possibility that different receptor subtypes have different temperature sensitivities thus requires further examination. Although the binding of benzodiazepines to GABA, receptors cannot simply be differentiated by the effects of tem-

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perature, it is likely that thermodynamic analysis will continue to provide valuable information on GABAA receptor interactions. I n a recent study, for example, it was reported that the anesthetics alphaxalone and propofol increased the entropy, but not the enthalpy, of [3H]flunitrazepam binding to rat brain membranes. Pentobarbitone, however, increased the enthalpy of binding and thus stimulates binding more effectively at lower temperatures (Prince and Simmonds, 1992). 3. Photoaffinity Labeling [3H]Flunitrazepam, although not a conventional photoaffinity ligand in that it lacks a specific photolabile group capable of generating a reactive species (Knowles, 1971), has been shown to be a very useful tool in photoaffinity labeling studies of benzodiazepine binding sites (Sieghart and Karobath, 1980; Mohler et al., 1980). Photoaffinity labeling of crude brain homogenates followed by analysis by SDS-gel electrophoresis resulted in labeling of mainly a 51-kDa protein (Mohler et al., 1980). Specificity of the labeling for the GABA, receptor was shown by the enhancement of labeling by GABA, an effect that was inhibited by bicuculline (Sieghart and Karobath, 1980). Detailed studies of the labeling patterns in different areas of the brain and during development, in addition to analysis of labeling patterns after tryptic cleavage, provided some of the first direct indications of receptor heterogeneity (Sieghart and Drexler, 1983). In photolabeling of affinity-purified receptor, in which only two major components were present, it was shown that [3H]flunitrazepam was incorporated into the 52-kDa a-subunit (Sigel and Barnard, 1984). More recently, however, multiple a-subunits have been detected by SDS-gel electrophoresis and [3H]flunitrazepam has been shown to label three different proteins with apparent molecular masses of 51, 53, and 59 kDa (Fuchs et al., 1988), all of which were recognized by an a-subunitspecific monoclonal antibody (see below). Later studies using subunitspecific antibodies have shown that the 51-, 53-, and 59-kDa proteins correspond to the a l - ,a*-, and a3-subunits, respectively (Stephenson et al., 1989; Fuchs et al., 1990). Although [3H]flunitrazepam labeling thus occurs on the a-subunits, these subunits are not the sole determinants of binding since, in studies of receptor expression, it has been shown that the presence of a y-subunit is required for both benzodiazepine binding and benzodiazepine modulation of GABAA receptor responses (Pritchett et al., 1989a). T h e position of the label within the a-subunit has not been determined but Olsen et al. (1991) have obtained partial sequences of proteolytic fragments of the labeled receptor to show that labeling occurs within residues 8-297, and most likely between residues 106 and 297. Using an alternative approach, Stephenson and Duggan ( 1989) have probed cyanogen bromide fragments with sequence-specific

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antibodies and have concluded that the binding site lies within residues 59 and 148 of the a,-subunit. [3H]Muscimol has also been used as a photoaffinity label for the GABA, receptor agonist sites although this again is not a conventional photoaffinity ligand (Cavalla and Neff, 1985; Asano et al., 1983). T h e major site of labeling in purified preparations was demonstrated to be the @-subunit (Casalotti et ad., 1986; Deng et al., 1986). More recently, several subunits of apparent molecular mass 51, 52, and 56 kDa were shown to be labeled by [3H]muscimol in purified receptors from rat brain and these were recognized by a P-subunit-specific antibody (Fuchs and Sieghart, 1989). [3H]Muscimol labeling has also been reported to occur on the a-subunits (Bureau and Olsen, 1988, 1990), suggesting either that agonist binding sites may occur at the interface(s) between subunits, as has been suggested for nicotinic acetylcholine receptors (Blount and Merlie, 1989; Middleton and Cohen, 1991), or that both a - and @-subunitscarry binding sites for [3H]rnuscimol. In this respect, possibly all subunits may be capable of binding GABAergic agonists since single subunits can be expressed to produce homoligomeric GABA-gated chloride channels (Section V,A).

C. PROTEIN MODIFICATION

Although the photoaffinity labeling studies described above have led to the identification of subunits that carry, at least in part, binding sites for receptor agonists and benzodiazepines, little is known about the amino acids within the subunit sequences that are involved in binding. These amino acids are currently being probed by sequencing of proteolytic fragments of photolabeled preparations and by site-directed mutagenesis (see below). In the absence of precise sequence information, chemical modification techniques have been used to provide some valuable information on the importance of certain amino acid residues in ligand binding. It has been shown that modification of receptors by reaction with diethyl pyrocarbonate (DEP) inhibited both benzodiazepine (Burch and Ticku, 1981; Sherman-Gold and Dudai, 1981; Burch et al., 1983) and P-carboline (Maksay and Ticku, 1984) binding without affecting the binding of other ligands. Flurazepam (Maksay and Ticku, 1984), but not a /3-carboline (Lambolez and Rossier, 1987), was able to protect the binding sites from DEP inhibition, indicating that the modified residues are close to, or are allosterically coupled to, the benzodiazepine binding site(s). Although DEP can react with both histidine and tyrosine residues, the pH dependencies of both benzodiazepine binding (Lambolez and

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Rossier, 1987; Maksay et al., 1991) and DEP inactivation (Maksay and Ticku, 1984; Lambolez et al., 1989) indicate that histidine residues are the major residues involved. In the early studies, both Bzl and Bz2 benzodiazepine receptors were shown to be sensitive to DEP (Maksay and Ticku, 1984). More recently, Binkley and Ticku (1991) have investigated the effects of DEP on diazepam-insensitive sites. These sites, which are enriched in the cerebellum and are now known to be associated with a4-and a6-subunits (Luddens et al., 1990; Wieland et al., 1992), can be investigated using the imidazobenzodiazepine Ro 15-4513, which binds to both diazepamsensitive and -insensitive sites (Sieghart et al., 1987). Treatment of cerebellar membranes by DEP decreased the number of sites for ['HI flunitrazepam and [3H]Ro15-1788 as expected, but had no effect on ['H]Ro15-4513 binding (Binkley and Ticku, 1991). Thus the binding of the latter ligand to both diazepam-sensitive and -insensitive sites does not appear to involve the histidine residue that is crucial for the binding of other benzodiazepines. Site-directed mutagensis has shown that the histidine residue that is essential for high-affinity agonist binding is residue 101 in the a,-subunit. In the a4-and a6-subunits (Wieland et al., 1992; Wisden et al., 1991; Korpi et al., 1993) there is an arginine residue at the equivalent position. Maksay (1992) has recently investigated the possible importance of this arginine in ['H]Ro15-45 13 binding by studying the effects of treating cerebellar membranes with the argininemodifying reagent 2,3-butanedione. Binding to the diazepam-insensitive sites was partially inhibited with an EC,, of 1.6 mM, compared to an EC,, of 3.5 mM for binding to the diazepam-sensitive sites. Another histidine residue that is conserved in diazepam-sensitive receptors is replaced by Y214 in as. The effects of tyrosine modification have, therefore, been investigated to assess the importance of this residue in benzodiazepine binding. Modification of cerebellar membranes with the tyrosine reagent tetranitromethane partially inactivated ['HIRo 154513 binding (Maksay, 1992) similar to the partial loss of ['Hlfluntrazepam binding that was observed in earlier studies (Maksay and Ticku, 1984). However, the receptor sites could not be protected from the effects of tetranitromethane by carrying out the modification in the presence of benzodiazepines, suggesting that the susceptible tyrosine is not located directly within the binding site (Maksay and Ticku, 1984). Cysteineicystine residues have also been shown to be important structural determinants for benzodiazepine binding. It has been shown that the treatment of brain membrane preparations with either the sulfhydry1 alkylating agent iodoacetamide or the disulfide reducing agent pmercaptoethanol results in reduced affinity for both ['Hldiazepam and [3H]muscimol (Marangos and Martino, 1981). Recently, de Bengtsson et

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al. (1993) reexamined these effects and showed that in bovine brain membranes, iodoacetamide alone had little effect on [3H]flunitrazepam binding but the affinity was much reduced if membranes were reacted with excess iodoacetamide after reduction with dithiothreitol (DTT). In another study, it was shown that whereas [3H]flunitrazepam binding is relatively insensitive to DTT reduction, the binding of the partial inverse agonist [3H]Ro15-4513 is inhibited by DTT (IC50of 4.6 mM), this being a consequence of a reduction in both affinity and number of sites. The binding sites for this latter ligand were protected if the reduction was carried out in the presence of either Ro15-4513 or flunitrazepam, suggesting that the disulfide that is important for the binding of [3H]Ro1545 13 is either located near a benzodiazepine binding site(s) or is involved in stabilizing the conformation of the site (Duncalfe and Dunn, 1993). The binding of both [3H]fli~nitrazepamand [3H]Ro15-4513 was inhibited by the sulfhydryl alkylatirig agent N-ethylmaleimide, although apparently by different mechanisms (Duncalfe and Dunn, 1993). Taken together these results suggest an important role for disulfide bonds and sulfhydryl groups in benzodiazepine binding and different benzodiazepines display differences in their sensitivity to modification of these groups, suggesting differences in their modes of binding. Protein modification studies have also been used to study the involvement of particular amino acid residues in the binding of GABA and other receptor agonists. Arginine residues have been implicated in [3H]muscimol binding, since exposure of membrane preparations or purified GABA, receptors to the arginine modification reagents 2,3butanedione or phenylglyoxal inhibited [3H]muscimol binding by up to 82% (Widdows et al., 1987). This was due to a loss of sites rather than a reduction in affinity and the sites could be protected by carrying out the reaction in the presence of GABA. It has also been reported that diazotized sulfanilate, a reagent that is fairly nonselective for histidine and tyrosine residues, inhibits the binding of [3H]GABA to its lowaffinity, but not its high-affinity, binding sites (Burch et al., 1983). The ability of GABA to potentiate benzodiazepine binding was lost in parallel, suggesting that the low-affinity sites are important for this allosteric property.

D. IMMUNOLOGICAL CHARACTERIZATION I n view of the large number of GABA, receptor subunits identified by cloning techniques (Section V), the potential number of receptor subtypes is very large. At the present time, however, we do not know the actual subunit compliment of any native receptor. In order to probe

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native receptor compositions and the regional distribution of receptor subtypes in the brain, panels of polyclonal and monoclonal antibodies have been raised against isolated receptors (Stephenson et al., 1986), specific subunits (Haring et al., 1985), and synthetic peptides whose sequences are unique to specific subunits (see below). In early studies, polyclonal (Stephenson et al., 1986) and monoclonal (Schoch et al., 1985; Haring et al., 1985) antibodies were raised against affinity-purified GABA, receptor preparations. These were used in immunoprecipitation and autoradiographic studies to further demonstrate the coexistence and colocalization of [3H]muscimol, [3H]benzodiazepine, and [35S]TBPS binding sites (Richards et al., 1986).Mamalaki et al. (1987) further demonstrated that a monoclonal antibody recognized both the a- and P-subunits in a purified receptor preparation, providing evidence for structural homology between these subunits as was soon after directly demonstrated by cDNA sequencing (Schofield et al., 1987). Although purified GABA, receptors were originally thought to contain only an a- and P-subunit (Section III,A,l), more detailed analysis has since revealed heterogeneity of these subunits. Monoclonal antibodies raised against partially purified receptors have been shown to recognize multiple bands on Western blots (Fuchs and Sieghart, 1989; Fuchs et al., 1990; Bureau and Olsen, 1990; Park and de Blas, 1991). In view of the structural homology of the GABA, receptor subunits, it is not surprising that such antibodies raised against the intact protein recognize multiple subunits. However, with the knowledge of subunit sequences that has been provided by molecular cloning, a number of laboratories have now succeeded in producing subunit-specific antibodies raised against either short synthetic peptides or fusion proteins containing putative intracellular receptor sequences as antigens. These antibodies are currently being used in quantitative immunoprecipitation and Western blotting analyses to identify receptor subtypes and to probe their subunit compositions. Using these techniques, a,-, a2-,a3-,and y2-subunits have been identified as integral components of affinity-purified receptors (see Stephenson, 1992). In the brain, the subunits that have been found to be most abundant are a , (Benke et al., 1991a; Duggan et al., 1991; Endo and Olsen, 1993, Liiddens et al., 1991; McKernan et al., 1991), &/p3 (Benke et al., 1991a), and y 2 (Benke et al., 1991a; Duggan et al., 1992). These subunits appear to associate to form a major receptor subtype (Benke et al., 1991a; Duggan et al., 1992), a finding that supports the conclusions drawn from in situ hybridization and immunocytochemical studies described below (see Sections V,B and V,C). Although initial immunopurification studies suggested that individual GABA, receptor complexes did not contain more than one type of subunit from the same class (Duggan and Stephenson, 1990), other stud-

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ies have contradicted this notion. An a,-subunit-specific antibody was able to precipitate a higher percentage of receptors than expected on the basis of the abundance of Bzl receptors (Liiddens et al., 1991; Section V,A, 1) and also the sum of cortical receptors precipitated by four individual anti-a-subunit (a1,a2,a3,and a4)antibodies was greater than 150% (Endo and Olsen, 1993). Using antibodies against a-subunit ( a l ,apta3, and as)sequences, it has been shown that only a single isoform is likely to be found in combination with a p- and y-subunit (McKernan et al., 1991). Thus receptors containing only a single a-subunit isoform appear to predominate. However, a minor population of receptors may contain more than a single isoform (McKernan et al., 1991). Stephenson and COworkers have used immunoblotting in conjunction with sequential antia-subunit immunoaffinity chromatography to demonstrate the presence 3, (Duggan et al., 1992), and a,a6 pairs (Pollard et al., of a I a 2 ,( ~ l ( ~ a2a3 1993).Although these results may be compromised by receptor aggregation occurring during purification, the authors used conditions to minimize such effects (Pollard et al., 1993). Initial attempts have been made to characterize the pharmacological profiles of individual receptor subtypes that have been purified by immunoaffinity chromatography. Receptors purified using an a,-subunitspecific antibody displayed binding properties of Bz 1 receptors, whereas receptors purified using anti-a, o r anti-as-subunit-specific antibodies displayed a Bz2 profile (McKernan et al., 1991; Zezula and Sieghart, 1991). These characteristics are in general agreement with the profiles of GABAA receptors in cells transiently transfected with these subunits in combination with a PI- and a y,-subunit (Section V,A). Such studies of native receptors will be important to complement the information obtained in expression studies. T h e continued development of this antibody repertoire and their use, in combination with in situ hybridization, will undoubtedly yield important information concerning the coordinate expression of individual subunits within specific cell populations. Further analysis of native receptors will then allow us to determine their oligomeric structures, thus providing access to the central question concerning the functional diversity of the GABAA receptor family. However, before considering these issues, we will first discuss the cloning of GABAA receptor genes. IV. Molecular Cloning of Receptor Subunits

A. INITIALISOLATIONOF RECEPTORSUBUNIT cDNAs

The partial amino acid sequence, obtained as a result of the purification of the GABAA receptor from bovine brain, allowed the design of

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oligonucleotide probes for the isolation of cDNAs encoding the a ] -and P,-subunits (Schofield et al., 1987). The deduced amino acid sequences of the encoded polypeptides were found to share approximately 35% identity. Comparison with the amino acid sequences of other neurotransmitter receptors, in particular the nicotinic acetylcholine and glycine receptors, also revealed sequence indentity and indicated that these receptors share certain conserved structural features, leading to the proposal that these receptors are members of a ligand-gated ion channel superfamily (Barnard et al., 1987; Schofield et al., 1987). T h e mature subunits contain four putative membrane spanning domains, the second of which is proposed to form the inner lining of the ion channel. The long N-terminal region contains a number of consensus sequences for N-linked glycosylation and is believed to be extracellular (Barnard et aE., 1987; Schofield et al., 1987). This region also contains the Cys-Cys loop, which has been proposed to play a role in agonist binding (Cockroft et al., 1990). Between the third and fourth transmembrane domains there exists a long loop that is presumed to be intracellular and can contain potential post-translational modification sites (Barnard et al., 1987; Schofield et al., 1987).This superfamily of ligand-gated ion channels currently includes the GABAA, glycine, nicotinic, and acetylcholine (both muscle and neuronal) and the recently described 5HT3 receptor families (Barnard, 1992). Further molecular biological studies revealed the existence of two other a-subunits (Levitan et al., 1988a). When an a,-, a*-,or a,-subunit was expressed in combination with the P,-subunit in Xenopus oocytes, functional receptors were produced that could be distinguished electrophysiologically by a 30-fold difference in their apparent sensitivity to GABA (Levitan et al., 1988a). A possible molecular basis for GABA, receptor heterogeneity was thereby suggested. I t was not possible, however, to demonstrate a robust benzodiazepine effect for these recombinant receptors, suggesting that an additional subunit, or factor, was necessary to produce GABA, receptors with the full range of characteristics as those found in viuo (Levitan et al., 1988b).

OF A cDNA ENCODING A y 2 SUBUNIT B. ISOLATION

In an effort to isolate further cDNA clones encoding GABAA receptor subunits, Seeburg and co-workers used a 96-fold degenerate pool of 23base oligonucleotides (Pritchett et al., 1989b; Ymer et al., 1989) designed to correspond to an octameric amino acid sequence (TTVLTMTT) that was found to be present in the second proposed transmembrane domains

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of the GABAA receptor a,-, a2-,as-,and P,-subunits (Schofield et al., 1987; Levitan et al., 1988a) and the glycine receptor a,-subunit (Grenningloh et al., 1987). This approach led the isolation of a number of clones, including one that encoded a polypeptide, named y2,that shares approximately 40% sequence identity with the previously described a,-and P1-subunits. When coexpressed with a,-and P,-subunits, the resultant receptor displayed a high-affinity binding site for centrally acting benzodiazepines and a potentiation of the GABA-mediated response (Pritchett et al., 1989b).

C. RECEPTOR HETEROGENEXTY REVEALED BY MULTIPLESUBUNITS A rapid explosion in the number and type of GABA, receptor subunit cDNAs soon resulted in the identification of a large gene family (reviewed in Wisden and Seeburg, 1092). In the rat, there are at least 13 separate subunits, which, on the basis of sequence identity, can be divided into four classes. Thus there are six a-subunits, three P-subunits, three ysubunits, and one &subunit (Wisden and Seeburg, 1992). In general, members of different classes share approximately 30-40% sequence identity, and this can rise to approximately 70% between members of the same class. A fifth subunit class has recently been described containing two members, p , (Cutting et al., 1991) and p 2 (Cutting et al., 1992), which are predominantly found in retina. It is not clear at present, however, whether these subunits form part of a functional GABAA receptor in vivo (Section V,A,5; Cutting et al., 1991, 1992). One confusing aspect that has arisen in the literature is the different names given to the same subunit sequence by different groups. Tobin and co-workers described the sequence of a rat subunit, which they termed a4 (Khrestchatisky et al., 1989). Two other groups published descriptions of the same subunit sequence, which they both termed a5 (Malherbe el al., 1990a; Pritchett and Seeburg, 1990). To avoid confusion in this review, we will use the latter nomenclature when referring to this subunit. T h e isolation of cDNAs encoding GABAA receptor subunits from nonmammalian species has revealed that GABAA receptor sequences have been more highly conserved through evolution than other members of this ligand-gated ion-channel superfamily. For example, the chicken GABA, receptor a,subunit is 98% identical to any mammalian a,subunit (Bateson et al., 1991a),whereas the chicken neuronal nicotinic acetylcholine receptor &-subunit (Nef et al., 1988) is only 85% identical to the rat homologue (Deneris et al., 1988). At least one subunit, termed p4,has

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been identified in avian species (Bateson et al., 1991b), the mammalian homologue of which has yet to be found. Cloning studies in invertebrate species have also identified GABA, receptor subunits that display significant sequence identities (up to 52%)with those of vertebrates (ffrenchConstant et al., 1991; Harvey et al., 1991).

D. FURTHER HETEROGENEITY ARISESFROM ALTERNATE SPLICING A further mechanism for the generation of receptor heterogeneity has been revealed by the identification of alternate splice variants of some GABA, receptor subunits. The 7,-subunit occurs as two variants that differ by the presence ( Y ~o~r absence ) (y2s)of 8 amino acids in the presumed intracellular loop region that lies between the third and fourth membrane-spanning domains. This has been shown to occur in bovine, human (Whiting et al., 1990), mouse (Kofuji et al., 1991), and chicken brains (Glencorse et al., 1990, 1992) and the &amino acid insertion present in the yZLvariant is absolutely conserved among these species. In the case of both the mouse gene (Kofuji et al., 1991) and the bovine gene (Whiting et al., 1990), this %amino acid insert is encoded by a separate exon of 24 bp. Based on the sequence conservation in this region between different species it has been proposed that a similar situation occurs for the chicken gene (Glencorse et al., 1992). T w o splice variants of the chicken P,-subunit that differ by the presence (P,') or absence (&) of four amino acids have also been demonstrated (Bateson et al., 1991b). Similar to the 7,-subunit, this occurs between the third and fourth membrane-spanning domains. These variants are generated, however, by a different alternative splicing mechanism, that of differential choice of 5' donor splice site. T h e Drosophila GABA, receptor gene (Rdl) transcript undergoes extensive alternative splicing. Two pairs of exons encoding portions of the N-terminal extracellular domain each undergo alternative usage such that four different forms of this subunit are generated (ffrench-Constant and Rocheleau, 1993).

V. Characterization of the Receptor Family

A. HETEROLOGOUS EXPRESSION REVEALSDIFFERENT FUNCTIONAL ATTRIBUTES OF GABA, RECEPTOR SUBUNITS Intense effort has been maintained in examining the ligand-binding characteristics and functional attributes of recombinant GABA, recep-

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tors expressed in heterologous expression systems. An initial hope was that by defining their properties, in comparison to those of in vivo receptors, equivalents could be found and thus the subunit compositions of in vivo receptors deduced. This has largely floundered in the face of the growing GABA, receptor gene diversity. If all possible subunit combinations were able to produce functional receptors, there would be over 500,000 different receptor subtypes. It is clearly impossible to characterize fully even a small percentage of this number of theoretical receptor subtypes and expect to be able to use such data to identify the composition of in vivo receptors. A number of potential difficulties should be considered when interpreting heterologous expression studies of recombinant receptors. First, although the subunit composition of the recombinant receptor is assumed to correspond to the nucleic acid species transfected or injected, no one has demonstrated that all of the subunit polypeptides expected are present and appropriately assembled. Furthermore, the question of subunit stoichiometry in recombinant receptors has not been addressed. Recently, it has been shown that certain subunit combinations may be preferentially formed over others (Angelotti and Macdonald, 1993; Angelotti et al., 1993; Verdoorn et al., 1990; Section V,D), a process that may be influenced by the particular heterologous expression system used (Angelotti and Macdonald, 1993; Angelotti et al., 1993). Macdonald and co-workers propose that some of the subunit combinations, formed in studies that have used high-level heterologous expression systems, may be somewhat artifactual in that the high levels of receptor proteins that are expressed in these cells may lead to inappropriate subunit association (Angelotti and Macdonald, 1993; Angelotti et al., 1993). It is also likely that some of the combinations used in these in vitro experiments may not exist in vivo. For example, most subunits have been shown to have the capacity to form homooligomeric GABA-gated channels in transfected cells; however, they generally display small currents, suggesting that these receptors are assembled with low efficiency. Even in cases where heterooligomeric receptors d o appear to be efficiently assembled, interpretation of apparent changes in GABA affinity andlor cooperativity is difficult, as these may arise from alterations in activation and/or desensitization kinetics (Mathers, 199 1). Despite these caveats, heterologous expression studies of GABA, receptors have clearly demonstrated the potential that exists for the generation of GABA, receptor heterogeneity and have revealed valuable information about the different contributions that subunits can make to a receptor’s pharmacological profile. When interpreted in the light of gene product localization (Sections V,B and V,C) and receptor purification studies (Section III,D), they have provided useful data to corroborate

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receptor subunit compositions proposed on the basis of nonfunctional approaches. A concept that is emerging from heterologous expression studies is that, in general, specific roles may be ascribed to individual subunit classes. However, there are clearly many characteristics of the receptor complex that are specified by interactions between subunits of different classes. 1. a-Subunit Class The a-subunit class is the largest, comprising six members, and it is believed that they specify the heterogeneity of the benzodiazepine binding site (see Doble and Martin, 1992, and Wisden and Seeburg, 1992, for review). Ligand-binding studies of membranes of HEK293 cells transiently transfected with a , 4 y 2 combinations revealed a correspondence between the presence of an al-subunitwith benzodiazepine Bzl pharmacology and an a,- or a,-subunit with that of Bz2 receptors (Pritchett et al., 1989a). Although the y,-subunit was required for the expression of the benzodiazepine site, changing the particular @-subunit present showed no effect. Furthermore, GABA potentiation of benzodiazepine binding was greater in a,-subunit-containing receptors (Pritchett et al., 1989a). Subsequently it was shown that the a,-subunit would also confer a Bz2 pharmacology, but one that was different from that conferred by a2- or a,-subunits with respect to zolpidem affinity (Pritchett and Seeburg, 1990). A third class of benzodiazepine pharmacology is displayed by a 4 y 2 recombinant receptors, in which the a-subunit is either a4 or a6. These receptors d o not bind benzodiazepine agonists, such as diazepam, but do bind certain antagonists and inverse agonists (Luddens et al., 1990; Wisden et al., 1991). Using site-directed mutagenesis, a single amino acid difference between al-and a3-subunits has been shown to confer Bzl or Bz2 benzodiazepine pharmacology. T h e exchange of a glutamic acid residue in the a,-subunit for a glycine residue, which appears in the corresponding position of the &,-subunit, confers on the hybrid receptor (a3G225,42y2) high affinity for CL2 18872, effectively changing the pharmacology of the receptor from Bz2 to Bzl (Pritchett and Seeburg, 1991). Similarly, a specific histidine residue present in some a-subunits has been shown (Wieland et al., 1992; Korpi et al., 1993) to be necessary for high-affinity benzodiazepine agonist binding. Homooligomers of al-,ap-,or a,-subunits form GABA-gated channels that have apparently normal channel properties which can be blocked by picrotoxin and display potentiation by barbiturates (Blair et al., 1988; Pritchett et al., 1988). T h e whole-cell currents are, however, small, suggesting that subunit assembly in these receptors may not be efficient.

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These data are in contrast to the biochemical evidence, discussed above, which indicated that the GABA agonist site might be located on the psubunit. The particular a-subunit present in heterooligomers affects not only the binding but also the functional properties of recombinant receptors. or a3-and the PIThus, receptor complexes formed with either a l - ,a,.-, subunit displayed u p to a 30-fold differing sensitivity to GABA (Levitan et al., 1988a). Similarly, the alp1combination is less sensitive to GABA than alpI (Khrestchatisky et al., 1989), whereas the a5pIcombination is more sensitive than a l p l or a3pI (Malherbe et al., 1990a; Sigel et al., 1990). There may also be differences between species in that rat alpI o r as@, combinations display similar GABA dose-response curves (Malherbe et al., 1990a), whereas the equivalent bovine combinations show a small (threefold) difference in GABA sensitivity (Levitan et al., 1988a). Addition of the y,-subunit to many of these combinations appears to maintain or even increase the influence of the a-subunit isoform present on GABA sensitivity (Sigel et al., 1990). The potency of steroid action has been shown to depend on the ti-subunit present: a l p l or asp1combinations display greater potentiation than aePI(Shingai et al., 1991). Surprisingly, the addition of the y,-subunit to the a l p l or the a2p1combinations produced an increase in steroid potency, whereas the a3p,y2combination was potentiated to lesser degree than that of aspl (Shingai et al., 1991). T h e use of y-subunit-containing ternary combinations allows an assessment to be made of exchanging a-subunits on the efficacy of benzodiazepine potentiation. Some discrepancies appear, however, between the results obtained from two comprehensive studies, one using rat subunits expressed in oocytes (Sigel et al., 1990) and the other using human subunits expressed in transfected cells (Puia et al., 1992). Ternary complexes (spy) containing an a,-subunit display the greatest potentiation of GABA-mediated currents by diazepam with a rank order of either a3 > a2 > a 1> a5 (Puia et al., 1992) or a , > a5 > a I (Sigel et al., 1990). In transfected cells (Puia et al., 1992) the GABA responses of these complexes are also differentially modulated by other compounds that act at the benzodiazepine site. For example, in contrast to the effects of diazepam, alpidem displays a rank order of efficacy on GABA-mediated currents of a 1 = a2 > a, % a5 (Puia et al., 1992). In support of this, Wafford et al. (1993) recently reported that a variety of benzodiazepine site ligands (full, partial, and inverse agonists) display differential potencand a 3 p I y 2 L combinations. The efficacy of ies and efficacies for a1/31y2L triazolam on GABA-gated currents was also shown to be greater than that of diazepam on a l P I y , and a,P1y2 combinations but not on the a3pIy2combination (Ducic et al., 1993).

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Only one study has reported in detail the coexpression of a number of subunit combinations that include more than one a-subunit with a pand a y-subunit (Sigel et al., 1990). They found that in contrast to a ffl(Y3(Y$I&y2 combination, omission of the q s u b u n i t (aI(~3plp2y2)resulted in a partial loss of cooperativity and affinity for GABA. In fact all combinations tested that included the a,-subunit produced receptors that displayed properties closest to those found in vivo (Sigel et al., 1990). The presence of at least one a-subunit was deemed necessary for the efficient production of functional GABA, receptors with large conductances (Sigel et al., 1990; Verdoorn et al., 1990). Significant benzodiazepine potentiation was obtained in at least one subunit combination lacking any a-subunits (p2y2),indicating that the a-subunits are not required for the formation of this site (Sigel et al., 1990). It is not clear, however, whether such a receptor is present in vivo, as other functional attributes of this particular combination do not reflect native receptor characteristics (Sigel et al., 1990; Verdoorn et al., 1990). Taken together, these data suggest that the a-subunits, in recombinant receptor complexes, play a major role (but not an exclusive one) in determining the nature of binding sites for GABA and some of the allosteric effectors. 2. p-Subunit Class There is little evidence to suggest that the particular P-subunit isoform influences the ligand-binding characteristics of recombinant receptors (Pritchett et al., 1989a; Wisden and Seeburg, 1992). Homooligomeric rat p,-subunit-containing channels can be formed in oocytes but the channels appear to open spontaneously in the absence of GABA (Sigel et al., 1989). In contrast, human P,-subunits expressed alone in HEK293 cells produced GABA-gated channels that could also be potentiated by barbiturates and blocked by picrotoxin (Pritchett et al., 1988). Analysis of heterooligomer combinations expressed in oocytes demonstrated that a @subunit is not essential for the formation of a picrotoxin site (Sigel et al., 1990). These ay complexes form functional GABA-gated channels that display GABA cooperativity and benzodiazepine sensitivity, but they exhibit relatively small currents (Sigel et al., 1990). In contrast, the a 1 y 2 combination expressed in cultured HEK293 cells produced large GABAactivated currents, equivalent to those seen for the a1P2y2combination (Verdoorn et al., 1990), highlighting the need for care in interpreting expression data from different experimental systems. In the latter study, the channel properties of alp2and a l y , combinations were found to be different, suggesting a role of the @-subunit (and the y-subunit) in determining channel characteristics (Verdoorn et al., 1990). In oocytes,

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the exchange of p1for @, in aXpYy2combinations generally resulted in a decrease in the apparent affinity for GABA but a marked increase in potentiation by diazepam (Sigel et al., 1990). This result is also in contrast to that obtained with similar combinations (a,P,y,, alP2y,, o r a l P 3 y 2 in ) HEK293 cells where no effect of the particular P-subunit was found on diazepam potentiation (Puia et al., 1992). These apparent differences in diazepam sensitivity may be a result of the expression system used, but may also arise from the use of differing diazepam concentrations (1 mM in the oocyte studies; Sigel et al., 1990; 10 mM in the HEK293 studies; Puia et al., 1992). Indeed, the most effective diazepam concentration was shown to be 0.3 to 1 mM for @,- or @,-containing combinations in oocytes (Sigel et al., 1990). Clearly, more detailed studies are required to test the possible effects of @-subunitson receptor channel properties and benzodiazepine potentiation. 3. y-Subunit Class

The y,-subunit can form homooligomeric channels in HEK293 cells that are reversibly potentiated by barbiturates (Shivers et al., 1989). Although the y,-subunit is required for expression of a benzodiazepine site, it is insufficient alone to achieve this (Pritchett et al., 1989b; Shivers et al., 1989). Unlike the @-subunitsthere are clear differences in both the binding and the functional properties of heterooligomeric recombinant receptors containing differing y-subunits. All three y-subunits, when combined with a-and @-subunits,can confer benzodiazepine binding on recombinant receptor complexes (Knoflach et al., 1991; Pritchett et al., 1989a,b; Ymer el al., 1990). I n combination with an a- and a @-subunit,the y l subunit produces a marked decrease in affinity for the antagonist flumazenil and the inverse agonist DMCM in comparison to 7,-containing combinations (Ymer et al., 1990). In contrast, an a1P2y3combination shows a marked decrease in benzodiazepine agonist affinity, relative to that of an al@,y2combination, whereas both combinations have similar affinities for antagonists and inverse agonists (Herb et al., 1992). Interestingly for these combinations, receptors containing y 3 displayed much greater differences in affinities for certain benzodiazepine site agonists (e.g., midazolam versus zolpidem) than did the corresponding y2containing combination (Herb et al., 1992). Functional differences between the different 7-subunit-containing receptor complexes are particularly striking. Most notably, exchanging the 7,-subunit for the y,-subunit in many aPy combinations changes the action of DMCM and @-CCM from that of inverse agonist to agonist (Puia et al., 1991). These data provide an explanation for the previous

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finding that these compounds function as agonists on GABAA receptors in astrocytes (Bormann and Kettenmann, 1988) since these cells have been shown to express the y I subunit gene to higher levels than those of other y-subunits (Bovolin et al., 1992a). In general, the higher affinities of benzodiazepine ligands for 7,-subunit-containing complexes is reflected in increased modulation of GABA-gated currents (Herb et al., 1992; Knoflach ct al., 1991; Puia et al., 1991). Some benzodiazepine effects, however, may be attributed to differential cooperative interactions between y- and a-subunits. For example, pCCM displays a similar degree of inverse agonist activity on a,/3,y, and a,p,y, combinations. It is also an inverse agonist of an a3/3,y,combination but it is an agonist of the a 2 P I y Icombination (Puia et al., 1991). The close interaction suggested by these studies between the a- and the y-subunits in the formation of the benzodiazepine site and the determination of its functional attributes indicate that this site may not reside solely on the a-subunit, as had been suggested by previous biochemical studies (Section III,B,3). Currently no site-directed mutagenesis studies have been reported investigating the possible role of the y-subunit either in inducing conformational changes in the a-subunit to allow binding or in directly forming part of the benzodiazepine site. Such studies may give a clearer understanding of the molecular interactions involved in these effects. The y,,-subunit alternative splice variant has been shown to be responsible for conferring ethanol enhancement in receptors expressed in oocytes from recombinant clones and brain mRNA (Wafford et al., 1991). Furthermore, the previously proposed phosphorylation site within the 8-amino acid insert of the y,,-subunit (Whiting et al., 1990) appears to be essential for this effect (Wafford and Whiting, 1992). No differences in benzodiazepine or barbiturate potentiation of receptors containing either the yZL-or the y,,-subunit forms have been detected (Wafford and Whiting, 1992; Wafford et al., 1991).

4. &Subunit Class There is virtually no information regarding the possible contribution of the 6-subunit to GABA, receptor characteristics. This subunit can form homooligomers in HEK293 cells that display GABA-gated channels with small currents that are sensitive to picrotoxin, bicuculline, and pentobarbitol (Shivers et al., 1989). These workers also cite unpublished data indicating that the b-subunit cannot replace the ?,-subunit in the generation of receptors with high-affinity benzodiazepine binding sites (Shivers et al., 1989).

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5. p-Subunit Class Two p-subunits have been identified and their expression is generally restricted to retina. The pl-subunit, in contrast to members of all other GABAA receptor classes, forms homooligomers that display large GABAgated currents (Cutting et al., 1991; Shimada et al., 1992). Furthermore, these channels display an unusual pharmacology that is not altered by coexpression with a-,p-, or y-subunits, picrotoxin-sensitive, barbiturate-, benzodiazepine-, bicuculline-, and baclofen-insensitive (Cutting et al., 1991; Shimada et al., 1992). I t appears likely that this subunit forms homooligomers in vivo in retina and has been classified as GABA, rather than GABAA (Shimada et al., 1992), although other workers argue for a separate classification entirely (Woodward et al., 1993).

B. GENEEXPRESSION A large body of literature attests to the intense effort that has gone into examining the expression characteristics of the GABAA receptor gene family. It was initially hoped that such studies, particularly those employing in situ hybridization techniques, would provide some insight into the possible subunit combinations that might exist in viuo. It soon became clear, however, that this gene family consisted of many members, each displaying specific temporal and spatial expression patterns. Nevertheless, these studies have been useful in defining the complexity of GABAA receptor gene expression and they have also provided some clues to the identity of possible in vivo receptor subunit combinations. Consequently, this section will deal with these latter conclusions and will not attempt a comprehensive review of this literature. 1 . Cell-Specific Expression

T h e rodent brain has been most widely used for the analysis of GABA, receptor gene expression by in situ hybridization with most studies focusing on a limited number of transcripts in particular brain regions. In addition, different groups have used a variety of probe types (oligonucleotides, DNA, or antisense mRNA) for the detection of GABAA receptor gene transcripts; consequently, although qualitative comparisons between these studies are possible, quantitative or even semiquantitative comparisons are difficult. Two groups have, however, made comprehensive studies of the distribution of GABAA receptor subunit mRNAs in the rat brain that do not suffer from these limitations. Seeburg and co-

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workers examined the distribution of all 13 known GABAA receptor genes (Laurie et al., 1992a; Wisden et al., 1992), whereas Richards and co-workers examined all hut those mRNAs encoding the ad-and the y3subunits (Persohn et al., 1992). The data from these studies are by and large consistent, not only with each other, but also with the more limited studies conducted by other workers. Seeburg and co-workers (Laurie et al., 1992a; Wisden et al., 1992) discuss in detail their colocalization data, with respect to previously reported ligand-binding autoradiographic and in vitro expression data, in an attempt to propose rationally possible subunit combinations of in vivo receptor subtypes. Some rat brain regions contain a large repertoire of GABAAreceptor transcripts, making any deductions about the possible subunit compositions in these areas difficult. For example, all subunit mRNAs except those encoding the a,-subunit are found in the dentate granule cells (Wisden et al., 1992). There are, however, a number of brain regions that express a more limited set of GABA, receptor genes, enabling restricted conclusions to he drawn about the subunit combinations that might give rise to specific receptor subtypes in these regions. Certain subunit genes are very often coexpressed at similar levels, such as a l p 2 , (~2p3, a& and asp,.The major GABAA receptor subtype is proposed to be a,P,y2 (Laurie et al., 1992a; Shivers et al., 1989; Wisden et al., 1992), which would correspond to the Bzl subtype (Pritchett et al., 1989a). Similarly, the major receptors constituting the Bz2 subtype are proposed to be a2p3y2,a3Pxy2 and a s p l y , combinations (Wisden et al., 1992). In certain brain regions other combinations are suggested that would represent specific receptor subtypes present at low overall levels. For example, the a,-subunit transcript is only found in cerebellar granule cells (Kato, 1990),in combination with a,-, p2-,p3-,y2-,and $-subunit mRNAs (Laurie et al., 1992a; Wisden et al., 1992). Given the evidence suggesting a pentameric structure for the GABAA receptor (Section V,D), the presence of six subunit mRNAs in these cells suggests at least two receptor subtypes there and Laurie et al. (1992a) propose a,&y2 and a,a,&y2 as likely combinations. Supporting evidence from immunoprecipitation studies for more than one a-subunit in a given receptor complex has been discussed previously (Section 111,D). Putative Bergmann glia, found in the Purkinje cell layer of the cerebellum, appear to contain only a2-and y,-subunit transcripts (Laurie et al., 1992a), a combination that has been shown to produce functional receptors in transfected cells (Verdoorn et al., 1990). In the thalamus, a region that has been proposed to contain GABAA receptors without an associated benzodiazepine binding site (Olsen et al., 1990), only a l - , a4-,and p2-subunit mRNAs are present, with the &subunit mRNA displaying a more restricted distribution pat-

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tern (Wisden et al., 1992). Thus, it is possible that arla4pnoccur together with the addition of 6 in some thalamic nuclei (Wisden et al., 1992). In the spinal cord, major subtypes have been proposed to comprise combinations of a,, a,,a3,p3, and y2 (Persohn et al., 1991, 1992). T h e large size of spinal motor and ganglionic neurons allowed ultrathin sections to be used for sequential probe analysis, demonstrating colocalization of subunit transcripts a I with a,, a, with p3, and p3 with y2 in individual cells (Persohn et al., 1992). Furthermore, the combination ~ t z p 3 ~was 2 shown to colocalize to individual spinal cord motoneurons (Persohn et al., 1992). T h e various subunit combinations suggested to exist in rat brain do not take into account the presence of the y l - and 7,-subunit transcripts that are found in a number of specific brain regions, albeit at lower levels than that of the y,-subunit (Laurie et al., 1992a; Wisden et al., 1992). Thus, it is possible that a number of the subunit combinations proposed as receptor subtypes that contain the y,-subunit may also exist as further combinations with the y,-subunit substituted with either a yI- or a y 3 subunit (Laurie et al., 1992a; Wisden et al., 1992). There are a few nuclei that contain lower levels of the y,-subunit mRNA relative to that of the y,-subunit (e.g., medial amygdaloid nucleus) or the y,-subunit (e.g., medial geniculate nucleus) transcripts (Wisden et al., 1992). In these regions, however, assignment of subunit combinations is difficult because of the large number of other subunit transcripts present (Wisden et al., 1992). T h e above studies used probes that detect both the long and the short alternate splice forms of the y,-subunit gene transcript and no in situ hybridization study has been reported to date describing the possible differential distribution of these two y,-subunit isoforms in adult rat brain. We have demonstrated that these variants are differentially expressed in the brains of l-day-old chicks (Glencorse et al., 1992) using oligonucleotide probes that specifically detect each of these mRNA species. These data demonstrate that certain brain regions either express one of these two transcripts or express both. It is possible, therefore, that cells expressing both mRNAs contain either two receptor subtypes, each containing a different y,-subunit isoform, or a single receptor subtype that incorporates both ?,-subunit isoforms (Glencorse et al., 1992). It is probable that a similar differential distribution of these alternative splice forms in the rat brain will be revealed by in situ hybidization, especially given the similarity in distribution patterns of both the 7,- and the &,-subunit transcripts between chick and rat brains (Bateson et al., 1991a; Glencorse et al., 1991). In support of this, Whiting et al. (1990) used the polymerase chain reaction to show differences in the relative

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amounts of these two alternate splice forms between gross regions of the rat brain. More recently, it has been shown that the y,,-subunit isoform is preferentially expressed, over that of the yZL,in rat pituitary cells (Valerio et al., 1992) and in the developing rat embryo (Poulter et al., 1993). As a consequence, therefore, of the differential expression of the 7,-subunit isoforms, it is most likely that each proposed receptor subunit combination that contains a y,-subunit actually represents either two (yZLor y Z s )or three (yZL,yZs,and yZLwith Y , ~ )receptor subtypes.

2 . Developmental Expression Given the large number of GABAA receptor genes, it is perhaps not surprising that fewer studies have been conducted tracing developmental expression. Nevertheless, it is clear that each subunit gene displays a specific developmental expression profile. For example, the overall levels of the +-subunit (Laurie et al., 1992b; MacLennan et al., 1991), agsubunit (Laurie et al., 1992b),a,-subunit (Laurie et al., 1992b; MacLennan et al., 1991), and a,-subunit (Laurie et al., 1992b) transcripts are higher in the brains of developing rats than in those of adult animals. In contrast, the a,-subunit (Beattie and Siegel, 1993; Bovolin et al., 1992b; Gambarana et al., 1991; Laurie et al., 1992b; MacLennan et al., 1991; Zhang et al., 1992) and +subunit (Laurie et al., 1992b) transcripts are undetectable before birth and reach peak expression in the adult rat brain. Similarly, differential expression profiles exist for the other subunit transcripts with those encoding the &-subunit (Beattie and Siegel, 1993; Gambarana et al., 1991; Laurie et al., 1992b), y,-subunit (Beattie and Siegel, 1993; Gambarana et al., 1991; Laurie et al., 1992b), and 6subunit (Laurie et al., 1992b) being present predominantly in the adult rat brain. The alternative splicing of the ?,-subunit transcript is also developmentally regulated, with levels of yps-subunit isoform remaining fairly constant from birth to adult whereas the y,,-subunit isoform rises from virtually undetectable levels at birth to peak expression in the brains of adult rats (Bovolin et al., 1992b) and mice (Wang and Burt, 1991). In brain regions that express only a subset of GABA, receptor subunit genes, it has been possible to trace a developmental switch from one subunit combination to another. Thus, the globus pallidus changes from a a2/a3&y,-subunit combination that that of a,p2y,/y2and the medial septum changes from a a,la3&y,-subunit combination to that of a,&y, (Laurie et al., 1992b). Such changes in expression are not universal, however, with some brain regions and cell types displaying differential developmental profiles. For example, in the cerebellum the levels of a,-, &-, &-, and y,-subunit transcripts in Purkinje cells did not alter from Postnatal Day 6 (when these cells could be resolved as a monolayer) to

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adulthood (Laurie et al., 1992b). In contrast, the large numbers of transcript types detected in postmigratory cerebellar granule cells could be classified into those (a,, a3, p,, yl, and y3) that showed little change in levels from Postnatal Day 6 to adulthood and those ( a l ,a6,p2, p3,y 2 , and 6) that showed a significant increase in their levels over the same period (Laurie et al., 199%). Thus, in two different cell types of this brain &-,p3-,and y,-subunit transcripts display differential region, the al-, developmental regulation (Laurie et al., 1992b).

C. IMMUNOCYTOCHEMICAL LOCALIZATION Despite the relative ease with which the cell-specific patterns and developmental profiles of GABA, receptor gene expression can be generated, these data d o not constitute proof that any of the subsequently proposed subunit combinations actually exist as in vivo receptor complexes. Detection of a particular mRNA species can be taken to imply only that the corresponding polypeptide is present in the same cell. Furthermore, even if the level of a particular mRNA species does reflect the level of the corresponding protein, there may be gross differences in intracellular localization (i.e., the mRNA residing in the cell body with the corresponding polypeptide being located in dendrites or axon terminals). T h e development of subunit-specific antibodies has, unfortunately, proved to be a difficult task and it is only recently that sufficient data have become available to assess the correspondence between the expression of a particular GABA, receptor gene and the presence of the translated mRNA product. Despite concerns about the use of mRNA localization studies to predict the localization of the corresponding subunit polypeptide, the data from immunocytochemical studies are largely a2-,a3in agreement with those of in situ hybridization. Thus, the al-, (Zimprich et al., 1991), a5-, a6-(Thompson et al., 1992),y 2 - (Benke et al., 1991a), and 6- (Benke et al., 1991b) subunits largely colocalize with their corresponding mRNAs. One of the major differences noted is with the y2-subunit, which has been detected by immunocytochemistry at high levels in the islands of Calleja and the substantia nigra (Benke et al., 1991a). In situ hybridization reveals that these regions contain y,-subunit mRNA but at levels lower than would be predicted from the corresponding polypeptide levels (Malherbe et al., 1990b; Shivers et al., 1989; Wisden et al., 1992). In the hippocampus the opposite situation is apparent with high levels of the ?,-subunit mRNA (Malherbe et al., 1990b; Shivers et al., 1989; Wisden et al., 1992) but only moderate levels of the polypeptide (Benke et al., 1991a). These results may be due to differences in the

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intracellular localization of mRNA and protein. Alternatively, some neurons may display differing gene transcription, mRNA turnover, mRNA translation, and/or protein turnover rates for the same gene or gene product. Further immunocytochemical studies will be required to determine whether the predominant equivalence of mRNA and protein distribution patterns seen to date holds true for all GABAA receptor subunits in all brain regions. A recent study by Fritschy et al. (1992), utilizing double and triple immunofluorescence labeling techniques, examined the colocalization of a,-, a3-,y2-, and &/p3 subunits of rat brain with polyclonal antibodies specific for the a l - ,a3-,and y2-subunits and a monoclonal antibody that detects both p2- and P3-subunits. Concentrating on regions that display a restricted repertoire of subunits, they identified five distinct patterns of colocalization; alfi2IP3y2, a&/&Y2, (Yla3&/@3Y29 a 3 7 2 3 and ala372. By examining the distribution on single cells they showed that the different subunits displayed local patterns of staining intensity, suggesting that these subunit combinations actually form complexes. Furthermore, in some cell types (Purkinje cells and hippocampal pyramidal cells) they found differential staining between cell bodies and dendrites, with the aI-and a,-subunit-specific antibodies, indicating the presence of more than one receptor subtype in these cells. One caveat to the immunocytochemical approach is that, just as the presence of a particular mRNA species does not prove the presence of the corresponding polypeptide, the presence of a specific set of polypeptides does not necessarily mean that they are formed into a functional receptor complex. However, as more data become available, it is becoming clear that these different methods of determining GABAA receptor gene product distribution largely produce compatible results.

D. ASSEMBLY OF SUBUNITS

Electron microscope imaging of uranyl actetate-stained GABAA receptors purified from pig brain has shown that this receptor exhibits the same pseudo-pentameric symmetry as the nicotinic acetylcholine receptor (Nayeem et al., 1994). Given the increasing evidence that a single neuron may express more than five GABAA receptor subunit genes, and thus more than one receptor subtype, a major question arises concerning the mechanism(s) by which subunits coassemble. At least in heterologous expression systems there does appear to be a preferential set of subunit combinations that associate. Using all possible homo- and heterooligomeric combinations of al-, p2, and y2-subunits, Verdoorn et al. (1990)

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found that only a l y 2 ,alp2,and a,P2y, combinations produced receptors displaying large GABA-gated currents, suggesting that these combinations assemble efficiently into functional receptors. More recently, Angelotti and co-workers (Angelotti and Macdonald, 1993; Angelotti et al., 1993) found that transfecting mouse L cells with al-,P1-,and y,,-subunit cDNAs resulted in alp1-and al@,yzs-containingreceptors, with the latter being the preferential form. In this experimental system a1y2sor PlyBs combinations did not form functional receptors. Thus, neurons that express a large number of GABA, receptor subunits may not display the full theoretical number of possible receptor subtypes. A recent study (Perez-Vetazquez and Angelides, 1993) has exmained differential GABA, receptor subunit localization within a single cell. When separately expressed in epithelial cells, GABA, receptor a l - and P,-subunits were targeted to the basolateral and the apical membranes, respectively. Coexpression of these subunits resulted in the al-subunit being rerouted to the apical surface. Different subunits, or subunit combinations, may therefore be targeted to different parts of a neuron (e.g., cell soma versus dendrites). Thus, individual subunits in a receptor complex may not only influence channel function, but also be important for receptor assembly andlor routing.

VI. The Future

T h e foregoing account attests to the remar..a le complexity of this important inhibitory neurotransmitter receptor. It is now clear that the GABAA receptor of the vertebrate brain is not a single entity but a family of receptors. The identification of the subunit composition of individual receptor subtypes that are present in the mammalian brain, either under normal physiological conditions or in disease, is a critical task. T h e differential topographical distribution of the mRNAs that encode the plethora of subunit isoforms that constitute this receptor presumably reflect the fine tuning of this complex receptor system. Recent evidence suggests that it is possible for individual cells to express more than one receptor subtype and further that these may be targeted to distinct locations within the cell. This would provide a useful modulation of the repertoire of cellular responses. However, it is also becoming clear that the system responds to usage. Chronic stimulation of GABAergic transmission, for example, by benzodiazepine treatment, results in changes to the expression profile of subunit isoforms. This could be responsible for the development of both tolerance and withdrawal seen with these

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agents clinically. If this is the case, then it may also be that similar changes underlie the pathophysiology of certain conditions in which inhibitory neurotransmission has been implicated. Thus, the regulatory mechanisms controlling GABA, receptor gene expression are at present an arena ripe for exploration. The molecular complexity that has been revealed over the past 5 years must have consequences for both the structure-function relationships of individual GABA, receptors and their roles in the integration of neuronal inhibition. To rationalize this complexity is the formidable task that lies ahead. It will require a synthesis of approaches culled from molecular biology, biochemistry, immunology, electrophysiology, and pharmacology. Perhaps it is not surprising that the vertebrate brain, which relies heavily on neuronal inhibition to support its functional integrity, has developed, thorough evolution, such a diverse GABA, receptor family to accommodate its needs.

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THE PHARMACOLOGY AND FUNCTION OF CENTRAL GABAB RECEPTORS

David D. Mott* and Darrell V. Lewist *

t

Departments of Pediatrics (Neurology) and Pediatrics (Neurology) and Neurobiology, Duke University Medical Center, Durham, North Carolina 2771 0

I. Introduction 11. Pharmacology of GABA, Receptors

A. GABAB Receptor Agonists B. GABAB Receptor Antagonists 111. Properties of GABAB Receptors A. GABAB Receptor Binding B. GABAB Receptor Distribution C. GABAB Receptor-Effector Systems 1V. Function of GABAB Receptors A. Postsynaptic GABAs Receptors B. Presynaptic GABA, Receptors on Excitatory Terminals C. Presynaptic GABA, Receptors on Inhibitory Terminals D. Opposing Effects o f Pre- and Postsynaptic GABA, Receptors V. Summary and Conclusions References

1. Introduction

It has become apparent that virtually all neurotransmitters that activate ligand-gated channels to produce fast synaptic transmission lasting for milliseconds also interact with a second class of receptors that produce slow synaptic responses that can last for seconds or minutes (Hille, 1992b). These two types of receptors serve very different functions. The receptors responsible for fast transmission are directly coupled to their effector channel and produce rapid, powerful responses allowing precise control of neuronal activity. In contrast, the receptors responsible for slow synaptic responses are indirectly coupled to their effector channels through guanosine 5'-triphosphate (GTP)-binding proteins [G proteins (Hille, 1992a)I. In addition to affecting ion channels, the G proteins coupled to these receptors can initiate protein phosphorylation, which potentially affects a variety of intracellular processes, including activation of certain genes. These G proteins can also interact with and modulate the outcome of other intracellular signaling pathways. This cascade of INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 36

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events enables G protein-coupled receptors to produce dramatic and prolonged changes in neuronal function, which can alter the responsiveness of neurons to subsequent synaptic signals. Potentially, these receptors can produce widespread changes in the state of neuronal activity. One such neurotransmitter that has been shown to activate receptors responsible for both fast and slow synaptic transmission is the inhibitory neurotransmitter y-aminobutyric acid (GABA). GABA is the primary inhibitory neurotransmitter in the mammalian central nervous system and exerts a powerful inhibitory influence in virtually every area of the brain. Thus, GABA has the potential to exert widespread regulation over neuronal function. GABA produces this inhibition through two different receptor types, termed GABAA and GABAB on the basis of their pharmacological properties. GABAA receptors are the better characterized of the two types. These receptors produce fast, powerful synaptic inhibition by directly increasing membrane chloride conductance and are responsible for classical feedforward (Alger and Nicoll, 1982a) and feedback [recurrent (Kandel et al., 1961; Andersen et al., 1963)] inhibition. In contrast, much less is known about GABA, receptors. In general, these receptor have been found to produce smaller, slower responses than GABAA receptors and are considered to be modulatory (Bowery, 1989, 1993; Ogata, 1990a). GABAB receptors are G protein-coupled to a number of cellular effector systems and so can have a variety of effects on neuronal activity (Wojcik and Holopainen, 1992; Bowery, 1993). Thus, in contrast to GABAA receptors, GABA, receptors enable GABA to produce a prolonged modulation of neuronal function. Furthermore, because of their modulatory action, these receptors may be a safe and effective site for the pharmacological manipulation of neuronal activity. Therefore, in this review we will focus on the pharmacological properties of GABA, receptors, the intracellular signaling systems to which these receptors are coupled, and their role in regulating synaptic transmission in the central nervous system.

11. Pharmacology of GABAs Receptors

A. GABA, RECEPTOR AGONISTS The GABA analogue baclofen (P-p-chlorophenyl GABA) was critical in the identification of GABAB receptors as a separate type of GABA receptor (Fig. 1).Baclofen was originally synthesized as a lipophilic GABA

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H2N-COOH

61 Baclofen

GABA

0 H~-!-H OH

3-APPA

0 YN

LCH~

6H

3-APMA

FIG. 1 . Structures of some selected CABABreceptor agonists.

analogue that could gain access to the brain and mimic the actions of GABA (Faigle and Keberle, 1972). However, it was subsequently observed that whereas baclofen and GABA produced many similar effects, baclofen did not mimic all of the actions of GABA. Furthermore, although most effects of GABA were potently antagonized by low concentrations of the plant alkaloid bicuculline the actions of baclofen were insensitive to this antagonist (Curtis et al., 1974; Fox et al., 1978). The reason for the discrepancy between these effects of GABA and baclofen was first put forth by Hill and Bowery (1981). On the basis of their observation that GABA depressed noradrenaline release from autonomic nerve terminals through a mechanism that was insensitive to bicuculline, but mimicked by baclofen, they suggested the existence of a second type of GABA receptor. They designated the classic GABA receptor as the GABA, receptor and the bicuculline-insensitive, bacolofen-sensitive GABA receptor, as the GABA, receptor. They found that these GABAs receptors were insensitive to isoguvacine and only weakly activated by muscimol, both GABAA receptor agonists. I n contrast, these receptors were activated by baclofen in a stereospecific manner with (-)baclofen having approximately equivalent potency with GABA, whereas ( + )baclofen was about 100 times less potent (Olpe et al., 1978; Hill and Bowery, 1981; Haas et al., 1985). The endogenous ligand at both GABAA and GABA, receptors is GABA (Fig. 1). T h e ability of GABA to bind to the recognition sites of both receptors arises from the ability of the GABA molecule to exist in

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a variety of conformations (Bowery, 1989; Kerr et al., 1990). It has been inferred from the structure of GABAA receptor ligands that in order for GABA to bind to GABAA receptors, it must assume an extended, planar conformation (Krogsgaard-Larsen et al., 1977,1988; Aprison and Lipkowitz, 1989). In contrast, the configuration of GABA binding to GABA, receptors is not yet known (Kristiansen et al., 1992). However, the structural requirements for GABAB receptor activation appear to be quite stringent, since few selective GABA, agonists have been found. In fact, for almost a decade, baclofen was the only known selective agonist at this receptor. The problem is that many of the compounds designed to be specific ligands at GABAB receptors have unfortunately also proven to show significant activity at GABA, receptors (Kerr et al., 1990). Recently, however, several phosphinic acid derivatives of GABA that are selective agonists at GABA, sites have been introduced. Two of the most potent of these compounds are 3-aminopropylphosphinic acid (3APPA) and its methyl derivative, 3-aminopropyl-methylphosphinicacid (3-APMA) (Fig. 1). They each appear to be 10-100 times more potent than baclofen and have been shown to act on both presynaptic and postsynaptic GABA, receptors (Ong et al., 1990a; Seabrook et al., 1990; Hills et al., 1991; Lovinger et al., 1992). However, 3-APPA appears to act as a partial agonist for some GABAB receptor-mediated effects (Bowery, 1989; Seabrook et al., 1990).For example, in neocortical neurons baclofen markedly suppressed the frequency of spontaneous burst discharges produced by removal of extracellular magnesium. In contrast, the frequency of bursting was insensitive to 3-APPA (Ong et al., 1990b). Similarly, baclofen has been shown to enhance P-adrenergic receptorstimulated adenosine 3' : 5'-cyclic monophosphate (CAMP)accumulation (see Section III,C,2). However, 3-APPA had no such effect (Scherer et al., 1988a; Pratt et al., 1989). Alternately, Raiteri (1992) reported that potassium-evoked [3H]GABArelease from spinal cord synaptosomes was suppressed by both GABA and 3-APPA, but not by (-)baclofen. All three agonists potently reduced [3H]GABA release in this preparation. The author concluded that GABA, receptors in cerebral cortex differ from those in spinal cord, the latter having a lower affinity for ( - )baclofen. These differences in the actions of baclofen and 3-APPA may indicate the existence of multiple GABAB receptor subtypes.

B. GABAB RECEPTOR ANTAGONISTS In order to determine the physiological role of GABAB receptors, it is apparent that receptor antagonists are necessary. However, in contrast

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to the GABA, receptor-channel complex for which the blockers bicuculline and picrotoxin are available, no naturally occurring compounds have been identified that specifically antagonize GABA, receptors. Attempts to synthesize GABA analogues that were antagonists at the GABAB receptor initially resulted in the production of 6-aminovaleric acid (DAVA) and 3-aminopropanesulphonic acid (3-APS) (Muhyaddin et al., 1982; Giotti et al., 1983; Schwarz et al., 1988) (Fig. 2). Although these compounds did block GABAB receptors, they did so with extremely low potency. Furthermore, they appeared to be agonists at GABA, receptors as well. Thus, they were of limited utility. Greater success was achieved through the synthesis of phosphonic analogues of baclofen, resulting in the production of phaclofen [3-amino2-(4-chlorophenyl)-propylphosphonicacid (Chiefari et al., 1987; Kerr et al., 1987)] (Fig. 2). Phaclofen appeared to be a weak but selective antagonist for GABAB receptors, requiring high concentrations (1mM) for effective GABAB receptor blockade. However, at these high concentrations, phaclofen also depresses GABA, receptor-mediated responses, possibly by acting as a partial agonist at GABAB receptors (Stirling et al., 1989; Kerr et al., 1990). Furthermore, phaclofen alone has been reported to stimulate the production of inositol 1,4,5-triphosphate in cultured neurons from chick tectum, indicating that it can act at a site other than GABAB receptors (Michler and Erdo, 1989). Interestingly, phaclofen appears to antagonize postsynaptic GABAB responses at lower concentrations than presynaptic GAHAB responses, suggesting that presynaptic and postsynaptic GABA, receptors may be pharmacologically distinguishable (Dutar and Nicoll. 1988a; Bonanno and Raiteri, 1992; Soltesz and Crunelli, 1992; Pende et al., 1993). However, given the low potency of phaclofen, further studies using more potent and selective antagonists are needed to confirm this possibility. Sulfonic analogues of baclofen have proven to be more potent GABAB receptor antagonists than phaclofen. Of these compounds, 2hydroxysaclofen (3-amino-2-(4-chlorophenyl)-2-hydroxypropylsulfonic acid) and saclofen (3-amino-2-(4-chlorophenyl)-propylsulfonic acid) are the most active, displaying approximatley 10 time greater potency than phaclofen (Curtis et al., 1988; Kerr et al., 1988, 1989) (Fig. 2). Binding studies have indicated that 2-hydroxysaclofen not only reduced the affinity of baclofen binding, but also reduced the apparent density of GABAB receptors, suggesting that the drug is a mixed competitive, noncompetitive antagonist (Al-Dahan et al., 1990). 2-Hydroxysaclofen effectively antagonizes both presynaptic and postsynaptic GABAB receptormediated effects. However, high concentrations of the antagonist are required to produce this blockade and at these higher concentrations,

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2-hydroxysaclofen has been reported to have small but significant effects on the binding of ligands to GABA,, serotonin, and muscarinic acetylcholine receptors (Al-Dahan et al., 1990). Similarly, we have found that high concentrations of 2-hydroxysaclofen (>400 / A M )partially suppresse GABA, receptor-mediated responses (Mott and Lewis, 1991). Thus, this antagonist appears to most effectively discriminate GABAB receptors when used in low concentrations. Similar to the strategy used to produce the potent agonists 3-APPA and 3-APMA, the most effective GABAB receptor antagonists to date are based on the phosphinic analogues of GABA. The first of these to be introduced was CGP 35348 [P-(3-aminopropyl)-P-diethoxymethylphosphinic acid (Bittiger et al., 1990; Oipe et al., 1990; Seabrook et al., 1990)] (Fig. 2). Binding studies reported that this compound was a competitive antagonist; however, the affinity of this compound for GABAB receptors was weak, similar to that of 2-hydroxysaclofen. CGP 35348 appeared to be very selective for GABAB receptors, as it has been shown not to act on GABA,, glutamate, muscarinic acetylcholine, a-adrenergic, serotonin (5-HT,), histamine (H,), or adenosine (A,) receptors. T h e major advantage of CGP 35348 over phaclofen and 2-hydroxysaclofen is its ability to cross the blood-brain barrier, allowing it to be systemically administered in vivo. A variety of other phosphinic analogues of GABA have recently been introduced, some of which are significantly more potent than previous GABA, receptor antagonists (for review see Froestl et al., 1992). Two of these compounds represent the first orally active GABAB receptor antagonists (Bittiger et al., 1992a; Lingenhohl and Olpe, 1993; Olpe et al., 1993a). These are P-(3-aminopropyl)-P-"-butyl-phosphinic acid (CGP 36742) and P-(3-aminopropyl)-P-cyclohexylmethylphosphinic acid (CGP 46381) (Fig. 2). Radioligand binding studies have shown that the potency of CGP 36742 in displacing bound 3-[3H]APPA is similar to that of CGP 35348; however, CGP 46381 is about seven-fold more potent. The higher potency of CGP 46381 is also evident after oral administration in vivo, where it substantially antagonizes the effects of baclofen at a dose (30 mg/kg) at which CGP 36348 is ineffective (Olpe et al., 1993a). Radioligand binding studies show that both of these compounds are selective for GABAB receptors, as they do not displace any of a variety of other receptor ligands (Bittiger et al., 1992a; Olpe et al., 1993a). Two of the most potent of the newly introduced GABAB receptor antagonists are 3-N-[l-{(S )-3,4-dichlorophenyl}-ethylamino]-2-(S )hydroxypropyl cyclohexylmethyl phosphinic acid (CGP 54626) and its a-(S)-methyl analogue, CGP 55845 (Bittiger et al., 1992b; Froestl et al., 1992; Jarolimek et aE., 1993) (Fig. 2). These compounds were each found

I03

CENTRAL CABA, RECEPTORS 0

H.#J-i.on 0

HaN-COOH

DAVA

3-APS

tl

CI

Phaclofen

2-Hydroxysaclofen

CGP 35348

CGP 46381

C G P 36742

C G P 54626

CGP 55845 FIG. 2. Structures of some selected GABA, receptor antagonists.

to displace 3-[3H]APPA binding to GABA, receptors with an IC,o of about 7 nM, making them approximately 15,000 times more potent than phaclofen and 5000 times more potent than CGP 35348. By comparison, the affinity of these compounds for the GABA, receptor is greater than that of bicuculline for the GABA, receptor as determined by either a [3H]muscimol assay [IC,, = 2 p M (Beaumont el al., 1978)] or ['HI( +)bi-

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DAVID D. MOTT A N D DARRELL V. LEWIS

cuculline methiodide assay[IC,o = 68 nh4 (Mohler and Okada, 1977)]. Because they have been recently introduced, little information is available regarding the selectivity of these compounds, although they have been reported to act exclusively at GABAB receptors in radioligand binding studies (Olpe et a1.,1993a). Similarly, little is known of their effects on synaptic responses, although a recent study by Jarolimek et al. (1993) reported that CGP 55845 potently antagonized postsynaptic GABAB receptor-mediated currents in hippocampal neurons. These new highly potent antagonists promise to provide many insights into the physiological role of GABAB receptors. In particular, the ability of several of these compounds to be orally active suggests that they will be useful tools for investigating the role of GABAB receptors in modulating behavior. Preliminary reports suggest that these new antagonists are not selective for GABAB receptor subtypes. As will be discussed below (see Section IV,D), GABAB receptors located postsynaptically can have different and often opposing effects on neuronal activity compared to those located presynaptically on axon terminals. Because of these opposing actions, generalized GABAB receptor antagonists often produce complex and contradictory effects on neuronal activity. Thus, antagonists that are selective for presynaptic or postsynaptic GABAB receptors are needed.

111. Properties of GABAB Receptors

A. GABAB RECEPTORBINDING GABAB receptor binding was first demonstrated in crude synaptic membranes using the tritiated form of baclofen as the prototype ligand (Hill and Bowery, 1981; Bowery et al., 1983). [3H]Baclofen was used because of its high potency and specificity for bicuculline-insensitive GABA receptors as well as its inability to be transported into neuronal tissue via the Nat-dependent GABA uptake process (Bowery et al., 1983). 3H-Baclofenbinding was stereospecific with the ( - )isomer being approximately 100 times more potent than the (+)isomer (Hill and Bowery, 1981; Robinson et al., 1989). Subsequent studies have commonly used [3H]GABA with addition of the GABAA agonist isoguvacine to prevent binding to GABAA receptors. These studies have reported that both GABA and (-)baclofen bind to a common GABAB receptor site with equal affinity (Hill and Bowery, 1981). In general, Hill coefficients close to unity and linear Scatchard plots have been reported, indicating a

CENTRAL G A B A s RECEPTORS

105

single population of GABA, receptors (Hill and Bowery, 1981; Majewska and Chuang, 1984; Bowery et al., 198513; Al-Dahan and Thalmann, 1989; Chu et al., 1990). The binding affinity ( K d )of [3H]GABA for both GABAB and GABAA receptors is similar and has typically been reported to be in the range of 30 to 80 nM (Bowery et al., 1983; Falch et al., 1986, but see Chu et al., 1990). However, several studies, using low concentrations of [3H](- )baclofen o r ['HIGABA, have reported both low-affinity (Kd 229-304 nM) and high-affinity (Kd 19-60 nM) forms of the receptor, suggesting either that the same receptor can exist in two different affinity states or that there are two populations of GABAB receptors (Karbon et al., 1983; Bowery et al., 1985b). In addition to GABA and baclofen, several other compounds have also been examined for their ability to displace 3H(-)baclofen from GABAB receptors. For example, 3-APPA is approximately 100 times more active than (-)baclofen at GABAB receptors (Pratt et al., 1989; Ong et al., 1990a). In contrast, the GABAA agonist isoguvacine and the GABAA antagonist bicuculline methobromide are devoid of any activity at the GABAB receptor, whereas the GABAA agonist muscimol is only weakly active, being over 300-fold less potent than (-)baclofen (Bowery et al., 1983). A summary of the relative potencies of several different compounds is given in Table I. Unlike binding to GABAA receptors, ligand binding to GABA, receptors is dependent on physiological concentrations of the divalent cations Ca2+ or Mg2+ (Hill and Bowery, 1981; Bowery et al., 1983; Kato et al., 1983; Majewska and Chuang, 1984). [3H]Baclofen or [3H] GABA binding is increased in a concentration-dependent fashion by divalent cations with the following order of potency Mn2+ = Ni2+ > Mg2+ > Ca2+> Sr2+> Ba" (Kato et al., 1983). T h e maximal effect of these divalent cations is not additive, suggesting that they act through a common binding site. Although these cations enhance GABA, receptor binding, other divalent cations, including Hg2+,Pb'+, Cd2+,and Zn2+, inhibit binding of [2H]baclofen to GABAB receptors (Drew et al., 1984; Turgeon and Albin, 1992). The opposing effects of these two groups suggest the existence of both excitatory and inhibitory cation sites for modulation of CABAB receptor binding. Another characteristic of CABAB receptors is their sensitivity to guanyl nucleotides. Guanyl nucleotides, such as GTP, do not affect GABAA receptor binding, but potently inhibit GABAB receptor binding (Hill et al., 1984; Asano et al., 1985; Ohmori et al., 1990). This effect is specific for guanyl nucleotides, as it is not mimicked by adenosine 5'-triphosphate (ATP). The inhibition of ligand binding produced by GTP is the result of a reduction in the affinity of the GABAB receptors rather than a

106

DAVID D. MOTT AND DARRELL V. LEWIS TABLE I POTENCY OF VARIOUSGABA ANALOGUES AT GABABSITES Analogue

GABA, binding (IC50; F M ) ”

GABA agonists GABA (-)Badofen (?)Badofen (+ )Badofen 3-APPA 3-APMA Muscimol Isoguvacine

0.026-0.04 0.03-0.08 0.18 15-33 0.00 1-0.007 0.014 2.4-12.3 > 100

GABA antagonists 3-APS 8-Aminovaleric acid Phaclofen 2-H ydroxysaclofen CGP 35348 CGP 36742 CGP 4638 1 CGP 54626 CGP 55845 Bicuculline methobromide

11 7-50 108-130 5.1-8.5 34 35-36 4.9 0.006-0.007 0.007 >lo0

Reference* 1-3 2-5 3

2, 3 4, 6, 7 4 2, 3, 7 3. 7 3 1-3 4, 5, 8 5, 8 9 10,Il 11 4, 12

4 3, 7

” Binding affinities were determined by measuring the displacement of bound [3H]baclofen (3,5,8), [3H]GABA + isoguvacine (1, 2, 6 ) , or [3H]3-APPA [CGP 27492, (4, 7, 9, 10, 11, 12)J from rat brain synaptic membranes. 1. Kristiansen et al. (1992); 2. Falch el al. (1986); 3. Bowery et al. (1983); 4. Froestl et al. (1992); 5. Al-Dahan et al. (1990); 6. Pratt et al. (1989); 7. Bittiger el al. (1988); 8. Drew et al. (1990); 9. Olpe el a/. (1990); 10. Bittiger et al. (1992a); 11. Olpe el a!. (1993a); 12. Bittiger et al. (1992b).

change in their binding capacity (Hill et al., 1984; Asano et al., 1985). Inhibition of GABAB receptor binding by GTP is blocked by pertussis toxin, suggesting that GABAB receptors are functionally coupled to inhibitory G proteins, either Gi and/or Go (see Section 111,C). Inhibition of ligand binding by guanyl nucleotides has been reported for other types of receptors, such as dopamine (Freedman et al., 19811, opiates (Pert and Taylor, 1980), a-adrenergic agonists (U’Prichard and Snyder, 1978), and serotonin (Mallat and Hamon, 1982), which are also coupled to GTP binding proteins. In addition to modulation of GABA, receptor binding by divalent cations and guanyl nucleotides, several other features also distinguish

CENTRAL GABA, RECEPTORS

107

GABA, from GABAA receptor binding sites. For example, numerous studies have shown that GABA binding to GABAA receptors may be enhanced by either benzodiazepines or barbiturates (Tallman et al., 1978; Willow and Johnston, 1980; Asano and Ogasawara, 1982; Skerritt et al., 1982). However, GABAB receptor binding is not affected by either of these classes of drugs (Doble and Turnbull, 1981; Bowery et al., 1988). Another difference in GABA, and GABAB receptors is in their reaction to receptor solubilization. Unlike GABA, reeptor binding, GABAB receptor binding is inhibited by exposure of the membranes to a variety of detergents, such as sodium deoxycholate o r Triton X-100 (Bowery et al., 1983; Ohmori et al., 1990), suggesting that GABAB receptor function is easily destroyed following solubilization. This reduction in GABAB receptor binding is produced primarily by a reduction in the number of binding sites, rather than any alteration in their affinity, suggesting that solubilization inhibits GABAB receptor binding either by removing the receptor and/or cation binding site from the membrane o r by denaturing the receptor (Bowery et al., 1983). T o date, GABAB receptor binding in both crude synaptic membranes and tissue slices has been examined exclusively using tritiated GABAB receptor agonists. However, Bittiger et al. (1992b), using crude membrane fractions from rat cerebral cortex, have recently reported the first GABA, binding study using a labeled GABAB receptor antagonist ([3H]CGP54626). Binding studies using labeled antagonist are of interest because they can potentially reveal different affinity states of the receptor or a different number of receptors or provide a more accurate determination of antagonist affinity than agonist binding studies. Using the labeled antagonist, Bittiger et al. (1992b) reported a maximum number of binding sites (BmaX)that was two to three times greater than that found for agonist binding. In addition, although the potencies of antagonists (CGP 35348 and CGP 36742) in displacing labeled antagonists were not different from those found in agonist displacement studies, agonists (GABA, (-)baclofen, and 3-APPA) were found to be much weaker. Taken together, these results suggest that GABAB receptor antagonists bind to different states of the GABAB receptor or receptor subtypes, which are only partially accessible to agonists. B. GABAB RECEPTORDISTRIBUTION GABA, receptors have been demonstrated on neurons in both the peripheral (For review see Ong and Kerr, 1990) and the central nervous system. In the central nervous system receptor autoradiography using [3H]GABA and [3H]baclofen (Gehlert et al., 1985; Bowery et al., 1987;

1 oa

DAVID D. MOTT AND DARRELL V. LEWIS

Chu et al., 1990) has demonstrated that, although most regions of the brain contain both GABA, and GABAs binding sites, GABAA sites predominated in the majority of these areas. For example, GABA, receptors were found to be in the highest concentration relative to GABAB receptors in areas such as frontal cortex, hippocampus, subiculum, amygdala, septum, the external plexiform layer of the olfactory bulb, and the granule cell layer of the cerebellum. Chu et al. (1990) reported that in these regions GABA binding to GABAA receptors accounted for about 70430% of total GABA binding. In contrast, in a few areas, including the molecular layer of the cerebellum, the interpeduncular nucleus of the brainstem, and certain thalamic nuclei, GABAB receptors were in higher concentration and in these areas GABAB sites could account for up to 90% of the total GABA binding. Finally, in other regions, such as the superficial gray of the superior colliculus and certain thalamic nuclei, the concentration of both GABAB and GABAAbinding sites was similar. The regions of the brain that exhibited the highest absolute concentrations of GABABbinding were frontal cortex, interpeduncular nucleus, the glomerular layer of the olfactory bulb, the superficial gray of the superior colliculus, and the molecular layer of the cerebellum. Intermediate levels of GABABbinding were found in the entorhinal cortex, molecular layer of the hippocampal dentate gyrus, amygdala, granule cell layer of the cerebellum, and various thalamic nuclei. Low levels of GABAB receptors were observed in hippocampus, subiculum, substantia nigra, hypothalamus, various thalamic nuclei, neostriatum, and dorsal raphe nucleus. Although the hippocampal formation contains a low level of GABAB receptors, the highest concentration of binding sites in this area is observed in the outer two-thirds of the molecular layer of the dentate gyrus, with lower levels of binding in the region of the granule cell layer. In the hippocampus GABAB sites are distributed throughout areas CA 1-CA4, with GABAB receptors being more highly concentrated in the dendritic than somatic layers (Bowery et al., 1985a, 1987; Chu et al., 1990). This result agrees well with the observation that GABAB receptor-mediated potentials are most readily produced by application of GABA in the dendritic layer (Newberry and Nicoll, 1985) (see Section III,C,5). Siniilarly, in cerebellum GABAB binding sites appear to be located almost entirely in the molecular layer (Wilkin et al., 1981). However, it should be noted that because of the small diameter of dendrites, the surface area of membrane in the dendritic layer is greater than that in the cell body layer. Thus, it is possible that even if the cell had an equal distribution of GABAB receptors over its entire surface, the receptor density determined by autoradiography may appear greater in the dendritic layer.

CENTRAL GABA, RECEPTORS

109

Although the above studies suggest that postsynaptic GABAB receptors may be preferentially located on dendrites in certain areas of the brain, receptor autoradiography coupled with selective lesions has been used to determine further the subcellular location of GABAB receptors. These studies have demonstrated that GABAB receptors can be located on nerve terminals as well as at postsynaptic sites. For example, electrolytic lesion of the habenular nucleus caused a 90% reduction in GABAB receptor binding in the interpeduncular nucleus. These results indicate that the majority of the GABAB receptors in the interpeduncular nucleus are on terminals of afferent fibers arising from the habenula (Price et al., 1984). In contrast, Bowery el al. (1985a) demonstrated that lesion of the perforant path, the primary afferent projection to the molecular layer of hippocampal dentate gyrus, caused no change in GABAB receptor binding in the molecular layer. They concluded that GABAB binding sites in the molecular layer were postsynaptic. This finding agrees with the observation that baclofen has only a minimal effect on excitatory transmission in the lateral perforant pathway (Lanthorn and Cotman, 1981; Ault and Nadler, 1982). However, it does not rule out the presence of GABAB sites on terminals of inhibitory interneurons. T h e presence of both presynaptic and postsynaptic GABAB receptors has recently been confirmed using monoclonal antibodies against the ( - )isomer of baclofen to visualize directly GABA, binding sites. Holstein et al. (1992) observed postsynaptic immunoreactivity on dendrites in the medial vestibular nucleus, indicating the presence of postsynaptic GABA, receptors. The immunostained dendrites received synapses from presumed GABAergic terminal. In addition, the authors observed immunostained terminals that synapsed onto both dendrites and axons. Similarly, Martinelli et al. ( 1992) reported postsynaptic immunoreactivity in both the molecular layer of the cerebellum and the substantia nigra, indicating the presence of postsynaptic GABAB receptors in these areas. These results suggest a morphological basis for both presynaptic and postsynaptic inhibition by GABAB receptors. It should be kept in mind that studies of GABA, receptor binding using labeled agonists are subject to a number of technical limitations. For example, because guanyl nucleotides lower the affinity of GABAB receptors for agonist, labeled agonist binding could be affected by any endogenous GTP remaining in the tissue. Similarly, endogenous GABA present in the tissue may be able to displace the labeled ligand. Thus, if different brain regions have different amounts of endogenous G T P or GABA, the ligand could bind with different kinetics in these different regions. These limitations may explain some of the variability observed between GABAB receptor binding in these studies. Perhaps, labeled antagonist binding might eliminate some of these problems.

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DAVID D. MOTT A N D DARRELL V. LEWIS

C . GABAB RECEPTOR-EFFECTOR SYSTEMS

GABAB receptors are coupled to a variety of intracellular effector systems. These effector mechanisms are the means by which GABA through GABAB receptors can transduce messages into the neuron. The effector systems so far described include (a)inhibition of adenylyl cyclase, (b) facilitation of the transmitter-mediated activation of adenylyl cyclase, (c) inhibition of agonist-induced inositol triphosphate (IP,) synthesis, (d ) inactivation of voltage-dependent calcium channels, and ( e ) activation of potassium channels. We will discuss each of these actions below. 1. Inhibition of Adenylyl Cyclase The observation that guanyl nucleotides reduce the affinity of GABA, receptors for GABA (see Section II1,A) suggested that these receptors played a role in regulating adenylyl cyclase activity. Indeed, both ( - )baclofen and GABA were found to inhibit basal adenylyl cyclase activity as measured by the conversion of [32P]ATPto cyclic [32P]AMP (Wojcik and Neff, 1984; Nishikawa and Kuriyama, 1989). Similarly, if forskolin was first used to ensure activation of adenylyl cyclase, GABAB receptor activation resulted in a greater reduction in cAMP levels. Inhibition of basal adenylyl cyclase activity was observed in cultured cerebellar granule cells (Xu and Wojcik, 1986) as well as membrane preparations from a variety of different brain regions (Wojcik and Neff, 1984). Inhibition of adenylyl cyclase was strongest in the striatum, followed by cerebellum, cerebral cortex, thalamus, hippocampus, and hypothalamus. In contrast, studies using slices of cerebral cortex or cerebellum and measuring cAMP accumulation within the tissue found that whereas GABA, receptor activation by ( - )baclofen inhibited forskolin-stimulated adenylyl cyclase activity, it failed to alter basal cAMP levels (Hill et al., 1984; Hill, 1985; Karbon and Enna, 1985). Inhibition of basal adenylyl cyclase activity in cerebellar membranes is produced by both ( - )badofen and GABA with half-maximal concentrations (EC,,) of 4 and 17 p M , respectively. As was reported for baclofen binding, the (-)isomer of the drug is the active form, being approximately 1000 times more potent than the (+)isomer at inhibiting adenylyl cyclase activity. In support of the role of GABAB receptors, the GABAB receptor antagonist phaclofen blocked the inhibition of adenylyl cyclase activity produced by (-)baclofen (Nishikawa and Kuriyama, 1989). In contrast, the GABA, receptor agonist isoguvacine did not alter adenylyl cyclase activity. Similarly, neither bicuculline methiodide, a GABA, receptor antagonist, nor diazepam, a benzodiazepine, modified the inhibition of adenylyl cyclase activity produced by GABA or ( - )badofen.

CENTRAL GABA, RECEPTORS

111

Finally, since the effects of agonists at GABAB and adenosine A, receptors were not additive, Wojcik et al. (1985) concluded that these two receptors share a common catalytic subunit on adenylyl cyclase. Adenosine A, receptors are known to couple to adenylyl cyclase through the inhibitory G protein G, (Dunwiddie, 1985); therefore, this finding suggested that GABAB receptors may do the same. Receptor coupling to adenylyl cyclase is accomplished through one of the many types of G proteins. These G proteins are composed of three subunits, a, p, and y, the a subunit of which contains an intrinsic GTPase. The major events in this transduction pathway are as follows (for review see Neer and Clapham, 1988; Sternweis and Pang, 1990): agonist binding to the receptor promotes the interaction of the receptor with G protein, catalyzing the exchange of guanosine 5'-diphosphate (GDP), which is normally bound to the a subunit, for GTP. This exchange of GTP for GDP promotes dissociation of the G protein from the receptor, which reverts back to its low-affinity conformation. The liberated G protein then separates into a and Py subunits. The a subunit can activate intracellular effector mechanisms. The By subunit may indirectly contribute to the inhibition by binding to free a subunits from stirnulatory G proteins to form the inactive heterotrimer. Hydrolysis of GTP back to GDP by the GTPase of the a subunit promotes dissociation of the a subunit from the adenylyl cyclase and reassociation with the Py subunit, thereby terminating the signal. GABABreceptors have been shown to be coupled to adenylyl cyclase through the inhibitory G proteins Gi and G, (Asano et al., 1985; Ohmori et al., 1990). T h e coupling of inhibitory G proteins with GABAB receptors was first reported by Asano et al. (1985),who found that binding of GABA to GABAB receptors in cortical membranes was decreased following incubation with pertussis toxin. Pertussis toxin selectively inactivates the inhibitory G proteins Gi and G, but not the stimulatory G protein, G,. The toxin catalyzes the transfer of adenosine 5'-diphosphate (ADP)ribose from nicotinamide adenine dinucleotide (NAD) to the a subunit of Gi and G,, thereby preventing association of these G proteins with the receptor, and thus causing the receptor to remain in the low-affinity conformation. Likewise, the reduction of GABAB receptor binding produced by pertussis toxin was the result of reduced receptor affinity, not receptor number, indicating that more of the receptors were in the low affinity conformation. As predicted, the subsequent addition of purified Gi/Goproteins restored high-affinity binding of GABA to GABAB receptors. Closer examination of the subtype of G proteins(s) necessary to restore high-affinity binding revealed that addition of Go, GZ or Gil, but not Gi2,was sufficient (Morishita et al., 1990). In addition, Go remained

112

DAVID D. MOTT A N D DARRELL V. LEWIS

effective, and Gi2 remained ineffective, even when the Py subunit complex was altered. These results indicate that coupling of the G protein to GABAB receptors is determined primarily by the (Y subunit. Furthermore, the ability of GABAB receptors to couple to different types of G proteins suggests that GABAB receptors may use different G protein subtypes to regulate separately the effectors to which they are coupled. Further evidence that GABAB receptors were coupled to G proteins was provided by Hill et al. (1989) and Bowery et al. (1989), who reported that (-)badofen and GABA increased the specific GTPase activity in rat brain membranes preparations with the largest increase occurring in areas known to contain high concentrations of GABAB receptors. This increase in GTPase activity was attributed to the receptor-mediated exchange of GDP for GTP on the (Y subunit of the G protein and the subsequent activation of the intrinsic GTPase activity of this subunit. This effect was reduced by prior exposure of the membranes to pertussis toxin, indicating that it was produced by an inhibitory G protein. Although these findings indicate that GABAB receptors are coupled to inhibitory G proteins, they d o not directly demonstrate a link between the GABAB receptor-coupled G protein and adenylyl cyclase. This link was first reported in cultured cerebellar granule neurons by Xu and Wojcik (1986) and in membranes of cerebral cortex by Nishikawa and Kuriyama (1989). These studies reported that pertussis toxin blocked the inhibitory effect of baclofen and GABA on forskolin-stimulated adenylyl cyclase activity. These results suggest that both the inhibition of forskolinstimulated adenylyl cyclase activity and the increase in GTPase activity are mediated by the (Y subunits of the same inhibitory G proteins that regulate GABAB receptor binding.

