INTERNATIONAL REVIEW OF
Neu rob iology VOLUME 21
Associate Editors W. R. ADEY
H. J. EYSENCK
D. BOVET
S. KETY
JOSE DELCADO
A. LAJTHA
SIRJOHNECCLES
0. ZANCWILL
Consultant Editors V. AMASSIAN
P. JANSSEN
R. BALDESSARINI
K. KILLAM
F. BLOOM
C. KORNETSKY
P. B. BRADLEY
B. A. LEBEDEV
0. CREUTZFELDT
V. LONCO
J. ELKES
P. MANDELL
K. FUXE
H. OSMOND
B. HOLMSTEDT
S. H . SNYDER
s. SZARA
INTERNATIONAL REVIEW OF
Neurobiology Editedby J O H N R. SMYTHIES Deportment of Psychiatry and the Neurosciences Program University of Alabama Medical Center Birmingham, Alabama
R O N A L D J. BRADLEY The Neurosciences Program University of Alabama Medical Center Birmingham, Alabama
VOLUME 21
1979
ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers
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COPYRIGHT @ 1979, 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, O R ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
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ISBN 0-12-366821-2 PRINTED IN THE UNITED STATES OF AMERICA 79 80 81 82
9 8 7 6 5 4 3 2 1
CONTENTS CONTRIBUTORS ..............................................................
vii
Relationship of the Actions of Neuroleptic Drugs to the Pathophysiology of Tardive Dyskinesia
.
Ross J BALDESSARINI AND DANIEL TARSY I . Introduction ......................................................... I1. Pathophysiology and Etiology of Tardive Dyskinesia ..................... I11. Conclusions .......................................................... References ...........................................................
1 12 36 37
Soviet Literature on the Nervous System and Psychobiology of Cetacea
THEODORE H . BULLOCK AND VLADIMIR S . GUREVICH I. I1. 111. IV .
Introduction ......................................................... Neuroanatomy ....................................................... Neurophysiology ...................................................... Summary ............................................................ Bibliography .........................................................
49 52 70 103 107
Binding and lontophoretic Studies on Centrally Active Amino AcidsA Search for Physiological Receptors
F . V. DEFEUDIS I. I1. 111. IV . V. VI . VII VIII .
.
Introduction ......................................................... Physiologic-Pharmacologic Studies in Vertebrates ....................... Physiologic-Pharmacologic Studies in Invertebrates ...................... Biochemical Studies with Vertebrate Preparations ....................... Biochemical Studies with Invertebrate Preparations ...................... Relevant Studies on Glial Cells ......................................... Relevant Studies with Tissue Cultures .................................. Concluding Remarks .................................................. References ...........................................................
130 133 150 154 189 192 198 202 204
Presynaptic Inhibition: Transmitter and Ionic Mechanisms
.
R. A. NICOLLA N D B E. ALCER
. Introduction .........................................................
I I1. 111. IV
.
Presynaptic Inhibition in Invertebrates ................................. Presynaptic Inhibition in Vertebrates ................................... Conclusion ........................................................... References ........................................................... V
217 218 233 251 253
vi
CONTENTS
Microquantitation of Neurotransmitters in Specific Areas of the Central Nervous System JUAN
I. I1. I11 IV .
.
M . SAAVEDRA
Introduction ......................................................... Methods ............................................................. Results. .............................................................. Conclusion ........................................................... References ...........................................................
259 261 270 273 273
Physiology of Glia: Glial-Neuronal Interactions
R. MALCOLMSTEWART AND ROGERN . ROSENBERC I . Introduction ......................................................... I1. Classification of Glial Cells ............................................. 111. Role of Glia in CNS Development ..................................... IV. Potassium and Neuroglial Function .................................... V. Putative Neurotransmitters and Glia .................................... VI . Conclusions .......................................................... References ...........................................................
275 276 278 291 297 304 304
Molecular Perspectives of Monovalent Cation Selective Transmembrane Channels
DANW. URRY
I . Introduction ......................................................... 1I:Channel Models ...................................................... 111. Structure and Relevance of the Gramicidin A Channel .................. IV A Molecular Theory of Electric-Field-Dependent Channel Formation ..... V Perspective of Ion Selectivity Derived from Gramicidin A and Poly-AAG . VI Polymorphism of Channel-Forming Peptides ............................ References ...........................................................
. . .
.
311 312 314 317 324 329 332
Neuroleptics and Brain Self-stimulation Behavior
ALBERTWAUQUIER
.
I Introduction ......................................................... I1 . General Methods ..................................................... I11. Test Procedures and Control Results ................................... IV . The Influence of Neuroleptics on Brain Self-stimulation Behavior ........ V . Implantation Site and Species Differences .............................. VI . Self-stimulation and Psychotropic Assays ................................ VII . Studies on Drug Interaction ........................................... VIII General Discussion .................................................... References ...........................................................
335 337 339 349 365 372 379 391 398
SUBJECT INDEX............................................................. CONTENTS OF PREVIOUS VOLUMES .............................................
405 41 1
.
CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.
B. E. ALGER,Departments o f Pharmacology and Physiology, University o f California, San Francisco, California 94143 (2 17) Ross J . BALDESSARINI, Department ofpsychiatry, Haward Medical School and Mailman Research Center, McLean Division o f Massachusetts General Hospital, I I5 Mill Street, Belmont, Massachusetts 021 78 (1)
THEODORE H. BULLOCK, Neurobiology Unit, Scripps Institution of Oceanography and Department of Neurosciences A-001, School o f Medicine, University o f Calqornia, San Diego, La Jolla, California 92093 (47) F. V. DEFEUDIS,Centre de Neurochimie du CNRS et Institut de Chimie Biologique, Faculti de Mtdecine, Strasbourg Cedex, France ( 129) VLADIMIR S. GUREVICH, Hubbs Sea World Research Institute, San Diego, California (47) R. A. NICOLL,Departments of Pharmacology and Physiology, University o f California, San Francisco, California 941 43 (2 17) ROGERN . ROSENBERG, Department of Neurology, Southwestern Medical School, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235 (275) JUANM. SAAVEDRA, Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20014 (259) R. MALCOLMSTEWART,Department o f Neurology, Southwestern Medical School, The University o f Texas Health Science Center at Dallas, Dallas, Texas 75235 (275) DANIELTARSY, Department o f Neurology, Boston VA Hospital and Boston University School o f Medicine and Division of Neurology, New England Deaconess Hospital and Department of Neurology, Haward Medical School, Boston, Massachusetts (1) DAN W. URRY,Laboratory of Molecular Biophysics and Cardiovascular Research and Training Center, University o f Alabama Medical Center, Birmingham, Alabama 35294 ( 31 1) ALBERTWAUQUIER, Department o f Pharmacology, Janssen Pharmaceutica, B-2340 Beerse, Belgium (335) vii
This Page Intentionally Left Blank
RELATIONSHIP OF THE ACTIONS OF NEUROLEPTIC DRUGS TO THE PATHOPHYSIOLOGY OF TARDIVE DYSKINESIA' By
Ross J. Baldessarini
Dmpadment of Piychiatry, Harvad Wkol School and lllbilman Roawrch Canter k h a n Division of k s w c h u m h n o m I hspitd blmont, Alhsaachuwlrs
~
and Daniel Tony lhpammnt of Nournlogy Boston VA Hosp'kl and Boston Uninnity School of W k i m and Divirbn of Noumbgy Now England Omconors Hopitol and lhpammnt of Noumbgy, Hawold Wkol school Boston, Massochudts
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introductory Comments on the Antipsychotic Drugs . . . . . . . . B. Actions of Neuroleptic-Antipsychotic Drugs . . . . . . . . . . . C. Neurologic Side Effects of Neuroleptic Drugs and the Problem of Tardive Dyskinesia . . . . . . . . . . . . . . . . . . . . . . . . 11. Pathophysiology and Etiology of Tardive Dyskinesia . . . . . . . . . A. Classical Neuropathologic Studies . . . . . . . . . . . . . . . . B. Dopamine Mechanisms . . . . . . . . . . . . . . . . . . . . . . C. Other Neurotransmitters . . . . . . . . . . . . . . . . . . . . . D. Experimental Neuroleptic Dyskinesias . . . . . . . . . . . . . . 111. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. .
. . .
1 2 3 8 12 12 15 30
35 36 37
1. Introduction
The aim of this review is to survey and synthesize the available information concerning late, apparently adaptive responses to the Partially supported by U.S. Public Health Service (NIMH) grants: MH-31154 and MH-30511 and NIMH Research Career Award (to R.J. Baldessarini): MH-47370. This material was, in part, gathered for an American Psychiatric Association task force study on tardive dyskinesia. The manuscript was prepared by Mrs. Mila Cason.