2 . Facilitation of Transmitter-Mediated Activation of Adenylyl Cyclase In contrast to its direct inhibition of adenylyl cyclase through Gi proteins, GABAB receptor activation also potentiates the accumulation of cAMP produced by other receptors (Karbon et al., 1984; Karbon and Enna, 1985; Hill, 1985; Watlingand Bristow, 1986, for review see Karbon and Enna, 1989). For example, addition of baclofen to the incubation medium markedly enhanced the increase in cAMP accumulation produced by norepinephrine (Karbon et al., 1984; Hill, 1985). Similarly, GABA or baclofen potentiated the increase in adenylyl cyclase activity caused by other G,-coupled neurotransmitters, such as adenosine A,, histamine, and vasoactive intestinal peptide (VIP) receptors (Karbon and Enna, 1985; Watling and Bristow, 1986; Scherer et al., 1988b; Schaad et al., 1989). In each case, baclofen caused a two- to three-fold increase in the accumulation of CAMP produced by the neurotransmitter. Potenti-

CENTRAL GABA, RECEPTORS

113

ation of the receptor-mediated increase in cAMP accumulation by baclofen is similar to the effects of a-adrenergic agonists on the increase in cAMP levels produced by P-adrenergic agonists (Daly et al., 1980; LeBlanc and Ciaranello, 1984). Interestingly, potentiation of CAMP accumulation has only been observed following receptor-mediated activation of adenylyl cyclase, not direct activation of adenylyl cyclase with forskolin. As discussed previously, following direct activation of adenylyl cyclase with forskolin, GABAB receptor activation inhibits cAMP formation (Karbon and Enna, 1985). Potentiation of transmitter-mediated cAMP accumulation was observed in slices from cerebral cortex, hippocampus, and corpus striatum, but not cerebellum (Karbon and Enna, 1985). The effect is thought to be GABAB receptor mediated because both baclofen and GABA are equally efficacious, with a concentration of about 3 P M (?)baclofen necessary to yield a half-maximal potentiations. In addition, potentiation by baclofen was stereoselective with the (-)isomer more potent that the (+)isomer. The effect was not sensitive to GABA, receptor antagonists, such as bicuculline methiodide (Karbon et al., 1984). Finally, except for the cerebellum, the intensity of the response in different brain regions paralleled the density of GABA, binding sites in these areas (Karbon and Enna, 1985; Bowery et al., 1987). In the cerebellum GABAB receptor activation failed to potentiate the CAMP response to norepinephrine, despite the high density of GABABbinding sites in this area. This discrepancy may be caused by the existence of subtypes of the GABAB receptor, only one of which is coupled to the augmenting response. Alternatively, it may be caused by multiple forms of adenylyl cyclase that are modulated differently by G protein interaction (Tang and Gilman, 1992). T h e mechanism by which GABAB receptors potentiate receptormediated adenylyl cyclase activity has not been conclusively demonstrated. It appears unlikely that baclofen interacts with the G,-linked receptor recognition site, since baclofen is able to augment the cAMP response to a variety of different G,-linked receptors. Further studies confirmed this for the P-adrenergic system by demonstrating that baclofen did not alter the affinity or number of P-adrenoceptors (Karbon and Enna, 1989). One possible mechanism by which baclofen could potentiate the effect of G,-linked receptors of cAMP levels is by synergizing the interaction of adenylyl cyclase with the stimulatory G protein, C, (Bourne and Nicoll, 1993). Like its inhibitory counterparts, G, is a heterotrimeric protein composed of an a,a p, and a y subunit. In most cases, receptor-mediated stimulation of adenylyl cyclase is mediated solely by the a subunit of the G, protein (Neer and Clapham, 1988; Sternweis and Pang, 1990).

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However, under certain conditions, Py subunits may also carry a signal. In light of this, multiple subtypes of adenylyl cyclase, which respond differently to the presence of Py subunits, have been identified (Tang and Gilman, 1992). Adenylyl cyclase type I, when activated by a,, can be inhibited by subsequent interaction with Py subunits. In contrast, adenylyl cyclase type 11, when stimulated by the a, subunit is potentiated up to five-fold by the subsequent binding of Py subunits (Tang and Gilman, 1991; Federman et al., 1992). Because of the similarity between their Py subunits, either G, or Gi,, proteins could provide these subunits. Thus, although ail,liberated by GABAB receptor-activation of Gi/, proteins could cause direct inhibition of adenylate cyclase, Py subunits liberated by this same interaction could synergize the effect of a, on adenylyl cyclase type 11, resulting in a net increase in cAMP in the neuron. Studies have shown that both type I and type 11 adenylyl cyclase are expressed in several different brain regions, including hippocampus (Gannon and McEwen, 1992; Mons et al., 1992), suggesting that this mechanism may occur in central neurons (Andrade, 1993) (see section IV). The potentiation of receptor-mediated adenylyl cyclase activity produced by baclofen is observed only in slice preparations (Karbon et al., 1984; Hill, 1985; Karbon and Enna, 1985). In contrast, in preparations of broken cells or membranes from these same brain regions, baclofen inhibits adenylyl cyclase activity (Wojcik and Neff, 1984; Xu and Wojcik, 1986; Nishikawa and Kuriyarna, 1989) (see Section III,C,I). One possible explanation for this difference is that the GABAB receptor-mediated potentiation of cAMP accumulation is dependent on cytosolic factors, such as phospholipase A, and arachidonic acid, which are not present in the membrane preparations. Phospholipase A, is a calcium-activated enzyme that catalyzes the formation of arachidonic acid from membrane phospholipids. Arachidonic acid is then metabolized into a number of substances, including prostaglandins and leukotrienes (Piomelli and Greengard, 1990; Shimizu and Wolfe, 1990). Potentially, either arachidonic acid or its metabolites could interact with the adenylyl cyclase system to increase cAMP levels; however, the actual molecular events by which this interaction may occur remain to be determined. In support of a role for phospholipase A, in the potentiating response of baclofen, Duman et al. (1986) found that nonspecific inhibitors of phospholipase A,, such as quinacrine and corticosterone, did not affect the potentiation of cAMP levels produced by isoproterenol alone; however, they prevented baclofen from augmenting isoproterenol-mediated cAMP accumulation. Furthermore, they found that inhibitors of arachidonic acid metabolism had no effect on the augmentation by baclofen, indicating that arachidonic acid itself mediates the potentiation. These results sug-

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gest that phospholipase A, plays a role in the augmenting response of GABA, receptor activation on G,-linked neurotransmitter stimulation of adenylyl cyclase. However, because the phospholipase A, inhibitors used in this study were not very specific, additional evidence is necessary to demonstrate conclusively a role for this enzyme.

3 . Inhibition of Agonist-Induced Inositol Trifihosphate (IP,) Synthesis Inositol phospholipid breakdown represents another intracellular signaling pathway that can be modulated by GABAB receptor activation. T h e mechanism of this pathway is as follows (for review see Berridge and Irvine, 1989): Binding of agonist to the receptor triggers the activation of phospholipase C through a G protein. This enzyme catalyzes the cleavage of phosphatidylinositol-4,5-bisphosphate (PIP,) resulting in the formation of diacylglycerol (DAG) and inositol l ,4,5-triphosphate (l,4,5.JP,). Both DAG and 1,4,5-IP3can then act as second messengers in the cell. 1,4,5-IP3 mobilizes calcium from intracellular stores, whereas DAG activates protein kinase C (PKC), a calcium-activated, phospholipiddependent enzyme. GABAB receptor activation does not affect basal [3H]inositol phosphate formation in cerebral cortex slices from rat or mouse (Crawford and Young, 1988, 1990; Godfrey et al., 1988,but see Hahner et al., 1991). However, in cerebral cortex both GABA and baclofen markedly inhibit the increase in [3H]inositol phosphate produced by histamine [H, receptors (Crawford and Young, 1988)] or serotonin [5-HT, receptors (Godfrey et al., 1988)] in a noncompetitive manner. Baclofen is effective at low concentrations and is stereospecific with the (-)isomer being markedly more potent than the (+)isomer. In addition, the GABA, agonist isoguvacine does not affect the histamine-induced increase in [3H]inositol phosphate formation (Crawford and Young, 1988).Similarly, bicuculline methiodide does not prevent the GABA-induced depression of the 5H T response (Godfrey et al., 1988). Taken together, these observations suggest that GABA acting on GABAB receptors may modulate both histamine and serotonin function. Unfortunately, the molecular mechanism through which GABA, receptors may modulate these receptors is currently unknown. 4. Inhibition of Voltage-Sensitive Calcium Channels In central neurons calcium influx through voltage-sensitive calcium channels has been found to be sensitive to a variety of dif€erent neurotransmitters, including norepinephrine, acetylcholine, adenosine, somatostatin, and opioid peptides (Carbone and Swandulla, 1989; Anwyl, 1991). Another neurotransmitter reported to inhibit calcium influx is

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GABA, acting through GABAB receptors. Electrophysiological studies reported that baclofen depressed excitatory postsynaptic potentials (EPSPs) in hippocampal neurons, possibly by inhibiting presynaptic calcium currents and thereby depressing transmitter release (Lanthorn and Cotman, 1981; Ault and Nadler, 1982). In support of this possibility, GABA and baclofen were reported to reduce the decrease in extracellular calcium concentration that occurred during repetitive stimulation in hippocampal area CA 1, presumedly by blocking calcium entry into presynaptic terminals of pyramidal neurons (Konnerth and Heinemann, 1983; Heinemann et al., 1984). Inhibition of calcium influx at presynaptic terminals was also suggested by the observation that baclofen depressed the K+-evoked release of transmitter from synaptosomes (Bonanno et al., 1989b). Similarly, direct measurements of calcium entry into cortical (Stirling et al., 1989) or cerebellar (Bowery and Williams, 1986) synaptosomes revealed that baclofen depressed calcium influx. Although these studies demonstrate that GABAB receptor activation can depress calcium entry into presynaptic terminals of central neurons, they do not reveal the mechanism of this action. Unfortunately, detailed study of calcium channels on presynaptic terminals is hampered by their electrophysiological inaccessibility. To overcome this problem voltage-sensitive calcium channels on neuronal cell bodies have been used as an experimental model in most cases. In particular, numerous studies have focused on sensory neurons in the dorsal root ganglion (DRG) because they are readily accessible and exhibit relatively large somatic calcium currents (Dunlap and Fischbach, 1978). In support of the usefulness of this model, it has been reported that many neurotransmitters, which depress calcium entry into central neurons, also decrease calcium current in somata of DRG neurons (Carbone and Swandulla, 1989; Anwyl, 1991). Examination of voltage-sensitive calcium currents in DRG neurons demonstrated that these currents were inhibited by GABA, receptor activation. The inhibition was first reported as a decrease in the calciumdependent plateau phase of the action potential in response to GABA or baclofen (Dunlap and Fischbach, 1978, 1981; Dunlap, 1981) with no change in the resting membrane potential. Subsequent studies using voltage clamp (Dunlap and Fischbach, 1981) o r whole cell patch clamp techniques (Deisz and Lux, 1985; Dolphin and Scott, 1986; Robertson and Taylor, 1986) confirmed that this apparent reduction in calcium current was not caused by an increase in an outward potassium current, but rather by a direct suppression of voltage-sensitive calcium channels. Several lines of evidence indicate that this effect was produced by GABAB receptor activation. For example, the effect of baclofen was stereospecific, with (-)baclofen being the active isomer (Dolphin and Scott, 1986; Rob-

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ertson and Taylor, 1986). T h e action of baclofen was blocked by phaclofen (Huston et al., 1990), but not bicuculline (Dunlap, 1981), and it was not mimicked by isoguvacine (Desarmenien et al., 1984). Multiple types of voltage-sensitive calcium channel have been shown to exist in neuronal membranes (for review see Nowycky et al., 1985; Miller and Fox, 1990; Bertolino and Llinas, 1992). In DRG neurons at least three classes of calcium channels have been reported: a low voltageactivated (LVA) T-type channel, a high voltage-activated (HVA) N-type channel, and a HVA L-type channel (Nowycky et al., 1985). It has been proposed that these different classes of voltage-sensitive calcium channel play different roles in neuronal function (for review see Bertolino and Llinas, 1992). For example, the N-type channel has been implicated in the control of neurotransmitter release, whereas the T-type channel has been suggested to control neuronal oscillatory activity, including spontaneous repetitive and burst firing. L-type channels are thought to be involved in the generation of action potentials and signal transduction. Because of the different physiological effects of these channels, it is of interest to determine which channel type is modulated by GABAB receptor activation. Unfortunately, the evidence indicating an effect of baclofen on a specific subtype of calcium channel in DRG neurons is inconclusive. Studies have found that baclofen will depress the LVA T-type channel by amounts ranging from 25 to 55% (Deisz and Lux, 1985; Dolphin et al., 1990; Formenti and Sansone, 1991). In addition, baclofen has been reported to slow the activation phase of the HVA calcium current and reduce the amplitude of both the HVA N-type calcium channel and the HVA L-type calcium channel, with the former channel being more sensitive to the drug (Dolphin and Scott, 1987; Green and Cottrell, 1988; Huston et al., 1990). Recently, however, Tatebayashi and Ogata (1992) reported a selective effect of baclofen on the HVA N-type calcium current with no effect of the drug on either the HVA L-type channel or the LVA T-type channel. They suggested that this preferential depression of the HVA inactivating N-type calcium current, which forms the rapid rising phase of the total HVA current, is responsible for the slowing of the activation phase of the total HVA calcium current. This result is of interest in light of the proposed role of N-type channels in regulating transmitter release. T h e mechanism by which GABAB receptor activation inhibits voltagesensitive calcium currents in DRG neurons involves inhibitory G proteins (Dolphin et al., 1990). The role of G proteins was demonstrated in studies (GDP-P-S),a GDP anashowing that guanosine 5 -0-(2-thiodiphosphate) logue that competitively inhibits the binding of GTP to G proteins, reduced the effect of baclofen on voltage-sensitive calcium currents.

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In contrast, guanosine 5 -0-(3-thiotriphosphate)(GTP-y-S), a nonhydrolyzable GTP analogue that irreversibly activates G proteins, enhanced the effect of baclofen and, if applied alone, mimicked the inhibitory effect of baclofen on the HVA N-type calcium current (Scott and Dolphin, 1986; Dolphin and Scott, 1987; Holz et al., 1989). Finally, it was observed that preincubation of DRG neurons with pertussis toxin blocked the inhibitory effect of baclofen on calcium influx, indicating that GABAB receptor activation inhibited voltage-sensitive calcium currents through one of the inhibitory G proteins, either Gi or Go (Dolphin and Scott, 1987; Holz et al., 1989). Using antibodies raised against the C terminus of the a subunit of Go and Gi proteins, Menon-Johansson and Dolphin (1992) found that the a subunit of Go, but not Gi, protein was responsible for the inhibition of voltage-sensitive calcium currents by baclofen. Similarly, the a subunit of the Go protein has been found to mediate inhibition of voltage-sensitive calcium channels by a variety of different neurotransmitters, including neuropeptide Y, (Ewald et al., 1988), opioid peptides (Hescheler et al., 1987), somatostatin (Kleuss et al., 1991), dopamine (Harris-Warrick et al., 1988), acetylcholine (Toselli et al,, 2989; Offermanns et al., 1991), and norepinephrine (McFadzean et al., 1989), suggesting an essential regulatory role for this G protein in calcium channel inhibition. It is not yet clear what role, if any, the dimer plays in the inhibition. Although it has been demonstrated that inhibitory G proteins are required for GABAB receptor-mediated inhibition of voltage-dependent calcium channels, it remains unclear whether these G proteins interact directly with the channel or whether another second messenger is involved. Several lines of evidence suggest the G protein may interact directly with the calcium channel (for review see Dolphin, 1991a). For example, using cell attached patches, Green and Cottrell (1988) found that baclofen inside the patch pipette was able to inhibit calcium channels, whereas external baclofen was not. These authors concluded that a second messenger capable of diffusing to calcium channels under the patch was not involved in the inhibition. A similar conclusion was reached by Dolphin et al. (1989), based on their inability to link the inhibition of voltage-dependent calcium channels in DRG neurons to any of the transduction systems known to be coupled to GABA, receptors. In contrast, in cultured spinal cord neurons Karnatchi and Ticku (1990) reported that inhibition of calcium currents by baclofen was antagonized by either activation of adenylyl cyclase by forskolin or the addition of 8-bromoCAMP.In addition, these authors found that activation of protein kinase C with phorbol esters also antagonized the action of baclofen. They concluded that both protein kinase A and C contribute to the inhibitory

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effect of baclofen on calcium currents in spinal cord neurons. A similar depression of calcium currents by phorbol esters or diacylglycerol has also been reported in other studies using DRG neurons (Rane and Dunlap, 1986; Gross and Macdonald, 1988). Although these results demonstrate that PKC activation mimics the effect of baclofen, they do not necessarily demonstrate that baclofen acts through this transduction mechanism to inhibit calcium currents. Furthermore, other studies have reported phorbol esters either to have no effect or to activate calcium currents (for review see Kaczmarek, 1987). Thus, the mechanism by which G proteins regulate calcium channel activity remains to be conclusively demonstrated. Interestingly, a recent study using DRG neurons has suggested that a continuous cycle of phosphorylation and dephosphorylation may tonically regulate activation of voltage-sensitive calcium channels. Dolphin (1991b, 1992) reported that CAMP-dependent phosphorylation, produced by direct addition of CAMPor activation of adenylyl cyclase with forskolin, was able to reverse the inhibition of calcium currents produced by G protein activation with GTP-y-S. Similarly, inhibition of calcium currents was reduced by addition of the active fragment of phosphorylated inhibitor 1, which prevented dephosphorylation by inhibiting phosphatase 1. T h e author concludes that the interaction between the G protein and voltage-dependent calcium channels may be regulated by phosphorylation, suggesting that GABAB receptor-mediated inhibition of these channels would be sensitive to the phosphorylation state. The potential site of this CAMP-dependent phosphorylation includes the calcium channel itself, the phosphatase l inhibitor, or the Go protein. In support of an action on the G protein, it was recently reported that phosphorylation of G,, protein by protein kinase C blocks its ability to inhibit adenylyl cyclase (Bushfield et al., 1991). In contrast to the large number of reports from DRG neurons, few studies in central neurons have examined calcium current modulation by GABA, receptors. Initial studies of central neurons reported baclofen to have no direct effect on calcium currents (Gahwiler and Brown, 1985; Howe, 1987; Howe et al., 1987). However, recent studies in these neurons have reported inhibition of calcium currents by baclofen (Scholz and Miller, 1991; Swartz and Bean, 1992; Mintz and Bean, 1993). The lack of an effect of baclofen in the report by Gahwiler and Brown (1 985) is most likely explained by their use of small depolarizing steps (+ 20 mV) to elicit the current. Indeed, in recent reports in hippocampus (Scholz and Miller, 1991) and cerebellum (Wojcik et al., 1990) baclofen had little to no effect on calcium currents evoked by small depolarizing steps (< + 30 mV), while significantly inhibiting calcium currents evoked by

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larger depolarizations. This inhibition was blocked by 2-hydroxysaclofen, indicating that it was mediated by GABA, receptor activation (Scholz and Miller, 1991). As in DRG neurons, central neurons express multiple types of calcium current, including an LVA T-type calcium current and two types of HVA calcium current: an o-conotoxin-sensitive N-type current and a dihydropyridine-sensitive L-type current. In addition, central neurons also express an HVA P-type calcium current that is resistant to both oconotoxin and dihydropyridines, but is sensitive to funnel-web spider toxin (for review see Bertolino and Llinas, 1992). In hippocampal neurons, as in DRG neurons, baclofen primarily suppressed the HVA N-type current; however, some inhibition of the HVA L-type current was also noted (Scholz and Miller, 1991). In addition, baclofen depressed the LVA T-type calcium current in interneurons in stratum lacunosummoleculare (Fraser and MacVicar, 1991) (see Section IV,A,P,b). Interestingly, in area CA3 baclofen was typically much more effective at inhibiting calcium currents in pyramidal cells than in nonpyramidal neurons in this same region (Wojcik et al., 1990). In cerebellar granule cells baclofen was found to inhibit an HVA L-type current, although an effect on another current type was not ruled out (Huston et al., 1990; Wojcik et al., 1990; Marchetti et al., 1991). Alternatively, in cerebellar Purkinje cells both baclofen and GABA were reported to suppress an HVA P-type current (Mintz and Bean, 1993). The reason for these differences in the effect of baclofen is unclear; however, they may reflect differences between cell types, differences in the subtype of calcium channel mediating the current, or differences in the mechanism coupling the receptor to the calcium channels. As in DRG neurons, baclofen appears to inhibit calcium channels in central neurons through activation of G proteins. Thus, in hippocampal neurons inclusion of GTP-y-S in the patch pipette enhanced calcium channel inhibition by baclofen whereas pretreatment of the cells with pertussis toxin blocked the actions of baclofen, indicating that baclofen acted through an inhibitory G protein (Wojcik et at., 1990).Furthermore, Sweeney and Dolphin (1992), using cortical and cerebellar membrane preparations, have recently examined the subtype of G protein involved in the interaction by using antibodies directed against the C-terminus of the a subunit of Gi and Go proteins. They found that, although baclofen activates both G, and Go proteins, the L-type calcium channel interacts selectively with the C-terminus of the a subunit of the Go protein. They suggested that, as in DRG neurons, GABA, receptor-mediated activation of Go proteins leads to inhibition of L-type calcium channels, whereas activation of Gi proteins causes inhibition of adenylyl cyclase.

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In summary, baclofen inhibits voltage-sensitive calcium currents in both DRG neurons and central neurons through activation of inhibitory G proteins. Inhibition of L-type calcium currents appears to occur by direct interaction of the C-terminus of the (Y subunit of Go proteins with the calcium channels, although the contribution of another transduction system has not been conclusively eliminated. In both DRG and hippocampal neurons baclofen appears to inhibit N-type calcium channels most effectively, whereas it inhibits L-type calcium channels in cerebellar granule cells and P-type calcium channels in cerebellar Purkinje cells. Although it is tempting to speculate that inhibition of calcium currents is responsible for the GABA, receptor-mediated inhibition of transmitter release, it must be remembered that these results have been collected indirectly by using somatic calcium channels as a model of calcium channels on neuronal terminals. Thus, direct evidence relating the GABA, receptor-mediated inhibition of calcium currents on neuronal terminals with depression of transmitter release has not yet been reported.

5. Activation of Potassium Channels In addition to its affect on voltage-sensitive calcium channels, GABA, receptor activation by baclofen also hyperpolarizes and reduces the input resistance of central neurons. This effect was first observed in the hippocampal formation (Klee et al., 1981; Misgeld et al., 1982) and has since been recorded in a variety of other brain regions including hypothalamus (Ogata and Abe, 1982), neocortex (Howe et al., 1987), lateral septum (Stevens et al., 1985), dorsal raphe (Colmers and Williams, 1988), and locus coeruleus (Shefner anti Osmanovic, 1991). In contrast, baclofen appears to produce no hyperpolarization in the neostriatum (Calabresi et al., 1992; Nisenbaum et al., 1992), the supraoptic nucleus (Ogata, 1990b), or sensory neurons in the DRG (Dunlap and Fischbach, 1981). Focal application of baclofen was found to hyperpolarize consistently pyramidal neurons in the hippocampus; however, this response was much stronger when baclofen was applied dendritically than when it was applied near the soma, suggesting that baclofen may act primarily in the dendrites (Newberry and Nicoll, 1984b, 1985). The hyperpolarization produced by baclofen was shown to persist during blockade of synaptic transmission with tetrodotoxin or cadmium, indicating that this effects was postsynaptic (Newberry and Nicoll, 198413, Nicoll and Newberry, 1984). Furthermore, baclofen acted in a stereospecific fashion with the (-)isomer being 100-200 times more potent than that (+)isomer (Newberry and Nicoll, 1984b, 1985; Nicoll and Newberry, 1984; Padjen and Mitsoglou, 1990). The GABA, receptor agonist 3-APPA also hyperpolarized central neurons, indicating that this effect was not specific to

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baclofen (Seabrook et al., 1990; Lovinger et al., 1992). Finally, the GABA, receptor antagonists phaclofen (Dutar and Nicoll, 1988b; Olpe el al., 1988), 2-hydroxysaclofen (Hasuo and Gallagher, 1988; Lambert et al., 1989), CGP 35348 (Olpe et al., 1990), and CGP 55845 (Jarolimek et al., 1993) blocked the effect of baclofen. These results demonstrate that the hyperpolarization and conductance increase were produced through activation of GABA, receptors. The ability of baclofen to hyperpolarize and increase the conductance of central neurons suggested that GABA should produce a similar postsynaptic effect. It had been previously shown that focal application of GABA onto the somata of hippocampal pyramidal neurons produced a hyperpolarization, due to activation of a bicuculline-sensitive GABA, receptor-mediated chloride conductance. Furthermore, focal application of GABA onto the dendrites of these same neurons resulted primarily in a large GABA, receptor-mediated depolarization (Alger and Nicoll, 1982b). However, blockade of these GABA, receptor-mediated responses with bicuculline exposed an underlying hyperpolarization similar to that produced by baclofen (Newberry and Nicoll, 1984b, 1985). Like the baclofen response, this GABA-induced hyperpolarization was evoked more readily in the dendrites than near the soma and was insensitive to pentobarbitone, a barbiturate that enhances the GABA, response (Newberry and Nicoll, 1985). Furthermore, it was not additive with the baclofen response, suggesting that both the GABA and the baclofen responses shared the same effector channel (Nicoll et al., 1989). Thus, the hyperpolarizing nature of the GABA response, its insensitivity to GABA, antagonists and barbiturates, and its nonadditivity with the baclofen response suggested that it was mediated through the same GABAB receptor-coupled mechanism as the baclofen response. This conclusion was strengthened when it was demonstrated that, in addition to blocking the baclofen response, GABA, receptor antagonists, such as phaclofen (Dutar and Nicoll, 1988b), CGP 35348 (Solis and Nicoll, 1992a), or CGP 55845A (Jarolimek et al., 1993), also blocked the postsynaptic action of GABA. However, despite the many similarities between the responses to baclofen and GABA, some pharmacological differences have been reported (Ogata et al., 1987; Dutar and Nicoll, 1988a; Muller and Misgeld, 1989; Segal, 1990a, but see Solis and Nicoll, 1992a). In particular, the available GABAB receptor antagonists have been found to be more effective at blocking the postsynaptic response to baclofen than the response to GABA. This difference leaves open the possibility that, whereas baclofen may be specific for a single GABA, receptor subtype, GABA may activate more than one GABA, receptor subtype with differing antagonist pharmacology (Johnston, 1986; Bowery, 1993).

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Several lines of evidence indicate that GABAB receptor activation hyperpolarizes neurons by increasing the conductance of the membrane to potassium ions. First, the bicuculline-insensitive dendritic response to both GABA and baclofen reversed at a hyperpolarized membrane potential, close to the equilibrium potential for potassium (Nicoll and Newberry, 1984; Gahwiler and Brown, 1985; Inoue et al., 1985c; Newberry and Nicoll, 1985). This reversal potential was similar to that of the postburst slow afterhyperpolarization [I,,, (Newberry and Nicoll, 1984b, 1985; Nicoll and Newberry, 1984; Inoue et al., 1985a)], which has been shown to be mediated by a calcium-activated potassium conductance (Alger and Nicoll, 1980; Schwartzkroin and Stafstrom, 1980). Second, altering the gradient of chloride ions across the membrane did not change the reversal potential of the baclofen response, as would he expected if it were mediated by an increase in chloride conductance, like the GABA, response (Newberry and Nicoll, 1984b, 1985; Nicoll and However, increasing the extracelluNewberry, 1984; Inoue et al.. 1985~). lar concentration of potassium did produce a depolarizing shift in the reversal potential of both the baclofen and the GABA responses by an amount close to that predicted by the Nernst equation (Nicoll and Newberry, 1984; Gahwiler and Brown, 1985; Inoue et al., 1985a; Newberry and Nicoll, 1985, but see Jarolimek and Misgeld, 1992).Third, external barium (Nicoll and Newberry, 1984; Gahwiler and Brown, 1985; Newberry and Nicoll, 1985; Connors et al., 1988) or internal cesium (Gahwiler and Brown, 1985), both of which block potassium channels, reduced the hyperpolarizing response. Finally, tetrahydroaminoacridine (THA), a potassium channel blocker, suppressed both the GABA- and the baclofen-induced conductance increase (Halliwell and Grove, 1989; Dutar et al., 1990; Lambert and Wilson, 1993). Taken together, these results demonstrate the GABAB receptor activation produces postsynaptic hyperpolarization by increasing the potassium conductance of the membrane. As with calcium channels, the potassium conductance activated by GABABreceptors is similar to that activated by a variety of other neurotransmitters including dopamine, serotonin, opiates, adenosine, and somatostatin (Andrade et ul., 1985; North et ul., 1987; Christie and North, 1988,Brown, 1990). In general, this conductance shows inward rectification such that the current becomes smaller on membrane depolarization (Gahwiler and Brown, 1985; Newberry and Nicoll, 1985, but see Premkumar et al., 1990a; Wagner and Dekin, 1993). This voltage dependence differentiates it from an M-type potassium current. T h e current is insensitive to potassium channel blockers such as tetraethylammonium chloride [TEA (Stevens et al., 1985)] and 4-aminopyridine [4-AP (Padjen and

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Mitsoglou, 1990; Solis and Nicoll, 1992a, but see Inoue et al., 1985a; Stevens et al., 1985)], but is blocked by the lidocaine derivative, lidocaine N-ethyl bromide [QX-314 (Nathan et al., 1990a; Andrade, 1991)l. In addition, several lines of evidence suggest that the GABAB receptoractivated potassium current is not dependent on calcium. First, the current is not blocked by inorganic calcium channel blockers, such as cadmium or cobalt, making it unlikely that entry of extracellular calcium is necessary (Gahwiler and Brown, 1985; Newberry and Nicoll, 1985; Stevens et al., 1985; Padjen and Mitsoglou, 1990, but see Blaxter et al., 1986). Second, intracellular injection of EGTA, a calcium chelator, fails to block the current, indicating that a rise in intracellular calcium, presumedly from intracellular stores, is also unnecessary (Thalmann, 1984; Andrade et al., 1986, but see Blaxter et al., 1986; Segal, 1990b). Finally, single-channel studies on cell-attached membrane patches from cultured hippocampal neurons have found that removal of calcium from the perfusion medium has no effect on potassium channel activity induced by baclofen or GABA (Premkumar et al., 1990a). Single-channel analysis of GABA, receptor-operated potassium channels has only recently been performed. Using cell attached patches from cultured hippocampal neurons, Premkumar et al. (1990a) found that 30- to 90-s exposure of the neuron to baclofen or GABA caused single-channel potassium currents to appear, which were sensitive to 2hydroxysaclofen, but not bicuculline. Single-channel current amplitudes varied considerably with the smallest having an amplitude of about 0.36 PA, which corresponded to a conductance of 5-6 pS. These channels were potassium-selective (P,,/P, ratio of 0.03-0.04) and opened in bursts. During these bursts, the channels flickered rapidly between subconductance states that were integral multiples of 5-6 pS. The authors concluded that GABAB receptor activation causes the opening of a single class of potassium channels that can be coupled together and open cooperatively, each cochannel having an “elementary” conductance of 5-6 pS. This conductance is three to four times smaller than the singlechannel conductance of 17-20 pS reported for GABA, receptormediated chloride channels (Bormann, 1988). GABA, receptors are indirectly coupled to potassium channels through G proteins. This conclusion is based on the observation that the hydrolysis-resistant GDP analogue, GDP-/3-S, reduced the action of baclofen, whereas the GTP analogue, GTP-y-S, mimicked the effect of baclofen (Andrade et al., 1986; Thalmann, 1988). In addition, pretreatment with pertussis toxin blocked the action of both baclofen and GABA, indicating that the coupling of GABA, receptors to potassium channels is achieved through the inhibitory G proteins, G, and/or Gi (Andrade

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et al., 1986; Colmers and Williams, 1988; Thalmann, 1988).Using excised

inside-out membrane patches from cultured hippocampal neurons, VanDongen et al. (1988) found that exposure of the membrane to purified or recombinant Go, but not Gi, would activate four types of potassium channels, one of which was also activated by serotonin. Since serotonin and GABAB receptors are coupled to the same G protein (Andrade et al., 1986),these results suggest that potassium channels are also coupled to GABAB receptors by Go protein. Despite the ability of Go proteins to activate directly neuronal potassium channels (VanDongen et d., 1988), it has not yet been conclusively demonstrated whether the Go protein-mediated coupling of GABAB receptors to neuronal potassium channels is direct or is mediated through a diffusible second messenger. A role for cAMP in this interaction has been ruled out based on the inability of 8-bromo-cAMP, a membrane-permeant cAMP analogue, or intracellular injection of cAMP to affect the baclofen-induced hyperpolarization (Andrade et al., 1986; Inniset al., 1988). It is also unlikely that phospholipase C plays a role in the GABAB receptor-mediated postsynaptic response because neither calcium (see above) nor protein kinase C activation (Andrade et al., 1986; Dutar and Nicoll, 1988a) is necessary for expression of the current. However, a role for phospholipase A, has been proposed. Premkumar et al. (1990b) found that application of arachidonic acid to the inner surface of excised inside-out membrane patches from cultured hippocampal neurons mimicked the effect of baclofen on single potassium channel currents. Fluctuation analysis revealed that the potassium channels activated are similar to those stimulated by baclofen. However, these results indicate only that arachidonic acid is capable of mimicking the effect of baclofen, not that it mediates this response in the normal cell. Thus, a role for arachidonic acid has yet to be conclusively demonstrated. Interestingly, inhibitors of PLA, do not block the EPSP depression produced by baclofen in hippocampal slices (Dunwiddie et d., 1990), suggesting that if presynaptic GABAB receptor-operated potassium channels play a role in the transmitter release process, they may not be modulated by arachidonic acid. GABAB receptors may also be coupled to other types of potassium channels. For example, Saint et al. (1990) reported that a high concentration of GABA or baclofen could alter the voltage dependence of an A-type potassium current in cultured hippocampal neurons. They concluded that by shifting the voltage dependence of inactivation of these channels to more positive potentials, GABAB receptor stimulation can enhance activation of the A-current at resting membrane potential. The authors suggest that by enhancing the A-current in presynaptic terminals,

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GABAB receptor activation could shorten action potential duration and thereby decrease transmitter release. Alternatively, Wagner and Dekin ( 1993) have reported that in cultured premotor respiratory neurons, GABAB receptors are coupled to a barium-insensitive outwardly rectifying potassium conductance. These channels are distinct from those underlying the response to GABA and baclofen described above, but have properties similar to the S-channel, which regulates transmitter release from Aplysia sensory neurons. In conclusion, GABAB receptors on a variety of central neurons are linked through an inhibitory G protein, presumedly Go protein, to an inwardly rectifying potassium conductance. It is not yet known whether Go proteins directly couple GABA, receptors to potassium channels o r whether an additional diffusible second messenger is involved; however, a role for phospholipase A, has been proposed. The Go protein that couples GABAB receptors to potassium channels is most likely the same G protein responsible for coupling GABAB receptors to voltage-sensitive clacium channels. In contrast, GABAB receptors are coupled through Gi protein to inhibition of adenylate cyclase and inhibition of transmitterstimulated adenylyl cyclase activation. The ability of a single cell to express all of these effector systems raises the possibility that either GABAB receptors are coupled to more than one subtype of G protein in that cell or that distinct subtypes of receptors are involved.

IV. Function of GAB& Receptors

The known actions of GABAB receptors can be attributed to the effector systems to which GABAB receptors are coupled. At present, a functional role for each of these effector systems has not been determined. However, several recent studies have suggested a potential physiological role for GABAB receptor-mediated potentiation of neurotransmitter-stimulated adenylyl cyclase activity. Andrade ( 1993) observed that baclofen enhanced the ability of norepinephrine, through P-adrenergic receptors, to increase intracellular cAMP and thereby reduce the calcium activated slow afterhyperpolarization (I,,,) in hippocampal neurons. However, in the absence of norepinephrine, baclofen had no effect on the reduction in the AHP produced by fJ-bromo-cAMP, a membrane-permeable cAMP analogue. Thus, the author concluded that GABAB receptor activation enhanced the action of norepinephrine on the AHP by potentiating the P-adrenergic stimulation of adenylyl cyclase. Similarly, Burgard and Sarvey (1991) found that baclofen was

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able to enhance the induction of long-lasting potentiation (LLP) of the excitatory response in the dentate gyrus produced by P-adrenergic agonists. They observed that a low concentration of isoproterenol had no effect; however, when isoproterenol and baclofen were applied together, at concentrations that alone produced no LLP, significant LLP was induced. Taken together, these results demonstrate that coactivation of GABAB receptors and G,-linked receptors can synergistically enhance several physiological effects, possibly by potentiating the stimulation of adenylyl cyclase (see Section III,C,2). In addition to interacting through adenylyl cyclase with G,-coupled receptors, GABAB receptors may also interact through inhibitory G proteins with GABAA receptors. Binding studies in whole cells from cerebellar primary culture have demonstrated that baclofen markedly reduces [3H]muscimol binding to GABAA receptors (Kardos and Kovacs, 1991). This effect was blocked by pretreatment with pertussis toxin, indicating that an inhibitory G protein was required. Similarly, Hahner et al., (199 1) found that in membrane vesicles from mouse cerebellum (and to a lesser extent from cerebral cortex) baclofen inhibits muscimol-stimulated 36Cluptake through nondesensitizing GABAA receptor/channels. This action of baclofen was stereoselective, calcium-dependent, and blocked by 2-hydroxysaclofen, indicating that baclofen was operating through GABAB receptors. The inhibitory action of baclofen was mimicked by GTP-y-S, but not GDP-P-S, suggesting that G proteins played a role in the inhibition. Furthermore, the action of baclofen was blocked by U73 122, an inhibitor of phospholipase C. These results suggest that GABAB receptors can inhibit the function of GABA, receptors through a G protein-mediated activation of phospholipase C and the subsequent phosphorylation of the GABAA receptor (Sigel and Baur, 1988; Browning et al., 1990; Hahner et al., 1991). Thus, it now appears that, through their transduction mechanisms, GABA, receptors can integrate with, and modulate the activity of other neurotransmitter systems. In contrast to this biochemical interaction, the majority of identified electrophysiological actions of GABAB receptors are mediated through modulation of potassium channels and voltage-sensitive calcium channels. These actions will be discussed in detail in the remainder of this review. We will first discuss postsynaptic GABAB receptor-mediated effects and then the presynaptic effects of GABAB receptors on both excitatory and inhibitory terminals. Because the majority of the studies of GABAB receptor function have been performed in the hippocampal formation, we will concentrate on the effects of GABAB receptors in this region and include results from other brain regions where appropriate.

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A. POSTSYNAPTIC GABA, RECEPTORS 1. Characteristics of GABA, Inhibit09 Postsynaptic Potentials

a. Late Inhibitory Postsynaptic Potentials. Orthodromic stimulation in the hippocampus evokes a synaptic response consisting of an EPSP followed by a biphasic inhibitory postsynaptic potential (1PSP) (Fig. 3). The early component of this IPSP, which peaks 10-20 ms after the stimulus, results from a GABAA receptor-mediated increase in the conductance of the membrane to chloride ions. It is blocked by GABAA antagonists, such as bicuculline or picrotoxin, and reverses at a membrane potential of about -70 mV, close to the equilibrium potential for chloride (for review see Bormann, 1988). In contrast, the late component of the biphasic IPSP is resistant to GABA, blockers. It peaks 150-200 ms after the stimulus and lasts for about 1 s (Fig. 3). A similar late IPSP has been recorded from neurons in many different brain regions, including neocortex (Howe et al., 1987; Connors et al., 1988; McCormick, 1989), thalamus (Soltesz et al., 1989; Crunelli and Leresche, 1991), locus coeruleus (Olpe et al., 1988), and septum (Stevens et al., 1987), indicating that the presence of these potentials is not restricted to the hippocampus. 6. Pharmacology and Conductance Mechanism of the Lute IPSP. The late 1PSP has many characteristics in common with the hyperpolarizing response to GABA or baclofen, suggesting that all are mediated through GABA, receptors. One similarity is that, like the hyperpolarizing GABA, receptor-mediated response, the late IPSP is associated with an increase in membrane potassium conductance (Nicoll and Alger, 1981; Thalmann and Ayala, 1982; Alger, 1984; Newberry and Nicoll, 1984b; Thalmann, 1984; Kehl and McLennan, 1985b; Hablitz and Thalmann, 1987). This conclusion is based on several observations. First, the late IPSP reverses at a membrane potential of about -90 mV, close to both the equilibrium potential for potassium and the reversal potential of the calcium-activated potassium conductance (Fig. 3). Second, the equilibrium potential of the late IPSP is sensitive to changes in the extracellular concentration of potassium, but not chloride. Finally, the response is blocked by agents know to block potassium channels, such as extracellular barium (Gahwiler and Brown, 1985; Newberry and Nicoll, 1985), intracellular cesium (Gahwiler and Brown, 1985; Otis and Mody, 1992), QX-314 (Nathan et al., 1990a; Otis and Mody, 1992),and THA (Halliwell and Grove, 1989; Lambert and Wilson, 1993). Finally, the hyperpolarization produced by baclofen and that produced by the late IPSP are not additive, indicating that the potassium conductance responsible for each of these responses is one and the same (Ogata, 1990b).

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The late IPSP is also similar to the GABAB receptor-mediated postsynaptic response in its dependence on G proteins. Thus, addition of GTPy-S mimics the hyperpolarization and conductance increase produced by the late IPSP. This effect is not additive with the late IPSP (Thalmann, 1988; Mott et al., 1993b). Furthermore, pertussis toxin blocks the late IPSP, indicating that as with the GABA, receptor-mediated hyperpolarization, the required G protein is either Goo r Gi (Thalmann, 1987, 1988; Dutar and Nicoll, 1988a). No conclusive evidence has been presented to demonstrate a requirement for a diffusible second messenger in the mechanism underlying the late IPSP; however, several potential candidate systems have been ruled out. For example, it is unlikely that G protein-mediated alterations in cAMP produce the late IPSP, since increasing the intracellular concentration of cAMP by applications of forskolin (Hablitz and Thalmann, 1987) or direct addition of the niembrane-permeant cAMP analogue 8bromo-CAMP (Newberry and Nicoll, 1984a) has no effect on the late IPSP in CA3 neurons. In addition, G protein activation of phosphiolipase C is unlikely to be required since neither of the reaction products of this enzyme mimicked the late IPSP. For example, 1,4,5 IP,, does not appear to be involved, since intracellular injection of EGTA has no effect on the late IPSP, indicating that the response is calcium-independent (Thalmann, 1984; Hablitz and Thalmann, 1987, but see Blaxter et al., 1986; Segal, 1990b). Alternatively, activation of protein kinase C with phorbol esters does not mimic, but rather blocks, the late IPSP (Baraban et al., 1985; Dutar and Nicoll, 1988a). Although these results suggest that phospholipase C is not responsible for the late IPSP, they do not exclude a modulatory role for protein kinase C. Thus, like the GABAB receptormediated response, the late IPSP appears to be mediated by a direct Go protein interaction with potassium channels, although a role for phospholipase A, has not been ruled out. T h e similarities in appearance and underlying mechanism between the late IPSP and the GABA, receptor-mediated postsynaptic response suggested that the late IPSP was produced by activation of GABABreceptors by synaptically released GABA. This conclusion was confirmed when it was demonstrated that the late IPSP was blocked by phaclofen (Dutar and Nicoll, 1988b; Karlsson et al., 1988; Solteszetal., 1988).Subsequently, studies in many different brain regions with a variety of GABABreceptor antagonists, including 2-hydroxysaclofen (Lambert et al., 1989), CGP 35348 (Olpe et al., 1990), and CGP 55845 (Jarolimek et al., 1993), have supported this finding (Fig. 3). c. Current-Voltage Relationship of IPSPIC,. The current-voltage (Z-V) relationship of the late IPSP (IPSPB) has generally been reported to show

A

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FIG. 3. (A) A typical perforant path-evoked response recorded from a dentate gyrus granule cell at resting membrane potential ( - 79 mV) showing the EPSP (solid triangle), the IPSP, (open circle), and the IPSPB (solid circle). Because of the hyperpolarized resting potential of granule cells the IPSP, is typically depolarizing. (B) Voltage dependence of the IPSP. (Left) Sample IPSPs recorded from a granule cell at the membrane potentials shown on the left. Responses were evoked by direct stimulation of interneurons in the dentate gyrus molecular layer. This monosynaptic IPSP was pharmacologically isolated by blocking the EPSP with DNQX and D-APV. (Right) Monosynaptic IPSP amplitude plotted against membrane potential for this cell. The amplitude of IPSP, (open circle) and IPSPB (solid circle) was measured at 14 and 200 ms, as indicated by the circles on the waveforms on the left. (C) Pharmacological characterization of the monosynaptic IPSP. (Top) Depolarization of a granule cell to - 60 mV caused the IPSP, to become hyperpolarizing. Addition

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no rectification (Thalmann, 1984; Hablitz and Thalmann, 1987; Rovira et al., 1990). Linear I-V curves over a range of membrane potentials from about - 110 to -50 mV have been reported. In granule cells of the dentate gyrus we found that, although IPSPB appeared to show inward rectification, this could largely be accounted for by the voltagedependent properties of the membrane (Mott et al., 1993b, also see Alger, 1984; Thalmann, 1984; Rausche et al., 1989; Crunelli and Leresche, 1991). Similarly, Otis et al. (1993) reported a linear current-voltage relationship for the isolated IPSCB in dentate granule cells. However, they also found that IPSCB displayed outward rectification in some cells when the extracellular potassium was raised. In contrast, other studies have reported that the late IPSP inwardly rectifies (Newberry and Nicoll, 1985; Soltesz et al., 1989). The reason for this discrepancy is not known; however, some of the apparent nonlinear behavior of the response may be caused by the voltage-dependent properties of the membrane. It is of interest to note that the current-voltage relationship generally reported for the response to GABAB receptor agonists shows inward rectification (see Section IlI,C,5).This raises the possibility that the population of potassium channels activated by the agonist may not be identical to those activated synaptically. d. IPSPIC, Kinetics. The kinetics of IPSPB are considerably slower than those of IPSPA, reflecting the G protein coupling of the GABAB receptor with the potassium channel. In general, the latency to onset of the GABAB receptor-mediated potential ranges from 30 to 50 ms (Connors et al., 1988; Davies et al., 1990; Mott et al., 1993b), whereas the onset latency of the underlying current is 12-35 ms (Hablitz and Thalmann, 1987; Otis et al., 1993). This latency is considerably longer than that of the GABAA receptor-mediated current [<3 ms (Mott et al., 1993b)], but is similar to the onset latency for other G protein-coupled neurotransmitters (Hille, 1992b). Since GABAB receptors are coupled to G proteins that must diffuse to their target channels, this onset latency puts certain constraints on the maximal dimensions of the signal system. Hille (1992b) observed that diffusion of transmitter to a receptor is fairly rapid; however, diffusion of second messenger components within the membrane

of picrotoxin blocked this IPSP. 'The remaining IPSP, was completely suppressed by the subsequent addition of 2-hydroxysaclofen, indicating that the response was entirely GABAergic. (Bottom) In another cell application of 2-hydroxysaclofen reversibly suppressed IPSP,. This cell was held depolarized to -63 mV with injected current. The stimulus intensity was increased in the presence of 2-hydroxysaclofen to compensate for the depression of IPSP, produced by the drug (see text). [Modified from Mott et al. (1992) with permission.]

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is several orders of magnitude slower. He concluded that for a fast signaling system, such as GABAA receptor-mediated responses, the receptor/channel complex must be within 0.9 pm of the transmitter release zones. In contrast, for a G protein-linked system that responds within 50 ms, the receptor molecules can be within 14 pm of the transmitter release zones, but the receptor and all necessary membrane molecules must be within approximately 0.35 pm of the effector channel. As the GABAB system responds faster than 50 ms and uptake of GABA may limit diffusional spread of the transmitter (Thompson and Gahwiler, 1992b),the maximal dimensions of this system may be smaller. Nevertheless, this conclusion raises the possibility that, whereas GABA, receptors are confined to the synaptic zone, GABAB receptors could be located some distance from the synaptic zone and still respond within the observed latency. The rise time and peak latency of IPSP/CBalso reflect the slow nature of this G protein-coupled response. Otis et al. (1993) reported that once activated, the rise time of the isolated IPSP, in dentate granule cells exhibited fourth-order activation kinetics. They suggested that these kinetics may result from the interaction of the four subunits thought to constitute neuronal potassium channels (also see Mackinnon, 1991). Numerous studies have reported values ranging from 130 to 200 ms for the latency to the peak of IPSP/CB (Kehl and McLennan, 1985b; Hablitz and Thalmann, 1987; Howe et al., 1987; Soltesz et al., 1989; Davies et al., 1990; Nathan et al., 1990a; Mott et al., 1993b). This latency is independent of membrane potential or stimulus intensity (Davies et al., 1990). It is considerably longer than the latency to peak of 14-30 ms reported for IPSP/C, (Howe et al., 1987; McCormick, 1989; Davies et al., 1990; Staley and Mody, 1992; Mott et al., 1993b) and is similar to the peak latency of other G protein-linked responses (Hille, 1992b).A peak latency of 130-200 ms for IPSP/CB suggests that GABAB receptor-mediated responses will have their greatest impact when the neuronal circuit is activated at frequencies from 5 to 10 Hz, in the range of hippocampal theta rhythm [3-10 Hz (Buzsaki, 1986; Vertes, 1986) (see Section IV,C,4,b)l. The duration of IPSP, shows considerable variability between neurons. In general, durations ranging between 400 and 1500 ms have been reported (Fujita, 1979; Alger, 1984; Howe et al., 1987; Rausche et al., 1989; Soltesz et al., 1989; Nathan et al., 1990a; Mott et al., 1993b). IPSCB duration is not sensitive to membrane potential but, as predicted for a metabotropic response, is temperature-sensitive, being prolonged by lower temperatures (Alger, 1984; Otis et al., 1993). In addition, the rate at which IPSCB decays is longer than its rate of activation, a necessary

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prerequisite for signal amplification in a G protein-linked system. The molecular mechanism underlying this slow decay rate is not known, but may reflect the duration of GABAB receptor occupancy by GABA or the rate of GTP hydrolysis. These observation suggest that signal amplification may occur in the Gf$BAB system, potentially enabling low levels of GABA to produce a functional response. e. Peak Conductance and Hyperpolarization of IPSPIC,. The peak conductance of IPSP/CB is considerably smaller than that of IPSP/CA. For example, in CA1 or CA3 hippocampal pyramidal neurons the peak GABAB conductance is approximately 13- 19 nS (Hablitz and Thalmann, 1987; Thalmann, 1987, 1988; Williams and Lacaille, 1990; Pacelli et al., 1991 ; Oleskevich and Lacaille, 1992). Similar values have been reported in neurons in the neocortex (Connors et al., 1988; Deisz and Prince, 1989; McCormick, 1989) and thalamus (Crunelli and Leresche, 1991). These conductance values are approximately 5-7 times smaller than peak GABA, conductance values of 90-140 nS recorded from these same neurons (Thalmann, 1987, 1988; Connors et al., 1988; Deisz and Prince, 1989; McCormick, 1989; Crunelli and Leresche, 199 1). Furthermore, peak GABAB conductance can vary between different types of neurons. For example, in dentate gyrus granule cells the peak GABAB conductance is only about 1.5-2.0 nS (Otis et al., 1993; D. D. Mott and D. V. Lewis, unpublished observations, 1993), approximately 10 times smaller than peak GABAB conductances in CAI or CA3 pyramidal neurons. However, in granule neurons the peak GABA, conductance is also about 10-fold smaller (Otis and Mody, 1993; D. D. Mott and D. V. Lewis, unpublished observations, 1993),such that the peak GABAB conductance is still 5-7 times smaller than peak GABAA conductance. Despite its small peak cAnductance, IPsP/CB produces a relatively large hyperpolarization from resting membrane potential in most neurons (10-20 mV). This occurs because the resting membrane potential of most neurons is closer to the equilibrium potential for chloride than for potassium. Therefore, despite the small peak GABAB conductance, the large driving force and long duration of IPSPB enable it to move a substantial amount of charge, similar to that carried during a GABAA response. For example, Otis et al. (1993) calculated that approximately 8 pC of charge leave a dentate granule cell during IPSCB, a value comparable to the range of 9-35 pC of charge moved during an IPSCA in these same cells. These characteristics of a small peak conductance and large hyperpolarization are consistent with the proposed function of IPSP, to produce hyperpolarizing inhibition and to reactivate voltageinactivated conductances (see Section IV,A,2). In contrast, at resting membrane potential in most neurons, IPSPA, which provides primarily

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shunting inhibition (Staley and Mody, 1992), produces a large conductance increase and relatively smaller hyperpolarization. f. Stimulus Dependence of IPSP/CB. Numerous studies have reported that IPSP/CB amplitude is markedly dependent on stimulus intensity, being evoked only by relatively strong stimuli (Newberry and Nicoll, 1984a; Stevens et al., 1987; Dutar and Nicoll, 1988a). Lower intensity stimuli, which will elicit GABAA responses, fail to evoke an IPSP/CB. Thus, IPSP/CB appears to require coactivation of a larger number of GABAergic terminals, and consequently a higher level of GABA release, than does IPSP/CA. This requirement could arise for several reasons. For example, GABAB receptors could have a lower affinity for GABA and so not be activated by concentrations of GABA that activate GABAA receptors. However, this possibility seems unlikely since GABA exhibits a similar affinity for both GABAA and GABAB receptors in binding studies (Hill and Bowery, 1981; Bowery et al., 1983) (see Section 111,A). Furthermore, in cultured neurons applied GABA activates presynaptic GABAB receptors at lower concentrations than GABAA receptors (Yoon and Rothman, 1991). Nevertheless, because of the potent GABA uptake system in brain slices (Dingledine and Korn, 1985), the affinity of GABA, receptors for GABA in this system has not been determined. An alternative, and more likely possibility is that, whereas GABA, receptors are located synaptically, GABAB receptors may be located extrasynaptically, so that GABA must diffuse to the receptor site. As previously discussed (see Section IV,A,l,d), GABAB receptors could be located at some distance from the point of GABA release and still respond within the observed onset latency. However, because of potent GABA uptake, adequate concentrations of GABA will diffuse to these extrasynaptic receptors only when high concentrations of GABA are present in the synaptic cleft or multiple GABAergic terminals are activated. This predicts that blockade of GABA uptake should markedly increase IPSPB. Indeed, several reports have confirmed this prediction (Dingledine and Korn, 1985; Thompson and Gahwiler, 1992b; Isaacson et al., 1993). GABA uptake blockers were reported to enhance dramatically IPSP/CB. However, they did not increase the amplitude or initial decay rate of a submaximal IPSP/CA, indicating that the uptake inhibitor was increasing GABA lifetime, but not peak GABA concentration. This observation suggests that the role of GABA uptake is to restrict the diffusion of GABA out of the synaptic cleft and therefore limit GABAB receptor activation. Thus, it appears that, because of GABA uptake, an adequate concentration of GABA reaches GABAB receptors only when the stimulus is strong enough to either burst fire interneurons or coactivate multiple GABAergic terminals and allow summation of GABA at the GABAB

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receptor. This stimulus dependence suggests that GABAB receptors will play a functional role only during periods of synchronized neural activation, such as during the induction of neuronal plasticity (see Section IV,C,4,b). GABA spill-over during repetitive stimulation has previously been proposed to produce depolarizing GABAA responses by activating extrasynaptic GABA, receptors (Alger and Nicoll, 1982b). T h e ability of a strong stimulus to activate GABAB receptors raises the question of the minimal stimulation required for activation of these receptors. For example, is the activation of one CABAergic interneuron capable of producing a unitary IPSPICB? One method of answering this question is to examine spontaneous inhibitory activity. Although numerous studies have observed spontaneous GABAA activity, spontaneous GABAB responses have not been reported (Doze et al., 1991; Otis and Mody, 1992).This lack of spontaneous IPSP/C,s suggests that GABA released from a single interneuron is not sufficient to activate GABAB receptors. However, the lack of spontaneous GABAB responses could also be attributed to other causes. For example, since the single-channel conductance of the GABA, receptor-operated potassium channel is very small, and because IPSP/CB has such a long time course, a small activation of the system, such as that produced by a single interneuron, might not produce a postsynaptic response that is detectable at the soma. Alternatively, it is possible that GABAA and CABAB responses are mediated by separate types of interneurons and that interneurons mediating GABAB responses are less spontaneously active than those responsible for GABAA responses (see Section IV,A,l,g). In support of this, Lacaille and Schwartzkroin (1988a,b) reported that GABAergic stellate cells in stratum lacunosum-moleculare of area CA 1 exhibit much less spontaneous activity than do GABAergic basket cells or GABAergic vertical cells of stratum oriendalveus. They suggest that these stellate cells mediate GABA, responses. Finally, it is possible that the overall level of spontaneous activity in vitro is less than that in vivo. A reduced frequency or amplitude of inhibitory events in vitro could decrease the probability that GABA concentrations will summate sufficiently to stimulate GABAB receptors. Thus, although spontaneous inhibitory events have primarily been studied in vitro, it is possible that recording in uivo may reveal spontaneous IPSP/CBs. In support of this, several studies have recently provided evidence for tonic GABA, receptor-mediated inhibition of spontaneous neuronal firing of at least some neurons in uivo (Andre et al., 1992; Lingenhohl and Olpe, 1993; Olpe et al., 1993a). Nevertheless, the lack of spontaneous IPSP/CBs in vitro raises the possibility that GABA released from spontaneously active interneurons is not able to activate GABA, receptors.

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In contrast to the lack of spontaneous IPSP/CBs under normal conditions, we (D. D. Mott and D. V. Lewis, unpublished observations, 1993) and others (N. A. Lambert and W. A. Wilson, unpublished observations, 1993) have observed a marked increase in spontaneous inhibitory activity and the appearance of spontaneous IPSP,s in CA3 hippocampal pyramidal neurons following epileptogenesis induced by repeated delivery of high intensity stimulus trains (Stasheff et al., 1985). Spontaneous IPSP/ C,s can also be recorded in the presence of agents that cause interneurons to burst fire, such as 4-AP, high extracellular potassium, or GABA, antagonists (Thalmann and Ayala, 1982; Alger, 1984; Muller and Misgeld, 1990; Segal, 1990a; Michelson and Wong, 1991; Muller and Misgeld, 1991). These findings suggest that the normal spontaneous activity of interneurons is unable to activate GABA, receptors; however, the increased GABA released by interneurons during a burst discharge is able to diffuse to, and activate, GABA, receptors. In this way GABA, receptors may be able to provide inhibition when GABAA receptormediated inhibition is depressed or when neurons are hyperexcitable (see Section IV,A,2,a). Finally, Bijak et al. (1991) found that in CA3 pyramidal cells application of a-adrenergic agonists, in concentrations that alone had no effect on membrane potential, would strongly potentiate the hyperpolarization produced by a submaximal concentration of baclofen. This effect was postsynaptic as indicated by its persistence during blockade of both sodium spikes with tetrodotoxin and fast synaptic transmission with both 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and picrotoxin. The enhancement of IPSPB by a-adrenergic agonists raises the possibility that norepinephrine may play a role in regulating GABA, responses in vivo, potentially enabling minimal activation of GABA, receptors to produce a functional response. g. Physaological Separation of GABA, and GABA, Responses. By analogy with other transmitter systems that have both fast and slow components, GABA, and GABA, receptors were initially thought to be colocalized at GABAergic synapses. In support of this, Segal and Furschpan (1990) observed that in cultured neurons activation of a presynaptic inhibitory cell produced both GABAA and GABAB IPSPs in the postsynaptic cell, demonstrating that in this system a single interneuron was responsible for both responses. Similarly, several studies have reported that application of agents, such as 4-AP or high potassium, that cause interneurons to burst fire, causes the appearance of large spontaneous IPSP/Cs that are mediated by both GABA, and GABA, receptors (McCormick, 1989; Michelson and Wong, 1991; Otis and Mody, 1992). This suggests that these spontaneous events are produced by the spontaneous activity of a

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single population of inhibitory neurons. This conclusion was supported by the observation that in many regions of the hippocampus a single stimulus coactivated both C;ABAAand GABA, receptors (Thalmann and Ayala, 1982; Newberry and Nicoll, 1984a; Dutar and Nicoll, 1988b). However, whereas both GABA, and GABAB responses can be evoked by stimulation in all strata of the hippocampus, GABAB responses appear to be more readily evoked in the dendritic than somatic layer (Dingledine and Langmoen, 1980; McChrren and Alger, 1985; Newberry and Nicoll, 1985) (see Section III,C,5). For example, Alger and Nicoll(1982a) found that in area CAl a late IPSP could be evoked by orthodromic stimulation in the dendrites, but not by alvear stimulation of the feedback inhibitory circuit. The interneurons mediating this alvear-evoked feedback inhibition, presumedly basket cells, project primarily to the soma and proximal apical dendrite of pyramidal cells (Schwartzkroin and Knowles, 1983; Schwartzkroin and Kunkel, 1985). Thus, the authors suggested that GABAB responses can be evoked primarily in a feedforward manner by stimulation of GABAergic neurons that project onto dendrites. In the dentate gyrus we have found that an IPSP/CB can be evoked in granule cells by either antidromic siimulation of granule cell axons (mossy fibers) or orthodromic stimulation in the molecular layer, indicating that in this area GABA, receptors can be activated in a feedback or feedforward manner (Mott et al., 1993b).However, the ratio between the amplitude of the IPSPB and IPSPA, following the antidromic and orthodromic stimuli, revealed that the GABAB response was relatively larger in response to stimulation of GABAergic axons in the molecular layer. There are several reasons why GABAB responses could be larger and more readily evoked in the dendritic than somatic layers. First, in the hippocampal formation immunocytochemical studies have localized the highest concentration of GABA transporters on GABAergic axons projecting to the soma of pyramidal and granule cells (Radian et al., 1990). This indicates that GABA uptake will be strongest in the cell body layer (Rovira et al., 1984) and suggests that diffusion of released GABA will be most restricted in this area. If GABAB receptors are located extrasynaptically, then the potent GABA uptake in this region may limit their activation. In support of this, Thompson and Gahwiler (1992b) reported that, under normal conditions, focal stimulation at the border of stratum pyramidale produced an IPSPAonly. However, when GABA uptake was blocked with tiagabine, this same stimulus now evoked an IPSP, as well. Second, binding studies have shown a greater density of GABAB receptors in dendritic than somatic layers, increasing the likelihood that dendritically released GABA will activate GABAB receptors (Bowery etal., 1985a, 1987) (see Section 111,B).Finally, electrophysio-

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logical and morphological studies have reported differences between GABAergic interneurons located in the cell body layer and those in the distal dendrites, which may contribute to the preferential dendritic activation of GABAB receptors (Lacaille et al., 1989). For example, in area CA1 basket cells in the somatic layer and vertical cells of stratum oriens/alveus both project primarily to postsynaptic targets within the cell body layer and have short duration action potentials (Knowles and Schwartzkroin, 1981; Lacaille et al., 1987, 1989). In contrast, stellate interneurons in stratum lacunosum-moleculare have long duration action potentials and axons that project diffusely to dendritic sites in the hippocampus and dentate gyrus (Lacaille and Schwartzkroin, 1988a,b). The longer duration of action potentials in stellate interneurons suggests that, when activated, these cells may release more GABA per action potential than do basket cells or vertical cells of the oriens/alveus. Because of the higher levels of GABA needed to stimulate GABAB receptors (see Section IV,A,l,f), these interneurons may be more likely to activate GABAB receptors. Thus, dendritic stimulation activates axons of interneurons (possibly stellate cells) projecting dendritically and causes the release of potentially larger amounts of GABA into a region with more GABAB receptors and less potent GABA uptake, increasing the probability of activation of GABAB receptors. In contrast, alvear stimulation o r direct stimulation in the cell body layer activates GABAergic axons with terminals located somatically and causes the release of a potentially smaller amount of GABA per action potential into a region with fewer GABAB receptors and more potent GABA uptake. GABA released in the somatic layer may be spatially restricted by the increased GABA uptake in this region and, therefore, less likely to activate GABAB receptors. The above observations raise the possibility that IPSP/C, and IPSP/ CB may be produced by separate GABAergic interneurons and that some interneurons that project dendritically may preferentially stimulate GABAB receptors. Indeed, several studies support this possibility. For example, in both area CAI and dentate gyrus isolated GABAB responses can occasionally be evoked by discrete stimuli delivered to the dendritic layer, whereas isolated GABA, responses can be produced by focal stimulation of all layers (Solis et al., 1992; Solis and Nicoll, 1992b). Similarly, focal application of glutamate in stratum lacunosum-moleculare of area CA1 could evoke IPSPBS (Williams and Lacaille, 1992), whereas glutamate application near stratum pyramidale produced IPSP,s (Madison and Nicoll, 1988; Samulack and Lacaille, 1991). A separation of the interneurons mediating GABA, and GABAB responses was also indicated by several studies that pharmacologically separated the two responses at a presynaptic level. For example, Doze et al. (1991) found

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that in area CA1 norepinephrine blocked IPSPA by suppressing synaptic excitation of inhibitory interneurons. In contrast, IPSPB was not affected by norepinephrine, suggesting that IPSPB was mediated by a separate population of interneurons. Similarly, Sugita et al. (1992) reported that in the lateral amygdala muscarine acted presynaptically on interneurons to suppress IPSPA, but not IPSPB. Serotonin was also reported to depress selectively IPSPB (Oleskevich and Lacaille, 1992; Sugita et al., 1992). The effect of serotonin was thought to be mediated by presynaptic 5-HT,, receptors, which suppressed the release of GABA onto GABAB receptors (Johnson et al., 1992). Finally, separation of GABAergic interneurons responsible for GABA, and GABA, responses was suggested by several studies examining the giant spontaneous inhibitory events produced during application of pharmacological agents designed to cause interneurons to burst [i.e., 4-AP or high potassium (Muller and Misgeld, 1990; Segal, 1990a)l. Although these giant spontaneous inhibitory events are typically mediated by coactivation of both GABAA and GABAB receptors (see above), several studies have reported the spontaneous appearance of dissociated IPSP, and IPSPB responses. These dissociated responses may be caused by the independent spontaneous activity of separate populations of interneurons. Thus, although both GABAA and GABA, responses can be coactivated in all strata of the hippocampus, the above results raise the possibility that some GABAergic interneurons projecting dendritically may be capable of producing purely GABAB receptor-mediated responses. However, it is not clear whether the appearance of an isolated IPSP/cB in the recorded neuron represents the stimulation of a GABAergic interneuron that projects exclusively to GABAB receptors at all sites or whether the axons of this interneuron can project to GABAB receptors on some neurons and GABA, receptors on others. Indeed, since GABAergic axons can synapse en passant (Schwartzkroin and Kunkel, 1985; Lacaille et al., 1987), it is even possible that the same axon could activate exclusively GABAB receptors at some sites, activate exclusively GABA, receptors at other sites, and/or co-activate both types of receptor at still other sites on the same or different neuron. Further studies will be needed to resolve this issue. Finally, it should be noted that, because of the slow time course of GABAB responses, it is not necessary to postulate that these receptors are synaptically located. Thus, as has been suggested for other G proteinlinked receptors (Hille, 1992b), such as atrial muscarinic receptors (Hartzell, 1980), GABA, receptors may be diffusely located rather than clustered synaptically. For example, if GABAB receptors are extrasynaptic, diffusely distributed, and have a high affinity for GABA, it is possible that isolated IPSP/CBs in the recorded neuron may represent GABA

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spill-over from neighboring GABAergic terminals on other neurons (Thompson and Gahwiler, 1992b; Isaacson et al., 1993). Thus, GABA, receptors may not be activated synaptically, but rather may be dependent on GABA spill-over in a manner similar to that of the GABA, receptors responsible for the dendritic depolarizing response to GABA (Alger and Nicoll, 1982b). h. GABA, Responses on GABAergzc Neurons. Interneurons in the hippocampal formation comprise a heterogeneous group of cells, which can be classified according to their morphology. The diversity of interneuronal types is especially apparent in the hilus of the dentate gyrus where at least 21 different classes of interneurons can be identified (Amaral, 1978).The majority of interneurons in both the dentate gyrus and hippocampus use GABA as their neurotransmitter (Ribak et al., 1978; Seress and Ribak, 1983; Babb et al., 1988; Woodson et al., 1989). Each of these GABAergic interneurons makes synaptic contact on many different excitatory neurons. For example, Li et al. (1992) estimated that in area CAl a single chandelier cell contacted 1214 pyramidal cells. On the other hand, each pyramidal cell can be synaptically contacted by many different interneurons (Miles and Wong, 1984). This high degree of connectivity endows interneurons with the ability to regulate powerfully excitatory transmission in the hippocampal formation. However, in addition to these synaptic contacts on excitatory neurons, anatomical studies also provide evidence for GABAergic inhibitory synapses on inhibitory interneurons in both the hippocampus (Schwartzkroin and Kunkel, 1985; Lacaille et al., 1987; Kunkel et al., 1988; Lacaille et al., 1989) and the dentate gyrus (Leranth and Frotscher, 1986; Misgeld and Frotscher, 1986).These inhibitory synapses on inhibitory neurons provide a basis for disinhibition in the hippocampal formation. Indeed, electrophysiological studies have recorded both GABA, and GABAB receptor-mediated responses in interneurons (Schwartzkroin and Mathers, 1978; Ashwood et al., 1984; Lacaille et al., 1989; Lacaille, 1991). GABAB responses were first demonstrated pharmacologically in studies using baclofen to hyperpolarize interneurons both in area CA1 (Madison and Nicoll, 1988) and in the dentate gyrus (Misgeld et al., 1989). Baclofen strongly hyperpolarized and increased the potassium conductance of interneurons in both of these areas. In the dentate gyrus the drug had a stronger postsynaptic inhibitory effect on interneurons than on granule cells (Misgeld et al., 1989), suggesting that in this area the net effect of the drug is disinhibitory (see Section IV,D). A physiological role for GABAB receptors on interneurons was recently demonstrated when it was observed that, in addition to evoking

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an IPSPA, afferent stimulation produced an IPSP, in spiny neurons in the hilus of the dentate gyrus (Scharfman, 1992) as well as in interneurons in stratum pyramidale of area CA1 (Lacaille, 1991). In area CA1, Lacaille (1991) found that the IPSP, in interneurons in stratum pyramidale was similar to that in pyramidal cells. For example, IPSPB in interneurons was stimulus-dependent, requiring higher stimulus intensities than IPSP,. It had a slow time course with a peak latency of about 131 ms and was antagonized by phaclofen. The response in interneurons in both CA 1 and dentate gyrus reversed at a very hyperpolarized potential, - 108 mV (Lacaille, 1991) and -82 mV (Scharfman, 1992), respectively, indicating that, like IPSPB in excitatory neurons, it was mediated by an increase in potassium conductance. In contrast to the effect of postsynaptic GABAB receptors in the dentate gyrus (Misgeld et al., 1989), in area CA1 the peak amplitude and conductance increase of IPSPB in interneurons was similar to that in pyramidal cells. Thus, IPSPB decreased resting input resistance in CA1 interneurons by about 8% compared to a decrease of 8-19% in pyramidal cells (Alger, 1984). GABAB responses in inhibitory interneurons are most likely produced by both local circuit interneurons and GABAergic projections from outside the hippocampus. In both area CA1 (Schwartzkroin and Kunkel, 1985; Lacaille et al., 1987, 1989; Kunkel et al., 1988) and dentate gyrus (Misgeld and Frotscher, 1986) anatomical studies have reported that inhibitory interneurons make inhibitory synaptic contact with each other, indicating that the IPSPBs are mediated partly by local inhibitory interneurons. In addition, Misgeld et al. (1992) observed that in the presence of 4-AP, spontaneous, late potassium-dependent IPSPs could be recorded from some aspiny and spiny interneurons in the hilus of the dentate gyrus, suggesting that these IPSPs were produced by burst discharges in local interneurons. However, anatomical studies have also provided evidence indicating that GABAergic neurons in other brain areas project onto GABAergic interneurons in the hippocampus. For example, GABAergic neurons in the septum were found to contact inhibitory neurons in several different hippocampal subfields (Freund and Antal, 1988; Gulyas et al., 1990). Thus both intra- and extrahippocampal GABAergic neurons are able to control the activity of interneurons in the hippocam pal formation through postsynaptic GABAergic inhibition mediated not only by GABA,, but also by GABAB receptors.

2. Functional Significance of IPSPIC, IPSPs mediated by GABAA and GABA, receptors have very different properties, suggesting that each serves a distinct inhibitory function. GABAAreceptor-mediated IPSPs are associated with a large conductance

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increase and so are very effective at inhibiting neurons from firing action potentials, even when excitatory stimulation is strong. These IPSPAShave a rapid onset and relatively short duration, providing a mechanism for precisely timed control of excitatory transmission (Connors et al., 1988; Staley and Mody, 1992). When GABA, receptor-mediated inhibition is blocked, epileptic seizures can develop (Dingledine and Gjerstad, 1980; Schwartzkroin and Prince, 1980), demonstrating the importance of this form of inhibition in controlling excitation. In contrast, under normal conditions, blockade of IPSPB does not cause the appearance of overt neuronal hyperexcitability (Karlsson et al., 1990; Olpe et al., 1990), indicating that postsynaptic GABAB receptors regulate excitatory transmission in a more subtle fashion. These receptors are thought to modulate synaptic transmission by providing hyperpolarizing inhibition and by reactivating voltage-inactivated conductances. We will discuss these functions below. a. GABA, Receptor-Mediated Inhibition. In contrast to IPSP,, which can produce powerful inhibition by markedly increasing membrane shunting conductance, GABA, receptor-mediated IPSPs are associated with a much smaller conductance increase, but a large hyperpolarization. Because of these properties, IPSPB was suggested to produce primarily hyperpolarizing inhibition (Connors et al., 1988; Misgeld et al., 1989; Crunelli and Leresche, 1991). Thus, although IPSPA powerfully inhibits spontaneous action potentials produced at all membrane potentials, IPSP, suppresses spontaneous cell firing produced by weak, but not strong, membrane depolarization (Connors et al., 1988; McCormick, 1989). Furthermore, Connors et al. (1988) observed that in neocortical cells, inhibition of spontaneous spike firing produced by baclofen could be partially counteracted by injecting depolarizing current into the cell to compensate for the hyperpolarization produced by baclofen. Alternatively, the inhibition produced by baclofen could be mimicked by injecting hyperpolarizing current into a control cell. In dentate gyrus granule cells we have found that IPSPB more strongly inhibits spontaneous action potentials produced by weak than by strong membrane depolarization (D. D. Mott and D. V. Lewis, unpublished observations, 1991). These observations suggest that membrane hyperpolarization contributes substantially to the inhibition produced by IPSPB. Because of the slow time course of IPSPB, GABAB receptor-mediated inhibition of spontaneous neuronal firing has a longer duration than that produced by IPSPA. For example, Lingenhohl and Olpe (1993), recording from neurons in the entorhinal-subicular region in vivo, found that spontaneous action potentials in these cells were inhibited for about 400 ms after a stimulus. This period of inhibition corresponded to the presence of an IPSPB in these neurons and both the IPSPB and the

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inhibition were reduced by the GABAB receptor antagonist CGP 4638 1. A similar inhibition of spontaneous action potentials has been observed in uitro (Kehl and McLennan, 198513; Connors et al., 1988; Misgeld et al., 1992). For example, in dentate granule cells we have observed that spontaneous action potentials, induced when the cell is depolarized with current injection, are inhibited during an evoked IPSPB. The duration of this inhibition ranged from about 400 to 1000 ms. Both the IPSPB and the inhibition of spontaneous spiking were blocked by 2-hydroxysaclofen (D. D. Mott and D. V. Lewis, unpublished observations, 1991).Alternatively, Kehl and McLennan (198513) found that spontaneous unit activity in area CA3 was inhibited for 200-500 ms after a stimulus, an interval corresponding to the duration of the late IPSP that they recorded from these neurons. Evidence for tonic GABAB receptor-mediated inhibition has been mixed. Olpe et al. (199 1) reported that in frontal cortex of choral hydrate anesthetized rats in uiuo intravenously applied CGP 35348 (30 mg/kg) reduced the depressant effects of iontophoretically applied baclofen, but had no effect on the spontaneous discharge rate of pyramidal neurons. However, in the same preparation iontophoretically applied CGP 35348 increased the spontaneous firing rate of these neurons. The authors suggested that the ability of iontophoretically applied CCP 35348 to increase the spontaneous firing rate may be due to the higher local concentration of CGP 35348 produced by this method of application, resulting in a more complete blockade of GABAB receptors. In support of this, Lingenhohl and Olpe (1993) found that the more potent antagonist CGP 46381, when injected intravenously (30 mg/kg), increased the discharge rate of some, but not all, neurons in the entorhinal-subicular region of chloral hydrate anesthetized rats in uiuo. When administered orally this drug caused a mild increase in the spontaneous discharge rate of cortical neurons (Olpe et al., 1993a). Finally, Andre et al. (1992) reported that iontophoretic application of CGP 35348 into rostra1 or caudal sensorimotor cortex increased the firing rate of spontaneously active neurons. In addition, they found that the excitatory response to iontophoretic application of acetylcholine and quisqualate was enhanced following GABAB receptor blockade. Taken together, these results suggest that at least some neurons are under the control of a weak, tonic GABAB receptor-mediated inhibition. The mild effect of GABAB receptor blockade on spontaneous activity is in marked contrast to the dramatic increase in spontaneous activity and the appearance of epileptiform discharges produced by blockade of GABA, receptors. I n addition to inhibiting spontaneous action potentials, IPSPB has been reported to depress evoked cell firing. Because of its slow onset latency, IPSPB does not inhibit the excitatory response to a single stimulus

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under control conditions. However, the extended duration of GABAB receptor-mediated inhibition suggests that it may regulate excitatory activity during repetitive stimulation. Measurements of paired pulse inhibition of evoked responses over a range of interstimulus intervals support this possibility (Kehl and McLennan, 1983, 1985b; Oliver and Miller, 1985; Karlsson and Olpe, 1989; Rausche et al., 1989; Olpe et al., 1993a) (see Section IV,C,4,a). When stimuli are delivered at short intervals (lo0 ms) a late and less powerful phase of inhibition of cell firing becomes apparent. In contrast to the early inhibition, this late inhibition is not blocked, but rather is enhanced by bicuculline. Studies in area CAI both in vitro (Karlsson and Olpe, 1989) and in uivo (Olpe et al., 1993a) have found that the inhibition is partially reduced by several highly potent GABA, receptor antagonists, suggesting that in this area IPSPB is at least partly responsible for the inhibition. In contrast, in the dentate gyrus IPSP, does not significantly contribute to the late phase of paired pulse inhibition. Considering the relatively small amplitude of IPSP, in granule cells (see Section IV,A, l,e), this minimal effect is not surprising. Thus, high concentrations of either phaclofen (1 mA4) or CGP 36348 (1 mM), which block the IPSP,, were unable to reduce significantly the late phase of paired pulse inhibition in vitro (Mott and Lewis, 1991, but see Rausche et al., 1989). A similar finding was reported in uiuo (Brucato et al., 1992). However, during blockade of GABAA receptor-mediated inhibition with picrotoxin, the late paired pulse inhibition in the dentate gyrus was increased (Mott and Lewis, 1989) (Fig. 4). The increase in paired pulse inhibition was greatest at an interstimulus interval of 150-250 ms, corresponding to the peak latency of IPSP,. Intracellular recording from granule cells revealed that picrotoxin markedly increased the amplitude of the polysynaptic IPSP,. Both the enhanced IPSP, and the increase in late paired pulse inhibition was suppressed by GABAB receptor antagonists. Thus, IPSPB in the dentate gyrus appears to produce only minimal inhibition of granule cells under normal conditions. However, when GABA, inhibition was blocked, postsynaptic GABA, receptor-mediated inhibition was facilitated. A similar increase in the late inhibition following blockade of GABA, inhibition was reported in area CA3 of the hippocampus (Kehl and McLennan, 1985a). In addition to being observed in the dentate gyrus, the enhancement of IPSP/C, during blockade of IPSP/CA has been reported in hippocampus (Newberry and Nicoll, 1984a; Scanziani et al., 1991), neocortex (McCormick, 1989), septum (Stevens et al., 1987), and thalamus (Soltesz

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ACSF

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1

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Picro toxin

ACSF

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+A 1

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va

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ZOO 3 ma 4

FIG. 4. GABA, receptor-mediated late inhibition of dentate gyrus granule cells is enhanced by blockade of IPSP,. (A) Responses to paired perforant path stimuli delivered 200 ms apart and recorded extracellularly in the dentate gyrus granule cell layer are shown. Application of phaclofen ( 1 m M ) does not prevent inhibition of the population spike on the second response, suggesting that GABA, receptors contribute only minimally to this inhibition. Throughout this figure population spikes are indicated by arrows. (B) In another slice application of picrotoxin (20 p M ) dramatically enhances the late inhibition. In the presence of picrotoxin, the perforant path stimulus intensity was reduced to compensate for the increase in the initial response produced by blockade of IPSP,&.The subsequent addition of phaclofen ( I mA4) reduces this enhancement of the late inhibition, suggesting that the increase in inhibition was produced by an increase in IPSP,. Note that phaclofen does not prevent the facilitation of the initial response by picrotoxin. (C) Intracellular recording from a granule cell reveals that picrotoxin (20 p M ) markedly enhances the polysynaptic IPSP, (solid circle). Subsequent blockade of GABA, receptors with 2-hydroxysaclofen (400 p M ) inhibits this enhanced polysynaptic IPSP,. The polysynaptic IPSP was evoked antidromically by mossy fiber stimulation. The cell was held depolarized to -65 mV with injected current causing IPSP, (open circle) to be hyperpolarizing. In addition to increasing IPSP,, blockade of IPSP, with picrotoxin also revealed a small NMDA receptor-mediated EPSP. Note that picrotoxin enhanced the amplitude of the polysynaptic IPSP,, but not the amplitude of the monosynaptic IPSP, (see Fig. 3).

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et al., 1989).Although the neuronal circuitry in these areas differs considerably, the enhancement is generally thought to occur in the following manner (also see Section IV,A,l,f). Blockade of GABAA receptormediated inhibition markedly increases the excitability of the neuronal population, causing excitatory neurons to synchronously burst discharge in response to a stimulus. This enhancement of excitation increases the excitatory drive of interneurons. In addition, blockade of GABAA receptors on the interneurons further increases their excitability. T h e enhanced excitatory drive and disinhibition of interneurons cause them to release a larger amount of GABA in response to a stimulus. This GABA can subsequently act on GABAB receptors to produce a larger IPSPB. The facilitation of interneuron firing, and thus the increase in IPSP,, is dependent on NMDA receptor activation. Thus, during blockade of GABA, receptors, application of D( - )-2-amino-5-phosphonovaleric acid (D-APV) suppresses the enhanced IPSPB (Scanziani et al., 1991). In contrast, under control conditions, IPSPB is insensitive to NMDA receptor blockade. The enhancement of IPSP, by bicuculline implies that postsynaptic GABA, receptors may be able to compensate partially for a reduction in GABA, receptor-mediated inhibition. This enhanced IPSPB may help suppress hyperexcitability in models of epilepsy that are associated with a reduction of GABAA inhibition (McCormick, 1989; Malouf et d., 1990; Karlsson et al., 1990, 1992b; Scanziani et al., 1991). For example, the enhanced IPSPB has been reported to contribute to the afterhyperpolarization (AHP) produced by epileptiform bursts during blockade of GABA, inhibition. Because of the long duration of these excitatory bursts, the later component of each burst overlaps the initial portion of IPSP,, enabling the IPSP, to help terminate the burst discharge (McCormick, 1989; Scanziani et al., 1991; Karlsson et al., 1992b). Consequently, in hippocampal pyramidal cells in both area CAI (Karlsson et al., 1992b) and area CA3 (Scanziani et al., 1991)CGP 35348 increased the duration of bicuculline-induced bursts by suppressing a component of the postburst AHP. A similar observation was reported in human neocortex (McCormick, 1989). Thus, although postsynaptic GABAB receptors do not normally suppress the excitatory response produced by a single stimulus, they can substantially contribute to the postburst AHP and consequently play a role in the termination of burst discharges. In addition to shortening the duration of burst discharges, postsynaptic GABAB receptors can also modulate the spontaneous burst frequency. Karlsson et al. (1990, 1992b) reported that CGP 35348 caused an increase in the frequency of bicuculline- or penicillin-induced spontaneous interictal bursts in hippocampal slices. T h e effect of CGP 35348 appeared

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to be greater in area CA3 than in area CA1. This proepileptic effect

is in marked contrast to the inability of postsynaptic GABAB receptor blockade to produce hyperexcitability under control conditions. Thus, the enhancement of IPSP, appeared to compensate partially for the loss of GABA, inhibition and in this manner contribute to the suppression of hyperexcitability. I n addition to reducing spontaneous interictal bursting produced by pharmacological reduction of GABAA inhibition, postsynaptic GABAB receptors also appear to regulate spontaneous interictal bursts in other experimental models of epilepsy. For example, CGP 35348 significantly increased the rate of bursting produced by lowering the extracellular concentration of magnesium (Karlsson et al., 1990, 1992b). Similarly, we have found that in area CA3 of the hippocampus, CGP 35348 alone does not cause control slices to burst spontaneously. However, in slices made epileptic by repeated application of high-intensity stimulus trains (Stasheff et al., 1985), CGP 35348 markedly enhanced the frequency of spontaneous interictal discharges. In some slices that received a lesser number of stimulus trains, spontaneous bursting would gradually cease. However, subsequent application of CGP 35348 would reinitiate bursting (D. D. Mott and D. V. Lewis, unpublished observations, 1993). These results indicate that postsynaptic GABAB receptors can regulate interictal bursting in several different experimental models of epilepsy. Interestingly, Chamberlin and Dingledine (1989) found that the frequency of spontaneous interictal bursts produced by raising extracellular potassium was not altered by phaclofen. The lack of an effect of IPSPB in this model is most likely caused by the reduction in IPSPB driving force produced by the increase in extracellular potassium concentration. This would reduce the hyperpolarization produced by IPSPB, rendering it less effective. In addition to suppressing interictal bursts, IPSPB has also been reported to modulate ictal activity. For example, in area CA3 of hippocampal slice cultures that were exposed to a low concentration of bicuculline, CGP 35348 not only caused burst duration to increase, but also caused the appearance of spontaneous electrographic seizures (Malouf et al., 1990; Scanziani et al., 1991). The proepileptic effect of CGP 35348 was attributed to its blockade of the enhanced IPSPB. These results indicate that the enhancement of IPSPB was able to compensate for the reduction in GABAA inhibition and suppress ictal activity. Morrisett el al. (1993) examined the effect of 2-hydroxysaclofen on electrographic seizures in area CA3 of hippocampal slices, which were made epileptic by repeated high-frequency stimulus trains. These authors found that blockade of GABAB receptors increased the intensity of evoked electrographic sei-

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zures and markedly lowered the stimulus threshold necessary for seizure expression. Thus, although blockade of GABAB receptors does not produce electrographic seizures in control slices, in these in uitro experimental models of epilepsy, postsynaptic GABAB receptors can modulate ictal activity. The regulation of ictal events by postsynaptic GABAB receptors in uitro raises the possibility that IPSPB may modulate ictal activity in uiuo. In support of this possibility, Karlsson et al. (1992b) found that systemic application of CGP 35348 did not produce hyperexcitability in control animals, but did increase the number of isoniazid-treated animals that displayed behavioral seizures. In addition, CGP 35348 significantly decreased the latency to seizure onset in animals pretreated with high doses of pentylenetetrazole (Karlsson et a/., 1990) o r isoniazid (Karlsson et al., 1992b). In contrast, blockade of GABAB receptors did not alter behavioral seizures in other epilepsy models. For example, seizures induced by picrotoxin, strychnine, NMDA, and electroshock were not consistently affected by CGP 35348. Furthermore, Karlsson et al. (1992b) found that CGP 35348 had no effect on the duration of behavioral seizures in kindled animals. The inability of CGP 35348 to facilitate behavioral seizures in these in uiuo models of epilepsy may be caused by differences in the method of seizure induction, which could occlude the subtle modulation by GABA, receptors, or by an insufficient blockade of GABA, receptors by the antagonist. Nevertheless, the enhancement of isoniazidinduced convulsions by CGP 35348 indicates that in certain models GABA, receptors may play an important role in regulating behavioral seizures. Since IPSPB is associated with a large hyperpolarization, it may be effective in preventing epileptiform activity, in part, by inhibiting the expression of voltage-sensitive conductances. Morrisett et al. ( 1993) found that in control hippocampal slices, when paired stimuli were delivered 100-600 ms apart, the second response of the pair, recorded in stratum pyramidale of area CA3, was only minimally inhibited. In contrast, when the slices were made epileptic by repeated high-frequency stimulus trains, each of the paired stimuli now evoked a burst response and the response to the second stimulus was markedly inhibited. This inhibition was reduced by application of phaclofen or 2-hydroxysaclofen. These results indicate that GABA, receptors more effectively inhibit burst activity than they inhibit nonepileptic excitatory responses. Since these burst responses were not sensitive to D-APV, the authors suggest that following epileptogenesis, the burst response is partially produced by non-N MDA receptor-mediated voltage-dependent conductances that are highly sensitive to the hyperpolarizing inhibition produced by IPSPB.

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Although postsynaptic GABAB receptors can modulate epileptiform activity in certain experimental models of epilepsy, blockade of these receptors does not produce hyperexcitability under normal conditions. Indeed, in nonepileptic tissue postsynaptic GABAB receptor-mediated inhibition seems only to suppress excitatory responses to weak afferent input and is rapidly overcome if the stimulus intensity is increased. What then, is the physiological role of IPSPB under normal conditions? We have found that in hippocampal slices postsynaptic GABA, receptormediated inhibition is especially effective at inhibiting responses mediated by NMDA receptors (Morrisett et al., 1991). NMDA receptoroperated channels are blocked in a voltage-dependent manner by magnesium. This blockade is relieved by membrane depolarization. Because of the voltage dependence conferred on the channel by the magnesium blockade, NMDA receptor-mediated responses are highly sensitive to hyperpolarizing inhibition, such as that produced by IPSPB. This inhibition is apparent in both the hippocampus and the dentate gyrus. For example, we found that NMDA receptor-mediated EPSPs (NMDA EPSPs) can be isolated by applying picrotoxin and 6,7-dinitroquinoxaline-2,3-dione (DNQX) to block GABA, and non-NMDA receptormediated responses, respectively. The time course of this NMDA EPSP is slow, causing the later portion of the response to overlap the initial portion of IPSPB. Thus, similar to the burst discharge previously discussed, the duration of the NMDA EPSP is increased by blockade of GABAB receptors. Furthermore, when paired stimuli are delivered 150-500 ms apart, the NMDA EPSP evoked by the second stimulus is markedly suppressed (Fig. 5). This inhibition is maximal when stimuli are delivered 150-250 ms apart, an interval corresponding to the peak of IPSPB. The inhibition is blocked by phaclofen, 2-hydroxysaclofen, or CGP 35348, indicating that it is mediated by GABA, receptors. In contrast, the non-NMDA receptor-mediated component of the EPSP, isolated using picrotoxin and D-APV, was not lengthened by blockade of GABAB receptors, nor was it inhibited when stimuli were paired over a range of intervals from 50 to 1600 ms. This indicates that the GABAB receptor-mediated inhibition is primarily selective for the NMDA receptor-mediated component of the EPSP and suggests that it is mediated by a postsynaptic effect of GABA, receptors (but see Section IV,B). The inability of IPSPB to inhibit the non-NMDA EPSP may reflect the voltage-independence of this excitatory response. Furthermore, the minimal effect of the IPSP, on normal synaptic transmission may reflect the lack of inhibition of the non-NMDA EPSP, since the non-NMDA EPSP is primarily responsible for the excitatory response under normal conditions.

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Interstimulus Interval (ms) 100 ms

FIG. 5. Postsynaptic GABABreceptors inhibit NMDA receptor-mediated responses. (A) Shown are NMDA EPSPs in a dentate granule cell in response to paired perforant path stimuli delivered 200 ms apart. NMDA EPSPs are isolated by application of DNQX (10 p M ) and picrotoxin (50 p M ) . Action potentials are truncated. 2-Hydroxysaclofen blocks IPSPBand consequently prevents inhibition of the second EPSP. Note the widening of the initial EPSP in the presence of 2-hydroxysaclofen, suggesting that IPSP, plays a role in truncating the NMDA response. Addition of D-APV blocks the EPSPs, confirming that they are NMDA receptor-mediated. (B) Time course of the paired pulse effects on NMDA (squares) and non-NMDA (triangles) receptor-mediated EPSPs. Note that NMDA EPSPs exhibited paired pulse facilitation at short intervals followed by inhibition at longer intervals. In contrast, the non-NMDA EPSP showed only a mild facilitation and no inhibition. Symbols and error bars represent mean C SE (n = 5-10 slices). The curves are significantly different by Kruskal-Wallis (P < ,001). Responses are dendritic EPSPs recorded in stratum radiatum of area CAI in response to paired stimulation of the Schaffer collaterals. Non-NMDA responses are isolated by application of D-APV (50 p M ) and picrotoxin (50 p M ) . [The graph is reprinted from Morrisett et al. (1991) with permission.] (C) Voltage dependence of the inhibition of the NMDA EPSP. Perforant path-evoked NMDA EPSPs are recorded from the same granule cell as in (A). Paired stimuli are delivered 200 ms apart while injected current is used to hold the cell at the potential indicated on the left. Note that hyperpolarization primarily reduced the amplitude of the initial NMDA EPSP, occluding further inhibition of the response evoked by the second stimulus.

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These observations suggested that GABAB receptors suppressed the NMDA EPSP through a postsynaptic mechanism. We attempted to confirm this using several additional lines of evidence. For example, we found that removal of extracellular magnesium reduced paired pulse inhibition of the NMDA EPSP. The magnesium-dependence of the inhibition suggests that the hyperpolarization produced by IPSP, suppresses the NMDA EPSP by preserving the magnesium block of the NMDA receptor-operated channel (Morrisett et al., 1991). Similarly, in dentate gyrus granule cells we found that membrane hyperpolarization with injected current reduced the amplitude of a single NMDA EPSP and substantially occluded the paired pulse inhibition (Fig. 5 ) . Presumedly, this occurred because the hyperpolarization reinforced the magnesium block of NMDA receptor-operated channels. Alternatively, GTP-y-S, when injected into granule cells, causes irreversible activation of G proteins and chronically opens the postsynaptic GABA, receptor-mediated potassium conductance (see Section III,C,5). Thus, we found that GTPy-S occluded the postsynaptic effects of baclofen in granule cells and substantially reduced paired pulse inhibition of NMDA EPSPs (D. D. Mott and D. V. Lewis, unpublished observation, 1991). These results demonstrate that the inhibition occurs postsynaptically through a G protein-coupled potassium conductance. Taken together, the above findings indicate that paired pulse inhibition of NMDA EPSPs is primarily mediated by IPSPB,which hyperpolarizes the cell and consequently prevents removal of the magnesium block from the NMDA receptor-operated channel. However, the inability of the above manipulations to suppress entirely paired pulse inhibition of NMDA EPSPs suggests that GABAB receptors may also suppress NMDA responses through an additional mechanism, possibly a presynaptic depression of transmitter release (see Section IV,B). By suppressing NMDA receptor-operated currents, GABAB receptors may modulate synaptic plasticity. Long-term potentiation (LTP) is a form of synaptic plasticity thought to model synaptic changes that occur during learning and memory (for review see Teyler and Discenna, 1987; Bliss and Collingridge, 1993).In many pathways LTP is dependent on calcium entry through N MDA receptor-operated channels and so requires both NMDA receptor activation and postsynaptic depolarization sufficient to lift the magnesium block of the NMDA receptor-operated channels (Wigstrom and Gustafsson, 1986; Nicoll et al., 1988; Bliss and Collingridge, 1993). For this reason, LTP is typically induced by highfrequency stimulation of afferent fibers sufficient to depolarize postsynaptic neurons. Postsynaptic GABA, receptor-mediated inhibition opposes the depolarization necessary for removal of the magnesium block

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and opening of the NMDA receptor-operated channels, suggesting that it may suppress LTP induction. Indeed, an inhibitory role for GABAB receptors in the induction of LTP has recently been demonstrated in area CA1 both in uitro (Olpe and Karlsson, 1990) and in uiuo (Olpe et al., 1993b). These studies reported that LTP induced by high-frequency stimulation was enhanced by blockade of GABAB receptors. T h e authors concluded that the enhancement of LTP induction was produced by a suppression of the underlying IPSPB, presumedly enabling the stimulus train to depolarize more effectively postsynaptic neurons. The ability of GABAB receptors to suppress LTP is consistent with the observation that the GABAB receptor antagonist CGP 36742 improves the cognitive performance of mice, rats, and rhesus monkeys in behavioral tests (Mondadori et al., 1992; Carletti et al., 1993). In addition to inhibiting the induction of NMDA receptor-dependent LTP, GABAB receptors may also modulate synaptic plasticity by suppressing seizure development during kindling. The kindling model of epilepsy refers to the gradual induction of seizures by the repeated application of stimulus trains that are initially subconvulsive (Goddard et al., 1969; McNamara, 1986). During kindling, the animal progresses through a series of characteristic behavioral stages (Racine, 1972). In support of a role for GABAB receptors in this process, Karlsson et al. ( 1992a) found that blockade of GABAB receptors accelerated the development of amygdala kindling. Thus, CGP 35348 reduced the number of stimulus trains required to reach the different behavioral seizure stages during kindling. The authors concluded that GABAB receptormediated inhibition may contribute to seizure suppression. Electrographic seizures can also be kindled in hippocampal slices in uitro by repeated application of stimulus trains that are initially subthreshold for production of an afterdischarge (Stasheff et al., 1985). We have found that CGP 35348 reduces the number of stimulus trains required to produce electrographic seizures in uitro (D. D. Mott and D. V. Lewis, unpublished observations, 1993). Suppression of epileptogenesis by GABA, receptors is consistent with the inhibitory effects of these receptors on various other experimental models of epilepsy, as discussed previously. However, numerous studies have shown that seizure induction by kindling both in uiuo (McNamara et al., 1988) and in vitro (Stasheff etal., 1989) is markedly or entirely suppressed by blockade of NMDA receptors. This raises the possibility that, similar to its effect on LTP, postsynaptic GABAB receptor-mediated inhibition may suppress epileptogenesis by inhibiting the NMDA receptor-mediated current. Additional studies are needed to test this possibility. Furthermore, it is important to keep in mind that a contribution by presynaptic GABAB receptors to regulation of either

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LTP induction or epileptogenesis has not been ruled out (see Sections IV,B and IV,C). I n conclusion, because of its relatively small conductance increase, but large hyperpolarization, GABA, receptor-mediated postsynaptic inhibition appears to serve a modulatory role in regulating excitatory activity. T h e efficacy of the inhibition depends on several factors. For example, it can vary depending on the brain area involved. Thus, under normal conditions, GABA, receptor-mediated inhibition is unable or only weakly able to inhibit evoked activity in dentate gyrus granule cells. In contrast, in areas CAS and CA 1, where IPSP, is associated with a larger hyperpolarization and conductance increase, the late GABAB receptormediated postsynaptic inhibition is stronger. Similarly, a strong postsynaptic GABA, receptor-mediated inhibition of cell firing has been observed in other brain areas, including neocortex (Howe et al., 1987; Connors et al., 1988),thalamus (Soltesz et al., 1989), and locus coeruleus (Olpe et al., 1988; Shefner and Osmanovic, 1991).T h e amount of inhibition also depends on the voltage-dependence of the excitatory current. Thus, NMDA EPSPs are strongly inhibited by IPSP,, as are some epileptiform burst discharges. In contrast, non-NMDA EPSPs, which constitute the excitatory response under normal conditions, are relatively insensitive to GABAB receptor-mediated inhibition. Alternately, the amount of inhibition depends on the intensity of the afferent input. For example, within a given brain area the inhibitory input must be of sufficient amplitude to activate postsynaptic GABAB receptors, but the excitatory input must not be strong enough to overcome the postsynaptic GABAB receptor-mediated inhibitory effect. The ability of IPSPB to inhibit more effectively cell firing produced by weak than by strong depolarizations led Connors et al. (1988) to suggest that this form of inhibition may enhance the neuronal signal-to-noise ratio by suppressing both background noise and responses to weak excitatory stimuli, but leaving responses to strong stimuli intact. Finally, postsynaptic GABAB receptors appear to be able to compensate for the reduction of inhibition during periods of hyperexcitability o r reduced GABAA receptor function. Thus, GABAB receptors may play an important role in modulating the induction and/or expression of epilepsy. Although postsynaptic GABA, receptors can inhibit the discharge of excitatory cells, GABAB receptors located on interneurons can suppress GABA release, thereby reducing both GABAA and GABAB receptormediated inhibition of excitatory cells on subsequent responses. As will be discussed below (see Section IV,D), the net outcome of these opposing GABAB receptor actions depends on the relative strength of GABA, receptor-mediated effects on excitatory and inhibitory neurons.

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6. Reactivation of Voltage-Inactivated Conductances. In addition to producing hyperpolarizing inhibition, IPSPB is well suited to reactivation of voltage-inactivated conductances. In particular, IPSPB is thought to play a role in regulating low-threshold calcium spikes. Low-threshold calcium spikes were initially observed in neurons of the inferior olive (Llinas and Yarom, 1981a,b) and have since been reported in a variety of areas of the brain, including thalamus (Jahnsen and Llinas, 1984a,b), neocortex (Friedman and Gutnick, 1987), lateral habenula (Wilcox et al., 1988), medial septum (Alvarez de Toledo and Lopez-Barneo, 1988), and pontine reticular formation (Greene et al., 1986). In addition, low-threshold calcium spikes have also been recorded in both pyramidal and nonpyramidal neurons in the hippocampus (Schwartzkroin and Slawsky, 1977; Lacaille and Schwartzkroin, 1988a,b; Fraser and MacVicar, 1991). These calcium spikes are associated with the ability of the neurons to discharge in rhythmic bursts and are generated by a LVA T-type calcium current [T current (Steriade and Llinas, 1988; Coulter et al., 1989b; Crunelli et al., 1989) (see section III,C,4)]. This T current is activated by membrane depolarization, following a period of membrane hyperpolarization to remove current inactivation. These properties suggest that by hyperpolarizing neurons, IPSPB would effectively deinactivate T currents. I n thalamocortical neurons IPSPB appears very suitable for this role, since its long duration potentially enables it to produce 50-100% removal of inactivation (Crunelli et al., 1989; Crunelli and Leresche, 1991). Indeed, when a thalamocortical neuron is held depolarized to the activation threshold for the calcium current, IPSPB is followed by a low-threshold calcium potential that often causes the cell to burst discharge. Thus, the hyperpolarization produced by IPSPB deinactivates the T current, enabling it to be activated as the neuron repolarizes. The burst of action potentials produced by the calcium potential is sufficient to stimulate GABAergic neurons, producing another IPSPB and perpetuating the cycle (for review see Crunelli and Leresche, 1991). Although this effect of IPSPB has so far been described only in thalamocortical cells, the coexistence of low-threshold calcium spikes and postsynaptic GABAB receptor-mediated responses in other brain areas suggests that IPSPB may serve a similar role in other neurons. Thus, postsynaptic GABAB receptors appear to prime low-threshold calcium currents and may contribute to the generation of rhythmic bursting activity. However, it should be noted that, although postsynaptic GABAB receptors can contribute to rhythmic burst firing activity, some types of burst firing can occur even during blockade of GABABreceptors (Leresche et al., 1990; Crunelli and Leresche, 1991).

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As discussed previously, GABA, receptor activation by baclofen can depress voltage-dependent calcium currents through a G proteinmediated mechanism (see section III,C,4). GABAB receptor activation seems to have a similar suppressive effect on T currents. Fraser and MacVicar (1991) have reported that interneurons from stratum lacunosum-moleculare of hippocampal area CA 1 express T currents that are entirely suppressed by baclofen. This raises the possibility that during IPSP, the T current may be simultaneously inhibited by both membrane hyperpolarization and a direct G protein-mediated mechanism. However, the membrane hyperpolarization would also remove inactivation of the current, potentially enabling it to be activated when the membrane repolarized. This dual inhibitory effect of IPSPB on T currents may contribute to neural synchronization. By modulating T currents, GABA, receptors may play a role in regulating absence epilepsy. Absence epilepsy in humans is associated with bilaterally synchronous rhythmic spike-wave discharges in the cerebral cortex and thalamus (Mirsky et al., 1986). Absence seizures are thought to be dependent on T currents. T currents have been implicated in the mechanism underlying absence seizures because these currents are associated with rhythmic oscillatory activity (DeschCnes et al., 1984; Steriade and Llinas, 1988), and because they are reduced by drugs used to treat absence epilepsy (Coulter et al., 1989a). Recent studies using both genetic and pharmacological models of absences epilepsy support a role for GABAB receptors in the regulating absence seizures (Hosford et al., 1992; Liu et al., 1992; Marescaux et al., 1992; Snead, 1992). For example, Hosford et al. (1992) reported that blockade of GABAB receptors with CGP 35348 or 2-hydroxysaclofen suppressed spontaneous absence seizures in lethargic mice, a mouse strain that is genetically predisposed to absence epilepsy. Furthermore, they found that baclofen enhanced the seizures in these mice. Similarly, Liu et al. ( 1992) reported that in rats with genetic absence epilepsy, bilateral injection of CGP 35348 into thalamic relay nuclei and reticular nuclei reduced the occurrence of seizures, whereas bilateral injection of baclofen at these sites exacerbated their occurrence. In contrast, they found that injection of these compounds into the midline thalamus had no effect. They concluded that GABAB receptors in thalamic relay nuclei and reticular nuclei play a role in regulating the occurrence of the seizures. Taken together, these findings indicate that GABAB receptors can contribute to the mechanism underlying absence epilepsy. However, although this contribution is most likely mediated through postsynaptic GABAB receptors, a role for presynaptic GABA, receptors cannot be excluded. Interestingly, one study

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has reported that absence seizures in at least one animal model are not suppressed by GABAB receptor antagonists. Qiao and Noebels (1992) found that in the stargazer mutant mouse, a genetically mutant mouse strain expressing absence epilepsy, absence seizures were insensitive to CGP 35348. This finding suggests that multiple mechanisms may underlie the expression of absence epilepsy and that GABAB receptors may not always play a role. In addition to exhibiting rhythmic spike-wave discharges during absence epilepsy, thalamocortical neurons also undergo rhythmic oscillations during slow-wave sleep (Fourment et ul., 1985; Steriade and Llinas, 1988). Based on the similarity between the hyperpolarizations recorded from these neurons during slow-wave sleep and that produced by IPSP,, it was suggested that GABAB receptors may play a role in modulating sleep (Crunelli et al., 1992). An initial study by Crunelli et al. (1992) reported that direct application of 2-hydroxysaclofen into the ventropostero-lateral nucleus of cats markedly increased light slow-wave sleep and decreased deep slow-wavesleep. The drug had no effect on paradoxical sleep o r total sleep time. On the other hand, Olpe et al. (1991) found that in adult rats, CGP 35348 increased the amount of slow-wave sleep during the second and third hours after drug administration. Although these results support a modulatory role for GABAB receptors in slowwave sleep, further studies, using more potent GABAB receptor antagonists, are needed to confirm these findings and to determine whether the effects are mediated by pre- or postsynaptic GABAB receptors.

B. PRESYNAPTIC GABAB RECEPTORSON EXCITATORY TERMINALS 1. Presynuptic Depression of Excitatory Responses A presynaptic effect of baclofen was first suggested in studies examining the antispastic effects of the drug. It was found that systemic administration of even a small amount of baclofen depressed mono- and polysynaptic reflex transmission in the spinal cord (Bein, 1972; Pierau and Zimmermann, 1973). At these small doses, baclofen produced presynaptic inhibition of primary afferent fiber terminals with no effects on inhibitory responses and only minimal effects on direct excitability of the neurons (Pierau and Zimmermann, 1973; Davidoff and Sears, 1974). This baclofen-induced presynaptic inhibition appeared to be mediated by a depression of transmitter release from excitatory terminals (Pierau and Zimmermann, 1973; Fox et al., 1978). Subsequent studies in spinal cord preparations have confirmed these observations (Curtis et al., 1974; Davies, 1981; Lev-Tov et al., 1988; Olpe et al., 1990; Wang and Dun,

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1990). In addition, a similar presynaptic depressant effect of baclofen on excitatory responses has been reported in a variety of different brain regions, including olfactory cortex (Cain and Simmonds, 1982; Collins et al., 1982), neocortex (Howe et al., 1987), neostriatum (Calabresi et a/., 1990, 1991, 1992; Seabrook et al., 1990; Nisenbaum et al., 1992), amygdala (Asprodini et al., 1992),dorsal raphe (Colmers and Williams, 1988), nucleus accumbens (Uchimura and North, 199l), thalamus (Crunelli et al., 1992; Soltesz and Crunelli, 1992), trigeminal nucleus (Fromm et al., 1992), and hippocampus (Lanthorn and Cotman, 1981; Ault and Nadler, 1982, 1983a; Olpe et al., 1982; Misgeld et al., 1984; Blaxter and Carlen, 1985; Inoue et al., 1985b). Electrophysiological studies support the existence of presynaptic GABA, receptors capable of regulating transmitter release because the suppressant effect of the drug could be localized to specific afferent pathways. This localization of GABA, receptors to specific afferent pathways is particularly evident in the hippocampal formation. For example, in area CA 1 baclofen inhibits the Schaffer collateral-evoked excitatory response with an IC,, of 3-4 p M (Lanthorn and Cotman, 1981; Olpe et al., 1982; Inoue et al., 1985b). In contrast, in this same area baclofen does not depress the response produced by stimulation of the perforant path [temporo-ammonic pathway (Ault and Nadler, 1982; Colbert and Levy, 1992)l. Similarly, in the dentate gyrus the response to medial and lateral perforant path stimulation is differently affected by baclofen (Lanthorn and Cotman, 1981; Ault and Nadler, 1983a) (Fig. 6). Baclofen depresses the excitatory response to medial perforant path stimulation with an IC,, of 3.5-5 p M . In contrast, the excitatory response to lateral perforant path stimulation is not reduced by even high concentrations of the drug (Kahle and Cotman, 1993; Lanthorn and Cotman, 1981). Finally, in area CA3 baclofen blocks the response to stimulation of recurrent excitatory collaterals of CA3 pyramidal neurons [associational fibers (Ault and Nadler, 1982, 1983a)l. However, reports of the effects of baclofen on the CA3 pyramidal cell response to mossy fiber stimulation have been less consistent with some studies finding no effect of baclofen (Ault and Nadler, 1982, 1983a; Inoue et al., 198513) and others reporting that the drug blocked mossy fiber-evoked excitatory responses (Lanthorn and Cotman, 1981; Misgeld et al., 1984; Hirata et al., 1992). Perhaps one reason for the discrepancy in the reports of the effect of baclofen on mossy fiber responses lies in the difficulty of stimulating mossy fibers in isolation. T h e effect of baclofen on paired pulse plasticity also indicated that GABA, receptors could act presynaptically. During paired stimulation of the lateral perforant path, the rising slope of the dendritic population

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ACSF

Baclofen

Baclofen + CGP 35340

0

.

9

25 rns

FIG. 6. GABA, receptor stimulation differently affects medial and lateral perforant path-evoked EPSPs in the dentate gyrus. Dendritic EPSPs evoked by paired stimuli delivered 50 ms apart to the medial (MPP, top) or lateral (LPP, bottom) perforant path are shown. The responses to medial perforant path stimulation show paired pulse depression, whereas the responses to lateral perforant path stimulation exhibit paired pulse facilitation. Baclofen (10 p M ) markedly depresses both the first and the second EPSPs evoked by medial perforant path stimulation and converts the paired pulse depression to a facilitation. In contrast, in the lateral perforant path baclofen increases the EPSP duration, most likely by suppressing the underlying inhibition (see Section IV,C). In addition, baclofen enhances paired pulse facilitation. CGP 35348 (1 mM) antagonizes the effects of baclofen in both pathways. All records are taken from the same hippocampal slice.

EPSP of the second response in the dentate gyrus is facilitated when stimuli are delivered 50 ms apart (Lgmo, 197 1; McNaughton and Barnes, 1977; McNaughton, 1980; Kahle and Cotman, 1993). We found that baclofen had little effect on the amplitude or slope of the response to the first lateral perforant path stimulus, but enhanced the response to the second stimulus (D. D. Mott, D. V. Lewis, W. A. Wilson, and H. S. Swartzwelder, unpublished observations, 1992, but see Kahle and Cotman, 1993) (Fig. 6). Since paired pulse facilitation appears to occur presynaptically, these results suggest that baclofen acted on GABA, receptors on the excitatory terminal. Alternatively, in the medial perforant path the response to the second of paired stimuli delivered 80-800 ms apart is normally depressed. This paired pulse depression is observed both in viuo and in uitro and is thought to be mediated by a reduction

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of transmitter release (McNaughton, 1980). As previously mentioned, baclofen depressed the medial perforant path response to the first stimulus, but during paired stimulation, the drug converted the paired pulse depression to a paired pulse facilitation (Harris and Cotman, 1985) (Fig. 6). T h e ability of baclofen to modulate paired pulse facilitation in these pathways suggests not only that GABA, receptors can act presynaptically, but also that they can play a role in regulating synaptic excitation during repetitive neuronal activation. Several lines of evidence indicate that these presynaptic inhibitory effects of baclofen are mediated through GABAB receptors. For example, the inhibitory effect of baclofen on transmitter release was found to be receptor-mediated, as it was stereoselective, with the ( - )isomer being about 100-200 times more potent than the (+)isomer (Ault and Nadler, 1982, 1983a; Olpe et al., 1982; Haas et al., 1985; Inoue et al., 198513). Furthermore, other agonists at GABAB receptors were also found to depress excitatory responses. For example, in striatal neurons both 3APPA and 3-APMA blocked EPSPs (Seabrook et al., 1990; Lovinger et al., 1992). In addition, exogenous GABA depressed excitatory transmission and the inhibitory effects of both GABA and baclofen were insensitive to GABA, antagonists, suggesting that they were produced by GABA, receptors (Ault and Nadler, 1982, 1983a; Yoon and Rothman, 1991;Jarolimek and Misgeld, 1992). Ultimately, mediation of these inhibitory effects by GABABreceptors was confirmed when it was found that they were blocked by 2-hydroxysaclofen or CGP 35348 (Curtis et al., 1988; Harrison, 1990; Randall et al., 1990; Seabrooket al., 1990; Thompson and Gahwiler, 1992a). These antagonists were reported to produce a concentration-dependen t antagonism of baclofen effects in a variety of different pathways. For example, we have found that depression of medial perforant path responses by 10 p M baclofen is blocked by CGP 35348 with an IC,, of 21 p M (D. D. Mott, D. V. Lewis, W. A. Wilson, and H. S. Swartzwelder, unpublished observations, 1993) (Fig. 6). In contrast to the potency of 2-hydroxysaclofen and CGP 35348 in blocking presynaptic GABA, receptors, phaclofen has not been consistently reported to antagonize the presynaptic inhibitory effect of baclofen. At concentrations that block postsynaptic GABAB receptor-mediated responses, phaclofen has only a weak effect (Seabrook et al., 1990) or no effect (Dutar and Nicoll, 1988a; Wang and Dun, 1990) on the depression of excitatory responses by baclofen. The differing effects of phaclofen at presynaptic and postsynaptic sites may reflect a pharmacological difference between GABAB receptors at these two sites, a difference in receptor density, or a difference in the efficacy with which receptors at these two sites are coupled to effector mechanisms.

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Release studies also support the existence of presynaptic GABA, receptors capable of depressing transmitter release. A depressant effect of baclofen on excitatory neurotransmitter release was first demonstrated in guinea pig cerebral cortex. Potashner (1978,1979)reported that baclofen inhibited the electrical stimulation-evoked release of endogenous 14Clabeled glutamate and aspartate. Similarly, Bowery and Hudson (1979) found that GABA depressed the evoked release of [3H]noradrenaline from sympathetic nerve terminals of the rat atria in a bicuculline- and picrotoxin-insensitive fashion. This depressant effect of GABA was mimicked by baclofen, suggesting that excitatory transmitter release was inhibited through activation of presynaptic GABA, receptors (Bowery et al., 1980). Subsequent studies in both spinal cord (Johnston et al., 1980; Raiteri, 1992) and brain (Kato et al., 1982) further confirmed the ability of baclofen to depress excitatory transmitter release. In the brain baclofen was found to depress transmitter release in a variety of different regions, including olfactory cortex (Collins et al., 1982), cerebral cortex (Bonanno and Raiteri, 1992; Raiteri, 1992; Raiteri et al., 1992; Pende et al., 1993), cerebellum (Zhu and Chuang, 1987), and the hippocampal formation (Olpe et al., 1982; Spencer et al., 1986, but see Burke and Nadler, 1988). In addition to suppressing release of the excitatory neurotransmitters glutamate and aspartate, GABA and baclofen have also been reported to depress calcium-dependent release of a variety of other neurotransmitters, including norepinephrine (Bowery and Hudson, 1979; Bowery et al., 1980; Suzdak and Gianutsos, 1985), serotonin (Bowery et al., 1980; Schlicker et al., 1984; Gray and Green, 1987), dopamine (Bowery et al., 1980),acetylcholine (Brown and Higgins, 1979), somatostatin (Bonanno et al., 1991), and GABA (see Section IV,C). Depression of transmitter release by baclofen or GABA was insensitive to GABAAblockade with bicuculline or picrotoxin, was not altered by the benzodiazepine diazepam, and was not mimicked by the GABAAreceptor agonists muscimol or THIP. The effect of baclofen was stereoselective, with the ( - )isomer being substantially more potent. Recently, several studies have used synaptosomes to examine directly whether GABA, receptor antagonists block the suppression of transmitter release caused by baclofen (Bonanno and Raiteri, 1992; Raiteri, 1992; Raiteri et al., 1992; Pende et al., 1993). CGP 35348 was found to be an effective antagonist of the ( - )badofen-induced inhibition of potassiumevoked release of both somatostatin and glutamate from cortical synaptosomes (Bonanno and Raiteri, 1992; Raiteri et al., 1992). However, similar to results from electrophysiological studies, phaclofen only weakly antagonized the suppression of glutamate release by baclofen. In contrast,

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phaclofen potently blocked the inhibitory effect of baclofen on somatostatin release (Bonanno and Raiteri, 1992; Raiteri, 1992). T h e differing potencies of phaclofen and CGP 35348 suggest that release of different neurotransmitters may be modulated by different subtypes of GABA, receptor. 2. Terminal Mechanism Neurotransmitter is released from the presynaptic terminal in response to an influx of calcium through voltage-sensitive calcium channels that are activated when the action potential invades the terminal. Calcium that enters the terminal increases the probability that transmittercontaining vesicles will fuse with the membrane, resulting in the release of transmitter into the synaptic cleft (Augustine et al., 1987). Transmitter release is susceptible to modulation by agents that affect any stage of this process. In addition to GABA, a number of neurotransmitters have been demonstrated to inhibit excitatory transmitter release, including adenosine (Fredholm and Dunwiddie, 1988), norepinephrine (Doze et al., 1991), acetylcholine (Hounsgaard, 1978), glutamate (Nicoll et al., 19901, and neuropeptide Y (Colmers et al., 1987, 1988). Despite the similarity of their action, these transmitters may each inhibit excitatory transmitter release by interacting with a different part of the release mechanism. Based on their known postsynaptic effects, presynaptic GABAB receptors could potentially suppress excitatory transmitter release by inhibiting voltage-sensitive calcium conductances or by increasing the potassium conductance in excitatory terminals. An increase in the potassium conductance of the presynaptic terminal would suppress transmitter release by shunting the invasion of the presynaptic action potential and thereby decreasing calcium entry into the terminal. However, studies in both the spinal cord (Allerton et al., 1989) and the hippocampus (Lambert et al., 1991b; Thompson and Gahwiler, 1992a)have demonstrated that barium, which blocks the baclofen-induced postsynaptic hyperpolarization, does not affect the suppression of excitatory transmitter release produced by baclofen. These results suggest that either GABAB receptors d o not decrease transmitter release by increasing potassium conductance or that different potassium conductances are modulated by presynaptic and postsynaptic GABA, receptors. In support of this latter possibility, baclofen has been reported to modulate more than one type of potassium current. Saint et al. (1990)have demonstrated that in cultured hippocampal neurons, in addition to stimulating a barium-sensitive potassium current, baclofen also shifts the voltage dependence of a somatic Atype transient potassium current, enhancing activation of this current at

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resting membrane potential (see Section III,C,5). If baclofen were to have a similar effect presynaptically, the enhanced A-current could speed action potential repolarization and consequently decrease transmitter release. Further studies are needed to test this possibility. Alternatively, presynaptic GABAB receptors could depress transmitter release by inhibiting voltage-dependent calcium currents. As previously discussed, GABABreceptors depress somatic calcium currents in sensory as well as central neurons. However, because most studies have examined the effects of baclofen on somatic calcium currents, evidence supporting the ability of presynaptic GABAB receptors on excitatory terminals to depress calcium currents is indirect. Several lines of evidence suggest that it is unlikely that presynaptic GABAB receptors entirely inhibit transmitter release by depressing voltage-dependent calcium currents. For example, in both sensory and central neurons higher concentrations of baclofen are required to depress somatic calcium currents than are necessary to inhibit transmitter release, suggesting that these effects may be pharmacologically separable (Zhu and Chuang, 1987). Alternatively, Potier and Dutar ( 1993) found that w-conotoxin depressed the evoked excitatory response, but did not prevent this response from being further depressed by baclofen. Thus, if baclofen does inhibit transmitter release by depressing calcium currents, at least part of this inhibitory action is independent of o-conotoxin-sensitive, N-type calcium channels. Calabresi et al. (1992) reported that in neostriatal neurons baclofen suppressed the excitatory response without reducing the duration of the calcium action potential, suggesting that the action of baclofen to block transmitter release was not through an inhibition of calcium influx. Taken together, these observations suggest that depression of evoked excitatory transmitter release by baclofen cannot be entirely attributed to a depression of voltage-dependent calcium currents. Thus, it appears that baclofen can, in part, suppress transmitter release through a mechanism that is independent of both calcium and postassium conductances. This raises the possibility that presynaptic GABAB receptors can suppress excitatory transmitter release through a direct interaction with the release mechanism. A direct interaction of GABAB receptors with the release mechanism was initially suggested in studies examining the effect of baclofen on the frequency of tetrodotoxin-insensitive miniature spontaneous EPSCs (Scanziani et al., 1992). These EPSCs were not affected by cadmium, indicating that they did not result from calcium entry through voltagesensitive calcium channels (Jarolimek and Misgeld, 1992; Scanziani et al., 1992). However, they were reduced in frequency by baclofen, suggesting that presynaptic GABA, receptors can decrease spontaneous

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transmitter release through a mechanism that is independent of calcium influx. Thus, it is possible that presynaptic GABA, receptors on excitatory terminals can interact directly with the release mechanism to decrease transmitter release. Alternatively, since the frequency of spontaneous transmitter release is dependent on the concentration of calcium in the presynaptic terminal, it is possible that GABAB receptors may suppress the release of intracellular calcium and so reduce the tonic level of calcium in the terminal (Scanziani et al., 1992). Whether either of these possible mechanisms can entirely account for the effect of baclofen on evoked excitatory transmitter release is, at present, unknown. The manner in which GABAB receptors are coupled to the transmitter release mechanism also remains unclear. Coupling of postsynaptic GABAB receptors to pertussis toxin-sensitive G proteins suggests that presynaptic GABA, receptors are coupled in a similar fashion. Indeed, in acutely isolated lamprey spinal cord baclofen was found to depress transmitter release through a pertussis toxin-sensitive mechanism (Alford and Grillner, 1991). Similarly, in cultured DRG neurons pertussis toxin blocked the inhibitory effect of baclofen on transmitter release (Holz et al., 1989). However, studies examining the effects of pertussis toxin on central neurons have not been as conclusive. For example, following intracerebroventricular (icv) injection of pertussis toxin, the inhibitory effect of baclofen on transmitter release persists, although the postsynaptic effect of baclofen is blocked (Colmers and Williams, 1988; Dutar and Nicoll, 1988a). T h e inability of pertussis toxin to block the effect of baclofen may reflect the poor diffusion of pertussis toxin in brain tissue (Van der Ploeg et al., 1991). Thus, it is possible that the toxin did not have adequate access to presynaptic terminals or that it was not taken up into these presynaptic sites. T o overcome this potential problem, pertussis toxin was injected directly into the hippocampus. Hippocampal slices prepared from animals pretreated in this fashion were either less sensitive (Potier and Dutar. 1993) or insensitive (Stratton et al., 1989) to the depressant effect of baclofen on excitatory transmitter release. These results suggest that a pertussis toxin-sensitive G protein is at least partly involved in the depressant effect of baclofen. Pertussis toxin was also reported to be effective at Mocking the effect of baclofen on transmitter release from cultured neurons in which diffusional barriers d o not hinder the access of the toxin to presynaptic terminals (Scholz and Miller, 1991; Travagli et al., 1991; Yoon and Rothman, 1991). In contrast, Thompson and Gahwiler (1992a) found that in hippocampal slice cultures pretreatment with pertussis toxin did not alter the inhibitory effect of baclofen on transmitter release. Thus, the coupling of presynaptic GABA, recep-

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tors to pertussis toxin-sensitive G proteins in both cultured neurons and acute slice preparations remains controversial. The conflicting results reported in different studies may reflect methodological differences or that the presynaptic effect of GABAB receptors is mediated by both pertussis toxin-sensitive and -insensitive G proteins. In addition to G proteins, other second messenger systems have been implicated in the mechanism underlying the suppression of transmitter release by presynaptic GABAB receptors. For example, presynaptic GABAB receptors have been reported to interact with the release mechanism through modulation of adenylate cyclase. Travagli et al. (1991) found that in cultured cerebellar granule cells 8-(4-chlorophenylthio)cyclic AMP (CPT-CAMP),a cAMP analogue and activator of protein kinase A, had no effect by itself on transmitter release, but blocked the inhibition of glutamate release produced by baclofen. The authors suggest that baclofen may inhibit transmitter release by decreasing cAMP synthesis and that CPT-CAMP could mimic an increase in cAMP and thus oppose the effect of baclofen. Similarly, protein kinase C activation may play a role in modulating the inhibitory effect of baclofen. Thompson and Gahwiler (1992a) found that phorbol esters inhibit both the presynaptic and the postsynaptic actions of baclofen. Protein kinase C is thought to produce this effect by phosphorylating, and thereby inactivating, certain critical proteins through which GABAB receptors are linked to the transmitter release process (Andrade et al., 1986). In contrast, Dunwiddie et al. (1990) reported that inhibitors of arachidonic acid and its metabolites did not interfere with the suppressant effect of baclofen on transmitter release, suggesting that these second messengers did not contribute mechanism underlying the inhibitory effect of presynaptic GABAB receptors. Thus, both protein kinase C and adenylate cyclase may contribute to the mechanism underlying the presynaptic effect of GABAB receptors on excitatory terminals. Since other presynaptic receptors can also regulate the activity of both protein kinase C and adenylate cyclase, these second messenger systems may represent a method for interaction between transmitter systems at a presynaptic level.

3 . Activity-Dependent Depression of Excitatoq Responses Although it is well established that pharmacological activation of presynaptic GABAB receptors will suppress excitatory transmitter release, a physiological role for these receptors has only recently been demonstrated. Calabresi et al. ( 1 990, 199 1) found that in neostriatum glutamatemediated synaptic responses were depressed during application of the GABA uptake blocker nipecotic acid. This action of nipecotic acid was

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mediated presynaptically and was not sensitive to bicuculline. Furthermore, it was mimicked by baclofen. T h e authors concluded that blockade of CABA uptake prolonged the action of synaptically released GABA, allowing it to stimulate presynaptic GABAB receptors on glutamatecontaining terminals. Furthermore, they found that during repetitive stimulation at 0.3- 10 Hz, the amplitude of the glutamate-mediated synaptic response was attenuated; however, the response to exogenously applied glutamate was unaffected. They suggested that synaptically released GABA was capable of feeding back onto presynaptic terminals to depress glutamate release. However, neither phaclofen nor S-hydroxysaclofen was able to antagonize this presynaptic effect, suggesting the existence of spare receptors or a different subtype of GABAs receptor on excitatory terminals. Activation of presynapt ic GABAB receptors by synaptically released GABA was demonstrated by Isaacson et al. (1993). They found that in area CA1 of the hippocanipus a brief train of conditioning stimuli to one pathway would produce heterosynaptic depression in the pyramidal cells of the subsequent excitatory response to stimulation of a second pathway. This heterosynaptic depression was blocked by CGP 35348, indicating that it was produced by stimulation of presynaptic GABA, receptors. Furthermore, they found that the GABA uptake inhibitor SKF 89976A markedly enhanced the presynaptic depression, enabling it to be detected at longer interstimulus intervals. They suggested that the “spill-over” of GABA onto excitatory terminals, which normally produces the depression, is dramatically enhanced by blockade of GABA uptake. Thus, synaptically released GABA is capable of diffusing to GABAB heteroreceptors on excitatory terminals to produce a presynaptic depression of transmitter release. This action of GABA appears to be restricted by GABA uptake. The strength of presynaptic regulation of excitation by GABA may therefore depend on the local strength of GABA uptake. 4. Functional Significance of Presynaptic Inhibition Few reports have addressed the physiological role of presynaptic GABAB receptors on excitatory terminals. However, the functional effects of GABAB receptor agonists suggest that endogenously released GABA acting at these same presynaptic sites will have similar effects. In particular, feedback of GABA onto these receptors may serve to limit the excess of glutamate released during repetitive stimulation. Thus, during repetitive stimulation these presynaptic GABA, receptors may regulate the induction of various forms of synaptic plasticity, such as long-term potentiation and epilepsy. Perhaps they may also modulate

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epileptiform bursting by inhibiting the release of excitatory transmitter during the burst. Asprondini et al. (1992) have reported that the sensitivity of presynaptic GABAB receptors is reduced following kindling. They suggest that this effect may contribute to the enhancement of excitatory transmission in kindled animals. In addition to these inhibitory effects, presynaptic GABAB receptors may also suppress inhibition by reducing excitation of inhibitory neurons (Collins et al., 1982), 2s has been previously demonstrated for a-adrenergic agonists (Doze et ai., 1991). Thus, it is possible that these presynaptic receptors may play important functional roles in the regulation of synaptic transmission in both normal and epileptic states. Confirmation of this possibility awaits further investigation. C. PRESYNAPTIC GABAB RECEPTORS ON INHIBITORY TERMINALS 1. Presynaptic Depression of Inhibitory Responses In addition to suppressing the release of excitatory neurotransmitter, GABAB receptors have also been reported to regulate the release of GABA. The existence of GABAB receptors capable of depressing GABAergic inhibition was initially observed as a baclofen-induced depression of IPSP,s in the hippocampal formation (Klee et al., 1981; Misgeld et al., 1982, 1984; Ault and Nadler, 1983b) and olfactory cortex (Cain and Simmonds, 1982; Collins et al., 1982; Scholfield, 1983). Subsequent studies demonstrated the ability of exogenously applied GABA as well as baclofen to reduce IPSP/C, and IPSP/CB (for review see Lambert and Harrison, 1993; Thompson et al., 1993) (Fig. 7). We found that in the dentate gyrus, baclofen typically depressed IPSPB more completely than it depressed IPSP,, most likely because baclofen also occluded IPSP, through postsynaptic GABAB receptor activation (Mott et al., 1993b). IPSP depression by baclofen was voltage-independent and was not associated with a change in the reversal potential of either component of the IPSP. Depression of inhibition by baclofen was stereospecific with activity in the (-)isomer (Howe et al., 1987; Mott et al., 1989) and was blocked by phaclofen, 2-hydroxysaclofen and CGP 35348, confirming the involvement of GABAB receptors (Dutar and Nicoll, 1988b; Olpe et al., 1988; Lambert et al., 1989; Davies et al., 1990, 1991).Baclofen depressed inhibition in a concentration-dependent fashion [IC,, 0.6 pM (Mott et al., 1989)] and at lower concentrations than it depressed excitation [IC,, 3.5-5 p M (Lanthorn and Cotman, 198l)] (Fig. 7). Although this difference in potency could be caused by multiple subtypes of GABAB receptors, it could also be explained by differences in the receptor-effector coupling of inhibitory and excitatory terminals.

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A

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8 A 100 ma

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-5

-t

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. 0

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FIG. 7. Depression of polysynaptic inhibition by baclofen. (A) Both IPSPA and IPSPB evoked in a dentate gyrus granule cell by mossy fiber stimulation are depressed by baclofen. The response in ACSF (artificial cerebrospinal fluid, dotted line) and baclofen (solid line) are shown superimposed on the right. The membrane potential was held at -66 mV with injected current in both control and drug. [From Mott et al. (1993b) with permission.] (B) Concentration-response curve for the suppression of recurrent inhibition by baclofen. Recurrent inhibition of the perforant path-evoked population spike in the dentate gyrus was evoked by stimulating the mossy fibers 5 ms before the perforant path. By suppressing the underlying IPSP,, as shown in (A), baclofen blocked recurrent inhibition with an estimated ECSoof 0.6 p M (n = 3). [The graph is reprinted from Mott et al. (1989) with permission.]

Initial studies examined the effect of baclofen on polysynaptic GABAA receptor-mediated inhibition, making it difficult to determine the site at which baclofen exerted its disinhibitory effect. Indeed, in a polysynaptic circuit there iire a number of potential sites at which baclofen could act to depress inhibition. For example, baclofen could reduce IPSPASby (a) suppressing excitation of inhibitory neurons, (6) inhibiting interneuronal discharge in response to synaptic excitation, (c) shunting the IPSP, by increasing the potassium conductance of the postsynaptic neuron, (d ) directly suppressing postsynaptic GABAA receptor function, and (e) decreasing GABA release from inhibitory terminals. We will discuss each of these possibilities below.

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First, baclofen could depress glutamate release from excitatory terminals and thereby reduce excitation of interneurons. This possibility was initially proposed by Collins et al., (1982) to explain the depression of inhibition produced by baclofen in olfactory cortex. However, subsequent studies in the hippocampus found that direct stimulation of interneurons during pharmacological blockade of excitatory transmission produced an isolated monosynaptic IPSP, that was depressed by baclofen (Davies et al., 1990). The ability of baclofen to suppress this monosynaptic IPSP indicated that, although a reduction of excitatory drive may contribute to the baclofen-induced depression of polysynaptic IPSPs, a reduction of excitation is not necessary for disinhibition to occur. Similarly, Misgeld et al. (1989) reported that baclofen depressed the mossy fiber-evoked polysynaptic IPSP, in dentate granule cells without reducing the mossy fiber-evoked EPSP recorded in the hilar inhibitory neurons thought to produce this IPSP,. Thus, in these neurons baclofen appeared to suppress inhibition by an action other than depression of interneuronal excitation. Second, as discussed previously (see Section IV,A, 1,h), baclofen could hyperpolarize and increase the potassium conductance of interneurons, thereby preventing their discharge in response to synaptic excitation. Indeed, baclofen has been shown to hyperpolarize interneurons in the dentate hilus (Misgeld et al., 1989) as well as area CA1 (Madison and Nicoll, 1988). However, depression of monosynaptic IPSPAS by baclofen is not antagonized by barium or THA, agents that block the GABA, receptor-linked potassium conductance (Lambert et al., 1991b; Lambert and Wilson, 1993, but see Thompson and Gahwiler, 1992a). Although it is possible that baclofen acts through a barium- and THA-insensitive potassium conductance, these results suggest that baclofen suppresses monosynaptic IPSPAsthrough some mechanism other than postsynaptic inhibition of interneurons. Thus, postsynaptic inhibition of inhibitory neurons does not appear to be required for suppression of monosynaptic IPSP,s by baclofen. However, these results do not rule out the possibility that this form of postsynaptic interneuronal inhibition by baclofen contributes to the depression of polysynaptic IPSPAS (Misgeld et al., 1989; Thompson and Gahwiler, 1992a; Mott et al., 1993b). Third, baclofen could increase the potassium conductance of the postsynaptic excitatory neurons and thereby shunt the IPSP. Although the GABA, receptor-mediated conductance increase is smaller than that produced by GABAA receptors, it can nevertheless be significant in certain neuronal types. In particular, in CA3 neurons baclofen can produce up to a 100% increase in the resting membrane conductance (Lambert and Wilson, 1993). However, blockade of this postsynaptic conductance

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increase by preloading CA1 or CA3 pyramidal cells with Cs' andlor QX-314 does not prevent suppression of the monosynaptic IPSP in these cells by baclofen (Lambert and Wilson, 1993). Similarly, blocking the postsynaptic conductance increase with THA or barium also has no effect on the suppression of monosynaptic IPSPs by baclofen (Lambert and Wilson, 1993). Alternatively, in dentate granule cells the GABAB receptor-mediated conductance increase is much smaller, making it unlikely that a conductance increase accounts for the substantial baclofeninduced depression of the lPSP in these cells. We have found that in these cells a low concentration of baclofen can produce a marked depression of the IPSP, while producing virtually no increase in postsynaptic conductance (Mott et al., 1993b). In addition, irreversible activation of the conductance by loading granule cells with GTP-y-S (Mott et al., 1993b) or blockade of the conductance with Cs' andlor QX-314 (Otis and Mody, 1992) does not prevent baclofen from suppressing the IPSP. Thus, an increase in postsynaptic conductance is not necessary for IPSP depression by baclofen. Fourth, GABA, receptor activation by baclofen could reduce IPSPs by interacting postsynaptically through second messengers with GABAA receptors. As discussed previously (see Section IV), activation of GABAB receptors in cerebellum has been reported to suppress GABAA receptor function through inhibitory G proteins. According to this mechanism, stimulation of G proteins activates phospholipase C, which causes phosphorylation of GABA, receptors with a subsequent reduction in GABAA receptor function (Sigel and Baur, 1988; Browning et al., 1990; Hahner etal., 1991). However, we have found that in hippocampal dentate granule cells maximal activation of postsynaptic G proteins by intracellular injection of GTP-y-S does not depress IPSPs, nor does it prevent their reduction by baclofen (Mott et al., 1993b). Similarly, injection of GTPy-S into hippocampal pyramidal neurons does not reduce IPSPA (Thalmann, 1988). Furthermore, baclofen suppresses IPSCA, but does not block the response to exogenously applied GABA, indicating that the function of postsynaptic GABAA receptors is not affected (Howe et al., 1987; Thompson and Gahwiler, 1989a; Harrison, 1990). Finally, it is possible that baclofen depresses inhibition by activating presynaptic GABAB receptors on inhibitory terminals and thereby reducing GABA release. Suppression of GABA release by baclofen would be similar to the effect of the drug on the release of transmitters from other synapses. Several lines of evidence support this possibility. For example, using inhibitory-excitatory cell pairs of cultured hippocampal neurons, Harrison (1990) found that stimulation of the inhibitory neuron produced an IPSC, in the excitatory cell. This IPSCA was markedly de-

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pressed by baclofen. In this culture system baclofen produced no postsynaptic effect on either the inhibitory or the excitatory cell, indicating that it did not shunt the IPSCA. Baclofen did not affect the response to exogenous GABA, indicating that the drug did not interact postsynaptically with GABA, receptors. Because the IPSCAwas produced by direct stimulation of the inhibitory neuron, baclofen could not have acted by depressing excitation of the inhibitory neuron or by preventing the inhibitory neuron from firing. Thus, the author concluded that baclofen must have suppressed the IPSCAby activating GABAB receptors on the axon or terminals of the inhibitory neuron, most likely by suppressing GABA release. Similar observations were also made in cell pairs of postnatal and embryonic cultured hippocampal neurons (Scholz and Miller, 1991; Yoon and Rothman, 1991). T h e reduction of IPSP/CA by baclofen in these cultured preparations was blocked by 2-hydroxysaclofen (Scholz and Miller, 1991; Yoon and Rothman, 1991), but not by phaclofen (Harrison, 1990). This differential effect of GABAB receptor antagonists may reflect a difference between the culture preparations or the existence of multiple GABAB receptor subtypes. The existence of GABAB receptors on inhibitory terminals was further supported by studies examining the effect of baclofen on GABA release. The existence of a GABA, autoreceptor capable of suppressing the release of GABA was first proposed by Anderson and Mitchell (1985), following their observation that the drug stereospecifically and in a concentration-dependent manner inhibited the potassium-evoked release of [ 3H]GABA from synaptosomes of median eminence. Subsequent studies examining the potassium-evoked release of both [3H]GABA and/ or endogenous GABA from cortical synaptosomes confirmed this observation (Pittaluga et al., 1987; Bonanno et al., 1989a,b; Giralt et al., 1990; Bonanno and Raiteri, 1992; Raiteri et al., 1992). However, whereas baclofen was found to depress the release of GABA, pharmacological differences were reported between this effect and the effect of baclofen on glutamate release. Using cortical synaptosomes, Bonanno and Raiteri (1992) found that, unlike its effect on glutamate release which was not sensitive to phaclofen but was blocked by CGP 35348, the effect of baclofen on GABA release was antagonized by phaclofen, but not by CGP 35348. Thus, they concluded that phaclofen-sensitive, CGP 35348insensitive GABAB receptors control the release of GABA, whereas phaclofen-insensitive, CGP 35348-sensitive GABAB receptors regulate glutamate release (see Section IV,B,l). Taken together, these results indicate that baclofen can suppress inhibitory transmission by stimulating GABAB receptors on inhibitory terminals to suppress GABA release. However, they do not rule out a

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contribution to the baclofen-induced disinhibition by GABAB receptors at other sites in the polysynaptic circuit. In fact, we have recently reported that in the dentate gyrus baclofen depressed polysynaptic IPSPs to a greater extent than it reduced monosynaptic IPSPs (Mott et al., 1993b). Baclofen potentially depresses polysynaptic IPSPs by acting on GABAB receptors unavailable in the monosynaptic inhibitory circuit. Thus, during polysynaptic transmission, baclofen most likely acted not only at inhibitory terminals, but also on GABA, receptors at other sites in the circuit to suppress inhibition. A similar observation was reported in area CA3 (Thompson and Gahwiler, 1992a). Further evidence supporting the contribution of GABA, receptors at multiple sites to the baclofen-induced depression of polysynaptic inhibition is provided by studies examining spontaneous inhibitory events. Spontaneous IPSP/Cs are composed of both action potential-dependent (TTX-sensitive) and action potential-independent (TTX-insensitive) events. Baclofen depressed both the amplitude and the frequency of action potential-dependent spontaneous events and this depression was antagonized by CGP 35348 (Lambert and Wilson, 1993). Although the decrease in IPSP/CA amplitude may be caused by GABA, receptors on inhibitory terminals, it is likely that the decrease in the frequency of spontaneous IPSP/C,s is caused by hyperpolarization of inhibitory neurons and the resulting decrease in their spontaneous firing rate. Thus, both GABA, receptors on inhibitory terminals and postsynaptic GABA, receptors on inhibitory neurons may contribute to the suppression of these action potential-dependent IPSP/CAs.However, it is also possible, although less likely, that this decreased frequency could result from other causes, such as a reduction in the amplitude of some spontaneous IPSP/ CAsbelow the limit of detection or an increase in the failure rate of GABA release at inhibitory terminals. Recordings from inhibitory neurons will be required to evaluate these possibilities.

2. Terminal Mechanism Presynaptic GABA, receptors appear to block inhibition through a mechanism different from the one through which they block excitation. Suppression of excitation by baclofen was apparent even when calciumindependent, TTX-insensitive, spontaneous, miniature EPSCs were examined, suggesting that baclofen depressed glutamate release by acting directly on the release mechanism (see Section IV,B,2). In contrast, baclofen did not reduce the frequency of calcium-independent, TTXinsensitive, spontaneous, miniature IPSCs, unless high concentrations of the drug were used (Otis and Mody, 1992, 1993; Pende et al., 1993). However, baclofen powerfidly suppressed the amplitude and frequency

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of TTX-sensitive, spontaneous IPSCs as well as evoked IPSCs, both of which were calcium-dependent (Otis and Mody, 1992; Pende et al., 1993). Thus, as opposed to its suppression of EPSP/Cs, baclofen appeared to block only those IPSP/Cs that were dependent on calcium influx into the terminal. The inability of baclofen to suppress calcium-independent IPSP/Cs suggests that the drug does not block GABA release by acting directly on the release mechanism, but rather it acts by interfering with calcium entry into the inhibitory terminal. Presynaptic GABA, receptors could depress calcium entry into inhibitory terminals either by directly suppressing calcium currents or by indirectly reducing calcium influx by increasing the potassium conductance of the terminal. An increase in potassium conductance would oppose terminal depolarization, causing a reduction in calcium influx and consequently a suppression of GABA release. However, the ability of baclofen to block the high potassium-evoked release of GABA from synaptosomes argues against this possibility (Raiteri et al., 1990). Alternatively, if an increase in the potassium conductance of the inhibitory terminal underlies the action of baclofen then blockade of this potassium conductance should prevent the disinhibitory effect of the drug. Barium has been shown to block the postsynaptic potassium increase produced by baclofen (see Section III,C,5). However, reports of the effects of barium on the disinhibitory effect of baclofen conflict. For example, barium had no effect on the baclofen-induced depression of monosynaptic IPSPs in area CA1 (Lambert et al., 1991b), but blocked the disinhibitory effect of baclofen in the dentate gyrus (Misgeld et al., 1989) and area CA3 (Thompson and Gahwiler, 1992a). The reason for this discrepancy is unclear. Fur ermore, by blocking potassium conductances in the terminal, barium would be expected to enhance action potential duration, thereby increasing calcium entry into the terminal and consequently facilitating GABA release. Thus, it is possible that barium occluded the effect of baclofen on GABA release by enhancing calcium influx into the terminal, rather than by directly blocking the potassium conductance. Another compound that blocks the postsynaptic potassium increase produced by baclofen is THA (see Section III,C,5). However, unlike barium, THA has no effect by itself on the monosynaptic IPSC,. Lambert and Wilson (1993) have used this drug to examine the contribution of a presynaptic potassium conductance to the disinhibitory action of baclofen in hippocampal CA3 pyramidal cells. They found that THA blocked the postsynaptic effect of baclofen, but did not prevent baclofen from depressing IPSC,. These results suggest that baclofen does not suppress inhibition by activating a presynaptic potassium conductance similar to that activated postsynaptically. Saint et al. (1990) have also suggested that baclofen could depress inhibition by altering the voltage

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dependence of activation/inactivation of a transient A-type potassium current in inhibitory terminals. Potentially, this could enable more current to be activated near resting membrane potential, speeding repolarization of the action potential and reducing calcium influx into the terminal (see Section III,C,5). However, in cultured neurons THA has been shown to suppress this current (Rogawski, 1987). Thus, the inability of THA to block the disinhibitory effect of baclofen suggests that baclofen did not depress inhibition by altering the voltage dependence of this potassium current (Lambert and Wilson, 1993). Although the above results suggest that an increase in potassium conductance is not responsible for the depression of inhibition, little evidence exists either supporting or refuting a role for a direct depression of calcium currents in the mechanism underlying baclofen-induced depression of inhibition. Baclofen has been shown to depress calcium currents in both pyramidal and nonpyramidal hippocampal neurons (Scholz and Miller, 1991; Swartz and Bean, 1992), but its effect on calcium entry into inhibitory terminals has not been reported. Further studies are needed to examine this possibility. The coupling mechanism responsible for the suppression of IPSP/Cs by baclofen appears to involve pertussis toxin-sensitive G proteins. Depression of IPSPs by baclofen in both cultured neurons (Scholz and Miller, 1991; Yoon and Rothman, 1991; Thompson and Gahwiler, 1992a, but see Harrison, 1990) and hippocampal slice preparations (Potier and Dutar, 1993) is blocked by pretreatment with pertussis toxin. Similarly, perfusion of the inhibitory neuron with GTP-yS, which irreversibly activates inhibitory G proteins, causes the baclofen-induced depression of IPSPAto become permanent (Lambert and Harrison, 1993). Scholz and Miller (1991) have reported that infusion of GTP-y-S also causes the baclofen-induced depression of calcium currents to become irreversible. Thus, baclofen depresses both voltage-sensitive calcium currents and IPSP/Cs through inhibitory G proteins. This G protein-coupled mechanism appears to be modulated by protein kinase C. Phorbol esters, which stimulate protein kinase C, block the disinhibitory effect of baclofen, possibly by phosphorylating inhibitory G proteins (Thompson and Gahwiler, 1992a). This is similar to the effect of phorbol esters on the baclofen-induced depression of excitatory transmitter release and may represent a mechanism by which other transmitter systems, which also stimulate protein kinase C, regulate the function of presynaptic GABAB receptors. 3. Activity-Dependent Depression of Inhibitory Responses It is well established that both IPSP, and IPSPB become depressed during repetitive stimulation of inhibitory pathways (for review see Al-

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ger, 1991; Prince et al., 1992). Indeed, inhibition is so labile that this activity-dependent depression of lPSP/Cs can be observed during lowfrequency stimulation or even after as little as a single conditioning stimulus (Fig. 8). In general, both pre- and postsynaptic mechanisms could contribute to this depression of inhibition. The decrease in IPSP conductance observed following repetitive stimulation was originally attributed to a reduction in the postsynaptic effectiveness of GABA. This decrease was thought to be caused principally by desensitization of GABAA receptors following prolonged exposure of the receptors to GABA released during the stimulus train (Ben-Ari etal., 1979). However, subsequent studies found little evidence to support this possibility (McCarren and Alger, 1985; Deisz and Prince, 1989; Thompson and Gahwiler, 1989b). Since desensitization of GABAA receptors typically requires prolonged GABA exposure (Wong and Watkins, 1982; Huguenard and Alger, 1986), the lack of a measurable contribution of desensitization most likely reflects the stimulus paradigm used to produce the disinhibition. In addition, GABAB receptor-mediated responses d o not desensitize. Thus, desensitization could not explain the observed activity-dependent depression of IPSP,. Another postsynaptic mechanism that was proposed to regulate inhibition was a shift in ionic gradients. It was observed that repetitive stimulation altered the reversal potential of IPSPA as well as in the reversal potential of the response to a focally applied GABA pulse (Wong and Watkins, 1982; McCarren and Alger, 1985; Thompson and Gahwiler, 1989b). This shift in reversal potential was most likely caused by an accumulation of intracellular chloride during the stimulus train. Since the magnitude of the inhibitory current is regulated by the chloride driving force, a change in the reversal potential for chloride, which alters its driving force, can potentially play an important role in modulating GABAA receptor-mediated inhibition. However, changes in the reversal potential for chloride were caused primarily by application of large amounts of exogenous GABA (Thompson and Gahwiler, 1989a) or highfrequency (10-1 00 Hz) stimulation (McCarren and Alger, 1985). In contrast, activity-dependent depression of both IPSPA and IPSPB has been reported following as little as a single conditioning stimulus (McCarren and Alger, 1985; Deisz and Prince, 1989; Davies et al., 1990). Indeed, the reduction in the IPSP produced by a single conditioning stimulus o r short burst of stimuli is caused almost exclusively by a decrease in inhibitory conductance, and is not associated with a change in IPSPA reversal potential (McCarren and Alger, 1985; Davies et al., 1990; Mott et al., 1993b). Furthermore, several studies have reported that during lowfrequency stimulation, the conductance of both 1PsPA and IPSPB, but

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A

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FIG. 8. Activity-dependent depression of inhibition in the dentate gyrus. (A) In a granule cell that is hyperpolarized to the reversal potential of IPSPBwith injected current ( - 106 mV), a mossy fiber stimulus evokes a depolarizing IPSP,. Addition of picrotoxin (50 p M ) completely blocks this response. (B) In another granule cell the IPSP, becomes depressed during mossy fiber stimulation at 5 Hz. The dotted line indicates the amplitude of the first IPSP, of the train for comparison. (C) In this same cell when mossy fiber stimuli are delivered 200 ms apart, the IPSP, evoked by the second stimulus is markedly depressed. The first and second IPSP,s of the pair are superimposed for comparison (right). In both (B) and (C) the membrane potential is hyperpolarized to - 97 mV, the reversal potential of IPSP, in this cell. [From Mott et al. (1993b) with permission.]

not the response to exogenous GABA, is depressed (McCarren and Alger, 1985; Deisz and Prince, 1989). These results suggested that the decrease in IPSP conductance produced by low-frequency stimulation is produced, not by a change in the postsynaptic effectiveness of GABA, but rather by a presynaptic depression of GABA release. Baclofen has been shown to depress inhibition, in part, by reducing the release of GABA from inhibitory terminals (Thompson et al., 1993) (see Section IV,C, 1). The possibility that GABA, acting on GABA, receptors, contributed to fading of inhibition during repetitive stimulation was initially suggested in studies comparing the activity-dependent de-

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pression of inhibition with that produced by baclofen (Deisz and Prince, 1989; Thompson and Gahwiler, 1989a). Thompson and Gahwiler (1989a,b) found that application of either GABA or baclofen to CA3 pyramidal neurons in slice culture mimicked the depression of IPSC, produced by repetitive stimulation and this disinhibition could not be accounted for by either GABA, receptor desensitization or a shift in the reversal potential of the inhibitory response. Similarly, Deisz and Prince ( 1989) found 'that activity-dependent depression of IPSPs in neocortical neurons was similar to the depression of IPSPs produced by application of exogenous GABA or baclofen. Furthermore, the GABA uptake inhibitor nipecotic acid enhanced depression of the IPSP, suggesting that accumulation of GABA might inhibit GABA release. Other studies have demonstrated that baclofen occludes the depression of monosynaptic IPSP/C,s produced by a single conditioning stimulus (Davies et al., 1990; Nathan and Lambert, 1991; Mott et al., 1993b). The inability of the conditioning stimulus to further reduce the IPSP/C, in the presence of baclofen suggested that both the conditioning stimulus and baclofen depress IPSP, through GABAB receptors. Taken together, these studies indicate that the reduction of the IPSP conductance during repetitive stimulation is due, in part, to feedback of synaptically released GABA onto GABA, receptor on inhibitory neurons. Direct evidence that a decrease of the IPSP/C could result from feedback of GABA onto a GABAB receptors was provided by studies demonstrating that GABAB receptor antagonists block activitydependent depression of inhibition. These drugs were effective in antagonizing the depression of inhibition produced by both low- and highfrequency stimulation. For example, we have reported that in the dentate gyrus recurrent inhibition is transiently reduced following a highfrequency stimulus train (Mott et a/., 1990a). This reduction of inhibition is partially antagonized by phaclofen, suggesting that GABA released during the train can feed back onto GABAB autoreceptors and suppress further GABA release. The lack of a complete block by phaclofen can most likely be attributed to the contribution of other mechanisms, such as a shift of ionic gradients, to the depression of inhibition. Similarly, during low-frequency stimulation or following a single conditioning stimulus, GABAB receptor antagonists have been reported to antagonize the depression of IPSP/C, (Davies et al., 1990, 1991; Mott et al., 1990a, 1993b; Calabresi et al., 1991; Lambert et al., 1991a; Deisz and Zieglgansberger, 1992; Fukuda et al., 1993; Lambert and Wilson, 1993) (Fig. 9). Antagonism of activity-dependent depression of inhibition by these drugs is concentration-dependent and reversible (Fig. 10). Interestingly, in some brain areas even a high concentration of CGP 35348 did not entirely

CENTRAL GABAB RECEPTORS

177

block activity-dependent depression of lPSP/CA (Deisz and Zieglgansberger, 1992; Lambert and Wilson, 1993). Similarly, in cultured neurons GABA, receptor antagonists are ineffective at blocking the depression of inhibition produced by a conditioning stimulus (Yoon and Rothman, 1991). T h e lack of an effect of these antagonists suggests that GABAB receptors responsible for depression of inhibition at these sites may express a different pharmacology or that GABA, receptors contribute only minimally to depression of inhibition at these synapses. This raises the possibility that GABA release from some interneurons is insensitive to GABA, receptor activation, a situation analogous to the lack of effect of GABABreceptor activation on glutamate release from some excitatory pathways (N. A. Lambert, personal communication, 1993). Nevertheless, GABA, receptor-dependent fading of inhibition has been observed in a number of brain regions, including the hippocampus (Davies et al., 1990, 1991; Lambert et al., 1991a; Lambert and Harrison, 1993),dentate gyrus (Mott and Lewis, 1991; Mott et al., 1993b), neocortex (Deisz and Zieglgansberger, 1992), and neostriatum (Calabresi et al., 1991), suggesting that this type of disinhibition may be of widespread importance. Unfortunately, it has not been possible to test whether GABAB receptor antagonists also block activity-dependent depression of iPsP/CB, since the antagonists block the postsynaptic GABAB receptor-mediated response in the same concentration range as they block activity-dependent depression of IPSC,. In all areas where it has been examined GABA, receptor-mediated fading of inhibition has a similar time course (Davies et al., 1990; Nathan and Lambert, 1991; Otis and Mody, 1992; Mott et al., 1993b). In the dentate gyrus we found that GABA, receptor-mediated fading of IPSCA was greatest when stimuli were delivered 100-400 ms apart, with a maximal effect at an interstimulus interval of 150-200 ms (Mott et al., 1993b) (Fig. 11). At shorter intervals, the second IPSCA was not reduced and temporally summed with the first. T h e depression of inhibition gradually returned to control amplitude by about 2-4 s. T h e time course of fading of IPSCA and IPSCB was similar, suggesting that depression of both IPsP/cA and IPsP/cB was mediated by a reduction of GABA release (Otis and Mody, 1992; Mott et al., 1993b; Otis et al., 1993). Although the peak of this time course corresponded to the peak of IPSC, in the postsynaptic cell, the decay rate of the disinhibition was slower than that of IPSCB. This difference in the time course between the action of presynaptic and postsynaptic GABAB receptors further suggests that the potassium conductance that mediates the postsynaptic 1PsCB cannot entirely account for fading of inhibition (Otis et al., 1993) (see Section IV,C,P).

A

1 mM CGP 35348

Control 0

0

0

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

Wash

0

0

0

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

k U k , 3

CGP 35340

0

0",.

0

0

O

0

3

100 ms

oo 00 p ~ ~ o o ~ 000

0:

0

0 0.

0 0

oooo

0

O. 00

.ooo 0 .

0 .

3.0.3, I""I""I""I""I""I""I""I""1

0

5

10

15

20

25

30

35

40

50

45

Time (min.)

B Control

1 PM CGP 55845

Superimposed

4

d

100 ms

FIG. 9. Paired pulse depression of IPSP, is mediated by GABAB receptors. (A) (Top) IPSPAsevoked by paired stimulation of mossy fibers 200 ms apart and recorded from a dentate gyrus granule cell are shown. The IPSPs are depolarizing because during the paired stimuli, the cell was hyperpolarized to -95 mV, the reversal potential of IPSP, in this cell. In control, the amplitude of the second IPSP, of the pair (IPSP,2, solid circle) is depressed relative to the amplitude of the first IPSPA(IPSP,,, open circle). This depression is blocked by application of 1 mM CGP 35348. After washing for 30 rnin, paired pulse depression of the IPSP, is again evident. Each waveform represents the average of three paired responses. Dotted lines indicate the amplitude of IPSP,,. (Bottom) In the same granule cell the amplitude of IPSPA, (open circles) and IPSPA2 (solid circles) of each pair throughout the experiment are shown. CGP 35348 (1 mM) was bath applied at the same time indicated by the solid bar. Note that during this time the amplitude of IPSP,, did not change, but the depression of IPSP,, was blocked. (B) Paired pulse depression of the mossy fiber-evoked IPSP, in another granule cell is shown. Depolarizing IPSq4s were observed because the cell was hyperpolarized to - 100 mV, the reversal potential of IPSPB, during the paired stimuli. Application of the potent GABAB receptor antagonist, CGP 178

A 400

pM

Control

2-OH Saclofen

Control

2-OH Saclofen

10

Wash

pM Wash

Jvma 100 m a

B Q,

a 3 2

g

130

-

120

-

I 2-OH Wash

110

-

100

-

d

a" 2

-E

Z

so: 80

:

a C

cu

Saclofen

80 70 50

i L

10 pM

40

pM

400 pM

FIG. 10. Blockade of paired pulse depression of IPSP, by 2-hydroxysaclofen. (A) IPSP,s produced by paired stimulation of mossy fibers 200 ms apart are shown. Application of 400 p M 2-hydroxysaclofen reversibly blocks the depression of IPSP, on the second stimulus (top). In a second cell 10 pM 2-hydroxysaclofen. which has no effect on the first IPSP, of the pair, reduces paired pulse depression of the second IPSP, (bottom). Responses from both cells are recorded with the membrane potential hyperpolarized to the reversal potential of IPSP, in that cell, causing the IPSP,s to be depolarizing. (B) Antagonism of paired pulse depression of IPSP, by 2-hydroxysaclofen is concentrationdependent. Bars and error bars represent mean SE(n = 4). Dotted line represents the amplitude of the first IPSP, of the pair. Asterisks indicate a significant antagonism of paired pulse depression by the drug (*P < .05). [From Mott et al. (1993b) with permission.]

*

55845 (1 p M ) blocked the depression of the second IPSPA of the pair. This effect was not reversible. The control and CGP 55845-treated responses are superimposed on the right for comparison. Note that in the presence of CGP 55845 the decay time of the first IPSP, increased (arrow), possibly because of the blockade of the underlying GABA, receptormediated conductance. Each waveform represents the average of four paired responses. 179

180

DAVID D. MOTT AND DARRELL V. LEWIS

A

b. 50 ms

a. 10 ms

c . 200 ms

d. 4 s

a..b..c 100 ma d. 2 s

B V

-a

t/)

a

1

90

/

*-.---

I

*

800

1000

4

a

4

zol 10

0

200

400

800

Interstimulus Interval (ms)

FIG. 11. Time course of paired pulse depression of inhibition. (A) Sample waveforms showing IPSCs evoked by paired stimulation of the mossy fibers at different interstimulus intervals and recorded in a granule cell. Membrane potential is held at -80 mV. Note the depression of both IPSC, and IPSCB at an interval of 200 ms. (B) Time course of paired pulse depression of IPSCA (open circles) and IPSCB (solid circles). Symbols and error bars represent mean ? SE (n = 6). Dotted line indicated the average amplitude of the initial IPSC. Asterisks indicate a significant depression of the second IPSC (*P < .05, **P < .01). Plus (+) indicates a significantly greater depression of IPSCB then IPSC, (P < .05). The inset shows the IPSCs evoked by mossy fiber stimuli delivered 10 ms apart. The labeled lines indicate the method used to measure the amplitude of the first and second IPSC when they overlapped. [From Mott et al. (1993b) with permission.]

CENTRAL GABAs RECEPTORS

181

Release studies also support a frequency-dependent depression of GABA release by GABAB autoreceptors. Waldmeier et al. (1988) found that electrical stimulation-evoked release of GABA from cortical slices was reduced when stimuli were delivered at frequencies between 0.25 and 8 Hz, with a maximal effect at frequencies between 1 and 4 Hz. This depression of GABA release was antagonized by phaclofen, CGP 35348, and CGP 55845 (Baumann et al., 1990; Waldmeier et al., 1993). However, these antagonists tended to increase release similarly at all frequencies, such that in the presence of the antagonist more GABA was released, but this release was still frequency-dependent. The authors concluded that synaptically released GABA can depress subsequent GABA release in a frequency-dependent manner by stimulating GABAB autoreceptors. However, they suggested that these autoreceptors may exist in both agonist- and antagonist-preferring states. Finally, they found that at frequencies higher than about 8-16 Hz GABA release increased to control levels, suggesting that GABAB autoreceptors were inoperative at these frequencies. The interval at which fading of IPSP/Cs was maximal corresponds to a stimulation frequency of about 5 Hz. Repetitive stimulation at lower frequencies depressed the IPSP to a lesser extent, whereas higher frequency stimulation caused IPSP summation during the train. In the dentate gyrus we found that, when a 5-Hz stimulus train was delivered to an inhibitory pathway, the IPSPA remained depressed through the train (Mott et al., 199313) (see Fig. 8). During the train, the decline in IPSP, was greatest on the second response with subsequent stimuli causing smaller reductions, until, after about three stimuli, the depression reached a steady state. Thus, nearly the maximal extent of fading was apparent after as little as a single stimulus. This depression of inhibition was antagonized by 2-hydroxysaclofen, confirming the involvement of GABAB receptors (Mott and Lewis, 1992) (see Fig. 15A). The extent of GABAB receptor-mediated fading of IPSCB (61%) was greater than that for IPSCA (37%).This difference in the magnitude of the depression of IPSCA and IPSCB could be caused by several factors, including separate GABAA and GABAs synapses (Lacaille et al., 1989; Segal, 1990a; Otis and Mody, 1992; Sugita et al., 1992), different affinities of GABAA and GABAB receptors for GABA (Bowery et al., 1983), or differences in the coupling mechanism of GABA, and GABA, receptors to effector channels. A number of studies have suggested that, similar to the postsynaptic GABAB receptor-mediated response (see Section IV,A, 1,f ), activitydependent depression of inhibition requires synchronous activation of interneurons. Indeed, when the stimulus intensity is reduced, the

182

DAVID D. MOTT A N D DARRELL V. LEWIS

percentage depression of the IPSC, declines, until little to no depression is evident at IPSCA threshold (McCarren and Alger, 1985; Davies et al., 1990). This stimulus dependence suggests that GABA released from a single interneuron is unable to stimulate GABAB autoreceptors. Otis and Mody (1992) found that CGP 35348 had no effect on the frequency o r amplitude of spontaneous IPSCAS in the dentate gyrus, suggesting that the spontaneous release of GABA was not sufficient to stimulate GABAB autoreceptors. Furthermore, Miles and Wong ( 1984), recording from inhibitory-excitatory cell pairs in area CA3, found that an action potential in the interneuron evoked an IPSP in the excitatory neuron. These IPSPs were of similar amplitude even when the action potentials in the interneuron occurred at intervals at which evoked IPSPs were depressed. Thus, GABA released following activation of a single interneuron did not appear capable of producing IPSP depression, suggesting that fading of inhibition required a larger amount of GABA to be released. Roepstorff and Lambert ( 1992) have recently provided evidence supporting this possibility by demonstrating that activity-dependent depression of inhibition in area CA 1 is enhanced by the GABA uptake blocker tiagabine. Similarly, lowering the extracellular calcium concentration, which decreased the release of GABA, reduced the amount of paired pulse depression of the IPSP. The requirement of activity-dependent depression of inhibition for a larger amount of GABA may indicate that the GABAB receptors responsible for the disinhibition have a lower affinity for GABA or that they are located remotely from the synaptic cleft. As discussed below, this stimulus dependence indicates that GABAB receptor-mediated disinhibition will most powerfully influence excitatory transmission when interneurons fire synchronously. Although the above studies demonstrate that GABAB receptor activation by released GABA can depress monosynaptic IPSP/Cs, they do not directly demonstrate the site of action of these GABAB receptors. In general, activity-dependent depression of monosynaptic IPSP/Cs could occur through activation of GABA, receptors located either on inhibitory neurons or postsynaptically on the recorded neuron. However, a substantial contribution by GABAB receptors on the recorded neuron seems doubtful, since blockade of the postsynaptic GABAB receptor-mediated conductance increase by injection of cesium and/or QX-314 into the recorded cell does not block fading of inhibition (Nathan and Lambert, 1991; Otis and Mody, 1992; Lambert and Wilson, 1993). In addition, paired pulse depression of IPSP/C, is not accompanied by a change in the decay kinetics of the second IPSP/CA, as would be expected if a conductance increase contributed to the depression (Nathan and Lambert, 1991; Mott et al., 1993b). Finally, we have found that fading of

183

CENTRAL GABA, RECEPTORS

inhibition is not occluded by irreversible activation of G proteins by injection of GTP-y-S into the recorded cell (Mott et al., 1993b) (Fig. 12). Thus, postsynaptic GABAB receptors do not contribute to fading of monosynaptic IPSP/Cs by increasing membrane shunting conductance or by a G protein-mediated alteration in the function of GABA, receptors. Activity-dependent depression of monosynaptic IPSP/Cs most likely occurs by stimulation of GABAB receptors on inhibitory interneurons. GABAB receptors on interneurons are located both on the soma, where they potentially inhibit cell discharge (Madison and Nicoll, 1988; Lacaille, 1991), and on or near axon terminals, where they suppress GABA release (Harrison, 1990). It is probable for several reasons that activitydependent depression of monosynaptic IPSP/Cs is mediated by GABAB autoreceptors on inhibitory terminals. First, because of the relatively few number of GABAergic neurons (Seress and Pokorny, 1981; Woodson et al., 1989), but the extensive axonal arbors of these cells (Li et al., 1992;

Control

GTP-7-S

-60

J

LOO m.

FIG. 12. Irreversible activation of postsynaptic C proteins does not prevent paired pulse depression of IPSP,. Polysyriaptic IPSPs, evoked by paired stimulation of the mossy fibers 200 ms apart, are shown. Responses are recorded from a granule cell using a recording electrode containing GTP-y-S (10 mM in 4 M potassium acetate). Immediately after impalement, the IPSP, evoked by the second stimulus was depressed. This could be seen clearly when the cell was hyperpolarized to - 94 mV, the reversal potential of IPSP, (left, bottom). However, when the cell was depolarized to - 60 mV, paired pulse depression of IPSP, was obscured by the IPSPB produced by the first stimulus (left, top). As GTPy-S diffused into the cell, the membrane potential hyperpolarized and the membrane conductance increased, occluding IPSP,. However, this did not prevent paired pulse depression of IPSP,, which could now be seen clearly at both depolarized (right, top) and hyperpolarized (right, bottom) potentials. Note the absence of an IPSPB in the responses recorded after diffusion of GTP-y-S. All responses are recorded from the same cell.

184

DAVID D. MOTT AND DARRELL V. LEWIS

Han et al., 1993), it is highly likely that more GABAergic axons than cell bodies are stimulated when a monosynaptic IPSP/C is evoked. Since monosynaptic IPSP/Cs are most likely evoked by stimulation of GABAergic axons, it seems probable that activity-dependent depression of these lPSP/Cs results from a GABAB receptor-mediated suppression of GABA release from inhibitory terminals. In support of this possibility, Lambert et al. (1991b) found that in area CA1 focal application of baclofen near the recording site suppressed the monosynaptic IPSP,, whereas focal application of baclofen near the stimulation site did not. They suggested that the somatic inhibition produced by activation of GABAB receptors was not sufficient to prevent the discharge of inhibitory neurons when they were directly stimulated. Thus, it seems unlikely that paired pulse depression of inhibition could be produced by GABAB receptors on interneuron somata. Alternately, THA, which is known to block the postsynaptic inhibitory actions of GABAB receptors, does not prevent paired pulse depression of IPSP/CA(Lambert and Wilson, 1993). Assuming that postsynaptic GABAB receptors on inhibitory interneurons were also blocked, these results suggest that stimulation of postsynaptic GABA, receptors on inhibitory interneurons is not necessary for activitydependent depression of monosynaptic IPSP/Cs. Thus, similar to the suppression of GABA release produced by baclofen, it is likely that released GABA acts through GABAB autoreceptors on inhibitory terminals to suppress monosynaptic IPSP/Cs. Intracellular recordings from inhibitory interneurons during paired stimulation are required to confirm this conclusion. Although GABAB autoreceptors appear to be responsible for activitydependent depression of monosynaptic lPSP/Cs, it is not clear whether GABAB receptors at other sites contribute to depression of polysynaptic IPSP/Cs. We have recently found that both monosynaptic and polysynaptic IPSPs in the dentate gyrus are depressed to a similar extent during paired pulse stimulation (Mott et al., 1993b). Since GABAB receptor-mediated depression of monosynaptic IPSPs is most likely mediated by GABAB autoreceptors, we suggested that presynaptic GABAB receptors on inhibitory terminals are sufficient to account for paired pulse depression of polysynaptic IPSPs. In contrast, both we (Mott et al., 1993b) and others (Thompson and Gahwiler, 1992a) have reported that baclofen produces a markedly greater depression of polysynaptic IPSPs than of monosynaptic IPSPs, suggesting that baclofen acts at a site@) outside the monosynaptic circuit to suppress inhibition. Direct recordings from inhibitory neurons during repetitive stimulation are necessary to confirm these findings and to determine the conditions under which GABAB receptors at other sites in the polysynaptic inhibitory circuit contribute to activity-dependent depression of inhibition.

CENTRAL GABA, RECEPTORS

185

4. Functional Significance of Presynaptic Disinhibition GABA, receptor-mediated inhibition plays a critical role in regulating excitatory synaptic transmission. Even partial blockade of IPSP/C, with agents such as bicuculline or picrotoxin can markedly enhance the spread of neural activity and facilitate the induction of long-term potentiation (for review see Stelzer, 1992). Strong disinhibition can result in the appearance of abnormal neuronal discharges and the induction and spread of epileptic seizures (Dingledine and Gjerstad, 1980; Schwartzkroin and Prince, 1980) (see Section IV,A,2). Similar effects are observed following administration of agents thought to act presynaptically to reduce GABA release, such as baclofen (Mott et al., 1989; Burgard and Sarvey, 1991) and opiates (Lee et al., 1990; Xie et al., 1992). For example, we have found that in the dentate gyrus baclofen, by suppressing the underlying IPSP,, can enhance the population EPSP, producing an increase in EPSP width and the appearance of multiple population spikes (Mott et al., 1989) (Fig. 13). Similarly, EPSP enhancement by baclofen-induced disinhibition has been reported in the dentate gyrus (Burgard and Sarvey, 1991),area CAI (Inoue et al., 198513; Peet and McLennan, 1986),neocortex (Howe et al., 1987), and olfactory cortex (Scholfield, 1983). Enhancement of the EPSP by baclofen in reversible and concentration-dependent with an EC,o of 1.0 p M , similar to the EC,, of 0.6 p M for depression of inhibition by baclofen (Mott et al., 1989). Although CGP 35348 has no direct effect on the EPSP or the underlying IPSP, (Davies et al., 1991), this antagonist blocks both the baclofen-induced enhancement of the EPSP and depression of IPSP,, indicating that baclofen produced these effects by stimulating GABA, receptors. Enhancement of the EPSP, but not the depression of IPSP,4,is blocked by D-APV, demonstrating that this enhancement is produced primarily by an increase in the NMDA component of the excitatory response (Burgard and Sarvey, 1991; D. D. Mott, D. V. Lewis, W. A. Wilson, and H. S. Swartzwelder, unpublished observations, 1991). 'This suggests that disinhibition produced by baclofen facilitates neural depolarization, which relieves the magnesium block of the NMDA receptor-operated channel, enabling current flow through these channels to contribute to the excitatory response. By enhancing the NMDA component of the excitatory response, baclofen can facilitate the induction of synaptic plasticity in both the ; and Sarvey, 1991) and area dentate gyrus (Mott et al., 1 9 9 0 ~Burgard CA1 (Olpe and Karlsson, 1990; Ballyk and Goh, 1992). We have found that in the dentate gyrus baclofen increases the maximal potentiation of the population spike that can be produced (Fig. 14). Furthermore, this drug lowers the stimulation threshold necessary for induction of longterm potentiation of the population spike (Mott et al., 1990~).However,

A

Superimp.