1 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 21
Copyright @ 1979 hy Academic Press, Inc. A11 rights of reproduction in any form reserved. ISBN 0-12-966821-2
2
ROSS J. BALDESSARINI AND DANIEL TARSY
neuroleptic drugs that may bear on the mechanisms underlying the late and persistent or “tardive” dyskinesias associated with their use in the treatment of patients with severe chronic neuropsychiatric illnesses. Before presenting a detailed review of the pathophysiology of this clinically important problem in Section 11, a very brief overview of the current use of the neuroleptic drugs, their neurological side effects, and their currently suspected mechanisms of action is provided in Section I. A. INTRODUCTORY COMMENTS ON
THE
ANTIPSYCHOTIC DRUGS
Antipsychotic agents include compounds proven effective in the management of a broad range of psychotic symptoms and particularly useful in the treatment of schizophrenia and mania. Nearly all produce neurological effects in animals and in patients. The evidence is now overwhelming that this class of substances has real and selective antipsychotic effects in schizophrenia and other disorders marked by abnormalities of thought associations, perceptions, and beliefs (Baldessarini, 1977a,b). Antipsychotic drugs are rarely, if ever, curative, although they are highly effective in hastening remissions of acute psychotic illnesses and also seem to prevent later exacerbations of psychotic symptoms. Since their effects in truly chronic and relentless psychoses such as schizophrenia are not always obvious, a judicious clinical evaluation of the sometimes limited benefits and long-term neurological risks is required. Although the antipsychotic drugs calm excited, agitated, or manic behavior, they are not merely a special kind of sedative, and their older appellation “tranquilizers” is a misnomer. The regular association between antipsychotic effects and extrapyramidal effects suggested the term neuroleptic for this class of drugs. However, the recent description of experimental agents that may have such desirable properties without adverse neurologic effects supports the conclusion that the more general and hopeful term antipsychotic is to be preferred while the search for drugs lacking neurological toxicity is pursued. Throughout this report the two terms “neuroleptic” and “antipsychotic” will be used more or less interchangeably. However, it should be made clear that the former term is less general and comprehensive, although it is appropriate for all antipsychotic agents available in current American medical practice, as they all also produce neurologic effects. The earliest antipsychotic drugs were the phenothiazines and the RauwoGfia alkaloids, notably reserpine ( 1952-53), although the usefulness of lithium salts for the management of excited manic patients had been described earlier ( 1949). The first antipsychotic phenothiazine, chlorpromazine (Largactil), was developed in France ( 1952), and intro-
NEUROLEPTIC DRUGS A N D TARDIVE DYSKINESIA
3
duced into American medicine as Thorazine (1954). At the present time, American practice accepts more than a dozen neuroleptic drugs of scientifically demonstrated clinical value for the treatment of psychoses. These include the phenothiazines of low-milligram potency (not efficacy) (with aliphatic or piperidine side chains in their chemical structures) that have a tendency to induce sedation, hypotension, and other autonomic side effects; the high-potency piperazine phenothiazines, which have relatively greater effects on extrapyramidal function; several nonphenothiazine tricyclic compounds (thioxanthenes and dibenzazepines); the butyrophenones and their still experimental analogues, the oral, relatively long-acting diphenylbutylpiperidines; an indolone; and the now rarely used Rauwoljia alkaloids (Baldessarini, 1977a). Although the antipsychotic o r neuroleptic drugs represent a wide variety of chemical structures, their pharmacology and spectrum of activity are remarkably similar (Baldessarini, 1977a). Thus, all the antipsychotic agents in current use in this country regularly produce a variety of presumably extrapyramidal disorders of the control of posture, muscle tone, and movement. A crucial question is whether the almost routinely encountered neurologic (“neuroleptic”) effects of the antipsychotic drugs are essential to their actions. T h e fact that several effective antipsychotic drugs have relatively little tendency to induce acute neurologic reactions (dystonias, parkinsonism, and restlessness) now strongly challenges the inevitability of the association of neurologic and antipsychotic effects. Such drugs include thioridazine (Mellaril), clozapine and sulpiride (both experimental agents), and their existence offers some hope that better antipsychotic agents with fewer neurologic side effects can be developed. Clozapine is of great theoretical interest as its extrapyramidal side-effect risk is extremely low, but its present status is in great doubt due to its association with agranulocytosis. Both clozapine and thioridazine are strongly anticholinergic; sulpiride is not. An important fact (or artifact) is that the methods of screening new substances for potential antipsychotic utility have essentially involved seeking neurologic reactions in laboratory animals, because there are no satisfactory animal tests for schizophrenia. This impasse, coupled with the current conservatism of the system for development and testing of new agents, particularly in the United States, has contributed to a repeated “rediscovery” of agents with very similar actions and limitations over the past 25 years. B. ACTIONS OF NEUROLEPTIC-ANTIPSYCHOTIC DRUGS In the past, a number of mechanisms had been proposed to explain the actions of the antipsychotic drugs. They differ from most other
4
ROSS J . BALDESSARINI AND DANIEL TARSY
depressants of the central nervous system (CNS) in several ways. Thus, they have limited ability to induce generalized sedative effects or coma until enormous overdoses are taken; in addition, tolerance to their antipsychotic effects is unknown, and addiction virtually does not occur. Unlike sedatives, they have been reported to have greater ability to diminish conditioned behavioral response than to depress unconditioned responses. They may have a selective ability to dampen the neurophysiologic effects of peripheral stimuli on the forebrain, while inhibiting to a much lesser extent the effects of stimulating electrodes placed in the brain stem. In addition to these distinctions from sedatives, antipsychotic drugs have striking inhibitory effects on autonomic and motoric expressions of arousal and strong affect in animals, presumably mediated by actions in the limbic forebrain and hypothalamus. T h e cellular and biochemical events underlying these behavioral and physiologic actions, however, have remained obscure until recently. It was proposed by European pharmacologists as long ago as the early 1960s that the neurologic, and possibly also the antipsychotic, effects may reflect the ability of neuroleptic drugs to interfere with synaptic transmission in the brain mediated by dopamine (Baldessarini, 1977b). This suggestion arose largely from the observation that among the biochemical consequences of giving a neuroleptic drug to an animal, there was a consistent increase in levels of the metabolites of dopamine, but variable effects on the metabolism of other candidate neurotransmitters. The possible importance of dopamine was given strong support by early histochemical studies of the normal distribution of aminecontaining neurons in the mammalian brain, which indicated a preferential distribution of dopamine fibers between midbrain and the basal ganglia (notably, the nigroneostriatal tract), and within the hypothalamus. More recently, anatomists have come to appreciate the existence of other dopamine projections from midbrain nuclei to forebrain regions that are associated with the limbic system and probably not primarily with the extrapyramidal motor system, as well as to temporal and prefrontal cerebral cortical areas closely interlinked with the limbic system (Fig. 1). A somewhat simplistic, but attractive, concept has been that many extrapyramidal neurologic effects of the antipsychotic drugs may be mediated by antidopamine effects in the basal ganglia, and that some of their antipsychotic effects may be mediated by the antagonism of “dopaminergic” neurotransmission in the limbic system, hypothalamus, or cortex. The latter supposition has been given indirect general encouragement by repeated “natural experiments” that have associated psychotic mental phenomena with lesions of the temporal lobe and other portions of the limbic system.
NEUROLEPTIC DRUGS AND TARDIVE DYSKINESIA
5
FIG. 1. Dopamine-containing neurons in the mammalian brain. The major systems involving dopamine are: the nigrostriulal pathway from the zona compacta of the midbrain substantia nigra to the neostriatum (caudate-putamen): mesolimbic projections from midbrain tegmentum through the lateral hypothalamus to limbic structures, including the septa1 nuclei (e.g., nucleus accumbens septi) and olfactory tubercle: and related mesocurtical projections, also arising in midbrain, and projecting particularly to prefrontal and temporal areas of the cerebral cortex: there is also a tuberoinfundibulur dopamine-containing (TIDA) system within the hypothalamus. The upper scheme is based on extensive studies in rat, but the anatomy is very similar in man, as depicted in the lower diagram.
6
ROSS J. BALDESSARINI A N D DANIEL TARSY
In recent years, a large body of data has accumulated to support the theory that the antagonism of dopamine-mediated synaptic neurotransmission is an important action of antipsychotic-neuroleptic agents (Baldessarini, 1977a); these effects on dopamine systems are summarized in Table I, and many aspects of them will be reviewed in detail below. Thus, antipsychotic agents, but not their nonantipsychotic congeners, are reported to increase the rate of production of dopamine metabolites (notably, dihydroxyphenylacetic and homovanillic acids), the rate of conversion of the precursor amino acid tyrosine through dopa to dopamine and its metabolites, and the firing rate pf presumably dopamine-containing neuronal cell bodies in midbrain. These effects have been interpreted as secondary or compensatory responses of plastic and adaptive neuronal systems attempting to maintain homeostasis in the face of what is assumed to be a primary interruption of synaptic transmission at the dopamine terminals in the caudate nucleus, septa1 nuclei, and cerebral cortex. Figure 2 shows the metabolic arrangements at such synapses. Evidence that a crucial primary event may be the blockade of postsynaptic dopamine receptor sites includes the ability of small doses of antipsychotic agents to block behavioral or neuroendocrine effects of dopamine agonists. Examples are stereotyped gnawing behavior in the rat induced by the putative direct dopamine agonist, apomorphine, possibly acting at the caudate nucleus; the locomotor excitement induced by the injection of dopamine into the nucleus accumbens septi of the limbic system; or the prolactin-decreasing response to apomorphine or L-dihydroxyphenylalanine(L-dopa, the immediate precursor of dopamine) believed to be mediated by hypothalamic or pituitary dopamine receptors. Such “tests” have been proposed as screening methods to detect even more agents of the kinds already available. More direct evidence of a receptor blockade has been provided by the antagonism of an apparently selective dopamine-sensitive adenylate TABLE I EFFECTS OF NEUROLEPTIC DRUGSON CNS DOPAMINE NEURONS~.~ 1. Block DA receptors 2. May alter DA release 3. Increase DA cell-firing rate in midbrain 4. Increase turnover of DA in forebrain (HVA increases) 5. TIDA-neuroendocrine effects (e.g., PL increases) a Abbreviations: DA: dopamine, HVA: homovanillic acid, PL: prolactin, TIDA: tuberoinfundibular hypothalamic DA system. For references, see Baldessarini (1977a) and Carlsson (1978).
NEUROLEPTIC DRUGS A N D TARDIVE DYSKINESIA
PRESYNAPTIC NEURON
POSTSYNAPTIC CELL
1 ( ‘Dopamine
I’
7
receptor
1) Hornovanillic acid
I Cerebrospinal Fluid and Circulation
FIG. 2. Metabolism at dopamine synapse in the brain. Dopamine is formed from L-tyrosine by hydroxylation (the rate-limiting step) to L-dihydroxyphenylalanine (dopa), which is rapidly decarboxylated. Dopamine is stored in presynaptic vesicles (shaded circle), from which release occurs into the synaptic cleft by neuronal depolarization in the presence of calcium. The released amine has a postsynaptic effect, possibly mediated by a recognition molecule (receptor), associated with adenylate cyclase, that converts adenosine triphosphate (ATP) to adenosine3’,5‘-(cyclic)monophosphate(cyclic AMP), which may, in turn, exert biochemical effects leading to altered neurophysiological sensitivity in the receptive cell. Neurotransmitter is inactivated largely by efficient high-affinity reuptake into the presynaptic terminals; excess dopamine that is not stored can be metabolized by monoamine oxidase (MAO) in the mitochondria and catechoI4-methyl-transferase (COMT), largely extraneuronal, to produce homovanillic acid from dihydroxyphenylacetic acid, an intermediary metabolite; the metabolites are removed in the cerebrospinal fluid and venous circulation by a probenecid-sensitive uptake, largely at the choroid plexus.
cyclase in homogenates of caudate or limbic tissue, and the interference with electrophysiological responses to dopamine iontophoretically applied to receptive cells in the caudate nucleus-a blockade overcome by presumed circumvention of the receptor sites on the cell surfaces by iontophoresed cyclic-AMP analogues (see Tarsy and Baldessarini, 1977; see also Fig. 2). A more recent development is the application of radioligand binding assays using homogenates of mammalian caudate nucleus and low concentrations (nanomolar or lo+’ M ) of intensely radioactive f3HI-dopamine, 13H]-labeledneuroleptic drugs (haloperidol or spiroperidol),or [3Hl-apomorphine(Creese and Snyder, 1978; Snyder et al., 1978). Pharmacologic evidence supports the suggestion that the binding of these ligands to brain tissue represents, at least partially, an
8
ROSS J. BALDESSARINI A N D DANIEL TARSY
interaction with a dopamine “receptor” site. Correlations are impressive between the in vitro potency of antipsychotic drugs of all types to interfere with the binding of such ligands and estimates of their potency to block the effects of dopamine agonists in animals or to produce clinical benefits in psychotic patients (Baldessarini, 1977b; Creese and Snyder, 1978). Analogues or isomers of the antipsychotic drugs that are clinically inactive lack this potent antagonistic effect against ligand binding. It is particularly interesting that two antipsychotic agerlis (thioridazine and clozapine) with relatively weak acute neurologic side effects seem to have antidopamine effects in the ligand-binding assays that correlate closely with their clinical potencies. Although their relative lack of extrapyramidal toxicity has been explained by a countervening antimuscarinic (antiparkinsonism?) action of these two drugs, this explanation is not satisfactory for a similar drug, sulpiride. These findings together strongly support the theory that antipsychotic agents interfere with the actions of dopamine as a synaptic neurotransmitter in the brain. Nevertheless, they do not prove that antidopamine effects are either necessary or sufficient for antipsychotic efficacy. They strongly suggest, however, that some of the extrapyramidal neurologic effects of this class of agents may be produced by antagonism of dopamine, largely on the basis of analogy to the demonstrated loss of dopamine in the caudate nucleus, and the beneficial responses to its precursor L-dopa in idiopathic parkinsonism. C. NEUROLOGICSIDEEFFECTS OF NEUROLEPTIC DRUGSAND PROBLEM OF TARDIVE DYSKINESIA
THE
T h e common and sometimes troublesome neurologic side effects of most antipsychotic drugs represent a unique constellation of syndromes not associated with other psychotropic agents. These reactions can be subdivided into several categories, as outlined in Table 11. Other classes of psychotropic drugs, including sedatives (e.g., barbiturates), tranquilizer-antianxiety agents (e.g., benzodiazepines such as diazepam [Valium]), and even tricyclic antidepressarits and lithium salts are more likely to exert toxic effects in the CNS, especially on overdosage, with generalized depressant effects, toxic delirium, and eventually coma and death. The antidepressants are also likely to produce atropine-like signs and symptoms on acute overdosage (Granacher and Baldessarini, 1975). Thus, for the antipsychotic agents, CNS toxicity is expressed in several characteristic syndromes. Except for parkinsonism, which is believed to reflect diminished functional activity of dopamine in the basal ganglia, the pathophysiology of these reactions is still poorly understood,
TABLE I1 NEUROLOGICAL SIDEEFFECTSOF NEUROLEPTIC-ANTIPSYCHOTIC DRUGS Reaction
Features
Maximum risk
Antiparkinson agents are diagnostic and curative (i.m. or i.v.; then p.0.)