Baclof en

Control

1 0 4 20

200 ms

ma

C

B 180

160

**

[

180 0

160

r

1

140

* * -L

0 4

g

c

100

80 60

40 2o 0

1 1

**

5 -

EPSP Width

2 o k

0

IPSP, Amp.

Bac

D-APV

+

Bac

CGP 35348

+

Bac

FIG.13. Baclofen enhances the EPSP by suppressing the underlying IPSP. (Aa) Perforant path-evoked responses recorded intracellularly from a granule cell at rest ( - 76 mV) are shown. I n control, note the appearance of a depolarizing IPSP,, as indicated by a deflection in the decay phase of the EPSP (see Fig. 3A), as well as a hyperpolarizing IPSPg. Baclofen (10 p M ) blocked both of these IPSPs, causing an increase in the width of the EPSP and the appearance of multiple action potentials. The EPSP in control and baclofen is superimposed on the right for comparison. (Ab) Blockade of excitatory transmission with 20 g M DNQX and 50 pM D-APV revealed the underlying monosynaptic IPSP, evoked by direct stimulation of inhibitory neurons. Because of the hyperpolarized resting membrane potential of this cell ( - 84 mV), the IPSPAis depolarizing while the IPSP, is hyperpolarizing. Baclofen (10 p M ) suppressed both IPSP, and IPSP,. Control and baclofen-treated IPSP,s are superimposed on the right for comparison. In both (Aa) and (Ab) the hyperpolarization produced by baclofen was counteracted by injection of a depolarizing current pulse during the stimulus. (B) Effect of baclofen (10 p M ) on EPSP width (n = 3) and IPSPA amplitude (n = 6). Bars and error bars represent mean SE. Dotted line 186

*

400

350

300

250

**

200

T

**

150

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

100

_........

__._.........____.,,...

50

0

ACSF

D-APV

MK-801

FIG. 14. Baclofen increases the maximal NMDA receptor-dependent potentiation that can be induced. Maximal potentiation of the population spike in the dentate gyrus was induced by delivering successive trains (50 pulses, 100 Hz) to the perforant path at 20min intervals. The test response recorded in the granule cell layer was measured 15 min after each train. Each train caused an increase in the test response amplitude until a maximal level (maximal potentiation) was reached. Following the train the test response was monitored for 2 h. The graph shows the effect of D-APV (100 p M ) and MK-801 (10 p M ) on maximal potentiation of the population spike in control ACSF (open bars) and in the presence of baclofen (10 p M , solid bars). Baclofen and D-APV were applied only during the stimulus trains. Because of its use-dependence, MK-801 was applied for at least 2 h prior to starting the experiment. Potentiation was assessed by comparing the amplitude of the population spike before and 2 h after stimulus trains. Bars and error bars represent mean SE (n = 5 slices in ACSF, 6 slices in baclofen, 3 slices in D-APV, 7 slices in D-APV and baclofen, 3 slices in MK-801, 3 slices in MK-801 and baclofen). Dotted line indicates the average amplitude of the population spike in control ASCF before the trains. Asterisks indicate a significant decrease in potentiation compared to baclofen alone (**P< .01).

*

indicates the average amplitude of the control response. Asterisks indicate a significant change from control (**P < .0 1). (C)Antagonism by D-APV (50 p M )or CGP 35348 (1 m M ) of the effect of baclofen on population EPSP (pEPSP) width. Perforant path stimulation was used to evoked pEPSPs in the molecular layer of the dentate gyrus. Bars and error bars represent mean 2 SE (n = 9). Dotted line indicates the average width of control pEPSPs. Asterisks indicate a significant increase in pEPSP width (**P < .01). 187

188

DAVID D. MOTT AND DARRELL V . LEWIS

although baclofen enables a previously subthreshold train to produce potentiation of the population spike, it does not enable this subthreshold train to produce a long-term enhancement of the dendritic population EPSP slope (Mott et al., 1993a). Thus, the primary effect of baclofen during a stimulus train is to enhance the potentiation of neural excitability. Baclofen enhances population spike potentiation through its disinhibitory action, which increases dendritic depolarization during the stimulus train. This depolarization enables greater current flow through NMDA receptor-operated channels, augmenting the development of potentiation. Indeed, application of D-APV during the stimulus train blocks both the enhanced depolarization during the train and the development of potentiation, indicating that these effects are dependent on NMDA receptor stimulation (Mott et al., 1990b). Interestingly, Burgard and Sarvey (199 1) have reported that in the dentate gyrus baclofen can potentiate the population spike in the absence of a stimulus train. They found that merely exposing the slice to baclofen was sufficient to produce an increase in population spike amplitude that persists following wash out of the drug. The ability of baclofen to enhance the NMDA receptor-mediated component of the excitatory response and facilitate population spike potentiation indicated that GABA, receptor-mediated disinhibition can have profound effects on excitatory transmission in the hippocampal formation. Similarly, w e have found that GABA, receptor-mediated activity-dependent disinhibition can also enhance excitatory responses, in particular the NMDA receptor-mediated component of the EPSP. This enhancement of excitation facilitates the induction of long-term potentiation and enhances the spread of neural act&ity. We will discuss these functions below. a. Enhancement of Excitatory Responses. Activity-dependent depression of IPSP/C,, produced when repetitive stimuli are delivered at 200-ms intervals, causes an enhancement of the corresponding excitatory response (Larson and Lynch, 1986; Mott and Lewis, 1992). Extracellularly, this enhancement of the excitatory response is reflected by an increase in EPSP duration and the appearance of multiple population spikes. Intracellular recordings show not only an increase in EPSP width and the appearance of multiple action potentials, but also a decrease in the IPSP, produced by the stimulus (Fig. 15). Similar to the depression of inhibition, enhancement of the excitatory response could be observed after as little as a single conditioning stimulus. This enhancement was evident when stimuli were delivered at intervals from about 50 to 2000 ms, with a maximal enhancement at an interval of 200 ms. This

CENTRAL GABA, KECEPTORS

189

time course is the same as that for depression of IPSP/C, (Mott and Lewis, 1991). In the dentate gyrus the enhancement of EPSP width and the occurrence of extra population spikes were almost completely blocked by D-APV, indicating that they were produced almost exclusively by NMDA receptor activation (Mott and Lewis, 1991) (Fig. 16). In area CA1 paired stimuli delivered 200 ms apart to the Schaffer collaterals produced a similar widening of the EPSP and appearance of multiple population spikes on the second response. Although D-APV significantly reduced the EPSP enhancement in area CA1, it did not block the effect as completely as in the dentate gyrus (Diamond et al., 1988; Larson and Lynch, 1988; Nathan et al., 1990b; Mott et al., 1991). This difference indicates that augmentation of the non-NMDA component of the EPSP constitutes a larger percentage of the enhancement of the EPSP in area CA1 than in the dentate gyrus (also see Nathan et al., 1990b). In contrast, in both area CAI and the dentate gyrus 2-hydroxysaclofen or CGP 35348 completely blocked the EPSP enhancement (Davies et al., 1991; Mott et al., 1991; Mott and Lewis, 1991) (Fig. 16). These antagonists had no effect on the response to ;I single stimulus or on the isolated NMDA receptor-mediated component of the EPSP, indicating that, during paired pulse stimulation, they blocked EPSP enhancement by antagonizing GABAB receptors. Thus, during paired or repetitive stimulation GABAB receptor-mediated disinhibition can enhance the EPSP. This effect has been reported both in vitro (Davies et al., 1991; Mott and Lewis, 1991) and in vivo (Brucato et al., 1992), suggesting that enhancement of excitation by activity-dependent disinhibition may play a role in behavior. Although activity-dependent disinhibition produced by GABAB receptors can produce an enhancement of the excitatory response, previous studies have well established the existence of another form of paired pulse facilitation (Lomo, 1971; Creager et al., 1980; McNaughton, 1980) (Fig. 17). This paired pulse facilitation is typically observed as an increase in EPSP slope and population spike amplitude on the second response. It is not usually associated with a marked increase in EPSP width, as has been reported during GABAB receptor-mediated enhancement of the EPSP. T h e time course of paired pulse facilitation indicates that it is maximal at shorter intervals (20-100 ms) than GABAB receptormediated enhancement of the EPSP (Lorno, 1971; Creager et al., 1980; McNaughton, 1980). In addition, paired pulse facilitation is not blocked by GABA, receptor antagonists (Mott and Lewis, 1991, 1992). Thus, these differences indicate that these two forms of facilitation are produced by separate mechanisms.

A ACSF

20 u M DNQX 60 U M D-APV

+ 400

+

uM

2-OH Saclofen 80

nV

100

mm

B

160

-

140

-

200 180

0

IPSCA Amplitude

120 100

-

80 60

-

0

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Interstimulus Interval (ms)

FIG. 15. Activity-dependent depression of inhibition enhances the EPSP width. (A) The first four intracellular responses evoked by a 5-&, 10-pulse stimulus train to the perforant path are shown. Subsequent responses appeared similar to the fourth response. In control ACSF burst responses developed by the second stimulus and persisted throughout the train (top). Blockade of excitatory transmission revealed a monosynaptic IPSP, that became depressed during the train with the maximal depression apparent by the second stimulus (middle). Application of 2-hydroxysaclofen antagonized the depression of IPSP, during the train. IPSPs collected before and after the addition of P-hydroxysaclofen are superimposed for comparison (bottom). All records are from a single cell with a resting membrane potential of - 76 mV. Monosynaptic IPSPs were recorded with the cell hyperpolarized to - 100 mV, the reversal potential of IPSP,. [From Mott etal. (1992) with permission. Copyright 1992 by the AAAS.] (B) Paired pulse enhancement of EPSP width

191

CENTRAL GABA, RECEPTORS

Int r a c ellular EPSP s Superimposed

D-APV

2-OH Saclofen 10 L J n v 50 ms 0 ms

FIG. 16. Paired pulse enhancement of the EPSP is bloc-xd by D-APV (50 pA , and by 2-hydroxysaclofen (400 p M ) . Granule cell responses to paired perforant path stimuli delivered 200 ms apart are shown. Responses at an expanded time base are superimposed for comparison on the right. All records are from the same cell, which was hyperpolarized by current injection to -91 mV, the reversal potential of IPSP,. Action potential are truncated. [From Mott and Lewis (1991) with permission. Copyright 1991 by the AAAS.]

(solid triangles) and paired pulse depression of IPSP, amplitude (open circles) share a similar time course. Symbols and error bars represent mean f SE (n = 5 slices for EPSP width, 6 cells for IPSC, amplitude). EPSP width was recorded extracellularly in the molecular layer of the dentate gyrus. EPSPs were evoked by paired stimuli delivered to the perforant path at different interstimulus intervals. The time course of paired pulse depression of IPSP, amplitude is taken from Fig. 1 1 . Dotted line indicates the amplitude ofthe initial response.

192

DAVID D. MOTT AND DARRELL V. LEWIS

Response to First ML Stimulus

Response to Second ML Stimulus Interstimulus Interval (ms) 20

50

200

800

10 mm

FIG. 17. Time course of paired pulse effects in the dentate gyrus. Extracellular fields were recorded in the granule cell layer (GCL) and the molecular layer (ML) in response to paired pulse stimulation of perforant path fibers in the molecular layer. A typical response to the first stimulus of the pair is shown on the left. The second response of the pair at different interstimulus intervals is shown on the right. At short interstimulus intervals (20 ms) the population spike of the second response was inhibited by GABA, receptor-mediated inhibition. At intermediate intervals (50 ms) the population spike was enhanced by paired pulse facilitation. At long intervals (200 ms) GABABreceptor-mediated disinhibition enhanced the width of the pEPSP and enabled the appearance of a second population spike. Finally, at longer intervals (800 ms) the population spike was inhibited by late paired pulse inhibition. All responses are from the same slice. Stimulus artifacts have been removed for clarity. [From Mott et al. (1992) with permission. Copyright 1992 by the AAAS.]

In addition to paired pulse facilitation, paired stimulation in the dentate gyrus at longer intervals reveals a late paired pulse inhibition (McNaughton, 1980) (see Section IV,A,2,a) (Fig. 17). This late inhibition is associated with a depression of population spike amplitude as well as a reduction of dendritic EPSP slope, suggesting that it is mediated, in part, by a suppression of excitatory transmitter release. The late inhibition begins about 100-200 ms after the initial stimulus, causing this period of inhibition to overlap the period of maximal GABAB receptormediated disinhibition. Thus, the enhancement of the EPSP and the appearance of multiple population spikes produced by the GABA, receptor-mediated disinhibition is often accompanied by a decrease in the initial population spike of the enhanced response (Mott and Lewis, 1991, 1992). Under control conditions, the late inhibition is little sensitive

CENTRAL GABA, RECEPTORS

193

to phaclofen or CGP 35348, indicating that GABA, receptors contribute only minimally to the inhibition (Brucato et al., 1992; Mott and Lewis, 1992, but see Rausche et al.. 1989). Thus, GABA, receptor antagonists, which block the EPSP enhancement during paired stimulation further reveal the opposing late inhibition. When paired stimuli are delivered 200 ms apart in the dentate gyrus, augmentation of the second response is most obvious when strong stimuli are used (Pacelli et aL., 1989; Mott and Lewis, 1991, 1992; Lewis et al., 1993). Weaker stimuli often d o not elicit much enhancement of the EPSP and at very low intensities the second response is sometimes depressed, presumedly by the late inhibition. Similarly, enhancement of the EPSP is most evident when the stimulating electrode is positioned close (<200pm) to the recording site. As the stimulating electrode is moved away from the recording site, the enhancement declines, until little to no increase is observed following perforant path stimulation on the subicular side of the hippocampal fissure (Mott et al., 1991; Mott and Lewis, 1992; Lewis et al., 1993). However, stimulation at this distant site produces marked enhancement of the response when short bursts of stimulation (2 to 4 pulses at 100 Hz) are delivered 200 ms apart. These observations indicate that EPSP enhancement requires strong neuronal activation. This requirement is most likely caused by the voltage-dependent block of the NMDA receptor-operated channels by magnesium, which is relieved only with sufficient depolarization. In conclusion, these data indicate that activity-dependent depression of inhibition is able to enhance excitatory responses. This enhancement consists primarily of an increase in the NMDA receptor-mediated component of the response. The increase in the NMDA receptor-mediated component of the excitatory response during repetitive stimulation at frequencies near 5 Hz is in rnarked contrast to the high frequency stimulation typically used to evoke these currents. b. Enhancement of LTP Induction. Long-term potentiation is typically induced with high-frequency stimulus trains (50-400 Hz, 1-2 s), sufficient to depolarize postsynaptic neurons and relieve the magnesium block of the NMDA receptor-operated channels (for review see Teyler and Discenna, 1987; Bliss and Collingridge, 1993). However, the enhancement of the NMDA component of the EPSP, when stimuli are delivered 200 ms apart, suggests that activity-dependent disinhibition would enable repetitive stimulation at a frequency of 5 Hz toinduce long-term potentiation. Indeed, several studies have previously reported that stimuli or groups of stimuli, delivered 200 ms apart, are extremely effective at inducing LTP. For example, Larson and Lynch (1986) reported that in area CA1 LTP could be induced by as little as four stimuli delivered at

A

I 10 ms

B

C

$

P P P4

E

Saclof en

2-oH

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120

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E

100

1

80

€+

e, $4

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8 ms

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60 40

20 0

FIG. 18. GABABreceptor-mediated activity-dependent disinhibition during 5-Hz stimulation enables induction of NMDA receptor-dependent LTP. (A) Extracellular responses were recorded from both the granule cell layer and the molecular layer during a 5-Hz, 10-pulse stimulus train to perforant path fibers in the molecular layer of the dentate gyrus. Only the first four responses recorded in the granule cell layer are shown, since subsequent responses appeared similar to the fourth response. Application of D-APV (50 pLM) or 2hydroxysaclofen (400 p M ) prevented enhancement of the response during the train. In contrast, in control ACSF the response became markedly enhanced during the train with an increase in pEPSP width and the development of multiple population spikes. All responses are from the same slice. (B) Responses evoked in the granule cell layer by a perforant path test stimulus before (left) and 30 min after (right) a 5-Hz, 10-pulse train to the perforant path. The perforant path test stimulus was set to evoke a population

CENTRAL GABAS RECEPTORS

195

100 Hz, when these stimuli were delivered 200 ms after a similar group of pulses, called the “priming” stimulus. Alternatively, LTP could be induced by a burst of 2 to 10 stimuli at 100 Hz delivered about 200 ms after a single priming stimulus (Rose and Dunwiddie, 1986; Diamond et al., 1988). This type of priming stimulation has been reported to effectively induce LTP in the dentate gyrus and area CA1 both in nitro and in vivo (Larson et al., 1986; Larson and Lynch, 1986; Rose and Dunwiddie, 1986; Diamond et al., 1988; Greenstein et al., 1988). T h e efficacy of this type of stimulation at inducing LTP resulted from the enhancement of the NMDA receptor-mediated component of the excitatory response during the stimulus train (Larson and Lynch, 1988). For example, in the dentate gyrus w e have found that a train of 10 stimuli delivered to the molecular layer at a frequency of 5 Hz effectivelyinduced LTP(Mott and Lewis, 1991)(Fig. 18).During this train, the excitatory responses become wider and developed multiple population spikes. Both the enhancement of the responses during the train as well as the development of LTP were blocked by D-APV, indicating that both effects were NMDA receptor-mediated. T h e enhancement of the NMDA receptor-mediated component of excitatory responses during the 5-Hz train, and thus the induction of LTP, was mediated by GABA, receptor activation. We found that in the dentate gyrus both 2-hydroxysaclofen and CGP 35348 had negligible effects on either a single stimulus-evoked response or an isolated NMDA receptor-mediated response. In contrast, these drugs blocked the augmentation of the NMDA receptor-mediated component of the EPSP during the 5-Hz train and prevented the development of LTP following the train (Mott and Lewis, 1991; Lewis and Mott, 1993) (Fig. 18). The drugs did this by antagonizing IPSP, depression during the stimulus train and thus inhibiting the postsynaptic depolarization that is necessary to enhance the NMDA receptor-mediated current and produce LTP. These results demonstrate that GABA, receptors can regulate LTP induction by modulating the level of inhibition. spike 25% of maximal before the train. Application of either D-APV (50 f i M ) or 2hydroxysaclofen (400 p M ) blocked the development of LTP following the train, whereas substantial LTP developed after an identical train in control ACSF. All responses are from the same slice. (C) Averaged data showing the effect of D-APV (50 p M ) and 2hydroxysaclofen (400 p M ) on the development of potentiation of the population spike amplitude (left) and dendritic pEPSP slope (right). Responses were measured 30 min after a 5-Hz, 10-pulse perforant path stimulus train. Bars and error bars represent mean k SE (n = 7). The dotted line indicates the amplitude of the pretrain test response. Asterisks indicate a significant increase in the response 30 min after the train (*P< .04, **P < ,001). [From Mott and Lewis (1991) with permission. Copyright 1991 by the AAAS.]

A

100

0.5

5.0

--I 10 ma

B 220 0

: 0

4

160

p:

140

.-d

120

Q)

Q

h b

l

k

a u 0

K FIG. 19. Frequency dependence of LTP induction. (A) Extracellular responses were recorded in the granule cell layer and the molecular layer during delivery of 10-pulse stimulus trains at 100, 0.5, and 5 Hz to the perforant path fibers in the molecular layer. Shown are all 10 granule cell responses during the 100-Hz stimulus train, but only the first five responses during the 0.5- and 5-Hz trains, as subsequent responses appeared similar to the fifth response. Note the enhancement of the response during the 5-Hz train, whereas responses were little changed during the 0.5-Hz train and became depressed during the 100-Hz train. All responses are from the same slice. Stimulus artifacts were removed for clarity. [From Mott et al. (1992) with permission.] (B) Averaged data showing the effect of perforant path stimulus trains of different frequency (10 pulses at 100, 0.5, or 5 Hz) on the amplitude of the population spike (left) and dendritic pEPSP slope (right) evoked by a perforant path test stimulus. The test stimulus was set to evoke a population spike 25% of maximal before the train. Extracellular responses were measured 30 min after the train in both the granule cell layer (population spike amplitude) and the molecular

CENTKAL GABA, RECEPTORS

197

T h e dependence of LTP induction on GABAB receptor-mediated disinhibition suggested that LTP induction should exhibit a frequencydependence that reflected the time course of the underlying disinhibition. Indeed, we found that EPSPs during a 5-Hz train became enhanced, whereas in the same slice EPSPs during either a 0.5- or 100-Hz train were not augmented. Furthermore, whereas the 5-Hz stimulation induced LTP, the same number of stimuli delivered at either 0.5 or 100 Hz did not produce LTP (Mott and Lewis, 1991, 1992) (Fig. 19). Subsequent examination of the underlying inhibition revealed that, during the 1 OO-Hz train, monosynaptic IPSP,s temporally summed, whereas they were virtually unaltered during the 0.5-Hz train. This contrasts with the marked depression of IPSP, amplitude observed during the 5-Hz train. Similarly, we have observed that perforant path stimulation on the subicular side of the hippocampal fissure effectively induced LTP when high-frequency bursts (100 Hz) of three to four stimuli were delivered at 5 Hz. LTP induced in this fashion was blocked by CGP 35348 (Mott et al., 1991; Lewis and Mott, 1993). In contrast, CGP 35348 did not block LTP induced by the same number of perforant path stimuli delivered as a single 100-Hz stimulus train (D. D. Mott and D. V. Lewis, unpublished observations, 1993). Thus, GABAB receptor-mediated disinhibition appears to enhance LTP induction primarily when stimuli or groups of stimuli are delivered at frequencies near 5 Hz. Similar studies in area CA 1 have demonstrated that GABAB receptors can regulate the development of LTP when it is induced using a priming stimulus (Davies et al., 1991; Olpe et al., 1993b). As in the dentate gyrus, LTP induction in area CAI is dependent on NMDA receptor activation secondary to an underlying GABAB receptor-mediated depression of inhibition. In addition to the dentate gyrus and area CA1, GABAB receptor-mediated disinhibition has been demonstrated in other areas of the brain (Calabresi et ul., 1991; Deisz and Zieglgansberger, 1992; Fukuda et al., 1993) that also show LTP (Teyler, 1989; Keller et al., 1990; Tsumoto, 1990; Uno and Ozawa, 1991; Vilhgi et ul., 1992), suggesting that regulation of LTP induction by these receptors is not limited to the hippocampal formation.

layer (pEPSP slope) of the dentate gyrus. Bars and error bars represent mean 2 S E (n = 6). Dotted line indicates the amplitude of the pretrain test response. Asterisks indicate a significant change in the amplitude of the response measured 30 min after the train (*P < .5, **P < .01). Note that the 5-Hz stimulus train produced potentiation of both the population spike and the pEPSP slope. In contrast, the 100-Hz train did not alter the test response and the 0.5-Hz train produced a lasting depression of the both the population spike and the pEPSP slope.

198

DAVID D. MOTT AND DARRELL V. LEWIS

In addition to inducing LTP in uitro, the delivery of groups of stimuli 100-200 ms apart has been shown to induce stable LTP in v i m in both area CA1 and the dentate gyrus (Staubli and Lynch, 1987; Diamond et al., 1988; Greenstein et al., 1988; Otto et al., 1991). Induction of LTP using stimuli or groups of stimuli 100-200 ms apart is of interest because this type of stimulation resembles the spontaneous activity of hippocampal neurons in rats during behavioral activity associated with exploration and learning. Indeed, in uiuo recordings in rats demonstrate that hippocampal neurons can fire complex bursts of two to seven action potentials in synchrony with the theta rhythm (Ranck, 1973; Otto et al., 1991). Theta rhythm is a period of rhythmic, slow-wave activity (3-12 Hz) that can be recorded in the hippocampal electroencephalogram (EEG) of rats during attentive or exploratory behaviors (Buzsaki, 1986; Vertes, 1986). Rhythmic bursting of CA1 pyramidal cells in synchrony with theta rhythm is thought to be important in certain behavioral learning tasks (Berry and Thompson, 1978; Winson, 1978; Otto et al., 1991). Alternatively, during awake immobility or slow-wave sleep, hippocampal neurons display rhythmic sharp wave activity, consisting of bursts of highfrequency discharge (Buzsaki, 1989). These hippocampal sharp waves have been proposed to be involved in memory consolidation (Buzsaki, 1989). The similarity between the pattern of neural activity exhibited during these periods and that used to produce LTP suggests that these types of spontaneous neural activity may provide a natural mechanism for the induction of long-term changes in synaptic transmission (Larson and Lynch, 1986; Diamond et al., 1988; Buzsaki, 1989; Otto et al., 1991). Furthermore, since GABA, receptors appear to be best activated during synchronized neural activity (see Section IV,C,3), the synchronous firing of inhibitory neurons during these rhythms may provide a mechanism for the stimulation of GABA, receptors. The similarity between the pattern of spontaneous activity during these rhythms and that used to maximize GABAB receptor-mediated disinhibition suggests that stimulation of GABAB receptors may contribute to the generation and/or maintenance of these rhythms or play a role in the induction of LTP during these types of neural activity. c. Enhancement of Signal Transmission. Inhibitory neurons regulate excitatory transmission. Thus, agents that modulate the function of these neurons can dramatically alter the synchrony and spread of neural activity. Chagnac-Amitai and Connors (1989) found that low concentrations of bicuculline could enhance the horizontal spread of activity in the neocortical slice. Slightly higher concentrations of bicuculline caused the appearance of epileptiform activity and enabled this activity to spread variably to further regions of the slice. This differed from epileptiform

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activity induced by high concentrations of bicuculline, which spread extensively throughout the preparation. The ability of even a small pharmacological reduction of inhibition to alter the spread of excitatory activity suggests that fading of inhibition produced by repetitive stimulation may also enhance the spread of neural activity. Indeed, w e have found that GABAB receptor-mediated fading of inhibition can enhance signal transmission through the dentate gyrus into area CA3 (Mott and Lewis, 1992; Mott et al., 199313). We found that a perforant path stimulus discharged granule cells, causing a subsequent response in area CA3 via the mossy fibers, the granule cell axons. The amplitude of this CA3 response corresponded to the amplitude of the population spike in the granule cell layer. Since the perforant path has been shown to project through area CA1 directly to area CA3 (Amaral, 1993), we made a cut through the perforant path in area CAI to prevent direct activation of CA3 neurons by the perforant path stimulus. When paired stimuli 10 ms apart were delivered to the perforant path, the population spike in the granule cell layer, which was evoked by the second stimulus, was inhibited by GABA, receptor-mediated inhibition. Similarly, the response in area CA3 was correspondingly reduced. However, when pairs of stimuli 10 ms apart were repeatedly delivered every 200 ms, paired pulse inhibition became depressed. This reduction in inhibition enabled the population spike of the second response of each pair to increase, causing a corresponding increase in the amplitude of the CA3 response (Fig. 20). T h e extent of the depression of paired pale inhibition observed during the train depended on the interval between stimulus pairs (Fig. 21). Depression of inhibition, and thus signal transmission into area CA3, was observed when pairs of stimuli were delivered between 50 and 2000 ms apart with a maximal effect when pairs of stimuli were delivered about 200 ms apart (Mott et al., 1993b).This time course parallel that for GABA, receptor-mediated fading of inhibition, suggesting that GABAB receptors contributed to the reduction of inhibition. Indeed, CGP 35348 blocked the depression of paired pulse inhibition during the train, and thus blocked the increase of signal transmission into area CA3 (Fig. 22). These results indicate that GABA, receptors confer filtering properties on the dentate gyrus, such that signal transmission is enhanced at frequencies at which disinhibition is maximal. GABA, receptor-mediated disinhibition has been reported to have a similar time course in other brain areas, suggesting that it may endow these areas with a similar filtering characteristics (Davies et al., 1990; Calabresi et al., 1991; Deisz and ZGglgansberger, 1992; Lambert and Wilson, 1993).

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DAVlD D. MOTT AND DARRELL V. LEWIS

B

A

A

cvr

DG Perf. Path Stim.

& I

CA3

--v

CA3

MF

Rec,

Stim.

D~

Stim.

MF Stim.

c+Fe

C Perf. Path Stim.

MF Stim.

T

TI 1

mv

3 mV 10 ma

FIG.20. Fading of inhibition facilitates signal transmission through the dentate gyrus into area CA3. (A) Diagram of a hippocampal slice showing the placement of electrodes and the location of the knife cut in area CAI, which was used to sever direct perforant path projections to area CA3. Stimulating electrodes were placed in the perforant path (Perf. Path Stim.) and the mossy fibers (MF Stim). Recording electrodes were placed in the granule cell layer in the dentate gyms (Rec. DG) and in stratum lucidum of area CA3 (Rec. CA3). (B) Responses to a perforant path stimulus of increasing intensity ( 1 10, 400, and 1000PA) were recorded in the dentate gyrus (DG, top) and in area CA3 (CA3, middle). Note that the amplitude of the CA3 response is dependent on the amplitude of the population spike in the dentate gyrus and also that the onset of the CA3 response occurs after this population spike. In contrast, mossy fiber stimuli of increasing intensity (200, 400, and 1000 PA) evoke a short latency response in area CA3 (bottom). (C) Shown are responses in the dentate gyrus (top) and area CA3 (middle) to paired stimuli 10 ms apart delivered to the perforant path at a frequency of 5 Hz. Note the depression of paired

CENTRAL GABA, RECEPTORS

20 1

T h e ability of burst stimulation at 5 Hz to reduce inhibition, and thereby enhance signal transmission, suggests that synchronized spontaneous activity could produce a similar effect in v i m In particular, spontaneous activity during both theta rhythm and hippocampal sharp waves is similar to the pattern of stimulation used to produce maximal disinhibition. Thus, GABAB receptor-mediated depression of inhibition during these endogenous rhythms may enhance the spread of the rhythmic activity. In addition, this type of disinhibition may enhance the onset and/or propagation of epileptiform activity that occurs at frequencies near 5 Hz (Ben-Ari et al., 1979). Taken together, the above results demonstrate that GABAB receptormediated fading of inhibition enhances both LTP induction and the spread of neural activity. This suggests that, when a polysynaptic circuit is stimulated every 200 ms with bursts of high-frequency stimulation, the increased transmission of the signal could produce LTP at multiple synapses in that circuit (Mott and Lewis, 1992). Development of LTP at polysynaptic sites would result in potentiation of an entire network, rather than of only a single synapse, as is typically observed following high frequency stimulation. Indeed, we have found that in the dentate gyrus high-frequency stimulation of the perforant path produces LTP of the monosynaptic granule cell response, but not of the polysynaptic response in area CA3. In contrast, when short bursts of three to four perforant path stimuli are delivered every 200 ms, both the monosynaptic response in the dentate gyrus and the polysynaptic response in area CA3 become potentiated (Mott and Lewis, 1992). Interestingly, in area CA3, following the perforant path stimulus train, the mossy fiber-evoked response is not potentiated. Rather, it is the CA3 response, evoked by stimulation of recurrent excitatory collaterals of CA3 neurons, that develops LTP. Development of this polysynaptic LTP is entirely blocked by CGP 35348, indicating that it is mediated by GABA, receptors (Fig. 23). T h e lack of mossy fiber potentiation may be caused by the NMDA receptor-independent form of potentiation known to occur at these synapses (Johnston et al., 1992). Indeed, LTP induction at mossy fiber synapses typically requires repeated long duration, high-frequency stimulus trains. Thus, we conclude that GABAB receptor-mediated

pulse inhibition of the population spike in the dentate gyrus during the train and the corresponding increase in the amplitude of the CA3 response. In contrast, a similar increase in the CA3 response during the train was not produced by an identical stimulus train delivered directly to the mossy fibers (bottom), indicating that a depression of paired pulse inhibition in area CA3 did not contribute to the enhancement of the CA3 response. [From Mott et al. (1993b) with permission.]

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A

10 hz

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TY

CA3

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-1

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Stimulation Frequency (Hz) FIG.21. Frequency dependence of signal transmission through the dentate gyrus into CA3. (A) Paired stimuli 10 ms apart were delivered to the dentate gyrus at different frequencies. Responses were recorded in the granule cell layer of the dentate gyrus (DG) and in stratum lucidum of area CA3 (CA3). Only the first two pairs of records from each five-pair train are shown. The stimulation frequency is indicated above each set of responses. Note that depression of paired pulse inhibition in the dentate gyrus and, thus, the enhancement of signal transmission into area CA3 were greatest when paired stimuli were delivered at a frequency of 5 Hz. (B) Averaged data showing the frequency dependence of signal transmission into area CA3. Trains of paired stimuli were delivered at different frequencies to the perforant path and responses recorded in the granule cell

C E N T R A L GABA, RECEPTORS

203

ACSF CA3

I

CGP 35340 CA3

J

2.5 mV

0.5 mV

10 ma

FIG.22. Signal transmission through the dentate gyrus into area CA3 is regulated by GABA, receptors. Paired perforant path stimuli 10 ms apart were delivered at a frequency of 5 Hz and recorded in the granule cell layer of the dentate gyrus (DG) and in stratum lucidum of area CA3 (CA3). Only the first three pairs of the four-pair train are shown. Addition of CGP 35348 (400 p M ) blocked both the depression of paired pulse inhibition in the dentate gyrus as well as the corresponding increase in the CA3 response during the train. [From Mott el al. (1993b) with permission.]

depression of inhibition enhances signal transmission into area CA3, thereby facilitating depolarization and the induction of NMDA receptordependent LTP. The similarity between the stimuli used to induce polysynaptic LTP and the pattern of spontaneous activity observed during both theta rhythm and hippocampal sharp waves suggests that GABA, receptors may play an important functional role during information storage in vtvo.

layer of the dentate gyrus and in stratum lucidum of area CA3. Dentate gyrus responses were measured by the amplitude of the population spike (solid circles) and CA3 responses were measured by the amplitude of the negative deflection (open circles).The amplitude of the second response of the pair, as a percentage of the first, was plotted against stimulation frequency. Only data for the second pair of the five-pair train are shown. Symbols and error bars represent mean 4 SE (n = 6). Dotted line indicates the amplitude of the first response of the pair. [From Mott el al. (1993b) with permission.]

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DAVID D. MOTT AND DARRELL V. LEWIS

Control

50 min a f t e r trains in 400uM CGP 35348

50 min a f t e r trains in ACSF

Reek DG

Perforant Path Stimulus

Rec

CA3--+v--0 . 2 3 10 ma

Mossy Fiber

Stimulus

Rec

cA3dP

10 ms

FIG. 23. GABAB receptor-mediated activity-dependent depression of inhibition enables the induction of polysynaptic LTP. Responses were recorded extracellularly in the granule cell layer of the dentate gyrus (Rec DG) and in the pyramidal cell layer of area CA3 (Rec CA3). Test stimuli were delivered to the perforant path, the mossy fibers, and the Schaffer collaterals. T h e amplitude of the test stimulus was set to evoke a response 25% of maximal before the train. LTP was induced by delivering 5 bursts of high-frequency stimulation (four pulses at 100 Hz) to the perforant path at a frequency of 5 Hz. This pattern of stimulation was repeated three times separated by 2-min intervals so that a total of 15 bursts was delivered. In the presence of CGP 35348 (400 p M ) this pattern of stimulation produced no change in the response to any of the test stimuli measured 50 min after the trains. In contrast, in this same slice, after washout of the CGP 35348, the stimulus trains produced marked potentiation of the response to the perforant path and Schaffer collateral test stimulus measured 50 min after the train. T h e response to the mossy fiber test stimulus was not potentiated.

CENTRAL GABA, RECEPTORS

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D. OPPOSING EFFECTSOF PRE-A N D POSTSYNAPTIC GABAB RECEPTORS

As is clear from previous sections of this review, GABA, receptors can produce diverse and often contradictory effects on excitatory transmission. These receptors can exert opposing inhibitory and disinhibitory actions that can interact in a complex fashion. Because of these contradictory actions, the overall effect of GABAB receptor stimulation can differ dramatically between brain regions, depending on the relative strength of the inhibitory and disinhibitory effects of GABA, receptors in that region. For example, in the dentate gyrus GABAB receptor stimulation with baclofen produces only a minimal postsynaptic inhibition of granule cells (Misgeld et al., 1989) and little suppression of glutamate release from perforant path terminals (Lanthorn and Cotman, 1981; Ault and Nadler, 1982). In contrast, the drug strongly suppresses the release of GABA from inhibitory neurons (Misgeld et al., 1989; Otis and Mody, 1992; Mott et al., 1993b). Thus, in this area baclofen has a net disinhibitory effect on the perforant path-evoked response that can enable the production of epileptiform bursts (Mott et al., 1989; Burgard and Sarvey, 1991). In contrast, in area (;A3 baclofen produces a robust inhibition of pyramidal cells as well as a strong suppression of excitatory transmitter release from Schaffer collaterals (Ault and Nadler, 1983a; Misgeld et al., 1984; lnoue et al., 1985b; Thompson and Gahwiler, 1992a). Therefore, despite the presence of GABAB receptor-mediated disinhibition (Misgeld et al., 1984; Thompson and Gahwiler, 1989a), the net effect of baclofen in this area is inhibitory and antiepileptic (Ault and Nadler, 1983b; Ogata et al., 1986; Swartzwelder et al., 1986a,b).Thus, it appears that, in general, the overall effect of GABA,, receptors in a given brain region depends on whether the receptors are more effective at suppressing inhibition or excitation in that area. T h e ability of baclofen to produce a different overall effect in different brain regions raises the question what determines the net effect of GABA, receptor stimulation within a given area. In general, the relative strength of the inhibitory and disinhibitory effects of GABA, receptors appears to be determined by several factors, including receptor distribution, access of GABA to the receptor, receptor-effector coupling, and the stimulation patterned used to evoke the response. We will discuss each of these below. Since GABABreceptors located on inhibitory and excitatory neurons can have dramatically different effects on excitatory transmission, the pattern of distribution of GABAB receptors within a given brain region may enable the net effect of GABA, receptor stimulation to differ depending on the neurons activated. For example, in area CAI GABAB

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receptors on Schaffer collateral terminals suppress excitatory transmitter release. Consequently, baclofen inhibits the pyramidal cell response evoked by stimulation of this pathway (Ault and Nadler, 1982). In contrast, perforant path terminals in this same area are not sensitive to GABAB receptor stimulation and so the pyramidal cell response evoked by stimulation of the perforant path is unaffected by baclofen (Ault and Nadler, 1982; Colbert and Levy, 1992). Thus, in this area the overall effect of GABA, receptor activation on the pyramidal cell response depends on the pathway stimulated. A similar separation of baclofensensitive and -insensitive inhibitory terminals may exist (N. A. Lambert, personal communication, 1993), suggesting that the magnitude of GABAB receptor-mediated disinhibition may also depend on the specific inhibitory interneurons that are activated. Alternatively, the net effect of GABAB receptors within a given brain region is determined by the access of synaptically released GABA to the receptor sites. GABA uptake appears to be a powerful regulator of the spread of released GABA (Thompson and Gahwiler, 1992b; Isaacson et al., 1993). Thus, GABA, receptors located farther from the release site are less likely to be activated by synaptically released GABA, especially if GABA uptake is robust (Thompson and Gahwiler, 1992b). For example, in area CA1 synaptically released GABA activates GABAB receptors on excitatory terminals only during strong stimulation or in the presence of a GABA uptake inhibitor (Isaacson et al., 1993). Consequently, under control conditions, the balance of GABA, receptor effects is determined primarily by GABAB receptors located on inhibitory interneurons o r postsynaptically on excitatory neurons. I n contrast, the ability of pharmacological agents to stimulate strongly receptors that are not activated o r only minimally activated by synaptically released GABA may cause these drugs to have effects different from those of released GABA. For example, in area CA 1 baclofen disinhibits pyramidal cells, but also suppresses excitatory transmitter release, producing a profound depression of the excitatory response (Ault and Nadler, 1982). Thus, the overall effect of the drug is inhibitory. In contrast, in this same area, during repetitive stimulation, GABA, receptor activation by synaptically release GABA causes little reduction of excitatory transmitter release and so has a predominantly disinhibitory effect (Nathan et al., 1990b; Davies et al., 1991; Nathan and Lambert, 1991). Thus, the function of the activated receptors plays a critical role in determining the overall effect of GABAB receptor stimulation. Finally, changes in the efficacy of receptor-effector coupling of either the presynaptic or postsynaptic GABAB receptor-mediated action may

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modulate the strength of these GABAB receptor-mediated effects. Second messenger-induced changes in the state of neuronal activity can potentially alter the functioning of GABAB receptors and thus the balance of GABAB receptor-mediated effects. For example, coactivation of other G protein-coupled neurotransmitter systems, such as norepinephrine, which can enhance the postsynaptic action of GABAB receptors (Bijak et al., 1991) (see Section IV,A,l,f), may alter the balance between disinhibitory and inhibitory actions of GABAB receptors. Thus, coactivation of noradrenergic terminals and GABAergic terminals may cause the net effects of GABABreceptor stimulation to become more inhibitory. This type of modulation may provide a mechanism €or other transmitter systems to regulate GABAergic inhibition. Another factor that can have an important influence on the overall effect of GABAB receptors is the stimulation paradigm used. A stimulation paradigm designed to maximize the disinhibitory effects of GABAB receptors can have dramatically different effects from one designed to maximize inhibitory effects. In general, during stimulation at frequencies of 5-7 Hz, presynaptic GABAB autoreceptors are optimally activated, causing GABA release to be maximally suppressed (Davies et al., 1991; Mott and Lewis, 1991, 1992; Mott et al., 1993b). Thus, at these frequencies GABAB receptor-mediated disinhibition plays the predominate role. However, during high-frequency stimulation (20-400 Hz), IPSP/C,s summate, producing strong GABAB receptor-mediated inhibition (Rausche et al., 1989). Furthermore, during high frequency stimulation, GABAB receptor-mediated depression of GABA release appears to be reduced (Waldmeier and Raumann, 1993) (see Section IV,C,3). T h e resulting increase in GABA release would enhance inhibition by more strongly stimulating both postsynaptic GABA, and GABAB receptors. Thus, GABA, receptors can have markedly different effects on inhibition, and consequently on excitatory transmission, depending on the stimulation paradigm used. In particular, by producing either disinhibition or inhibition, depending on the stimulation paradigm, GABAB receptors can have opposing effects on NMDA receptor-mediated currents and thereby on the development of NMDA receptor-dependent LTP (Olpe and Karlsson, 1990; Burgard and Sarvey, 1991; Davies et al., 1991; Morrisett et at., 1991; Mott and Lewis, 1991, 1992; Olpe et al., 1993b). For example, we have shown that in the dentate gyrus, when stimuli are delivered at theta frequencies, the disinhibitory effects of GABAB receptor activation enhances NMDA receptor-mediated responses and thereby facilitates LTP induction (Mott and Lewis, 1991, 1992). Similarly, in area CA1

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Davies et al. (199 I ) have reported that primed burst potentiation is dependent on GABAB receptors. In contrast, we have found that postsynaptic inhibition produced by GABAB receptors suppresses NMDA receptormediated currents (Morrisett et al., 199l), suggesting that during highfrequency stimulation GABAB receptors would have an inhibitory effect on synaptic plasticity. In agreement with this, Olpe and Karlsson (1990) found that, during high-frequency stimulation in area CA1, GABAB receptor stimulation retards LTP, possibly through postsynaptic inhibition. The stimulus dependence of GABAB receptor-mediated effects on LTP induction in a single brain area was recently demonstrated by Olpe et al. (199313). These investigators found that in area CA1, LTP induced by high-frequency stimulation was facilitated by CGP 35348, whereas in this same area, under identical conditions, LTP induced by primed burst stimulation was impaired by the same concentration of the drug. Similarly, the effects of GABAB receptors on the spread of neural activity may also depend on the stimulation parameters used. We have shown that in the dentate gyrus GABA, receptor-mediated disinhibition produced during 5-Hz stimulation facilitates the spread of neural activity (Mott and Lewis, 1992; Mott et al., 1993b). However, the predominance of GABA, receptor-mediated inhibition at higher frequencies of stimulation suggests that GABA, receptors may inhibit the spread of activity during high-frequency stimulation. Support for this possibility was provided by Dreier and Heinemann (1991), who found that baclofen, most likely through a postsynaptic inhibitory action, suppressed the spread of epileptiform activity from entorhinal cortex through the dentate gyrus into area CA3. Thus, it is possible that, whereas GABAB receptormediated disinhibition enhances the spread of activity during stimulation at theta frequency, GABA, receptor-mediated inhibition may inhibit the spread of neural activity during higher frequency or more prolonged burst activity. These differences in the net effects of GABAB receptors both within a single brain region and in different brain regions indicate that pharmacological manipulation of GABAB receptors with agents that are not specific for the inhibitory or disinhibitory actions of these receptors may have complex and contradictory behavioral effects. The actions of these agents may depend not only on the brain area or areas to which they are applied, but also on the stimulation paradigm used and the activity of other transmitter systems. These complex actions of GABAB receptor activation point out the need for compounds specific for the differing actions of these receptors.

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V. Summary ond Conclusions

In conclusion, GABAB receptors enable GABA to modulate neuronal function in a manner not possible through GABA, receptors alone. These receptors are present at both pre- and postsynaptic sites and can exert both inhibitory and disinhibitory effects. In particular, GABAB receptors are important in regulating NMDA receptor-mediated responses, including the induction of LTP. They also can regulate the filtering properties of neural networks, allowing peak transmission in the frequency range of theta rhythm. Finally, GABAB receptors are G protein-coupled to a variety of intracellular effector systems, and thereby have the potential to produce long-term changes in the state of neuronal activity, through actions such as protein phosphorylation. Although the majority of the effects of GABAB receptors have been reported in vitro, recent studies have also demonstrated that GABA, receptors exert electrophysiological actions in vivo. For example, GABAB receptor antagonists reduce the late IPSP in vivo and consequently can decrease inhibition of spontaneous neuronal firing following a stimulus (Lingenhohl and Olpe, 1993). In addition, blockade of GABAB receptors can increase spontaneous activity of central neurons, suggesting the presence of GABA, receptor-mediated tonic inhibition (Andre et al., 1992; Lingenhohl and Olpe, 1993). Despite these electrophysiological effects, antagonism of GABA, receptors has generally been reported to produce few behavioral actions. This lack of overt behavioral effects most likely reflects the modulatory nature of the receptor action. Nevertheless, two separate behavioral studies have recently reported an enhancement of cognitive performance in several different animal species following blockade of GABAB receptors (Mondadori et al., 1992; Carletti et al., 1993). Because of their small number of side effects, GABAB receptor antagonists may represent effective therapeutic tools for modulation of cognition. Alternatively, the lack of overt behavioral effects of GABAB receptors may indicate that these receptors are more important in pathologic rather than normal physiological states (Wojcik et al., 1989). For example, a change in receptor affinity or receptor number brought on by the pathology could enhance the effectiveness of GABA, receptors. Of significance, CGP 35348 has been shown to block absence seizures in genetically seizure prone animals, while inducing no seizures in control animals (Hosford el al., 1992; Liu et al., 1992). Thus, GABAB receptors may represent effective sites for pharmacological regulation of absence seizures. Perhaps further behav-

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ioral effects of these receptors will become apparent only after additional studies have been performed using the highly potent antagonists that have been recently introduced. Alternatively, because of the conflicting inhibitory and disinhibitory effects of the drugs, further elucidation of the role of GABA, receptors in behavior may have to await the development of receptor antagonists that are specific for either the inhibitory or the disinhibitory effects of the receptor.

Acknowledgments

We thank Drs. Cui-Wei Xie and Nevin A. Lambert for helpful comments and suggestions and Patsy Martin for secretarial assistance. This work was supported by National Institute of Health Grant NS27488.

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THE ROLE OF THE AMYGDALA IN EMOTIONAL LEARNING

Michael Davis Ribicoff Research Facilities of the Connecticut Mental Health Center, Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut 06508

I. Introduction 11. Morphology 111. Electrophysiology IV. Anatomical Connections between the Amygdala and Brain Areas Involved in Fear and Anxiety V. Elicitation of Fear by Electrical or Chemical Stimulation of the Amygdala VI. Effects of Amygdala Lesions on Conditioned Fear VII. Effects of Amygdala Lesions on Unconditioned Fear VIII. Effects of Local Infusion of Drugs into the Amygdala on Measures of Fear and Anxiety IX. T h e Role of the Amygdala i n Attention X . T h e Amygdala Is Critical for the Fear-Potentiated Startle Effect XI. Are Aversive Memories Actually Stored in the Amygdala? XII. Is the Amygdala Absolutely Essential for Fear-Potentiated Startle? XIII. Can Initial Fear Conditioning Occur without the Amygdala? XIV. T h e Role of Excitatory Amino Acid Receptors in the Amygdala in Fear Conditioning A. Formal Similarities between Classical Fear Conditioning and Associative LongTerm Potentiation B. The Role of NMDA Receptors in the Amygdala in the Acquisition vs Expression of Fear Conditioning C. Effects of Non-NMDA Antagonists 011 the Expression of Conditioned Fear D. Effects of NMDA Antagonists on Second-Order Conditioning E. NMDA Antagonists Delay Extinction of Fear-Potentiated Startle XV. Conclusions References

I. Introduction

A major goal of neurobiology is to understand the neural basis of emotion and learning. A great deal of evidence now suggests that a single structure in the brain, the amygdala, is critical for attaching emotional significance to formally neutral stimuli through the process of Pavlovian fear conditioning. T h e present chapter will review evidence to support

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this assertion. Initially, a brief overview of amygdaloid morphology and electrophysiology will be given. Next, anatomical targets of the central nucleus of the amygdala that appear important for various signs of fear will be enumerated, along with studies looking at the effects of lesions and electrical or chemical stimulation of the amygdala. The possible role of the amygdala in attention and data showing that an intact amygdala is necessary for fear conditioning will be mentioned. Finally, the role of excitatory amino acid transmitters in the amygdala in the acquisition and expression of conditioned fear will be reviewed.

II. Morphology

The amygdala is an almond-shaped area of the brain lying between the external capsule and the hypothalamus. It has a number of subnuclei that have been distinguished on the basis of morphology and connectivity. Nuclei with a cortex-like appearance include the basolateral and lateral nuclei (which together comprise the basolateral complex), and the accessory basal and cortical nuclei. The noncortex-like nuclei include the central and medial nuclei (for review see McDonald, 1992). Immunohistochemical and cytoarchitectural studies indicate the cortex-like amygdala nuclei are contiguous with each other and show considerable dendritic overlap at most of their mutual borders (McDonald, 1982a,b, 1984). These nuclei contain two main cell types (a) spine-dense pyramidal projection neurons that constitute the majority of neurons within each nuclei and (b) spine-sparse nonpyramidal neurons that are a heterogeneous population of presumed local circuit neurons. The high spine density of the projection neurons suggests that they can receive and integrate a vast array of synaptic contacts from both intrinsic and extrinsic afferent inputs. The projection neurons use glutamate as a neurotransmitter, whereas the aspiny neurons stain for vasoactive intestinal peptide (VIP), y-aminobutyric acid (GABA), and/or somatostatin (SOM) (McDonald, 1985; Roberts, 1992; Nitecka and Ben-Ari, 1987). The borders separating the cortex-like nuclei from the noncortexlike nuclei are very sharp with little or no dendritic overlap. In the lateral subdivision of the central nucleus the principal neuron is a mediumsized ovoid or fusiform neuron, which is reported to have the highest spine density of any cell type in the amygdaloid complex. The primary neurons of the medial subdivision of the central nucleus are also fusiform but have a moderate to sparse density of dendritic spines.

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111. Electrophysiology

Recent intracellular recordings from in vitro brain slice preparations have shown that stimulation of afferent pathways to the lateral and basolateral amygdala evoke both excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs), which are mediated by amino acid neurotransmitters. Hence, stimulation of the stria terminalis, the lateral amygdala, the external capsule, or the ventral endopyriform nucleus evokes a glutamate-mediated EPSP and subsequent GABA-mediated fast and slow IPSPs in the basolateral amygdala (Rainnie et al., 1991a,b; Washburn and Moises, 1992a; Gean and Chang, 1952). Similarly, focal stimulation of the basolateral amygdala evokes a glutamate-mediated EPSP and GABA-mediated IPSPs in neurons of the lateral nucleus (Sugita et al., 1992, 1993). The EPSP consists of a fast component mediated by activation of AMPAIkainate receptors (i.e., blocked by the specific antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline, CNQX) and a slower component mediated by activation of N-methyl-D-aspartate (NMDA) receptors (i.e., blocked by the specific antagonist 2-amino-phosphonopentanoic acid, AP5; Rainnie et al., 199la). T h e fast, GABA-mediated IPSP results from activation of GABA, receptors and the slower IPSP is mediated by activation of GABAB receptors (Rainnie et al., 1991b). Blockade of the fast IPSP with the GABA, receptor antagonist bicuculline results in epileptiform burst firing of normally quiescent basolateral neurons, suggesting that the fast IPSP probably dictates the primary state of excitability in the nucleus. It is possible that GABA, and GABAB responses may be mediated by activat.ion of a heterogeneous population of GABA interneurons (Rainnie et al., 1991b; Sugita et al., 1992). In addition, repetitive stimulation of the external capsule evokes a long-duration slow EPSP in the basolateral nucleus, which is enhanced by the acetylcholinesterase inhibitor eserine and blocked by the specific muscarinic acetylcholine receptor antagonist, atropine. This slow cholinergic EPSP shows marked summation with increased frequency of stimulation (Washburn and Moises, 199213). High-frequency stimulation of the external capsule has been reported to evoke long-term potentiation (LTP) at synapses within the basolateral nucleus (Chapman et al., 1990), which appears to be resistant to AP5 (Chapman and Bellevance, 1992). In contrast, high-frequency stimulation of the endopyriform nucleus results in LTP in the basolateral nucleus that is blocked by AP5 (Gean et al., 1993). LTP produced by stimulating these amygdaloid afferents may be modulated by norepinephrine because activation of postsynaptic P-adrenergic receptors results in an enhancement of NMDA-mediated

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EPSPs evoked by stimulation of the endopyriform cortex (Gean et al., 1992). T h e uniform distribution of noradrenergic input from the locus coeruleus to the amygdala suggests that these results may be physiologically relevant. Together, these results are especially interesting because fear conditioning measured with the inhibitory avoidance paradigm is blocked by local infusion of NMDA antagonists into the amygdala (see Section XIV) and is positively modulated by P-adrenergic receptors in the amygdala (cf. McGaugh et al., 1992). Much less is known about the integrative properties of the central nucleus of the amygdala. Afferents from the projection neurons of the lateral and basolateral nuclei are glutamatergic and stimulation of the basolateral amygdala evokes an EPSP in the central nucleus that has both quisqualate/kainate and NMDA receptor-mediated components (Nose et al., 1991). Hence, repetitive stimulation via this pathway may be expected to produce LTP similar to those observed in the basolateral complex. This study also demonstrated that a strychnine-sensitive long-lasting IPSP occurs following repetitive stimulation of the dorsal lateral subdivision of the central nucleus. This presumed glycinergic IPSP probably results from an input arising in the brainstem. Repetitive stimulation also reveals a slow GABA,-mediated IPSP. Furthermore, glutamatergic transmission was blocked by presynaptic activation of either GABA, receptors or A, adenosine receptors. Although the central nucleus contains mainly peptidergic neurons, no peptide-mediated synaptic transmission has been reported in the nucleus. However, exogenous application of corticotropin releasing factor (CRF), a peptide found in neurons of the central nucleus, evokes a membrane hyperpolarization that would reduce excitability of the nucleus, possibly by an action at CRF autoreceptors. In contrast CRF acts as an excitatory transmitter in the basolateral nucleus (Rainnie et al., 1992).

IV. Anatomical Connections between the Amygdala and Brain Areas Involved in Fear and Anxiety

The amygdala receives highly processed sensory information from all modalities through its lateral and basolateral nuclei (Amaral, 1987; LeDoux et al., 1990; Ottersen, 1980; Turner, 1981; VanHoesen, 1981). In turn, these nuclei project to the central amygdala nucleus (Amaral, 1987; Aggleton, 1985; Krettek and Price, 1978b; Millhouse and DeOlmos, 1983; Nitecka and Frotscher, 1989; Nitecka et al., 1981; Ottersen, 1982; Roberts et al., 1982; Russchen, 1982; Smith and Millhouse, 1985), which then projects to a variety of hypothalamic and brainstem

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target areas that directly mediate specific signs of fear and anxiety. A great deal of evidence now indicates that the amygdala, and its many efferent projections, may represent a central fear system involved in both the expression and the acquisition of conditioned fear. (Davis, 1992; Gray, 1989; Gloor, 1960; Kapp and Pascoe, 1986; Kapp et al., 1984, 1990; LeDoux, 1987; Sarter and Markowitsch, 1985). Figure 1 summarizes work done in many different laboratories indicating that the central nucleus of the amygdala has direct projections to hypothalamic and brainstem areas that might be expected to be involved in many of the symptoms of fear or anxiety. Direct projections from the central nucleus of the amygdala to the lateral hypothalamus (Krettek and Price, 1978a, Price and Amaral, 1981; Shiosaka et al., 1980) appear to be involved in activation of the sympathetic autonomic nervous system seen during fear and anxiety (cf. LeDoux et al., 1988). Direct projections to the dorsal motor nucleus of the vagus (Hopkins and Holstege, 1978; Schwaber et al., 1982; Takeuchi et nl., 1983; Veening et al., 1984) may be involved in several autonomic measures of fear or anxiety, because the vagus nerve controls many different autonomic functions. Projections of the central nucleus of the amygdala to the parabrachial nucleus (Hopkins and Holstege, 1978; Krettek and Price, 1978a; Price and Amaral, 1981; Takeuchi et al., 1982) may be involved in respiratory changes during fear, because electrical stimulation o r lesions of this nucleus are known to alter various measures of respiration. Projections from the amygdala to the ventral tegmental area (Beckstead et al., 1979; Phillipson, 1979; Simon etnl., 1979; Wallace et al., 1989) may mediate stress-induced increases in dopamine metabolites in the prefrontal cortex (Thierry et al., 1976). Direct amygdala projections to the locus coeruleus (e.g., Cedarbaum and Aghajanian, 1978; Wallace et al., 1989),or indirect projections via the paragigantocellularis nucleus (Aston-Jones et al., 1986), or perhaps via the ventral tegmental area (e.g., Deutch et al., 1986), may mediate the response of cells in the locus coeruleus to conditioned fear stimuli (Rasmussen and Jacobs, 1986), as well as being involved in other actions of the locus coeruleus linked to fear and anxiety (cf. Redmond, 1977). Direct projections of the arnygdala to the lateral dorsal tegmental nucleus (e.g., Hopkins and Holstege, 1978) and parabrachial nuclei (see above), which have cholinergic neurons that project to the thalamus (cf. Pare et al., 1990), may mediate increases in synaptic transmission in thalamic sensory relay neurons (Pare et al., 1990; Steriade et al., 1990) during states of fear. This cholinergic activation, along with increases in thalamic transmission accompanying activation of the locus coeruleus (Rogawski and Aghajanian, 1980), may thus lead to increased vigilance and superior signal detection in a state of fear or anxiety. In addition, release of norepinephrine onto motor neurons via amygdala activation of

Conditioned Fear Stimulus

/

Lateral Hypothalamus

AMYGDALA

-

Parabrachial Nucleus

Parasympathetic Activation

Tachycardia, Galvanic skin response, Paleness, pupil dilation, blood pressure elevation Ulcers, urination, defecation, bradycardia

-

Activation

__c

Increased Respiration

Panting, respiratory distress

Activation of Dopamine. Norepinephrine and Acetylcholine

Behavioral and EEG arousal, increased vigilance

Pontine reticular formation-

Increased Reflexes

Increased Startle

Central Grey

Cessation of behavior Analgesia

Ventral Tegmental Area Locus Coeruleus Dorsal Lateral Tegmental N.

Trigeminal, Facial Motor N Fear Stimulus

Sympathetic

Dorsal Motor N. of Vagus-

f Nucleus Ambiguous

\

Behavioral Test or Sign of Fear or Anxiety

Effect of Amygdala Stimulation

Anatomical Target

~

--t

--

Freezing, Conflict test, CER, Social Interaction Conditioned analgesia Facial Expressions of Fear

Mouth open, jaw movements

Paraventricular N. (Hypothal.) + ACTH Release

t

Corticosteroid Release ("Stress Response")

FIG. 1 . Schematic diagram showing direct connections between the central nucleus of the amygdala and a variety of hypothalamic and brainstem target areas that may be involved in different animal tests of fear and anxiety. [From Davis (1992) with permission from Wiley-Liss, Inc.]

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the locus coeruleus, or via amygdala projections to serotonin containing raphe neurons (Magnuson and Gray, 1990), could lead to enhanced motor performance during a state of fear, because both norepinephrine and serotonin facilitate excitation of motor neurons (e.g., McCall and Aghajanian, 1979; White and Neuman, 1980). Projections of the amygdala to the nucleus reticularis pontis caudalis (Inagaki et al., 1983, Rosen et al., 1991) probably are involved in fear potentiation of the startle reflex (Hitchcock and Davis, 1991). The central nucleus of the amygdala projects to a region of the central gray (Beitz, 1982; Gloor, 1978; Hopkins and Holstege, 1978; Krettek and Price, 1978a; Post and Mai, 1980) that has been implicated in conditioned fear in a number of behavioral tests (Borszcz et al., 1989; Hammer and Kapp, 1986; LeDoux et al., 1988; Liebman et al., 1970), as well as a critical part of a general defense system (cf. Adams, 1979; Bandler and Depaulis, 1988; Blanchard et al., 1981; Fanselow, 1991; Graeff, 1988; LeDoux et al., 1988; Zhang et al., 1990). Direct projections to the trigeminal and facial motor nuclei (Holstege et al., 1977; Post and Mai, 1980; Ruggiero et al., 1982) may mediate some of the facial expressions of fear. Finally, direct projections of the central nucleus of the amygdala to the paraventricular nucleus of the hypothalamus (Gray, 1989; Silverman et al., 1981; Tribollet and Dreifuss, 198 l), or indirect projections by way of the bed nucleus of the stria terminalis and preoptic area, which receive input from the amygdala (DeOlmos et al., 1985; Krettek and Price, 1978a; Weller and Smith, 1982) and project to the paraventricular nucleus of the hypothalamus (Sawchenko and Swanson, 1983; Swanson et al., 1983), may mediate the prominent neuroendocrine responses to fearful or stressful stimuli. At present, the identity of the transmitters released onto these target sites by amygdala neurons is just beginning to emerge. Gray (1989) estimates that 25% of the neurons in the central nucleus of the amygdala and bed nucleus of the stria terminalis contain known neuropeptides, as well as glutamate. The main output neurons of the amygdala contain corticotropin-releasing factor, somatostatin, and neurotensin, with smaller contributions from substance P and galanin-containing cells.

V. Elicitation of Fear by Electrical or Chemical Stimulation of the Amygdala

Importantly, it has also been shown that electrical stimulation of the amygdala can produce a complex pattern of behavioral and autonomic changes that, taken together, highly resembles a state of fear. Thus,

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electrical stimulation of the amygdala can alter heart rate and blood pressure, both measures used to study cardiovascular changes during fear conditioning (cf. Davis, 1992). Using chemical, rather than electrical, stimulation in unanesthetized rats, Iwata et al. (1987) found that infusion of L-glutamate into the central nucleus elicited increase in blood pressure and heart rate. Ohta et al. (1991) found that infusion of the cholinergic muscarinic agonist carbachol into the amygdaloid complex elicited a pressor response along with immobilization, body shaking, searching, and rearing. Infusion of norepinephrine or serotonin had no effect. The largest pressor effect of carbachol was found after infusion into the central nucleus of the amygdala. Bradycardia was produced by infusion into the “dorso-central” part of the amygdala, whereas tachycardia was produced by infusion into the “medio-ventral” parts of the amygdala. Low doses of arginine-8-vasopressin infused into the central nucleus also enhanced the normal bradycardiac response and immobilization seen during stress, whereas oxytocin appears to block these effects (Roozendaal et al., 1992b). Interestingly, these bradycardiac and immobilization responses only occurred in rats bred to have low rates of avoidance in an active avoidance task, consistent with the hypothesis of this group that the central nucleus of the amygdala is primarily involved in “passive” rather than “active coping styles.” Using very small infusion cannulas, Sanders and Shekhar (1991) found increases in blood pressure and heart rate when the GABAA antagonist bicuculline methiodide was infused into the basolateral but not the central nucleus. Equimolar concentrations of the GABA, agonist baclofen, the GABA, antagonist phaclofen, o r the glycine antagonist strychine did not have these cardiovascular effects when infused into either nucleus. Importantly, the pressor effects of blocking GABA, receptors in the amygdala were associated with increases in locomotor activity rather than immobilization. We have found very similar effects in our own laboratory (Y. Lee and M. Davis, unpublished, 1992). Amygdala stimulation can also produce gastric ulceration (Henke, 1980; Henke, 1982; Innes and Tansy, 1980; Sen and Anand, 1957), which may result from chronic fear or anxiety. Electrical stimulation of the amygdala also alters respiration (Anand and Dua, 1956; Applegate et al., 1983; Bonvallet and GaryBobo, 1972; Harper et al., 1984),a prominent symptom of fear, especially in panic disorders. Electrical or chemical stimulation of the central nucleus of the amygdala produces a cessation of ongoing behavior (Applegate et al., 1983; Gloor, 1960; Kaada, 1972; Ohta et al., 1991; Roozendaal et al., 1992b; Ursin and Kaada, 1960; Willcox et al., 1992), a critical component in several animal models such as freezing, the operant conflict test, the conditioned emotional response,

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and the social interaction test. Electrical stimulation of the amygdala also elicits jaw movements (Applegate et al., 1983; Gloor, 1960; Kaku, 1984; Ohta, 1984) and activation of facial motoneurons (Fanardjian and Manvelyan, 1987), which probably mediate some of the facial expressions seen during the fear reaction. These motor effects may be indicative of a more general effect of amygdala stimulation, namely that of modulating brainstem reflexes such as the massenteric (GaryBobo and Bonvallet, 1975; Bonvallet and GaryBobo, 1975), baroreceptor (Lewis et al., 1989; Schlor et al., 1984; Pascoe et al., 1989), nictitating membrane (Whalen and Kapp, 1991), and the startle reflex (Rosen and Davis, 1988), which is also increased by local infusion of N-methyl-D-aspartate into the central nucleus of the amygdala (Koch and Ebert, 1993). Electrical stimulation of the amygdala has been shown to increase plasma levels of corticosterone, indicating an excitatory effect of the amygdala on the hypothalamo-pituitary-adrenal axis (Dunn and Whitener, 1986; Feldman et al., 1982; Mason, 1959; Matheson et al., 1971; Redgate and Fahringer, 1973; Smelik and Vermes, 1980; Setekleiv et al., 1961; Yates and Maran, 1974). As mentioned above, some of these excitatory effects may be mediated through the preoptic area and bed nucleus of the stria terminalis, which receive input from the amygdala (DeOlmos et al., 1985; Krettek and Price, 1978a; Weller and Smith, 1982) and project to the paraventricular nucleus of the hypothalamus (Sawchenko and Swanson, 1983; Swanson et al., 1983). In addition, direct projections from the medial nucleus of the amygdala to the hypothalamus exist (Gray et al., 1989; Silverman et al., 1981; Tribollet and Dreifuss, 1981), and these projections may also mediate some of the excitatory effects of the amygdala on the hypothalamic-pituitary axis. Finally, in humans electrical stimulation of the amygdala elicits feelings of fear or anxiety as well as autonomic reactions indicative of fear (Chapman et al., 1954; Gloor et al., 1981). Although other emotional reactions occasionally are produced, the major reaction is one of fear or apprehension. Viewed in this way, the highly correlated set of behaviors seen during fear may result from activation of a single area of the brain (the amygdala, especially its central nucleus), which then projects to a variety of target areas which themselves are critical for each of the specific symptoms of fear, as well as the perception of anxiety. Moreover, it must be assumed that all of these connections are already formed in an adult organism, because electrical stimulation produces these effects in the absence of prior explicit fear conditioning. Thus, much of the complex behavioral pattern seen during a state of “conditioned fear” has already been “hard wired” during evolution. In order for a formerly neutral stimulus to produce the constellation of behavioral effects used to define a state of

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fear or anxiety, it is only necessary for that stimulus to now activate the amygdala, which in turn will produce the complex pattern of behavioral changes by virtue of its innate connections to different brain target sites. Viewed in this way, plasticity during fear conditioning probably results from a change in synaptic inputs prior to or in the amygdala, rather than from a change in its efferent target areas. The ability to produce LTP in the amygdala (Clugnet and LeDoux, 1990; Chapman et al., 1990, Chapman and Bellevance, 1992; Gean et al., 1993) and the finding that local infusion of NMDA antagonists into the amygdala blocks the acquisition (Fanselow et al., 1991; Izquierdo et al., 1992; Kim and McGaugh, 1992; Miserendino et al., 1990) and extinction (Falls et al., 1990) of fear conditioning are consistent with this hypothesis (see Section X).

VI. Effects of Amygdala lesions on Conditioned Fear

Many studies indicate that lesions of the amygdala block the effects of a conditioned stimulus in a variety of behavioral test situations. Lesions of the amygdala eliminate or attenuate conditioned freezing normally seen in response to a stimulus formerly paired with shock (Blanchard and Blanchard, 1972; Helmstetter, 1992a,b; Roozendaal et al., 1990, 1991a,b; Kiernan and Cranney, 1992; LeDoux, 1992; LeDoux et al., 1988; Lorenzini et al., 1991; Phillips and Ledoux, 1992; Romanski and LeDoux, 1992) to a dominant male rat (Bolhuis et al., 1984; Luiten et al., 1985) o r during a continuous passive avoidance test (Slotnick, 1973). Lesions of the amygdala counteract the normal reduction of bar pressing in the operant conflict test (Kopchia et al., 1992; Shibata et al., 1986) and the conditioned emotional response paradigm (Kellicut and Schwartzbaum, 1963; Spevack et al., 1975). In birds, lesions of the archistriatum, believed to be homologous with the mammalian amygdala, block the development of a conditioned emotional response (Dafters, 1976) or heart rate acceleration in response to a cue paired with a shock (Cohen, 1975). In both adult (Gentile et al., 1986; McCabe et al., 1992; Kapp et al., 1979; Roozendaal et al., 1990, 1991a,b) and infant mammals (Sananes and Campbell, 1989) lesions of the central nucleus block conditioned changes in heart rate. Ibotenic acid lesions of the central nucleus of the amygdala (Iwata et al., 1986), localized cooling of this nucleus (Zhang et al., 1986), or lesions of the lateral amygdala nucleus (LeDoux et al., 1990; Romanski and Ledoux, 1992) also block conditioned changes in blood pressure. Ablation of the central nucleus can reduce the secretion of adrenocorticotropin (Beaulieu et al., 1987), corticosteroids

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(Roozendaal et al., 1992a; Van de Kar et al., 1991) and prolactin (Roozendaal et al., 1992a). Lesions of the central nucleus o r of the lateral and basolateral nuclei of the amygdala block fear-potentiated startle (Hitchcock and Davis, 1986, 1987; Kiernan and Cranney, 1992; Sananes and Davis, 1992). This, along with a large literature implicating the amygdala in many other measures of fear such as active and passive avoidance (for reviews see Kaada, 1972; Sarter and Markowitsch, 1985; Ursin et al., 1981 and recent papers by Coover et al., 1992; Roozendaal et al., 1993; Tassoni et al., 1992) and evaluation and memory of emotionally significant sensory stimuli (Bennett et al., 1985; Bresnahan and Routtenberg, 1972; Ellis and Kesner, 1983; Gallagher e t a l . , 1980; Gallagher and Kapp, 1978, 1981; Gold et al., 1975; Handwerker et al., 1974; Kesner, 1982; Liang et al., 1985, 1986; McCaugh et al., 1990; Mishkin and Aggleton, 1981), provides strong evidence for a crucial role of the amygdala in fear.

VII. Effects of Amygdola lesions on Unconditioned Fear

Lesions of the amygdala are known to block several measures of innate fear in different species (cf., Blanchard and Blanchard, 1972; Ursin et al., 1981). Lesions of the cortical amygdala nucleus and perhaps the central nucleus markedly reduce emotionality in wild rats measured in terms of flight and defensive behaviors (Kemble et al., 1984, 1990). Large amygdala lesions or those that damaged the cortical, medial, and in several cases the central nucleus dramatically increase the number of contacts a rat will make with a sedated cat (Blanchard and Blanchard, 1972). In fact, some of these lesioned animals crawl all over the cat and even nibble its ear, a behavior never shown by the nonlesioned animals. Following lesions of the archistriatum birds become docile and show little tendency to escape from humans (Phillips, 1964, 1968), consistent with a general taming effect of amygdala lesions reported in many species (cf. Goddard, 1964). Finally, lesions of the amygdaloid complex inhibit adrenocortical responses following olfactory or sciatic nerve stimulation (Feldman and Conforti, 1981) and attenuate the compensatory hypersecretion of ACTH that normally occurs following adrenalectomy (Allen and Allen, 1974). Lesions of the central nucleus have been found to attenuate significantly ulceration (Henke, 1980) or elevated levels of plasma corticosterone produced by restraint stress (Beaulieu et al., 1986, 1987) and lesions of the medially projecting component of the ventroamygdalofugal pathway, which carries the fibers connecting the central

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nucleus of the amygdala to the hypothalamus, attenuate the increase in ACTH secretion following adrenalectomy, whereas lesions of the stria terminalis do not (Allen and Allen, 1974). Finally, lesions of the amygdala have been reported to block the ability of high levels of noise, which may be an unconditioned fear stimulus (cf. Leaton and Cranney, 1990), to produce hypertension (Galeno et al., 1984) or activation of tryptophan hydroxylase (Singh et al., 1990). Other data indicate that the amygdala appears to be involved in some types of aversive conditioning, but this depends on the exact unconditioned aversive stimulus that is used. For example, electrolytic lesions of the basolateral nucleus (Pellegrino, 1968), o r fiber-sparing chemical lesions of most of the amygdaloid complex (Cahill and McGaugh, 1990), attenuate avoidance of thirsty rats to approach an electrified water spout through which they were previously accustomed to receiving water. Importantly, however, these same lesioned animals did not differ from controls in the rate at which they found the water spout over successive test days o r their avoidance of the water spout when quinine was added to the water (Cahill and McCaugh, 1990). This led Cahill and McGaugh to suggest that “the degree of arousal produced by the unconditioned stimulus, and not the aversive nature per se, determined the level of amygdala involvement” (p. 541). I t is especially interesting in this regard that, although many studies have shown that electrolytic lesions of the amygdala can interfere with taste aversion learning, an elegant series of experiments have now shown that these effects result from an interruption of gustatory fibers passing through the amygdala en route to the insular cortex (Dunn and Everitt, 1988). In these studies, ibotenic acid lesions of the amygdala fail to block taste aversion learning, whereas ibotenic acid lesions of the gustatory insular cortex do. Once again, the amygdala does not seem critical for all types of aversive conditioning, but instead conditioning that involves an obvious fear component such as that produced by aversive shocks.

VIII. Effects of Local Infusion of Drugs into the Amygdala on Measures of Fear and Anxiety

Clinically, fear is regarded to be more stimulus specific than anxiety, despite very similar symptoms. Figure 1 suggests that spontaneous activation of the central nucleus of the amygdala would produce a state resembling fear in the absence of any obvious eliciting stimulus. In fact, fear and anxiety often precede temporal lobe epileptic seizures (Gloor et al.,

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198l ) , which are usually associated with abnormal electrical activity of the amygdala (Crandall et ul., 1971). An important implication of this distinction is that treatments that block conditioned fear might not necessarily block anxiety. For example, if a drug decreased transmission along a sensory pathway required for a conditioned stimulus to activate the amygdala, then that drug might be especially effective in blocking conditioned fear. However, if anxiety resulted from activation of the amygdala not involving that sensory pathway, then that drug might not be especially effective in reducing anxiety. On the other hand, drugs that act specifically in the amygdala should af-fect both conditioned fear and anxiety. Moreover, drugs that act at various target areas might be expected to provide selective actions on some but not all of the somatic symptoms associated with anxiety. It is noteworthy in this regard that the centrai nucleus of the amygdala is known to have high densities of opiate receptors (Goodman et al., 1980), whereas the basolateral nucleus, which projects to the central nucleus (Aggleton, 1985; Krettek and Price, 1978b; Millhouse and DeOlmos, 1983; Nitecka et al., 1981; Ottersen, 1982; Smith and Millhouse, 1985; Russchen, 1982), has high densities of benzodiazepine receptors (Niehoff and Kuhar, 1983). In fact, local infusion of opiate agonists into the central nucleus of the amygdala blocks the acquisition of conditioned bradycardia in rabbits (Gallagher et al., 1981, 1982) and has anxiolytic effects in the social interaction test (File and Rodgers, 1979). Furthermore, local infusion of benzodiazepines into the amygdala has anxiolytic effects in the operant conflict test (Hodges et al., 1987; Nagy et ul., 1979; Petersen and Scheel-Kruger, 1982; Petersen et al., 1985; Scheel-Kruger and Petersen, 1982; Shibata et al., 1982, 1989; Thomas et al., 1985), tests of conditioned analgesia and freezing (Helmstetter, 1993); the light-dark box measure in mice (Costalletal., 1989)and antagonizes the discriminative stimulus properties of pentylenetetrazol (Benjamin et al., 1987).The anticonflict effect can be reversed by systemic administration of the benzodiazepine antagonist flumazenil (Hodges et al., 1987; Petersen et al., 1985; Shibata et al., 1989) o r coadministration into the amygdala of the GABA antagonist bicuculline (Scheel-Kruger and Petersen, 1982), and mimicked by local infusion into the amygdala of GABA (Hodges et al., 1987) or the GABA agonist muscimol (ScheelKruger and Petersen, 1982). In general, anticonflict effects of benzodiazepines occur after local infusion into the lateral and basolateral nuclei (Petersen and Scheel-Kruger, 1982; Petersen et al., 1985; Scheel-Kruger and Petersen, 1982; Thomas et al., 1985) (the nuclei of the amygdala that have high densities of benzodiazepine receptors) and not after local infusion into the central nucleus (Petersen and Scheel-Kruger, 1982;

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Scheel-Kruger and Petersen, 1982). Taken together, therefore, these results suggest that drug actions in the amygdala may be sufficient to explain both fear-reducing and anxiety-reducing effects of various drugs given systemically. i n fact, local infusion into the amygdala of the benzodiazepine antagonist flumazenil significantly attenuated the anticonflict effect of the benzodiazepine agonist chlordiazepoxide given systemically (Hodges et al., 1987). This is a very powerful experimental design and strongly implicates the amygdala in mediating the anxiolytic effects of benzodiazepines. importantly, however, benzodiazepines can still have anxiolytic effects in animals with lesions of the amygdala (Yadin et al., 1991; Kopchia et al., 1992, M. Kim and M. Davis, unpublished, 1992), indicating that structures which mediate anxiogenic behavior after amygdala damage can also be affected by benzodiazepines. Recently, a new class of anxiolytic compounds acting as 5-HT, receptor subtype antagonists have been reported to produce anxiolytic effects after local infusion into the amygdala (Costall et al., 1989; Higgins et al., 1991). Such infusions also can block some of the signs of withdrawal following subchronic administration of diazepam, ethanol, nicotine, or cocaine (Costall et al., 1990) or increases in levels of dopamine or the serotonin metabolite 5-HIAA in the amygdala after activation of dopamine neurons in the ventral tegmental area (Hagan et al., 1990). i n addition, local infusion of a 5-HT,, antagonist into the central nucleus has been reported to have an anticonflict effect (Takao et al., 1992). Activation of neuropeptide Y receptors in the central nucleus has been reported to produce selective anxiolytic effects in the conflict test (Heilig et al., 1993). Finally, in a very interesting study mentioned earlier looking at the general issue of individual differences in how animals respond to stress (e.g., passive vs active coping styles), local infusion into the central nucleus of very low amounts of arginine-8-vasopressin increased stressinduced bradycardiac and immobility responses, whereas oxytocin had an opposite effect (Roozendaal et al., 1992b). This effect was seen in rats bred for low rates of avoidance behavior but not the more aggressive rats that show high avoidance rates.

IX. The Role of the Amygdala in Attention

More recently it has been emphasized (Kapp et al., 1992) that in addition to its direct connections to the hypothalamus and brainstem, the central nucleus of the arnygdala also has the potential for indirect widespread effects on the cortex via its projections to cholinergic neurons

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located within the sublenticular substantia innominata (Price and Amaral, 1981; Grove, 1988; Russchen et al., 1985), which in turn project to the cortex. In fact, the rapid development of conditioned bradycardia during Pavlovian aversive conditioning, critically dependent on the amygdala, may not simply be a marker of an emotional state of fear, but instead a more general process reflecting an increase in attention. I n the rabbit, low-voltage, fast EEG activity, generally considered a state of cortical readiness for processing sensory information (Steriade and McCarley, 1990), is acquired during Pavlovian aversive conditioning at the same rate as conditioned bradycardia (Yehle et al., 1967). Electrical stimulation of sites in the central nucleus that produce bradycardia also produce low-voltage fast EEG activity (Kapp et al., 1990). In addition, electrical stimulation of the central nucleus elicits pupillary dilation and pinna orientation (Applegate et al., 1983; Ursin and Kaada, 1960), both of which would be associated with an increase in sensory processing. In fact, an attention or orienting reflex was the most common response elicited by electrical stimulation of the amygdala in cats described by Ursin and Kaada (1960). These and other observations have led Kapp et al. (1992) to hypothesize that the central “nucleus and its associated structures function, at least in part, in the acquisition of an increased state of nonspecific attention or arousal manifested in a variety of CRs which function to enhance sensory processing. This mechanism is rapidly acquired, perhaps via an inherent plasticity within the nucleus and associated structures in situations of uncertainty but of potential import; for example, when a neutral stimulus (CS) precedes either a positive or negative reinforcing, unexpected event (US)” p. 241. Gallagher et al. (1990) have found results consistent with this attentional hypothesis. In their studies, a CS such as a light or a tone is paired with receipt of food. Initially rats rear when the light goes on or show small startle responses when the tone goes on, both of which habituate with stimulus repetition. When these stimuli are then paired with food, these initial orienting responses return (CS-generated CRs) along with approach behavior to the food cup (US-generated responses). Neurotoxic lesions of the central nucleus of the amygdala severely impair CSgenerated responses without having any effect on unconditioned orienting responses o r US-generated responses. On the basis of these and other data, Gallagher and Holland (1992) have concluded that the central nucleus of the amygdala modulates attention to a stimulus that signals a change in reinforcement. Similar conclusions have been reached by Halgren (1992) based on recording stimulus-evoked electrical activity in the amygdala in epileptic patients. In these studies subjects are presented with a series of visual

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or auditory stimuli, some of which they are instructed to ignore and other to attend. Averaged evoked responses show a prominent negative-positive component occurring roughly 200-300 ms after stimulus onset (N200/P300). These components, especially N200, are prominent within the amygdala and are much larger when elicited by a stimulus to which the subject is asked to attend. Halgren summarizes the cognitive conditions that evoke the N200/P300 as being stimuli that are novel or signals for behavioral tasks and hence necessary to attend and process. Moreover, these components, along with other autonomic measures of the orienting reflex, seem to form an overall reaction of humans to stimuli that demand their evaluation.

X. The Arnygdala I s Critical for the Fear-PotentiatedStartle Effect

Brown et al. (195 1) demonstrated that the amplitude of the acoustic startle reflex in the rat can be augmented by presenting the eliciting auditory startle stimulus in the presence of a cue (e.g., a light) that has previously been paired with a shock, a phenomenon termed the “fearpotentiated startle effect” (Davis and Astrachan, 1978). In this test a central state of fear is considered to be the conditioned response. Conditioned fear is operationally defined by elevated startle amplitude in the presence vs the absence of a cue previously paired with a shock. Our laboratory has been using this test to ask a number of questions about the role of the amygdala in Pavlovian fear conditioning. Selective destruction of cell bodies in the lateral and basolateral amygdala nuclei (Sananes and Davis, 1992) o r electrolytic lesions of the central nucleus of the amygdala (Hitchcock and Davis, 1986) following training completely block fear-potentiated startle. Low-level electrical stimulation of the amygdala markedly increases acoustic startle amplitude (Rosen and Davis, 1988) with no obvious signs of behavioral activation during stimulation at these same stimulation currents and durations. Consistent with other studies, we have found a direct connection between the central nucleus of the amygdala and the exact part of the nucleus reticularis pontis caudalis that is critical for startle using anterograde and retrograde tracing techniques (Rosen et al., 199 1). Electrolytic lesions at various points along this output pathway completely block fear-potentiated startle, whereas lesions of the other major output of the amygdala through the stria terminalis and bed nucleus of the stria terminalis d o not (Hitchcock and Davis, 1991).

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XI. Are Aversive Memories Actually Stored in the Amygdala?

Since the description of patient H.M., who developed profound amnesia following bilateral resection of the medial temporal lobe including the hippocampus and the amygdala (Scoville and Milner, 1957), these structures have been extensively studied for their roles in learning and memory. A prominent effect of lesions of the hippocampal formation (i.e., the hippocampus proper and its surrounding cortices) is to produce temporally graded memory impairment for premorbid information. That is, recent memories are selectively impaired, whereas remote memories remain intact (Squire, 1992). This temporal gradient of retrograde amnesia supports the idea of a gradual consolidation or reorganization of memory over time (McGaugh, 1966; Squire, 1992) in which memory is temporarily established in the hippocampal formation at the time of learning, but then is gradually established permanently somewhere else and thus becomes independent of the hippocampal formation (ZolaMorgan and Squire, 1990; Squire, 1992). Regarding the amygdala, Liang et al. (1982) showed that retention of an inhibitory avoidance response in rats was impaired when the amygdala was lesioned 2 but not 10 days after learning. In addition, infusion of a local anesthetic, lidocaine, or the glutamate antagonist CNQX, into the amygdala 5 min before the retention test impaired inhibitory avoidance performance in rats when the retention test was given 1-5 days, but not 12 or 21 days, after learning (Liang, 1991, K. C. Liang, personal communication, April 29, 1993). These findings suggest that the amygdala, like the hippocampal formation, plays a temporally limited role in memory processing and is not the permanent memory storage site for an avoidance response (Liang et al., 1982; McGaugh, 1989). It is important to emphasize, however, that inhibitory avoidance involves both conditioned fear (i.e., fear of the place where shock was received) and operant behavior (i.e., inhibition of the natural tendency to go from a lighted area to a dark area). Initially, fear may be required to motivate the operant behavior. Later on, the operant behavior may not depend on an antecedent level of fear. It is conceivable, therefore, that the fear memory is actually stored in the amygdala, whereas the memory of the operant response is stored elsewhere. Lesions of the amygdala shortly after training would disrupt operant performance by disrupting the fear motivating aspect in this test. However, once the memory of the operant response has became permanently established in some other structure, lesions of the amygdala would no longer have an effect.

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This line of reasoning would predict that lesions of the amygdala would always block fear memories regardless of when they were performed. In order to test this, groups of rats were trained for potentiated startle and then given lesions of the amygdala or sham lesions either 6 or 30 days after training (Kim and Davis, 1993b). Figure 2 shows that lesions of the amygdala completely blocked the expression of fearpotentiated startle when given either shortly or long after training. Hence, there was no evidence of a temporal gradient of conditioned fear after amygdala lesions compared to the steep temporal gradients

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reported with inhibitory avoidance. These data are at least consistent with the idea that the amygdala is the actual site of storage for conditioned fear memories. Clearly, however, other interpretations are equally plausible. Thus, the central nucleus may simply be an obligatory part of a neural pathway that relays the fear memory from some other structure to the startle circuit. Alternatively, the central nucleus may play an obligatory permissive role in modulating the effects of fear memories relayed from some other structure to the startle pathway. At any rate, it is clear that conditioned fear measured in this way differs considerably from that measured with inhibitory avoidance vis-a-vis amygdala lesions, indicating the two paradigms are measuring different aspects of an aversive experience.

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XII. Is the Amygdala Absolutely Essential for Fear-Potentiated Startle?

As mentioned earlier, the amygdala plays a crucial role in both the acquisition and the expression of fear-potentiated startle. However, there are examples in the literature where some effects of amygdala lesions on aversively motivated conditioning are dependent on the degree of learning achieved before surgery. For example, post-training lesions of the amygdala impair the retention of an active avoidance response in weakly trained animals, but not in overtrained animals (Brady, et al., 1954; Fonberg et al., 1962; Horvath, 1963; Thatcher and Kimble, 1966). Accordingly, w e tested whether overtraining would interact with the effect of amygdala lesions on fear-potentiated startle (Kim and Davis, 1993a). T o determine the number of training trials required to produce an asymptotic level of fear-potentiated startle, an acquisition curve was obtained by giving rats two training trials (light CS paired with 0.6-mA footshock US) and six fear-potentiated startle test trials per day for 30 days. To prevent the rats from learning that the shocks were only given at a fixed time point in the daily experimental session, the order of presenting the training vs test trials was alternated daily. Figure 3 shows the acquisition of fear-potentiated startle using this procedure. The magnitude of fear-potentiated startle gradually increased to reach a significant level on Day 6 (after 10 training trials) and an asymptotic level by about Day 12 (after 22 training trials). On Days 32-33, the rats were given either sham or electrolytic lesions of the amygdala aimed at the central nucleus. Five to six days later, all animals were retrained and tested for 7 more days, as described above. Figure 4 shows that even though the rats were overtrained for roughly 20 days beyond asymptote before surgery, lesions of the amygdala still totally blocked the expression of fear-potentiated startle, consistent with the earlier study in which a 30-day interval of no training intervened between training and testing. These results provide strong evidence that, unlike the findings from active avoidance tasks, overtraining does not alter the ability of amygdala lesions to block the expression of fearpotentiated startle. The fact that lesions of the central amygdala nucleus have very similar effects in non-overtrained (Hitchcock and Davis, 1986, 1987) and overtrained rats strongly suggests that the central nucleus of the amygdala is necessary for the expression of fear-potentiated startle, regardless of the degree of learning. Most surprisingly, however, these lesioned rats quickly reacquired fear-potentiated startle when retrained (Fig. 4).This unexpected result suggests that an intact central nucleus of the amygdala is not necessary for reacquisition of potentiated startle, or for its expression following

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reacquisition. Subsequent studies replicated this effect in animals where histological analysis showed 100% damage to the central nucleus of the amygdala. These results clearly indicate that the central nucleus is not absolutely essential for the expression of fear-potentiated startle, indicating that there must be another brain system that can compensate for the loss of the central nucleus, project to the startle circuit, and support reacquisition of fear-potentiated startle. Moreover, potentiated startle in rats retrained after amygdala lesions is selectively reduced by anxiolytic drugs like diazepam and buspirone (Fig. 5 ) , similar to fear-potentiated startle with an intact amygdala. Hence, receptors in the amygdala are not required to obtain a reduction of fear-potentiated startle with these compounds, at least in animals that have been given amygdala lesions.

XIII. Can Initial Fear Conditioning Occur without the Amygdala?

If there is a secondary system that can compensate for the loss of the central nucleus of the amygdala to support reacquisition, it would be important to know whether rats without an intact central nucleus could

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acquire fear-potentiated startle by using this secondary system. To test this, rats were given either lesions of the central nucleus of the amygdala or sham lesions and 10 days later presented with two training and six test trials per day, as described above. As shown in Fig. 6, rats with the central nucleus lesions did not show any indication of fear-potentiated startle, even with extensive training, whereas the controls showed a normal acquisition curve. Because the lesioned rats did not acquire fearpotentiated startle, the shock intensity for this group was increased from 0.6 to 1 mA on Day 15. The shock intensity for the controls was kept at 0.6 mA throughout the experiment. The lesioned rats still did not learn, even using this much higher shock intensity. Because it was obvious that these rats could not learn using these parameters, the experiment was terminated on Day 24 (i.e., after 46 training trials). In these experiments, the rats' reactivity to shock was also measured during training. Consistent with earlier results (Hitchcock and Davis, 1986), rats with central nucleus lesions tended to react less to the shock compared to controls. However, this reduction in shock reactivity was only significant on a few of the many test days. Moreover, the somewhat reduced shock reactivity of the lesioned rats cannot account for the finding that the lesions blocked initial acquisition but not reacquisi-

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tion. That is, if the central nucleus-lesioned rats did not acquire fearpotentiated startle because their shock sensitivity was impaired by the lesions, it is difficult to understand how rats lesioned after initial acquisition could have so readily reacquired fear-potentiated startle. Thus, we

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believe that the slight reduction of shock reactivity caused by the central nucleus lesions was not a major factor for the blockade of initial acquisition of fear-potentiated startle. However, it is still possible that central nucleus-lesioned rats might learn if shock levels higher than 1 mA were used. T h e findings that overtraining did not alter the effects of central nucleus lesions on the expression of fear-potentiated startle and that the central nucleus lesions totally blocked the initial acquisition of fearpotentiated startle are consistent with the proposal that the central nucleus is the essential efferent pathway through which conditioned fear exerts its modulatory effects on the acoustic startle reflex circuit in the brainstem (Davis, 1992). Despite the seemingly critical role of the central nucleus in the initial acquisition and expression of fear-potentiated startle, the fact that these same lesions did not prevent reacquisition indicates the presence of a secondary brain system that can compensate for the central nucleus under certain conditions. However, the fact that the central nucleus lesions still prevented initial acquisition of potentiated startle indicates that this secondary brain system cannot compensate for the central nucleus in all cases. It would appear that the central nucleus either induces some sort of functional change in this secondary brain system during initial training or plays a permissive role so that this secondary system, alone, can support fear-potentiated startle when the central nucleus is subsequently removed. This idea implies that the central nucleus is not simply an efferent pathway for the expression of fearpotentiated startle, but also plays an important role in acquisition by affecting other brain structures during training. At the present time, we have no direct information on the identity of a secondary brain structure that could compensate for the central nucleus of the amygdala. Our first assumption is that it is probably a forebrain structure that projects directly to the same part of the startle pathway as the central nucleus does. As mentioned earlier, the central nucleus directly projects to the startle pathway through the ventral amygdalofugal pathway (Rosen el al., 1991) and electrolytic lesions of this pathway at several levels of the brainstem block the expression of fearpotentiated startle (Hitchcock and Davis, 1991). Preliminary data suggest that reacquisition is not observed in rats with lesions of the ventral amygdalofugal pathway performed after 60 training trials, whereas once again, reacquisition is still observed in rats with lesions of the central nucleus (M. Kim and M. Davis, unpublished, 1992). Thus, the secondary brain system that can compensate for the central nucleus in reacquisition may be a forebrain structure that projects to the brainstem startle reflex circuit through the ventral amygdalofugal pathway.

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XIV. The Role of Excitatory Amino Acid Receptors in the Amygdala in Fear Conditioning

These data indicate that the amygdala appears to be critical for formation of Pavlovian fear-conditioned measured with the fear-potentiated startle test. In view of this, it is important to know what neural mechanisms in the amygdala might actually mediate the learning process.

BETWEEN CLASSICAL FEARCONDITIONING AND A. FORMAL SIMILARITIES ASSOCIATIVE LONG-TERM POTENTIATION

As suggested previously (Kim et al., 1991; Miserendino et al., 1990) fear conditioning might be mediated by associative LTP, given the formal similarities between the two phenomena. In Pavlovian fear conditioning, a weak stimulus (CS), which has little behavioral effect by itself, is consistently paired with a strong aversive stimulus (US). Following a small number of pairings, sometimes only one, the weak stimulus comes to produce effects formerly produced only by the strong, aversive stimulus. This change is not seen when the stimuli are presented in an unpaired fashion. In associative LTP, activation of a weak input to a given postsynaptic cell is paired with activation of a second, strong input projecting to the same cell. Following a small number of pairings, the initially weak synaptic input is potentiated. This potentiation is not seen when an equal number of the weak and strong inputs are presented in an unpaired sequence (cf. Brown et al., 1988). In the CA1 region of the hippocampus, activation of the weak input releases excitatory amino acids, such as glutamate, which bind to both NMDA and non-NMDA receptors on the postsynaptic neuron. Binding to the non-NMDA receptor results in weak synaptic transmission. Binding to the NMDA receptor has little consequence, because the highly Ca2+permeable NMDA channel is normally blocked by Mg2+.However, if the postsynaptic neuron is depolarized further by a sufficiently strong input, the Mg2+ block is relieved and glutamate binding to the NMDA receptor allows Ca2+ to enter the cell, which triggers a series of events that leads to an enduring potentiation of synaptic efficacy in the formerly weak input. If glutamate is prevented from binding to the NMDA receptor by giving a competitive NMDA receptor antagonist such as AP5 shortly before pairing the weak and strong stimulus, associative LTP does not occur. However, once LTP has been established, administration of an NMDA antagonist does not prevent its expression, whereas blockade of fast-synaptic transmission with a non-NMDA antagonist such as CNQX does.

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I n classical fear conditioning, a neutral stimulus (CS) might release glutamate onto neurons in the amygdala, which could bind to both NMDA and non-NMDA receptors. However, this might not produce much of a behavioral response because of only weak activation of nonNMDA receptors and the Mg2+blockade of NMDA channel permeability at resting membrane potentials. However, presentation of a strong aversive stimulus (US) at about the same time could depolarize the neuron, relieve the Mg2+ block, and allow Ca2+ to enter the cell. This could trigger events that would increase the ability of the CS to activate that neuron, allowing the CS to produce an effect similar to that previously produced by the shock.

IN THE AMYGDALA IN THE B. THEROLEOF NMDA RECEPTORS ACQUISITION vs EXPRESSION OF FEARCONDITIONING

As mentioned earlier, recent studies have shown that both NMDAdependent (Gean et al., 1993) and NMDA-independent (Chapman and Bellavance, 1992) LTP can occur in amygdala brain slices or in viuo following tetanic stimulation of the part of the medial geniculate nucleus that projects to the lateral nucleus of the amygdala (Clugnet and LeDoux, 1990).If convergence between the conditioned stimulus and shock occurs at the amygdala, and an NMDA-dependent process is involved in the acquisition of conditioned fear, then local infusion of NMDA antagonists into the amygdala should block the acquisition of conditioned fear. 1. Fear-Potentiated Startle

To test this prediction, Miserendino el al. (1990) implanted rats with bilateral cannulae aimed at the basolateral nucleus of the amygdala, which is known to have high levels of NMDA receptors (Monaghan and Cotman, 1985) and 1 week later infused with ACSF or various doses of AP5. Five minutes later they were presented with the first of 10 lightshock pairings presented at an average intertrial interval of 4 min, creating a 45-min training session. T h e infusions and training procedures were repeated the following day. One week later, all animals were tested for fear-potentiated startle without any drug infusions. Figure 7 shows that AP5 caused a dose-related attenuation of fear-potentiated startle, with a total blockade at the higher doses. Observation of the animals during training found no evidence of catalepsy or ataxia (cf. Leung and Desborough, 1988). T h e effect did not seem to result from a decrease in sensitivity to footshock, because local infusion of AP5 into the amygdala did not alter either overall reactivity to footshock or the slope of

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reactivity as a function of different footshock intensities. In contrast to the blockade of acquisition, AP5 did not block the expression of fearpotentiated startle because infusion of APV immediately before testing did not block potentiated startle in animals previously trained in the absence of the drug. Other studies showed that propranolol, which has local anesthetic effects (Weiner, 1985) and alters one-trial inhibitory avoidance conditioning (Liang et al., 1986), did not block or even attenuate fear-potentiated startle after local infusion into the amygdala prior to training. In addition, AP5 given after training but 1 week before testing did not block potentiated startle, ruling out any permanent damage to the amygdala or receptor blockade caused by residual drug during testing. Local infusion of AP5 did not affect visual prepulse inhibition, a sensitive measure of visual processing in rats (Wecker and Ison, 1986). Finally, infusion of AP5 into deep cerebellar nuclei did not block acquisition even at a dose eight times that required to block acquisition after

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local infusion into the amygdala. These data indicate, therefore, that AP5’s blockade of conditioning probably did not result from local anesthetic effects, permanent damage to the amygdala, or blockade of visual transmission, and did not occur when AP5 was infused into a different part of the brain. Similar results have also been found using an auditory rather an a visual conditioned stimulus (Campeau et al., 1992). 2. Freezing Using doses very similar to those outlined above, Fanselow et al. (1991) have found that local infusion of AP5 into the basolateral nucleus of the amygdala before training blocked conditioned freezing measured 24 h later when the CS was the stimulus of the testing cage. This effect was highly localized, because infusion into the immediately adjacent central nucleus had no effect. Interestingly, infusion of AP5 into the hippocampus also was able to block conditioned freezing measured 24 h later (Young et al., 1992),consistent with recent work showing the dependence of contextual, compared to explicit cue, fear conditioning on both the amygdala and the hippocampus (Fanselow et al., 1992; Phillip and LeDoux, 1992).

3 . Inhibitoly Avoidance Measured 48 h afer Training In an extensive series of experiments, Kim and McGaugh (1992) D - A P ~or , CPP prior to found that intraamygdala infusion of D,L-AP~, training caused a dose-dependent impairment of performance measured 48 h later using a multiple-trial step through avoidance paradigm. The potency of the drugs was consistent with their relative affinities at the NMDA receptor. This effect was not seen when AP5 was infused into the striatum, immediately above the amygdala. Intraamygdala infusion of D,L-AP~ did not affect footshock sensitivity or locomotor activity and the blockade of memory formation could not be attributed to statedependent effects. More recently, Izquierdo et al. (1992) found that immediate posttraining infusion of AP5 into the amygdala, medial septum, or hippocampus blocked step-down inhibitory avoidance measured 18 h after training. In an important extension of this work, this same group showed that AP5 was amnestic when infused into either the hippocampus or the amygdala immediately after training, but not thereafter (Jerusalinsky et al., 1992). However, the AMPAIkainate antagonist CNQX was amnestic if infused into the hippocampus o r amygdala immediately or 90 or 180 min after training. In contrast, AP5 infused into the entorhinal cortex was amnestic when given 90 or 180 min but not immediately or 360 min after training (Ferreira et al., 1992; Jerusalinsky et al., 1992).

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These data suggest that a process sensitive first to AP5 and then to CNQX in the amygdala and hippocampus is critical for post-training memory processing of this step-down inhibitory task. Later on, an AP5sensitive process in the entorhinal cortex may come into play. At present, it is not clear how these post-training manipulations relate to LTP because in hippocampal slices, AP5 blocks LTP only if given before the strong tetanizing stimulus. Nonetheless, this exciting approach is beginning to describe the timing and sequence of brain structures involved in the formation of long-term memories and the role of excitatory amino acid receptors during this process. C. EFFECTSOF NON-NMDAANTAGONISTS ON THE EXPRESSION OF CONDITIONED FEAR 1. Fear-Potentiated Startle

As mentioned earlier, once learning has occurred, local infusion of AP5 into the amygdala does not block the expression of either fearpotentiated startle or inhibitory avoidance. In contrast, Fig. 8 shows that local infusion of the AMPA antagonist CNQX into the amygdala dosedependently blocks the expression of fear-potentiated startle using either a visual (top panel) or an auditory (bottom panel) conditioned stimulus (Kim et al., 1993). At the highest doses, the blockade is complete because the small residual effect seen using an auditory conditioned stimulus represents a slight unconditioned effect because it is of the same magnitude as one would find following unimpaired noise-shock pairings (Campeau and Davis, 1992). Importantly, CNQX blocked the expression of fear-potentiated startle but had no systematic effect on baseline startle after local infusion into the amygdala. 2. Inhibitory Avoidance Using a step-down inhibitory avoidance paradigm, Izquierdo et al. (1993) found that local infusion of CNQX at doses similar to those described above blocked the expression of inhibitory avoidance when infused directly into the amygdala or the dorsal hippocampus right before testing. Liang (1991) also reported that CNQX infused in the amygdala before testing blocked the expression of step-through inhibitory avoidance. This blockade occurred when testing was carried out 24 h after training but not 21 days after training. This suggests, at least for inhibitory avoidance, that non-NMDA receptors in the amygdala are critical for the expression of recently formed memories but not older

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ones, perhaps because brain areas in addition to the amygdala become recruited as eventual storage sites over time. Consistent with this hypothesis, infusion of CNQX in both the amygdala and the hippocampus blocks the expression of inhibitory avoidance when infused 6, 13, or 20 days after training (Biachin et al., 1993).

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D. EFFECTSOF NMDA ANTAGONISTSON SECOND-ORDER CONDITIONING As mentioned earlier, icv administration of AP5 or local infusion into the amygdala does not alter pain sensitivity as measured by the hot-plate test or the threshold for shock induced activity o r vocalization. Although these measures would seem to rule out any gross effects of AP5 on peripheral pain sensitivity, it is not clear how these measures relate to the transmission of shock information in the amygdala, which is probably critical for the formation of conditioned fear. Without a careful analysis of changes in shock-induced single-unit activity in the amygdala, it is difficult to rule out the possibility that AP5 blocks fear conditioning by reducing the ability of footshock to activate neurons in the amygdala. This problem can be partly addressed using second-order conditioning, which consists of first pairing a neutral stimulus, such as light, with a shock (first-order association) and then pairing a second neutral stimulus, such as a tone, with the light (second-order association). Second-order fear conditioning is defined by increased fear (e.g., increased startle) in the presence of the tone, relative to groups that had the tone and light presented together in a nonpaired fashion or that had the tone and light paired, but the light and shock presented in a nonpaired fashion. William A. Falls and I now have preliminary evidence that local infusion of AP5 into the amygdala blocks the formation of second-order conditioning. Because this procedure does not involve stock presentation during the second-order conditioning phase, the blockade of this type of excitatory conditioning cannot be attributed to an interruption of shock information at the level of the amygdala. Furthermore, because these same doses of AP5 d o not block the expression of fear-potentiated startle to the light (e.g., Miserendino et al., 1990), the blockade of secondorder conditioning would be difficult to ascribe to a blockade of amygdala activation by the light during the second-order conditioning phase. Taken together, these data provide further evidence that AP5 in the amygdala blocks excitatory conditioning without affecting gross sensory transmission in the amygdala.

E. NMDA ANTAGONISTS DELAY EXTINCTION OF FEAR-POTENTIATED STARTLE Clinically, the inability to eliminate fear and anxiety ranks as one of the major problems in psychiatry. Although a good deal is known about neural systems involved in the acquisition of fear, much less is known

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about the neural systems that might be involved in extinction of conditioned fear (cf. LeDoux et nl., 1989). To begin to approach this problem, we measured whether blockade of excitatory amino acid receptors at the level of the amygdala would alter the process of experimental extinction (Falls et al., 1992). Rats were implanted with bilateral cannulae aimed at

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FIG. 9. AP5 blocks extinction after local infusion into the basolateral nucleus of the arnygdala. Mean amplitude startle response o n Noise-Alone trials (black bars) and LightNoise trials (white bars), and the difference between these two trial types ( + standard error of the mean, hatched bars) prior to (Pre) or following (Post) presentation of 60 lights (A) or a n equivalent amount of exposure to the experimental context (B) in rats infused with either 50 nmol AP5 or its vehicle. (A) Rats that received vehicle immediately before Light-Alone presentations showed a significant reduction in fear-potentiated startle (i.e., extinction), whereas rats that received AP5 did not. (B) Exposure to the experimental context alone was not sufficient to produce extinction. [From Falls el al. (1992)with permission from the Society for Neuroscience.]

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the basolateral nucleus of the amygdala and trained for potentiated startle in the usual way. One week later, all animals were given an initial short test session and subsequently matched into four groups each having equivalent levels of fear-potentiated startle. The next day half the animals were presented with 30 lights not followed by shocks. Five minutes before this extinction session, one group was infused with AP5 and one group with the ACSF vehicle. The two remaining groups were treated identically, except no lights were presented. They were placed into the test cages immediately after receiving either AP5 o r ACSF. Twenty-four hours later, all rats were tested for fear-potentiated startle. Figure 9 shows that animals infused with AP5 or ACSF, but not given light-alone extinction trials had levels of potentiated startle equivalent to that observed in their initial test. Animals infused with ACSF immediately before the light-alone trials had very little potentiated startle on their second test, indicating that extinction had occurred. In contrast, animals infused with AP5 immediately before their light-alone trials displayed levels of potentiated startle that did not differ from their initial test or from the groups not given light-alone trials, and significantly higher levels than the group given ACSF and light-alone trials. These data indicate, therefore, that AP5 infused into the amygdala blocked extinction of fear-potentiated startle, suggesting that an NMDA-dependent mechanism in the vicinity of the amygdala may be important for extinction of conditioned fear.

XV. Conclusions

An impressive amount of evidence from many different laboratories using a variety of experimental techniques indicates that the amygdala plays a crucial role in conditioned fear and anxiety, as well as attention. Many of the amygdala projection areas are critically involved in specific signs that are used to measure fear and anxiety. Electrical stimulation of the amygdala elicits a pattern of behaviors that mimic natural or conditioned states of fear. Lesions of the amygdala block innate or conditioned fear, as well as various measures of attention, and local infusion of drugs into the amygdala has anxiolytic effects in several behavioral tests. Lesions of the amygdala block the expression of fear-potentiated startle, regardless of when they are performed after training. Animals cannot acquire this form of conditioned fear if the lesions of the central nucleus are given prior to any training. However, if they are first trained with an intact amygdala, then lesioned, and then retrained, they can

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require fear-potentiated startle. This suggests that the central nucleus of the amygdala is critical for the acquisition of conditioned fear. However, when it is present, other systems, yet to be identified, seem to learn in parallel. When the central nucleus is then lesioned, these other systems can take over to support fear conditioning. This suggests a critical role of the amygdala in the initial acquisition of conditioned fear. It is possible that LTP in the amygdala may mediate this form of plasticity because an NMDA-dependent form of LTP has been observed in the amygdala and local infusion of NMDA antagonists into the amygdala blocks the formation of conditioned fear memories measured with several different tests of fear. A better understanding of brain systems that inhibit the amygdala as well as the role of its very high levels of peptides may eventually lead to the developoment of more effective pharmacological strategies for treating clinical anxiety disorders and perhaps memory disorders as well.

Acknowledgments

Research reported in this chapter was supported by NIMH Grant MH-25642, MH47840, Research Scientist Development Award MH-00004, a grant from the Air Force Office of Scientific Research, and the State of Connecticut.

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EXClTOTOXlClTY AND NEUROLOGICAL DISORDERS: INVOLVEMENT OF MEMBRANE PHOSPHOLIPIDS

Akhlaq A. Farooqui and Lloyd A. Horrocks Department of Medical Biochemistry, The Ohio State University, Columbus, Ohio 43210

I. Introduction 11. Classification of Excitatory Amino Acid Receptors

111.

IV.

V. VI.

VII. VIII. IX.

A. The NMDA Receptor and Its Multiplicity B. T h e AMPA Receptor and Its Multiplicity C. T h e Kainate Receptor and Its Multiplicity D. The L - A P ~Receptor E. T h e Metabotropic Receptor and Its Multiplicity Excitatory Amino Acid Receptors and Neural Membrane Phospholipid Metabolism A. Metabotropic Receptors and Phospholipase C B. Ionotropic Receptors and Phospholipase A2 Role of Enhanced Excitatory Amino Acid-Mediated Phospholipid Metabolism in Developing Brain Possible Mechanism of Cell Injury Caused by Excitatory Amino Acids Excitatory Amino Acid Receptors, Phospholipid Metabolism, and Neurological Disorders A. Alzheimer Disease B. Ischemia C. Spinal Cord Injury D. Traumatic Brain Injury E. Epilepsy F. Guam-Type Amyotrophic Lateral SclerosislParkinsonism-Dementia G . Huntington Disease H. Domoic Acid Neurotoxicity I. AIDS Dementia Complex Excitatory Amino Acid Receptor Antagonists and the Treatment of Neurological Disorders Conclusion Summary References

1. Introduction

Phospholipids form the backbone of neural membranes and provide the membranes with suitable fluidity and permeability (Porcellati, 1983; Farooqui and Horrocks, 1985). The distribution of phospholipids is normally asymmetric across the plane of the plasma membrane, with phosINTERNATIONAL REVIEW OF NEUROBlOLOGY, VOL. 36

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phatidylethanolamine and phosphatidylserine concentrated in the inner leaflet and phosphatidylcholine and sphingomyelin concentrated in the external leaflet (Porcellati, 1983). Normal membrane phospholipid homeostasis is based on a balance between phospholipid catabolism and resynthesis by reacylation and de novo synthesis pathways (Porcellati, 1983). The phospholipid bilayer is penetrated to varying degrees by receptors, enzymes, and ion channels that protrude differentially through the membrane or are localized predominantly on the intracellular or extracellular membrane surface. One of the important functions of the biomembrane is the regulation of ion homeostasis. Neural membranes are highly interactive and dynamic and therefore the interaction of ligand with receptor markedly affects their phospholipid metabolism (Porcellati, 1983; van Rooijen et at., 1986; Nahorski et al., 1986; Loffelholz, 1989; Farooqui and Horrocks, 1985). This in turn regulates the microenvironment for the activities of the membrane-bound enzymes and ion channels (Farooqui and Horrocks, 1985). Excitatory amino acids, such as glutamate and aspartate, are major neurotransmitters in the mammalian central nervous system. Although these amino acids are responsible for normal excitatory transmission, they also represent a potential source of neurotoxicity. For this reason the concentration of glutamate surrounding its receptors is regulated by several mechanisms (Mayer and Westbrook, 1987).Abnormally low levels of glutamate can compromise normal levels of excitation, whereas excessive levels can produce toxic effects (Rothman and Olney, 1986). During the past decade, significant information has accumulated on the importance of excitatory amino acids and their receptors in neural membrane phospholipid catabolism (Nicoletti et al., 1986a,b, 1987b, 1988a, 1990). Degradation of membrane phospholipids, induced by excitatory amino acid receptors, results in generation of second messengers that transduce extracellular signals into the nerve cell, and this process may be involved in neuronal survial, synaptogenesis, neuronal plasticity, and learning and memory processes (Muller et al., 1988; Wood et al., 1990; Collingridge and Bliss, 1987). Current interest in excitatory amino acids is also based on the belief that excitatory amino acid receptors are involved in neural cell injury in a variety of neurological disorders. T h e molecular mechanisms of neural cell injury remain unknown; however, cellular injury is often accompanied by marked degradation of membrane phospholipids, with the generation of free fatty acids, lysophospholipids, lipid peroxides, and eicosanoids. The accumulation of free arachidonic acid, a fatty acid normally stored exclusively in membrane phospholipids, has been found to be a sensitive indicator of altered phospholipid metabolism and membrane injury. This chapter ( a ) at-

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tempts to integrate evolving concepts of the relationship between excitatory amino acid receptors and phospholipid metabolism and (b) discusses and evaluates the importance of excitatory amino acid-induced changes in phospholipid metabolism in neurological disorders. It is hoped that this discussion will promote further studies on the molecular mechanism of cellular injury that may involve overstimulation of excitatory amino acid receptors, abnormal membrane function, and marked degradation of membrane phospholipids.

II. Classification of Excitatory Amino Acid Receptors

T h e dicarboxylic amino acids, glutamate and aspartate, are putative excitatory amino acid neurotransmitters in the central nervous system. Treatment of neurons with glutamate, aspartate, and their related analogs causes cell swelling, vacuolization, and eventual cell death (Olney, 1989). This observation prompted Olney and his associates to propose an “excitotoxin” concept of neuronal cell death (Rothman and Olney, 1986, 1987; Olney, 1989). Excitotoxicity refers to a paradoxical phenomenon whereby the neuroexcitatory action of glutamate and related compounds becomes transformed into a neuropathological process that can rapidly cause neuronal cell death (Olney, 1989). The mechanism by which glutamate and its analogs (Fig. 1) kill neurons is not clear. However, it has been shown that these metabolites produce their effects by interacting with certain receptors on the cell surface. These receptors are called glutamate receptors or excitatory amino acid receptors (Mayer and Westbrook, 1987; Monaghan et al., 1989). Based on their transduction mechanisms, glutamate receptors are classified into two subclasses, ionotropic and metabotropic. Ionotropic glutamate receptors are coupled directly to cation channels and mediate fast excitatory synaptic responses at various synapses throughout the central nervous system. Metabotropic glutamate receptors are coupled to the second messenger system through GTP-binding protein (G-protein) and are involved in the generation of slow synaptic responses and modulation of neuronal excitability (Monaghan et al., 1987; Schoepp et al., 1990a; Farooqui and Horrocks, 1991). Ionotropic glutamate receptors are further classified according to their preferential agonists into ( a )N-methyl-D-aspartate (NMDA) receppropionate (AMPA) tors; (6) a-amino-3-hydroxy-5-methyl-4-isoxazole receptors; (c) kainate receptors; and ( d ) the AP4 receptor, which is identified by the antagonistic action of 2-amino-4-phosphonobutyrate.

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AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

L-Glutamic acid

NMDA

Kainic acid

COOH

I%

Quisqualic acid

AMPA

lbotenic acid

L-AP4

BMAA

Domoic acid

FIG. 1 . Structures of glutamate and its analogs that induce excitotoxicity.

NMDA receptors are selectively antagonized by a-amino-w-phosphonocarboxylic acids, including 2-amino-5-phosphonovalerate (APV). AMPA and kainate receptors are markedly antagonized by 6-cyano7-nitroquinoxaline-2,3-dione(CNQX) and 6,7-dinitroquinoxaline-2,3dione (DNQX). Selective antagonists of kainate receptors (2-amino-3[3-(carboxymethoxy)-5-methylisoxazol-4-yl] propionic acid (AMOA) and 2-arnino-3-[2-(3-hydroxy-5-methyl-isoxazol-4-y1) methyl-3-oxisoxazoline-4-ylI propionic acid (AMNH)) have also been reported. The four receptor subtypes can also be distinguished on the basis of electrophysiological studies showing differences in cell firing patterns

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

27 1

and membrane conductance (Mayer and Westbrook, 1987; Monaghan et al., 1989). Regional distribution studies have indicated that the highest density of kainate receptors occurs in the stratum lucidum of the hippocampus (mossy fiber system) and in the i m e r and outer layers of the neocortex. T h e highest density of NMDA receptors is found in the hippocampus (stratum radiatum) and in the striatum, thalamus, and cerebral cortex. Distribution of AMPA receptors is similar to NMDA receptors, but in the cerebellum AMPA receptors predominate in the molecular layer and NMDA receptors predominate in the granule cell layer (Monaghan et al., 1983; Olverman et al., 1986). Nothing is known about the regional distribution of the L-AP4 receptor. The kainate and AMPA receptors have also been purified (Henley and Barnard, 1989; Hampson and Wenthold, 1988). High-energy radiation inactivation of membrane preparations has indicated that kainate, AMPA, and NMDA receptors have approximate molecular masses of 76, 52, and 209 kDa, respectively (Honor6 and Drejer, 1988). T h e three receptor subtypes are linked to membrane cation channels that open as agonist binds to the receptor site (Holopainen et al., 1990; Murphy and Miller, 1988; Holopainen et al., 1989). The cDNAs for NMDA, kainate, and AMPA receptors have recently been cloned (Sakimura et al., 1990; Sato et al., 1993).

A N D ITS MULTIPLICITY A. THENMDA RECEPTOR

T h e discovery of potent and selective agonists and antagonists (Figs. 1, 2, and 3) has resulted in extensive information on the NMDA receptor-channel complex (Wood et al., 1990; Fagg and Baud, 1988). It consists of four domains: (1) the transmitter recognition site with which NMDA and L-glutamate interact; (2) a cation binding site located inside the channel where Mg2+can bind and block transmembrane ion fluxes; (3) a PCP binding site that requires agonist binding to the transmitter recognition site, interacts with the cation binding site, and at which a number of dissociative anesthetics (PCP and ketamine), 2 opiate Nallylnormetazocine (SKF-10047), and MK-80 1 bind to function as open channel blockers; and (4) a glycine binding site that appears to modulate allosterically the interaction between transmitter recognition site and PCP binding site (Fagg and Baud, 1988). Radioligand binding studies have indicated the presence of a polyamine binding site in the NMDA receptor complex (Singh et al., 1990; Ransom and Stec, 1988). Like the glycine binding site, the polyamine modulatory site is not fully activated under normal conditions. In addition there is evidence that ZnZi, acting

272

AKHLAQ A. FAROOQUI A N D LLOYD A . HORROCKS

HO

OH

HO

OH

Y

‘P’

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)-OH

0

0

D-AP5

d

D-AP7

b k O H 0

%H

CPP

D-ASP-AMP

HO

OH

CGP 37849

CGS 19755

FIG. 2. Structures of competitive antagonists of the NMDA receptor.

at a separate site near the mouth of the ion channel, acts as an inhibitory modulator of channel function (Westbrook and Mayer, 1987). The NMDA receptor complex also contains an arachidonic acid binding site. The amino acid sequences of this binding site resemble fatty acid binding proteins (Petrou et al., 1993). Other modulators of NMDA receptors include sulfhydryl redox reagents and H ions. Normal functioning of the NMDA receptor complex depends on a dynamic equilibrium among various domain components. Loss of equilibrium during membrane perturbation may cause the entire system to malfunction and result in an expression of excitotoxicity (Olney, 1989). An important consequence of excitatory amino acid activation is the influx of Ca2+ into neurons (Murphy and Miller, 1988; Holopainen et al., 1989, 1990). Some studies have indicated that the NMDA receptor site is primarily site-coupled to Ca“ influx (MacDermott et al., 1986), but the other receptor subtypes +

EXCITOTOXICITY A N D NEUROLOGICAL DISORDERS

PCP

SKF 10.047

Demxadrol

Dextrorphan

LY 154045

MK 801

273

0

MLV 5860

Ketarnine

FIG. 3. Structures of noncompetitive antagonists of the NMDA receptor.

have also been shown to increase Ca2+ influx into cells (Murphy and Miller, 1988; Holopainen et al., 1989, 1990). Collective evidence suggests that when the membrane is depolarized, the Mg2+block is relieved and the receptor can be activated by glutamate. Activation of the NMDA receptor therefore requires the association of two synaptic events: membrane depolarization and glutamate release. This associative property provides the logic for the role of the NMDA receptor in sensory integration, memory function, and the coordination and programming of motor activity (Collingridge and Bliss, 1987; Lester et al., 1988). In addition,

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AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

the Ca2+influx through the NMDA receptor is, in part, responsible for the neurotoxic actions of glutamate. Multiple subtypes of NMDA receptors exist (Barnes and Henley, 1992; Monyer et al., 1992). Molecular cloning studies have identified at least four complementary DNA species in rat brain, encoding NMDA receptor subtypes NMDARl (NRl), NMDAR2A (NR2A), NMDAR2B (NR2B), and NMDAR2C (NRPC). NRSA, NRSB, and NR2C channels resemble each other 50 to 70% in amino acid sequence. NRPA and NRPC channels differ in gating behavior, magnesium sensitivity, and regional distribution in rat brain. All NMDA receptor subtypes contain phosphorkinase and protein kinase ylation sites for Ca2+-calmodulin-dependent C at the cytoplasmic domains. These kinases play a crucial role in the induction and maintenance of long-term protentiation (Barnes and Henley, 1992; Gasic and Hollman, 1992).

B. THEAMPA RECEPTOR AND ITS MULTIPLICITY Electrophysiological studies have indicated that the AMPA receptor may be involved in fast excitatory synaptic transmission (Mayer and Westbrook, 1987; Monaghan et al., 1989). AMPA-activated channels are voltage-independent and selective for monovalent cations (Mayer and Westbrook, 1987). Unavailability of a specific ligand has resulted in a lack of pharmacological and biochemical information of the AMPA receptor. Using the most specific radioligand [3H]AMPA,the AMPA receptor was solubilized from chick brain (Henley and Barnard, 1989). The rank order of potency for competitive ligands in displacing ['HI AMPA binding is reported to be AMPA = quisqualate > 6-cyano-7-nitroquinoxaline2,3-dione > L-glutamate > kainate. A purified AMPA receptor from rat brain has a molecular mass of 105 kDa. Treatment of rat brain membranes with phospholipase A, significantly increases the binding of [3H]AMPA to the AMPA receptor. Kinetic analysis has indicated that phospholipase A, treatment increases the affinity of the AMPA receptor without changing the maximum number of sites. In contrast, phospholipase A, treatment does not modify the binding of kainate to the kainate receptor nor of glutamate and glycine to the NMDA receptor (Massicotte and Baudry, 1990). T h e AMPA receptor shows increased affinity for AMPA in the presence of SCN- ions. High-energy radiation inactivation analysis has shown that a regulatory unit is coupled to the AMPA receptors (Honore et al., 1985). Quinoxalinediones (CNQX, DNQX, and NBQX) are selective and potent competitive antagonists and the 2,3benzodiazepine muscle relaxant GY K 1 52466 ([ 1-(4-aminophenyl)-4-

275

EXCITOTOXICITY A N D NEUROLOGICAL DISORDERS

methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine HC1) is a highly selective noncompetitive antagonist. A biochemical analysis of the AMPA receptor channel purified from rat brain has indicated that this receptor is a pentameric complex composed of combinations of the GluR1-4 subunits (see below), and the occurrence of additional subunits is very unlikely (Barnes and Henley, 1992). However, the subunit composition of the native AMPA receptors involved in single neuron responses remains unknown. Collective evidence from binding studies indicates that AMPA receptors mediate those neurotoxic events that involve postsynaptic neuronal membranes (Mayer and Westbrook, 1987). C. THEKAINATERECEPTOR AND ITS MULTIPLICITY Very little is known about the kainate receptor at the molecular level. Detailed pharmacological studies have been hindered by a lack of specific agonists and antagonists (Fig. 4). Ligand binding studies with [3H]kainate have demonstrated specific saturable and high-affinity binding to brain

H

H

OZN& Nc

02N* I

I Hz-2s

02N

H

H

CNQX

%

OzN

I

H

DNQX

NBQX COOH

HO 0

N H>

COOH

(1S,3R)-ACPD

“2

E

(RS)-4-C ar boxy-3-hydroxyphenylglycine FIG.4. Structures of selective competitive antagonists of the AMPAikainate and transACPD receptors. CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DNQX, 6,7-dinitroquinoxaline-2.3-dione; NBQX, 6-nitro-7-sulfamoylbenzo ( f ) quinoxaline-2,3-dione; (lS,SR)-ACPD, (1S,3R)-l-aminocyclopentane-l,3-dicarboxylic acid; (RS)-4-carboxy-3hydroxyphenylglycine, a competitive trans-ACPD antagonist.

276

AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

membranes. Displacement studies with analogs of kainate have indicated a good correlation between excitatory and neurotoxic potencies of kainate (Coyle, 1983). In spinal cord C-fiber afferents, kainate receptors display an agonist potency of domoate > kainate > AMPA >> glutamate (Evans el al., 1987). The same pattern has been reported for the displacement of [3H]kainate binding to rat brain membranes (London and Coyle, 1979). Kainate binding sites have recently been solubilized and purified from rat and frog brain (Hampson et al., 1987; Hampson and Wenthold, 1988). Gel filtration on Sepharose 6B indicates a molecular mass of 550 kDa. Polyacrylamide gel separations give a molecular mass of 48 kDa. The large differences in molecular mass determined by the above procedures may result from the breaking of the kainate receptor complex into subunits. AMOA and A M N H have been reported to be selective antagonists for the kainate receptor (Frandsen et al., 1990). Electrophysiological studies have indicated that rat hippocampal neurons show two different types of current response to kainate. In the type I response, kainate causes little increase in membrane Ca2+ permeability and there is an outward rectification in the current-voltage plots. T h e type I1 kainate response is characterized by prominent Ca2+Permeability hnd an inward rectification in the current-voltage plots (Ozawa et al., 1991). Xenopus brain is an exceptionally rich source of both kainate and AMPA binding sites, and the relationship between these sites has been studied in detail (Barnes and Henley, 1992). In this tissue the functional interaction between kainate and AMPA can be unequivocally correlated with a physical colocalization of the two types of sites in a single protein complex. The two sites coexist in a 1 : 1 ratio and cannot be separated by various physical and biochemical procedures (Barnes and Henley , 1992; Miller, 1991). The purified protein shows high-affinity binding for both AMPA and kainate and they are mutually and fully competitive with K, values identical to the Kd values for the radioligand (AMPA 34 nM and kainate 15 nM). Molecular cloning studies have identified at least six kainate/AMPA receptor subtypes (named GluR1, GluR2, GluR3, GluR4, GluR5, and GluR6). The AMPA and kainate receptor subunits are large, with a subunit molecular mass of about 100 kDa, and show little sequence homology to subunits of other receptors. All of these subtypes exhibit an individual distribution in the nervous system and also show alteration in expression during development. It is interesting to note that GluR6 is markedly stimulated by kainate, but not by AMPA, and is therefore, strictly speaking, a kainate rather than a kainate/AMPA receptor (Barnes and Henley, 1992; Sat0 et al., 1993; Gasic and Hollman, 1992).

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

277

D. THEL - A P ~ RECEPTOR In contrast to NMDA, AMPA, and kainate receptors, the L - A Precep~ tor is characterized by the agonistic action of L - A Pon ~ certain glutamateusing synapses (Monaghan et al., 1989). Superfusion of rat hippocampal slices with L - A P markedly ~ blocks lateral perforant path-evoked excitation of dentate gyrus granule cells in a stereospecific manner (Koerner and Cotman, 1981). Based on various pharmacological studies, it has been suggested that L - A P ~ may act at both pre- and postsynaptic sites (Lanthorn et al., 1984). Quanta1 analysis has demonstrated that in the case of the mossy fiber-CA3 synapse of guinea pig hippocampus, L-AP4 may act only by a presynaptic mechanism (Monaghan et al., 1989). Studies with cultured hippocampal neurons have also indicated L - A P inhibition ~ of synaptic response by a presynaptic mechanism that is mimicked by Lglutamate (Lanthorn et al., 1984), suggesting that L - A P is~ a presynaptic receptor agonist. Recent pharmacological studies have indicated that the AP4 receptor is a G-protein-coupled glutamate receptor that is distinct from the metabotropic glutamate receptor in the following ways. First, it does not stimulate inositol phospholipid turnover in several different mammalian preparations (Trombley and Westbrook, 1992) o r in oocytes injected with whole-brain mRNA or in the cloned metabotropic receptor transcript. Second, staurosporine does not block the response of L-AP4, indicating that activation of protein kinase C is not required (Trombley and Westbrook, 1992). Although the occurrence of the L-AP4 receptor has been clearly established in various preparations by physiological studies, its biochemical characterization has been elusive (Monaghan et al., 1989). Thus, very little is known about the molecular properties of this receptor (Monaghan et al., 1989).

E. THEMETABOTROPICRECEPTOR A N D ITS MULTIPLICITY

This receptor acts through a GTP-binding protein-dependent mechanism to elicit polyphosphoinositide hydrolysis (Monaghan et al., 1989). Its activation leads to increased cellular levels of inositol 1,4,5trisphosphate and diacylglycerols with mobilization of Ca2+from intracellular stores. It responds to quisqualate but not to AMPA; thus it is AMPA-insensitive (Sladeczek et al., 1985, 1988). Polyphosphoinositide hydrolysis can also be enhanced by ibotenate, a compound that also has affinity for NMDA ionotropic excitatory amino acid receptors (Sladeczek et al., 1988). However, NMDA does not mimic this effect of ibotenate on the metabotropic receptor, and selective NMDA antagonists do not

278

AKHLAQ A. FAROOQUI A N D LLOYD A . HORROCKS

attenuate ibotenate stimulation in the rat hippocampus (Sladeczek et al., 1988). Thus, ibotenate does not stimulate polyphosphoinositide hydrolysis via activation of ionotropic NMDA receptors but may act on the metabotropic excitatory amino acid receptor that is also activated by quisqualate (Schoepp et al., 1990b). This well-characterized metabotropic receptor is the Q p receptor (Sladeczek et al., 1988; Manzoni et al., 1990a). Potent stimulation of polyphosphoinositide turnover in mammalian brain slices also occurs with the rigid glutamate analog (*)trans1-aminocyclopentyl- 1,3-dicarboxylate (ACPD) (Palmer et al., 1989; Desai and Conn, 1990). The Q p receptor has recently been renamed the transACPD receptor (Monaghan et al., 1989; Desai and Conn, 1990). T h e available preparations of (-+)trans-ACPDare a racemic mixture of 1S,3R-, 1R,3S-, lR,3R-, and lS,3S-stereoisomers (Fig. 5). Of the four compounds, lS,SR-ACPD is responsible for the stimulation of metabotropic receptors. At micromolar concentrations, lS,3R-ACPD potently mobilizes intracellular calcium in neurons by stimulating polyphosphoinositide hydrolysis. 1R,3R-stereoisomer is a potent selective NMDA receptor agonist. 1S,3S-isomer has a similar but very weak activity on NMDA receptors. lR,SS-ACPD is a weak partial agonist for metabotropic receptors (Schoepp et al., 1990a). T h e trans-ACPD receptor is also stimulated

HOOC,/O\EOOH H

NH2

HOOC

COOH

1R,3R

1S , 3 S

1R.3S

1S,3R

trans-(dicarboxylFACPD FIG. 5. Structures of stereoisomers of 1 -aminocyclopentane-1,3-dicarboxyIic acid; 1R,3R, (lR,3R)-l-arninocyclopentane1,3-dicarboxylic acid; 1S,3S, (1S,3S)-l-arninocyclopentane-l,3-decarboxylic acid; 1R,3S, (1R,3S)-l-aminocyclopentane-l,3-dicarboxylic acid; acid. and 1S,3R, (1S,3R)-l-aminocyclopentane-1,3-decarboxylic

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

279

by (2S, 3S, 4 s ) a-(carboxycyclopropyl) glycine (L-CCG-1) in hippocampal synaptoneurosomes (Nakagawa et al., 1990). Two prominent trans-ACPD receptor-mediated electrophysiological effects have been identified in hippocampal neurons (Stratton et al., 1989, 1990). They are membrane depolarization and blockade of the slow after hyperpolarization mediated by a calcium-activated potassium conductance. The most effective inhibitors of metabotropic receptors are 2-amino-3-phosphonopropionate (AP3) and 2-amino-4-phosphonobutyrate(AP4), both of which block responses in a noncompetitive fashion (Schoepp et al., 1990a). The competitive antagonists for the trans-ACPD receptor include (RS )-4-carboxyand 3-hydroxyphenylglycine, (RS)-a-methyl-4-carboxyphenylglycine, (S)-4-~arboxyphenylglycine (Fig. 4). A cDNA for the metabotropic glutamate receptor has recently been cloned from the rat cerebellum cDNA library (Masu et al., 1991; Barnes and Henley, 1992). These studies have demonstrated that the cloned receptor (mGluR1) stimulates phosphatidylinositol turnover through a pertussis toxin-sensitive G-protein. In transfected CHO cells, the rank of phosphatidylinositol hydrolysis potency is quisqualate > Lglutamate 2 ibotenate > L-homocysteine sulfinate 2 (*)trans-ACPD. This receptor also evokes the stimulation of CAMP formation and arachidonic acid release with comparable agonist potencies. The mRNA for this receptor is predominantly expressed in hippocampal and cerebellar neuronal cells. This receptor shares no sequence similarity with other members of G-protein-coupled receptors and possesses a unique structure (molecular mass 1 13 kDa) with a large hydrophilic sequence preceding the seven putative transmembrane domains (Masu et al., 1991; Barnes and Henley, 1992; Aramori and Nakanishi, 1992). The occurrence of four additional subtypes of mGluR (mGluR2, mGluR3, mGluR4, and mGluR5) has also been reported recently (Barnes and Henley, 1992; Aramori and Nakanishi, 1992; Tanabe et al., 1992). The four newly identified receptors share a high degree of sequence similarity with mGluR1 and possess a large extracellular domain preceding the seven putative transmembrane segments. mGluR2, mGluR3, and mGluR4, unlike mGluR1, are coupled to an inhibitory CAMP cascade. Although the precise signaling pathway of mGluR3 and mGluR4 remains to be elucidated, no linkage has been found between these receptors and polyphosphoinositide turnover. mGluR5 has 60% sequence homology with mGluR1 and it is coupled to phosphoinositide response. Thus, among the five metabotropic receptors reported, mGluRl and mGluR5 are the only ones capable of stimulating polyphosphoinositide turnover (Gasic and Hollman, 1992).

280

AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

In sztu localization studies have indicated that mRNA encoding for different metabotropic receptors is differentially distributed in brain. For example, mRNA for mGluRl is localized in virtually all neuronal cells of the dentate gyrus and areas CA2 through CA4 of rat hippocampus, whereas mRNA for mGluR5 is specifically found in pyramidal cells throughout areas CAI through CA4 of the hippocampus and granule cells of the dentate gyrus (Barnes and Henley, 1992; Masu et al., 1991; Gasic and Hollman, 1992). mRNA encoding mGluRl is concentrated in Purkinje cells of the cerebellum, whereas mRNA for mGluR5 is found in a small population of Golgi cells in this brain region. The differential distribution of metabotropic receptors suggests a highly specialized role for them, possibly related to the stimulation of specific second-messenger systems performing specific functions (Masu et al., 1991; Barnes and Henley, 1992; Gasic and Hollman, 1992). The function of the trans-ACPD receptor remains unknown. However, there is growing speculation that it plays a key role in synaptic growth and synaptic stabilization (Monaghan et al., 1989; Manzoni et al., 1990b). The metabotropic excitatory amino acid receptors are also implicated in long-term potentiation (LTP), a long-lasting increase in synaptic efficacy induced by high-frequency stimulation of specified pathways in the hippocampus. It is well established now that protein kinase C is necessary for the maintenance of LTP (Kaczmarek, 1987). T h e observation that metabotropic excitatory amino acid receptors are able to activate protein kinase C suggests that they may be involved in the initiation of the late component of LTP (Manzoni et al., 1990b).

II1. Excitatory Amino Acid Receptors and Neural Membrane Phospholipid Metabolism

In neural membranes the degradation of membrane phospholipids is linked to various types of receptors (van Rooijen et al., 1986; Nahorski et al., 1986; Loffelholz, 1989; Pelech and Vance, 1989). The initial event following receptor stimulation is the activation of specific phospholipases A,, C, or D. The activation of these enzymes results in generation of second messengers such as diacylglycerol, inositol 1,4,5-trisphosphate, and arachidonic acid (Berridge, 1984; Nishizuka, 1984b, 1986, 1989). Diacylglycerol activates protein kinase C; inositol 1,4,5-trisphosphate mobilizes calcium from intracellular stores; and arachidonic acid and its oxygenated metabolites modulate neuronal activity by regulating activities of a number of enzymes including some isozymes of protein kinase C and membrane-bound Na' , K+-ATPases (Berridge, 1984; Nishizuka,

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

28 1

1984b, 1986, 1989; Farooqui et al., 1988a; Shimizu and Wolfe, 1990; Axelrod, 1990; Volterra, 1989).

A N D PHOSPHOLIPASE C A. METABOTROPICRECEPTORS

Stimulation of membrane phospholipid degradation by excitatory amino acid agonists has been reported to occur in a variety of neuronal preparations. These include mouse striatal cultures (Sladeczek et al., 1985, 1988), cerebellar granule cell cultures (Nicoletti et al., 1986b), rat hippocampus slices (Schoepp and Johnson, 1988; Schoepp, 1989; Ambrosini and Meldolesi, 1989), and rat brain synaptoneurosomes (Recasens et al., 1987). It is now well established that the trunsACPD receptor is involved in the turnover of polyphosphoinositide through the activation of phospholipase C (Fig. 6). Thus, in primary neuronal cultures from rat brain, excitatory amino acids stimulate polyphosphoinositide hydrolysis with a rank order of potency of quisqualate > ibotenate > glutamate > kainate, NMDA > a-amino-3hydroxyl 5-methyl-4-isoxazole propionate (Patel et al., 1990). A similar rank order of potency was also obtained in striatal neurons (Sladeczek et al., 1985, 1988), but a different rank order of potency (glutamate > quisqualate > kainate) was reported in cerebellar granule cell cultures (Nicoletti et al., 1986b). These differences may be contributed by regional differences in the excitatory amino acid reuptake process and/or regional heterogeneity of excitatory amino acid receptor regulation of phospholipase C (Patel et al., 1990). In cerebellar granule cells, glutamate, NMDA, and kainate can induce the translocation of protein kinase C into the plasma membrane from the cytosol and promote the phosphorylation of membrane proteins, receptors, and ion channels as yet unidentified (Vaccarino et al., 1987). Phencyclidine, APV, and Mg2+block the action of glutamate and NMDA, whereas the action of kainate is insensitive to Mg2+and APV, but inhibited by PDA. T h e translocation of protein kinase C by excitatory amino acids is also dependent on calcium influx (Manev et al., 1989).

B. IONOTROPIC RECEPTORS AND PHOSPHOLIPASE A, The occurrence of an NMDA-sensitive glutamate receptor that activates the release of arachidonic acid from membrane phospholipids of striatal neurons (Dumuis et al., 1988)and cerebellar granule cells (Lazarewicz et al., 1988) has been independently reported. Phospholipase A,

282

AKHLAQ A. FAROOQUI A N D LLOYD A. HORROCKS

Postsynaptic

KA and

Ca2+ I

Eicosanoids & free radicals

I

Activation of intracellular phospholipases, lipases, proteases and endonucleases

*

Cell death

FIG. 6. Hypothetical diagram illustrating the involvement of glutamate receptors, calcium ions, and membrane phospholipid degradation during neurotrauma and neurodegeneration. NMDA, N-methyl-~-aspartate;KA, kainate; AMPA, a-2-amino-3-hydroxy-5methyl-4-isoxazole-4-propionate; trans-ACPD, trans- 1-aminocyclopentyl-1,3-dicarboxyIate; PL. phospholipids: PLA,, phospholipase A,; AA, arachidonic acid; LIPO, lipoxygenase; CYCLO, cycloxygenase; PIP,, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; G, G-protein; DAG, 1,2-diacylglycerol;PKC, protein kinase C; IPS,inositol 1,4,5trisphosphate; Ca2+, intracellular calcium; ER, endoplasmic reticulum; and VSCC, voltage-sensitive calcium channels. IP3 mobilizes Ca2+ from ER. Ca2' entering through the VSCC, the NMDA channels, and from intracellular stores may stimulate PLA,, PLC, PKC, lipases, proteases, and endonucleases. Modified from Farooqui and Horrocks (1991).

may be the main effector responsible for the massive arachidonate release in response to the stimulation of the ionotropic NMDA receptor (Lazarewicz et al., 1988) (Fig. 6). Unlike the a-adrenergic receptor, where phospholipase A, is coupled to the receptor complex through a G-protein (Axelrod et al., 1988; Axelrod, 1990), in cerebellar granule cells the coupling of the NMDA receptor to phospholipase A, is not mediated by a G-protein (Lazarewicz et al., 1990). Thus it seems likely that the elevated intracellular concentration of Ca2+ ions, triggered by the opening of NMDA receptor-coupled cation channels, may serve the secondmessenger function in activating intracellular phospholipase A, (Lazarewicz et al., 1990). Similarly, the exposure of rat hippocampal neurons to

283

EXCITOTOXICITY A N D NEUROLOGICAL DISORDERS

NMDA causes a dose-dependent stimulation of arachidonic acid release from membrane phospholipids (Sanfeliu et al., 1990). This elevation in the release of arachidonic acid can be blocked in a dose-related manner by the NMDA receptor antagonist 2-amino-5-phosphovaleric acid or by Mg" , which blocks NMDA receptor-linked Ca2+ ion channels. T h e removal of external Ca2+inhibits NMDA-induced release, whereas treatment with calcimycin greatly stimulates the arachidonic acid release. T h e inhibitors of phospholipase A,, nordihydroguaiaretic acid and mepacrine, decrease the NMDA-dependent arachidonic acid release in a doserelated manner (Sanfeliu et d.,1990). Our studies with neuron-enriched cultures from fetal mouse spinal cord have indicated that kainate has no effect (Fig. 7), whereas glutamate and NMDA stimulate the activities of diacylglycerol and monoacylglycerol lipases in a dose- and time-dependent manner (Fig. 81, and this increase can be blocked by dextrorphan or MK-801 (Farooqui et al., 1993) (Fig. 9). At present it is not clear how the NMDA receptor is coupled to diacylglycerol and monoacylglycerol lipases. However, marked enrichment of polyphosphoinositide turnover is observed in striatal neurons after NMDA treatment. This suggests the involvement of phospholipase C. Diacylglycerol lipase probably hydrolyzes much of

25--

-

-f5

01

I

I

I

I

" I

I

I

I

I

O

Y

I

1

1 I

i

FIG. 7. Time-dependence of the effect of kainate on the activities of diacylglycerol (open square) and monoacylglycerol (solid square) lipases in neuron-enriched cultures from fetal mouse spinal cord. Dose-dependence was studied at 15 min. Time-dependence was studied at 100 p M . Each point is the mean of three experiments. Specific activity is expressed as nmol/min/mg protein.

284

AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS 40-

A

30 ,-

20

0

40

60 Concentration (PM)

100

80

35

30

10

5 I

0

I

1

1

4

8

1

1

1

1 1

t

12

16

20

24

28

Time (min) FIG. 8. Dose-dependence (A) and time-dependence (B) of the effect of glutamate on the activities of diacylglycerol (open square) and monoacylglycerol (solid square) lipases in neuron-enriched cultures from fetal mouse spinal cord. Dose-dependence was studied at 15 min. Time-dependence was studied at 50 p M . Each point is the mean of three experiments with the SEM indicated by the bar. Specific activity is expressed as nmol/min/ rng protein. From Farooqui et al. (1993).

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

285

30 -r

c ._ 5 ._ c

20

0

m

0 ..c

0

g (u

10

0 1

2

3

4

Treatment

FIG.9. Effect of glutamate and MK-80 1 on diacylglycerol (open bar) and monoacylglycerol (hatched bar) lipases. Each point is the mean of three experiments with the SEM indicated by the bar. Specific activity is expressed as nmol/min/mg protein. Incubations were done for 15 min. Bars: 1 , control; 2, glutamate (100 p M for 15 min); 3, MK-801 (150 p M for 15 min); 4, glutamate (100 p M ) + MK-801 (150 F M ) . From Farooqui etal. (1993).

the diacylglycerol released by the action of phospholipase C. Thus, both phospholipase A, and diacylglycerol lipase and monoacylglycerol lipase pathways participate in the release of arachidonate in response to glutamate and its analogs. However, the relative contributions of these pathways are still obscure. Phospholipase A, may be associated with the signal transduction process and diacylglycerol lipase activity may be involved in neuronal plasticity. Unlike glutamate, which becomes excitotoxic only after it is released extracellularly, arachidonic acid may elicit its effects in both the extracellular and the intracellular spaces. It produces a variety of detrimental effects on membrane structures, activities of membrane-bound enzymes, and neurotransmitter uptake systems (Chan et al., 1983; Yu et al., 1986). The addition of arachidonic acid to the rat dentate gyrus causes a slow onset and persistent increase of synaptic activity, which is accompanied by a marked elevation in the release of glutamate (Lynch and Voss, 1990). It seems likely that arachidonic acid and its metabolites, which are generated by NMDA-receptor stimulation at the postsynaptic level, may cross the synaptic cleft to act at the presynaptic level and thus may act as retrograde messengers for the activation of protein kinase C ( y -

286

AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

form) and long-term potentiation (Axelrod, 1990; Lynch and Voss, 1990; Piomelli and Greengard, 1990). The stimulation of polyphosphoinositide turnover by excitatory amino acids does not occur exclusively in neurons. Several studies have indicated that rat cortical astroglia can also respond to AMPA, ibotenate, and kainate with increased polyphosphoinositide hydrolysis (Pearce et al., 1986; Milani et al., 1989). Other investigators have failed to observe the stimulation of polyphosphoinositide turnover in astroglia (Nicoletti et al., 1986~).In studies (Pearce et al., 1986; Milani et al., 1989) where stimulation of phosphoinositide metabolism was observed in astroglial cells with quisqualate, ibotenate, and kainate, no effect was detected with NMDA or quinolinic acid. The nonselective glutamate receptor antagonist y-D-glutamylglycinefully inhibited glutamate agonist-induced polyphosphoinositide breakdown. Pretreatment of the astroglial cells with phorbol esters negated the effects of excitatory amino acid receptor agonists, suggesting a feedback role for protein kinase C in glutamate receptor-linked signal transduction (Gonzales et al., 1987; Milani et al., 1989). Because few studies have been performed on the stimulation of polyphosphoinositide hydrolysis in astrocytes (Milani et al., 1989; Sanfeliu et al., 1990; Nicoletti et al., 1990),it is premature to compare the pharmacology and distribution of excitatory amino acid receptors between neurons and astrocytes. However, the trans-ACPD receptor on astroglial cells seems to display some features different from those of the receptor expressed by neurons. DNQX, an AMPA antagonist, failed to block inositol phospholipid breakdown by quisqualate applied to astroglia, but was effective with granule cells (Milani et al., 1989). Differences between the trans-ACPD receptor types on neurons and glia can also be seen in the cyclic GMP response to an excitatory amino acid agonist (Novelli et al., 1987). Despite the above differences, trans-ACPD receptors appear to share a common molecular signaling mechanism. The differences in excitatory amino acid receptor subtype expression between neurons and glial cells may be responsible for neural behaviors such as the resistance of astroglial cells to glutamate neurotoxicity (Rothman, 1984; Vibulsreth et al., 1987). Because astroglia in culture possess glutamate receptors linked to polyphosphoinositide metabolism, it is possible that glial cells in brain slices may contribute, at least partly, to the receptor-stimulated accumulation of inositol phosphates in such preparations, especially when challenged with AMPA or ibotenate (Milani et al., 1989). Thus all of the above studies indicate that in mammalian brain, multiple forms of trans-ACPD receptors (Furuya et al., 1989; Nicoletti et al.,

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

287

1986a, 1988b; Schmidt et al., 1987; Sugiyama et al., 1987) are linked directly to polyphosphoinositide hydrolysis and CAMP synthesis. All other excitatory amino acid receptors operate through cation channels and affect phospholipid metabolism indirectly. In the latter case, changes in phospholipid metabolism may also depend on the coupling mechanisms (Axelrod, 1990; Axelrod et al., 1988).

IV. Role of Enhanced Excitatory Amino Acid-Mediated Phospholipid Metabolism in Developing Brain

Susceptibility of the central nervous system to excitatory amino acidmediated injury changes markedly during development (McDonald et al., 1988, 1990; McDonald and Johnston, 1990). Susceptibility peaks near postnatal day 7 in rats, and the severity of excitotoxic injury at this stage is approximately 60 times greater than the injury observed in adult rat brain on an equal basis (McDonald et al., 1988, 1990; McDonald and Johnston, 1990). Similarly, in rat hippocampus and neocortex, excitatory amino acid-stimulated degradation of phosphoinositides transiently peaks at postnatal day 6 (1000 times the response at postnatal day 35) and declines during postnatal development (McDonald and Johnston, 1990). What does this similarity between the development of excitotoxicity and the stimulation of phosphoinositide hydrolysis mean? Does it mean that phosphoinositide turnover plays a role in adaptive regulation by providing second messengers for recovery during and after excitotoxic injury or is it rather an epiphenomenon closely associated with excitotoxicity that can be used as an index to monitor the injury process? Answers to those questions are not known. However, an increase in glutamate-induced phosphoinositide metabolism is seen following deafferentation, during the time period in which neuronal regeneration is taking place and new synapses are being formed (Nicoletti et al., 1987a). Furthermore, the developmental patterns of phosphoinositide metabolism in cerebellum, olfactory bulb, and hippocampus correspond to intense periods of synaptogenesis in these regions (Palmer et al., 1990; Crain et al., 1973; Markus and Petit, 1987; Dudek et al., 1989), indicating that excitatory amino acid-induced phosphoinositide metabolism may play an important role in synaptic growth and stabilization. Another question that is important from a developmental neurobiology viewpoint is the role of neuron-neuron and neuron-glia interactions in excitatory amino acid-mediated neurotoxicity during brain development. The survival of developing neurons and their synaptic activity

288

AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

depends on the availability and exchange of trophic factors (nerve growth factor, fibroblast growth factor, epidermal growth factor, etc.) (Horrocks el al., 1990). Neurons contacting glia survive for longer time periods than do neurons not contacting glia (Mattson and Rychlik, 1990). Furthermore, hippocampal glial cells can protect neurons against excitatory amino acid-mediated cell death. This protection is mediated by fibroblast growth factor (Mattson and Rychlik, 1990).The role of the phosphoinositide second-messenger system in neuronal survival deserves further investigation.

V. Possible Mechanism of Cell Injury Caused by Excitatory Amino Acids

The molecular mechanisms of cell injury caused by excitatory amino acids are becoming evident. In uitro studies (Rothman and Olney, 1986; Choi, 1988a,b; Olney, 1989; Choi and Rothman, 1990) indicate that excitotoxin-induced neuronal injury may involve two distinct events. First, the exposure of neurons to an excitotoxin (30 min) may cause an acute neuronal swelling (acute neurotoxicity) resulting from the depolarization-mediated influx of Na+, C1-, and water (Rothman and Olney, 1986; Choi, 1988a,b).This process is reversible if the excitotoxin is removed from the system. The degree to which this event contributes to neuronal injury is unclear but it has been suggested that water entry causes osmotic lysis. The second event is characterized by excessive calcium influx primarily via NMDA receptor-channel activation. This is brought about by exposing neurons to glutamate for briefer periods (5 min) and observing neuronal degeneration over a 24-h period (delayed neurotoxicity). A rise in intracellular Ca2+ may affect the activities of a number of enzymes (Table I), including the activation of lipolytic (lipases and phospholipases) and proteolytic (Calpain I and other calcium-dependent proteases) enzymes. The activation of lipolytic enzymes releases arachidonic acid from neuronal membrane phospholipids and sets in motion “the arachidonic acid cascade.” The latter includes the synthesis of prostaglandins, leukotrienes, and thromboxanes. The arachidonic acid cascade also potentiates the formation of free radicals and lipid hydroperoxides. The latter are known to inhibit reacylation of phospholipids in neuronal membranes (Zaleska and Wilson, 1989). This inhibition may constitute an important mechanism whereby peroxidative processes contribute to irreversible neuronal injury and death (Fig. 6).

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

289

TABLE I STIMULATORY EFFECT OF CaZt ON ENZYMATIC ACTIVITIES INVOLVEDI N EXCITATORY AMINOACIDNEUROTOXICITY Enzyme

Reference

Phospholipase A,

Farooqui and Horrocks (1988)

Phospholipase C

Farooqui and Horrocks (1988)

Diacyglycerol lipase

Farooqui and Horrocks (1988)

Plasmalogenase

Arthur et al. (1985)

Calpain

Siman and Noszek (1988)

Protein kinase C

Nishizuka (1986)

Calmodulin-dependent protein kinase

Chin et al. ( 1 985)

Guanylate cyclase

Novelli et al. (1987)

Nitric oxide synthase

Gally et al. ( 1 990)

Calcineurin

Halpain et al. (1990)

Endonuclease

Siesjo (1990)

T h e activation of proteases by calcium may cause a breakdown of the cytoskeleton, leading to severe cellular damage (Melloni and Pontremoli, 1989). Thus, exposure of hippocampal neurons to an excitotoxin also causes the degradation of spectrin and MAP2 which correlates well with subsequent neuronal injury (Siman et al., 1989). The cytosolic protease calpain induces the conversion of xanthine dehydrogenase to xanthine oxidase (Dykens et al., 1987) and may help in production of free radicals. Another target of calcium-induced injury may be protein kinase C. This enzyme catalyzes phosphorylation of many synaptic membrane proteins and is regulated by Ca2+,diacylglycerol, free fatty acids, and phosphatidylserine (Nishizuka, 1984a; Berridge, 1984; Farooqui et al., 1988a). Protein kinase C plays an important role in delayed neurodegeneration (Mattson, 1990a); its activators are toxic to cultured human cortical neurons and its inhibitors diminish neuronal loss (Mattson, 1990a). Calcium may also stimulate calcineurin, a calcium/calmodulin-dependent phosphatase that is involved in dephosphorylation of DARPP-32 (a dopamineand CAMP-regulated 32 kDa phosphoprotein) and MAP2 (microtubuleassociated protein) (Halpain et al., 1990; Girault et al., 1990; Halpain and Greengard, 1990; Chin et al., 1985). A direct interaction between dopamine and glutamate receptors at the molecular level may be involved in regulation of the functional state of cytoskeletal proteins that participate in the control of neuronal morphology (Halpain and Greengard, 1990).

290

AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

A recent advancement in understanding the role of Ca2+in delayed neurotoxicity is the observation that inhibitors of nitric oxide synthase block the development of delayed NMDA-induced neuronal death in cortical neurons (Dawson et al., 1991) and hippocampal slices (Izumi et al., 1992). Earlier studies have indicated that NMDA receptor activation promotes the Ca2+-dependentrelease of nitric oxide in CNS neurons (Garthwaite et al., 1988). Nitric oxide is a short-lived, intra- and intercellular second messenger, whose release is dependent on increases in intracellular Ca2+.For nitric oxide to play an important role in delayed neurodegeneration would require that this second messenger activate other longer-lived processes o r be released in an ongoing fashion for a critical period of time. Nitric oxide stimulates guanylate cyclases, leading to increases in intracellular cGMP. Thus, cGMP could provide a means for longer lasting changes in intracellular function initiated by nitric oxide (Moncada et al., 1991). Nitric oxide can also induce neuronal degeneration by virtue of its free radical property. It remains to be seen how nitric oxide affects membrane phospholipid degradation. Finally, calcium may even activate endonucleases that catalyze the fragmentation of DNA, a process that may be involved in programmed cell death (Nicotera et al., 1989). Thus, two major processes may be involved in neuronal injury caused by the overstimulation of excitatory amino acid receptors. One is the large Ca2+ influx (neuronal injury occurring when a certain threshold intracellular Ca2+ concentration is attained for a certain duration) and the other is the accumulation of free radicals and lipid peroxides as a result of neural membrane phospholipid degradation. Another mechanism that may be involved in glutamate neurotoxicity is the inhibition of cysteine uptake by glutamate and its analogs. A reduced availability of cysteine reduces the availability of glutathione, an important component of cellular defense against free-radical injury. Thus inhibition of cysteine uptake increases vulnerability to oxidative stress (Kato et al., 1992; Bridges et al., 1991; Vignes et al., 1992). Free radicals can disrupt membrane integrity by reacting with proteins and unsaturated lipids in the plasma membrane. These reactions lead to a chemical cross-linking of membrane proteins and lipids and a reduction in membrane unsaturated lipid content. This depletion of unsaturated lipids may be associated with alterations in membrane fluidity and permeability and changes in activities of membrane-bound enzymes and receptors (Farooqui et al., 1988b, 1989a, 1990). Calcium and free radicals may act in concert to induce neuronal injury (Pellegrini-Giampietro et al., 1990). Overstimulation of other receptor types (muscarinic M , and M,, a I adrenergic, and histamine H I ) also results in a stimulated neural mem-

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

29 1

brane phospholipid metabolism and a transient increase in intracellular Ca2+(Berridge, 1984,1987)but this Ca2+comes from intracellular stores and its level is not so high as that produced by the overstimulation of NMDA receptors (Cairdo and Meldolesi, 1991; McMillian et al., 1990). Furthermore, as stated above, the duration of exposure to calcium ions is equally important in producing neuronal injury (Tanaka et al., 1989; Mattson, 1990a). This may be one of several reasons for the lack of injury during the overstimulation of nonexcitatory amino acid receptors. Despite the above developments, the precise molecular mechanism by which stimulation of excitatory amino acid receptors and accumulation of intracellular calcium contribute to neuronal injury and death remains poorly understood. However, calcium-induced alterations in neuronal membrane phospholipid metabolism may be an important part of neuronal injury and death. A major challenge of future studies is to define the sequence of molecular events set in motion by high concentrations of intracellular Ca2+ that lead to neuronal injury and degeneration. Another important process that may be involved in neuronal degeneration in neurodegenerative diseases is the impairment of energy metabolism. Experimental evidence for impaired energy metabolism in excitotoxicity has recently been obtained (Novelli et al., 1988). Inhibitors of oxidative phosphorylation or Naf , K+-ATPase allow glutamate or NMDA to become neurotoxic at concentrations that ordinarily produce no toxicity. How the delayed onset and slow progression of neurodegenerative disease relate to a defect in energy metabolism and excitotoxicity remains to be seen. At present little is known about phospholipid metabolism in membranes during the repair process after excitotoxic injury. However, based on physicochemical and metabolic studies on liposomal and biological membranes, it has been proposed that some phospholipases and methyltransferases may be involved in the repair of membrane damage caused by lipid peroxidation (Dawson, 1978; Scott, 1984; van Kuijk et al., 1987).

VI. Excitatory Amino Acid Receptors, Phospholipid Metabolism, and Neurological Disorders

Biochemical and mechanical trauma, as well as chronic neurodegenerative diseases, may involve excitatory amino acid neurotoxicity. The following section describes the importance of excitatory amino acids and their receptors in inducing abnormal phospholipid metabolism in biochemical and mechanical trauma as well as chronic neurodegenerative

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diseases (Table 11). In most of the neurological disorders discussed below, NMDA and trans-ACPD receptors play a major role in mediating glutamate-induced neurotoxicity. However, kainate and AMPA receptors may also contribute to the injury of neuronal and glial cells (Olney, 1989).

A. ALZHEIMER DISEASE

Excitatory amino acid receptors are implicated in learning and memory processes (Collingridge and Bliss, 1987).There is a loss of neocortical and hippocampal terminals in Alzheimer disease (AD), as determined by the measurement of sodium-dependent binding and active uptake of ~-[~H]aspartate (Palmer et al., 1986; Hardy et al., 1987; Cowburn et al., 1988, 1989). The concentration of free glutamate is reduced in several cortical and subcortical areas in Alzheimer disease (Arai et al., 1984; Sasaki et al., 1986; Hyman et al., 1987). The number of NMDA receptors is also decreased in neocortical and hippocampal regions, according to one group of investigators (Greenamyre et al., 1985, 1987; Greenamyre

TABLE I1 INVOLVEMENT OF EXCITATORY AMINOACIDRECEPTORS I N ACUTETRAUMA AND NEURODEGENERATIVE DISEASES Pathologic condition

Excitotoxin

lschemia

Glutamate

Alzheimer disease

EAA receptor involved

Reference" (1)

Glutamate

NMDA and trans-ACPD NMDA, AMPA

(2)

Spinal cord injury

Glutamate

NMDA

(3)

Traumatic head injury

Glutamate

(4)

Epilepsy

Glutamate

NMDA NMDA, AMPA

Huntington disease

Quinolinate

NMDA

(6)

Guam-type amyotrophic lateral sclerosis/Parkinsondementia

P-N-Methylamino L-alanine

NMDA

(7)

Olivopontocerebeller atrophy

Quinolinate

trans-AC PD

(8)

(5)

a (1) (Seren et al., 1989; Haba et al., 1991); (2) (Greenamyre et al., 1987; Dewar et al., 1990, 1991); (3) (Faden and Simon, 1988); (4) (Hayes et al., 1992a); ( 5 ) (McDonald et al., 1991; Lothman, 1990); (6) (Bruyn and Stoof, 1990; Heyes et al., 1991); (7) (Copani et al., 1991a; Taylor, 1991); (8) (Feinstein and Halenda, 1988).

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and Young, 1989; Penney et al., 1990; Shimihama et al., 1990). A significant and selective reduction of AMPA receptors also occurs in the molecular layer of the cerebellum from patients with Alzheimer disease compared to normal subjects (Dewar et al., 1990). In adjacent sections from the same brain of Alzheimer patients, there was no alteration in the level of kainate- or NMDA-sensitive binding in the molecular layer (Dewar et al., 1990). In contrast, another group of investigators (Geddes et al., 1986; Monaghan et al., 1987; Cotman et al., 1989) has stated that the number of NMDA receptors remains relatively stable in Alzheimer disease. T h e reason for this discrepancy is not known, but the severity of the cases examined or differences in methodology may be responsible (Cotman et al., 1989). Little is known about the relationship between excitatory amino acid receptors and the neuropathological changes seen in Alzheimer disease. Neurofibrillary tangles, the hallmark of the disease, are composed of paired helical filaments (Terry and Katzman, 1983). Incubation of cultured human neurons with aspartate or glutamate causes the formation of paired helical filaments (DeBoni and CrapperMcLachlan, 1985; Mattson, 1990b). Treatment of hippocampal neurons with glutamate in the presence of Ca2+not only induces their degeneration but also increases T and ubiquitin immunostaining (Mattson, 1990b). This increase can be prevented by the removal of extracellular calcium. The collective evidence suggests (Mattson, 1990b) that the Ca2+ influx caused by glutamate can lead to modifications of cytoskeleton-associated proteins similar to those seen in the neurofibrillary tangles of patients with Alzheimer disease. T h e formation of neurofibrillary tangles, in Alzheimer disease and as a result of exposure of neurons to glutamate, may involve calcium-dependent proteases and protein kinases (Siman et al., 1989; Mattson, 1990b). At present there is no evidence that neurofibrillary tangles are involved in the degeneration of neurons, but they may be a consequence of the degenerative process (Mattson, 1990b). The P-amyloid protein found in the neuritic plaques of Alzheimer disease, although not toxic to 14-day-old cultures of cortical neurons when used alone, increases the vulnerability of these cultures to excitatory amino acids (Koh et al., 1990), suggesting that excitotoxicity may play an important role in the pathogenesis of Alzheimer disease. It has also been reported recently that impaired energy metabolism induced by glucose deprivation in vitro markedly enhances the susceptibility of cultured cortical neurons to P-amyloid neurotoxicity (Copani et al., 1991b; Kelleher et al., 1993). Furthermore, cysteine, a nonessential amino acid, has been found to produce extensive excitotoxic damage via an effect on NMDA and AMPA receptors. This may be pertinent to Alzheimer disease because elevated

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AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

plasma cysteine to sulfate ratios are found in Alzheimer disease patients. Thus, although excitotoxicity may not be the initiating event, it may interact with an underlying neuronal vulnerability or inherited metabolic profile to fuel continued neurotoxicity and progression of the disease process (Murphy et al., 1987). Alterations in excitatory amino acids in Alzheimer disease (Greenamyre and Young, 1989; Shimihama et al., 1990; Dewar et al., 1990) may be related to the disturbance in Ca2+ homeostasis in cultured skin fibroblasts from patients with this disease (Peterson et al., 1985, 1988; Peterson and Goldman, 1986). Fibroblasts and frontal cortex also show reduced protein kinase C activities in the particulate fraction and elevated protein kinase C levels in the soluble fraction (Shimihama et al., 1990; Huynh et al., 1989), suggesting the involvement of altered protein phosphorylation (Vincent and Davies, 1990; Parks et al., 1991) and secondmessenger cascades (Shimihama et al., 1990). Abnormalities in excitatory amino acid receptors and calcium levels may induce abnormalities in neural membrane phospholipid metabolism. Determination of the phospholipid composition of different regions of human brain has indicated that levels of glycerophospholipids, plasmalogens, and polyphosphoinositides are markedly decreased in patients with Alzheimer disease compared to age-matched controls (Horrocks et al., 1978; Stokes and Hawthorne, 1987; Suzuki et al., 1965; Suzuki and Chen, 1966; Farooqui et al., 1988b; Gottfries, 1990). This decrease in glycerophospholipids is accompanied by marked elevations of phospholipid degradation metabolites, such as glycerophosphocholine, phosphocholine, and phosphoethanolamine, in autopsy samples from AD patients (Barany et al., 1985; Miatto et al., 1986; Pettegrew et al., 1988a,b; Nakada and Kwee, 1990). Furthermore, marked increases have been reported in levels of prostaglandins and lipid peroxides in Alzheimer brains (Iwamot0 et al., 1989; Jeandel et al., 1989; Subbarao et al., 1990; Volicer and Crino, 1990). The marked changes observed in these phospholipids and their catabolic metabolites may be coupled to the elevated activities of lipolytic enzymes in Alzheimer brains. Thus we found markedly higher activities of diacylglycerol and monoacylglycerol lipases (Figs. 10 and 11) in plasma membrane and synaptosomal plasma membrane fractions prepared from nucleus basalis and hippocampal regions of Alzheimer brain, compared to age-matched normal human brain (Farooqui et al., 1988c, 1990). Cytosolic fractions obtained from the nucleus basalis and hippocampal regions of Alzheimer disease patients also had four- to sixfold higher specific activities of lysophospholipase than corresponding fractions from normal human brain (Fig. 12). A 63% lower phospholipase D activity in brain homogenates from Alzheimer patients compared

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295

f

NB

CN

FC

PC

OC

HC

cc

Region FIG. 10. Specific activities of monoacylglycerol lipase in PM and SPM fractions from different regions of normal and AD autopsy brain. Enzymatic activity was determined as described. Statistically significant differences in enzymatic activity were observed in AD compared to the normal control human brain. *P < .05; **P< .01 (Student's t test); NB, nucleus basalis; CN, caudate nucleus; FC, frontal cortex convexity; PC, parietal cortex convexity; OC, occipital cortex convexity; HC, hippocampus; CC, corpus callosum; open bar, control SPM; hatched bar, control PM; stippled bar, A D SPM; solid bar, AD PM. From Farooqui et al. (1990).

to controls has been shown by others (Kanfer et al., 1986).T o date no one has determined the activities of phospholipases A, and C in Alzheimer disease. Both of these enzymes are coupled to the excitatory amino acid receptors and are involved in the turnover of neural membrane phospholipids (Dumuis et al., 1988; Lazarewicz et al., 1988) with the generation of diacylglycerols, free fatty acids, and lipid peroxides. At high concentration these metabolites are cytotoxic to the neuronal membranes. The degradation products of membrane phospholipids, phosphoethanolamine and phosphocholine, may act as false neurotransmitters and compete with NMDA and glutamate for excitatory amino acid binding sites (Pettegrew et al., 1988a). Thus it is clear that there may be a link between excitatory amino acids and abnormal phospholipid metabolism in Alzheimer disease. Further studies are required into the

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AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

Region FIG. 11. Specific activities of diacylglycerol lipase in PM and SPM fractions from

different regions of normal and AD autopsy brain. Enzymatic activity was determined as described. Statistically significant differences in enzymatic activity were observed in A D compared to the normal control human brain. *P < .05; **P< .01 (Student’s t test); NB, nucleus basalis; CN, caudate nucleus; FC, frontal cortex convexity; PC, parietal cortex convexity; OC, occipital cortex convexity; HC, hippocampus; CC, corpus callosum; open bar, control SPM; hatched bar, control PM; stippled bar, AD SPM; solid bar, AD PM. From Farooqui et al. (1990).

enzymatic and molecular aspects of neural membrane phospholipids in this disease. This discussion does not claim that Alzheimer disease is caused by an overstimulation of excitatory amino acid receptors. However, during the progress of the disease, glutamatergic neurons may be damaged by an excitotoxic mechanism and this process may involve changes in neural membrane phospholipids at an early stage. There are many other neurotransmitter systems that are abnormal in Alzheimer disease (Farooqui et al., 1988b; Palmer and Gershon, 1990). These systems are also linked to neural membrane phospholipid metabolism. So the changes in cholinergic, dopaminergic, and GABAergic systems may also contribute to the abnormal phospholipid metabolism (van Rooijen etal., 1986; Nahorski et al., 1986).At present it cannot be decided whether the changes in neural membrane phospholipid metabolism are primary o r secondary in nature. Even if these changes are secondary, they may

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297

35 :.