5-30 days
DA blockade
Antiparkinson agents help (p.0.)
Motor restlessness; not anxiety or agitation
5-60 days
Unknown
Reduce dose or change drug; antiparkinson agents or benzodiazepines may help
Oral-facial dyskinesia; choreoathetosis
Mos.-yrs. DA excess (worse on withdrawal)
Prevention best; treatment unsatisfactory
Perioral tremor (late Parkinsonism variant?)
Mos.-yrs.
Antiparkinson agents may help
Spasm of muscles of tongue, face, neck, back; may mimic seizures; not hysteria
1-5
Parkinsonum'
Bradykinesia, rigidity, variable tremor, mask-facies, shuffling gait
Akathisia' Tardive dyskinesiaa.'
Perioral tremor ('t-abbit" syndrome) a*d
Treatment
Unknown
Acute dystaiaag
Marsden et al. (1975). Garver et al. (1976). ' Baldessarini and Tarsy (1978). Jus el al. (1974).
days
Proposed mechanism
Unknown
10
ROSS J. BALDESSARINI AND DANIEL TARSY
although it is suspected that effects on dopamine-mediated systems in the basal ganglia are involved (Table 11). The first clinical reports recognizing the occurrence of late and persistent dyskinesias following prolonged treatment with phenothiazines were published in the late 1950s (Schocker, 1957; Sigwald et al., 1959). Thereafter, a number of reports appeared describing the occurrence of similar persisitent dyskinesias, nearly always among chronic psychiatric patients, in the course of treatment with phenothiazine antipsychotic drugs (Uhrbrand and Faurbye, 1960; Druckman et al., 1962; Hunter et al., 1964; Schmidt and Jarcho, 1966; Degkwitz, 1967). Initial reports emphasized the orofacial distribution of the dyskinesia and referred to it as a “bucco-linguo-masticatory syndrome” (Sigwald et al., 1959; Uhrbrand and Faurbye, 1960); it was later referred to as “terminal extrapyramidal insufficiency syndrome” (Haddenbrock, 1964); the now commonly used term “tardive dyskinesia” (late and persistent dyskinesia) was introduced by Faurbye and his colleagues in the 1960s (Faurbye et al., 1969). The subject has been extensively reviewed (Crane and Gardner, 1969; Kazamatsuri et al., 1972; Klawans, 1973a,b; Marsden et al., 1975; Tarsy and Baldessarini, 1976, 1977; Baldessarini and Tarsy, 1978). Tardive dyskinesia was initially regarded as a complication of antipsychotic drug therapy largely restricted to elderly, chronically institutionalized, frequently brain-damaged patients receiving prolonged drug treatment. In recent years, however, the problem of tardive dyskinesia as a more general public health problem of major proportions, which may interfere with the ultimate rehabilitation of the patient in his community, has been given strong emphasis (ACNP-FDA, 1973; Crane, 1973a,b). T h e syndrome of tardive dyskinesia consists of involuntary or semivoluntary movements of a choreiform (ticlike) nature, sometimes with an athetotic or dystonic component. These classically affect the tongue, facial, and neck muscles but often also affect the extremities, digits, and muscles that control posture and sometimes those used in breathing. Early signs of tardive dyskinesia are movements of the tongue or extremities. Oral-lingual-masticatory movements are common, especially in older patients; it is usual to find abnormalities of posture and at least subtle choreiform movements of the fingers as well, especially in younger patients. The movements of tardive dyskinesia are much less voluntary and purposeful and more classically choreoathetotic than the stereotyped mannerisms and posturing that occur spontaneously in schizophrenia (Marsden et al., 1975). They usually become worse if the antipsychotic agent is withdrawn and can be suppressed, at least tem-
NEUROLEPTIC DRUGS AND TARDIVE DYSKINESIA
11
porarily, by readministering a neuroleptic drug or an amine-depleting agent. At the present time, it seems clear that the prolonged use of antipsychotic (neuroleptic) drugs can lead to the development of late dyskinetic disorders which may be either transient or persistent (Marsden et al., 1975). Tardive d yskinesia has been reported in patients exposed to virtually all of the phenothiazines (although relatively infrequently with piperidines such as thioridazine; Tarsy and Baldessarini, 1976), occasionally a thioxanthene (Anath and Costin, 1977; Firestein, 1977) or a butyrophenone (Castaigne et al., 1969; Jacobson et al., 1974), but has only rarely been associated with exclusive exposure to reserpine (Wolf, 1973; Tarsy and Baldessarini, 1977). While similar involuntary movement disorders occur in various known or presumed brain disorders, repeated exposure to neuroleptic agents by itself is probably adequate to induce the syndrome of tardive dyskinesia. Evidence that antipsychotic drugs are directly responsible for tardive dyskinesia is mainly epidemiologic, but appears to be quite convincing (Crane, 1973a,b; Baldessarini, 1974; Tarsy and Baldessarini, 1976). Epidemiologic studies strongly support an association between the use of neuroleptic drugs and the development of persistent or relatively transient forms of tardive dyskinesia. While there is some evidence that prolonged exposure or exposure to large total amounts of drug may be a contributing factor, this is not established, and a correlation with the size of average doses is extremely weak. Older patients, and possibly females, may be at somewhat higher risk and may have a somewhat poorer prognosis for eventual remission following prolonged removal of the suspected offending agents, and the aging brain may present an increased risk for neuroleptic-related tardive dyskinesias, especially of the oral region. In a general way, prolonged neuroleptic exposure among chronic psychiatric populations and the recent tendency to use relatively high doses of antipsychotic agents for prolonged periods in the United States may contribute to prevalence rates here that seem to be higher than those in many other countries. On the other hand, specific predictors of risk (such as type of drug and dose, timing of exposure, prior experience of acute reversible extrapyramidal symptoms, use of antiparkinson agents, or sex) do not reveal clinically wuseful risk factors, with the possible exception of advanced age, that might guide medical practice. Moreover, it is clear that similar exposure to neuroleptics neither produces tardive dyskinesia in all patients nor are any groups (as defined by age, drug history, etc.) exempt from risk. Although the association between neuroleptic agents and late tran-
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ROSS J. BALDESSARINI AND DANIEL TARSY
sient or persistent dyskinesias is now quite clear, the mechanisms by which these drugs might induce such neurotoxic actions remain unknown (Baldessarini and Tarsy, 1976; Tarsy and Baldessarini, 1977). One of the leading hypotheses concerning the basis of tardive dyskinesia is that prolonged blockade of dopamine receptors may lead to a paradoxical increase in the functional activity of dopamine in the basal ganglia of the brain (Klawans, 1973a; Tarsy and Baldessarini, 1973, 1974), as will be discussed in detail below. This altered functional state might come about through the phenomenon of disuse supersensitivity of dopamine receptors (Schelkunov, 1967; Klawans, 1973a; Tarsy and Baldessarini, 1973, 1974). T h e principal clinical support for this hypothesis is provided by several aspects of the natural history of the disorder: late onset after prolonged exposure to neuroleptics, tendency to worsen (at least temporarily) on abrupt discontinuation of the neuroleptic, and a differential response to drugs (Table 111) that is virtually opposite to that of parkinsonism, a condition widely held to be due, at least in part, to a deficiency of the availability of dopamine to its receptors in the caudate nucleus. Thus, drugs that decrease the availability or activity of dopamine tend to decrease the signs of tardive dyskinesia, but to worsen parkinsonism, while dopamine agonists tend to have the opposite effects in each condition, respectively. Anticholinergic agents, while generally moderately helpful in parkinsonism, tend to make tardive dyskinesia worse, although effects on cholinergic systems are probably of secondary importance in both conditions (Marsden et al., 1975).
II. Pathophysiology and Etiology of Tardive Dyrkinesia
A. CLASSICAL NEUROPATHOLOGIC STUDIES The prolonged and frequently irreversible course of tardive dyskinesias strongly suggests that permanent structural alterations of the brain may be responsible for this disorder. However, neuropathologic studies following acute or prolonged administration of antipsychotic drugs in laboratory animals (Mackiewicz and Gershon, 1964; Popova, 1967; Romasenko and Jacobson, 1969; Colon, 1975) have not demonstrated specific or localized pathologic changes in the brain beyond those which might be produced secondarily by the diverse systemic effects of these drugs (Roizin et al., 1959; Jalou et al., 1968). A report (Pakkenberg et al., 1973) of an 18% reduction of neuronal cell counts in the neostriatum of rats treated for 1 year with perphenazine enanthate is of
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TABLE 111 THEDIFFERENTIAL PHARMACOLOGY OF TARDIVE DYSKINESIA".~ Agents that may partially suppress tardive dyskinesia Dopamine antagonists Apomorphine (in low dose) Butyrophenones Clozapine Papaverine (mechanism uncertain) Phenothiazines Pimozide Amine-depleting agents Reserpine Tetrabenazine Blockers of catecholamine synthesis a-Methyldopa a-Methyltyrosine Blockers of catecholamine release Lithium salts Cholinergic agents Deanol (mechanism uncertain) Physostigrnine Choline and Lecithin "GABA agonists" Valproate (mechanism uncertain) Baclofen (mechanism uncertain) Agents with variable, negligible, or uncertain effects a-Meth yldopa Amantadine Antihistamines Barbiturates Benzodiazepines Methylphenidate Penicillamine Physostigmine Pyridoxine (B,) Tryptophan Agents that worsen tardive dyskinesia Anticholinergic agents Antiparkinsonism agents (e.g., benztropine) Dopamine agonists Amphetamines L-dopa Other Agents Phenytoin Some drugs appear in more than one category, reflecting ambiguity in the literature. Note that while apomorphine is usually classed as a dopamine agonist, it actually has complex mixed actions, may antagonize DA at low doses, and has clear antidyskinetic effects. For references, see Baldessarini and Tarsy (1978).
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uncertain significance since gliosis and degenerative changes of remaining neurons were not present. Moreover, subsequent studies using much larger doses of perphenazine enanthate for 6 months had no effect on cell counts in neostriatum or substantia nigra (Gerlach, 1975; Fog et al., 1976).
Human neuropathologic studies have been few and sometimes limited in design as regards including tissue from age-matched control subjects, the use of quantitative and objective methods (as for cellcounting), the inappropriate inclusion of patients with known progressive dementing neurologic diseases, which occasionally can be confused with schizophrenia with a movement disorder (e.g., Huntington’s disease) (Jellinger, 1977; Kaufman, 1977), and problems of discounting degenerative effects of aging (Hunter et al., 1968; Christensen et al., 1970; Jellinger, 1977). In man, postmortem neuropathologic changes following chronic neuroleptic treatment in patients without extrapyramidal syndromes have usually consisted of scattered areas of neuronal degeneration and gliosis in the basal ganglia and other brain regions without convincing localization (Roizin et al., 1959; Forrest et al., 1963). Individual patients with drug-induced parkinsonism or tardive dyskinesia have been reported to have abnormalities in the globus pallidus and putamen (Poursines et al., 1959), caudate nucleus and substantia nigra (Gross and Kaltenbach, 1968), and inferior olive (Griinthal and Walther-Buel, 1960). Hunter et al. (1968) reported no significant neuropathologic abnormality in three patients with tardive dyskinesia, although two of these showed, among other lesions, neuronal degeneration in the substantia nigra which was believed to be consistent with their advanced age. In one of the few neuropathologic studies of a large number of patients with tardive dyskinesia, Christensen et al. (1970) reported neuronal degeneration and gliosis of the substantia nigra in 27 of 28 brains from elderly patients (mean age, 74 years) with chronic oral dyskinesias, 2 1 of which were attributed to antipsychotic drugs; only 7 of 28 control brains matched for age and psychiatric diagnosis, but lacking a history of dyskinesia, showed similar changes. Other careful and controlled neuropathologic studies of large numbers of patients with tardive dyskinesia are still rare, but several have failed to replicate these findings, especially among younger patients, while one found evidence of degenerative changes in the caudate nucleus (Jellinger, 1977). Since prolonged administration of perphenazine enanthate to rats reportedly produced no changes in cell counts or morphology in the substantia nigra (Gerlach, 1975; Foget al., 1976), and since degenerative changes of the basal ganglia and midbrain occur in some elderly individuals without tardive dyskinesia (Jellinger, 1977), it has been suggested
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15
that preexisting nigral changes, unrelated to effects of neuroleptic drugs and possibly accompaniments of aging, predispose in some poorly understood way to the subsequent appearance of tardive dyskinesia (Christensen et al., 1970; Gerlach, 1975). In view of the fact that even in several other extrapyramidal neurologic diseases, relatively moderate or apparently absent light microscopic histopathologic changes occur, it should probably not be surprising that to date conventional neuropathologic studies as well as computerized axial tomography (CAT scanning) of the brain of such patients (Gelenberg, 1976) have not revealed obvious structural defects in tardive dyskinesia. Although one Turkish group reported pneumoencephalographic evidence of cortical atrophy (without dementia) in 1 1 out of 30 chronic schizophrenics under the age of 50 exposed to prolonged neuroleptic treatment (and in several other patients treated with antidepressants for prolonged periods) (Sabuncu et al., 1977), this observation is quite surprising as it is not consistent with earlier neurologic evaluations of chronic schizophrenics, and it requires further critical evaluation. Electron microscopic studies of postmortem brain tissue in tardive dyskinesia has scarcely been initiated up to this time (Jellinger, 1977). Very recently, newer biochemical approaches have been made to the problem of postmortem changes in the brain tissue of patients exposed to neuroleptic drugs, including assays of neurotransmitter concentrations, activities of their synthesizing enzymes, and of the binding of radiolabeled ligands to their presumed receptor sites. As these studies have grown out of the dopamine supersensitivity hypothesis, they will be discussed below. B. DOPAMINE MECHANISMS Several clinical observations have indicated that the syndrome of tardive dyskinesia may be associated with a pathophysiologic state of relative dopaminergic overactivity. Thus, it tends to worsen on withdrawal of the antipsychotic-neuroleptic drugs, which are believed to be antagonistic to dopamine receptors (Carlsson and Lindqvist, 1963; York, 1972; Matthysse, 1973; Karobath and Leitich, 1974; Bunney et al., 1975; Seeman et al., 1975, 1976; Costal1 and Naylor, 1976; Creese et al., 1976a,b; Siggins et al., 1976; Baldessarini, 1977a; Iversen, 1977) and may also have a lesser effect of antagonizing the release of this central synaptic neurotransmitter (Seeman and Lee, 1975). Furthermore, tardive dyskinesia often resembles the dyskinesias and ticlike disturbances produced by L-dopa in patients with Parkinson’s disease, and the dyskinesias and tics produced by amphetamines (Mattson and Calverly,
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1968) or other stimulants (indirect dopamine agonists) in some patients with underlying brain dysfunction or tic disorders (Golden, 1977), or in amphetamine addicts (Rylander, 1972). Moreover, administration of d-amphetamine (Smith et al., 1977) or of L-dopa, the immediate precursor of dopamine, usually exacerbates tardive dyskinesia (Hippius and Lange, 1970; Chase, 1972; Gerlach et al., 1974; Paulson et al., 1975), although low doses (Carroll et al., 1977) o r repeated treatment (Alpert and Friedhoff, 1976) may have apparently paradoxical beneficial effects. T h e fact that tardive dyskinesia may be suppressed by treatment with drugs that deplete or block the action of dopamine (see Table 111), such as tetrabenazine, reserpine, phenothiazines, and butyrophenones (Kazamatsuri et al., 1972, 1973), as well as a-methyl-p-tyrosine (Chase, 1972; Gerlach et al., 1974), provides additional support for a state of relative excess of dopaminergic function in the CNS. Since improvement after use of antidopaminergic agents may occur without the appearance of clinically significant parkinsonism (Kazamatsuri et al., 1972), it is not clear that the benefits are due solely to the superimposition of a hypokinetic state. One possible mechanism to account for the apparent functional overactivity of central dopaminergic mechanisms in tardive dyskinesia is the development of denervation or disuse supersensitivity of dopamine receptors (Schelkunov, 1967; Klawans, 1968, 1973a,b; Gyorgy et al., 1969; Carlsson, 1970; Tarsy and Baldessarini, 1973, 1974). Since drugs responsible for tardive dyskinesia appear to produce pharmacologic blockade of transmission at dopamine-mediated synapses, disuse supersensitivity-mediated alterations of receptors may be a long-term consequence of their use. There is a good deal of experimental evidence to support the occurrence of synaptic supersensitivity in the CNS in general (Sharpless, 1964, 1975; Tarsy and Baldessarini, 1974) and in striatal dopamine-mediated synapses in particular (Baldessarini and Tarsy, 1976; Tarsy and Baldessarini, 1974, 1977). 1. Denmation or Disuse Supersensitivity Behavioral Evidence Supporting
the Concept A number of behavioral studies lend support to the existence of this phenomenon. After unilateral destruction of the nigrostriatal tract in rats (Ungerstedt, 1971) or the striatum in mice (Pycock et al., 1975; Thornburg and Moore, 1975), L-dopa o r low doses of the putative direct dopamine-receptor agonist apomorphine produced contralateral turning behavior consistent with enhanced sensitivity of the denervated striatum. Intraventricular administration of 6-hydroxydopamine to newborn rats resulted in the appearance of stereotyped behavior at 3
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17
months following doses of apomorphine found to be ineffective in control rats (Creese and Iversen, 1973). Stereotyped behavioral responses to apomorphine were also increased by 6-hydroxydopamineinduced nigrostriatal lesions of adult rats (Price and Fibiger, 1974). Furthermore, the administration of L-dopa or apomorphine to monkeys pretreated with intraventricular o r intracaudate 6-hydroxydopamine resulted in the appearance of abnormal lip and tongue movements, chorea, dystonia, and hemiballism that did not appear in animals given a control injection (Ng et al., 1973). Further behavioral evidence accumulated in several animal species in recent years (Miiller and Seeman, 1978) indicates that prolonged administration of antipsychotic drugs (Schelkunov, 1967; Stolk and Rech, 1968; Klawans and Rubovits, 1972a; Tarsy and Baldessarini, 1973, 1974; Moller-Nielson et al., 1974; Gianutsos et al., 1974; Jackson et al., 1975; Von Voigtlander et al., 1975; Sayers et al., 1975a,b, 1976; Sahakian et al., 1976; Smith and Davis, 1976; Waldmeir and Maitre, 1976; Gianutsos and Moore, 1977; Scatton, 1977; Costal1et al., 1978) or other agents (Tarsy and Baldessarini, 1974) antagonistic to dopamine, followed by their discontinuation, also produces a behavioral state of increased responsiveness to dopamine agonists. These effects appear to represent the development of functional supersensitivity to dopamine in the brain, and this state persists for at least several weeks (Klawans and Rubovits, 1972a; Tarsy and Baldessarini, 1974; Sahakian et al., 1976). T h e duration of supersensitivity may to some extent parallel the duration of pretreatment with a neuroleptic agent (Muller and Seeman, 1978). Additional studies reveal that prolonged exposure of animals to a neuroleptic agent leads to a prolonged (weeks) requirement for increased doses of the same agent (“tolerance”) (Asper et al., 1973) or a dissimilar neuroleptic agent (“cross-tolerance”) (Moller-Nielson et al., 1974) to block the behavioral effects of apomorphine. It has also been noted recently that repeated treatment with a neuroleptic agent led to increasing tolerance toward the ability of the same or dissimilar neuroleptics to block electrically induced selfstimulation of the brain through an electrode in the nigrostriatal pathway of the rat (Eichler et al., 1979). Moreover, on discontinuation of treatment, there was a marked and prolonged (more than 1 month) increase in the self-stimulation response (Eichler el al., 1979). The development of increased rates of self-stimulation during treatment with neuroleptics has also been noted in frontal cortex (Eichleretal. , 1977),but development of such tolerance to neuroleptic blockade in the nucleus accumbens remains uncertain (Eichler et al., 1979); the latter region is currently considered to be mainly “limbic” rather than extrapyramidal in
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function, and suspected of being an important site of mediation of the antipsychotic effects of neuroleptic drugs (Stevens, 1973; Baldessarini, 1977a; Bunney and Aghajanian, 1978).Inconsistent with this hypothesis, however, is some recent evidence which strongly suggests that limbic supersensitivity can follow prolonged neuroleptic pretreatment, albeit perhaps less easily than in the striatum (Scatton, 1977). This evidence arises from evaluation of behavior by local injection of dopamine into the nucleus accumbens (Jackson et al., 1975; Ungerstedt and Ljungborg, 1977),or by the elicitation of locomotor activity with low systemic doses of apomorphine (Ungerstedt and Ljungborg, 1977). It is also evident that accumbens supersensitivity to a direct dopamine agonist follows lesioning of accumbens dopamine terminals by local microinjections of 6-hydroxydopamine (Ungerstedt and Ljungborg, 1977), sometimes given after protection of norepinephrine cells by desmethylimipramine (Kelly and Moore, 1977). The development of tolerance to neuroleptic blockade of self-stimulation induced by an electrode in frontal cortex (Eicher et al., 1979) also seems to fail to support a crucial role of cortical dopamine projections in the mediation of antipsychotic effects of these drugs. These findings suggesting limbic or cortical tolerance or supersensitive dopamine responses apparently fail to support a dopamine hypothesis for antipsychotic drug action (Baldessarini, 1977a) because tolerance to these drugs in their clinical use in the psychoses, and gradual worsening of psychosis (Ungerstedt and Ljungborg, 1977) (“tardive psychosis”) are unknown. These several behavioral observations concerning responses after repeated exposure to neuroleptic drugs in animals do, overall, seem to be consistent with the phenomenon of increased sensitivity to dopamine, possibly mediated by increased sensitivity of postsynaptic receptor and effector mechanisms in response to disuse (or frank denervation) of dopamine-mediated synapses. Such behavioral experiments have led to applications with potential practical, and even clinical, significance. Thus, they have been applied in a growing number of studies aimed at predicting the clinical efficacy of experimental therapeutic approaches to the dyskinesias. Many examples of the application of this strategy will appear later in this review. However, as one intriguing example of this approach, it has been reported recently that lithium given simultaneously with prolonged neuroleptic treatment in the guinea pig (Klawans et al., 1977) or rat (Pert et al., 1978) can prevent the evolution of supersensitive behavioral responses to dopamine agonists, but not block them once developed (Klawans et al., 1977). Furthermore, lithium treatment can prevent the increased binding of labeled neuroleptic drugs to presumed receptor sites in brain tissue that will be discussed
NEUROLEPTIC DRUGS AND TARDIVE DYSKINESIA
19
presently (Pert et al., 1978). Lithium salts have little effect on tardive dyskinesia, although it is not known whether lithium might have a protective effect in man if given with neuroleptics before tardive dyskinesia develops. A second practical point to be emphasized arises from the growing behavioral or biochemical evidence demonstrating impressive regional differences in the extrapyramidal and limbic effects of typical neuroleptic agents and those “atypical” antipsychotic agents (such as clozapine and sulpiride) with less tendency to induce neurological side effects. An implication of such observations is that they may permit the detection of new experimental agents that act selectively on limbic tissues, permitting the development of new drugs that are selectively antipsychotic and less neurotoxic than existing neuroleptics (Costal1 and Naylor, 1976; Ungerstedt and Ljungborg, 1977; Eichler et al., 1979).