30

-

c

2

. P Q

.-K

25-

E

**

T

Region

FIG. 12. Specific activity of lysophospholipase in cytosolic fractions from different regions of normal and AD autopsy brains. Enzymatic activity was determined as described. Statistically significant differences in enzymatic activity were observed in A D compared to the normal human brain. *P < .05; **P < .01 (Student’s t test); open bar, controls; hatched bar, AD; for abbreviations see Fig. 10 legend. From Farooqui et al. (1990).

be useful for monitoring therapeutic responses or developing diagnostic tests. B. ISCHEMIA Ischemic insults are caused by a shortage of oxygen (and substrate) and failure to remove metabolic waste products (Auer and Siesjo, 1988). Ischemic brain injury is accompanied by a decline in high-energy phosphates and an accumulation of lactate (Auer and Siesjo, 1988). T h e earliest biochemical events that occur during ischemic insult are the release of free fatty acids from various phospholipids (Bazan, 1970, 1989; Bazan et al., 1986) and the accumulation of lysophospholipids and diacylglycerols (Abe et al., 1987). A significant decrease in various phospholipids has been reported in ischemic brain (DeMedio et al., 1980; Yoshida et al., 1980). Stimulation of phospholipase A, and A, activities has been reported in extracts from acetone-derived powders of ischemic

298

AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

gerbil brain and in homogenates of dog brain (Edgar et al., 1982; Hirashima et al., 1984; Rordorf et al., 1991). The actions of phospholipase C and diacylglycerol lipase may be responsible for the release of free fatty acids (Abe et al., 1987; Umemura et al., 1992). Thus, it is now well accepted that during ischemic injury, free fatty acids are derived from membrane phospholipids through the activation of phospholipases and lipases (Bazan, 1976, 1989; Bazan et al., 1986; Galli and Petroni, 1990; Umemura et al., 1992). During early stages of ischemia (within 1 min), inositol glycerophospholipids are the main source of the increase in free arachidonic acid and diacylglycerol (Ikeda et al., 1986). This release of arachidonic acid may result from the transneuronal breakdown of inosito1 glycerophospholipids triggered mainly by the release of glutamate in the synaptic cleft during the ischemic insult (Benveniste et al., 1989). The pathway by which glutamate is released during ischemic injury is not clear. However it has been suggested that three mechanisms may be involved during this process: (a) calcium induced release of neurotransmitter pools of glutamate, (b) reversal of the sodium/glutamate cotransporter and (c) leakage of glutamate from the cytosol of damaged cells (Ikeda et al., 1989). Furthermore, the molecular mechanism by which glutamate exerts its neurotoxic effect is also not fully understood. However, it has been reported that presynaptic calcium accumulation precedes the postsynaptic calcium rise (Van Reempts and Borgers, 1985) and that excitatory amino acids increase the calcium conductance of the postsynaptic neuron (Berdichevsky et al., 1983). This may stimulate calcium-dependent enzymes (especially phospholipase A,, which degrades membrane phospholipids) in the postsynaptic membrane. The addition of calcium to synaptosomes in uitro causes destruction of the synaptic membrane (Moskowitz et al., 1984). Within minutes after ischemia, a marked accumulation of glutamate occurs (Benveniste et al., 1989). The source of this extracellular glutamate is the calciumstimulated release of neurotransmitter-containing presynaptic vesicles (Drejer et al., 1985). Magnesium, which inhibits synaptic transmitter release, also inhibits cell death in uitro (Drejer et al., 1985). In uitro studies have also shown that a brief exposure of neurons to glutamate produces widespread neuronal death that can be attenuated by the removal of extracellular calcium (Choi, 1988a). This finding, together with reports that glutamate-operated channels of the NMDA type have high conductance (MacDermott et al., 1986), suggests that calcium entry via the NMDA receptor channel and the intracellular accumulation of calcium are the main events. A speculative description of glutamate neurotoxicity was proposed recently (Choi, 1990). Glutamate neurotoxicity may have three stages:

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(1) induction (overstimulation of glutamate receptors leading to a set of immediate cellular derangements); (2) amplification (events that intensify these derangements and promote the excitotoxic involvement of additional neurons); and (3) expression (the destructive cascade directly responsible for neuronal cell degeneration) (Choi, 1990). In vivo the activation of polyphosphoinositide hydrolysis by excitatory amino acids is observed in hippocampal and cortical, but not striatal, slices from rats that underwent 30 min of transient global ischemia (Seren et al., 1989). In hippocampus and cerebral cortex this potentiation is present at 24 h and 7 days after reperfusion. However, in cortical slices no enhanced responsiveness to excitatory amino acids is observed during occlusion nor at short intervals after reperfusion. These observations suggest that postischemic recirculation leads to a slow and progressive increase in the coupling of the trans-ACPD receptor to phospholipase C activity (Seren et al., 1989) and this process may be involved in the delayed form of cell death. Similar results have been obtained in gerbil hippocampus following transient ischemia (Haba et al., 199 1). A molecular mechanism by which glutamate exerts its neurotoxic effect has recently been proposed (Wieloch and Westerberg, 1990). According to this mechanism, the glutamate-induced stimulation of tramACPD receptors activates the polyphosphoinositide cycle through the activation of phospholipase C and results in the generation of diacylglycerol and inositol 1,4,5-trisphosphate (Fig. 6). The latter mobilizes Ca2+ from intracellular stores. T h e diacylglycerol is hydrolyzed by the diacylglycerol and monoacylglycerol lipases to arachidonic acid and glycerol (Farooqui et al., 1989b). Presynaptically arachidonic acid inhibits glutamate uptake (Chan et aE., 1983; Yu et al., 1986),leading to a slow clearing of glutamate from the interstitial space. Further, free radicals formed from the oxidation of arachidonic acid may also trigger the release of glutamate in the hippocampus (Pellegrini-Giampietro et al., 1988). This causes a persistent stimulation of NMDA receptors, resulting in rapid influx and accumulation of calcium and activation of phospholipase A,. Under these conditions NMDA receptor antagonists may prevent the release of arachidonic acid and stimulate glutamate uptake, thereby terminating glutamate receptor stimulation and inhibiting neuronal degeneration (Wieloch and Westerberg, 1990). Postsynaptically, arachidonic acid, along with Ca2+,may modulate the activity of some isozymes of protein kinase C by enhancing its translocation from cytosol to plasma membrane. At the plasma membrane level, protein kinase C performs a variety of functions, including the regulation of neurotransmitter release, activation of proton-sodium exchanges, opening of calcium channels (Kaczmarek, 1987; Kikkawa et al., 1989),and regulation of gene expres-

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AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

sion (Nishizuka, 1989). During the very early phases of ischemic injury, both protein kinase C and calcium/calmodulin-dependent kinase are inhibited, whereas CAMP-dependent protein kinase (protein kinase A) is not affected (Zivin et al., 1990). This observation suggests that protein phosphorylation abnormalities during ischemia are not generalized (Zivin et al., 1990; Churn et al., 1990). The time course of protein kinase C inhibition closely corresponds to irreversible loss of neurologic function and there is evidence that an endogenous inhibitor of protein kinase C is generated (Zivin et al., 1990). These results, along with others (Churn et al., 1990),suggest that protein phosphorylation, particularly by protein kinase C, is critical to the maintenance of neurologic function during ischemia (Zivin et al., 1990). Another important event during ischemic injury is the generation of lipid peroxides and free radicals from arachidonic acid (Benveniste et al., 1989). Free radical formation and excitatory amino acid release are mutually related and cooperate in a series of molecular events that link ischemic injury to neuronal cell death (Pellegrini-Giampietro et al., 1988). Furthermore, lazaroids (2 l-aminosteroids), powerful antioxidants, can attenuate the neuronal injury induced by the exogenous application of glutamate or NMDA (Monyer et al., 1990). Free radicals are attractive candidates for mediating the expression of excitotoxicity (Monyer et al., 1990). Thus excitotoxic injury and free radical-mediated injury may involve molecular events that overlap substantially but not completely (Monyer et al., 1990). T h e above changes in neural membrane phospholipids and their metabolites suggest that the breakdown of neuronal membrane phospholipids induced through excitatory amino acid receptors plays a major role in the pathophysiology of cerebral ischemia. C. SPINALCORDINJURY Injury to the spinal cord triggers autodestructive processes that lead to varying degrees of tissue necrosis and paralysis, depending on the severity of the injury. Neurochemical changes include degradation of membrane phospholipids with the generation of free fatty acids, diacylglycerols, eicosanoids, and lipid peroxides (Demediuk et al., 1985b,c; Saunders and Horrocks, 1987; Xu et al., 1990); a rise in intracellular free Ca2+within minutes after injury (Stokes et al., 1983); and activation of phospholipases and lipases (Demediuk et al., 1985b,c; Taylor, 1988). Spinal cord injury is also associated with marked changes in tissue levels of excitatory amino acids within the injury regions (Demediuk et al., 1988; Panter et al., 1990). The mechanism by which mechanical trauma

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

30 1

releases excitatory amino acids during spinal cord injury is unknown. However, based on studies in vitro, it has been suggested that mechanical trauma per se induces synaptic and nonsynaptic release of glutamate from the injured neurons and glial cells (Tecoma et al., 1989), producing swelling, granularity, and degeneration. T h e injury to the neurons is markedly reduced when the mechanical insult is delivered in the presence of dextrorphan, an NMDA antagonist (Tecoma et al., 1989). It is interesting to note that mechanical trauma also perturbs cell membranes in neuron-enriched cultures from fetal mouse spinal cords and results in the generation of high levels of free fatty acids, diacylglycerols, and lipid peroxides (Demediuk et al., 1985a). High levels of arachidonic acid inhibit glutamate uptake (Yu et al., 1986), which causes continuous stimulation of NMDA receptors, leading to neuronal death. NMDA antagonists have been reported to improve the neurological outcome after spinal cord injury in rats (Faden et al., 1988; McIntosh et al., 1990). Because cellular damage after injury results in part from delayed events, including ischemia (Faden, 1983) and an influx of calcium (Stokes et al., 1983), it has been suggested that NMDA receptors may contribute to secondary injury following spinal cord trauma (Faden and Simon, 1988). Intrathecal injections of high concentrations of an excitatory amino acid have indicated that AMPA and kainate, but not NMDA, produce marked damage to spinal cord neurons (Urca and Urca, 1990). This neurotoxic effect is nonselective and is accompanied by a loss of sensory and motor functions. NBQX (Fig. 4),a potent AMPA/ kainate receptor antagonist, restores functional deficits resulting from a standardized contusive injury in rats (Wrathall et al., 1992). Thus, the collective evidence suggests that excitatory amino acid receptor-induced membrane damage may contribute to the pathogenesis of spinal injury.

D. TRAUMATIC BRAININJURY Numerous investigations have indicated that traumatic brain injury is accompanied by widespread neuronal depolarization, accumulation of glutamate in extracellular space, and increased levels of unesterified arachidonic acid and eicosanoids, as well as leukotrienes (Hayes et al., 1992a,b). Two mechanisms may be mainly involved in the release of arachidonic acid from membrane phospholipids during head injury: direct activation of phospholipase A, (Shohami et al., 1989) and stimulation of phospholipase C (Wei et al., 1982) followed by diacylglycerol and monoacylglycerol lipases. Both phospholipases are activated after experimental brain injury (Shohami et al., 1989; Wei et al., 1982). T h e

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AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

chain of events following traumatic head injury may involve accumulation of arachidonic acid because of the activation of phospholipases A, and C. The high levels of arachidonic acid inhibit the uptake of glutamate into neurons. Excitatory amino acid receptors are therefore exposed to higher levels of glutamate that overstimulate the NMDA receptors, leading to neuronal degeneration. The protective effect of NMDA antagonists after head injury may be of clinical value in the management of head injury (McIntosh et al., 1989; Shapira et al., 1990). This suggests that glutamate-induced degradation of membrane phospholipids may play an important role in the pathophysiology of head injury.

E. EPILEPSY

Recent neurochemical studies on epileptic human brain have shown increased concentrations of glutamate and aspartate in the focal, compared to nonfocal, regions of the cerebral cortex (Nadi et al., 1987; Sherwin et al., 1988). Furthermore, a marked increase has been reported in the number of NMDA receptors in the epileptic focus (Sloviter and Dempster, 1985; Wyler et al., 1987; McDonald et al., 1989; Hosford et al., 1989). Intracerebral injections of glutamate and its analogs produce seizure activity and brain damage in rats (Bradford and Dodd, 1975) that resemble the seizure-related brain damage in patients with epilepsy (Olney et al., 1986). T h e mechanism by which persistent seizure activity results in degeneration of neurons and brain damage is not understood. However, circumstantial evidence suggests that alterations in excitatory amino acids and their receptors may contribute to the pathogenesis of epilepsy and brain damage (Dichter, 1989). Changes in NMDA and AMPA receptors (Wyler et al., 1987; McDonald et al., 1989; Hosford et al., 1989) may be coupled to the accumulation of Ca2+in rat hippocampus during seizures (Griffiths et al., 1983). Calcium ion accumulation triggers a variety of biochemical processes, including the degradation of membrane phospholipids, proteolysis of cytoskeletal proteins, and protein phosphorylation (Bazan, 1976; Bazan and Rodriguez de Turco, 1980). Seizure activity induces marked alterations in membrane phospholipid metabolism, resulting in an accumulation of free fatty acids, diacylglycerols, and the oxygenated products of arachidonic acid (prostaglandins and leukotrienes) (Bazan and Rodriguez de Turco, 1980; Siesjo and Wieloch, 1986). Accumulation of free fatty acids and diacylglycerols during seizure activity may result from the stimulation of phospholipases A,, A,, and C and diacylglycerol and monoacylglycerol lipases (Bazan, 1976; Bazan and Rodriguez de Turco, 1980). Thus the loss of key fatty

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acids from neural membrane phospholipids because of the action of phospholipases and lipases may cause changes in membrane fluidity and in the activities of membrane-bound enzymes and ion channels. Furthermore, the lysophospholipids generated by the action of phospholipase A, may not only produce a detergent-like effect on the neural membranes but also affect mitochondria1 function (Lenzen et al., 1989). These studies suggest that excitatory amino acid receptors may alter neural membrane phospholipid metabolism and contribute to the brain damage in epilepsy (Dubeau et al., 1992).

F. GUAM-TYPE AMYOTROPHIC LATERAL SCLEROSIS/ PARKINSONISM-DEMENTIA Guam-type amyotrophic lateral sclerosis/Parkinspnism-dementia (ALS/PD) is a disease of the motor neurons and corticospinal tract. It is characterized by muscle weakness, progressive muscular atrophy, paralysis, and spasticity and is remarkably common in the Chamorro population of the Mariana Islands of Guam and Rota (Garruto and Yase, 1986). Abnormal glutamate metabolism with elevated plasma glutamate levels has been reported in patients with ALS/PD (Plaitakis and Caroscio, 1987). The pathogenesis of the disease remains unknown, although p-Nmethylamino-L-alanine (L-BMAA),a nonprotein amino acid toxin found in the seed of the false sago palm (Cycas circinalis L.), has been implicated (Spencer et al., 1987). The neurotoxic effects of L-BMAA have been ascribed to the activation of excitatory amino acid receptors (Ross et al., 1987). L-BMAAstimulates the NMDA-gated ion channel in the presence of bicarbonate ions that interact with the toxin to produce the molecular configuration needed for the activation of excitatory amino acid receptors (Weiss and Choi, 1988; Weiss et al., 1989). The action of L-BMAA involves the activation of the trans-ACPD receptor, which is coupled to inositol phospholipid hydrolysis (Copani et al., 1990). This suggests that the neurotoxic action of L-BMAA mediated through the trans- ACPD receptor may induce the degeneration of neurons accompanied by increased membrane inositol phospholipid degradation. Cerebrospinal fluid (CSF) from ALS patients is markedly neurotoxic. T h e treatment of rat cortical neuronal cultures with diluted (50%) CSF for 24 h resulted in 53% degeneration of neurons. The neurotoxic effect of CSF from ALS patients can be blocked by CNQX but not by MK801 o r AP7, suggesting the involvement of AMPA/kainate receptors (Couratier et al., 1993).

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AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

The neurotoxic action of glutamate may contribute to the selective degeneration of neurons in the substantia nigra in Parkinson disease (Turski et al., 1991). T h e strongest evidence in support of this hypothesis is provided by the finding that NMDA antagonists protect neurons in the substantia nigra from the neurotoxic effects of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine(MPTP). T h e role of glutamate in selective dopaminergic toxicity seems to be indirect. The metabolic products of MPTP probably produce neurotoxicity by blocking mitochondrial energy metabolism (Mizuno et al., 1987). Impaired energy metabolism may cause membrane depolarization and release of the Mg2+ block of NMDA receptor-operated ion channels so that even normal neuronal concentrations of glutamate become toxic (Novelli et al., 1988). Impairment of mitochondrial energy metabolism also occurs in Parkinson disease, suggesting that glutamate may become toxic via a mechanism similar to that operative in MPTP-induced Parkinsonism. It is interesting to note that marked changes have been reported in phospholipid metabolism with deposition of lipofuscin pigments in Parkinson disease (Clausen, 1984). Another pathological condition in which the trans-ACPD receptor may be involved is olivopontocerebellar atrophy. This disease is characterized by Purkinje cell degeneration (Blackstone et al., 1989) and reduction in inositol 1,4,5-trisphosphate receptors (Kish et al., 1989). These results indicate that the inositol 1,4,5-trisphosphate-linkedexcitatory amino acid receptor system may be particularly active in Purkinje cells and may be involved in olivopontocerebellar atrophy. G. HUNTINGTON DISEASE

Huntington disease is a rare and progressive movement disorder caused by an autosomal dominant mutation on chromosome 4. The nature of the defective gene and its encoded protein is still unknown (Gusella et al., 1983, 1987). T h e disease is characterized by the selective degeneration of striatal neurons as well as intellectual deterioration and psychiatric symptoms (a form of dementia). Evidence for the role of excitatory amino acids in the pathogenesis of Huntington disease is based on results obtained from a rodent model in which a single injection of kainate into the striatum produced a pattern of degeneration similar to that seen in Huntington disease (Stone et al., 1987). Furthermore, a 93% reduction of NMDA binding has been reported to occur in putamen from Huntington brain, compared to normal (Young et al., 1988). More recent evidence indicates that quinolinic acid and kynurenic acid, endogenous excitotoxins, may be involved (Beal et al., 1986; Connick et al.,

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1989). It has been suggested that increased concentrations of these excitotoxins in the areas of the cortex concerned with feedback regulation of the striatum may be responsible for cell death (Connick et al., 1989). Changes in cation levels (Na', K', and Ca2+),along with endogenous excitotoxins, may play a major role in neuronal damage (Korf et al., 1986). It has been hypothesized that an influx of large quantities of Ca2+ into the susceptible neurons may be responsible for neuronal necrosis in Huntington disease (Schwarcz and Shoulson, 1987). At present no information is available on phospholipid metabolism in this disease; however, a significant reduction in phosphoethanolamine and ethanolamine is reported. Based on the measurement of these primary amines, it has been suggested that alterations in membrane phospholipids may be a consequence of the pathological process (Ellison et al., 1987).

H. DOMOICACIDNEUROTOXICITY Domoic acid is a rigid structural analog of glutamate. It produces its excitatory action by interacting with kainate receptors. Domoic acid toxicity was first reported in Canada in 1987 when 107 individuals consumed cultured blue mussels (Mytilus edulis L.) and displayed symptoms including gastrointestinal distress, cardiovascular instability, convulsions, agitation, confusion, disorientation, and coma. Chemical analysis of the mussels indicated that they contained high concentrations (900 p g / g wet wt) of domoic acid. T h e source of the domoic acid was traced to a species of plankton (Nitzschia pungens forma multiseries) which, in high bloom, synthesizes large amounts of domoic acid. Filter-feeding mussels ingest the plankton as a food source (Teitelbaum et al., 1990). T h e brains of the individuals who died because of neurotoxicity had lesions distributed throughout the hippocampus, amygdala, thalamus, and cerebral cortex. The survivors became demented. Domoic acid produces toxic effects on hippocampal neurons in cultures. Ionic currents induced in hippocampal neurons by domoic and kainic acid are identical and are markedly different from those induced by NMDA. Domoic acid currents are not blocked by NMDA antagonists but are blocked by CNQX and DNQX. Subcutaneous injections of domoic acid (2.5 to 3 mg/kg body wt) to adult rats produce an acute seizure-brain damage syndrome almost identical to that induced in rats by kainate (12 mg/kg body wt) and with features analogous to the neurotoxic syndrome seen in the human food poisoning victims (Stewart et al., 1990). Domoic acid markedly stimulates cGMP synthesis in neuronal cultures in the absence of Mg2+ (Novelli et al., 1992). This suggests that subtoxic

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concentrations acting at the kainate receptor may enhance NMDA agonist-mediated neurotoxicity by reducing the Mg2+ block at the NMDA receptor channel. At present no information is available on phospholipid changes in domoic acid toxicity.

I. AIDS DEMENTIA COMPLEX The CNS manifestations of acquired immune deficiency syndrome (AIDS) include a substantial loss of neurons in cortex and retina. The mechanism of neuronal degeneration in AIDS is not known. Human immunodeficiency virus type I (HIV-1) sheds a coat glycoprotein called gp 120. This protein, like glutamate and its analogs, increases intracellular calcium concentration and produces neurotoxicity in neuronal cultures at picomolar concentrations (Lipton et al., 1991). Treatment with NMDA antagonists and L-type calcium channel antagonists attenuates both the increased intracellular calcium concentration and delayed neuronal death. Nitric oxide also contributes to gp 120 toxicity, since nitroarginine, an inhibitor of nitric oxide synthase, also prevents neurotoxicity. Arachidonic acid and cytokines are produced during HIV- 1 infection and act synergistically with endogenous glutamate to activate neuronal NMDA receptors. Ca2+then enters NOS neurons to stimulate the formation of NO which is neurotoxic to adjacent neurons (Dawson et al., 1993). Degradation of endogenous glutamate by glutamate-pyruvate transaminase also protects neurons from gp 120 related neurodegradation, suggesting that gp 120 and glutamate are necessary for neurodegeneration as synergistic effectors. This is particularly important in light of recent reports indicating that the levels of quinolinic acid, an endogenous NMDA agonist, are markedly increased in cerebrospinal fluid of patients with AIDS complex (Heyes et al., 1989, 1991). High levels of quinolinic acid are known to induce changes in physicochemical properties of human erythrocyte membranes and stimulate lipid peroxidation in rat brain homogenate (Stone, 1993; Rios and Santamaria, 1991). These processes may be involved in neurodegeneration in AIDS complex. At present nothing is known about membrane phospholipid changes in AIDS dementia. However, viral infections are known to stimulate calcium influx and activities of phospholipases (Suzuki and Matsumoto, 1982). Thus abnormal phospholipid metabolism, altered calcium homeostasis and excitotoxicity are probably involved in neuronal loss in AIDS dementia complex.

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VII. Excitatory Amino Acid Receptor Antagonists and the Treatment of Neurological Disorders

Acute biochemical and/or mechanical trauma causes neuronal cell death either immediately (primary injury) or after some delay (secondary injury). Cell death in delayed injury is more extensive than in primary injury and results from metabolic modifications around or at a distance from the primary site of trauma (Leprince et al., 1990). It can happen as long as 8 days after the acute injury. Such a delay opens the possibility of therapeutic intervention to prevent secondary cell death (Leprince et az., 1990). Different strategies have been used to attenuate acute neuronal trauma and the neurodegenerative condition caused by excessive stimulation of the excitatory amino acid receptors. These include use of (a)competitive or noncompetitive antagonists of the ionotropic excitatory amino acids, (b) drugs that prevent the sustained activation of Ca2+dependent enzymes, (c) drugs or substrates that improve the intracellular energy balance, and ( d ) reducing agents or free radical scavengers. A list of excitatory amino acid antagonists that have been used successfully for the treatment of injury in animal models of neurological disorders appears in Table 111. T h e rationale behind using excitatory amino acid receptor antagonists for therapeutic intervention seems sound (Albers et al., 1992). However, these antagonists may produce behavioral and physiological side effects that result in complications (Buchan, 1992). Competitive NMDA antagonists, such as 2-amino-5-phosphonovalerate (APV) and 2-amino-7-phosphonoheptanoate (APH), directly block glutamate recognition sites (Choi, 1988a; Olney, 1989). These drugs are very polar and hence do not cross the blood-brain barrier very well. T h e NMDA noncompetitive antagonists (ketamine, SKF 10047, MK801, dextrorphan) prevent neuronal loss after ischemic and mechanical injury. These drugs are psychotomimetic and may cause anxiety, depression, and/or narcolepsy (Svennerholm et al., 1990). Recent reports on the antagonism of AMPA receptors by the GYKl series of 2,3benzodiazepine are very important. Because these derivatives cross the blood-brain barrier rapidly (Palmer and Lodge, 1993), they may protect neurons from damage. Tetrahydroaminoacridine (THA)! an anticholinesterase drug, produces some beneficial effects in Alzheimer disease by modulating the action of glutamate on NMDA receptors (Greenamyre and Young, 1989). MK-801, the noncompetitive NMDA antagonist, has been used to treat epileptic seizures in animal models and results are encouraging (Dichter, 1989). Use of excitatory amino acid receptor an-

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TABLE 111 EXCITATORY AMINOACID-RELATED THERAPEUTIC AGENTSUSEDTO TREAT NEUROLOGICAL DISORDERS IN HUMAN AND ANIMAL MODELS Therapeutic agent used” Neurological disorder Alzheimer disease

Human T H A , gangliosides (1)

Ischemia Spinal cord trauma Traumatic head injury

Gangliosides (3) Gangliosides (9) -

Animal models Gangliosides (2) MK-80 1, 2 APH, gangliosides (4)

MK-801, NBQX (10) (5) Glutamate antagonists (6)

Epilepsy

-

M K-80 1

Huntington disease

-

MK-801

(7) (8) (1) (Ala et al., 1990; Greenamyre and Young, 1989; Svennerholm et al., 1990); (2) (Favaron et al., 1988); (3) (Collingridge and Bliss, 1987); (4) (Albers et al., 1989; Fagg and Baud, 1988); (5) (Faden et al., 1988; McIntosh et al., 1990; Wrathall et al., 1992); (6) (Bullock and Fujisawa, 1992); (7) (Dichter, 1989); (8) (Beal et al., 1988);(9) (Geisler et al., 1991); (10) (Wrathall et al., 1992).

tagonists to slow the progress of Alzheimer disease cannot be justified because acute administration results in harmful side effects. Furthermore, the role of excitatory amino acids in learning and memory processes (Wood et d., 1990) suggests that the direct antagonism of excitatory amino acid receptors may further impair Alzheimer disease patients. In cerebellar granule cells, overstimulation of excitatory amino acid receptors becomes neurotoxic when intracellular energy levels are reduced (Novelli et al., 1988; Favit et al., 1992).Cultured cerebellar granule cells degenerate spontaneously within 12 to 15 days. This form of “agerelated” degeneration results from the progressive depletion of energyproviding substrates in the culture medium, which enables the toxic action of the endogenously released glutamate. The degeneration of neurons can be prevented by supplementing the medium either with glucose or with NMDA receptor antagonists. The addition of ubiquinone also protects cerebellar granule cultures against spontaneous degeneration (Favit et al., 1992).Ubiquinone exhibits great potency as a neuroprotective agent and is equally active in preventing neuronal degeneration induced by an acute pulse with a toxic concentration of glutamate. The

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neuroprotective activity of ubiquinone may be mediated by two independent mechanisms: (1) an improvement in energy balance and (2) removal of free radicals and inhibition of lipid peroxidation (Favit et al., 1992). Gangliosides, a novel class of glycosphingolipids with neurotrophic and neuritogenic properties, have been used to treat Alzheimer disease (Ala et al., 1990; Bassi et al., 1984) and ischemia (Porsche-Wiebking, 1989). T h e mechanism for the beneficial effects of gangliosides is not known. However, it has been suggested that gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures by inhibiting the consequences of uncontrolled and persistent glutamate receptor activation (Favaron et al., 1988) and thus reduce secondary brain damage. Gangliosides may prolong the life of degenerating neurons by incorporation into their plasma membranes and modulation of calcium ion homeostasis. Because of their neurotrophic and neuritogenic character, the gangliosides can also potentiate endogenously occurring neuronotrophic factors (such as nerve growth factor) and thus facilitate the survival of the degenerating neurons. Furthermore, gangliosides are known to inhibit activities of a number of enzymes (Table IV) that are associated with signal transduction and neurotoxicity. It must be clearly understood, however, that ganglioside therapy of Alzheimer disease and ischemia is not likely to reverse the disease process. Another avenue of therapeutic interest is the use of growth factors. Several growth factors with neurotrophic effects have recently been isolated and characterized. Epidermal growth factor (EGF), fibroblast growth factor (FGF), and nerve growth factor (NGF) have been reported to produce attenuating effects on excitotoxicity (Horrocks et al., 1990; Freese et al., 1992; Abe and Saito, 1992). Cycloheximide inhibits the

TABLE IV EFFECTOF GANGLIOSIDES ON ENZYMES ASSOCIATED WITH SIGNAL TRANSDUCTION A N D NEUROTOXICITY Enzyme

Effect

Reference

Ca2+/CaM-dependentprotein kinase I1

Inhibited

Higashi and Yarnagata, 1992

Myosin light chain kinase

Inhibited

Fukunaga el al., 1990

Cyclic nucleotide phosphodiesterase

Inhibited

Yates et al., 1989

Phospholipase A,

In hi hi ted

Yang et al., 1994 a,b

Calcineurin

Stimulated

Fukunaga et al., 1990

Proteases (calpains)

Inhibited

Croall and Dernartino, 1991

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AKHLAQ A. FAROOQUI AND LLOYD A. HORROCKS

protective effect of EGF against neurotoxicity (Abe and Saito, 1992), suggesting that EGF promotes the synthesis of some factor(s) essential for neuronal survival. It must be noted that lipocortin- 1 , a phospholipase A, inhibitory protein, inhibits NMDA receptor-mediated neuronal damage in rat striatum (Black et al., 1992). Lipocortin-1 is also a potent calcium-binding protein and may therefore inhibit the rise in intracellular Ca2+that follows NMDA receptor stimulation. Although in the past decade considerable progress has been made toward understanding excitatory amino acid-induced cell death and its blockade by the excitatory amino acid receptor antagonists, this knowledge is not enough to develop treatments for neurological disorders. More experimental work is required on the pharmacology and synthesis of new excitatory amino acid antagonists. These drugs should lack side effects and be able to cross the blood-brain barrier easily. Furthermore, attempts should be made to develop an early assessment of the severity and prognosis of neurological disorders so that excitatory amino acid antagonists can be administered at the proper time.

VIII. Conclusion

Glutamate is a major excitatory transmitter in the mammalian central nervous system. This amino acid produces its effects by acting on at least five receptor subtypes that are designated kainate, AMPA, NMDA, LAP4, and trans-ACPD receptors. Abnormalities in glutamate neurotransmitter systems are believed to be involved in neurological disorders such as epilepsy (Sloviter and Dempster, 1985; Wyler et al., 1987), in certain neurodegenerative diseases such as Alzheimer disease and ALSiPD (Greenamyre and Young, 1989; Wyler et al., 1987), and in brain and spinal cord damage following ischemia and spinal cord trauma (Panter et al., 1990; McIntosh et al., 1990). The cytotoxicity of glutamate in cultured cerebral cortical neurons, as well as in cerebellar slices (Choi, 1988a,b),is apparently related to increased intracellular Ca2+that stimulates and regulates phospholipases, proteases, protein kinases, and endonucleases (Meyer, 1989). During intermittent transsynaptic stimulation of excitatory amino acid receptors, the resulting increased intracellular free calcium concentration is rapidly reversed by the intervention of several mechanisms that regulate intracellular Ca2+homeostasis (Meyer, 1989). However, under pathological situations, the resulting increased extracellular glutamate concentration elicits a constant stimulation of the

EXCITOTOXICITY AND NEUROLOGICAL DISORDERS

31 1

excitatory amino acid receptors. This results in a persistent increase in intracellular free calcium that may be responsible for the abnormal metabolism of neural membrane phospholipids and the generation of high levels of free fatty acids, diacylglycerols, eicosanoids, and lipid peroxidation. These lipid metabolites, along with abnormal ion homeostasis and lack of energy generation, may be responsible for cell death in a variety of neurological disorders such as Alzheimer disease, ischemia, epilepsy, spinal cord trauma, and ALS/PD. It is interesting to note that all of these neurological disorders show an increase in intracellular free Ca2+,abnormal phospholipid metabolism, and accumulation of free fatty acids, diacylglycerols, and prostaglandins and related compounds (Table V). This suggests that excitotoxin-induced membrane phospholipid metabolism may be a common mechanism involved in cellular response in the above neurological disorders. It is not known at this time whether the involvement of membrane phospholipids in neural trauma is a primary process or is induced by secondary factors. T h e fact that abnormalities in membrane phospholipid metabolism occur in a variety of neurological disorders can be used to monitor disease processes in human subjects and animal models. This information will be valuable for developing diagnostic tests and measuring therapeutic responses (Farooqui, 1987; Farooqui et al., 1988b). Our emphasis on excitatory amino acids and their receptors in cell trauma does not rule out the participation of other mechanisms involved in cell injury and degeneration. However, it is timely and appropriate to apply the concept of excitotoxicity to the degradation of membrane phospholipids. Excitotoxin-stimulated membrane phospholipid degradation results in the generation of high levels of free fatty acids, diacylglycerol, and eicosanoids. At low levels these lipid metabolites act as second messengers (Axelrod, 1990; Volterra, 1989), but at high concentrations produce cytotoxicity that may be involved in cell injury and death. In ischemia and spinal cord trauma, the excitotoxin-induced brain damage may be rapid (days) because of the sudden lack of oxygen, decrease in ATP levels, and collapse of ion gradients. In neurodegenerative diseases, however, in which oxygen and nutrients are available to the nerve cell, the damage may gradually accumulate for years. T h e evidence described above showing the involvement of excitatory amino acids and their receptors in neural membrane phospholipid metabolism is, for the most part, indirect and circumstantial. Further work is required into the molecular mechanisms of excitotoxin-induced abnormalities in neural membrane phospholipid metabolism.

3 12

AKHLAQ A. FAROOQUI A N D LLOYD A. HORROCKS TABLE V STATUS OF INTRACELLULAR CALCIUM, PHOSPHOLIPIDS, A N D LIPIDMETABOLISM IN NEUROLOGICAL DISORDERS INVOLVING EXCITATORY AMINOACID RECEPTORS ~

Neurological disordeP

Parameter Intracellular calcium Lipolytic enzymes Phospholipid metabolism Diacylglycerol Free fatty acids Prostaglandins Lipid peroxides

Alzheimer disease Increased (1) Increased (24) Abnormal (2) ND Increased (3) Increased (4) Increased (5)

Ischemia

Spinal cord trauma

Traumatic head injury

Increased (6) Increased (25) Abnormal (7) Increased (8) Increased (9) Increased (10) Increased

Increased (12) Increased (26) Abnormal (13) Increased (14) Increased (15) Increased (16) Increased

Increased (27) Increased (28) ND

( 1 1)

(17)

ND Increased (29) Increased (30) Increased (31)

Epilepsy Increased (18) ND Abnormal (19) Increased (20) Increased (4) Increased (22) Increased (23)

Note: ND. Not determined. ‘ ( I ) (Peterson et al., 1985, 1988; Peterson and Goldman, 1986): (2) (Farooqui et al., 1988b; Gottfries, 1990); (3) (Nakada and Kwee, 1990); (4) (Iwamoto et al., 1989); ( 5 ) (Subbarao et al., 1990; Berti-Mattera et al., 1985); (6) (Deshpande et al., 1987; Dienel, 1984); (7) (Bazan et al., 1986; DeMedio et al., 1980); (8) (Abe et al., 1987; Ikeda et al., 1986; Bazan et al., 1986); (9) (Bazan el al., 1986; Bazan, 1970; Bazan et al., 1989); (10) (Bazan et al., 1986; Griffiths et al., 1983); (1 1) (Bazan et al., 1986; Bazan, 1989; Haba et al., 1991); (12) (Stokes and Somerson, 1987; Stokes et al., 1983); (13) (Demediuk et a1 , 1985b,c, 1987); (14) (Demediuk etal., 1985b,c, 1987); (15) (Demediuk etal., 1985b,c, 1987); (16) (Saunders and Horrocks, 1987; Xu eta[., 1990); (17) (Demediuk et al.. 1985a.c; Anderson and Means, 1985); (18) (Criffiths et al., 1983); (19) (Bazan and Rodriguez de Turco, 1980; Bazan, 1989); (20) (Bazan and Rodriguez de Turco, 1980; Bazan et al., 1986; Bazan, 1989); (21) (Bazan and Rodriguez de Turco, 1980; Bazan, 1989); (22) (Bazan and Rodriguez de Turco, 1980; Bazan, 1989); (23) (Singh and Pathak, 1990); (24) (Farooqui el al., 1990; Tocco et al., 1991); (25) (Edgar et a/., 1982; Rordorf ef al., 1991); (26) (Taylor, 1988); (27) (Hayes et al., 1992a); (28) (Shohami et al., 1989; Wei et al., 1982); (29) (Shohami et al., 1989; Wei et al., 1982); (30) (Ellis et al., 1981; Shohami et al., 1987); (31) (Hayes el al., 1992b).

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IX. Summary

Excitatory amino acids and their receptors play an important role in membrane phospholipid metabolism. Persistent stimulation of excitatory amino acid receptors by glutamate may be involved in neurodegenerative diseases and brain and spinal cord trauma. The molecular mechanism of neurodegeneration induced by excitatory amino acids is, however, not known. Excitotoxin-induced calcium entry causes the stimulation of phospholipases and lipases. These enzymes act on neural membrane phospholipids and their stimulation results in accumulation of free fatty acids, diacylglycerols, eicosanoids, and lipid peroxides in neurodegenerative diseases and brain and spinal cord trauma. Other enzymes, such as protein kinase C and calcium-dependent proteases, may also contribute to the neuronal injury. Excitotoxin-induced alterations in membrane phospholipid metabolism in neurodegenerative diseases and neural trauma can be studied in animal and cell culture models. These models can be used to study the molecular mechanisms of the neurodegenerative processes and to screen the efficacy of therapeutic drugs.

Acknowledgments

Supported in part by NIH Grants NS-10165 and NS-29441

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I NJURY-RE LATED BEHAVI0R A N D NEURONAL PLASTICITY: A N EVOLUTIONARY PERSPECTIVE ON SENSITIZATION, HYPERALGESIA, AND ANALGESIA

Edgar T. Walters Department of Physiology and Cell Biology, University of Texas Medical School at Houston, Houston, Texas 77030

I. Introduction 11. Evolutionary Considerations

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IV.

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VI.

VII.

VII I.

A. T h e Special Place of Injury among Selection Pressures B. Evolutionary Relationships among Injury-Related Mechanisms Adaptive Behavioral Reactions to Injury A. Previous Functional Models of Injury-Related Behavior B. A General Adaptive Model of Injury-Related Behavior C. Fast Reactions to Injury and the Threat of Injury D. Risk Assessment and Behavioral Choice during Injury E. Delayed Reactions to Injury Classes of Injury-Related Behavioral Modifiability A. Behavioral Modifications following Actual Injury B. Definition of Nociceptive Sensitization C. Sensitization Targeted to a Site of Injury D. General Sensitization, Arousal, and Anxiety E. Do Some Animals Lack a Capacity for Nociceptive Sensitization? F. Antinociception, Competing Response Inhibition, and Analgesia G. Conditioned Fear Injury Signals A. Activation of Nociceptors B. Tissue Damage and Inflammation C . Neuronal Damage Mechanisms of Rapid Nociceptive Sensitization A. Incremental Sensitization Mechanisms and Imminent Injury B. Rapid Sensitization Mechanisms in Vertebrates C. Rapid Sensitization Mechanisms in Invertebrates Mechanisms of Long-Term Nociceptive Sensitization A. An Adaptive Hypothesis about Reactions to Axotomy B. Persistent Neural Sensitization in Vertebrates C. Persistent Neural Sensitization in Invertebrates Conclusions References

1. Introduction

Neuronal reactions to bodily injury in humans are associated with pain and suffering. Understandably, these reactions have been studied INTERNATIONAL REVIEW OF NEUROBIOLOCY. VOL. 36

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far more intensively by clinically oriented researchers than by comparative neurobiologists, ethologists, or evolutionary biologists. In most human settings, pain and some of its behavioral consequences are considered problems to be controlled or eliminated by any means available. Pressing as such clinical and social problems are, injury-related behavioral alterations in humans and other species may also offer clues about fundamental mechanisms underlying sensation and behavior. Indeed, research in many areas of psychology and neuroscience (for example, studies of learning and memory) routinely use experimental paradigms that simulate aspects of actual or threatened injury. In addition, because injury-related behavior in virtually all animals continues to be subject to many of the same selection pressures that have been present since the dawn of animal life, and because many of the neurons that are altered during injury-related behavior are relatively accessible, these behaviors and their underlying neuronal mechanisms offer a special opportunity to relate identifiable neuronal mechanisms to evolutionary and ecological forces, and perhaps to identify primitive mechanisms of memory that still operate in our own nervous systems. Much of the research on neural reactions to bodily injury has focused on changes in pain perception. Pain and related terms have been defined in many ways, but the following definitions from the International Association for the Study of Pain (Merskey et al., 1986) are now widely accepted. Pain is a sensory o r emotional experience associated with actual o r potential tissue damage. Hyperalgesia is an increased response to a stimulus that is normally painful, whereas the related term, allodynia (which is sometimes considered a subclass of hyperalgesia), is pain due to a stimulus that does not normally provoke pain. Analgesia is defined as the absence of pain in response to stimulation that would normally be painful, whereas hypoalgesia refers to a decrease in pain evoked by the same type of stimulation. Each of these effects is commonly, although not exclusively, produced by serious peripheral injury. Because pain is defined in terms of subjective human experience (Merskey et al., 1986; DeGrazia and Rowan, 1991), it is difficult, if not impossible, to conclude that these experiences occur in other species. However, pain in humans is accompanied by characteristic behaviors, such as withdrawal reflexes and vocalization, as well as distinctive autonomic changes. Observation of similar reactions to noxious stimulation in rats, monkeys, and other mammals has led to animal models of pain (Dubner, 1989). Hyperalgesia and allodynia are modeled in animals by states of increased responsiveness termed nociceptive sensitization. Analgesia and hypoalgesia are modeled by states of decreased responsiveness termed antinociception. Animal models have been responsible for much of our expanding knowledge of neural mechanisms that may contribute to pain and hyperalgesia.

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T h e present review begins with a consideration of evolutionary aspects of injury-related behavior, and then examines behavioral, physiological, and molecular aspects of potentially adaptive behavioral reactions to injury. Common behavioral alterations produced by noxious stimulation in vertebrates and invertebrates are noted and placed into a tentative adaptive framework. Particular emphasis is placed on the defensive functions and underlying mechanisms of sensitization, which is defined broadly and applied at three different levels: behavioral sensitization, network sensitization, and cellular sensitization. Finally, emerging results from quite separate lines of research on cellular and molecular correlates of nociceptive sensitization (network and cellular) in vertebrates and invertebrates are compared.

II. Evolutionary Considerations

Common neuronal reactions to bodily injury are likely to represent evolutionary adaptations to a hostile environment. T o begin to understand how these adaptations function, it is necessary to consider the selection pressures that shaped them. By also considering evidence about the early evolutionary history of animals, some clues about the origin and diversity of mechanisms underlying adaptive neuronal reactions to bodily injury might be found.

A. THESPECIAL PLACEOF INJURY AMONG SELECTION PRESSURES Contemporary organisms are the products of more than 3 billion years of biological evolution. Biologists disagree about the relative contributions of chance and natural selection to broad patterns of evolution (e.g., Gould and Vrba, 1982). However, there is no question that natural selection is the preeminent force that shapes and maintains adaptations (Ridley, 1985; Skelton, 1990). An evolutionary adaptation is defined as a heritable trait that enhances an organism’s reproductive success, i.e., a trait that has been selected during the course of evolution by promoting the possessor’s survival or fecundity. In an influential recent theory of adaptation, Vermeij (1987) divided selection pressures affecting survival into nonbiological hazards (e.g., extremes of temperature, radiation, and turbulence) and biological hazards (competition, predation, parasitism and disease). Of these hazards, he agrees with Darwin (1872b) and many other authorities (e.g., Fisher, 1958) that biological enemies provide the most important selection pressures. By this view, nonbiological hazards

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sometimes cause greater mortality than biological hazards, but they generally have less influence on the evolution of adaptations. Major catastrophes (e.g., volcanic eruptions, asteroid impact) can cause massive extinctions, but organisms have much less opportunity to evolve adaptations to infrequent, catastrophic events. More common nonbiological calamities (e.g., storms) shape many adaptations, but much of this pressure is actually due to an accentuation of biological selection pressures. For example, high winter mortality in some species has been linked to intraspecific fights over scarce food resources, and severe storms have been shown to dislodge various marine animals from shelter, exposing them to increased predation (see Vermeij, 1987). Bodily injury is the final mediator of many of the selection pressures affecting survival. Competition can directly injury combatants, and intraspecific combat can attract the attention of predators. Successful predation always involves some form of injury to prey. However, predatory encounters are often unsuccessful (e.g., Vermeij, 1982), and in many cases injuries inflicted on prey during such encounters are not lethal (e.g., Vermeij, 1987). Similarly, intraspecific combat and nonbiological hazards often produce sublethal injuries. As discussed below, injury that does not quickly kill an animal can reduce survival and fecundity in many ways. These effects represent extremely strong pressures which select for mechanisms that minimize injury-induced vulnerability and disruption of reproductive functions. Because virtually all animals are subject to predation and/or parasitism at some stage in their lives (Lapage, 1958; Curio, 1976; Endler, 1986; Crawley, 1992), and all organisms are subject to nonbiological hazards, one can assume that strong injury-related selection pressures have shaped all contemporary animals. Although such pressures would seem likely to result in adaptive behavioral reactions to injury, this need not always be the case. For example, anatomical adaptations (e.g., strong armor) or very short generation times, might in principle make behavioral adaptations to injury unimportant (e.g., Guthrie, 1975). It is important to find out what kinds of animals show behavioral adaptations to injury and, if one is interested in finding potentially primitive behavioral adaptations, to ask at what stage in biological evolution the features associated with adaptive behavioral reactions to injury seen today may have emerged.

RELATIONSHIPS AMONG B. EVOLUTIONARY

INJURY-RELATED MECHANISMS

Attempts to reconstruct the evolutionary history of processes that leave little or no fossil record have obvious limitations. However, paleobi-

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ological evidence coupled with comparisons of contemporary animals can provide useful clues about early events. Although evidence about the early evolution of adaptive reactions to injury is currently meager, it seems likely that systematic comparisons of nociceptive behavior and its cellular underpinnings in contemporary animals can be joined with paleobiological information to suggest plausible sequences for the evolution of some nociceptive mechanisms. Perhaps the most basic questions that can be asked are: T o what extent are common mechanisms used in adaptive reactions to bodily injury throughout the animal kingdom, and how closely are these mechanisms related to each other? Plausible arguments can be made for both recent origins and very early origins of neuronal mechanisms underlying adaptive reactions to injury, and the safest bet is that both types of mechanism are represented in the animal kingdom. The case for recent origins of many mechanisms underlying adaptive behavioral reactions to bodily injury is based on two premises. First, virtually all contemporary animals are susceptible to some form of injury, suggesting that such injury-related pressures have long been widespread and have provided many opportunities during evolutionary history for the selection of new mechanisms to support adaptive responses to bodily injury. Second, different groups of animals have evolved an enormous diversity of life styles and, presumably, correspondingly diverse reactions to injury. Multifarious reactions to injury are likely to involve a significant number of recently evolved mechanisms specialized for different injuryrelated needs in different animals. Although common injury-related selection pressures exist and can result in apparently similar mechanisms in different animal groups, by this view these are most likely to be analogous (independently derived) adaptations. T h e opposing view is that many adaptive neuronal reactions to bodily injury in contemporary animals are mediated by primitive mechanisms shared with most other animals. This would be true if the last common ancestor of existing animal phyla already possessed fundamental neuronal adaptations to bodily injury and these mechanisms were largely conserved during subsequent evolution. This hypothesis would be supported by evidence that common ancestors of modern animals also faced strong, injury-related selection pressures. The last common ancestor of today’s major animal groups lived at least 570 million years ago, prior to the dramatic evolutionary radiation in the early Cambrian period when most of the basic body plans characteristic of modern phyla originated (McMenamin and McMenamin, 1990). This ancestral animal is thought to have been small and soft-bodied, perhaps with affinities to today’s flatworms (Fedonkin, 1990; Signor and Lipps, 1992). The extent to

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which it and its ancestors were preyed upon is unknown. Some authorities think that predators were present in the late Precambrian (Vendian) era, and that growing predation pressure may have been a major cause of the mass extinction of many animal groups during this period (Stanley, 1973; Fedonkin, 1990). However, the rarity of fossils (because mineralized skeletons and shells had not yet evolved) makes firm conclusions difficult (Hutchison, 196 1).Other authorities suggest that predators were uncommon during this early stage of metazoan evolution (McMenamin and McMenamin, 1990), which, if true, would indicate that predatory pressures had relatively little effect on the development of adaptations by these primitive animals. Another way in which diverse animals could share homologous mechanisms of injury-related plasticity is by parallel evolution. In this scenario similar injury-related pressures (in divergent lineages) repeatedly led to parallel modifications of preexisting, homologous mechanisms that had a different function in the last common ancestor of these groups. This primitive mechanism would have had to be highly “preadapted” for minimizing deleterious aftereffects of injury. Cooptation of preexisting solutions to different problems (a process termed “preaptation” or “exaptation” by some authors (see Gould and Vrba, 1982; Campbell, 1990) appears to be a frequent occurrence in evolution. It is much easier to refine existing mechanisms for new functions than to create entirely new mechanisms (e.g., Jacob, 1977). Moreover, cooptation of preadapted mechanisms can facilitate the gradual evolution of novel designs through a series of stages, with intermediate designs being adaptive for each stage (Campbell, 1990). In principle, the relative times of origin of different physiological mechanisms can be inferred by systematic comparative studies across lineages having known times of divergence. Identification of key molecules involved in injury-related neuronal and behavioral plasticity (although a very difficult task) will increase the power of such comparisons, and may eventually offer opportunities for the application of methods used in the study of molecular evolution to questions about the evolution of adaptive reactions to injury.

111. Adaptive Behavioral Reactions to Injury

If it is assumed that selection pressures acting on injury-related behavior have been very strong during most of evolutionary history, it follows that common patterns of behavioral and neuronal plasticity observed after injury are likely to have widespread adaptive value. Discerning the

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adaptive function(s) of a biological trait can be extremely difficult, and some traits probably lack any true biological function (e.g., Could and Vrba, 1982). However, strong evidence for the adaptiveness of most anatomical and physiological traits suggests that the most conservative assumption is that a given trait has a function shaped by natural selection, even if that function is not obvious (cf. Tinbergen, 1951; Alcock, 1989; Skelton, 1990; Williams and Nesse, 1991). Because natural selection acts at the behavioral level rather than the cellular or molecular levels (e.g., Arbas et al., 1991), this assumption may be particularly good for behavioral traits. Recognition of the adaptive functions of prominent injuryinduced changes in behavior will be necessary for a full understanding of the biology of pain, analgesia, and nociceptive memory, and may prove useful for guiding investigations into some of the underlying mechanisms.

MODELSOF ~ J U R Y - R E L A BEHAVIOR TED A. PREVIOUS FUNCTIONAL At first glance, the adaptive functions of behavioral alterations following injury may seem so obvious on the basis of our personal experience not to merit detailed examination. This evident transparency of function (in addition to ethical dilemmas inherent in controlled studies of injury in mammals) may explain why relatively little has been written about the biological significance of common behavioral reactions to injury, and why much of the supporting evidence for function is anecdotal. At second glance, the adaptive functions of behavioral reactions may not be obvious, especially with regard to the functions of the most distressing reaction to injury-pain. Until about 15 years ago the dominant perspectives on the function of pain were variants of (a) the traditional view of physiologists (e.g., Hardy et al., 1952) that pain is a simple sensation that serves as a warning device to prevent further injury and (b) a longstanding view of experimental psychologists that pain is a motivational device that serves to condition, reinforce, or punish behavior (e.g., Mowrer, 1939; Solomon and Brush, 1956). Neither of these views explains the dramatic variation seen in pain reports and injury-related behavior among different subjects or in the same subject at different times after injury (e.g., Melzack and Wall, 1983). Variation in pain-related behavior with time following noxious stimulation has long been noted. For example, more than a century ago Charles Darwin pointed out that pain has different effects depending on the time since a traumatic event: Pain, if severe, soon induces extreme depression or prostration; but it is at first a stimulant and excites to action, as we see when we whip a horse. . . (Darwin, 1872a, p. 81.)

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Attempts to account for sequential changes in pain sensitivity following noxious stimulation are central to two functional models of injuryrelated behavior that have greatly influenced recent research on pain and fear. In 1979 Patrick Wall argued against the view that pain is primarily a sensory phenomenon (signaling injury), claiming instead that the most important function of pain is to drive recuperative behavior. Wall divided the period after injury into immediate, acute, and chronic stages. In the immediate stage escaping, fighting, and obtaining aid take priority over treating the injury, which explains why the organism is analgesic. The acute stage is a transitional phase marked by pain, agitation, and aggressiveness. Recuperation takes place during the chronic stage, which is characterized by intense, often unremitting pain, as well as prolonged sleep, inactivity, depression, and disturbances of eating, grooming, and social behavior. The intensity and duration of this phase depend on the amount of damaged tissue. At about this time Robert Bolles and Michael Fanselow (1980)developed a “perceptual-defensive-recuperative” (PDR) model of fear and pain that displayed several similarities to Wall’s model. It divided postinjury behavior into the three phases for which the model was named, and based the third phase on Wall’s idea that the function of delayed pain is to promote recuperation from injury. A novel part of the PDR model was the initial “perceptual” phase. Bolles and Fanselow pointed out that an organism is continuously perceiving aspects of its environment, and the perceived context of an injurious event (including the attacker, if present) will be associated with an injury. Thus the most immediate effect of injury, and the most important function of the perceptual phase, is learning: specifically, learning to fear the source of the injury and the context in which the injury occurred. This fear will be rapidly triggered in subsequent encounters with stimuli that had been associated with the injury. Fear also occurs following injurious stimulation, during the defensive (second) phase. Like Wall’s model, the PDR model emphasizes the inhibition of pain-related recuperative behavior during and shortly after a noxious encounter, i.e., when escape andfor fighting take priority (this occurs in the first phase of Wall’s model). Unlike Wall, they suggested that this analgesia and other aspects of defensive behavior (freezing, fighting) are primarily a response to fear, rather than a response to the injury itself or to pain. In the PDR model, postinjury analgesia occurs because the organism fears the context (including the attacker) in which it was just injured, and this fear inhibits pain. Conversely, they suggest that hyperalgesia during the recuperative phase may involve inhibition of fear by pain. Both of these functional models have been refined over the years, but remain essentially intact (e.g., Wall, 1989; Fanselow and Lester,

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1988). Both continue to influence research, with Wall’s model making a larger impact on researchers studying the physiology and pathophysiology of pain and analgesia (e.g., Price, 1988; Kiyatkin, 1990), and the PDR model influencing research on the behavior and behavioral pharmacology of conditioned fear and analgesia (e.g., Siegfried et al., 1990). Although each model is useful, neither provides a truly comprehensive account of common behavioral reactions to injury observed in mammals, and neither is concerned with identifying the most fundamental adaptive behavioral patterns (i.e., those that are likely to be shared with nonmammalian species). Identification of fundamental behavioral and neuronal adaptations to bodily injury requires a close inspection of behavioral and neuronal reactions to bodily injury in diverse groups of animals.

ADAPTIVE MODELOF INJURY-RELATED BEHAVIOR B. A GENERAL Before general patterns of injury-related behavior are reviewed, a more comprehensive model of injury-related behavior is presented (Table I). This model summarizes the main conclusions to be drawn from behavioral research that will be reviewed below and provides a basis for selection of injury-related changes in neural properties to be reviewed. As was just seen, most interpretations of the functions of injury-related behavior have been based on clinical reports, anecdotal observations of the behavior of wounded mammals, and data collected from rats using aversive electric shock as an injury-like stimulus. Although useful, these sources provide a restricted perspective on injury-related behavior. This perspective can be broadened by incorporating findings from ethology and behavioral ecology, and by including observations on invertebrates. In subsequent sections, injury-related behavior is reviewed from this broader biological perspective. The general adaptive model of injuryrelated behavior shown in Table I includes features that have been incorporated into various other behavioral models of injury and defense, including an invertebrate model of injury-induced behavior (Walters, 199 l), the Wall (1979) model (specifically, the recuperative phase and analgesia during escapelfighting behavior), the PDR model (specifically, an immediate perceptual phase during which conditioned fear is acquired-Bolles and Fanselow, 1980; Fanselow and Lester, 1988), and models of anti-predator and agonistic behavior in wild rats (specifically, a risk assessment phase during the initial injurious encounter and postencounter vigilance-Blanchard and Blanchard, 1988; Blanchard et al., 1990). As will be seen, however, the general adaptive model of injuryrelated behavior differs from earlier models by including (a) rapid risk assessment and behavioral choice based on properties of the noxious

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EDGAR T. WALTERS TABLE I GENERALADAPTIVEMODELOF INJURY-RELATED BEHAVIOR Immediate

A. Injury detection 1. Location 2. Severity (actual or imminent) 3. Rate of change (if imminent injury)

B. Withdrawal, startle-like responses C. Imprint attacker and COI in memory

Rapid

Delayed

A. Passive defense (mild or A. Wound-related protection 1. Seek shelter and hide potential injury) (immobility, vigilance) 1. Freeze or slowly retreat 2. Risk assessment 2. Sensitize wounded sites 3. Prepare for active defense 3. Prime appropriate defensive actions 4. Protect stimulated site 4. Remember dangerous B. Active defense (severe or context, warning signals likely injury) 1. Escape and/or retaliate

2. Suppress competing responses

C. Tonic immobility (when active defense fails)

B. Recuperative behavior 1. Clean, disinfect wound 2. Immobilize injured part 3. Conserve energy 4. Monitor healing

stimulus itself, and (b) an emphasis on defensive functions during the delayed, recuperative phase following injury.

C. FASTREACTIONS TO INJURY A N D THE THREAT OF INJURY The focus of this article is on adaptive reactions to injurious stimulation of the body surface. Behavioral reactions to injury often occur within the context of defense against an attack by a predator or an aggressive conspecific. Ethological and ecological aspects of defensive behavior have been studied in diverse species (e.g., Edmunds, 1975; Curio, 1976; Endler, 1986; Vermeij, 1987; Blanchard et d., 1990; Crawley, 1992). Although these studies have paid little attention to the effects of injury on behavior, they provide an important backdrop for interpreting functional aspects of injury reactions. Clearly, an animal is most likely to survive and reproduce if it escapes a potential attack before it develops, and the function of most defensive mechanisms is to prevent physical contact with an attacker. Consequently most prey animals have welldeveloped “primary defenses,” such as crypsis (camouflage, immobility) to minimize the prey’s detectability by predators, or aposematism (e.g., warning colors), which advertises the prey’s toxicity or distastefulness (Edmunds, 1975). Many prey also have very sensitive predator recognition capabilities that allow prey to move out of danger before the predator can attack (Tinbergen, 1951; Blanchard and Blanchard, 1988; Curio, 1993). Once a predator is recognized, a large variety of “secondary defenses” may come into play, including passive defenses (e.g., armor,

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distastefulness, toxicity) and active defenses (withdrawal into a shelter, flight, startle displays, diversion behavior, feigning death, aggressive retaliation) (Edmunds, 1975; Endler, 1991).When physical contact is made, and the predatory encounter enters the subjugation phase (Endler, 1986), injury becomes likely. However, even at this stage, death may be avoided. In a survey of 60 predator species Vermeij (1982) found that predators attacked only 19% of their prey with an efficiency (after detection) greater than 90%. In most cases a predator’s detection, pursuit, and subjugation of prey are unsuccessful, at least in part because of the efficacy of the defensive behavior of prey. Although defensive behavior during an attack shows almost unlimited variety across the animal kingdom, several features are quite common. Freezing (e.g., Hediger, 1955; Ratner, 1967; Myer, 1971; Hirsch and Bolles, 1980; Fanselow and Lester, 1988) is a widespread response to detection of a predator at a distance. T h e sudden immobility eliminates many of the visual and auditory cues used by predators to locate and track prey. Startle responses (Eaton, 1984) also occur in many animals prior to a potential attack, especially to sudden, rapidly looming stimuli. Startle-like, ballistic responses such as jumping can also be an immediate response to noxious stimulation of the body surface in some animals (e.g., Fanselow, 1982; Kiyatkin, 1990), but such reactions to noxious (as opposed to visual and auditory) stimuli have received little scientific attention. T h e most common overt responses to noxious contact are withdrawal (of the stimulated body part or sometimes the entire body) and escape locomotion (flight). Examples of defensive withdrawal abound throughout the animal kingdom (see also Kavaliers, 1988a) and include, for example, the flexion reflexes of humans and other mammals (Sherrington, 1947), leg withdrawal in caterpillars (Weeks and Jacobs, 1987), bending and shortening reflexes of leeches (Kristan et al., 1982), various withdrawal reflexes in the gastropod mollusc Aplysia (Walters and Erickson, 1986), withdrawal of sea anemones (Pantin, 1950), and contraction of ciliate protozoans (Wood, 1970). These reflexes are extremely rapid (often involving short, mono- or disynaptic pathways between sensory and motor neurons) and are largely automatic. Locomotion away from a noxious stimulus has been observed in virtually all organisms that can move (e.g., Eisemann et al., 1984; Kavaliers, 1988a), including the simplest animals, such as jellyfish (Donaldson et al., 1980) and flatworms (Corning and Kelly, 1973),as well as protozoans (e.g., Kung et al., 1975) and even bacteria (Berg, 1975). In some cases escape locomotion may be the most immediate response to a noxious stimulus. However, the most rapid locomotor responses described thus far are startle-like responses that appear to be activated preferentially

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by distal (visual and auditory) stimuli. Examples include the fast turning and running responses of cockroaches to wind puffs (Camhi and Tom, 1978), the giant-axon-mediated tail flip responses of crayfish to visual stimuli or light taps (e.g., Wine and Krasne, 1982),and “fast-start” swimming responses of goldfish to rapidly looming visual stimuli (Eaton et al., 1981). In contrast, when the eliciting stimulus is intense and activates nociceptive o r somatosensory receptors, escape locomotion often begins with a slight delay (beginning after immediate withdrawal reflexes). For example, local withdrawal precedes escape (swimming, crawling, or running) in the leech (Kristan et al., 1982), the nudibranch mollusc Trztonza (Getting, 1977), and Aplysia (Walters and Erickson, 1986). This delay may simply reflect the longer time needed for signals to get through the more complex circuitry involved in generating rhythmic locomotor patterns. However, delays may in some cases function to increase response flexibility. For example, pinching a crayfish tends to activate the relatively slow non-giant-mediated tail flip sequence rather than the fast giant-axon-mediated tail flip (Wine and Krasne, 1982). The latter is a ballistic response that cannot be modified once initiated. T h e former begins later, but allows directional control and evasive manuevers, which may be adaptive if an attacker is close enough to pinch or bite the crayfish. In general, a slight delay may be particularly useful when the source of the noxious stimulus is uncertain; the time gained may be used to quickly find out more about the nature and disposition of the attacker. Aggressive retaliation (fighting back) is also a common form of defense in vertebrates (Edmunds, 1975; Endler, 1986). Retaliation using diverse weapons occurs in invertebrates as well, as anybody who has received a bee sting or ant bite knows. Retaliation with noxious chemical substances is particularly common in insects (Evans and Schmidt, 1990). Aggressive retaliation, as well as escape locomotion and withdrawal, should benefit from rapid priming of the appropriate motor and sensory systems at the earliest sign of danger. In mammals such mobilization of the body’s defensive resources during or prior to an emergency is familiar as the “fight or flight” reaction (Cannon, 1929).General defensive arousal has also been implicated in arthropods (e.g., Kravitz, 1988)and gastropod molluscs (e.g., Walters et al., 1981; Walters and Erickson, 1986). Another important means of optimizing defensive efforts during an encounter with a predator or aggressive competitor is to suppress behavior that may interfere with fighting or fleeing. This is probably the most important function of attack-related analgesia in mammals (e.g, Wall, 1979; Bolles and Fanselow, 1980 and see below). Inhibition of potential competing responses during defensive behavior has also been described in gastro-

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pod molluscs (e.g., Davis and Mpitsos, 1971; Marcus et al., 1988; Illich et al., 1994a) and crayfish (e.g., Kuwada and Wine, 1979).

D. RISKASSESSMENT AND BEHAVIORAL CHOICE DURING INJURY Clearly, it is adaptive to perceive and assess a threat as early as possible during an attack. Any error in predator detection is costly. In answering the question-is an attack occurring?-the cost of a false negative answer is probably death. The cost of a false positive answer is, at the very least, disruption of ongoing behavior. Frequent disruptions will reduce energy intake and reproductive activity. More dramatic costs are incurred if a false positive answer triggers active defenses. Active responses use more energy, but, more importantly, visual and auditory stimuli associated with vigorous defensive actions are very effective in attracting predators. For example, a struggling fish attracts sharks, and a hare’s distress call attracts birds of prey (Curio, 1976). Indeed, distress calls may be an adaptive defense in part because newly attracted predators may distract or even attack a predator that has seized prey that is emitting calls, thus providing additional opportunities for the prey to escape. Although these considerations have guided various analyses of defensive behavior in response to distant (visual, auditory, chemical) danger signals (e.g., Tinbergen, 1951; Endler, 1986; Fanselow and Lester, 1988; Blanchard et al., 1990; Caro and Fitzgibbon, 1992), they apply equally well to aspects of defensive behavior triggered by somatosensory stimuli (touch and pain). Ethologists and experimental psychologists have focused largely on the behavior of highly visual and auditory animals that rely primarily on these modalities (and smell) for warning. However, even in these animals, attacks can occur under conditions in which visual and auditory information is inadequate. For example, many predators (e.g., owls and various snakes, fish, spiders, insects, and centipedes) move silently and hunt at night (Pfeffer, 1989). Other attackers, including ectoparasites such as fleas, lice, and mosquitos, may be too small to be noticed before they come into contact and provide noxious stimulation. Furthermore, even an animal with excellent distance receptors may not perceive an attack before contact is made because the animal is distracted or asleep. It should also be remembered that most invertebrates (with the notable exceptions of many arthropods and the cephalopod molluscs) lack image-forming eyes and sensitive auditory systems. Although many of these animals use chemical senses for distance reception, chemical stimuli may provide little or no warning of a fast-moving attack. The behavior of simple blind and deaf animals may seem far removed from

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that of humans, but at some point during evolution our early ancestors (and the ancestors of all other animals) had not developed sensitive visual and auditory systems, and thus probably depended on cutaneous sensation for the earliest detection of an attack. Adaptive responses to potential or actual injury depend on the nature of the attack and the context in which it occurs. One of the propositions of this article is that many animals rapidly utilize somatosensory information present at the onset of injurious stimulation to assess the risk of further injury and to choose the most appropriate behavioral response. In particular, the location, severity, and rate of change of injurious stimulation are evaluated, and this information is combined with other information to decide, initially, on passive or active defense (Table I). These defenses are usually initiated after very rapid (a)reflexive withdrawal of the stimulated body part and (b) startle-like responses in some cases. Passive defense is likely to be chosen if the injury is slight, the stimulus is brief or decreasing in intensity, and the possible costs of active defense are substantial (e.g., local predator density is high and movement is likely to attract predatory attention). Active defense will be chosen if the injury is severe, the stimulus is prolonged (or repeated), and/or the costs of active defense are low. In later sections of this chapter behavioral and neuronal plasticity contributing to rapid risk assessment and behavioral choice after injury will be discussed. Risk assessment is proposed to occur in part on the basis of information derived from properties of the noxious stimulus (location, severity, rate of change, repetition), and defensive choices can be made even if there is little or no additional information available for assessing risk. By this view, the noxious stimulus is a danger signal, warning of additional (perhaps much worse) potential injury. This view is consistent with previous suggestions that some forms of pain act as danger signals (e.g., Hardy et al., 1952) and that pain or injury can directly activate complex ensembles of defensive behavior which sometimes include analgesia (Melzack and Wall, 1983; Blanchard and Blanchard, 1988; Siegfried et al., 1990). It differs from assertions that a noxious or injurious stimulus (the unconditioned stimulus or US in the PDR/conditioning model of fear and pain, see Bolles and Fanselow, 1980) is not itself a danger signal.

E. DELAYED REACTIONS TO INJURY Animals that escape during the subjugation phase of a predatory encounter are likely to have sustained injuries, as are animals that have engaged in intense intraspecific combat (e.g., Blanchard et al., 1985).

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Sometimes animals can recover from extremely severe injuries. For example, Schaller described a deer that had survived an attack by a tiger: One adult sambar stag at Kanha was apparently straddled by a tiger and severely lacerated before managing to escape. The wounds healed, leaving large scars on the shoulders, sides, and rump, some of them 12 to 15 inches long and 2 inches wide. There were eleven such scars on his right side and nine on the left. His movements were stiff and slow. (Schaller, 1967, p. 290.)

Another anecdotal testament to the remarkable ability of some animals to survive appalling injury in the wild comes from hunters who have killed game and found earlier bullet wounds or the stumps of limbs previously amputated in traps. While severe injury can sometimes be overcome in the wild, it is obvious that injury poses significant risks. Even an injury that is not immediately life threatening, such as was produced experimentally in the field by clipping toads’ toes off, can signfiicantly reduce survival rates (Clarke, 1972). Controlled experiments have also shown that injury reduces reproductive capacity. For example, after autonomy of their tail, salamanders show a delay in sexual maturity and a reduction in fecundity (Maiorana, 1977). Reduced fecundity is probably due to a diversion of physiological resources into recuperation and regeneration (during a time when energy intake is often reduced, see below). Why d o survival rates decrease? The most obvious answer is that injury weakens an animal, leaving it less able to care for itself. Thus, it may not be able to get enough to eat or drink. It may also be less able to defend itself against predators and aggressive conspecifics, some of whom may be attracted by signs of injury (see below). Following Wall (1979),most commentators on injury-related behavior explicitly or implicitly recognize the importance of optimizing an animal’s recuperative efforts after an injurious encounter by using persistent pain to motivate behavior that minimizes disturbance and promotes treatment of the wound (e.g., Melzack and Wall, 1983; Price, 1988). However, there has been less recognition of the importance of defense during recuperation. Indeed, the PDR model asserts that recuperative behavior and defensive behavior are mutually antagonistic and do not overlap (Bolles and Fanselow, 1980). However, Bolles and Fanselow based their conclusions on experiments using a noxious stimulus, electric shock, that typically produces very little actual injury. As will be discussed below, tonic signals coming from damaged tissue are likely to amplify and maintain behavioral alterations following noxious stimulation. There are at least three significant threats to an injured region of an animal. First, the strains inherent in normal activity may cause additional damage to body parts weakened by injury (e.g., Melzack and Wall, 1983).

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Thus an injured region needs to be protected from self-inflicted damage. Behavioral protection (e.g., inactivity, guarding responses) may be particularly important in animals, such as most vertebrates, some molluscs, and many worms, that lack a hard exoskeleton. The exoskeleton of insects and crustaceans provides a strong cast to protect damaged tissue and keep it immobilized during repair and regeneration. Arthropods and other segmented animals also benefit from having large numbers of appendages. Severe damage to an appendage, or even its loss, is of little consequence since remaining appendages can maintain behavioral function. Many arthropods readily sacrifice appendages, autotomizing limbs that have been injured or poisoned (e.g., Sherrington, 1947; Eisner and Camazine, 1983). In these animals a completely new limb is often regenerated to replace the autotomized limb (Guthrie, 1975). The second threat occurs when a wound breaks the integrity of the body surface. An open wound not only permits the entry of infectious microorganisms (microparasites), but may also attract macroparasites. Disease and parasites directly increase mortality, and also increase the risk of predation (Curio, 1976; Dobson et al., 1992; Moller et al., 1993). Indeed, some parasites change the appearance and behavior of intermediate hosts in order to increase the probability of being eaten by the final host (Curio, 1976). Following escape, many vertebrates (Wall, 1979; Bolles and Fanselow, 1980) and perhaps some insects (Hentschel and Penzlin, 1982) spend time grooming their wounds. Licking cleans the wound, and, at least in mammals, the saliva delivers to the wound antimicrobial factors (including immunoglobulins, lysozyme, peroxidase, myeloperoxidase, lactoferrin, and thiocynate-see Sherertz, 1988; Hart and Powell, 1990), and growth-promoting factors [including epidermal growth factor (EGF) and nerve growth factor (NGF)-see Glantz et al., 19891. In rodents licking has been shown to accelerate wound healing (Hutson et al., 1979; Bodner et al., 1991). Moreover, wound healing is accelerated by application of EGF (Niall et al., 1982; S. Noguchi et al., 1991) or NGF (Li et al., 1980; Lawman et al., 1985). The third threat results from the attractiveness of injured animals to predators. Curio (1976) has reviewed a number of studies suggesting that injured animals are preyed upon preferentially by wolves and various birds. He also points out that the “broken-wing trick” commonly used by birds to distract predators from their young strongly indicates the efficacy of injury-like behavior in attracting predators. Oddity of movement, often a consequence of injury, is particularly effective at provoking attack by many predators, including birds, fish, mammals, and octopus (Curio, 1976).A wounded site on the body may be preferentially targeted by some predators because it is relatively weak and vulnerable. Predators

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and parasites may not be the only threats an injured animal faces. Various birds (especially corvids) will attack and kill disabled conspecifics (or even disabled members of other species that are not their prey), presumably to remove the potent predatory attractant from the home area of the aggressive nonpredators (Curio, 1993). Somewhat similar pressures may be faced by some invertebrates. For example, injured cockroaches maintained under social conditions in the laboratory are usually attacked and devoured by conspecifics within 2 days of injury (Guthrie, 1975). Chemosensory signs of injury are potent attractants for many different predators. For example, domestic cats have been shown to be aroused by the smell of rodent blood, and this arousal is particularly dramatic if the donor is stressed with noxious shock prior to collection of the blood (Cocke and Thiessen, 1986). Injury-related chemoattractants may be particularly important in an aquatic environment (the environment in which most of biological evolution occurred). Tissue damage releases a large number of water-soluble molecules that are normally found inside cells, and these are very potent stimulants to many aquatic predators. Various fish as well as lobsters and shrimp are excited by extracts of marine animals, and this stimulation can be mimicked by low concentration mixtures of common, low-molecular-weight constituents of cells and blood, such as amino acids, nucleotides, and quartenary amines (Carr and Derby, 1986; Carr, 1988). This effect is used to advantage by spear fishermen, who break apart common marine creatures such as sea urchins to attract elusive fish. Because so many predators respond to chemoattractants found in wounds, an aquatic animal that has been wounded will be at great risk for additional attacks if fluid from inside the body continues to leak out of the wound, o r if there is extensive superficial tissue damage. Because many predators and parasites use chemical senses to guide their attacks, a wounded site is particularly likely to be the target of any additional attacks. If the injury is severe, all of these injury-related threats may persist for a long period of time while the animal recuperates. Although most animals have powerful hemostatic mechanisms to stop blood loss after injury, these mechanisms may not be completely effective, particularly in animals having high blood pressures (e.g., invertebrates possessing hydrostatic skeletons). High internal pressures will act to drive blood out of any perforations in injured integument. Because wounded tissue may be weaker than normal, wounds can reopen if active behavior begins before healing is complete. This would expose the animal once again to the spectrum of injury-related threats. All of these considerations point to the adaptiveness of defensive vigilance during recuperation, and the special importance of guarding the wounded region (Table 1). For some

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period after an attack, the attacker (or other members of its group) is likely to be nearby, and the injured animal will be well served by seeking shelter, hiding, remaining immobile, and maintaining an intense, general vigilance. The special vulnerability of the wounded region suggests that defenses should be focused on this part of the body. This can be accomplished by making the wounded site particularly sensitive, so that local protective reflexes are rapidly activated at the first sign of disturbance. It should also be useful to have specific defensive motor patterns appropriate for protecting the wounded region primed and ready for action. Long-term behavioral alterations targeted to a site of injury can be considered to be one example of a much larger class of “inducible defenses.” These are defined by ecologists as responses “activated through a previous encounter with a consumer or competitor that confer some degree of resistance to subsequent attacks” (Harvell, 1990).Inducible defenses are characterized by the properties of specificity,amplification, and memory and include immune responses, the growth of spines or armor, and fear conditioning. As time passes the likelihood that the original attacker is nearby will decrease and the costs of immobility and intense viligance will increase. Normal behavior can resume gradually if the general vigilance and tendency to freeze are relaxed. However, until healing is completed, it should be adaptive to minimize movement, and to maintain local, wound-specific hypersensitivity and guarding behavior. Localizing these protective effects to the injured part of the body will reduce interference with other behaviors that periodically become necessary (e.g., feeding, drinking). On the other hand, even wound-specific protective behavior will have costs, so it will be beneficial to monitor the wound and its recovery so that completely normal behavior can be resumed as soon as healing is adequate.

IV. Classes of Injury-Related Behavioral Modifiability

An enormous number of studies (largely by experimental psychologists) have examined modifications of behavior by noxious stimulation. However, with a few exceptions, little of this research has considered the functional significance of these modifications from an injury-related perspective. Most behavioral studies have addressed the effects of noxious stimulation on associative modifications of test responses such as bar-pressing, running in a shuttle box, or analgesia, and have generally ignored questions about how an animal detects, interprets, and reacts to a noxious unconditioned stimulus in the absence of prior experience

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with that stimulus. Furthermore, such studies almost never involve explicit peripheral injury; typically, aversive cutaneous shock is used. Despite these caveats, it is likely that several classes of behavioral modifiability studied with aversive shock represent injury-related responses. Aversive peripheral shock can be considered noxious, even when it does not damage tissue, if it causes high-frequency activation of nociceptors similar to that produced by actual injury. Low-threshold cutaneous afferents (and other fibers too, such as sympathetic axons and motor neuron and proprioceptor axons if the electrode is in muscle) are likely to be activated as well, but these fibers would also be activated transiently by actual injury if their peripheral branches were damaged. The major difference from actual injury is probably that electric shock does not lead to the induction of persistent activity or to slow axonal injury signals in neurons innervating the region of noxious stimulation (see below). This consideration suggests that noxious electric shock applied to skin and muscle may be useful for investigating rapid effects of injury, but may not trigger some of the important long-term effects of actual injury. In this section reflex modifications produced by actual injury will be described, followed by discussion of classes of behavioral modifiability that have been defined primarily on the basis of studies involving electric shock.

A. BEHAVIORAL MODIFICATIONS FOLLOWING ACTUAL INJURY

There have been very few experimental studies of the effects of actual injury on behavioral responses of animals. This may be due to ethical considerations, and perhaps to a lack of recognition of the important role that injury and potential injury play in the behavioral biology of most animals. Nevertheless, two studies using explicit peripheral injury illustrate behavioral modifications consistent with the general adaptive model of injury-related behavior proposed above (Table I ) . Clifford Woolf (1984) avoided the ethical problems attendant on the deliberate production of serious injury in an experimental animal by using chronic decerebrate rats to examine long-term alterations in the hindlimb flexion reflex. In the absence of injury he found that moderately intense mechanical stimulation produced “wind-up”-successively greater reflex responses (having successively lower thresholds) to repeated stimuli. Moderate thermal injury of the side of the foot caused a large decrease in the flexion reflex threshold (to calibrated von Frey hair stimuli) near the injury and a small decrease in threshold for contralateral test stimuli. The injury also decreased the latency and increased the vigor of responses to

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immersing the injured foot in a hot water bath. These effects occurred within 10 min and remained for at least 3 h. In most cases no tissue necrosis occurred and the swelling subsided within 24 h. In every case the reflex responses also returned to normal within 24 h. Reflexes evoked by stimulating the contralateral foot showed a slight, statistically insignificant enhancement. More severe thermal contact injuries to the foot (which produced tissue necrosis) caused much larger increases in reflex sensitivity to mechanical (but not heat) stimuli, and this dramatic enhancement persisted for 4-7 weeks. The severe injury also produced a smaller, but statistically significant, enhancement of reflexes evoked by stimulating the contralateral foot. Complex effects of severe injury on defensive responses of rats have been reported from Eastern Europe (Kiyatkin, 1990). Limb injury caused a decrease in the threshold for evoking vocalization by tail shock, while increasing the latency for tail withdrawal to tail shock. Unfortunately, the injured limb does not seem to have been tested, so it is not known whether that limb showed woundspecific sensitization, as would be predicted by the general adaptive model (Table I). Somewhat similar effects of peripheral injury on a defensive reflex have been observed in the mollusc Aplysia (Walters, 1987a). T h e injury procedure was designed to mimic the biting pattern observed in attacks on Aphsia by a carnivorous mollusc, Pburobranchaea, in the lab (Walters et al., 1993; see also Clatworthy and Walters, 1993b). A rapid series of 10 brisk pinches was delivered to one side of the tail and then a small “bite” of tissue was removed during the last pinch. The defensive reflex used to monitor alterations in behavior was a siphon response that is involved in directing defensive ink secretions toward the tail (Walters and Erickson, 1986). When tested between 2 h and 1 week after injury, cutaneous test stimulation near the site of injury produced enhanced siphon responses. Cutaneous test stimulation of the contralateral side of the tail produced weak, shorter-lasting facilitation of the same responses. Essentially identical results were found when strong, noxious shock was substituted for the pinch and bite sequence (see below). The main finding both in the decerebrate rat and in Aplysia was that the body surface near an injury became effectively hypersensitive (as indicated by enhanced reflexes evoked in that region), and this hypersensitivity lasted for days or weeks while the injury healed. Because the effective hypersensitivity was greatest near the site of injury, this effect was termed site-specific sensitization (Walters, 1987a). In addition, each study found more general facilitatory effects that were expressed by responses to test sites contralateral to the injury. This general sensitization was weaker than the site-specific sensitization. These and other

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behavioral alterations produced by actual injury are likely to represent previously defined classes of behavioral modification that have been examined extensively by experimental psychologists using stimuli (usually electric shock) that mimic injury. It should be noted that in the absence of additional information (ideally, recordings from the involved neurons), even the most sensitive behavioral studies cannot distinguish conclusively between changes in ( a ) the sensitivity of the sensory system representing the injured region and (b) the sensitivity or responsiveness of the motor systems mediating defensive responses specific for the injured region. However, as will be discussed below, several mammalian and invertebrate preparations have begun to yield information on the localization of plastic changes and other issues concerning the neuronal mechanisms underlying injury-related behavioral modifications.

B. DEFINITION OF NOCICEPTIVESENSITIZATION 1. Hyperalgesia, Allodynia, and Sensitization

Hyperalgesia and allodynia may be considered aspects of nociceptive sensitization in humans. Human injury produces “primary” hyperalgesia within the injured tissue itself and “secondary” hyperalgesia in the surrounding, undamaged tissue (Lewis, 1942; Hardy et al., 1952; Treede et al., 1992). Both classes of hyperalgesia are expressed as an increase in the magnitude of pain evoked by painful stimulation and a decrease in pain threshold. H yperalgesia is often accompanied by allodynia, in which touches or temperatures that normally are not painful evoke pain. Thus, both hyperalgesia and allodynia reflect an increase in sensitivity of the injured region. These effects, accompanied by facilitated responses that serve to protect the injured site, often last about as long as it takes the injury to heal. However, chronic pain that persists long after healing is complete occurs in some patients (e.g., Melzack and Wall, 1983). As discussed above, hyperalgesia and allodynia are often not apparent until some time after an injury (Wall, 1979; Bolles and Faneslow, 1980), but under some conditions they are expressed almost immediately (Hardy et al., 1952; Melzack and Wall, 1983). T h e term sensitization has had different meanings in different disciplines involved in the study of injury-related behavior and neuronal plasticity. For example, two forms of sensitization of protective behauioral responses have been mentioned in this article as a consequence of noxious stimulation of the decerebrate rat and Aplysia. This use of “sensitization” at the behavioral level is consistent with its use by psychologists studying

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learning and memory, who usually restrict the term to behavioral modifications. On the other hand, psychologists studying pain and hyperalgesia typically use the term “sensitization” to refer to an increased sensitivity of neural systems, e.g., “peripheral sensitization” and “central sensitization” of nociceptive pathways. These different uses of “sensitization” by investigators in two fields that have largely been separate from each other present a difficulty for any attempt to integrate knowledge from both fields. One way to overcome this difficulty is to define “sensitization” in a manner that permits it to be applied consistently to injury-related data at both the behavioral and the neural levels of analysis.

2 . Types of Nociceptive Sensitization: Definitions Sensitization is defined here simply as an increase in the effective sensitivity of a response system. An increase in sensitivity is identified by one or more of the following changes in a response elicited by a constant stimulus: an increase in the magnitude, duration, speed, or frequency of occurrence of the response, or a decrease in its threshold or latency. The response system may be of any type, but this chapter is concerned with sensitization of responses observed at the behavioral, neural network, and single neuron (cellular) levels. The latter are both considered forms of neural sensitization. This article is also solely concerned with nociceptive sensitization, defined as sensitization (behavioral or neural) triggered by noxious stimulation of the body. In general, nociceptive sensitization is assumed to enhance defensive rather than appetitive responses (e.g., Walters et al., 1981), although this may not always be true (e.g., Kupfermann and Weiss, 1981). Under these broad definitions, nociceptive sensitization can be divided into various overlapping types, distinguished by the level of analysis, the operations used to enhance the sensitivity of the response system, and the specificity of the enhancement (Table 11). In this section behavioralsensitization is discussed, and possible contributions of various subtypes of behavioral sensitization to adaptive injury reactions are proposed (Table 111). In Sections VI and VII neural sensitization (network and cellular) will be discussed. Although instances of neural sensitization in appropriate networks may appear likely to provide mechanisms for a given example of behavioral sensitization, it must be kept in mind that showing correlations between neural and behavioral sensitization is not sufficient to prove a causal relationship. Indeed, unobserved neural systems or even changes in nonneural systems (e.g., warming up of muscles) may be responsible for observed behavioral facilitation. Behavioral sensitization can be divided into at least two forms that are distinguished by the operations used to induce and test it. Simple

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T A B L E I1 TYPES OF NOCICEPTIVE SENSITIZATION Level

Experimental operation

Specificity

~

1. General sensitization 1. Behavioral sensitization 1. Simple sensitization * Separate sensitizingand test pathways Site-specific sensitization 2. Neural sensitization * Can be expressed after a single a , Network sensitization 3. Modality-specific b. Cellular sensitization sensitizing sensitization Peripheral 2. Incremental sensitization Central * Sensitizing and test pathways the 4’ Response-specific sensitization same 4. Mixed 5 . Mixed * Repeated sensitizing stimulus

.

3. Mixed

sensitization is produced by the presentation of one or more sensitizing stimuli and is monitored by test stimuli applied to a separate pathway. Because the test stimuli are different from the sensitizing stimuli, simple sensitization can sometimes be seen after only a single sensitizing stimulus. Incremental sensitization is both produced and monitored by repeated application of the sensitizing stimulus. Because the sensitizing and test pathways are the same, the sensitizing stimulus must be repeated at least once in order to recognize this form of sensitization, and more repetitions

T A B L E 111 CLASSESOF BEHAVIORAL PLASTICITY ASSOCIATED WITH

INJURY

Immediate

Rapid

Delayed

1. Incremental, site-specific sensitization (warm-up, wind-up) 2. General sensitization (arousal, dishabituation)

A. During passwe defense 1. Incremental, site-specific sensitization (hyperalgesia, allodynia) 2. General sensitization (arousal, anxiety) 3. Response-specific sensitization (reflex dominance, pseudoconditioning) 4. Unconditioned fear

A. Wound-related protection I. Site-specific sensitization (hyperalgesia. allodynia) 2. General sensitization (anxiety) 3. Response-specific sensitization (reflex dominance, pseudoconditioning) 4. Conditioned fear

3. Fear conditioning

B. Dut-ing active defense 1. General inhibition (analgesia, hypoalgesia) 2. Response-specific inhibition 3. Site-specific depression (“fatigue”)

B. Recuperative behavior 1. Response-specific inhibition 2. Recovery of function

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may be necessary. It should be noted that this distinction is purely operational, and the same mechanisms may be involved in both simple and incremental sensitization. In principle, nociceptive sensitization might be expressed as a completely general sensitization of defensive responsiveness, affecting all somatosensory test stimuli, all sites on the body, and all defensive responses. General sensitization can be considered a form of somatosensory vigilance. In many cases, however, behavioral sensitization shows at least partial specificity to a site on the body, to a particular sensory modality or submodality (e.g., to innocuous or noxious tactile stimuli), and/or to particular defensive responses. Because most behavioral responses habituate (decrement) with repeated stimulation, sensitization often occurs against a background of behavioral habituation. Because of this, two forms of response enhancement are sometimes distinguished (e.g., Marcus et al., 1988; Rankin and Carew, 1988). Sensitization is defined in this context as an increase in a test response above its initial (nondecremented) value following the presentation of a modulating stimulus (a noxious stimulus in the case of nociceptive sensitization). Dishabituation is defined as the rapid restoration (partial or complete) of a decremented test response to its initial level (but no higher) following the presentation of a modulating stimulus. In most cases, a noxious stimulus probably produces both sensitization and dishabituation. If separate mechanisms are involved (see Section VI,C,2,a), these would be expected to work together to enhance appropriate defensive responses. Few investigators have distinguished among different types of behavioral sensitization, assuming implicitly that nonassociative response enhancement reflects simply, general sensitization (e.g., Mackintosh, 1983; Byrne, 1987). However, some subtypes of behavioral sensitization have been recognized previously. Incremental sensitization (also called “warmup” or “wind-up”) has been distinguished from simple sensitization by several authors (e.g., Hinde, 1970; Razran, 1971; Lockery and Kristan, 199 I; Ehrlich et al., 1992). Distinctive features of site-specificsensitization (Walters, 1987a) and response-specific sensitization (see Section IV,B,5) have also been recognized. Below, the various types of nociceptive sensitization are divided into two general classes: those that are targeted to a site of injury and those that increase the general somatosensory vigilance of the animal (Table 111).

C. SENSITIZATION TARGETED TO A SITEOF INJURY

Many of the different types of sensitization that have been described following noxious stimulation appear to be targeted to a site of injury.