2 . Disuse Supersensitivity Behavioral Results That May Not Be Consistent with the Hypothesis There are several recent observations that may or may not call into question the significance of drug-induced behavioral supersensitivity to dopamine agonists as a possible “laboratory animal model” of mechanisms underlying tardive dyskinesia, but that do provide further insights into the characteristics of tissue responses to agents that interact with catecholamine-mediated synaptic transmission in the CNS. One type of observation questions the requirement of prolonged exposure to neuroleptics to induce supersensitivity. There are recent studies demonstrating biochemical and behavioral evidence of clear increases in this sensitivity after even a single dose of a neuroleptic (Hyttel, 1977; Martres et al., 1977)-increases that may persist for many hours or even several days (Martres et al., 1977) and which are prevented by drugs that block protein synthesis (Costentin et al., 1977). Such rapid changes in catecholamine receptor sensitivity are not unprecedented, as diurnal changes of considerable physiological significance have been documented in the /3-adrenergic receptors of the pineal gland, evidently as a reflection of diurnal changes in availability of norepinephrine from sympathetic fibers innervating this organ (Kebabian et al., 1975). Even more puzzling are seemingly paradoxical increases in responsiveness to dopamine after prolonged (Costentin et al., 1975; Klawans and Margolin, 1975; Friedman et al., 1975; Segal, 1975; Rebec and Segal, 1977) or even brief (Martres et al., 1977) exposure to certain dopamine agonists. This phenomenon remains to be explained in light of other recent studies that indicate that dopamine supersensitivity induced by neuroleptics can be prevented o r reversed by treatment with
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an agonist or precursor such as L-dopa and that agonists alone, if anything, may slightly decrease binding of ligands supposedly indicative of dopamine receptors (Friedhoff et al., 1977). T h e sensitivity-enhancing effect of agonists (amphetamine and apomorphine have most commonly been studied) might reflect presynaptic actions of such drugs as amphetamine and apomorphine, which may reduce the synthesis, turnover, and release of dopamine (Carlsson, 1975; Harris et al., 1975), possibly at lower doses than required for postsynaptic effects (Carlsson, 1975; Martres et al., 1977). Amphetamine, at least, can exert this effect for many hours after its initial dopamine-enhancing and behaviorally stimulating actions (Rebec and Segal, 1977).Alternatively, apomorphine and other newer dopamine agonists may be “partial agonists” at dopamine receptors, possibly leading to a net reduction in effects in competition with the natural agonist, dopamine (Goldberg, 1975; Baldessarini et al., 1976). Apomorphine might also act by selectively reducing sensitivity of presynaptic receptors to dopamine, so as to facilitate the turnover and release of dopamine (Martres et al., 1977; Worms and Scatton, 1977). The paradoxical effect of dopamine agonists to induce further supersensitivity suggests the need for a cautious attitude toward recently suggested therapeutic approaches to tardive dyskinesia (and even schizophrenia) using low doses of dopamine agonists (notably, L-dopa or apomorphine) for their apparent antidopamine effects (Carroll et al., 1977), or by repeated and gradually increasing exposure to dopamine agonists such as L-dopa in the hope of “resuppressing” dopamine receptors and thus diminishing their sensitivity (Alpert and Friedhoff, 1976). Since it has recently been found that, in contrast to apomorphine itself (Sahakian et al., 1976), long-acting ester “prodrugs” of apomorphine (Baldessarini et al., 1977) induce tolerance to their behavioral and dopamine-turnover suppressing effects (Worms and Scatton, 1977), they may suppress dopamine receptor sensitivity, and so be potentially useful for the treatment of tardive dyskinesia, or even of psychoses. Another feature of doparnine supersensitivity that must be considered in the attempt to evaluate animal models of mechanisms underlying tardive dyskinesia is the differential ability of various classes of neuroleptics to induce supersensitivity. Since certain “atypical” neuroleptics (notably thioridazine, clozapine, and sulpiride) are relatively less likely to induce acute extrapyramidal effects in patients, or presumably analogous actions in animals (such as inducing catalepsy or antagonizing dopamine receptor agonists), it has been suggested that they may produce tardive dyskinesia less often than other neuroleptic agents. This prediction seems to be supported by clinical experience with thioridazine as corn-
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21
pared with many other antipsychotic agents, and clozapine and sulpiride have not been associated with tardive dyskinesia, although the latter experimental drugs have not yet been used for prolonged periods in many patients. T h e ability of one or more of these agents after repeated administration to induce behaviorally supersensitive responses (Smith and Davis, 1976; Gianutsos and Moore, 1977; Scatton, 1977) to a dopamine agonist such as apomorphine (or tolerance to dopamine turnoverenhancing actions of neuroleptics [Waldmeir and Maitre, 1976; Scatton, 19771) has been reported, but this appears to be an inconsistent or relatively weak capability (Sayers et al., 1975a; Waldmeir and Maitre, 1976; Scatton, 1977). Moreover, the suggestion that these “atypical” agents have less impact on the extrapyramidal motor system because of relatively strong anticholinergic actions (Snyder et al., 1974) is probably not entirely correct, and they may simply have regional selectivity, with a preference for limbic dopamine receptors (Costal1 and Naylor, 1976). Thus, while thioridazine and clozapine are strongly anticholinergic [their antimuscarinic potencies approach those of tricyclic antidepressants (Snyder et al., 1974) and they can induce a toxic state similar to atropine poisoning on overdosage (Granacher and Baldessarini, 1976)], sulpiride is not especially anticholinergic (Nishiura, 1976). Furthermore, the biochemical and behavioral actions of the “atypical” neuroleptics are not mimicked by combining a more typical neuroleptic with a centrally active antimuscarinic agent (Sayers et al., 1975a, 1976), nor is there any clinical evidence that anticholinergic-antiparkinson agents tend to diminish the risk of tardive dyskinesia (Tarsy and Baldessarini, 1976). Rather, there are some speculations that they may even increase the risk (Kiloh et al., 1973; Klawans, 1973a), based largely on the tendency of anticholinergic drugs to worsen existing signs of tardive dyskinesia (Kiloh et al., 1973; Klawans, 1973a; Tarsy and Baldessarini, 1976); in addition, anticholinergic agents may variously increase (Sayers et al., 1975a, 1976), decrease (Smith and Davis, 1976) or have no effect (Tarsy and Baldessarini, 1974) on dopamine supersensitivity induced by neuroleptics in laboratory animals. Further, repeated cotreatment with choline plus a neuroleptic drug may reduce the subsequent supersensitive response to a dopamine agonist (Davis et al., 1978). While denervation or disuse supersensitivity is a reasonable explanation of much of the above evidence indicating enhanced behavioral effects of dopamine agonists following treatment of animals with neuroleptics, the synaptic mechanisms by which the phenomenon is produced remain open to question. The enhanced responses to apomorphine, which is believed to act primarily directly on dopamine receptors (AndCn et al., 1967; Tarsy and Baldessarini, 1974), and the
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persistence of the enhanced behavioral responses for several weeks in most of these studies (Klawans and Rubovits, 1972a; Tarsy and Baldessarini, 1974) suggest that changes in receptor sensitivity rather than in presynaptic metabolism of dopamine may account for the findings. 3. Disuse Supersensitivity: Neurophysiological and Biochemical Evidence Several studies have attempted to identify changes in the postsynaptic sensitivity to dopamine following denervation more directly than in behavioral experiments. In neurophysiological studies, destruction of dopamine-containing neurons of the nigrocaudate pathway of cats with intraventricular (Feltz and DeChamplain, 1972) or intranigral 6-hydroxydopamine (Siggins et al., 1974, 1976; Schultz and Ungerstedt, 1978) or repeated pretreatment with haloperidol (Yarbrough, 1975; Bunney, 1977) all produced a significant increase (up to 100-fold; Schultz and Ungerstedt, 1978) in the sensitivity of caudate neurons to microiontophoretically applied dopamine, although in one study (Spehlmann and Stahl, 1974) electrolytic lesions of the ventral tegmentum (which reduced levels of all caudate biogenic amines) depressed rather than enhanced the effect of microiontophoretically applied dopamine upon caudate neurons. Several biochemical approaches have also been employed. A biochemical strategy to detect possible receptor changes produced by denervation has been the study of dopamine-sensitive adenylate cyclase in denervated brain regions based on the proposal that hormonesensitive cyclase activity reflects receptor stimulation (Kebabian et al., 1972; Iversen, 1977). An increase in adenylate cyclase response to norepinephrine in rat cerebral cortex in 6-hydroxydopamine- (Palmer, 1972; Kalisker et al., 1973; Skolnick et al., 1978), as well as reserpinepretreated (Palmer et al., 1976) animals has been demonstrated, but the effect of 6-hydroxydopamine is sometimes inconsistent and may not bear a close relationship to small changes in binding of /3-receptor labeling agents (Skolnick et al., 1978). Unilateral destruction of the nigrostriatal tract (Mishra et al., 1974; Premont et al., 1975; Krueger et al., 1976) and prolonged treatment of the rat with trifluoperazine or haloperidol (Burkhard and Bartholini, 1974; Von Voigtlander et al., 1975; Friedhoff et al., 1977) have been reported to induce an increase in responsiveness of dopamine-sensitive adenylate cyclase in some preparations of neostriatal tissues that may reflect supersensitivity of dopamine receptors. However, these effects of drugs or even lesions on catecholamine-sensitive adenylate cyclase reactions have usually been relatively small (two- to tenfold decrease in agonist EC50values) (Mishra et al., 1974; Palmer et al., 1976; Skolnick et al., 1978) and in some