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This targeting takes the form of a selective increase in sensitivity of the injured region, and a preferential enhancement of motor responses that serve to protect the injured site (Tables I and 111). 1. Incremental, Site-Specific Sensitization and Short-Term Hyperalgesia Incremental sensitization of defensive responses to somatosensory stimulation has been described in diverse animals. For example, repeated tactile stimulation of the foot of the snail Helix causes a clear increase in withdrawal amplitude to the second and third stimuli, followed by progressive habituation of the withdrawal response (Balaban, 1983).Similar patterns of an initial increase in response followed by depression of the response have been observed during repeated elicitation of escape swimming in the nudibranch mollusc Tritonia (Frost et al., 1988b), the local bending reflex of the medicinal leech (Lockery and Kristan, 1991), the running escape response of a crab (Romano et al., 1991), and the eyestalk withdrawal reflex in another crab species (Appleton and Wilkens, 1990). Incremental sensitization without any habituation has been reported (over hundreds of trials) in the wiping reflex of spinal frogs (Franzisket, 1963) and (over 5-10 trials) in the tail withdrawal reflex in Aplysia (Walters et al., 1983b). Although the possible site specificity was not tested in any of these studies, the fact that the sensitized responses were evoked from the site of sensitizing stimulation suggests that site specificity should be examined. Site-specific sensitization of siphon and tail withdrawal responses following a brief sequence of noxious tail shock has been demonstrated in Aplysia (Walters, 1987b, and see below). Perhaps the most extensive observations of incremental sensitization were made in a series of studies initiated by Thompson and Spencer (1966) on habituation of the hindlimb flexion reflex in the acute spinal cat. They found that the flexion response to repeated test shocks applied to a cutaneous nerve often displayed an initial increase in amplitude, which they termed “sensitization,” prior to the habituation phase. This incremental sensitization was subsequently found to have two forms, which could be observed sequentially in the same preparation under appropriate conditions (Groves et al., 1969). An immediate, transient sensitization (lasting a few seconds at most) was produced by highfrequency nerve stimulation (>8 Hz), even at relatively low shock intensities. With higher intensities a slowly developing “long-term” sensitization appeared, but it was monitored only for a few minutes. T h e general adaptive model of injury-related plasticity (Table I ) suggests that there are different functions for early and late sensitization (Table 111). T h e widely recognized function of late hyperalgesia and site-specific sensitization is to protect the wounded site (see below). On

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the other hand, the early sensitization of a stimulated site may reflect an injury detection and risk assessment process. For the reasons given earlier, it may be adaptive under some conditions for an animal to give only a limited response to a stimulus that is neither clearly noxious nor innocuous. If a quasi-noxious stimulus is repeated, however, and shows no sign of diminishing, the safest assumption is that injury is imminent. By immediately sensitizing a stimulated site, an animal can ensure that rapid, vigorous defensive responses are made if the quasi-noxious stimulus fails to weaken or occurs again. As will be discussed below, rapid sensitization can lead to positive feedback effects that yield explosive reactions to maintained or slowly increasing stimulation. Injury detection and risk assessment should encompass a wide range of potentially noxious patterns. If site-specific sensitization plays an important role in this process, the sensitization must begin within a fraction of a second to trigger defensive responses to rapidly strengthening stimuli. Conversely, the sensitization should persist long enough (perhaps for minutes) to contribute to the recognition and assessment of threatening stimulation patterns that develop very slowly. Stimuli that are only weakly noxious are often used in the study of animal models of pain and analgesia (Dubner, 1989). The proposed injury detection function predicts that in studies of pain modulation by painful stimulation, early hyperalgesia (sensitization) is most likely to be observed under conditions where (a) tests for pain are applied to the same site, or close to the site, where the modulating painful stimulus is delivered, (6) the modulating painful stimulus is not too severe, and (c) tests for pain are given seconds or minutes after the modulating painful stimulus. As described below, most studies of pain modulation have not met these conditions and have revealed analgesia (antinociception) rather than hyperalgesia. However, several studies of rodent models of pain have shown hyperalgesia under conditions similar to those predicted by the general adaptive model. For example, Kelly (1982) showed that foot shock facilitated subsequent responses to electrical stimulation of the foot (lowering the threshold on “flinch” and “jump” tests) while decreasing responses to radiant stimulation of the dorsal surface of the tail (“tailflick” test). Illich and Grau (1990) found that moderately intense tail shock enhanced vocalization and the force of tail movements evoked by a brushing stimulus applied to the tail 2 and 8 min after the shock. Similarly, moderate intensity tail shock enhanced the force of tail movements in response to weak shocks delivered thorugh the same electrodes 2 min after the moderate tail shock (Illich, 1993). In separate studies Illich (1993) found that moderate intensity tail shock caused a transient increase in vocalization and a decrease in the theshold for eliciting tail

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35 1

movements with weak tail shock. These effects were observed 2 and 8 min but not 32 or 128 min after the moderate tail shock. When radiant heat applied to the tail was used as both the modulating noxious stimulus and the test stimulus the facilitatory effects were much weaker, but there was still a significant increase in vocalization 2 min after the moderately intense stimulus (Illich, 1993). Various other observations have shown less hyperalgesia or sensitization with thermal than mechanical stimuli. For example, radiant heat was found to be less effective than mechanical stimulation in producing incremental sensitization in the decerebrate rat (Woolf, 1984). Indeed, repeated application of radiant heat produced wind-down of flexion responses, in contrast to the windup produced by repeated mechanical stimulation. Heat injury in the monkey produces both primary and secondary mechanical hyperalgesia, but only primary heat hyperalgesia (Raja et al., 1984). Taken together, these observations suggest that injury-induced sensitization and hyperalgesia affect mechanosensory pathways more than thermal pathways. This difference should not be surprising, since mechanosensory stimuli rather than thermal stimuli are associated with the most common biological threats-attacks from predators, parasites, and conspecifics, as well as accidental, self-inflicted injury.

2 . Long-Term Site-Specific Sensitization and Hyperalgesia Long-term site-specificsensitization and hyperalgesia (Table 111) have been produced using noxious stimuli that mimic aspects of actual injury. In mammals cutaneous shock at intensities that do not cause tissue damage produces short-term (see above) but not long-term facilitation of defensive responses. However, shock applied directly to peripheral nerves can cause much longer lasting effects. Woolf and Wall (1986a) found in the decerebrate rat that shocking peripheral nerves produced facilitation of the hindlimb flexion reflex that lasted for hours. In order to produce facilitation the shock had to be intense enough to activate small diameter C-fibers, which largely comprise axons of nociceptors. In addition, the facilitation was prevented by pretreatment with the C-fiber neurotoxin capsaicin. Facilitation was also observed when contralateral test stimuli were delivered, but it was much weaker, showing that weak general sensitization accompanied the dramatic site-specificsensitization. Two observations suggested that the degree and duration of site-specific sensitization depended on the apparent severity of the “injury” signaled by the nerve activity. First, the requirement for C-fibers suggested that nociceptors mediating “slow pain” had to be activated. Second, the effects were considerably more pronounced when C-fibers from deep tissue (muscle or joints) were activated. T h e most dramatic effects occurred

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after injection of mustard oil, a chemical irritant that strongly activates C-fibers, into the ankle o r knee joint capsule. Massive activation of nociceptors in deep tissues would normally be associated with serious injury. A particularly prominent effect of injury in vertebrates is inflammation in the damaged region. Several studies have shown that artificially induced peripheral inflammation leads to long-lasting site-specificsensitization. For example, in the decerebrate rat Woolf (1984) found that massive inflammation produced by injecting turpentine into the foot enhanced ipsilateral reflex sensitivity for 2 weeks. A number of inflammatory models of chronic pain have been utilized, but some of these produce quite general, rather than site-specific, physiological and behavioral alterations (see Dubner, 1989). An interesting comparison of the behavioral effects of several locally acting inflammatory agents was described by Iadarola and colleagues (1988a). When injected into the hindfoot in the rat, Freund’s adjuvant (containing dried mycobacterium cells), carrageenan, yeast extract, and a phorbol ester all produced inflammation (monitored by the degree of swelling) and sensitization (monitored by the latency to withdraw the food to a radiant heat stimulus). The sensitization was site-specific, since no facilitation was observed in tests of the contralateral, uninjected foot. Freund’s adjuvant and carrageenan produced the most persistent site-specific sensitization, lasting more than 4 days. Facilitation produced by phorbol ester lasted less than 1 day. In general, the time course of reflex facilitation paralleled that of the inflammation. During the local inflammation the animals protected the injected foot (keeping it elevated) and showed a reduction in feeding, but they did not alter the amount of their spontaneous locomotor behavior. In terms of the general adaptive model for injury-related behavior, the latter observation suggests that the rats are behaving as if they d o not expect imminent attack. This might reflect an apparent lack of severity of the “injury” because of restriction of the inflammation to relatively superficial tissues. Another possibility is that many of the danger signals associated with actual injury (i.e., the signals causing an animal to hide and restrict its activity) are not triggered by inflammation alone. Site-specific sensitization lasting at least 1 week has been produced in the mollusc Aplysiu by applying a 45-s train of strong shock to one side of the tail (Walters, 1987a). Siphon withdrawal responses and tail withdrawal responses were facilitated when elicited by weak shock applied near the site of strong shock, but not when the weak shock was applied to the contralateral side of the tail. N o sensitization was observed when the strong shock was delivered under conditions of transient, local anesthesia, which shows that the behavioral changes were not due to possible damage of the skin by the shock. Although the effects of

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inflammatory-like processes on defensive responsiveness have not yet been examined at the behavioral level in invertebrates, a local inflammatory-like reaction has been shown to enhance the excitability of sensory neurons activated by tail stimulation in Aplysia (Clatworthy et al., 1994, and see Section VII,B,4).

3. Response-Specific Sensitization, Reflex Dominance, and Pseudoconditioning One way to protect an injured region is to increase the sensitivity of that region to all potentially threatening stimuli so that any protective reflex evoked by stimulation of the region will be activated quickly and vigorously (Table I). Another way is to prime or sensitize specific defensive responses that protect the region, even if these responses are evoked by stimulation outside the injured region. Such response-specific sensitization (Table 111) would bias behavioral choice toward responses that protect the wounded region. Because the most obvious reflexive responses to tactile stimulation usually involve contraction of muscles underlying the area of stimulation (i.e., reflexes exhibit “local sign”Sherrington, 1947), it is usually difficult to distinguish site-specific sensitization from response-specific sensitization at the behavioral level. However, the sensitization of specific motor responses is strongly indicated when a stimulus that will not evoke a particular behavioral response before noxious stimulation (regardless of the intensity of the test stimulus) does so after noxious stimulation (Erickson and Walters, 1988). Such response transformation has been termed “pseudoconditioning” by experimental psychologists because it presents nonassociative confounds in experiments on associative learning (Grether, 1938; Kimble, 1961). T h e idea of altering behavior by sensitizing specific motor responses is also equivalent to Ukhtomsky’s concept of “reflex dominance” (see Kandel and Spencer, 1968; Razran, 1971). The transformation of responses to a test stimulus into responses resembling the response to a noxious unconditioned stimulus has not been investigated extensively, but is probably quite common. Indeed, Kimble (1961) has suggested that pseudoconditioning may be a part of all conditioning involving a noxious unconditioned stimulus. Examples of pseudoconditioned defensive responses that have come to resemble the response to noxious stimulation (usually electric shock) include human finger withdrawal (Harris, 194 l), goldfish escape swimming (Harlow and Toltzien, 1940), frog wiping responses (Franzisket, 1963), whole-body withdrawal in polychaete worms (Evans, 1966), and directional siphon responses in the mollusc Aplysia (Erickson and Walters, 1988). The last study found that transformation of siphon responses occurred within 30 s after noxious stimulation and could persist for at least 1 day.

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4. Long-Term Hyperalgesia and Neuropathic Pain

An inevitable consequence of significant bodily injury is nerve damage. Nerve damage may be restricted to the endings of sensory neurons innervating the injured tissue, or may be massive (e.g., if a limb is severed and major nerves are transected). Nerve damage in humans is often associated with intense, prolonged pain and profound behavioral adjustments (e.g., Melzack and Wall, 1983). Although the pain and behavioral changes that follow major nerve damage can be persistently incapacitating, and thus apparently maladaptive, it seems likely that these effects are extreme manifestations of mechanisms that were selected during evolution to protect injured peripheral tissue that was denervated by the injury. Alterations of sensation due to general tissue injury and to nerve damage are often distinguished from each other because of apparent differences in the peripheral triggering events (e.g., Coderre et al., 1993). However, both would be expected to be produced by serious injury, and both are characterized by strong hyperalgesia and allodynia (e.g., Treede et al., 1992).Recent rat models of neuropathic pain based on loose ligation of the sciatic nerve (Bennett and Xie, 1988; Attal et al., 1990), tight partial ligation of the sciatic nerve (Seltzer et al., 1990),and tight ligation of spinal nerves (Kim and Chung, 1992; Palecek et al., 1992b) all showed a decrease in threshold for evoking foot withdrawal to mechanical stimulation (i.e., allodynia), enhanced responses to heat (i.e., thermal hyperalgesia), and signs of spontaneous pain such as frequent licking of the affected foot. These sensitizing effects should aid in the defense of a wounded body part against parasitic attacks, self-inflicted injury, and perhaps some predatory threats. One relatively common effect of neuropathic pain that may not appear adaptive is autotomy (self-inflicted wounding) of the painful appendage (e.g., Coderre and Melzack, 1987; Kingery and Vallin, 1989; Seltzer et al., 1991a,b). One possible function of mammalian autotomy is suggested by a demonstrated role of autotomy in invertebrates. Eisner and Camazine (1983) showed that shedding of a leg by spiders after the leg was stung by certain insect prey prevented the systemic spread of otherwise lethal venom. Interestingly, this response was triggered not by the puncture wound itself, but by various venoms (which contain neuroactive substances such as serotonin that produce pain in man and sensitization in diverse species-see Section VI). These observations suggest that some neuropathic pain models may trigger neural activity that has been associated during evolution with the detection of lifethreatening poisons in peripheral tissues (particularly appendages). Directed, self-inflicted wounding under natural conditions may cause either the rapid removal of a dispensible body part containing dangerous toxins

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or local bleeding in a poisoned body part that reduces the concentration of toxins entering the systemic circulation.

AND ANXIETY D. GENERAL SENSITIZATION, AROUSAL,

1. Short-Term General Sensitization and Arousal

If an animal escapes from an attack, the likelihood will be high for some period of time that the attacker remains in the area. Clearly, during times of danger it is beneficial for an animal to optimize the performance of perceptual and response systems that are involved in defensive behavior. This can be done by “arousing” either the entire nervous system or the appropriate sensory and motor systems. A relatively general enhancement of an animal’s sensitivity to potentially threatening stimuli (Tables I and 111) probably occurs in many species. General sensitization, like response-specific sensitization, is difficult to observe in isolation in mammals because of their capacity for rapid associative learning; any aversive stimulus is likely to be associated by a mammal with the context and thus changes in response observed after noxious stimulation may be due largely to conditioned fear of the situation rather than nonassociative sensitization. Sensitization can also be difficult to distinguish from dishabituation of previously habituated responses, which almost always accompanies general sensitization. Although dishabituation and sensitization have been suggested to reflect the same underlying process (Groves and Thompson, 1970), recent evidence shows that they can be separated, at least in Aplysia (Hochner et al., 1986b; Marcus et al., 1988; Rankin and Carew, 1988; Carew, 1989; Wright et al., 1992). In mammals general sensitization may be easiest to demonstrate in spinal animals lacking the ability to react to contextual cues. Sensitization expressed as facilitation of flexion reflexes occurs readily in the spinal cat (e.g., Thompson and Spencer, 1966; Durkovic, 1975).A clear example of general sensitization in an intact mammal was reported by Davis (1989), who showed that foot shock sensitizes the acoustic startle reflex in the rat. Sensitization of heart rate acceleration to visual stimuli following noxious shock has been demonstrated in the pigeon (Cohen, 1974). General sensitization following noxious stimulation is prominent in some invertebrates, where it has been suggested to serve the same functions that aversive associative learning does in mammals (Wells, 1968; Razran, 1971). Sensitization appears to be very common in molluscs and annelids. Responses that have been used to show short-term general sensitization (following noxious shock) include gill and tail withdrawal reflexes in Aplysia (Carew et al., 1971; Walters et al., 1983b), withdrawal of the snail Physa into its

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shell (Wells, 1975),and the shortening and local bending reflexes of the medicinal leech (Boulis and Sahley, 1988; Lockery and Kristan, 1991). Escape swimming in the nudibranch mollusc Tritonia, evoked by delivery of concentrated NaCl solution to the tail, was prolonged by prior delivery of a fish liver extract to the head (Brown and Willows, 1990). An interesting question is whether this extract (which is full of products of tissue damage) might contain cues that have been used during evolution by prey such as Tritonia to detect the presence of actively feeding predators (cf. Crow1 and Covich, 1990). Another example of general sensitization was reported recently for movements of the tiny nematode worm Caenorhabditis elegans, which were sensitized by mechanical stimulation (Rankin et al., 1990).

2. Long-Term Sensitization, Vigtlance, and Chronic Anxiety Although defensive arousal and general sensitization normally are transient events, prolonged unsignaled noxious stimulation may produce persistent general sensitization (Table 111). Seligman (1 968) has shown that unsignaled shock delivered to rats causes long-lasting defensive arousal (as monitored by the suppression of appetitive behavior). Although some of this effect might be due to conditioned fear of the context, part of it may reflect long-term general sensitization. Long-term general sensitization produced by repeated unsignaled shock has been described for Aplysia siphon withdrawal (Pinsker et al., 1973; Frost et al., 1985) and escape locomotion (Walters, 1980). Long-term general sensitization resembles long-term site-specific sensitization (Walters, 1987a)but requires a much more prolonged sequence of noxious stimulation (i.e., hours or days of periodic stimulation rather than tens of seconds). There appear to be interesting parallels between the persistently aroused states produced by unsignaled shock in these experimental animals with chronic anxiety in humans (Seligman, 1975; Walters, 1980; Kandel, 1983).A functional interpretation of such effects is that actual or threatened injury triggers increased vigilance (including somatosensory vigilance). The more frequent and less predictable injurious attacks are, the longer an animal needs to maintain a high level of continuous vigilance so that it will be prepared for the next attack. Sustained vigilance becomes less important when attacks can be predicted by reliable signals (fear conditioning, see below). E. Do SOMEANIMALS LACKA CAPACITY FOR NOCICEPTIVE SENSITIZATION?

Not all animals have been found to sensitize readily, and those that do may show only some types of sensitization. Of particular significance

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for attempts to understand the evolution of nociceptive plasticity is the rarity of reports of sensitization in the largest group of animals-the arthropods. Nociceptive sensitization has been described occasionally in crustaceans (see above) but in some cases where it has been looked for, sensitization has been difficult to find. For example, a shock-induced decrease in the threshold of the lateral-giant-axon-mediated escape response has been described in crayfish (Krasne and Glanzman, 1986). However, the authors report that this sensitization, which was expressed in response to electrical test stimuli delivered by implanted electrodes to interganglionic connectives, has not been observed under more natural conditions despite many efforts in their laboratory and others to find it. The difficulty in finding natural defensive sensitization in arthropods (general or site-specific, short- or long-term) might be due to our limited understanding of the conditions under which nociceptive sensitization is expressed in arthropods. It may also be the case that arthropods have evolved other adaptations that make nociceptive sensitization unnecessary. For example, adult arthropods, unlike worms and many molluscs, are covered completely by a hard exoskeleton. Many molluscs have shells, but even shelled molluscs often have exposed soft parts that can be attacked and injured (e.g., by siphon biting fish). The possibility that sensitization is particularly important for soft-bodied animals (such as mammals and opisthobranch molluscs) should be tested systematically. One test would be to see whether arthropods show greater sensitization during molting, before their new exoskeletons have hardened. Do other heavily armored invertebrates, such as echinoderms, lack nociceptive sensitization? Additional potentially important factors are life span and size. Many arthropods, especially the insects, have very short adult life spans and are quite small. These attributes may make nociceptive memory and compensatory growth of receptive fields of little consequence. The fact that sensitization and other adaptive responses to injury have not been apparent in insects (e.g., Guthrie, 1975; Eisemann et al., 1984) suggests that these features may be more common in larger, longer-lived animals. One test of these ideas would be to see whether larger, softerbodied, relatively long-lived larval forms of some insects display defensive sensitization. Interestingly, preliminary results (E. T. Walters, P. A. Illich, P-N. P. Le, S. Bell, and J. C. Weeks, 1993, unpublished observations) show that large, relatively soft-bodied larvae of the hawkmoth Manduca sexta display sensitization of a defensive striking response lasting at least 20 min. Thus, defensive sensitization can occur in insects, but generalizations about the functions and evolutionary history of sensitization and injury-related behavior will require the careful study of many more species in all the major phyla.

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F. ANTINOCICEPTION, COMPETING RESPONSE INHIBITION, A N D ANALGESIA Because of society’s interest in controlling pain, analgesic and antinociceptive processes have probably been studied more intensively than any other aspect of injury-related behavior and neurobiology. A pn’on’, antinociception and behavioral inhibition would appear to be an extremely important part of adaptive reactions to injury (Tables I and 111). Sherrington (1947) pointed out nearly 90 years ago that singleness of action is critical for effective behavior, and requires the inhibition of responses incompatible with intended behavior. As has now been emphasized by many authors, if injury is produced by a predator or aggressor, recuperative reflexes that are normally elicited by injury need to be inhibited (i.e., the organism must be made analgesic) to prevent interference with the more urgent need of continued defense (Liebeskind et al., 1976; Wall, 1979; Bolles and Fanselow, 1980; Amit and Galina, 1988; Randall and Rodgers, 1988) (Table I). In addition, an animal may have several alternative defensive responses at its disposal and those that are not selected need to be inhibited so that they d o not interfere with the chosen response. Inhibition of defensive responses by noxious stimulation has been demonstrated in mammals (see reviews by Fanselow and Lester, 1988; Blanchard et al., 1990) and invertebrates (e.g., Marcus et al., 1988; Romano et al., 1991; Illich et al., 1994a). The proposal of Bolles and Fanselow (1980) that analgesia in mammals is produced by contextual stimuli perceived at the time of injury (i.e., that “fear inhibits pain,” see Section II1,A) has been supported by numerous demonstrations of conditioned hypoalgesia in rats (e.g., Fanselow and Baackes, 1982; Ross and Randich, 1985; Crau, 1987). On the other hand, the relative importance of contextual stimuli in initiating and maintaining analgesic reactions during an injurious encounter is open to question. Although it can be very difficult to exclude a role for contextual stimuli, various lines of evidence show that noxious stimulation directly elicits antinociceptive responses in mammals (i.e., that “pain inhibits pain”). For example, in mice attacked by conspecifics the degree and duration of hypoalgesia depend on the number of bites received under conditions where the context is constant (e.g., Randall and Rodgers, 1988; Siegfried et al., 1990). Furthermore, when tested in the same context (which included a nonaggressive mouse), previously attacked mice have been reported to show no conditioned hypoalgesia even though memory of the previous attack was expressed by other behavioral alterations, such as submissive postures (Siegfried et al., 1990). Strong evidence for direct antinociceptive effects of noxious stimulation in mammals comes from simplified preparations lacking the capacity to perceive

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the visual, olfactory, and auditory context. For example, Meagher and Grau (Meagher and Grau, 1988) have shown that strong shock inhibits defensive responses in the spinal rat. In addition, noxious stimuli applied to one part of the body of anesthetized rats and monkeys produces powerful inhibition of neural activity in nociceptive pathways from other parts of the body (e.g., Le Bars et al., 1979a,b; Chung et al., 1984). Another inhibitory effect may occur with prolonged noxious stimulation-site-specific depression, which is sometimes identified as fatigue or habituation to the noxious stimulus (Table 111).Although nociceptive responses and pain initially show incremental sensitization, if a noxious stimulus is repeated enough times or maintained for a long enough period the behavioral and sensory responses begin to weaken (e.g., Thompson and Spencer, 1966; Lockery and Kristan, 199 1). Depression may involve two processes. First, action potential discharge in sensory neurons activated by noxious stimulation eventually begins to decline during repeated stimulation. This has been shown in mammalian Cpolymodal nociceptors (Beitel and Dubner, 1976; Adriansen et al., 1984) and myelinated nociceptors (Perl, 1968),and in nociceptive sensory neurons of Aplysia (Clatworthy and Walters, 1993b). Second, in many neurons, including interneurons that mediate habituation in the spinal rat (Thompson and Spencer, 1966) and sensory neurons that mediate habituation in Aplysia (Castellucci et al., 1970), repeated activation produces synaptic depression. This depression is often overshadowed initially (during incremental sensitization) by concomitant facilitatory processes (Groves and Thompson, 1970). Neither aspect of activity-dependent depression will be likely to have developed very far until after the CNS has been well informed about the noxious event in the sensory neuron’s receptive field, and the animal has begun appropriate defensive action. At this point activity-dependent depression of discharge and synaptic transmission, along with extrinsic inhibitory influences (e.g., opiate release), may transiently reduce the sensitivity of the wounded region to minimize the activation of recuperative reflexes that might interfere with defensive behavior. Activity-dependent depression in a nociceptor may also reflect dynamic homeostatic processes that limit potentially maladaptive effects of excess activity on the cell itself or on the neural network it excites (Clatworthy and Walters, 199317; LeMasson et al., 1993). It seems clear that various antinociceptive processes exist in mammals, serving several overlapping functions, and that both fear-like and painlike stimuli can be triggers for antinociception, depending on the situation and the species. Fear-like mechanisms appear to be responsible for anticipatory hypoalgesia that prepares an animal for fleeing or fighting, e.g., the hypoalgesia produced in mice by the mere sight of a nearby

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weasel (Kavaliers, 198813) or produced in rats by the smell of shocked conspecifics (Fanselow, 1985).On the other hand injury-induced or paininduced hypoalgesia may be very important for preventing movement during what is probably the defense of last resort for rodents and many other animals-tonic immobility (Amir, 1986), also termed “feigning death” o r thanatosis (Edmunds, 1975). There is ample evidence that prey movement is a key releasing stimulus for predatory attack and killing responses (e.g., Fox, 1969; Herzog and Burghardt, 1974; Fanselow and Lester, 1988). Thus, if prior defenses prove ineffective (e.g., anticipatory freezing, flight, aggressive retaliation) there may still be a chance that a predator will lose interest if the prey ceases to move during the attack (Sargeant and Eberhardt, 1975). Diversion of a predator’s attention from an immobile prize may be particularly likely if the predator is young and hunting for practice rather than to eat-a pattern familiar to many cat owners (Leyhausen, 1979). Tonic immobility after capture is a widespread phenomenon, having been reported in insects and birds, as well as various mammals (Edmunds, 1975; Sargeant and Eberhardt, 1975). Although much has been learned about the neuropharmacology of antinoception in mammals (particularly the opiate system), neuronal mechanisms underlying antinociception are still not well understood. This is probably because the opiate and nonopiate systems underlying antinociception are extremely complex-there are several different pain modulatory systems and each one exerts inhibitory influences on pain transmission at various levels of the neuraxis, including the first afferent synapse (Mudge et al., 1979; Watkins and Mayer, 1982; Besson and Chaouch, 1987; Fields and Basbaum, 1989; Kiyatkin, 1990; Light, 1992). i t is probably safe to assume that short-term inhibition of neural signaling in nociceptive pathways involves, at least to some extent, the same basic pre- and postsynaptic inhibitory mechanisms that operate throughout the mammalian nervous system (e.g., Nicoll and Alger, 1979; Besson and Chaouch, 1987), but much remains to be learned about cellular mechanisms of antinociception. Under some conditions long-term depression of synaptic transmission involving postsynaptic induction mechanisms may be involved (Randic et al., 1993; see also Artola et al., 1990). Antinociceptive responses that may involve the actions of endogenous opioid peptides have also been implicated in birds, reptiles, amphibians, fish, and some invertebrates (reviewed by Kavaliers, 1988b). For example, the terrestrial snail Cepaea nemoralis displays a foot lifting response to a hot substrate that is delayed by morphine, this effect is blocked by opiate antagonists such as naloxone, and repeated application of morphine induces tolerance (Kavaliers et al., 1985; Kavaliers, 1987, 1988a). A brief

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inhibitory effect of tail shock on siphon withdrawal in Aplysia has been described (Mackey et al., 1987; Marcus et al., 1988), part of which involves presynaptic inhibition of nociceptive sensory neurons by the release of a neuropeptide, Phe-Met-Arg-Phe-NH, (FMRFamide) (Belardetti et al., 1987; Mackey et al., 1987). Interestingly, FMRFamide has structural affinities with opiates (Greenberg and Price, 1983).

FEAR G. CONDITIONED There is no doubt that associative learning permits mammals to recognize and avoid situations similar to those in which they have been injured previously (Tables I and 111). Indeed, the ease with which rats can learn to fear stimuli associated with noxious stimulation has made conditioned fear the paradigm of choice among many psychologists using rats to study principles of learning (e.g., Rescorla, 1988). Some of the roles of associative learning during and after injury have been discussed extensively by Fanselow and Bolles (Bolles and Fanselow, 1980; Fanselow, 1991). The fear elicited in rats by recognition of stimuli previously associated with injury is usually expressed as a freezing response (which interrupts appetitive behavior such as feeding or bar-pressing for food) accompanied by anticipatory analgesia (Fanselow and Lester, 1988 and see above) and by a potentiation of startle responses (Brown et al., 1951; Davis and Astrachan, 1978). Conditioned fear-like states are not unique to rodents o r mammals. The mollusc Aplysia exhibits a fear-like state when presented with a chemosensory stimulus that has been paired previously with strong head shock (Walters et al., 1979, 1981). This state is associated with ( a ) immobility; ( 6 ) facilitation of withdrawal, escape, and defensive inking responses; and (c) inhibition of feeding behavior.

V. Injury Signals

An important task in analyzing adaptive behavioral reactions to injury in different groups of animals is to identify the signals used by animals to recognize, localize, and assess the severity of actual and imminent injury. These include activation of nociceptors, products of general tissue damage and inflammation, and slow signals initiated within damaged axons.

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A. ACTIVATIONOF NOCICEPTORS In principle, the simplest way that an animal can tell that it is being injured is to rely on the activation of specialized nociceptive sensory neurons that detect potential or actual damage to their receptive fields (e.g., Head et al., 1905; Sherrington, 1947). Primary nociceptors have been characterized in mammals (reviewed by Besson and Chaouch, 1987; Light, 1992), amphibians (Spray, 1976), leeches (Nicholls and Baylor, 1968; Johansen et al., 1984), and Aplysia (Byrne et al., 1974; Walters et af., 1983a). Myelinated nociceptors and C-polymodal nociceptors in mammals respond selectively to intense mechanical stimuli. Many Cpolymodal nociceptors are also activated by injury-related chemical stimuli (see below) and by high temperatures. Activation of myelinated nociceptors is associated with “fast pain,” whereas activation of C-polymodal nociceptors is thought to mediate “slow pain” in humans (see reviews by Besson and Chaouch, 1987; Light, 1992). Ostensible nociceptors (the “dorsal cells”) have been reported in lamprey (Martin and Wickelgren, 197 l ) , but the nociceptive function of these neurons has been questioned because they also respond to very light pressures (Christenson et al., 1988). However, if one defines a nociceptor as a primary sensory neuron showing responsiveness to potential damage, or as a sensory neuron exhibiting maximal responsiveness to stimuli that cause actual damage (Sherrington, 1947), the lamprey dorsal cells may still qualify. The skin of the lamprey is extremely delicate so even light touch might be threatening if repeated to the same site. In addition, although they fire in response to light pressure, the dorsal cells show greater activation (and afterdischarge) to stimuli that injure the skin (Christenson et al., 1988). Similarly, mechanosensory neurons innervating the siphon of Aplysia were not initially recognized as nociceptors because they respond weakly to innocuous pressure (Byrne et al., 1974). However, these cells, like identified mechanosensory neurons innervating the tail of the same animal (Walters et al., 1983a), have a very wide dynamic range and they respond maximally to crushing stimuli that injure their receptive field (P. A. Illich and E. T. Walters, unpublished observations, 1994). Whereas the N cells in the leech (Nicholls and Baylor, 1968),like high-threshold nociceptors in mammals, respond only to intense or injurious stimuli, the P cells resemble the wide-dynamic-range nociceptors in Aplysiu by firing weakly to light pressure and maximally to high-pressure stimulation that may threaten tissue destruction. A partial nociceptive function for the P cells is supported by observations that swimming (a response used for escape) can often be evoked by intracellular stimulation of single P o r N cells (Debski and Friesen, 1987). Furthermore, repeated application of stimuli that

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selectively activate P cells causes incremental sensitization of the local bending reflex (Lockery and Kristan, 1991). T h e S cell in the nudibranch Tritonia (Getting, 1976) can also be considered a nociceptor, but with an interesting twist. It is not known yet whether these cells are primary or secondary sensory neurons but, like the sensory neurons in mammals, lamprey, leech, and Aplysiu mentioned above, they have a centrally located cell soma, a peripheral axon, and a well-defined excitatory receptive field on the body surface that responds to intense mechanical stimulation with a rapidly adapting discharge of action potentials. The unusual feature of these sensory neurons is that they also respond to external chemical stimuli. The S cells are selectively tuned to chemosensory cues from the major predator on adult Tritonia, the voracious sea star, Pycnopodia. Gentle touch anywhere on the body by the tube feet of this fast-moving echinoderm causes massive, very slowly adapting sensory neuron discharge that can persist for many seconds. Activity in these cells, in turn, excites interneurons that produce local withdrawal and vigorous escape swimming (Getting, 1977). In each of these sensory systems the identified nociceptors provide a labeled line from a restricted peripheral receptive field to neurons controlling protective reflexes specific for that region of the body. In each case, synaptic connections are also made to neurons involved in other behaviors. The labeled line organization accounts for the identification and localization of'a noxious stimulus but does not explain how an animal assesses its severity or rate of change (Table I). When receptive fields are quite small, as they are for many mammalian nociceptors (Perl, 1968, see Light, 1992), the severity of injury could be indicated solely by the number of sensory neurons activated, which would indicate the spatial extent of the injury. When receptive fields are larger (e.g., in Aplysia a single receptive field can cover half the tail or siphon), the same number of sensory neurons might be activated by a tiny, trivial injury and by a larger, more serious wound. Perhaps for this reason, known nociceptive systems appear to use a frequency code for injury in addition to the labeled line. In every case, a stronger noxious stimulus causes higher frequency, more prolonged activation of the affected nociceptors than does a weaker stimulus (see references listed above). Because the rate of change of a noxious stimulus influences the instantaneous firing frequency of nociceptors, a frequency code also provides information on the rate of development of a potential or actual injury. Nociceptors have been identified and studied in vertebrates, annelids, and molluscs, but have not yet been reported in the largest animal phylum: Arthropoda. Although many workers have been impressed with the apparent lack of effect of bodily injury on ongoing behavior of some

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arthropods (e.g., Eisemann et al., 1984),there are suggestions that intense somatosensory stimulation can be perceived by insects and crustaceans as aversive. A large number of studies have employed electric shock as a training stimulus to produce avoidance learning in cockroaches and crabs (e.g., Horridge, 1962; Punzo, 1983), and aversive conditioning in fruit flies (reviewed by Dudai, 1988). Although the behavioral changes observed in these studies might be artifacts of the unnatural training stimulus, the training shocks might also exert their effects by activating nociceptors that have not yet been identified. It is also possible that arthropods use pattern or frequency codes that do not require high threshold nociceptors to represent noxious stimuli. Preliminary observations on large caterpillars of the hawkmoth Manduca sexta indicate that brief, strong pinch to the body causes a dramatic striking response of the head directed at the pinch site (E. T. Walters, P. I. Illich, J. Le, S. Bell. and J. C. Weeks, unpublished observations, 1993). Weak, brushing stimuli that are known to activate identified mechanoreceptors in the same area fail to elicit the head strike. An interesting question is how the caterpillar’s CNS discriminates noxious from innocuous stimuli. Possible sources of information about injury include (a) currently unidentified high-threshold nociceptors and (b) high-frequency, sustained activity in low-threshold, wide-dynamic-range mechanoreceptors, perhaps because of rapid modulation by chemical factors released from tissue damaged by the pinch.

B.

TISSUE

DAMAGE AND

INFLAMMATION

An animal might recognize that it has been injured by detecting substances released during general tissue damage. Such substances could be intracellular constituents released from ruptured cells, or substances released by cells involved in inflammatory processes. The term “inflammation” is used here in its most general sense as the “local reaction of living tissue to injury” (Ritchie, 1990). Inflammation normally protects an organism against infection and promotes repair of injured tissue. In humans and other mammals inflammation produces pain and hyperalgesia which, by aiding in the defense of the injured site, also contribute to tissue repair and protection against infection (e.g., by minimizing movement of injured tissue and the likelihood of reopening the wound). T h e potent behavioral and neural sensitization produced by inflammation has been used to advantage in several experimental models of pain and hyperalgesia based on peripheral injections of various inflammatory agents. These include Freund’s adjuvant (which contains a mycobacte-

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rium), carrageenan, yeast extract, mustard oil, and turpentine (e.g., Woolf, 1984; Iadarola et al., 1988a). The moderate but long-lasting pain produced in the popular formalin test is based at least partly on the inflammatory reaction to formalin injection (reviewed by Tjolsen et al., 1992). In mammals, bradykinin, serotonin, histamine, hydrogen ions, potassium ions, and perhaps prostaglandin E, can directly activate nociceptors (see reviews by Handwerker, 1991a,b; Treede et al., 1992; Dray and Perkins, 1993; Levine et at., 1993). Bradykinin’s ability to activate nociceptors involves the generation of diacyl glycerol and the activation of PKC (Levine et al., 1993; Schepelmann et al., 1993). As discussed below, most of these substances and several others can also sensitize mammalian nociceptors to mechanical stimulation. Several of these substances can be released from ruptured animal cells. These include hydrogen and potassium ions, adenosine, and various substances synthesized from arachidonic acid (e.g., prostaglandins, leukotrienes) since arachidonic acid is released during membrane injury (Brigham, 1989). Almost all the information on signals released during tissue damage has come from studies of mammals. Bradykinin is generated from proteins (kininogens) circulating in plasma. Tissue damage exposes negatively charged surfaces and lowers pH, activating kallikrein enzymes, which cleave the kininogens into the nonapeptide bradykinin (Stewart, 1989). In peripheral tissue serotonin is largely found in platelets, and histamine in mast cells. A widespread role for both of these substances in triggering pain and aversive responses is suggested by their presence in numerous stings and venoms (e.g., Erspamer, 1966). Other substances that have been shown to alter nociceptor function include interleukin-1, a cytokine released by leukocytes when they are attracted to sites of injury (Ferreira et al., 1988), and NGF, the synthesis of which is increased in surrounding nonneuronal tissue by nerve injury (Heumann et al., 1987; Lewin and Mendell, 1993). The potential importance of injury signals derived from inflammatory cells is suggested by the observation that regeneration of sensory axons into the dorsal spinal root can be enhanced by the production of an inflammatory reaction in rat dorsal root ganglia by injection of either Corynebacterium parvum or isogenous macrophages into the ganglion (Lu and Richardson, 1991; see also, Illich et al., 1994b). Chemical signals released from damaged tissue that carry noxious information or modulate nociceptor signaling have not yet been reported in invertebrates. However, during an “inflammatory” reaction to a foreign body (cotton string) implanted around a peripheral nerve in Aplysza, nociceptive sensory neurons show a prolonged increase in excitability that may be dependent on substances released from host defense cells

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that are attracted to and encapsulate the foreign body (Alizadeh et al., 1990; Clatworthy et al., 1994a). In addition, strong noxious stimulation of Aplysia body wall releases humoral factors that can inhibit the gill withdrawal reflex, increase heart rate, and contract body wall muscle, but neither the identities of these factors nor their effects on nociceptors are known yet (Cooper et al., 1989; Krontiris-Litowitz et al., 1989).

DAMAGE C. NEURONAL Any significant injury will directly damage some neurons, with sensory neurons innervating skin and superficial muscle being the most exposed and the most likely to be damaged. Damage to axons will cause a transient (seconds to minutes) injury discharge (Wall et al., 1974; Berdan et al., 1993; Spira et al., 1993), and in the case of sensory neurons the injury discharge will propagate to synaptic terminals that excite neurons within the CNS or neurons outside the CNS that project to the CNS. This initial injury discharge has been shown to play an important role in triggering long-term behavioral and neuronal changes during transection of peripheral nerves in rats. For example, Seltzer et al. (199 la) found that behavioral effects of nerve section (autotomy) could be delayed and decreased by artificially blocking the injury discharge and, conversely, that the autotomy was accelerated and enhanced by artificially activating the nerve (at C-fiber strength) immediately before nerve section (see Section VII,B,Z). The importance of early injury-related discharge is also suggested by a variety of findings showing that blockade of afferent activity prior to injury (“preemptive analgesia”) attenuates hyperalgesia (e.g., Woolf and Wall, 1986a; McQuay et al., 1988; Woolf and Thompson, 1991; see review by Coderre et al., 1993). Afterdischarge generated in sensory neuron somata may be important for amplifying immediate injury discharge and triggering long-term alterations in Aply3ia (Clatworthy and Walters, 1993a) and perhaps in mammals (Devor and Wall, 1990). Following the immediate injury signal carried by transient spike activity, slower injury signals are sent to the sensory neuron’s soma, synapses, and thence to the CNS. The most extensively investigated type of slow axonal injury signal is a negative one-the interruption of continuous retrograde flow of trophic substances (e.g., NGF) from target organs and satellite cells along the nerve (e.g., Purves, 1988; Titmus and Faber, 1990). T h e importance of such signals has been repeatedly supported by demonstrations that disrupting axonal transport (by depolymerizing

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microtubules or cooling) produces alterations that are similar to those produced by axotomy (e.g., Pitman et al., 1972; Purves 1976; Fawcett and Keynes, 1990; Wu et al., 1993). However, positive injury signals (Cragg, 1970) are also involved in triggering adaptive reactions to axonal injury. For example, disruption of axonal transport has been shown to prevent or delay some injury-induced alterations in neurons from mammals (Singer et al., 1982; Soiefer et al., 1988) and Aplysia (Gunstream et al., 1994). In Aplysia several axonal proteins have nuclear localization sequences that, when exposed, cause the proteins to be transported retrogradely to the soma and then imported into the nucleus (Ambron et al., 1992; Schmied et al., 1993). Direct evidence for positive axonal injury signals in Aplysia has been provided by the demonstration that intracellular injection of axoplasm from previously injured nerves into individual sensory neuron somata produced persistent hyperexcitability of the soma (Ambron, et al., 1994; Walters et al., 1994) similar to that which follows axonal injury (Walters et al., 1991). What initially activates positive injury signals is not yet known, but damage to the axon causes many ionic and biochemical perturbations (e.g., Fishman et al., 1990; Berdan et al., 1993; Ziv and Spira, 1993) that might lead to unmasking of nuclear localization sequences and access of injury signal proteins to the retrograde transport/nuclear import pathway (Walters et al., 1994). T h e activation, production or uptake of retrograde signals might also be secondary to a release of growth factors (e.g., Richardson and Ebendal, 1982; Grothe and Unsicker, 1992; Bonni et al., 1993; Clatterbuck et al., 1993) or cytokines (Frisen et al., 1993; Clatworthy et al., 1994a,b; Matsuda-Nakagawa et al., 1993) from surrounding cells in the injured nerve. Another form of delayed injury signal might be produced by spontaneous firing of sensory neurons following injury of their axons (e.g., Russell and Burchiel, 1988; Kajander and Bennett, 1992; Kajander et al., 1992; Wall and Gutnick, 1974; Wall and Devor, 1983; reviewed by Devor, 1989; Snow and Wilson, 1991) and, perhaps, by injury of their peripheral receptive fields. Spontaneous activity in sensory neurons is prominent in neuropathic pain models involving loose ligation of the rat sciatic nerve (see Section VIl,B,l).

VI. Mechanisms of Rapid Nociceptive Sensitization

The studies reviewed above suggest that some behavioral reactions to bodily injury appear formally similar in very distantly related animals,

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such as rats, leeches, and Aplysza. Evaluation of the relative roles of homologous processes (i.e., those that descended from a common ancestor) and analogous processes (i.e., those that were derived independently in chordates, annelids, and molluscs in response to common selection pressures) in accounting for behavioral similarities requires detailed comparisons of the underlying mechanisms. Because of the complexity of the involved neural networks, the mechanisms underlying some aspects of injury-related behavior are resistant to cellular and molecular analysis. However, sufficient progress has been made in the analysis of certain aspects of injury-related behavior to allow preliminary comparisons in diverse groups of animals that can set the stage for considering possible evolutionary patterns. Below I consider mechanisms involved in rapid sensitization and long-term sensitization following noxious stimulation. For more information on mechanisms related to nociceptive sensitization in mammals, the reader should consult recent reviews by Dubner and Ruda (1992), Coderre et al. (1993), Simone (1992), Handwerker and Kobal(1993), Treede et al. (1992),and Willis (1992).Additional information on mechanisms related to nociceptive sensitization in invertebrates can be found in reviews by Carew and Sahley (1986), Byrne (1987), Castellucci and Schacher (1990), Hawkins et al. (1993), and Walters (1991).

A. INCREMENTAL SENSITIZATION MECHANISMS A N D IMMINENT INJURY In Section IV,B,3 it was argued that incremental behavioral sensitization may reflect a general mechanism for recognizing imminent injury and rapidly activating appropriate defenses. The major premise was that it is adaptive for animals to assume that a quasi-noxious stimulus is dangerous if it is repeated or maintained without a decrease in intensity. The results to be reviewed below suggest that a quasi-noxious stimulus triggers a rapid increase in sensitivity of the nociceptive system (neural sensitization-see Table 11). This ensures that a subsequent stimulus of equal or greater magnitude delivered to the same region will evoke an enhanced defensive response and produce an additional increase in sensitivity. If the quasi-noxious stimulus is prolonged, the sensitivity of the defensive system will increase during the stimulus until a defensive response is initiated. If the degree of increment in sensitivity of the system during quasi-noxious stimulation depends on the perceived intensity of the stimulus, positive feedback results; i.e., the more sensitive the system, the more sensitive it becomes with additional stimulation. This property would cause an accelerating response to sustained or increasingly intense

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stimuli that would lead very rapidly to the production of maximal defensive responses. An accelerating sensory response to sustained, quasinoxious stimuli would also counteract, at least for awhile, the depressive effects of prolonged stimulation (e.g., sensory adaptation, response habituation, fatigue) that are properties of virtually all sensory systems (e.g., Dusenberry, 1992; Treede et al., 1992). Several mechanisms of neural sensitization have now been identified in mammalian and invertebrate preparations that can contribute to incremental behavioral sensitization. Again, neural sensitization is defined as an increase in the effective sensitivity of a neural system, and is expressed as an increase in the magnitude, duration, speed, or frequency of occurrence of neural responses, or a decrease in their threshold or latency (see Section IV,B,2).Neural sensitization can be mediated by sensitization of individual nerve cells (“cellular sensitization”) or by alterations of the network (“network sensitization”) that do not involve an increase in the sensitivity of individual neurons to their inputs. For example, the synaptic output of neurons within the network may be facilitated, increasing the response of the network to its inputs even if the sensitivity of individual neurons within the network remains the same. In the next two sections mechanisms of neural sensitization that might contribute to incremental sensitization in mammals and invertebrates are briefly reviewed. Because sensitization of responses to mechanical stimuli appears to be more important than sensitization to thermal stimuli during predatory, parasitic, and agonistic encounters (not to mention the fact that thermal sensitization has no significance for most aquatic animals), this discussion will be limited largely to sensitization of mechanosensory pathways.

B. RAPIDSENSITIZATION MECHANISMS I N VERTEBRATES 1. Peripheral Mechanisms

Most nociceptors in mammals show cellular sensitization in the periphery during persisting or repeated noxious stimulation (Treede et al., 1992). This neural sensitization has been examined most extensively in response to noxious heat stimulation, which lowers the heat threshold and increases heat-evoked activity in myelinated and C-polymodal nociceptors in humans, monkeys, cats, rabbits, and rats (reviewed by Treede et al., 1992). Sensitization of mechanosensory neurons has been harder to document, which is somewhat surprising in view of the prominence of mechanical sensitization during cutaneous hyperalgesia in man (Lewis, 1942; Hardy et al., 1952). In part this may be because of the difficulty

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in standardizing and controlling noxious mechanical stimuli (Light, 1992) and because of limitations on the typical testing methods (see below), but it may also reflect the important role played by central sensitization mechanisms (see Section VII,A,3). The first report of sensitization of rnechanosensory responses described decreases in mechanical thresholds in cat C-polymodal nociceptors after heat injury (Bessou and Perl, 1969). Reeh and colleagues (1987) showed that dramatic mechanical sensitization of myelinated nociceptors occurs in the rat tail following mild mechanical trauma of the skin on the tail. Sensitization was manifested as the appearance of spontaneous activity in the nociceptor, lowering of its mechanical threshold, and expansion of its receptive field. C-polymodal nociceptors on the tail also showed spontaneous activity after mild mechanical trauma, but no decrease in threshold or expansion of their receptive fields. A related mechanism for mechanical sensitization is implicit in the properties of recently discovered “mechanically insensitive afferents” (MIAs) in rat, monkey, and cat (Schaible and Schmidt, 1988; Handwerker et al., 1991; Meyer et al., 1991). In the monkey, nearly half of the myelinated nociceptors and a third of the C-polymodal nociceptors that could be identified by electrical stimulation of hairy skin were unresponsive to mechanical stimuli (Meyer et al., 1991). However, injection into the electrical receptive field of an “inflammatory soup” containing histamine, bradykinin, prostaglandin El, and serotonin often caused mechanical sensitivity to appear in these neurons. Since mechanical trauma causes the local release of these inflammatory agents, these results suggest that an important mechanism of mechanical sensitization is the recruitment of previously silent nociceptors. Because most studies of peripheral sensitization have examined sensory neurons initially identified by their responses to nontraumatic mechanical stimulation (a procedure that would have overlooked the MIAs), these results may help explain the paucity of reports of mechanical sensitization in mammals. A large number of substances associated with inflammation and injury can sensitize mammalian nociceptors. Directly acting mediators include serotonin, adenosine, prostaglandins E, and I,, a lipoxygenase product of arachidonic acid, and lowered pH (reviewed by Treede et al., 1992; Levine et al., 1993). In addition, many substances released during inflammation and injury can produce indirect sensitization of nociceptors. These effects require additional cellular elements, such as sympathetic postganglionic neuron terminals and white blood cells. Indirect mediators of peripheral nociceptor sensitization include bradykinin, leukotriene B,, nerve growth factor-derived octapeptide, interleukin- I/3 and interleukin-8, and peptide fragments from bacterial cell wall proteins and the complement cascade (see Cunha et al., 1991; Treede et al., 1992;

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Levine et al., 1993). It has been reported that foot withdrawal latency is decreased following subcutaneous injections into the rat paw of the neuropeptides neurokinin A, substance P, and calcitonin gene-related peptide (Nakamura-Craig and Gill, 1991). Among the most potent sensitizing substances are serotonin (Taiwo et al., 1992; Taiwo and Levine, 1992; Hua and Yaksh, 1993) and bradykinin (Beck and Handwerker, 1974; Steranka etal., 1988; Handwerker, 1991a; Dray and Perkins, 1993). Serotonin, adenosine, prostaglandin E,, and the lipoxygenase product 8R,15S, diHETE exert their direct sensitizing effects on nociceptors by activating adenylyl cyclase, increasing cyclic AMP synthesis (Taiwo and Levine, 1991). The indirectly acting sensitizing mediators appear to converge on this same second-messenger system. For example, bradykinin and interleukin- 1/3 cause the release of directly acting prostaglandin E, from sympathetic postganglionic neurons, whereas leukotriene B, and various peptide fragments cause the release of directly acting 8R, 15SdiHETE from neutrophils (Levine ei! al., 1993). This scheme does not explain certain observations though. For example, bradykinin causes thermal hyperalgesia without producing significant mechanical hyperalgesia in humans (Manning et al., 1991). Several differences between thermal and mechanical hyperalgesia/sensitization have yet to be explained (e.g., Treede et al., 1992).

2. Central Mechanisms Hardy and colleagues ( 1952) proposed that secondary hyperalgesia in humans is due to hyperactivity (sensitization) within the CNS rather than peripheral sensitization of nociceptors. Their evidence was that hyperalgesia was expressed in skin outside the region served by the local axon reflex (indicated by the flare response) and could be prevented by anesthetic blocks that prevented sensory activity from reaching the CNS. These observations have been extended in human volunteers by LaMotte et al. (1991),who used capsaicin injections to produce strong activation of C-polymodal nociceptors. The induction of hyperalgesia due to capsaicin injection required that the initial C-fiber activity reach the CNS, but once established, the hyperalgesia did not need continuing input from the periphery for its maintenance. Studies using experimental animals have revealed injury-related neural sensitization within the spinal cord and perhaps in higher centers. Neural sensitization persisting for hours or longer will be discussed in Section V l I . In this section mechanisms contributing to central sensitization of rapid onset (i.e., within seconds or minutes will be discussed. As mentioned earlier (Section IV,A), injury or nerve stimulation that mimics injury causes a rapid, long-lasting enhancement of foot with-

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drawal responses in the decerebrate rat. Cook and colleagues (1986) found evidence that this enhancement does not depend on changes in the excitability of primary sensory neurons or the motor neurons. Mendell and Wall (1965) showed that repeated stimulation of peripheral nerves at intervals of a few seconds produced a progressive increase (“wind-up”)in the number of evoked action potentials in many neurons located within the dorsal horn of the cat spinal cord. The dorsal horn contains second-order interneurons that project to the thalamus and midbrain, as well as making local connections in the spinal cord (e.g., Willis, 1985; Light, 1992). Wind-up of dorsal horn cell activity occurred only if the nerve stimulus was intense enough to activate C-fibers. These observations were extended by intracellular recordings from dorsal horn neurons during repeated nerve stimulation. In the cat (Price et al., 197 1) about 50% of the dorsal horn neurons showed wind-up of discharge, which was associated with recruitment of additional fast EPSPs and, in some cases, slow depolarization and prolonged discharge. Similarly, in the rat (Woolf and King, 1987) wind-up to repeated nerve shock at C fiber strength was observed in nearly 50% of the dorsal horn neurons examined. In this study only dorsal horns with a wide dynamic range were examined; i.e., cells that responded to both low- and high-intensity mechanical stimuli, but responded best to intense (pinching) stimuli. Most of these cells showed slow depolarizing responses that could last for many seconds after the last nerve shock. Although the depolarization was sometimes large enough to cause spike inactivation at the peak of the response, it would normally be expected to bring these neurons closer to spike threshold and thus make them more excitable. However, it is not known yet whether these cells show actual cellular sensitization during and after noxious stimulation of the body surface. It will be interesting to examine changes in input conductance and in the spiking responses to intracellular injection of depolarizing current pulses to see whether the cell’s electrical excitability actually changes. Suggestive evidence for the ability of noxious input to increase the excitability of dorsal horn interneurons was obtained by Yoshimura and Jessell (1989), who showed in an in uitro spinal cord slice preparation that repetitive stimulation of dorsal roots at an intensity that activated C-polymodal nociceptors caused a slow, 20- to 120-s EPSP in many dorsal horn cells that was often accompanied by an increase in membrane input resistance. An increase in input resistance (a decrease in input conductance) will increase the responsiveness of a cell to its inputs by causing a larger depolarization to be produced by a given synaptic current (e.g., Carew and Kandel, 1976; Schulman and Weight, 1976).

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Other alterations have begun to suggest that cellular sensitization may occur in dorsal horn interneurons, and demonstrate that noxious stimulation rapidly sensitizes the network to which these neurons belong. Kenshalo and colleagues (1982) found that noxious heating increased the peak activation of dorsal horn interneurons that project to the thalamus by mechanical stimuli delivered just outside of the area damaged by the heat. Dorsal horn interneurons receive convergent synaptic input from a large number of primary sensory neurons, and thus have excitatory receptive fields that are larger than those of individual sensory neurons (Willis, 1985). Intracellular recordings indicate that the peripheral receptive fields of dorsal horn cells are composed of two zones: an inner impulse-generating zone in which mechanical stimulation activates the dorsal horn cell, and a large subthreshold or “low probability firing” fringe in which mechanical stimulation evokes fast EPSPs in the dorsal horn cell that usually fail to reach threshold for generating an action potential (Woolf and King, 1989). Noxious cutaneous or nerve stimulation causes a rapid expansion of the impulse-generating zone, apparently at the expense of the subthreshold fringe, and decreases its threshold (e.g., McMahon and Wall, 1984; Cook et al., 1987; Cervero et al., 1988; Woolf and King, 1990; Laird and Cervero, 1989). Similar changes in receptive field size and threshold have been described in trigeminal neurons of the rat after injection of mustard oil into the deep masseter muscle (which closes the jaw) (Hu et al., 1992). One consequence of receptive field expansion during wind-up and incremental sensitization is that an increasing number of local circuit and projection interneurons should be activated by successive stimuli delivered within the same region (Dubner and Ruda, 1992). This would be expected to increase the strength of the nociceptive signals that ( a ) evoke local flexion reflexes and ( b ) are transmitted to higher centers. The same effect would be expected from expansion of sensory neuron receptive fields (Billy and Walters, 1989a, see Section VII,C, 1 ) . The increase in size and responsiveness of the cutaneous receptive fields of dorsal horn cells during incremental sensitization might be a direct consequence of slow, long-lasting EPSPs in the dorsal horn cells produced by sensory neurons activated by noxious stimulation. Other potential mechanisms include increased activation of sensory neurons, presynaptic facilitation of fast EPSPs from sensory neurons, increased “reverberatory” activity feeding back from excitatory interneurons, and decreased pre- or postsynaptic inhibition from local or higher order inhibitory interneurons. T o date slow synaptic potentials from sensory neurons have received the most attention as potential mechanisms for

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rapidly developing sensitization. Slow EPSPs in dorsal horn cells (Woolf et al., 1988; Yoshimura and Jessell, 1989) can be produced by various neuropeptides and excitatory amino acids present in dorsal root ganglion cells (the sensory neuron somata) and their central terminals (reviewed by Levine et al., 1993). For example, substance P (SP) and calcitonin gene-related peptide (CGRP) produce slow, long-lasting depolarizations of dorsal horn cells, with the primary function of CGRP possibly being to potentiate the effects of SP (Biella et al., 1991). Coadministration of SP and CGRP facilitate the foot withdrawal reflex in the decerebrate rat (Woolf and Wiesenfeld-Hallin, 1986). The excitatory amino acid glutamate can also produce slow, longlasting depolarizations of dorsal horn cells. One component of this slow depolarization, lasting several seconds, depends on the N-methyl-Daspartic acid (NMDA) receptor (Murase et al., 1989a,b; Gerber et al., 1991), whereas a second component, lasting several minutes, does not (Gerber et al., 1991). Activation of the NMDA receptor-gated channel requires both the binding of glutamate and sufficient depolarization to remove a block of the channel by Mg2+ (Mayer et al., 1984). This depolarization could be produced by neuropeptides such as SP, as well as by glutamate's fast excitatory actions that are mediated by non-NMDA receptors (e.g., Dougherty and Willis, 1991b; Woolf and Thompson, 1991; Dubner and Ruda, 1992). Intracellular studies (Thompson et al., 1990) and extracellular studies (e.g., Davies and Lodge, 1987; Dickenson and Sullivan, 1987; Ren et al., 1992) show that wind-up and the rapid expansion of dorsal horn interneuron receptive fields are blocked by NMDA antagonists. The use of NMDA agonists and antagonists has supported a role for this receptor in the increased activation of dorsal horn cells and enhanced nociceptive behavior occurring shortly after noxious stimulation in several mammals (e.g., Aanonsen et al., 1990; Haley et al., 1990; Dougherty and Willis, 1991b; Murray et al., 1991; Woolf and Thompson, 1991; Dougherty et al., 1992a; Dougherty and Willis, 1992), including humans (e.g., Kristensen et al., 1992). An important finding was reported by Randic et al. (1993), who showed that brief, high-frequency stimulation of primary afferent fibers in a transverse slice preparation of rat spinal cord produced long-term potentiation (LTP) of monosynaptic and polysynaptic EPSPs monitored in dorsal horn interneurons. This LTP, which showed no decrement over the 1-2 h testing period, appears remarkably similar to LTP in the hippocampus and cerebral cortex. In particular, it was blocked by an NMDA receptor antagonist during the induction phase but not during the maintenance phase of LTP. In addition, the same dorsal horn cell would express LTP when tetanization occurred with the cell kept relatively

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depolarized, whereas it would express LTD when tetanization occurred with the cell kept hyperpolarized (cf. Artola et al., 1990). Recent findings have provided strong support for the suggestion that SP enhances the effects of NMDA receptor-mediated discharge in dorsal horn cells (Dougherty and Willis, 1991a; Nagy et al., 1993) and that SP-mediated effects and NMDA receptor-mediated effects work synergistically to facilitate flexor reflexes (Xu et al., 1992). However, SP can also enhance the responses to excitatory amino acids mediated by non-NMDA receptors, and SP has been observed to inhibit the responses of some dorsal horn neurons to microiontophoretic application of glutamate or aspartate (Dougherty et al., 1993). Several second messengers have been implicated (using pharmacological methods) in wind-up of dorsal horn cell responses. Unfortunately, identification of the actual contributions of various intracellular signaling pathways to central sensitization will be very difficult because of the heterogeneity of the dorsal horn neuronal population and the obstacles to intracellular analysis presented by the small size of the dorsal horn neurons and their sensitivity to anoxic and mechanical damage (Yoshimura and Jessell, 1989). The involvement of NMDA receptors in windup suggests a second messenger role for the Ca2+ that enters through NMDA-receptor-gated channels, and the potential involvement of various Ca2+-dependentprotein kinases. Evidence has been presented for contributions of PKC (Coderre, 1992; Coderre et al., 1993). Apparently there has been little investigation of the roles in central sensitization of other Ca2+-dependentprotein kinases such as Ca2+/calmodulin-dependent protein kinases (CaM kinase) and tyrosine kinases. Cyclic AMPdependent protein kinase (PKA) may also be involved in sensitization since application of a membrane-permeant cyclic AMP analog (8-Br cyclic AMP) facilitated monosynaptic inputs evoked by stimulating dorsal roots, and potentiated the effects of excitatory amino acids (Cerne et al., 1992). Furthermore, in this same study intracellular injection of a protein inhibitor of protein kinase A (PKI) into dorsal horn neurons blocked the potentiation of the NMDA response of these cells produced by application of a cell-permeable cyclic AMP analog. Recently another Ca2+dependent (and NMDA receptor dependent) messenger has been implicated in rapid central sensitization-the highly diffusible gas, nitric oxide (NO), which is synthesized by the Ca'+dependent enzyme nitric oxide synthase. This enzyme appears to be localized in sensory neurons and in regions of the spinal dorsal horn containing sensory neuron terminals (reviewed by Meller and Gebhart, 1993). Behavioral sensitization produced by spinal (intrathecal) administration of NMDA was found to be blocked by nitic oxide synthase antago-

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nists (Haley etal., 1992; Kitto et al., 1992; Meller etal., 1992)and extracellular hemoglobin (Kitto et al., 1992), which suggests that the N O has to diffuse out of the neuron of origin in order to be effective. One of the targets of N O is guanylate cyclase, which synthesizes cyclic GMP. Administration of an inhibitor of guanylate cyclase (methylene blue) also reversibly blocks the behavioral sensitization produced by NMDA application (Meller et al., 1992).

C. RAPIDSENSITIZATION MECHANISMS IN INVERTEBRATES Mechanisms of nociceptive sensitization have been examined in only a few invertebrates, with the bulk of the research being performed on a single species, the mollusc Aplysia calqornica. All of the initial studies, and much of the current work on this species, has focused on central mechanisms underlying general sensitization. However, as described below, mechanisms of general sensitization have a broader significance because, at least in Aplysia, their activity-dependent amplification provides a basis for site-specificsensitization and perhaps a simple form of associative learning. 1. Peripheral Mechanism Relatively little is known about peripheral mechanisms contributing to injury-related changes in defensive behavior of invertebrates. Facilitation (lasting several minutes) of motor responses activated during noxious stimulation can be produced by post-tetanic potentiation (PTP) at the neuromuscular junctions of still active or recently active motor n-Lurons in crayfish (e.g., Lnenicka and Atwood, 1985; Atwood and Wojtowicz, 1986)and the mollusc, Aplysia (Jacklet and Rine, 1977; Frost et al., 1988a; Hickie and Walters, 1992; see also Section VI,C,2,h). Facilitation of motor neuron synapses during sensitization may also involve heterosynaptic mechanisms in the periphery (e.g., Lukowiak and Colebrook, 1988a,b). Evidence in various invertebrate preparations suggests that neuromuscular transmission can be modified by the local release of neuromodulators such as serotonin (e.g., Weiss et al., 1975; McPherson and Blankenship, 1992), octopamine (e.g., O’Shea and Evans, 1978; Hoyle, 1984),and neuropeptides (e.g., Bishop et al., 1987; Hall and Lloyd, 1990; Whim and Lloyd, 1989) that are known to be localized to neuronal processes near neuromuscular junctions or contained in the motor neuron terminals themselves. Nociceptive sensitization in some invertebrates involves an enhancement of the peripheral sensitivity of mechanosensory neurons. In Aplysia, noxious shock to the tail was observed to enhance the number of action potentials recorded intracellularly in wide-dynamic range VC sensory

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neurons during taps close to the site of noxious stimulation (Clatworthy and Walters, 1993a), and extracellular recordings suggest that the tail shock may also increase the responsiveness of other afferent populations (Walters, 1987b). Furthermore, moderate intensity tail shocks repeated at 5-s intervals cause a progressive “wind-up” of the number of action potentials evoked in VC cells by each shock (Clatworthy and Walters, 1993a). A small increase in the number of spikes evoked in LE sensory neurons by siphon tap was seen after either stimulation of an interganglionic connective or application of serotonin to the siphon (Klein et al., 1986). Similarly, injection of serotonin or the molluscan neuropeptide, small cardioactive peptide, (SCP,), into the tail reduced the mechanical threshold of VC nociceptors innervating that region (Billy and Walters, 1989b). Serotonin, which plays an important role in central sensitization in Aplyszu (see next section), also facilitates peripheral synapses of LE siphon sensory neurons onto peripheral motor neurons located within the siphon (Clark and Kandel, 1984). Serotonin immunoreactive axons and varicosities are found in peripheral tissues as well as in the CNS (Goldstein et al., 1984; Longley and Longley, 1986). Depletion of serotonin throughout the animal by injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine causes a dramatic reduction in both the dishabituation of gill withdrawal and the sensitization of spontaneous gill contractions produced by noxious tail shock (Glanzman et al., 1989). Although these defects were interpreted in terms of central effects, they might also reflect a perturbation of peripheral functions of serotonin. For example, the decreased duration of gill contractions (and some of the deficit in sensitization) evoked by tail shock after serotonin depletion (Glanzman et al., 1989) might be partly due to a loss of rapid facilitatory mechanisms that normally amplify peripheral and central sensory discharge to noxious stimulation (see Clatworthy and Walters, 1993a). Interestingly, peripheral serotonin application also increases the number of spikes evoked by mechanical stimulation in P and T mechanosensory neurons of the leech (Gascoigne and McVean, 1991). Depletion of serotonin in the leech with the neurotoxin 5,7-dihydroxytryptamine prevents sensitization of the touch-elicited shortening reflex (Ehrlich et al., 1992). In principle, noxious stimulation might also modulate signaling in invertebrate sensory and motor neurons via circulating humoral factors. Modulation of invertebrate nociceptors by injury-related humoral factors has not yet been described. However, humoral modulation of the stretch receptor in the lobster has been described in a system where the possibility of neural modulation can be clearly excluded. Stretch receptors of the lobster oval organ, which lack any efferent innervation, are modulated by serotonin, octopamine, and the neuropeptide proctolin circulating in

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the hemolymph (Pasztor and Bush, 1987).Given the evidence for defensive sensitization in crustaceans and the importance of peripheral neuroendocrine actions of serotonin and octopamine for agonistic behavior of lobsters (see review by Kravitz, 1988), this observation suggests that it will be interesting to see whether these circulating amines enhance peripheral sensitivity of nociceptive sensory neurons in crustaceans. Motor systems are also modulated by humoral factors in crustaceans. Of particular interest is a mechanism observed in crayfish that could cause a selective enhancement of responses mediated by motor neurons that continue to fire after noxious stimulation. This response-specific sensitization mechanism is suggested by the observation that very low concentrations of octopamine cause presynaptic facilitation at the neuromuscular junction, and that these effects are augmented and prolonged by background activity in the motor neuron (Breen and Atwood, 1983). Apparently similar activity-dependent extrinsic modulation (ADEM) of central synaptic transmission has been observed in Aplysia sensory neurons and might also contribute to facilitation of the neuromuscular junction in Aplysia, although the extrinsic modulators in these cases are probably released primarily from local nerve terminals (see next section).

2. Central Mechanisms a. Facilitation of Central Sensory Neuron Synapses in Aplysia. T h e first clue about sensitization mechanisms in invertebrates was revealed in a study of habituation and dishabituation in Aplysia by Castellucci and colleagues ( 1970) before behavioral sensitization had yet been demonstrated in this animal. They found that intense stimulation of the neural connectives that carry noxious information to the abdominal ganglion from the pleural ganglia (the ganglia containing the somata of the VC wide-dynamic-range sensory neurons, which innervate most of the body) facilitated central synaptic connections between LE siphon sensory neurons and a gill motor neuron. Although they looked at EPSPs that had decremented during repeated stimulation, the connective stimulation sometimes increased the EPSP amplitude above its initial value. Again, behavioral sensitization has been defined as the increase above the initial value of a habituated or unhabituated test response by a modulating stimulus, whereas dzshabituation is usually defined as the rapid recovery (partial or complete) of a habituated test response following a modulating stimulus (e.g., Marcus et al., 1988; see Section IV,B,2). Behavioral sensitization was first reported in Aplyia by Carew et al. (1971), who showed that transient, general sensitization of the gill withdrawal reflex to siphon stimulation could be produced by strong tactile stimulation of the head o r neck. To study neural mechanisms these authors used an isolated

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ganglion preparation and substituted stimulation of an interganglionic connective for the tactile sensitizing stimulus. This neural stimulus produced synaptic facilitation of nondecremented monosynaptic EPSPs from siphon sensory neurons to a gill motor neuron. They found that facilitation of the sensorimotor synapse was not due to an increase in the input resistance of the motor neuron and was not a consequence of motor neuron activation. In addition, it was truly heterosynaptic (due to the actions of an extrinsic modulator released from facilitatory interneurons) rather than homosynaptic (due simply to activation of the sensorimotor synapse), since the sensory neuron was not activated by the sensitizing stimulus. Similar heterosynaptic facilitation was observed for connections between wide-dynamic-range tail sensory neurons and tail motor neurons following tail shock (Walters et al., 1983b). The implication that heterosynaptic facilitation is due to presynaptic alterations within sensory neuron terminals was supported by the results of a quanta1 analysis of the connection between LE sensory neurons and a gill motor neuron under conditions of low transmitter release (Castellucci and Kandel, 1976). b. Increased Excitability o j Sensory Neuron Soma in Aplysia. In addition to increasing the excitability of the peripheral arbor of the sensory neuron and enhancing synaptic transmission at its peripheral and central terminals, noxious cutaneous stimulation (Walters, 198713) and nerve stimulation (Klein et al., 1986; Walters and Byrne, 1985) cause a rapid increase in the excitability of the sensory neuron soma. Although changes expressed within the readily accessible soma have proven very useful for analysis of molecular correlates of sensitization (see below), the function of these changes in excitability has until recently been unclear. The cell body represents a “dead end” off the direct path between the peripheral receptors and the central synaptic terminals, and, at least in the case of the pleural VC sensory neurons, the soma is too distant from most of the terminals to affect directly synaptic transmission (Walters et al., 1983a; Hammer et al., 1989). An answer to this mystery was provided by the discovery that noxious cutaneous or nerve stimulation often produces an afterdischarge of spikes that is generated in or near the soma (Clatworthy and Walters, 1993a). The centrally located nociceptor soma thus acts as an amplifier that supplements and prolongs spike trains initiated by noxious peripheral stimulation. c. Serotonin as a Sensitizing Modulator in Aplysia. The finding that bath application of serotonin mimicked facilitation of LE synapses produced by connective stimulation (Brunelli et al., 1976) and facilitation of VC synapses produced by tail shock (Walters et al., 1983b) led to many studies of the effects of this neuromodulator on wide-dynamic-range sensory

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neurons of Aplysia. Serotonin was also found to facilitate synapses or produce spike broadening in most but not all of the other mechanosensory populations that have been identified in this species (Rosen et al., 1989; Dubuc and Castellucci, 1991). An important role of serotonin in defensive sensitization was supported by several findings: ( a ) serotonin depletion reduces behavioral dishabituation and sensitization, as well as facilitation of sensory neuron EPSPs (Glanzman et al., 1989, and see above); (6) a pair of identified serotonergic interneurons is activated by noxious cutaneous stimulation and, when stimulated intracellularly, causes facilitation of sensory neuron synapses and broadening of sensory neuron spikes (see below) (Mackey et al., 1989); and (c) the serotonergic antagonist cyproheptadine blocks sensory neuron spike broadening and synaptic facilitation produced by stimulating a peripheral nerve (Mercer et al., 1991). Interestingly, serotonin-containing varicosities are found not only within the neuropil near synaptic contacts (Goldstein et al., 1984; Kistler et al., 1985; Longley and Longley, 1986), but also on the somata of pleural sensory neurons (Zhang et al., 1991), where serotonin might be involved in generating afterdischarge (Clatworthy and Walters, 1993a) and alterations dependent on gene regulation (see Section VII,C,4). It should be kept in mind, however, that the necessity of serotonin for defensive sensitization has only been examined under a very limited set of conditions (Glanzman et al., 1989), and some evidence also exists for sensitizing roles for both the neuropeptide SCP, (Abrams et al., 1984; Pieroni and Byrne, 1992; Schacher et al., 1990) and an unidentified neuromodulator released by facilitatory interneurons (the L29 cells) onto synapses formed by LE sensory neurons (Hawkins and Schacher, 1989). d. Effects of Serotonin on Central Sensory Neuron Properties in Aplysia. Serotonin has a number of direct effects on LE and VC sensory neuron somata: the membrane slowly depolarizes; input conductance, spike threshold, and spike accommodation decrease; and spike duration increases (Klein and Kandel, 1978; Walters et al., 1983b; Baxter and Byrne, 1990). The former effects increase excitability (and perhaps afterdischarge) and, with the increase in spike duration, tend to increase the safety factor for conduction through regions where conduction failure occurs (Clatworthy and Walters, 1993a,b).The increase in spike duration also increases transmitter release when it occurs in or near presynaptic terminals (Klein and Kandel, 1978; Hochner et al., 1986a; Blumenfeld et al., 1990). These changes involve a depression by serotonin of at least three different K + currents: (1) the S-type K + current, which is partially active at resting potential and shows slow additional activation with depolarization to between -30 and 0 mV, and little or no inactivation during sustained depolarization (e.g., Klein et al., 1982; Baxter and Byrne, 1989);

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(2) a transient, steeply voltage-dependent “delayed rectifier” K + current that is inactive at rest, and activates rapidly and then inactivates with depolarizations to - 10 mV and above (Baxter and Byrne, 1989; Goldsmith and Abrams, 1992; Hochner and Kandel, 1992); and (3) a steadystate Ca2 + -activated K + current (Walsh and Byrne, 1989). In addition, serotonin increases a slowly inactivating, L-type Ca2+current, which does not directly affect synaptic transmission (Edmonds et al., 1990). The function of this Ca2+current remains unknown, but it might be involved in activity-dependent mobilization of transmitter that serves to counteract synaptic depression (Edmonds et al,, 1990) o r contribute to PTP (Eliot, 1991). It might also contribute to the central or peripheral afterdischarge produced by injurious stimulation of VC nociceptors’ receptive fields (Clatworthy and Walters, 1993a). The lack of an effect of serotonin on the N-type Ca2+ current during synaptic facilitation that is associated with Ca2+ influx into the sensory neuron terminals, coupled with the lack of an effect of the L-type Ca2+ current on transmitter release, provides strong evidence for a role of spike broadening due to depression of K + currents in this facilitation (Eliot et al., 1993). e. Second Messengers, Mediating Central Serotonin Effects on Sensory Neurons in Aplysia. General sensitization and the effects of serotonin in Aplysia have been linked, thus far, to two second-messenger systems in widedynamic-range sensory neurons. Serotonin (Bernier et al., 1982; Ocorr and Byrne, 1985; Bacskai et al., 1993) and tail shock (Ocorr et al., 1986) increase cyclic AMP levels in LE and VC neurons. Some of the shortterm sensitizing alterations in sensory neurons depend on continuously elevated cyclic AMP levels (Castellucci et al., 1982), which depend on continuous activity of adenylyl cyclase (Yovell et al., 1987), which, in turn, may depend on continuing activity of serotonergic interneurons activated by noxious stimulation (Mackey et al., 1989). Serotonin also activates an isoform of PKC (Sacktor et al., 1988; Sacktor and Schwartz, 1990; Sossin and Schwartz, 1992). Cyclic AMP, which activates PKA, appears to be responsible for depression of both the S-type K + current (e.g., Shuster et al., 1985; Baxter and Byrne, 1989) and the Ca2+-activated K + current (Walsh and Byrne, 1989). The depression of the voltagedependent, transient K + current observed shortly after serotonin application appears to be largely due to activation of PKA (Goldsmith and Abrams, 1992; Hochner and Kandel, 1992), but after several minutes the depression of this current may be mediated in part by PKC (Sugita et al., 1992). Activation of PKC also appears to be primarily responsible for the enhancement of the L-type Ca2+ current (Braha et al., 1993). The serotonin-induced depression of the S-type K + current (Baxter and Byrne, 1990; Goldsmith and Abrams, 1992) and the Ca2+-activated K +

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current (Walsh and Byrne, 1989) contribute to the increased soma excitability. The decreases in both the voltage-dependent, transient K + current and the S-type K + current are responsible for spike broadening, which contributes to presynaptic facilitation, especially when the synapse has not been depressed (e.g., Hochner et al., 1986b; Goldsmith and Abrams, 1992; Hochner and Kandel, 1992).Spike duration-independent processes also contribute to serotonin-induced synaptic facilitation (perhaps directly modulating transmitter mobilization rather than indirectly modulating Ca2+levels as occurs during spike broadening) (Gingrich and Byrne, 1985; Hochner et al., 1986b). The spike durationindependent facilitation produced by serotonin may be mediated by several different kinases (Ghirardi et al., 1992; Goldsmith and Abrams, 1992; Pieroni and Byrne, 1992),including PKC (Sugita etal., 1992; Braha et al., 1993). f. Activity -Dependent Sensitizing Effects on Central Components of Aplysia Sensory Neurons. Nociceptive sensory neurons activated by a noxious stimulus will show intrinsic, activity-dependent plasticity in addition to the plasticity induced by extrinsic modulators such as serotonin. A brief, high-frequency burst of action potentials triggered by intracellular stimulation produces homosynaptic post-tetanic potentiation (PTP) in VC and LE sensory neurons lasting about 5 min (Clark and Kandel, 1984; Walters and Byrne, 1984). In addition, intrinsic activity-dependent effects interact with extrinsic modulatory effects. An extensively studied interaction in Aplysia is ADEM, in which spike activity occurring immediately prior to binding of an extrinsic modulator to the neuron causes an amplification of the extrinsic modulator's effects. Upon its discovery, ADEM was initially proposed to be a mechanism for associative learning (Hawkins et al., 1983; Walters and Byrne, 1983a). However, both ADEM and PTP in nociceptors activated during injury may function primarily as mechanisms of site-specific sensitization to protect a wounded region (Walters, 1987a,b). A cellular mechanism contributing to ADEM in Aplysia sensory neurons is the activation of adenylyl cyclase by Ca2+, which enters the cell during spike activity (Ocorr et al., 1985; Yovell et al., 1987; Abrams and Kandel, 1988; Abrams et al., 1991). This enhances cyclic AMP synthesis and should amplify all the cyclic AMP-mediated effects normally produced by the extrinsic modulator. In addition to amplifying extrinsic synaptic facilitation, ADEM causes a rapid, persistent depolarization of the sensory neuron that may increase resting entry of Ca2+ into the cell (Walters and Byrne, 1983b) and contribute to the generation of afterdischarge (Clatworthy and Walters, 1993a). All forms of activity-dependent cellular sensitization in Aplysia sensory neurons (including ADEM and homosynaptic PTP and LTP-see Section

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VII,C,S) should contribute to site-specific sensitization around an injury Tables I-III), since sensory neurons innervating the injured region will show the greatest activation during an injurious encounter. g. Interneuronal Contributions to Sensitization in Aplysia. Nociceptive sensitization involves an increase in both the magnitude and the duration of defensive responses. The first suggestion that interneurons are a locus of plasticity underlying sensitization came from the observation that increases in the duration of withdrawal responses and motor neuron activity on tests given after noxious stimulation appeared far greater than could be accounted for by observed changes in the duration of sensory neuron activity during testing (Eberly and Pinsker, 1984). Indeed, although sensitization is sometimes accompanied by an afterdischarge to test stimulation in wide-dynamic-range sensory neurons, afterdischarge only increases the duration of sensory neuron firing by 1 to 2 s (Clatworthy and Walters, 1993a), whereas the increase in duration of siphon responses can be many tens of seconds (Pinsker et al., 1973; Frost et al., 1985; Walters, 1987a). At least some of the increase in duration after sensitization appears to be due to the activation by each test stimulus of a pattern generator (“Interneuron 11”) that drives periodic pumping movements of the siphon and mantle organs (Byrne 1983; Eberly and Pinsker, 1984; Frost et al., 1988a; Koester, 1989). Evidence for a contribution of other interneurons to the increase in magnitude of withdrawal responses during nociceptive sensitization in Aplysia was obtained by Frost and colleagues (Frost et al., 1988a), who showed that the synapses of a set of interneurons (the L29 cells) which excite siphon motor neurons display PTP for 5-10 min following their strong activation by noxious tail shock. However, because .the L29 cells also cause powerful excitation of potent inhibitory interneurons, the net effect of PTP in these cells is uncertain (Fischer and Carew, 1993). Recent evidence indicates that the prolonged interneuronal excitation and facilitation of phasic responses involves the release of neuropeptides such as SCP, (Trudeau and Castellucci, 1992). Although serotonin application facilitates sensory to motor connections and can, under some conditions, enhance tail withdrawal responses (Walters et al., 1983b), the dominant effect of bath-applied serotonin observed thus far on siphon responses and interneurons in the siphon withdrawal network is inhibitory (Fitzgerald and Carew, 1991). Interestingly, an important source of enhanced central excitation during sensitization comes from disinhibition; dramatic decreases have been observed in the inhibition of excitatory interneurons in the siphon withdrawal reflex following sensitizing connective stimulation (Frost et al., 1988a; Trudeau and Castellucci, 1993). T h e potential complexity of interneuronal mechanisms of sensitization is underscored

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by optical recordings which have shown that a few hundred neurons may be activated in the Aplysia abdominal ganglion during tactile stimulation of the siphon (Zecevic et al., 1989; Nakashima et al., 1992; Falk et al., 1993). Thus, as in the leech (Lockery and Kristan, 1990), some of the processing of nociceptive information may be distributed across a relatively large number of interneurons. However, as optical recording methods improve, they should aid in the identification and functional characterization of excitatory and inhibitory interneurons that are particularly important for altering defensive responsiveness. h. Motor.Neurona1 Contributions to Sensitization in Aplysia. Jacklet and Rine (1977) discovered a form of neuromuscular potentiation in the gill of Aplysia produced by tonic, relatively low-frequency background firing of the gill motor neuron. Because noxious stimuli cause long-lasting activation of gill motor neurons, this effect would be expected to enhance gill withdrawal responses and, indeed, Hawkins (1992) has shown that this peripheral potentiation is primarily responsible for the early phase of sensitization and dishabituation of the gill withdrawal reflex. Similar peripheral potentiation was shown to cause strong potentiation of siphon responses during prolonged elevation of background firing in siphon motor neurons following tail shock (Frost et al., 1988a; Hickie et al., 1992). Although the increases in background firing of gill and siphon motor neurons may be largely a consequence of a prolonged increase in synaptic input from interneurons (see above), some motor neurons may show intrinsic changes that increase their excitability or spontaneous firing rates after noxious stimulation. Such changes have been observed in ink motor neurons, opaline motor neurons, and some siphon motor neurons after electrical or pharmacological stimuli that mimic aspects of noxious stimulation (Carew and Kandel, 1977; Tritt and Byrne, 1980; Walsh and Byrne, 1985; Frost et al., 1988a). Ink and opaline release by Aplysia, like withdrawal responses, can be sensitized by noxious tail shock (Walters et al., 1993; Illich et al., 1994a). i. Central Sensitization Mechanism in Other Invertebrates. Central neuronal correlates of rapid defensive sensitization have also been described in other invertebrates. Incremental sensitization of a withdrawal reflex in the land snail Helix has been associated with depolarization and increased excitability of premotor “command” neurons (Balaban, 1983). Sensitization of the escape swim in the nudibranch mollusc Tritonia involves an increased excitability of interneurons and facilitation of interneuronal synapses (Frost et al., 1988b; Brown and Willows, 1991). Mechanosensory neurons that appear to be homologous with the pleural VC sensory neurons in Aplysia have been described in the closely related genus Bursatella. Although the effects of noxious stimulation have not been described

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in Bwsatella, serotonin produced similar increases in spike duration and soma excitability in the pleural sensory neurons (Wright and Kirschman, 1992). In the leech, neuronal processes associated with sensitization of shortening and bending reflexes display several apparent similarities to, and some differences from, sensitization processes reported in Aplysia. In particular, Sahley (1987) found that cutaneous shock increases spike duration in low-threshold T sensory neurons and increases the number of spikes evoked by depolarizing the soma of P sensory neurons. Peripheral serotonin application has been reported to increase the number of spikes evoked in T and P cells by a mechanical stimulus (Gascoigne and McVean, 1991), but bath application of serotonin does not reproduce the spike broadening and increased excitability in T and P cells produced by cutaneous shock, nor does depletion of serotonin with 5,7-DHT block the sensitizing effects of cutaneous shock on these sensory neurons (Ehrlich et al., 1992 and C. L. Sahley, personal communication, 1994). Although the sites of action are not yet known, it has become clear that serotonin plays an important role in sensitization in the leech. Serotonin depletion blocks behavioral sensitization, but not dishabituation, of the shortening reflex (Ehrlich et al., 1992). Application of serotonin agonists (Lockery and Kristan, 1987) or intracellular stimulation of either of two serotonin-containing interneurons can produce brief sensitization of the local bending reflex (Lockery and Kristan, 1991). Serotonin has also been implicated in the leech as an important factor in the increase in activity in neurons involved in inducing shortening following noxious stimulation (e.g., Belardetti et nl., 1982; Catarsi et al., 1987).These effects of serotonin may depend, at least in part, on the synthesis of cyclic AMP (Belardetti et al., 1982; Biondi et al., 1982). An involvement of serotonin in nociceptive sensitization in arthropods is less certain. A role for serotonin has been suggested in sensitization of the eyestalk withdrawal reflex in the crab (Appleton and Wilkens, 1990).Sensitization of the lateral giant escape reaction in the crayfish is associated with facilitated transmission between primary afferents and first-order interneurons, but in this case the sensitizing neuromodulator appears to be octopamine rather than serotonin (Glanzman and Krasne, 1983; Krasne and Glanzman, 1986). Although published reports of defensive behavioral sensitization in insects are unknown to this author, it is interesting that flexion and extension reflexes in the locust can be “dishabituated” and “sensitized” by iontophoresis of octopamine into central neuropil (Sombati and Hoyle, 1984). In addition, it will be interesting to see whether defensive networks show sensitization similar to the “central excitatory state” that underlies enhancement of feeding and drinking behavior in the fly after stimulation with sucrose (Dethier et al., 1965; Vargo, and Hirsch, 1985).

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VII. Mechanisms of long-Term Nociceptive Sensitization

In principle, long-term neural sensitization might be produced by maintenance of the alterations underlying rapid sensitization. As will be seen, some of the alterations that are expressed shortly after noxious stimulation are observed days and weeks later in both mammals and some invertebrates. Unlike rapid sensitization, persistent nociceptive sensitization might also involve neuronal growth (and degeneration). Indeed, results to be reviewed below suggest that growth of peripheral and central processes of mechanosensory neurons is induced by bodily injury in both mammals and invertebrates. Such growth might have a number of sensitizing effects. Growth of peripheral processes could enlarge the sensory neuron’s receptive field, increasing the number of sensory neurons activated by a given stimulus. Peripheral growth might also increase receptor density within a receptive field, which could decrease mechanosensory threshold. Growth of central sensory processes could increase the number of postsynaptic targets that are excited by a sensory neuron, which would increase the effective receptive field sizes of the secondorder cells. Central growth could also increase the density of synaptic contacts onto existing synaptic targets, which would increase the strength of synaptic transmission to those cells. In this section evidence is reviewed concerning mechanisms of neural sensitization underlying adaptive reactions to injury of skin and muscle. Although much has been learned about mechanisms of neural sensitization involved in the inflammation ofjoints (see e.g., Schaible and Schmidt, 1988; Sessle and Hu, 1991), these studies, which are directed more toward understanding the effects of progressive diseases such as arthritis than the effects of traumatic injury, are beyond the scope of this chapter.