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instances have not been found at all in the striatum (Rotrosen et al., 1975; Von Voigtlander et al., 1975; Krueger et al., 1976). In spite of failure to demonstrate supersensitivity consistently by this method, the possibility remains that alteration of the dopamine receptor occurs at some other point in the receptor or “effector” mechanisms which may not be adequately reflected in changes of adenylate cyclase activity. In another biochemical test, it has been noted that prolonged treatment with a neuroleptic drug altered the ability of a dopamine agonist to depress the synthesis rate and turnover of dopamine in rat corpus striatum. This pretreatment led to a greater dopamine-turnoverinhibiting effect of a dopamine agonist alone, and enhanced the ability of such an agonist to block the dopamine-depleting effect of a-methyltyrosine (Gianutsosand Moore, 1977).These observations have been considered to be consistent with supersensitivity to dopamine of central receptors believed to modulate the neurophysiologic and biochemical activity of nigrostriatal dopamine neurons (Gianutsos and Moore, 1977). In yet another biochemical approach to the study of receptors, several groups have recently reported the labeling of dopamine receptors in mammalian brain (Burt et al., 1975; Seeman et al., 1975) with tritiated dopamine or apomorphine, and haloperidol or spiroperidol, which might label distinct agonist and antagonist “states” of the receptor, respectively (Snyder and Creese, 1978), or, more likely, similar but different binding sites (Cohen and Lipinski, 1978; Lee et al., 1979). It has been found very recently that kainic acid, which destroys many “GABAergic” and cholinergic neurons in the caudate nucleus believed to be receptive to dopamine, reduces the binding of the radioligand PHIhaloperidol markedly and almost completely removes the activity of dopamine-sensitive adenylate cyclase (Creese et al., 1977b).These observations thus support the idea that both biochemical measures at least in part reflect changes occurring at postsynaptic dopamine receptors. In rats, 6-hydroxydopamine-induced lesions of dopamine-containing neurons in the substantia nigra led to enhanced binding of some of these labeled ligands (Creese et al., 1977a; Fuxe et al., 1978a; Goldstein et al., 1978), presumably to dopamine receptors, but decreased labeling by [3H]-apomorphine (possibly a selective presynaptic agent) (Muller and Seeman, 1979)-a phenomenon also observed with tissue from brains of patients with Parkinson’s disease (Muller and Seeman, 1979b).Similar increases have followed prolonged treatment with neuroleptic drugs (Burt et al., 1977; Friedhoff et al., 1977; Muller and Seeman, 1977; Friend et al., 1978a; Kobayashi et al., 1978). Moreover, these changes have recently been found to correlate well with the degree of behavioral
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supersensitivity to apomorphine appearing in the same animals (Rotrosen et al., 1975; Creese et al., 1977a), and to be prevented by simultaneous treatment with the dopamine precursor L-dopa (Friedhoff et al., 1977). Further support for the usefulness of this approach was obtained by the survey of changes in the level of labeling with a variety of tritiated putative receptor-labeling ligands following prolonged treatment of the rat with haloperidol (Muller and Seeman, 1977; Friend et al., 1978a). Thus, there was a significant increase in binding to presumed dopamine or neuroleptic sites in striatal and limbic tissue, although not in pituitary tissue (Muller and Seeman, 1977; Friend et al., 1978a) and little change in binding to serotonin, norepinephrine (a-receptor), or acetylcholine (muscarinic) sites in the brain (Muller and Seeman, 1977). The latter result regarding muscarinic sites seems inconsistent with a recent report of enhanced behavioral responsiveness to atropine, and decreased responsiveness to physostigmine, in haloperidol-pretreated mice, consistent with a diminished sensitivity of central muscarinic acetylcholine receptors (Dunstan and Jackson, 1977). Other recent data, in addition to increases in binding site density of dopamine-receptor labeling ligands, also suggest the involvement of macromolecular mechanisms in supersensitivity to dopamine agonists following pretreatment with antidopamine agents. In one study, protein synthesis inhibitors prevented induction of such supersensitivity by a-methyltyrosine (Costentin ct al., 1977). This result is paralleled by the ability of cycloheximide, an inhibitor of protein synthesis, to block the CNS denervation supersensitivity to serotonin agonists after lesioning with a selective serotonin-neuron toxin (Sperk et al., 1978). It has also been suggested recently that a Ca2+-binding protein activator of dopamine-sensitive adenylate cyclase may contribute to the supersensitivity to dopamine induced by typical neuroleptics (but not by clozapine or the inactive isomer of butaclamol) (Gnegy et al., 1977). Thus, the various biochemical studies reviewed provide strong supporting evidence of possible receptor-macromolecular changes in neuronal membranes that might underlie the behavioral supersensitivity to dopamine that follows prolonged exposure to neuroleptics. 4. Attempts to Evaluate the Disuse Supersensitivity Concept an Man
Some of the newer biochemical approaches to the study of receptors of neurotransmitters and transmitter levels in the brain have begun to be applied to postmortem studies of neuropathology in human brain tissue. For example, if, as reported, dopamine levels in the basal ganglia normally decrease with advancing age (McGeer et al., 1977), this might contribute to increased sensitivity of dopaminergic mechanisms to
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neuroleptic agents, and hence predispose older patients to tardive dyskinesia, and might somehow contribute to an increased likelihood of long-lasting oral-facial dyskinesias. Interestingly, older rats seem to be more susceptible to development of supersensitivity to dopamine agonists after treatment with neuroleptic drugs (Smith, 1977; Smith et al., 1978). It has also been reported recently (Bird et al., 1977), but not confirmed (Lee et al., 1979), that levels of dopamine or those of its metabolite homovanillic acid may be increased in brains of chronic schizophrenic patients, almost all of whom have been exposed to prolonged treatment with neuroleptic-antipsychotic drugs. Dopaminesensitive adenylate cyclase activity was not different from normal in one postmorten study of brain tissue of similar patients (Carenzi et al., 1975). In the brains of patients with Huntington’s disease (Enna et al., 1976; Fields et al., 1977) and Parkinson’s disease (Enna et al., 1976), receptorlabeling techniques with radioactive ligands have been applied recently. In Huntington’s disease, the number of [3H]-neuroleptic binding sites per milligram of tissue or protein sampled from the basal ganglia was reported to be decreased more than 50% (Fields et al., 1977), a finding that is consistent both with a postsynaptic location of many dopamine receptors on cholinergic or GABA-ergic neurons (Creese et al., 197713) and with the degenerative loss of such cells in Huntington’s disease (Enna et al., 1976). In parkinsonian caudate nucleus tissue, [3H]neuroleptic binding sites were also somewhat diminished in one study (Reisine et al., 1977), but increased in another, suggesting the presence of denervation supersensitivity (Muller and Seeman, 1979). While the first observation may seem to contradict the prediction that a loss of nigrostriatal dopamine, as occurs in Parkinson’s disease (Bernheimer et al., 1973), would increase the density or efficiency of dopamine receptors, it may reflect the modern prolonged treatment of such patients with L-dopa or other dopamine agonists (Reisine et al., 1977). More recently, it has been reported that the basal ganglia and limbic tissues of chronic schizophrenic patients contain increased binding sites for some ([3H]-labeled neuroleptics), but not all ([3H]-apomorphine, which may label presynaptic sites preferentially; Lee et al., 1979; Muller and Seeman, 1978a,b) of the currently employed radioactive ligands proposed to label dopamine receptors (Lee et al., 1979). This phenomenon, if it can be replicated (and preliminary results from another laboratory using [3H]-spiroperidol seem to be disconfirmatory; Creese, 1977), may reflect changes induced by prolonged treatment with antipsychotic agents, although in a few patients said not to have been exposed to such treatments, there was a similar increase in binding of radioactive haloperidol (Owen et al., 1978; Lee et al., 1979).
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Another strategy aimed at the evaluation of possible supersensitive dopamine receptor mechanisms in patients has been the study of endocrine responses believed to be regulated in part by small tuberoinfundibular dopamine-containing neurons of the hypothalamus that influence the function of the anterior pituitary or adenohypophysis. Most of these studies have included assays of blood levels of prolactin or growth hormone in schizophrenic subjects with or without signs of tardive dyskinesia but with prolonged exposure to antipsychotic drugs, and compared basal levels or responses to a dopamine agonist in such subjects with normal controls (Meltzer et al., 1978). The results of this approach in animals or man have not supported a dopamine supersensitivity hypothesis in tardive dyskinesia (Ettigi et al., 1976; Smith et al., 1977; Tamminga et al., 1977; Burnett et al., 1979). On the other hand, one need not expect this approach to be productive since it has been demonstrated repeatedly that hypothalamic dopamine neurons are peculiarly unable to develop tolerance to the dopamine-turnoverenhancing (Bowers and Rozitis, 1976) and endocrine-altering (e.g., prolactin-increasing) (Beaumont et al., 1974; Gruen et al., 1978) actions of neuroleptic drugs, whereas evidence of tolerance to their dopamine antagonistic effects in the forebrain is abundant (e.g., Asper et al., 1973; Moller-Nielsen et al., 1974). Since, elsewhere in the CNS, receptor supersensitivity is commonly associated with increased tolerance to the prolonged antagonism of dopamine’s actions (Martres et al., 1977) although almost never in the tuberoinfundibular system (Gianutsos and Moore, 1977), the neuroendocrine approach may not be an appropriate one for the evaluation of changes in the dopamine receptor. In addition to this theoretical objection, artifacts due to effects of residual neuroleptic drugs in the brain are extremely hard to eliminate in experiments of this type. Interestingly, it has recently been noted that increased binding of [3Hl-haloperidolafter prolonged neuroleptic treatment in rat was found in striatal, but not in pituitary, tissues (Friend et al., 1978b). One other clinical approach to the evaluation of central effects possibly mediated by dopamine has been the measurement of nauseaproducing threshold doses of intravenous apomorphine in normals and drug addicts compared with chronic schizophrenic subjects (not necessarily with tardive dyskinesia) previously exposed to neuroleptics. Again, no differences were found (Angrist and Gershon, 1978). Although denervation or disuse supersensitivity may be produced experimentally in animals and may be an important mechanism in transient and reversible clinical forms of tardive dyskinesia, especially in younger patients (Tarsy and Baldessarini, 1976), it seems improbable that this mechanism alone is responsible for the development of the
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more persistent forms of tardive dyskinesia. T h e usually prolonged and sometimes irreversible course of the syndrome seems inconsistent with a relatively transient, purely pharmacologic state of disuse supersensitivity ; it suggests instead that significant structural or other neurotoxic changes have taken place, possibly at the postsynaptic receptor and related to known effects of neuroleptic agents on cell membranes or cellular respiratory mechanisms, for example (Seeman, 1972; Faurbye, 1976). One might also speculate that antipsychotic drugs may interfere with synaptic transmission of some unidentified “trophic” factor, thereby producing alterations in the postsynaptic membrane analogous to those described after denervation of the neuromuscular junction (Guth, 1974). Available clinical approaches to the detection of supersensitive responses to dopamine agonists in vim, or to the demonstration of altered dopamine receptors in brain tissue, have so far not produced convincing support for the disuse supersensitivity hypothesis for tardive dyskinesia, although, clearly, such studies are still at a very preliminary stage of development.
5. Presynaptic Mechanisms Although alterations in postsynaptic receptor mechanisms in the nigrostriatal system have generally received greater attention, the possibility that prolonged exposure to neuroleptics may permanently alter presynaptic mechanisms is also worthy of consideration. A possible explanation for the apparent increase in dopamine activity in tardive dyskinesia might be its increased availability through the well-known increase of dopamine turnover in response to neuroleptic drugs (Carlsson and Lindqvist, 1963; Matthysse, 1973). This biochemical response is paralleled by an increase in the firing rate of dopamine neurons in the midbrain on acute systemic administration of these agents (Bunney et al., 1975). These effects seem to represent acute, possibly adaptive or compensatory responses to the blockade of dopamine-mediated synaptic transmission by neuroleptic agents. However, in animal experiments, accelerated turnover of dopamine in response to neuroleptic drugs is reported to be only a transient effect, at least in the caudate nucleus and.to some extent in portions of the limbic system, although not in the hypothalamus (Asper et al., 1973; Stille and Lauener, 1974; Scatton et al., 1975, 1976; Bowers and Rozitis, 1976); in some studies prolonged treatment of the rat with neuroleptic agents was actually associated with decreased striatal dopamine turnover 24 hr after termination of the neuroleptic treatment (Scatton et al., 1975, 1976). There is currently some debate as to whether “atypical” antipsychotic drugs such as clozapine and sulpiride induce tolerance to their
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dopamine-turnover-enhancing effects, and especially whether they do so in the mesolimbic and mesocortical systems; evidently they can increase turnover if large doses are used, and this effect is most evident in the nigrostriatal system (Waldmeir and Maitre, 1976; Scatton, 1977). In man, however, there is evidence that increases in the lumbar cerebrospinal fluid (CSF) levels of homovanillic acid (HVA, a major metabolite of dopamine, and presumably an index of dopamine turnover) persist during several months of treatment with neuroleptic agents (Bowers, 1974; Post and Goodwin, 1975; Post et al., 1975; Post, 1978), although diminishing elevations have been observed after the first few weeks of treatment (Gerlach et al., 1975; Post and Goodwin, 1975). Attempts to evaluate dopamine turnover in patients with tardive dyskinesia include several studies of dopamine metabolites in the lumbar CSF that have yielded conflicting results. In three studies, the rise in lumbar CSF concentrations of HVA measured after probenecid (to block its exit into the venous blood) was normal (Pind and Faurbye, 1970; Gerlach et al., 1975; Bowers et al., 1979), while in another study (Chase, 1973), dopamine turnover, as estimated by this probenecid method as well as by the response of HVA levels in the CSF to haloperidol treatment, was diminished in patients with tardive dyskinesia. Chase (1973) suggested that low CSF levels of HVA in these patients might reflect either structural changes in dopamine neurons or functional responses to altered sensitivity of dopamine receptors. It has been suggested (Korczyn, 1972) that increased sensitivity to dopamine in tardive dyskinesia might occur because of impaired presynaptic reuptake and inactivation of dopamine, either due to preexisting subclinical disease of the nigrostriatal system (Christensen et al., 1970) or to toxic effects of neuroleptic drugs insufficient to produce extrapyramidal signs but sufficient to interfere with reuptake mechanisms. According to this hypothesis, however, one might expect choreoathetotic dyskinesias to occur early in the course of nigrostriatal damage associated with idiopathic Parkinson’s disease, but this does not occur. It has been reported recently that treatment with chlorpromazine may even increase the numbers of presynaptic vesicles in some neurons (Kaiya et al., 1977). Several recent studies of the effect of prolonged administration of neuroleptic drugs on presynaptic dopamine metabolism have produced somewhat conflicting results but are worthy of consideration. As already discussed, in at least two studies, prolonged administration of neuroleptics to rats led to a reduction in dopamine turnover in the basal ganglia but not in limbic areas or cerebral cortex when measured 24 hr after
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discontinuation of the treatment (Scatton et al., 1975, 1976). In addition, Velley et al. (1975) have shown that prolonged treatment of newborn rats with a neuroleptic agent led to reduced dopamine synthesis and turnover in the striatum at 40 days of age. Although this reduction in dopamine turnover could be explained on the basis of supersensitivity of dopamine receptors leading to feedback inhibition of nigrostriatal activity (Asper et al., 1973), a direct effect of neuroleptics on dopamine neurons could not be excluded (Velley et al., 1975). In contrast to studies of the effect of sustained and prolonged exposure to neuroleptic agents to decrease dopamine turnover, a recent study found that upon termination of long-term administration of penfluridol, a hyperkinetic syndrome appeared in the rat together with evidence of increased receptor sensitivity and an increased rate of dopamine turnover (Engel et al., 1975). Another similar study failed to find an increase in dopamine turnover after prolonged treatment with haloperidol, and in addition found increased sensitivity to the dopamine-turnover-reducing action of apomorphine, another effect that may be mediated by supersensitive receptors (Gianutsos et al., 1975). These animal studies involving relatively brief exposure (weeks) to neuroleptics and carried out under varying circumstances are subject to several interpretations and at present must be regarded as of uncertain relevance to an understanding of tardive dyskinesia in patients exposed to antipsychotic agents for many months o r years. An additional presynaptic mechanism to be considered is that nigrostriatal neurons may contain dopamine-sensitive “autoreceptors” important for feedback inhibition of nigrostriatal dopamine activity (Aghajanian and Bunney, 1974; Kehr et al., 1975; Martres et al., 1977). Recently, evidence has been presented that the dendrites of dopaminergic neurons in the substantia nigra contain, and can take up, dopamine (Bjorklund and Lindvall, 1975), apparently in a releasable form (Geffen et al., 1976; Korf et al., 1976). It has been suggested that these dendrites may mediate self-inhibition of dopamine neurons at dendro-dendritic synapses (Groves et al., 1975; Geffen et al., 1976; Korf et al., 1976). However, this concept fails to account for observations that although intravenously administered neuroleptics reversed the inhibitory effect of dopamine agonists on nigral firing rates, chlorpromazine applied directly to dopaminergic neurons in the substantia nigra had little or no effect and did not block the inhibitory action of dopamine (Aghajanian and Bunney, 1974; Bunney and Aghajanian, 1976). Moreover, the observation that lesions which remove most of the dopamine-sensitive adenylate cyclase from midbrain do not injure nigrostriatal neurons (but
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do remove GABA and substance P) further calls this hypothesis into serious question (Gale et al., 1977), although the dendrites may modulate the activity of other nearby cells in the substantia nigra. The observation that low doses of apomorphine depress locomotor activity while high doses increase it has been explained on the basis of activation of dopamine autoreceptors (location, e.g., in the nigra or striatum, not specified) such that motility changes produced by apomorphine may reflect a balance between presynaptic and postsynaptic effects (Kehr et al., 1975; Strobom, 1976). The observation that, unlike the dyskinesia-enhancing effect of L-dopa (Gerlach et al., 1974), apomorphine (Carroll et al., 1977) and several other dopamine agonists (Chase and Shoulson, 1975) may suppress tardive dyskinesia and other forms of choreoathetosis (Baldessarini et al., 1976; Carroll et al., 1977) can be interpreted along similar lines. Whether prolonged administration of neuroleptic drugs might damage this hypothetical self-regulatory system, thereby leading to inappropriately increased dopaminergic activity, is unknown. Two other presynaptic mechanisms should also be considered. One is the phenomenon of neuronal “sprouting” following partial neuronal lesions that leave the perikaryon intact, and which has been observed in central catecholamine neurons following both electrolytic (Katzman et al., 197 1) and 6-hydroxydopamine-induced lesions (Jonsson et al., 1974). A second mechanism is suggested by evidence that partial lesioning of nigrostriatal neurons leads to increased dopamine turnover in the remaining cells (Agid et al., 1973). Whether these experimental phenomena, the clinical relevance of which is uncertain, can contribute to an understanding of the pathophysiology of tardive dyskinesia is only speculative at present. The phenomenon of sprouting and excessive regeneration might now be approached neuropathologically in postmortem studies of brain by the application of recently developed immunohistochemical techniques to visualize central dopamine projections (Fuxe e l al., 1978b). C. OTHERNEUROTRANSMITTERS The possibility that changes in other neuronal systems may account for dopamine sensitivity or offer independent contributions in tardive dyskinesia has not been widely studied. For example, tardive dyskinesia could represent toxic or destructive effects on striatal interneurons on which dopamine usually exerts an inhibitory effect, and which in turn may have a physiological feedback influence on nigrostriatal dopamine neurons, or even serve as an output pathway for nigrostriatal projections (Garcia-Munoz et al., 1977). Such interneurons may utilize acetylcholine
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(ACh) (McGeer et al., 1971, 1976) or y-aminobutyric acid (GABA) (Harris et al., 1975; McGeer and McGeer, 1975; Roberts et al., 1976), or a peptide such as substance P (Gale et al., 1977) as their neurotransmitter. A similar phenomenon has been suggested to occur in Huntington’s disease (Carenzi et al., 1975; McGeer et al., 1976; Bird et al., 1977). Although postmortem biochemical and histochemical studies of neuronal systems that utilize ACh or GABA are now feasible (Domino et al., 1973; Carenzi et al., 1975; McGeer et al., 1976; Bird et al., 1977; McGeer and McGeer, 1977; Smith, 1977), they have not yet been reported in brains from patients with tardive dyskinesia. Such studies in patients with chronic schizophrenia who have also received long-term treatment with neuroleptic drugs so far reveal little consistent evidence for deficiency in enzymes used in the metabolism of ACh (Domino et al., 197.3; McGeer and McGeer, 1977). There are at least two postmortem studies that suggest that the activity of the enzyme that produces GABA (glutamic acid decarboxylase) may be decreased in several areas of brain of chronic schizophrenics exposed to antipsychotic drugs (McGeer and McGeer, 1977; Smith, 1977), although two other very recent reports failed to confirm this result (Crow et al., 1978; Perry et al., 1978). If centrally effective agonists of GABA become available for clinical use (Lenman et al., 1976; Roberts, 1976; Shoulson et al., 1976), they might be worthy of trial in tardive dyskinesia, particularly in light of these recent neuropathological findings (McGeer and McGeer, 1977; Smith, 1977) and the evidence that neurons utilizing GABA as a neurotransmitter may have inhibitory effects on nigrostriatal dopamine neurons (Harris et al., 1975; Roth and Bunney, 1976; Tarsy et al., 1976). Sodium valproate has recently been claimed to be of moderate but inconsistent benefit in various dyskinesias and had been proposed as such a GABA-facilitating agent (Lenman et al., 1976; Symington et al., 1978), although there is now considerable doubt about its interactions with this inhibitory neurotransmitter (Perry and Hansen, 1978). So far there is little evidence for deficits in other neurotransmitter systems or of benefits of agents that modify their action in patients with tardive dyskinesia, with the possible exception of ACh-potentiating drugs (Tarsy and Baldessarini, 1976, 1977). There is considerable clinical, animal behavioral, physiologic, and biochemical evidence for a reciprocal functional relationship between dopaminergic and cholinergic mechanisms in the basal ganglia (Roth and Bunney, 1976; Tarsy, 1976). Among extrapyramidal disorders, this relationship is most apparent in idiopathic Parkinson’s disease (Duvoisin, 1967) and neuroleptic-induced parkinsonism (Ambani et al., 1973). In animal behavioral models of both hypokinetic and hyperkinetic motor
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activity, anticholinergic drugs are well-known to potentiate several effects of dopamine agonists (for references, see Rubovits and Klawans, 1972; Tarsy, 1976; Baldessarini et al., 1977). Moreover, this reciprocal relationship is illustrated by biochemical studies which demonstrate both dopaminergic regulation of striatal cholinergic activity (Stadler et al., 1973; Sethy and Van Woert, 1974; Trabucchiet al., 1974), and cholinergic regulation of striatal dopaminergic activity (OKeefe et al., 1970; Bowers and Roth, 1972; Javoy et al., 1975). In addition to the above findings, recent studies suggest that there may also be cholinergic regulation of nigrostriatal dopaminergic activity at the level of the substantia nigra (Aghajanian and Bunney, 1974; Javoy et al., 1974). With regard to the first of these mechanisms, acute blockade of dopamine neurotransmission by antipsychotic drugs increases the turnover and release of acetylcholine within the striatum, while dopamine agonists exert the opposite effects (Stadler et al., 1973; Trabucchi et al., 1974), suggesting that the nigrostriatal dopamine system may exert an inhibitory effect on striatal cholinergic interneurons (Hattori et al., 1976). Viewed in terms of this possible relationship, the effect of a dopamine agonist to disinhibit (facilitate) activity in striatal cholinergic neurons may be mimicked by administration of a cholinergic drug, whereas, conversely, the inhibitory effect of a dopamine agonist on striatal cholinergic activity may be mimicked or potentiated by an anticholinergic drug. By the same token, the effect of a dopamine agonist or antagonist may be blocked by cholinergic or anticholinergic drugs, respectively (see Tarsy, 19’76, for further discussion). It has recently been reported that repeated treatment with a neuroleptic drug can diminish behavioral effects believed to be mediated by central muscarinic ACh receptors (Dunstan and Jackson, 1977). This result is thus consistent with the view that some diminution of ACh function may contribute to apparent functional overactivity of dopamine. In addition, prolonged treatment with a neuroleptic agent may also modify noradrenergic mechanisms in the CNS (Dunstan and Jackson, 1976). Although, as indicated above, a reciprocal balance between dopaminergic and cholinergic effects in the basal ganglia is evident in spontaneous and drug-induced parkinsonism (Duvoisin, 1967; Ambani et al., 1973), evidence for a similar relationship in choreiform disorders in general and tardive dyskinesia in particular has been less consistent (Tarsy and Baldessarini, 1976, 1977). When neuroleptics are withdrawn, as drug-induced parkinsonism becomes less prominent, signs of tardive dyskinesia increase (Crane, 1973a). When anticholinergic drugs are used to treat drug-induced parkinsonism in patients also manifesting tardive dyskinesia, reduction of parkinsonism is associated with a reciprocal