A. AN ADAPTIVE HYPOTHESIS ABOUT REACTIONS TO AXOTOMY

Noxious stimulation that is intense enough to induce long-term behavioral alterations is likely to injure peripheral branches of sensory neurons and sympathetic neurons innervating skin and muscle, and might injure peripheral branches of somatic motor neurons as well. Long-term plasticity of sensory and motor neurons following serious wounding of the body surface has received very little experimental study in mammals. One study in decerebrate rats using extracellular recording techniques found some evidence for brief (lasting less than 20 min)

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increases in the excitability of motor neurons and no evidence for changes in sensory neuron terminals in the dorsal horn, but this study only examined alterations occurring within 30 min after superficial injury produced by injection of mustard oil underneath the skin of the paw (Cook et al., 1986). On the other hand, a large number of studies have examined long-term changes in these and other neurons following section o r crush of a peripheral nerve containing the neuron’s axon. Regeneration often occurs after nerve crush, but generally fails after complete section of a major nerve trunk. In both cases, a number of electrophysiological and morphological alterations develop over several days in the axotomized neuron (see recent reviews by Titmus and Faber, 1990; Snow and Wilson, 1991). Of interest here is the possibility that some of these alterations represent adaptive reactions in the neuron that would normally help the animal cope with aftereffects of injury. A specific adaptive hypothesis is that some of the electrophysiological and morphological reactions observed in undamaged parts of a neuron during regeneration of a peripheral branch function to ( a ) compensate for partial loss of peripheral function and (b) contribute to the defensive sensitization of a region that has been seriously injured. Both functions should be served by long-lasting enhancement of the signaling effectiveness of the partially injured neuron. Although limited adaptive interpretations have occasionally been offered for electrophysiological alterations observed after axotomy (e.g., Kuno and Llinas, 1970), the dominant view in this field has been that such alterations have little or no biological function-they are merely side effects of a profound change in state of the cell as it regenerates its axon (Baker et al., 1981; Titmus and Faber, 1990). A common form of this view is that regenerative growth requires that the neuron regress to a less differentiated growth state (e.g., Carpenter and Bergland, 1957; Watson, 1974; Gurtu and Smith, 1988; Denburg, 1989; Chiu et al., 1993). The more prolonged action potentials and smaller afterhyperpolarizations of many axotomized neurons (reviewed by Titmus and Faber, 1990) resemble early features of some developing neurons (e.g., Spitzer and Lamborghini, 1976; Fitzgerald and Fulton, 1992). In addition, differences between the excitability of the soma/dendritic region and the initial segment of the neuron, or between different classes of motor or sensory neurons, tend to be reduced by axotomy, suggesting regression to an earlier, less differentiated state (Titmus and Faber, 1990). It has been suggested that maintenance of the differentiated state of a neuron requires continuous retrograde transport of trophic substances (e.g., NGF) from the target and satellite cells along the nerve, and that axotomy

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causes dedifferentiation by interrupting the flow of trophic substances (e.g., Purves, 1988; Titmus and Faber, 1990; Wu et al., 1993; but see Singer et al., 1992; Traynor et al., 1992; Gunstream et al., 1994; Walters et al., 1994). T h e increased excitability of soma and dendritic regions, as well as hyperexcitability of the neuroma, after axotomy has been explained as a “damming effect” in which Na’ channels destined for peripheral parts of the neuron accumulate at the stump and become inserted into whatever membrane is available, including soma membrane (e.g., Titmus and Faber, 1990; Devor et al., 1993). That some electrophysiological alterations following axotomy may be byproducts of other effects, such as loss of trophic signals or damming of Na+ channels, does not mean that these “incidental” alterations are not also subject to natural selection. If one assumes that any injuryinduced effect could have been selectively reinforced during evolution if it promoted the survival of the injured animal, it follows that common changes in neuronal signaling following injury may be adaptive during injury-related behavior. The clearest test of this hypothesis would come from experiments in which (a) relatively “natural” injuries are produced (i.e., wounding of the body surface in the absence of anesthesia), (6) changes in neuronal signaling are monitored intracellularly, and (c) the neuronal alterations are correlated with simultaneously measured behavioral changes. Because of technical limitations and obvious ethical problems, this experiment has not been performed in a mammal. Instead, most studies of peripheral neuronal injury involve forms of trauma that would not occur in nature, e.g., a clean transection of a major nerve with no immediate neural activation and little or no entry of microorganisms through the wound. The total loss of a very large number of peripheral receptive fields as a consequence of major nerve transection mimics the neural consequences of the most massive injuries-injuries that are very likely to prove fatal because of hemorrhage and infection. Injuries involving large-scale loss of axons appear to be associated with an aftereffect that does not seem adaptive-degeneration and death of a significant fraction of the sensory neurons sustaining axonal damage. Various studies have reported that anywhere from 0 to 65% of sensory neurons with axons in a sectioned nerve are slowly lost after transection of their axons (reviewed by Snow and Wilson, 1991). What is needed to evaluate potentially adaptive long-term reactions of sensory and motor neurons to injury of their peripheral processes are experimental models of natural trauma that mimic injury serious enough to cause long-lasting behavioral alterations, without being so severe that extreme, abnormal, and possibly maladaptive effects are produced.

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B. PERSISTENT NEURAL SENSITIZATION IN VERTEBRATES 1. Adaptive Reactions to Axon Injury in Peripheral Neuroru Despite the limitations just listed, some interesting hints of adaptive reactions to axonal injury may be seen in various studies of axotomy. If one accepts the behavioral evidence for the adaptiveness of site-specific sensitization around a wound (see above), it follows that increases in the sensitivity and output of sensory neurons innervating an injured area will be adaptive. In addition, it might be adaptive to increase the responsiveness of motor neurons controlling muscles that withdraw or protect a badly injured body part. In an extensive review of the effects of axotomy on neuronal physiology, Titmus and Faber (1990)showed that vertebrate sensory neurons, motor neurons, and sympathetic and parasympathetic postganglionic neurons display general tendencies for the excitability of the soma to increase after axotomy. The increased soma excitability was often associated with a reduction in the amplitude and/or duration of the spike afterhyperpolarization (AHP). In sensory neurons (Gallego et al., 1987; Gurtu and Smith, 1988) and other neurons (see Titmus and Faber, 1990) the reduction of the AHP and broadening of the spike appear to be caused by depression of K + conductances, including a Ca2+-dependent K + conductance. Sensory neurons from different sources show considerable variation in their reactions to axotomy. As pointed out by Titmus and Faber, relatively excitable (based on rheobase values) cat dorsal root ganglion (DRG)cells show little additional increase in soma excitability after axotomy (Czeh et al., 1977), whereas less excitable hamster DRG cells (Gurtu and Smith, 1988) and cat cranial sensory neurons (Gallego et al., 1987; Belmonte et al., 1988) show clear increases in excitability after axotomy. A related alteration after axotomy of-sensory neurons is the appearance of spontaneous activity in normally silent sensory neurons. These spontaneous action potentials are generated within the sensory neuron somata in the DRG and in sensory neuron endbulbs in the neuroma on the stump of a transected nerve (Wall and Gutnick, 1974; reviewed by Devor, 1989; Snow and Wilson, 1991 ; see also Russell and Burchiel, 1988). Spontaneous activity generated in sensory neuron somata also occurs in the loose ligation model of neuropathic pain, beginning within a day after loosely ligating the rat sciatic nerve and persisting for at least several days (Kajander and Bennett, 1992; Kajander et al., 1992). Increased excitability of sensory neurons would normally only be adaptive if it enhanced signaling between surviving peripheral branches and the sensory neuron’s synaptic terminals. Various investigators have suggested that electrophysiological properties of the

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sensory neuron soma parallel those of the peripheral spike generating zone (e.g., Handwerker, 1991b) andlor central synaptic terminals (e.g., Kandel, 198l), but this assumption has not been tested directly in sensory neurons. Not all regions of an axotomized neuron show increased excitability; the initial segment (between the soma and the axon) often shows a decrease in excitability and a decreased safety factor for conduction (Titmus and Faber, 1990). Nevertheless, the possibility that hyperexcitability of the input and output components of a partially damaged sensory neuron would produce adaptive sensitization, combined with evidence for mechanical, chemical, and electrical hyperexcitability in sensory neuron endbulbs within neuromas on transected nerves (reviewed by Devor, 1989; Snow and Wilson, 1991), as well as evidence for adaptive, injury-induced hyperexcitability in an invertebrate preparation (see below), suggests that long-term enhancement of peripheral and central signaling in vertebrate sensory neurons may occur as a direct reaction to axonal injury. A second effect of peripheral neuronal injury that could contribute to defensive sensitization and to partial restoration of function in tissue denervated by the injury is collateral sprouting from surrounding sensory neurons. There is little evidence for functional sprouts mediating low-threshold responses (Snow and Wilson, 1991, but see Kinnman, 1987). However, collateral sprouting of myelinated and unmyelinated nociceptors has now been demonstrated many times in various species, including rats, rabbits, and humans (e.g., Weddell et al., 1941; Devor et al., 1979; Nixon et al., 1984; Doucette and Diamond, 1987; Inbal et al., 1987; Kingery and Vallin, 1989).Collateral sprouting begins within about 4 days, stops after 2-4 weeks, and usually does not completely reinnervate a region denervated by nerve transection. The sprouts appear to be displaced eventually by regenerating branches of the damaged nerve. Of great interest, given that Ca2+-dependent mechanisms in the soma may be involved in the induction of long-term neural sensitization (see below), is that the collateral sprouting of myelinated and unmyelinated nociceptors into skin on the back of the rat is activity dependent and, moreover, that the locus for the required activity appears to be in or near the sensory neuron soma. This was first shown in a study where a single application of 50 pinches to the cutaneous receptive field of the nerve containing the sprouting sensory neurons accelerated the collateral sprouting, causing significant sensitivity to pinch in the denervated field to return within 10 rather than 14 days (Nixon et al., 1984).Acceleration of sprouting was also produced by electrical stimulation of the intact nerve or skin, and it was prevented by blocking centrally conducted action potentials with tetrodotoxin. Similar precocious expansion of C-

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polymodal nociceptors into a denervated field in the same preparation was observed after noxious heating of the innervated skin (Doucette and Diamond, 1987; see also Pertovaara, 1988). Interestingly, collateral sprouting but not regenerative sprouting of the damaged branches requires NGF, since the former but not the latter was inhibited by treatment with an antiserum to NGF (Diamond et al., 1987). A third effect of peripheral neuronal injury that could contribute to defensive sensitization was suggested by the finding that about a week after damaging the nerve innervating a rabbit ear, 20% of the surviving C-polymodal nociceptors became excitable by sympathetic stimulation or norepinephrine (NE) application (Sato and Perl, 1991).T h e activation of these nociceptors occurred at the sensory terminals rather than the site of nerve injury. C-polymodal nociceptors normally are not activated by sympathetic stimulation or NE. Indeed, sympathetic stimulation and NE suppress the incremental sensitization of these nociceptors to repeated application of noxious heat. Following nerve injury, these investigators found not only that sympathetic stimulation activated C-polymodal nociceptors, but that the suppression of thermal sensitization by sympathetic stimulation was abolished. These effects suggest that long-term behavioral sensitization can involve a dramatic chemical sensitization in which nociceptors become sensitive to the release of neuromodulators that they are normally insensitive to. The dependence of some forms of long-lasting pain and hyperalgesia in humans on the sympathetic innervation of the afflicted area has long been recognized in the clinical conditions of causalgia and reflex sympathetic dystrophy. Campbell and colleagues ( 1992) have suggested that the most troublesome consequence of the acquired capacity of N E to activate nociceptors is a central sensitization (see below) that causes innocuous stimuli to become painful (i.e., sympathetic stimulation becomes capable of exciting nociceptors, which, in turn, trigger central allodynia). Not surprisingly, peripheral sensitization and injury-induced collateral and regenerative sprouting of sensory neurons have been associated with a large number of potential molecular signals. For example, the growth-associated protein GAP-43 increases in sensory neuron somata after sciatic crush and is transported to both peripheral and central terminals (Hoffman, 1989; Woolf et al., 1990). Nerve section induces a dramatic increase in the number of N O synthase mRNA-positive cells in the DRG, suggesting an involvement of NO in the reactions of sensory neurons to axotomy (Verge et al., 1992).Axotomy also increases ornithine decarboxylase activity in DRG cells (Wells, 1987; Soiefer et al., 1988) and the expression of various transcription factors, including Jun and Jun D, and the cyclic AMP response element binding protein CREB (Jenkins

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and Hunt, 1991; Leah et al., 1991; Herdegen et al., 1992). There has apparently been very little investigation of possible roles of cyclic AMP, Ca2+,and other second messengers in the initial induction of long-term growth and cellular sensitization of vertebrate sensory neurons.

2 . Central Sensitization Mechanisms Woolf (1983) obtained some of the first strong hints that relatively long-lasting neural sensitization in the spinal cord can be produced by peripheral tissue injury. He found in decerebrate rats that thermal injury to one foot caused an expansion of the receptive fields for activating alpha motor neurons innervating that limb, and that expansion of the receptive field to the contralateral paw persisted when local anesthetic was injected into the injured foot to block sensory activity coming from the injury. The sensory block was made 1 h after the injury, but the motor neurons were only tested for 15 min after the block. Although these results did not demonstrate central neural sensitization lasting longer than 15 min, they helped to stimulate a rapidly growing number of investigations into long-term central sensitization. For example, Coderre and Melzack (1987) found in the rat that 24 h after thermal injury to one foot the latency for withdrawal of the injured foot was dramatically reduced and the withdrawal latency for the contralateral foot showed a smaller but significant decrease. The expression of the long-term facilitation of the contralateral withdrawal response was not blocked by anesthetizing the foot 15 min before testing. However, the facilitation was prevented by spinal anesthesia applied a few minutes after the injury, suggesting that early activation of neurons in the spinal cord is critical for long-lasting sensitization. Evidence has also been obtained for longlasting central sensitization produced by mechanical injury. Laird and Cervero (1989) found that noxious 2-s pinches delivered to the rat tail caused large expansions of the mechanosensory receptive fields of widedynamic-range interneurons (but not of high-threshold interneurons) in the dorsal horn that appeared to persist for an hour or longer. In many of these cells the threshold for responses to mechanical stimulation decreased. Although these authors did not try to block potentially altered sensory input, the large degree of expansion of the receptive fields was taken as evidence that the underlying mechanisms were central rather than peripheral. Many clinical observations (including phantom limb pain) are also consistent with long-lasting neural sensitization within the CNS (see review by Coderre et al., 1993). Much of the recent research into long-term central sensitization has focused on alterations produced in experimental models of cutaneous and muscular inflammation (e.g., ladarola et al., 1988a), and peripheral

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neuropathy (especially the loose ligation model developed by Bennett and Xie, 1988).Studies of articular inflammation have also demonstrated central sensitization (e.g., Schaible et al., 1987; Dougherty et al., 1992b), but these studies lie outside the scope of this chapter. Inflammation of the hindlimb of the rat produced by subcutaneous injection of Freund’s adjuvant caused an increase in the responsiveness of dorsal horn interneurons to peripheral stimulation, an increase in their background activity, and enlargement of their receptive fields (in the absence of a change in the receptive field size of primary sensory neurons) that lasted for at least 5 days (Hylden et al., 1989). Partial nerve injury produced by Ioose ligation of the rat sciatic nerve (selectively injuring the myelinated afferents) caused an increase in the responsiveness of dorsal horn interneurons to innocuous mechanical stimuli (but not to noxious mechanical stimuli), an increase in their background activity, and the generation of afterdischarges to mechanical and thermal stimuli (Palecek et al., 1992a). Unlike injury to skin and muscle (see above) or inflammation of the hindlimb, the partial nerve injury did not produce changes in the receptive fields of dorsal horn interneurons. Nerve injury produced by tight ligation of a spinal root in the monkey had similar effects on the responses of dorsal horn interneurons, increasing their responsiveness to peripheral stimulation (mechanical, heat, and cooling), and increasing their background activity (Palecek et al., 199213). The long-lasting enhancement of responsiveness observed in dorsal horn interneurons following peripheral injury, peripheral tissue inflammation, and partial nerve injury could be due to one or more of several basic mechanisms, including: (1) sustained excitation by continuing, high levels of activity in primary sensory neurons (in this case the alterations expressed by central interneurons would be due to peripheral sensitization), (2) persistently decreased inhibition (due to either central or peripheral changes), ( 3 )enhanced excitability of the dorsal horn interneurons (central cellular sensitization), and (4) presynaptic facilitation of synapses from primary sensory neurons or excitatory interneurons (causing network sensitization). As discussed below, each of these mechanisms might make a contribution to long-term, central sensitization in mammals, at least under some conditions. T h e first potential mechanism for enhancing central responsiveness to phasic peripheral inputs is tonic excitation from afferents. A role for continuing afferent input after peripheral injury is supported by observations of increased spontaneous activity in primary sensory neurons following subcutaneous injection of the inflammatory agent carrageenan (Kocher et al., 1987) and continuing spontaneous activity generated in sensory neuron somata following loose ligation of the rat sciatic nerve

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(Kajander and Bennett, 1992; Kajander et al., 1992).Behavioral sensitization associated with continuing afferent activity may depend on activation of central NMDA receptors by excitatory amino acids released from sensory neurons. Behavioral sensitization due to inflammation o r partial nerve injury is blocked by repeated application of NMDA antagonists (Seltzer et al., 1991b; Mao et al., 1992a; Ren et al., 1992; Yamamoto and Yaksh, 1992). In the case of partial nerve injury produced by loose nerve ligation, NMDA antagonists block not only the induction of sensitization but also its maintenance (tested 3 days after onset of the ligation) (Ma0 et al., 1992b). Similar effects were shown in the decerebrate rat with peripheral chemical injury. Administration of NMDA antagonists 30 min after applying mustard oil to the foot rapidly reversed the facilitation of the hindlimb flexion reflex, whereas pretreatment with the antagonists largely prevented the development of this facilitation (Woolf and Thompson, 1991). Evidence that continuing high levels of sensory input maintain central alterations causing allodynia has also been found in human patients with reflex sympathetic dystrophy (Gracely et al., 1992). A second potential mechanism contributing to long-term central sensitization after peripheral injury is disinhibition. Evidence for a decrease in inhibition of defensive responses has come from studies of partial nerve injury produced by loose ligation. This partial nerve injury preferentially destroys myelinated fibers (e.g., Basbaum et al., 1991; Coggeshall et al., 1993), which might result in a loss of inhibitory effects that are normally mediated by the large fibers (Melzack and Wall, 1965; Melzack and Wall, 1983). However, behavioral sensitization is not closely correlated with the time course of destruction of the unmyelinated fibers (Coggeshall et al., 1993). Disinhibition might also occur if inhibitory interneurons are damaged or destroyed after peripheral injury. Loose ligation nerve injury has been shown to produce trans-synaptic degeneration of small, presumably inhibitory interneurons in the dorsal horn under some conditions (Sugimoto et al., 1988).This effect was particularly dramatic when amplified by subconvulsive doses of an agent, strychnine, which itself causes disinhibition but no neuronal damage. This led Sugimoto and colleagues ( 1988) to propose that continuing spontaneous activity in surviving C-polymodal nociceptors caused prolonged depolarization that was sufficient to cause excitotoxicity and death in small inhibitory interneurons. It was later shown that partial nerve injury by loose ligation decreased extracellularly recorded potentials associated with primary afferent depolarization and presynaptic inhibition (Laird and Bennett, 1992).These data suggest that small inhibitory interneurons mediating presynaptic (and perhaps postsynaptic) inhibition are particularly

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vulnerable to excitotoxicity. It will be interesting to see whether such persistent disinhibitory effects also occur with other, more natural forms of injury. T h e third potential mechanism for persistently enhancing central responsiveness to peripheral inputs is long-term cellular sensitization expressed as an enhancement of the intrinsic excitability of dorsal horn interneurons. Although neither an enhancement of electrical excitability nor chemical excitability (postsynaptic facilitation) has yet been demonstrated with intracellular techniques in dorsal horn interneurons during long-term central sensitization, such changes seem likely to be involved. One reason is that prolonged postsynaptic facilitation is a notable consequence of intense afferent activity in other parts of the brain, notably the hippocampus during long-term potentiation. One possible intrinsic alteration involves the opioid peptide dynorphin, which may produce postsynaptic facilitation (as well as presynaptic facilitation, see below) of dorsal horn interneurons (see Dubner and Ruda, 1992). Thus, a prolonged enhancement of dynorphin release (a presynaptic effect) might cause a continuing postsynaptic facilitation of dorsal horn interneurons that relay nociceptive input. Dynorphin is found in local circuit and projection interneurons in regions of the dorsal horn involved in pain transmission (Nahin et al., 1989). Within 4 h of injecting an inflammatory agent into a rat hindpaw, messenger RNA for dynorphin increases in parts of the ipsilateral dorsal horn receiving direct sensory input from the inflamed paw and remains elevated for 10 to 14 days (Iadarola et al., 1988a,b). The dynorphin peptide levels increase in the same region within 2 days (Iadarola et al., 1988a); reviewed by Dubner and Ruda, 1992). Dynorphin immunoreactivity in dorsal horn also increases following high-frequency activation of unmyelinated sensory neurons (Hutchinson et al., 1990) and partial nerve injury produced by loose ligation (Draisci et al., 1991). A potential contribution of the increased dynorphin levels to central sensitization has been suggested by observations of expanded dorsal horn neuron receptive fields and increased neuronal responsiveness to peripheral cutaneous test stimuli following spinal administration of exogenous dynorphin or an agonist that binds to the same kappa-opiate receptor (Hylden et al., 1991). A large fraction of dorsal horn interneurons showing increases in dynorphin gene expression exhibit Fos protein in their nuclei shortly after the onset of inflammation (K. Noguchi et al., 1991). Fos is a transcriptional regulator encoded by the immediate early gene c-fos and is expressed in dorsal horn cells within 30 min of the onset of inflammation. T h e activation of c-fos in spinal dorsal horn has also been seen in other studies (e.g., Hunt et al., 1987;

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Menetrey et al., 1989; Williams et al., 1990a,b). Because c-fos is activated by Ca2+ (Morgan and Curran, 1991), it has been suggested that Ca2+ influx through NMDA receptor-gated channels may be important for regulating transcription in dorsal horn neurons, as it has been shown to be in other parts of the brain (see reviews by Dubner and Ruda, 1992; Coderre et al., 1993). A fourth general mechanism that may contribute to long-term central sensitization is presynaptic facilitation of sensory neuron synapses in the spinal cord. Because of the difficulty in examining synapses in the dorsal horn with electrophysiological techniques, no direct evidence for presynaptic facilitation has been reported using intracellular recording methods following peripheral injury. Nevertheless, indirect evidence for presynaptic alterations has come from recent electrophysiological and morphological studies. Following high frequency afferent stimulation, glutamate and aspartate release into the spinal slice perfusate increases (Rusin et al., 1993). Furthermore, if transmitter release were increased after LTP, one would expect all pharmacologically isolated components of the potentiated EPSP to be increased and, indeed, both the AMPA receptormediated EPSP and the NMDA receptor-mediated components are potentiated (Randic et al., 1993). Recent morphological alterations in the dorsal horn following experimental nerve transaction suggest that longterm synaptic facilitation, involving the growth of new terminals, might occur after peripheral injury. Although the most obvious effect of sectioning a major nerve is often degeneration and death of some of the sensory neurons (see Section VII ,A), several reports have described apparent sprouting of primary sensory neuron terminals into partially denervated regions of the spinal cord after nerve section or interruption of retrograde transport from the periphery. However, the short distances over which such sprouting was thought to occur, combined with the limited resolution of the assays used, made these claims of anatomical plasticity controversial (see reviews by Snow and Wilson, 1991; MacMahon, 1992). More recently, Woolf and colleagues (1992) found convincing evidence for sprouting of central sensory neuron terminals after nerve injury in the adult rat by taking advantage of the highly ordered topographic distribution of these terminals to discrete laminae of the dorsal horn. Six to 9 weeks after sectioning and ligating a sural nerve they injected horseradish peroxidase (HRP) into single sensory neuron axons (myelinated, low-theshold, AP fibers that would normally innervate hair follicles). T h e terminal arbors of these cells showed several differences from controls, including a broader branching pattern and penetration of branches into lamina I1 of the dorsal horn, which contains high-

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threshold interneurons that normally receive monosynaptic connections from C-polymodal nociceptors. Similar results were obtained when an HRP-lectin conjugate selective for myelinated afferents was applied to the whole nerve. These findings suggested that allodynia following peripheral nerve injury might be due to the formation of novel synaptic connections between low-threshold sensory neurons and high-threshold dorsal horn interneurons. Recent evidence also suggests that fine afferents (likely to be nociceptors) sprout the other way-from lamina I1 into deeper laminae after nerve section (Cameron et al., 1992). Both sets of findings indicate that sensory neurons can grow new synaptic terminals after peripheral injury. Furthermore, they raise the possibility (which will be more difficult to demonstrate) that additional synaptic terminals can be added to already established targets, thereby facilitating existing synaptic connections. Growth of new terminals onto new and existing targets may be promoted by the increase in GAP-43 observed in central terminals and growth cone-like structures of axotomized sensory neurons (Woolf et al., 1990; Coggeshall et al., 1991). Recent evidence also suggests that NGF may play an important role in central hyperalgesia. NGF levels increase dramatically at a site of peripheral injury or inflammation (Heumann et al., 1987), and enhanced retrograde transport of NGF occurs (Donnerer et al., 1992). Enhanced peripheral NGF levels can produce hyperalgesia (Lewin et al., 1993), perhaps by upregulating the synthesis of neuropeptides that are released at central terminals of primary nociceptors (Donnerer et al., 1992; Lewin and Mendell, 1993). Many of the features associated with short- and long-term nociceptive sensitization in the spinal cord (e.g., the involvement of NMDA receptors, PKC, and NO) are similar to features that have been described elsewhere in the mammalian nervous system, most notably in LTP in the experimentally favorable hippocampal slice. Thus, some clues about mechanisms that might contribute to nociceptive sensitization may come from developments in the rapidly moving area of LTP research. Several recent findings in the hippocampus that have apparently not yet been explored in the spinal cord as potential mechanisms of sensitization include an enhancement by histamine of postsynaptic sensitivity to glutamate in the absence of a change in electrical excitability (Bekkers, 1993), the involvement of cyclic AMP in a late stage of LTP (Frey et al., 1993), a requirement for Ca2'-calmodulin-dependent kinase I1 (e.g., Malenka et al., 1989; Malinow et al., 1989; Silva et al., 1992) and tyrosine kinase in LTP (Grant et al., 1992), and the involvement of the gases N O and CO in activity-dependent presynaptic facilitation during LTP (e.g., Zhuo et al., 1993).

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C. PERSISTENT NEURAL SENSITIZATION IN INVERTEBRATES Both peripheral and central alterations have been implicated in longterm reactions to bodily injury in invertebrates. 1. Peripheral Alterations There has been very little explicit study of the effects of peripheral injury on neuronal properties in invertebrates. In Aplysia, a cut injury on one side of the tail, or a 90-s sequence of strong shock to the center of the tail caused a decrease in the mechanosensory threshold of widedynamic-range VC sensory neuron receptive fields overlapping the traumatized site, and this increased sensitivity was present 1 to 3 weeks after the trauma (Billy and Walters, 1989a). In addition, the same receptive fields expanded their area by about 50%, suggesting that the injury triggered growth of the peripheral sensory arbor. The expansion of receptive fields in an injured region could contribute to site-specific sensitization by increasing the overlap of receptive fields and thus the number of sensory neurons activated by a given stimulus (Billy and Walters, 1989a). Although this type of experiment has apparently not been reported in other invertebrates, some evidence in the leech suggests that growth of peripheral receptive fields can occur under some conditions that might be similar to those produced by severe injury. Blackshaw and colleagues (Blackshaw et al., 1982) found that destruction of 3 and 4 N sensory cells within a ganglion (by intracellular injection of pronase) caused the receptive field of the surviving N cell to expand into the denervated area. Injury of peripheral axons of N and T cells by crushing or cutting nerves caused exuberant sprouting of the injured cells at the injury site and within the central ganglion (Bannatyne et al., 1989). Evidence from both molluscs (e.g., Rehder et al., 1992; Ziv and Spira, 1993) and insects (e.g., Spira et al., 1987) suggests that Ca2+influx is an important early signal for triggering regrowth of axon tips and, perhaps, sprouting neurites. The capacity for sprouting and regeneration of peripheral and central neuronal processes is quite common in the animal kingdom (see review by Hulsebosch and Bittner, 1980) and is probably especially well developed in groups such as amphibians and gastropod molluscs that can regenerate entire appendages (e.g., Aguilar et al., 1973; Chase and Kamil, 1983; Moffett and Snyder, 1985). Although molluscs and annelids have centrally located mechanosensory neurons such as the LE, VC, T, P, and N cells, they also have numerous, small, arthropod-like sensory neurons that have their somata located in the periphery, near the receptive field (e.g., Janse and Swigehem, 1975; Peinado and Zipser, 1987;

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Xin et al., 1992). Peripheral sensory neurons may increase in number during adult life in animals that continue to grow and increase their surface area (e.g., Peinado and Zipser, 1987; Purves, 1988). It has been shown that, in contrast to central sensory neurons in Aplysia and mammals, peripheral mechanosensory neurons in the cricket show a loss rather than gain of excitability following axonal injury (Chiba and Murphey, 1991). These observations, and the likelihood that the exposed peripheral sensory neuron soma will often fail to survive serious injury to its nearby receptive field, suggest that adaptive reactions to local injury involving peripheral sensory neurons may rely more on an addition of new neurons than a cellular sensitization of surviving peripheral sensory neurons. 2. Central Alterations Persistent central alterations associated with defensive sensitization and peripheral injury have only been reported in a few invertebrates. With the exception of work on Aplysia, these reports represent isolated findings that have not yet been closely linked to either behavioral function or molecular mechanisms. One example is a report that noxious stimulation causes persistent depolarization of “command” interneurons controlling pneumostome closure in a snail (Beregovoi et al., 1988). A more extensive literature exists on the effects of axotomy on neural excitability, which is reviewed briefly in Section VII,B,4. Another example of a finding of potential significance for mechanisms of nociceptive sensitization was reported by Bulloch (1984). He found that a small incision in the body wall of anesthetized snails, which caused some blood loss, triggered transient (lasting only about 3 days) sprouting of distant undamaged central neurons. Mechanisms of long-term nociceptive sensitization have been studied systematically in Aplysia calzfornica. Indeed, work on nociceptive sensitization in this species has yielded what is perhaps the most detailed mechanistic understanding of long-term memory in any animal. a. Persistent Physiolopal Alterations in Aplysia Sensory Neurons following Sensitizing Signals. The physiological alterations in Aplysia sensory neurons that have been associated with long-term general sensitization and site-specific sensitization are similar to the changes that have been described during short-term sensitization (see Section V1,C). A synaptic correlate of long-term general sensitization was found 1 day after the delivery of 64 intense shocks to the tail or neck (spread over 4 days): the amplitude of monosynaptic EPSPs from LE siphon sensory neurons to a gill motor neuron was doubled (Frost et al., 1985). Similar synaptic facilitation was found 24 h after five repeated 5-min applications of

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serotonin to dissociated LE siphon sensory neurons and a gill motor neuron in culture (Montarolo et al., 1986). Quanta1 analysis of long-term synaptic facilitation in pairs of sensory and motor neurons in dissociated culture indicated that this facilitation is due to a presynaptic enhancement of transmitter release (Dale et al., 1988). One day after cutaneous shock delivered to the intact animal in a site-specific sensitization procedure, EPSPs from tail (VC) sensory neurons to tail motor neurons were facilitated; in this case, repeated shocks were delivered for only 90 s, but the shocks were delivered to the receptive fields of the tested neurons (Walters, 1987b). Pairing of intracellular activation with noxious shock outside the receptive field of tail sensory neurons also produced synaptic facilitation lasting at least 1 day (Buonomano and Byrne, 1990). In addition, site-specific training and testing protocols revealed a decreased threshold for initiating an action potential in the VC sensory neuron soma and a large increase in the likelihood of generating afterdischarge following the initiation of a soma action potential (Walters, 1987b), as well as a decrease in an outward current in the soma resembling the S-type K + current (Scholz and Byrne, 1987). As with short-term sensitization, all the activity-dependent alterations would be expected to contribute to site-specific sensitization of an injured region. In addition to activitydependent heterosynaptic facilitation, increasing evidence points toward long-term, activity-dependent, homosynaptic facilitation in nociceptive pathways in Aplysia. Ten intracellularly driven bursts at 5-s intervals in a single tail (VC) sensory neuron can produce LTP lasting 1-2 h (Walters and Byrne, 1985). Although this mechanism would appear to be homosynaptic (dependent only on activation of the synapse showing the potentiation), the possibility that the activated sensory neuron triggered recurrent release of extrinsic modulators from postsynaptic targets could not be ruled out in this preparation. Conclusive evidence for homosynaptic LTP at Aplysia sensorimotor synapses was recently reported by Lin and Glanzman (1993), who showed in dissociated cell cultures lacking interneurons or other sources of modulatory factors that LTP was produced by high-frequency stimulation of the sensory neurons. They then showed that the LTP in culture depends on postsynaptic depolarization for its induction; injection of hyperpolarizing current into the motor neuron prevented the potentiation (Lin and Glanzman, 1994a). The critical difference between these experiments and earlier ones that failed to find LTP in culture (Schacher et al., 1990) was probably the higher frequency and greater number of sensory neuron spikes initiated during successful LTP induction. Of great interest in this regard is the recent evidence reported by Dale and Kandel ( 1993) indicating that glutamate

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40 1

is the neurotransmitter released by the sensory neurons and that both the channel structure and the transmitter recognition site on the postsynaptic cells may have properties resembling those of the NMDA receptor-gated channels in vertebrates. A primitive origin for NMDA receptor-gated channels is also supported by observations of similar channels in visual interneurons of crayfish (Pfeiffer-Linn and Glantz, 1991). Lin and Glanzman (1994b) found that LTP of Aplysia sensorimotor synapses is significantly reduced by an NMDA receptor antagonist, and completely blocked by postsynaptic injection of a Ca2+chelator. It is not yet known if Ca2+ influx in the motor neuron causes the production of nitric oxide, but recent reports of nitric oxide synthase in the CNS of Aplysia and other molluscs (e.g., Lukowiak et al., 1993; Winlow et al., 1993) support this possibility. Taken together, these unexpected results suggest that LTP mechanisms in nociceptive pathways of Aplysia may be closely related to LTP mechanisms in the mammalian hippocampus and spinal cord. b. Persislent Morphologwal Alterations in Aplysia Sensory Neurons Produced by Cutaneous Shock. T h e most extensive analysis of central mechanisms associated with long-term defensive sensitization has been on the LE and VC wide-dynamic-range sensory neurons of Aplysia. The first observations of long-term central changes in sensory neurons were morphological. Bailey and Chen (1983) found that 2 days following either 4 or 10 days of noxious, repeated cutaneous shock, the number, size, and vesicle complement of active zones from LE sensory neurons were larger in the abdominal ganglion of animals showing long-term general sensitization than in unshocked controls. This training also produced an increase in the total number of synaptic varicosities (Bailey and Chen, 1988a) and in the number of presynaptic contacts onto an identified motor neuron (Bailey and Chen, 1988b). The increased number of varicosities and active zones lasted 1-3 weeks, whereas the active zone size and vesicle complement returned to normal within a week (Bailey and Chen, 1989). In dissociated cell culture repeated applications of serotonin to sensory and motor neurons were found to produce long-term synaptic facilitation (see below). Under these conditions parallel growth of sensory neuron neurites and varicosities occurred, which depended on the presence of an appropriate postsynaptic target cell (Glanzman et al., 1990). An early stage of this growth appears to involve the internalization and degradation of Aplysia cell adhesion mole-cules (“ApCAMs,” which are related to mammalian neural cell adhesion molecules or NCAMs) localized on the sensory neuron membrane (Bailey et al., 1992a and see below).

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3 . Role of Protein Synthesis in the Induction and Maintenance of Long-Term Cellular Sensitization Given the profound morphological changes in wide-dynamic-range sensory neurons of Aplysia associated with nociceptive sensitization, it would be expected that alterations in protein synthesis would occur. Indeed, long-term synaptic facilitation in cultured Aplysia neurons is associated with several shifts in protein synthesis, including a very early increase, followed by a slight decrease, and a later increase in overall synthesis (Barzilai et al., 1989). Changes in synthesis of a number of specific proteins have been described following application of serotonin or cyclic AMP (see below) (Castellucci et al., 1988; Barzilai et al., 1989; Eskin et al., 1989) or nerve stimulation (Noel et al., 1991), and a few of these proteins have now been identified. One of the proteins that is down-regulated shortly after prolonged application of serotonin is an ApCAM (see above), which appears to be homologous with mammalian and arthropod cell adhesion molecules (Mayford et al., 1992). The reduction of ApCAM levels may relieve an inhibition on growth of neurites and new synapses. One of the late proteins whose synthesis is enhanced is calreticulin, the major Ca*+-binding protein within the endoplasmic reticulum (Kennedy et al., 1992). Another newly identified protein is the Aplysia homolog of BiP, a “heat shock protein” or “stress protein” found in the endoplasmic reticulum, where it is involved as a “chaperone” in the folding and assembly of secretory and membrane proteins (Kuhl et al., 1992). Behavioral training increased the steady-state level of BiP mRNA and the synthesis of BiP protein within 3 h. The folding of proteins and assembly of protein complexes are likely to be necessary for structural changes involved in long-term memory (Kuhl et al., 1992). Increased synthesis of a heat shock protein has also been reported in the somata of mammalian neurons shortly after an injury-related procedure: peripheral axotomy (New et al., 1989). It will be interesting to see whether sensitization, reactions to axotomy, and learning involve common “stressrelated” proteins. The very early increase in protein synthesis during sensitization is of considerable interest because it may reflect, in part, the synthesis of immediate-early gene products (transcription factors) that in turn stimulate late-onset gene expression necessary for producing long-term alterations in the sensory neuron (Barzilai et al., 1989). Consistent with this possibility, Montarolo and colleagues (Montarolo et al., 1986) found that long-term but not short-term facilitation of sensorimotor synapses produced by repeated serotonin application in culture was selectively prevented by the presence of inhibitors of RNA and protein synthesis during

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and shortly after serotonin application. Protein synthesis during such “training” (but not several hours afterward) was also shown to be necessary for ( a ) long-term, tail-shock-induced sensitization of the gillwithdrawal reflex in an isolated reflex preparation containing the tail, siphon, and gill (Castellucci et al., 1989);( 6 ) long-term, serotonin-induced changes in excitability of the soma of VC sensory neurons in dissociated cell culture (Dale et al., 1987); ( c ) long-term facilitation of sensorimotor connections produced by prolonged application of a cell-permeable cyclic AMP analog in dissociated cell culture (Schacher et al., 1988 and see below); and ( d ) long-term increases in the number of sensory neuron varicosities in dissociated cell culture (Bailey et al., 1992b). As described below, specific transcription factors involved in the induction of longterm sensitization that have been examined thus far are regulated by the cyclic AMP cascade. 4. Role of Cyclic A M P in the Induction and Maintenance of Long-Term Cellular Sensitization A prolonged elevation of cyclic AMP in the cell soma has been shown to be a primary signal for inducing long-term cellular sensitization in Aplysia sensory neurons in both the intact nervous system and dissociated cell culture. In the whole animal, injection of cyclic AMP into the soma of pleural VC sensory neurons resulted in a reduction of a current resembling the S-type K + current 1 day later (Scholz and Byrne, 1988). Injection of cyclic AMP also caused morphological alterations within 24 h, doubling the number of central synaptic varicosities in the VC sensory neurons (Nazif et al., 1991). In dissociated cell culture, a membrane-permeant cyclic AMP analog caused long-term (24 h) facilitation of sensorimotor connections (Schacher et al., 1988). Using imaging techniques in culture, it was found that prolonged serotonin application increases cyclic AMP levels throughout the sensory neuron and causes a detectable translocation of the catalytic subunit of PKA into the nucleus (Bacskai et al., 1993). Dash et al. (1990) discovered an important link between the elevation of cyclic AMP and the dependence of long-term synaptic facilitation on protein synthesis. They found that Aplysia sensory neurons contain a pr-otein similar to mammalian transcription factors that are regulated by PKA and increase transcription by binding to the cyclic AMP-responsive element (CRE) in DNA. T h e importance of this pathway for nociceptive memory in Aplysia was shown by the ability of excess CRE sequence, microinjected into the sensory neuron nucleus, to prevent long-term synaptic facilitation in culture, presumably by binding the endogenous

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CRE-binding transcription factors. Recently, similar experiments have implicated a necessary role in long-term facilitation for another cyclic AMP-dependent transcription factor previously found in mammals, a member of the C/EBP immediate-early gene family (Alberini et al., 1994). Direct evidence for transcriptional activation of CRE-binding factors was reported by Kaang and colleagues (Kaang et al., 1993), who used gene transfer into individual sensory neurons to show that serotonin activates a reporter gene driven by the CRE and that this induction requires CREbinding proteins. It seems likely that the CRE drives the transcription of immediate-early genes (including C/EBP) and, thus, the synthesis of regulatory proteins that are transiently expressed in sensory neurons following prolonged application of serotonin or cyclic AMP (Barzilai et al., 1989; Eskin et al., 1989). The cyclic AMP pathway plays a role not only in the induction of long-term sensitization but also in maintaining it for many hours after induction. This possibility was suggested by the observation that a single application of either serotonin or a cell-permeable cyclic AMP analog to isolated clusters of pleural sensory neurons caused the phosphorylation of 17 substrate proteins, which was not dependent on transcription or translation (Sweatt and Kandel, 1989). Repeated or prolonged application of either serotonin or the cyclic AMP analog resulted in long-term (24 h) phosphorylation of the same 17 proteins, but in contrast to the short-term phosphorylation, the long-term phosphorylation required transcription and translation. The possible importance of at least some of these phosphorylated proteins was suggested by the finding by Montarolo and colleagues (1992) that application of a specific PKA inhibitor (RpCAMPS)reduced synaptic facilitation in culture 1.5 and 12 h after repeated serotonin application (although, curiously, this study did not find an effect at 24 h). An explanation for both the long-lasting phosphorylation and the continuing dependence of synaptic facilitation on PKA activity was suggested by the observation that PKA activity could be persistently enhanced by a decrease in the amount of regulatory subunits relative to the catalytic subunits of the enzyme following repeated cutaneous shock in the intact animal (Greenberg et al., 1987). It was later found that the loss of regulatory subunits was evident 24 h after prolonged exposure to serotonin or a cell-permeable cyclic AMP analog in isolated pleural VC sensory clusters, and that this loss depended on protein synthesis (Bergold et al., 1990). T h e loss of regulatory subunits was due to accelerated proteolysis rather than reduced synthesis (Bergold et al., 1992). Interestingly, the proteolysis was found to occur in an ATPubiquitin-proteasome-dependent pathway (Hegde et al., 1993), which

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also degrades other proteins that control cell growth, such as the immediate-early gene product, c-Fos. 5. Central Alterations of Invertebrate Neurons Produced by Axonal Injury and Peripheral ‘7nflammatio.n” An unexpected finding in Aplysia, given the evidence that serotonin and spike activity are important signals for inducing long-term alterations in sensory neurons during sensitization, was that the same alterations can be triggered under conditions in which neither serotonin (or other neurotransmitters) nor spike activity are likely to contribute. Pedal nerves containing axons of the wide-dynamic-range VC sensory neurons were crushed in the intact animal while it was cooled to 0°C and infused with a high concentration of Mg*+ ions to block synaptic transmission and most spike activity (Walters et al., 1991). Under these conditions there were no signs of either behavioral o r neural sensitization for about 2 days. However, beginning within 3 days, and persisting for 1-2 months, the sensory neurons showed robust cellular sensitization (Walters et al., 1991; Dulin and Walters, 1993). This was expressed as decreases in action potential threshold, accommodation, and afterhyperpolarization, and increases in action potential duration, afterdischarge, and synaptic transmission. In addition, axonal injury induces growth of new sensory neuron neurites within the CNS (Dulin and Walters, 1993; Dulin et al., 1994). The similarity of these effects to those produced in LE and VC sensory neurons during general sensitization, site-specific sensitization, and aversive classical conditioning suggested that slow signals carried by retrograde axonal transport from the site of axonal injury (Gunstream et al., 1994) activate a common molecular program for cellular sensitization and growth. By this hypothesis, the same program can be activated after a delay by slow axonal signals and rapidly by extrinsic neuromodulators such as serotonin or by interactions of neuromodulator effects with intrinsic spike activity (e.g., ADEM) (see also Walters, 1992). Long-term cellular sensitization following axonal injury is not unique to defensive mechanosensory neurons. T h e finding that similar alterations occur in cerebral mechanosensory neurons (Clatworthy and Walters, 1994) suggested this plasticity is not restricted to cells primarily having defensive functions, since a substantial fraction of this cerebral sensory population is likely to be involved in feeding behavior (e.g., Rosen et al., 1989). It was also discovered that delayed hyperexcitability following axon injury is not restricted to sensory neurons. Soma hyperexcitability occurred within a few days of nerve crush in the giant mucus gland motor neuron R2, and within about a week in pedal motor neurons (Dulin and Walters,

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1992). Interestingly, in R2 nearby axotomy has been shown to alter rapidly the synthesis of several nuclear proteins (Buriani et al., 1990). One of the up-regulated proteins found in this study bears interesting similarities to CRE-binding proteins (see preceding section). Peripheral injury in invertebrates, as in vertebrates, results in “inflammatory” or “immune” responses that function to protect the animal from microorganisms entering through a wounded body surface (e.g., Roitt et al., 1989). An interesting possibility is that signals such as cytokines released from immunocytes or microglia (Masuda-Nakagawa et al., 1993) attracted to an injured site may be recognized by nearby neurons and used to trigger long-term adaptive reactions to bodily injury in the recipient neurons. This possibility was supported by the finding that an inflammatory reaction induced by implanting a foreign body (cotton string) next to a peripheral nerve in Aplysia caused the same set of sensitizing alterations in wide-dynamic-range VC sensory neurons that has been associated with general sensitization, site-specificsensitization, associative learning, and axonal injury in these cells (Clatworthy et al., 1994). The changes occur in the absence of detectable axonal injury, and after a very long latency (about a week), during which time the foreign body and nerve become covered with a thick capsule containing numerous immunocytes (Clatworthy et al., 1994). Immunocytes have not yet been investigated in Aplysia, but in the bivalve mollusc Mytilus edulis immunocytes are attracted to the cut ends of transected nerves, possibly in response to the release of opiate-like neuropeptides (Stefan0 et a!., 1989).Of particular interest is that some of these immunocytes appear to contain serotonin, which is an important extracellular signal for inducing long-term cellular sensitization in Aplysia sensory neurons (see above). The central effects of peripheral axon injury have also been examined in T and N sensory neurons of the leech (Bannatyne et al., 1989). Axotomy induced sprouting of neurites within central ganglia and connectives (as well as peripherally-see preceding section). I n addition, a tendency for the duration of the spike afterhyperpolarization in the soma to decrease was noted. It would be interesting to test the effects of axotomy on repetitive firing properties, such as spike accommodation, but these properties were not examined. It would also be interesting to examine effects of axotomy on the P cells, which seem most similar functionally to the wide-dynamic-range sensory neurons in Aplysia that are altered by axonal injury. Axotomy has been shown to increase the soma excitability of another neuron in the leech, the “anterior Pagoda” cell, which shows increases in spike height, spike duration, and input resistance after nerve section (Pellegrino et al., 1984). The increase in

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soma excitability following nerve crush was reversed after axonal regeneration (Matteoli et al., 1986). Increased soma excitability has also been described in a number of insect and crustacean motor neurons and interneurons. These cells normally have nonspiking somata, but express overshooting spikes for several days or weeks after peripheral axotomy (Pitman et al., 1972; Goodman and Heitler, 1979; Kuwada and Wine, 1981; Roederer and Cohen, 1983). In each case the alterations were consistent with an increased density of Na' channels in or near the soma, perhaps due to accumulation by a damming effect (see review by Titmus and Faber, 1990, and Section VII,A). Although the inducing signals are not yet known, it has been suggested that increased Ca2+entry into the soma is an important signal for triggering both the soma hyperexcitability and growth that follow axotomy in nonspiking insect neurons (Roederer and Cohen, 1983). Axon injury may also alter central neuronal connections to produce network sensitization. For example, Bulloch and Kater (1982) demonstrated a widespread formation of electrical connections among regenerating neurons in the snail Helisoma. It has been suggested that these new connections may contribute to a hypersensitivity that has been described anecdotally for some snails during early stages of regeneration (Moffett and Snyder, 1985). T h e facilitation of chemical synapses observed after axotomy of pleural (Walters et al., 1991) and cerebral (Clatworthy and Walters, 1994) sensory neurons in Aplysza should also contribute to central network sensitization.

VIII. Conclusions

Injury-related behavioral and neuronal plasticity has been studied intensively by investigators in various scientific disciplines. However, several of these fields have had little contact with each other, and thus there has been relatively little integration of findings across these fields. For example, few investigators of aversive learning and behavioral sensitization have tried to relate their work to discoveries emerging from the study of pain and hyperalgesia or axonal injury and regeneration. A major goal of this article is to point out that a consideration of the functional and evolutionary context of injury-related plasticity can begin to tie together diverse findings and foster broad, multidisciplinary approaches to physiological processes, such as pain and memory, that are of considerable interest to society. Although few scientists would deny

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the role played by natural selection in shaping the biological processes underlying pain and memory, evolutionary and adaptive considerations have played little if any part in the design and interpretation of most experiments on injury-related plasticity. In an attempt to make sense out of a very large and diverse scientific literature, this review was organized around an explicit adaptive hypothesis: that the common behavioral and neural alterations observed after bodily injury have been shaped by well-defined selection pressures that have been present through most of the course of metazoan evolution. Although a consideration of evolutionary arguments and paleobiological data support this general assumption, it must be kept in mind that it can never be proven. Adaptive hypotheses are useful for organizing complex sets of biological data and posing explicit experimental questions, but such hypotheses need to be advanced cautiously and abandoned gracefully in the face of contrary evidence (cf. Gould and Vrba, 1982; Alcock, 1989; Williams and Nesse, 1991). The basic adaptive hypothesis proposed for injury-related behavioral and neural plasticity had several consequences. First, it extended previous adaptive views of injury-related behavior by examining the consequences of different degrees of injury and by considering nociceptive behavior patterns in invertebrates as well as vertebrates (Table I). Application of this general model to diverse animals raised various questions that will be interesting to pursue. For example, is nociceptive sensitization particularly important for soft-bodied animals? Is long-term nociceptive memory better developed in longer-lived animals? Is injury-related compensatory growth of peripheral sensory arbors unimportant in very small animals? Second, the need to relate potentially adaptive reactions to injury across behavioral, neural network, and cellular levels of analysis led to a reformulation of the concept of “sensitization,” so that it could be applied consistently at each level without losing the most commonly held meanings of the term (Table 11). This led to a selective reappraisal of an extensive literature on behavioral modifications produced by actual injury, the appearance of injury (usually caused by artificial activation of nociceptors through electric shock), and the threat of injury (Table 111). Third, to begin to dissect out general and specialized mechanisms underlying adaptive behavioral reactions to injury, findings on nociceptive neural sensitization were juxtaposed from two very active but traditionally separate fields of research: mammalian hyperalgesia and invertebrate sensitization. T o add relevant data from another area that has rarely been associated with nociceptive sensitization, a specific adaptive hypothesis about reactions to peripheral axotomy was proposed, which offered an explanation for patterns of hyperexcitability seen in various neurons following injury to their axons.

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A comparison, across diverse species, of neural reactions to bodily injury, or to signals likely to be associated with bodily injury, revealed similarities and differences between vertebrates and invertebrates. Table I V lists features of neural sensitization associated with injury or injuryrelated signals that have been reported or strongly implicated in at least one mammal and one invertebrate. This relaxed criterion provides a very preliminary suggestion of potentially general mechanisms involved in injury reactions. Clearly, these and other potentially general features need to be examined much more thoroughly in order to determine how widespread they actually are-some will almost certainly be found to be uncommon or only superficially similar in diverse animal groups. A

TABLE IV FEATURES OF NOCICEPTIVE NEURALSENSITIZATION REPORTED I N BOTH VERTEBRATES A N D INVERTEBRATES Rapid sensitization

Long-term sensitization

Activation of nociceptors

Sustained activity in nociceptors

Peripheral cellular sensitization of nociceptors

Modulation of nociceptors by extrinsic inflammatory signals

Peripheral sensitization mediated by serotonin and cyclic AMP

-

Modulation of nociceptors by intra-axonal in-jury signals

Cellular sensitization in wide dynamic range neurons

Peripheral cellular sensitization of nociceptors

Wind-up of responses to quasi-noxious stimulation expressed in second-order nociceptive interneurons

Cellular sensitization in sensory neurons involving decreased after hyperpolarization and reduced K +

Central sensitizing actions from release of neuropeptides

Collateral sprouting by peripheral processes Of nocicePtors

Central sensitizing actions mediated by cyclic AMP

Central sprouting of sensory neuron synaptic terminals (presynaptic facilitation?) Activity-dependent induction signals requiring Ca2+influx in soma and neurites

Central sensitizing actions mediated by PKC Central sensitizing actions from &inhibition of excitatory interneurons

-

Sensitizing actions due to Ca2+ influx through channels gated by NMDA receptors (classical LTP?) Alterations in protein synthesis following transient activation of immediate early genes Up-regulation of cellular stress proteins

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detailed look at molecular mechanisms should begin to reveal the extent to which specific nociceptive mechanisms that are outwardly similar have diverged or been conserved during evolution. Table V lists features that are apparently important for nociceptive neural sensitization but that have only been reported in either vertebrates or invertebrates. It is far too early to suggest that any of these features is unique to specific animal groups. Indeed, many may be relatively general. For example, until very recently it was assumed that NMDA receptor-dependent LTP mechanisms are unique to higher levels of the vertebrate brain. However, the recent discoveries by Randic et al. (1993), Lin and Glanzman (1993, 1994a,b) and Dale and Kandel (1 993) suggest the fascinating possibility that LTP mechanisms currently being defined in the mammalian hippocampus and cerebral cortex (where they presumably have cognitive functions) reflect a very primitive adaptive neuronal process that continues to function not only in the mammalian spinal cord (where it may contribute to a distributed memory of injury), but also in invertebrate nervous TABLE V FEATURES OF NOCICEPTIVE NEURALSENSITIZATION NOT YET REPORTEDI N BOTH VERTEBRATESAND INVERTEBRATES Rapid sensitization

Long-term sensitization

Modulation of nociceptors by extrinsic inflammatory signals (vertebrates) Peripheral sensitization mediated by prostaglandins and lipoxygenase products (vertebrates)

Sensitization of nociceptors by interruption of retrograde transport of trophic factors (vertebrates)

Expansion of nociceptive interneuron receptive fields (vertebrates)

Central sensitizing actions from disinhibition of excitatory interneurons (vertebrates)

Peripheral sensitization of nociceptor responses to chemical signals (vertebrates)

Central network sensitization from release of N O (and CO?) (vertebrates)

Transient formation of electrical synapses (invertebrates)

Facilitation of neuromuscular transmission (invertebrates) Cellular sensitization of nociceptors mediated by reduction of K + currents (invertebrates) Presynaptic facilitation of central synapses of mechanosensory neurons (invertebrates) Central sensitization mediated by biogenic amines (invertebrates) Activity-dependent enhancement of sensitizing actions of extrinsic modulators (invertebrates)

-

Long-term alterations triggered by cyclic AMP via nuclear CREB protein and C/ EBP factors (invertebrates) Down-regulation of cell adhesion molecules (invertebrates)

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systems.If molluscan and mammalian LTP do, in fact, share molecular mechanisms, interesting questions are raised about the role of injury-related selection pressures in the evolution of this fundamental mnemonic process. Although we do not yet know whether broader biological considerations can really help us answer pressing questions about pain and hyperalgesia, an evolutionary perspective may prove useful for interpreting existing data and suggesting new lines of research. For example, from an evolutionary perspective, injury-induced behavior is quite unusual because both the selection pressures and the initiating cellular events (signals of incipient tissue damage) may be quite similar for all animals, and may have been similar in primitive common ancestors of existing animals. If these events depend on common consequences of cellular damage (e.g., on an influx of Ca2+ or disruption of the cytoskeleton), some may have been utilized as signals for defensive behavior and cellular repair very early in evolution, even before the advent of multicellular organisms. This raises the possibility that some existing mechanisms of cellular sensitization are extremely primitive, predating multicellularity. Such mechanisms could thus be even more primitive than some of the developmental mechanisms characteristic of multicellular organisms that appear to be shared with mechanisms of learning and memory (e.g., Hebb, 1949;Jessell and Kandel, 1993; Shatz, 1992). Given the possibility that some mechanisms of cellular sensitization originated in single-celled organisms in response to injury and stress-related selection pressures, it may be particularly interesting to see whether stress-related proteins that appear to play a role in long-term sensitization in Aplysia, such as BiP and ubiquitin, are involved in long-term neuronal reactions to bodily injury in other species. These proteins are found in all cells, from bacteria to humans, so their involvement in nociceptive sensitization and other forms of memory would support the possibility that some alterations involved in nociceptive sensitization, hyperalgesia, and various forms of memory originated in primitive adaptive reactions to cellular injury and stress (see also Walters, 1991; Walters et al., 1991). Our only evidence for or against the possibility that a given physiological feature is primitive lies in the distribution of that feature across the animal kingdom. Thus it will be very important to compare injury-related plasticity at the behavioral, neuronal, and molecular levels across the broadest possible range of species and cells.

Acknowledgments

The author is grateful to Drs. Thomas Abrams, Paul Illich, Christine Sahley, and William Willis for helpful comments o n parts of this chapter. The preparation of the article

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was supported by Grants MH38726 from the NIMH, and BNS-9011907 and IBN-9210268 from the NSF.

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INDEX

second-order conditioning and extinction, 256-258 Acquired immunodeficiency syndrome AMPA, see cu-Amino-3-hydroxy-5-methyl4-isoxazole dementia complex, 5, 11-12, 22-23, 306 and EAA receptors and phospholipid Amygdala metabolism, 306 basolateral nucleus, benzodiazepine neuronal loss in central nervous system, receptors, 237-238 3-4 and brain areas involved in fear and Adaptation anxiety, anatomical connections, to potential or actual injury, 338 228-231 related hypothesis on reactions to central nucleus axotomy, 386-388 electrical or chemical stimulation related reactions effects, 232-233 axonal injury in peripheral neurons, EPSP evoked in, 228 389-392 neuropeptides, 231 neuronal injury, 366-367 opiate receptors, 237-238 Adenylyl cyclase EAA receptors in, role in fear inhibition by GABA, receptor, 110-1 12 conditioning, 250-258 transmitter-mediated activation, electrical or chemical stimulation, fear facilitation, 112-1 15 elicitation, 23 1-234 AIDS, see Acquired immunodeficiency electrophysiology, 227-228 syndrome lesions, effects Allodynia, definition, 326, 345-346 conditioned fear, 234-235 ALS/PD, see Guam-type amyotrophic unconditioned fear, 235-236 lateral sclerosis/ local infusion of drugs, and measures Parkinsonism-dementia of fear and anxiety, 236-238 Alzheimer disease morphology, 226 association with intracranial roles amyloidosis, 30-3 1 attention, 238-240 and EAA receptors and phospholipid aversive conditioning, 236 metabolism, 292-297 fear-potentiated startle effect, 240, familial, 29-30, 37-39 244-245 treatment with gangliosides, 309 initial fear conditioning, 245-249 y-Aminobutyric acid, see GABA and storage of aversive memories, ru-Amino-3-hydroxy-5-methyl-4-isoxazole 241-243 receptor P-Amyloid, in body fluids, 36-37 multiplicity, 274-275 P-Am yloidogenesis regional distribution, 271 molecular factors, 42-44 2-Amino-phosphonopentanoic acid, process, 34-35 effects Amyloidosis acquisition/expression of fear familial cerebral, P-arnyloid precursor conditioning, 25 1-254 protein mutations in, 37-39

A

429

430

INDEX

intracranial, Alzheimer disease associated with, 30-31, 4 3 P-Amyloid precursor protein processing via enzymatic and subcellular pathways, 32-34 structure, 31-32 Analgesia and antinociception and competing response inhibition, 358-361 definition, 326 Antinociception animal models, 326 and competing response inhibition and analgesia, 358-361 Anxiety and general sensitization and arousal, 355-356 perception of, 233 representation by amygdala and efferent projections, 228-23 1 AP5, see 2-Amino-phosphonopentanoic acid Aplysia defensive reflex, peripheral injury effects, 344 neuronal contributions to sensitization, 383-384 sensory neurons persistent alterations, 399-40 1 sensitization, 378-383 site-specific sensitization, 352-353 L - A P receptor, ~ activity at pre- and postsynaptic sites, 277 Arousal, as defensive behavior, 355-356 Arthropods and evolution of nociceptive plasticity, 356-357 and nociceptors, 363-364 Astrocyte P-adrenergic stimulation, role of gp120, 14 and macrophages, feedback loop, 9-10 role in HIV-related neuronal damage, 12-13 Astrogliosis, in HIV infection, 2 Attack, risk assessment during, 337-338 Attention, role of amygdala, 238-240 Aversive conditioning amygdala role, 236 Pavlovian, 239

Aversive memory, storage role of amygdala, 241-243 Avoidance, inhibitory measurement after training, 253-254 non-NMDA antagonist effects, 254-255 Axonal injury peripheral neurons, adaptive reactions, 389-392 produced central alterations of invertebrate neurons, 405-407 Axotomy, reactions to, adaptive hypothesis, 386-388

B Baclofen ability to hyperpolarize, effect on central neurons, 121-123 depression of polysynaptic inhibition, 167-17 1 effect on calcium-independent IPSP, 172- 173 effect on population spike potentiation, 188 as GABAB receptor agonist, 98-100 induced disinhibition, 185- 188 presynaptic depressant effect, 156- 161 Barbiturates, effects on GABA, receptor binding, 58-59 Behavior, see also Adaptation defensive, see also Defense aggressive retaliation as, 336 arousal as, 355-356 effects of amygdala lesions, 235 escape locomotion as, 335 freezing as, 335 startle as, 335 withdrawal as, 335 injury-related general adaptive model, 333-334 previous functional models, 331-333 nociceptive, 329 related choice, and risk assessment, during injury, 337-338 related effects of GABAB receptors, 209-2 10 Behavioral modification, after actual injury, 343-345 Benzodiazepine receptor, on amygdala basolateral nucleus, 237-238

INDEX

Benzodiazepines, recognition site of GABAA receptor, 55-58 Bicuculline, block of GABA, receptor, 52,55 Brain areas GABA, receptor effects, 205-208 involved in fear and anxiety, anatomical connections with amygdala, 228-23 1 developing, EAA-mediated phospholipid metabolism, 287-288 mRNA encoding metabotropic receptors, differential distribution, 280 Xenopus, kainate and AMPA binding sites, 276 Brain injury, traumatic, and EAA receptors and phospholipid metabolism, 301-302

C Calcium channel intracellular, gp120 effects, 4 voltage-dependent activation, 2 effect on neurotoxicity, 21 L-type, 3 voltage-sensitive, inhibition by GABA, receptor, 115-121 Calcium current T-type, deinactivation, 154- 156 voltage-dependent, inhibition by presynaptic GABA, receptor, 162 Calcium ion in AIDS-related neuronal injury, 2-3 [Ca2'I,, increase in neurological disorders, 31 1-312 role in delayed neurotoxicity, 290-291 Cell injury, induced by EAAs, mechanism, 288-291 Central nervous system, AIDS patients, neuronal loss, 3-4 Cerebrospinal fluid AIDS dementia complex patients, increased quinolinic acid levels, 306 ALS/PD patients, neurotoxicity, 306 Channel conductance, GABA, receptor, 54

43 1

Chemoattractants, injury-related, 34 1 CNQX, see 6-Cyano-7-nitroquinoxaline2.3-dione Conditioning aversive Pavlovian, 239 role of amygdala, 236 fear, see Fear conditioning second-order, NMDA antagonist effects, 256 Conductance IPSP peak, 133-134 reduction during repetitive stimulation, 176-177 voltage-inactivated, reactivation, 154- 156 Corticotropin releasing factor, evoked membrane hyperpolarization in amygdala, 228 6-Cyano-7-nitroquinoxaline-2,3-dione, effects conditioned fear expression, 254-255 gp 120-induced neuronal injury, 6 Cyclic AMP, role in long-term cellular sensitization, 403-405 D

Defense associated sensitization, role of peripheral neuronal injury, 390-392 choice according to risk, 338 importance during recuperation, 339, 341-342 primary and secondary, 334-336 reflex, peripheral injury effects, 344 Dementia complex, AIDS-associated, 306 Depolarization dendritic, increase, 188 dorsal horn cells by glutamate, 374 facilitation, 203-204 neural, role of disinhibition, 185 Depression, site-specific, 359-36 1 Dextromethorphan, sites of action in HIV neurotoxicity, 19 Dextrorphan effect on NMDA receptor-operated channels, 22 sites of action in HIV neurotoxicity, 19

432

INDEX

Diazepam, binding site on GABA, receptor, 56-57 Disease, and parasites, as result of injury, 340 Disinhibition presynaptic, 185-204 role in long-term central sensitization after peripheral injury, 394-395 Dizocilpine prevention of gpl20-induced neuronal injury, 6 sites of action in H I V neurotoxicity, 18-19 Domoic acid, neurotoxicity, 305 Dorsal root ganglion, in studies of voltage-sensitive calcium channels, 116-121

induced cell injury, mechanism, 288-29 1 mediated phospholipid metabolism in developing brain, 287-288 Excitatory postsynaptic potential in Aplysia, 378-380 duration increase, 188- 189 en hancement, 192- 193 evoked in amygdala, 227-228 fast and slow, in dorsal horn cells, 372-374 Excitotoxicity growth factor effects, 309-310 and membrane phospholipid degradation, 31 1 Extinction, fear-potentiated startle, NMDA antagonist effects, 256-258

E EAA, .see Excitatory amino acids Epidermal growth factor, effects on excitotoxicity, 309-3 10 Epilepsy, and EAA receptors and phospholipid metabolism, 302-303 Epileptiform activity bicuculline effects, 198-199 role of disinhibition, 201, 205 EPSP, see Excitatory postsynaptic potential Evolution and neuronal reactions to bodily injury, 327-330 nociceptive plasticity, 356-357 related perspective on pain and hyperalgesia, 41 1 Excitation, tonic, from afferents, 393-394 Excitatory amino acid receptor in amygdala, role in fear conditioning, 250-258 antagonists, and treatment of neurological disorders, 307-310 classification, 269-280 and phospholipid metabolism, 280-287, 291-307 role in neural cell injury, 268-269 Excitatory amino acids, see also specific EAAs and [Ca’+], levels, 2-3 excitotoxic effect, 23

F

Fading of inhibition GABA, receptor-mediated, 199-201 and GABA, receptor-mediated conductance, 182-183 role of potassium conductance, 177 Fear conditioned, 361 effects of amygdala lesions, 234-235 expression, effects of non-NMDA antagonists, 254-255 elicitation by stimulation of amygdala, 231-234 in motivation of operant behavior, 241-243 representation by amygdala and efferent projections, 228-23 1 unconditioned, effects of amygdala lesions, 235-236 Fear conditioning, and associative long-term potentiation, similarities, 250-25 1 Fibroblast growth factor, effects on excitotoxicity, 309-3 10 Freezing amygdala lesion effects, 234 benzodiazepine effects, 237 as defensive behavior, 335 in response to fear, 361 role of NMDA receptors, 253

lNDEX G

GABA effect on mammalian central nervous system, 52 as GABAB receptor agonist, 98- 100 mediated fast and slow IPSPs evoked in amygdala, 227-228 synaptically released, feedback onto GABA, receptor, 176-177 GABA,+ receptor electrophysiological studies, 53-55 and GABA, receptor, IPSPs, physiological separation of responses, 136-140 gene expression, 81-85 heterogeneity, 73-74 immunocytochemical localization, 85-86 immunological characterization, 69-7 1 pharmacology, 52-59 photoaffinity labeling, 66-67 purification, 59-61 radiolabeled ligand binding studies, 62-64 reconstitution, 61-62 subunit cDNAs, isolation, 71-73 subunit classes assembly, 86-87 functional attributes, 74-8 1 thermodynamics, 64-66 GABAB receptor activation during synchronized neural activity, 198 adenylyl cyclase activation, 112- 1 15 adenylyl cyclase inhibition, 110- 112 agonists, 98-100 antagonists, 100-104 binding, 104- 107 distribution, 107-109 and GABA, receptor, IPSPs, physiological separation of responses, 136- 140 mediated inhibition of spontaneous neuronal firing, 142-153 mediated potentiation, 126- 127 pharmacology, 98- 104 postsynaptic, 128-156 potassium channel conductance activation, 121-126

433

pre- and postsynaptic, opposing effects, 205-208 presynaptic on excitatory terminals, 156- 166 depression of excitatory responses, 156-161, 164-165 terminal mechanism, 161-164 on inhibitory terminals, 166-204 depression of inhibitory responses, 166-171, 173-184 functional significance of presynaptic disinhibition, 185-204 terminal mechanism, 171- 173 regulation of long-term potentiation, 195-197 responses on GABAergic neurons, 140-14 1 role in inositol triphosphate synthesis, 115 voltage-sensitive calcium channel inhibition, 115-121 GABAB receptor-effector systems, 110-126 Galactosyl ceramide, gp120 binding, 13 Gangliosides pharmacological use, 22 in treatment of Alzheimer disease and ischemia, 309 Gene, chromosome 14 FAD, 44 Glutamate and [CaZt], levels, 2-3 depolarization of dorsal horn cells, 374 mediated EPSP evoked in amygdala, 227-228 neurotoxicity, 298-300 role in final common pathway of neuronal injury, 16 Glutamate receptor, ionotropic, 269-27 1 Glycoprotein, myelin-associated, gp 120 binding, 13-14 Glycoprotein gp 120 binding to galactosyl ceramide, 13 sulfatide and myelin-associated glycoprotein, 13-14 induced neuronal injury, 4-5 role of NMDA, 6-8 role in P-adrenergic stimulation of astrocytes and microglia, 14

434

INDEX

G proteins coupling to postsynaptic GABA, receptors, 163-164 inhibitory, and baclofen, 173 postsynaptic, irreversible activation, 183 Guam-type amyotrophic lateral sclerosis/ Parkinsonism-dementia, and EAA receptors and phospholipid metabolism, 303-304 H

Heart rate, conditioned changes, block by central nucleus lesions, 234 HIV, see Human immunodeficiency virus Human immunodeficiency virus infected monocytic cells, role in indirect neuronal injury, 8-12 related gp120, induced neuronal injury, 4-5 type 1, proteins, neurotoxicity, 14 Huntington disease, and EAA receptors and phospholipid metabolism, 304-305 Hyperalgesia definition, 326, 345-346 evolutionary perspective, 4 1 1 long-term, and neuropathic pain, 354 and long-term site specific sensitization, 351-353 short-term, and incremental site-specific sensitization, 349-35 1 Hyperpolarization baclofen-induced, effect on central neurons, 121-123 IPSP, 133-134 large, associated IPSP, 148 H ypoalgesia anticipatory, 359-360 definition, 326 I

Immobility, tonic, after capture, 360 Inflammation articular, 392-393 produced central alterations of invertebrate neurons, 405-407 as result of injury in vertebrates, 352-353 and tissue damage, 364-366

Inhibition competing response, and antinociception and analgesia, 358-361 fading of, see Fading of inhibition Inhibitory avoidance measurement after training, 253-254 non-NMDA antagonist effects, 254-255 Inhibitory postsynaptic potential activity-dependent depression, 184, 188-191 characteristics, 128-14 1 conductance, reduction during repetitive stimulation, 176-177 evoked in amygdala, 227-228 functional significance, 14 1- 156 GABA, and GABAB responses, physiological separation, 136- 140 GABA, receptor-mediated inhibition, 142-153 kinetics, 131-133 late component current-voltage relationship, 129-13 1 pharmacology and conductance mechanism, 128-129 peak conductance and hyperpolarization, 133- 134 reactivation of voltage-inactivated conductances, 154-156 stimulus dependence, 134- 136 Injury, see also specific types adaptive behavioral reactions to, 330-342 evolutionary considerations, 327-330 imminent, and incremental sensitization mechanisms, 368-369 related behavioral modifiability, classes, 342-361 risk assessment and behavioral choice during, 337-338 signals, 361-367 site, sensitization targeted to, 348-355 and threat’of, fast reactions, 334-337 Inositol triphosphate, synthesis, role of GABA, receptor, 115 Interferon-y, released by HIV-infected macrophages, 9-12 Interleukin- lp, released by HIV-infected macrophages, 9-12

INDEX

Interneuron contributions to sensitization in Aplysia, 383-384 dorsal horn intrinsic excitability, 395-396 responses to nerve injury, 393-395 GABA release, 177 synchronous activation, 181-182 lnvertebrates persistent neural sensitization, 398-407 rapid sensitization mechanisms, 376-385 and vertebrates, nociceptive neural sensitization features, 409-4 10 Ionotropic receptor, and phospholipase A,, 281-287 IPSP, see Inhibitory postsynaptic potential Ischemia and EAA receptors and phospholipid metabolism, 297-300 treatment with gangliosides, 309

K

Kainate receptor multiplicity, 275-276 regional distribution, 271 Ketamine, sites of action in HIV neurotoxicity, 19

L

Labeling, photoaffinity, see Photoaffinity labeling Lesion, amygdala effects on conditioned and unconditioned fear, 234-236 role in fear-potentiated startle expression, 244-245 Locomotion, escape, as defensive behavior, 335 Long-term potentiation associative, similarities with fear conditioning, 250-25 1 enhanced induction by fading of inhibition, 201 evoked at synapses within amygdala basolateral nucleus, 227-228 as form of synaptic plasticity, 15 1- I53

435 induction, role of GABA, receptors, 209 induction with high-frequency stimulus trains, 193-198, 201 IPSP blockade role, 185 metabotropic EAA role, 280 monosynaptic and polysynaptic EPSPs, 374 NMDA receptor-dependent, 410-41 1 induction, 203-204

M Macrophage HIV-infected, direct effects on neurons, 15 role in neurotoxic effects of gp120, 8-12 Magnesium ion, effect on open NMDA channels, 19 Memantine, as NMDA open-channel blocker, 19-20, 22 Memory, aversive, storage role of amygdala, 241-243 Metabotropic receptor multiplicity, 277-280 and phospholipase C, 281 N-Methyl-D-aspartate antagonists, sites of action, 17 open-channel blockers, sites of action, 17-20 N-Methybaspartate receptor in amygdala, role in acquisition/ expression of fear conditioning, 25 1-254 antagonists, in prevention of neuronal degeneration, 308 dependent long-term potentiation, 203-204,410-411 mediated currents, suppression by postsynaptic inhibition, 208 multiplicity, 27 1-274 and non-NMDA receptors on postsynaptic neuron, 250-25 1 operated channel, magnesium block, 185, 193 overstimulation, 15-16 redox modulatory site, 20-21 regional distribution, 27 1 role in gp120-induced neuronal injury, 6-8

436

INDEX

Microglia, P-adrenergic stimulation, gp120 role, 14 Model injury-related behavior, 331-334 pain animal, 326 experimental, 364-366 Mutation, P-amyloid precursor protein, 37-39

N Nerve growth factor, effects on excitotoxicity, 309-3 10 Neurofibrillary tangles in Alzheimer disease, 30, 293 formation, 40 Neuron, see also Interneuron central effects of baclofen ability to hyperpolarize, 121-123 spontaneous activity, role of GABAs receptors, 209 degeneration prevention, 308 direct effects of HIV-infected macrophages, 15 dorsal horn, noxious input effects, 372-376 GABAergic, GABA, receptor responses on, 140-141 inhibitory and excitatory, differential effects of GABA, receptors, 205-208 GABAB receptor on, 176-177 invertebrate, central alterations, 405-407 loss

in central nervous system of AIDS patients, 3-4 gpl20-mediated, 4-5 motor, contributions to sensitization in Aplysm, 384 peripheral, axon injury, adaptive reactions, 389-392 projection, spe Projection neuron sensory Aplysia persistent alterations, 399-40 1 sensitization, 378-383

synapses, presynaptic facilitation, 396-397 spontaneous firing, GABA, receptormediated inhibition, 142-153 Neuronal injury adaptive reactions, 366-367 gp120-induced, 4-5 role of NMDA, 6-8 indirect, 8-1 2 nirnodipine effects, 4 NMDA-induced, 290-291 role of astrocytes, oligodendrocytes, and HIV-1 proteins, 12-15 Neuropathy, axonal, 13 Neurotoxicity cerebrospinal fluid from ALSiPD patients, 303 domoic acid, 305 glutamate, 298-300 HIV-1 protein, 14 NMDA-mediated, 19-2 1 Neurotransmitter, see spec@ neurotransmitters

Nimodipine, effect on neuronal injury, 4, 6 Nitric oxide synthase, effect on gp120induced neuronal injury, 6 Nocjceptors, activation, 362-364 0

Octopamine, and rapid sensitization mechanism in invertebrates, 376-378 Opiate receptor, on arnygdala basolateral nucleus, 237-238

P Pain definition, 326 evolutionary perspective, 4 I 1 experimental models, 364-366 modulation studies, 350-35 1 neuropathic, and long-term hyperalgesia, 354 Paired pulse, facilitation, 189- 193 Parasite, infestation, as result of injury, 340-34 1 Phencyclidine, sites of action in HIV neurotoxicity, 18- 19

1NL)EX

Phospholipase A, and ionotropic receptors, 281-287 Phospholipase C , and metabotropic receptors, 28 1 Phospholipids EAA-mediated metabolism in developing brain, 287-288 membrane degradation, 268 metabolism and EAA receptors, 280-287 metabolism, and EAA receptors and neurological disorders, 29 1-307 Phosphorylation, protein role in neurologic function during ischemia, 300 signal transduction via, 39-42 Photoaffinity labeling, GABA, receptor, 66-67 Plasticity behavioral, injury-related, 347 and neuronal, 330,407-408 during fear conditioning, 234 nociceptive, evolution, 356-357 synaptic, long-term potentiation as, 151-153 Platelet-activating factor, release from HIV-infected macrophages, 9- 11 Postsynaptic potential, see Excitatory postsynaptic potential; Inhibitory postsynaptic potential Potassium channel, activation by GABA, receptors, 12 1- 126 Potentiation, see also Long-term potentiation long-lasting, 127 Predation, heightened, in response to injury. 340-341 Projection neuron afferents, glutamatergic activity, 228 spine-dense pyramidal, 226 Projections, from amygdala direct to hypothalamus, 233 role in fear and anxiety, 229-231 Protection behavioral, as result of injury, 340, 342 injured region, 353 Protein kinase C, in protein phosphorylation. 39-42

437

Proteins G, see G proteins HIV- 1 , neurotoxicity, 14 modification studies, GABA, receptor, 67-69 phosphorylation role in neurologic function during ischemia, 300 signal transduction via, 39-42 Protein synthesis, role in induction and maintenance of long-term cellular sensitization, 402-403 Pseudoconditioning, and reflex dominance and response-specific sensitization, 353 Pulse, paired, see Paired pulse

Quinolinate from macrophages, effect on neurons, 15 relationship to degree of dementia in AIDS patients, 6 role in EAA-induced neurotoxicity, 11-12 Quinolinic acid, increased levels in cerebrospinal fluid from AIDS patients, 306

R Receptor coupling, postsynaptic GABA, receptors to G proteins, 163-164 Receptor-effector coupling, 206-207 Recuperation, from injury, importance of defense, 339, 341-342 Redox modulatory site, on NMDA receptor-channel complex, 20-2 1 Reflex, dominance, and response-specific sensitization and pseudoconditioning, 353 Reproductive capacity, injury effects, 339 Respiration, fear-related changes, role of amygdala, 229-230 Retaliation, aggressive, as defensive behavior, 336 Reversal potential, GABA, receptor IPSP, 174-1 76

438

INDEX

Risk assessment, and behavioral choice during injury, 337-338 RNA, messenger, metabotropic receptor, differential distribution, 280

S p-Secretase, in p-amyloidogenesis, 36-37 Selection pressures, injury-related, 328-330,408-409 Sensitization general, and arousal and anxiety, 355-356 incremental site-specific,and short-term hyperalgesia, 349-35 1 long-term nociceptive, mechanisms, 386-407 long-term site-specific,and hyperalgesia, 351-353 nociceptive animal models, 326 in arthropods, 356-357 definition and types, 345-348 nociceptive neural, in vertebrates and invertebrates, 409-4 10 rapid nociceptive, mechanisms, 367-385 response-specific, and reflex dominance and pseudoconditioning, 353 Serotonin and rapid sensitization mechanism in invertebrates, 376-378, 384-385 as sensitizing modulator in Aplysia, 379-382 Shock, cutaneous, and persistent neuronal alterations, 401 Signal transduction, via protein phosphorylation, 39-42 Signal transmission, enhancement, 198-204 Spinal cord injury, and EAA receptors and phospholipid metabolism, 300-301 long-lasting neural sensitization, 392-393 sensory neuron synapses, presynaptic facilitation, 396-397 Sprouting injured cells at injury site, 398

in response to peripheral neuronal injury, 390-392 Startle as defensive behavior. 335 fear-potentiated acquisition in absence of amygdala, 245-249 extinction, NMDA antagonist effects, 256-258 non-NMDA antagonist effects, 254-255 role of NMDA receptors, 251-254 Startle effect, fear-potentiated, role of amygdala, 240, 244-245 Startle reflex fear-potentiated, 23 1 NMDA effects, 233 Steroids, binding site on GABA, receptor, 59 Stimulation electrical, central nucleus, 239 electrical or chemical, amygdala, 231-234 paradigm, role in effects of GABAB receptors, 207 prolonged, depressive effects, 369 repetitive amygdala external capsule, 227 associated reduction in IPSP conductance, 176- 177 Subnuclei, in amygdala, 226 Sulfatide, gp120 binding, 13-14 Survival rate, injury effects, 339

T

Thermodynamics, GABA, receptor, 64-66 Tissue injury and inflammation, 364-366 peripheral, role in long-lasting neural sensitization, 392-393 Training degree, role in fear-potentiated startle expression, 244-245, 247-249 subsequent inhibitory avoidance, 253-254 Tumor necrosis factor-a, released by HIV-infected macrophages, 9-12

INDEX

V Vertebrates inflammation in response to injury, 352-353 and invertebrates, nociceptive neural sensitization features, 409-4 I0 persistent neural sensitization, 389-397 rapid sensitization mechanisms, 369-376

439

Vigilance, and long-term sensitization and chronic anxiety, 356 W

Withdrawal, as defensive behavior, 335

X Xenopw, brain, kainate and AMPA binding sites, 276

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CONTENTS OF RECENT VOLUMES

Volume 26

Eye Movement Dysfunctions and Psychosis Philip S. Holzman

T h e Endocrinology of the Opioids Mark J . Millan and Albert Herz

Peptidergic Regulation of Feeding J . E . Morley, T . J . Badness, B . A. Gornell, and A. S. Leuine

Multiple Synaptic Receptors for Neuroactive Amino Acid Transmitters-New Vistas Najam A . Shartf

Calcium and Transmitter Release Ira Cohen and William Van der Kloot

Muscarinic Receptor Subtypes in the Central Nervous System Wayne H o s ~and John Ellis

Excitatory Transmitters Related Brain Damage John W . Olney

Neural Plasticity and Recovery of Function after Brain Injury John F . Marshall

Potassium Current in the Squid Giant Axon John R. Clay

From Lmmunoneurology to Imrnunopsychiatry: Neuromodulating Activity of AntiBrain Antibodies Branulazl D. JanklouiC

INDEX

and

Epilepsy-

Volume 28

Effect of Trernorigenic Agents on the Cerebellum: A Review of Biochemical and Electrophysiological Data V . G. Longo and M . Massotti INDEX

Volume 27

T h e Nature of the Site of General Anesthesia Keith W . Miller T h e Physiological Role of Adenosine in the Central Nervous System Thomas V . Dunwiddie Somatostatin, Substance P, Vasoactive Intestinal Polypeptide, and Neuropeptide Y Receptors: Critical Assessment of Biochemical Methodology and Results Anders Undkn, Lou-Lou PeterJon, and Tainas Bartfaz

Biology and Structure of Scrapie Prions Michael P . McKinley and Stanley B . Prusiner Different Kinds of Acetylcholine Release from the Motor Nerve S . Thesleff Neuroendocrine-Ontogenetic Mechanism of Aging: Toward an Integrated Theory of Aging V . M . Dilman, S. Y . Reuskloy, and A . G.Golubev

T h e lnterpeduncular Nucleus Barbara J . Morlpy 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 Does Receptor-Linked Phosphoinositide Metabolism Provide Messengers Mobilizing Calcium in Nervous Tissue? John N . Hawthorne

441

442

CONTENTS OF RECENT VOLUMES

Short-Term and Long-Term Plasticity and Physiological Differentiation of Crustacean Motor Synapses H . L. Atwood and J . M . Wojtowicz Immunology and Molecular Biology of the Cholinesterases: Current Results and Prospects Stephen Brimijoin and Zoltan Rakoncray INDEX

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Volume 30

Biochemistry of Nicotinic Acetylcholine Receptors in the Vertebrate Brain Jakob Schmidt

Volume 29

T h e Neurobiology of N-Acetylaspartylglutamate Randy D. Blakely and Joseph T. Coyle

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

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

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

Targeting Drugs and Toxins to the Brain: Magic Bullets Lance L. Simpson

Neurotoxin-Binding Site on the Acetylcholine Receptor Thonios L. Lentz and Pan1 T. Wilson Calcium and Sedative-Hypnotic Drug Actions Peter L. Carlen and Peter H . w u Pathobiology of Neuronal Storage Disease Steven CI. Walkley

Neuron-Glia Interrelations Antonia Vernadakis Cerebral Activity and Behavior: Control by Central Cholinergic and Serotonergic Systems C. H . Vandemolf INDEX

Thalamic Amnesia: Clinical and Experimental Aspects Stephen G . Waxman

Volume 31

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

Animal Models of Parkinsonism Using Selective Neurotoxins: Clinical and Basic Implications Michael J . Zigmond and Edward M . Stricker

Retinal Transplants and Optic Nerve Bridges: Possible Strategies for Visual Recovery as a Result of Trauma or Disease James E. Turner, J e n y R. Blair, Magdalene Seiler, Robert Aramant, Thomas W . Laedtke, E . Thomas Chappell, and Lauren Clarkson Schizophrenia: Instability in Norepineph-

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443

Mechanisms of Chemosensory Transduction in Taste Cells Myles H . Akubas Quinoxalinediones as Excitatory Amino Acid Antagonists in the Vertebrate Central Nervous System Stephen N . Davies and Graham L. Collingridge Acquired Immune Deficiency Syndrome and the Developing Nervous System Douglas E. Brenneman, Susan K . McCune, and Illana Gozes INDEX

Volume

33

INDEX

Olfaction S. G . Shirley Volume 32

On 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. Ushemood Coinjection of Xenopw Oocytes with cDNAProduced and Native mRNAs: A Molecular Biological Approach to the Tissue-Specific Processing of Human Cholinesterases Shlomo Seidman and Hemona Soreq Potential Neurotrophic Factors in the Mammalian Central Nervous System: Functional Significance in the Developing and Aging Brain Dalia M . Aruujo, Jean-Guy Chabot, and Rbmi Quirion Myasthenia Gravis: Prototype ofthe Antireceptor Autoimmune Diseases Simone Schonbeck, Susanne Chrestel, and Reinhard Hohlfeld Presynaptic Effects of Toxins Alan L. Hamey

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Volume 34

Neurotransmitters as Neurotrophic Factors: A New Set of Functions Juan P. Schwartz Heterogeneity and Regulation of Nicotinic Acetylcholine Receptors Ronald J . Lukm and Merouane Bencherzf

444

CONTENTS OF RECENT VOLUMES

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INDEX

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Volume

35

Biochemical Correlates of Long-Term Potentiation in Hippocampal Synapses Satoru Otani and Yehezkel Ben-An Molecular Aspects of Photoreceptor Adaptation in Vertebrate Retina Satoru Kauramura

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