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increase in severity of tardive dyskinesia (Bordeleau et al., 1967; Fann and Lake, 1974), an effect that is not surprising in view of the worsening effect of anticholinergic-antiparkinson agents in Huntington’s chorea (Klawans and Rubovits, 1972b) as well as in tardive dyskinesia (Klawans and Rubovits, 1972b, 1974), and their occasional capacity to induce dyskinesias in Parkinson’s disease (Fahn and David, 1972; Birket-Smith, 1974). While oral administration of anticholinergic drugs frequently, though inconsistently, worsens tardive dyskinesia (Kiloh et al., 1973; Gerlach et al., 1974), attempts to produce improvement with intravenous physostigmine, a centrally active anticholinesterase, have, as in the case of Huntington’s disease, produced mixed results. Two studies (Klawans and Rubovits, 1974; Davis et al., 1976) reported mild improvement in tardive dyskinesia, but in other studies (Gerlach et al., 1974; Tarsy et al., 1974) no consistent beneficial effects were observed following intravenous physostigmine. Although an oral preparation of physostigmine is now available, it has evidently not yet been evaluated in the treatment of dyskinesias. It is possible that the effect of drugs given intravenously may simply not parallel their oral activity or exert sufficiently long-lasting effects. Thus, for example, intravenous anticholinergic drugs have variously been reported to produce some worsening in tardive dyskinesia (Klawans and Rubovits, 1974) or no consistent change (Granacher et al., 1975; Tarsy and Bralower, 1977). T h e possibility that a provocative pharmacologic test might reveal early (and hopefully more readily reversible) stages of tardive dyskinesia, such as by the use of a test dose of an antiparkinson agent, has been suggested (Granacher et al., 1975), although it does not yet seem to be a useful clinical approach. Since physostigmine depends for its cholinergic activity on the presence of intact endogenous cholinergic neurons, its variable effects in tardive dyskinesia might be on the basis of damage to cholinergic neurons or receptors (Smith, 1977), as is possibly the case in Huntington’s disease (Hiley and Bird, 1974; McGeer and McGeer, 1976; Bird et al., 1977). The fact that in preclinical studies cholinergic mechanisms have been described that either facilitate or inhibit dopaminergic activity (Lenman et al., 1976; Tarsy, 1976) may offer yet another explanation for ambiguous effects of cholinergic drugs in choreiform ayndromes, depending on the functional balance that is obtained between these potentially opposite effects. It is also possible that clinical attempts to demonstrate cholinergic responsiveness in tardive dyskinesia have been limited simply by the relatively small number of practical and effective centrally active cholinergic drugs which are available. Efforts at the treatment of tardive dyskinesia by oral administration of physostigmine or of large doses of presumed acetylcholine precursors such as
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dimethylaminoethanol (deanol) (Re, 1974)and choline (Daviset al., 1975, 1976; Growdon et al,, 1977) or lecithin (Growdon et d.,1978),although not dramatically or consistently effective, are worthy of continued study, as is the suggestion that an oral, centrally active, reversible anticholinesterase agent, tetrahydroaminoacridine (THA) (Granacher and Baldessarini, 1976), may have antidopamine effects in the rat (Cali1 et al., 1978). Whether such treatments all consistently increase the levels or functional availability of acetylcholine in the brain is another point that requires further study. For example, deanol may not do so (Zahniser et al., 1977). The possibility has been raised that administration of anticholinergic drugs may increase the incidence of tardive dyskinesia (Kilohetal., 1973; Klawans, 1973a) by altering the threshold for appearance of these movements (Klawans, 1973a).At present there is actually little evidence to support this assumption, although it seems clear that oral anticholinergic drugs generally worsen the syndrome once it has already appeared. Possibly relevant in this regard is the observation that the enhanced sensitivity to apomorphine produced by prolonged neuroleptic treatment in animals is only slightly (Sayers et al., 1976) and inconsistently (Tarsy and Baldessarini, 1974) enhanced (Smith and Davis, 1976) by the simultaneous administration of an anticholinergic agent. A contributory role of anticholinergic drugs has also been considered in attempts to address the question of whether acute extrapyramidal effects of neuroleptic agents are strongly associated with the later development of tardive dyskinesia. Such an association would also be consistent with the hypothesis that both the early and late extrapyramidal disorders depend on direct or indirect effects of the blockade of dopamine receptors, respectively. An association between drug-induced parkinsonism and tardive dyskinesia was suggested in at least one study (Crane, 1972), although it has also been reported that tardive dyskinesia also occurs in some patients without previous parkinsonism (Klawans, 1973b;Jus et al., 1976), and it is clear that the majority of patients experiencing acute neuroleptic-induced extrapyramidal reactions never go on to develop tardive dyskinesia. One suggestion to explain the apparently inconsistent correlation of early and late neurological signs takes note of microiontophoretic electrophysiological studies of the caudate nucleus that have disclosed two populations of neurons, respectively inhibited or facilitated by dopamine (Cools and Van Rossum, 1976). Thus, in order to explain the appearance of tardive dyskinesia without previous parkinsonism, Klawans ( 1973a,b) has proposed that these two receptors may not be equally susceptible to blockade with antipsychotic drugs. Electrophysiological evidence to support such a differential response has been presented (Feltz, 1971) but has not always been confirmed in
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subsequent studies (York, 1972), although a growing body of physiological and pharmacological data that support such a concept has been reviewed recently (Cools and Van Rossum, 1976). D. EXPERIMENTAL NEUROLEPTIC DYSKINESIAS An important experimental test of the drug-etiology theory of tardive dyskinesia would be to reproduce the neurological phenomena in laboratory animals following prolonged administration of neuroleptic drugs. Attempts to reproduce tardive dyskinesia in animals generally have had only limited success. Oral dyskinesias have been produced in monkeys by prolonged administration (months) of antipsychotic drugs (Deaneau and Crane, 1969; Paulson, 1972; Bkdard et al., 1977). In five of 15 rhesus monkeys fed large doses of chlorpromazine daily for up to 9 months, buccolingual dyskinesias, biting behavior, and self-destructive acts occurred that were maximal several hours after each dose but that ceased when chlorpromazine was discontinued (Paulson, 1972). In another study (Bkdard et al., 1977), two of six monkeys (Macaques) treated with haloperidol for 6 months developed choreoathetosis and oral dyskinesias that, in one case, persisted for 6 months after cessation of treatment. More recently, three Cebus monkeys developed buccolingual and some limb dyskinesias following prolonged treatment with haloperidol (Gunne and Barany, 1976; Gunne, 1977). These reactions had many descriptive and pharmacologic similarities to clinical tardive dyskinesia. They differed from acute dystonias, which also occur in Cebus monkeys (Weiss and Santelli, 1978) and the baboon (Meldrum et al., 1977) and are typically reversible with antiparkinson agents. The Cebus model of tardive dyskinesia is currently undergoing intensive study for new therapeutic approaches to tardive dyskinesia (Gunne and Barany, 1976; Gunne, 1977). Although some dyskinesias produced in these studies more closely resemble the acute dyskinesias (dystonias) seen early in the treatment of patients with antipsychotic agents, others appear to be a valid animal model for tardive dyskinesia. A possibly less relevant model of tardive dyskinesia is the production of striking and grotesque oral-facial dyskinesias after repeated treatment of Rhesus monkeys with a combination of chlorpromazine and an amphetamine, or an amphetamine and the opioid methadone, which is not known to be associated with late dyskinesias during prolonged use in addict rehabilitation programs (Carlson, 1977). Smith and Davis (1975) have reported an emergence of spontaneous, as well as enhanced, dopamine agonist-induced, stereotyped behavior during administration of very high doses of haloperidol to rats for a period of 2 months. As in the case of the above primate studies, this
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effect was a transient one that followed each dose of neuroleptic and did not appear during the final withdrawal period, and so may have more similarity to the acute dystonic reactions to antipsychotic drugs in patients. Furthermore, a more recent study in the rat treated weekly for 9 months with a long-lasting potent experimental neuroleptic agent (flupenthixol in oil) revealed minimal neurological impairment during or at the end of this prolonged treatment (Nielsen, 1977).
111. Conclusions
An open-minded attitude concerning the etiology of tardive dyskinesias is still called for. More work can and should be done to study the possible relationship of drug effects and the tardive dyskinesia syndrome. More epidemiological studies in countries where antipsychotic drugs are still not extensively used would be helpful. Since dyskinesias follow treatment with neuroleptic-antipsychotic agents after various lengths of time, are manifest in various patterns of anatomical involvement, and show varying degrees of persistence, they may not necessarily represent a single disease with a unitary pathophysiology. While similar dyskinesias can occur independently in association with senile brain changes and many other overt or presumed states of CNS disease, usually there are no organic, metabolic, or neurological factors found on clinical examination that might contribute to the development of neuroleptic-related tardive dyskinesias. More attempts should be made to reproduce the dyskinetic phenomena by prolonged administration of drugs to intact animals, as well as those with selective brain lesions, compromised cerebral circulation, or old age, and to make maximum use of the newer primate models of the disorder. There is also a need for further carefully controlled neuropathologic studies in animals and man following prolonged exposure to neuroleptic agents, using classical neurohistology as well as newer techniques such as chemical assays of transmitters and their metabolites, as well as biochemical and immunohistologic assays of neuron-specific enzymes, and the new labeling assays for neurotransmitter and drug receptors. Such methods are applicable to postmortem human brain tissue for a more direct evaluation of the current pathophysiologic hypotheses concerning the response of the brain to prolonged exposure to neuroleptic drugs, and the development of tardive dyskinesia. At the present time, the conclusion seems inescapable that an important and relatively selective action of the antipsychotic-neuroleptic drugs is to block the actions of dopamine as a neurotransmitter in
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various regions of the CNS. Acute extrapyramidal and sustained neuroendocrine side effects of these agents are almost certainly, in part at least, reflections of this action in the basal ganglia and hypothalamus or pituitary, respectively. Antipsychotic effects may further in part reflect antidopamine effects in limbic portions of the forebrain, although this hypothesis remains highly tentative. There is also excellent evidence that prolonged exposure to antidopamine drugs can lead to a variety of secondary or partially compensatory adjustments in the physiology and biochemistry of dopamine neurons and other cells with which they interact in the animal or human CNS. Among these adjustments, some tend to increase the effectiveness of dopamine as a neurotransmitter, particularly in the basal ganglia. These effects may help to explain the clinical observation that the risk of acute clinical extrapyramidal reactions diminishes in time, as the risk of tardive dyskinesia increases. Another crucial source of support for a “dopamine supersensitivity” hypothesis in tardive dyskinesia is the now considerable amount of clinical pharmacologic evidence to suggest that functional overactivity of extrapyramidal mechanisms mediated by dopamine is an important aspect of the clinical pathophysiology of tardive dyskinesia. An explanation for the prolonged and even irreversible course of some cases of tardive dyskinesia awaits further research, especially as it suggests that irreversible neurotoxic or degenerative effects of neuroleptic agents may occur. While the precise pathophysiology of tardive dyskinesia remains uncertain, the study of neurological, behavioral, and endocrinological effects of the neuroleptic agents on the CNS has contributed to an improved understanding of their actions, at least equal to that of many other drugs used in medicine. Moreover, insights gained from studies of the antidopamine effects of the antipsychotic drugs in various brain regions promise to lead the way to a more rational basis for developing new, less neurotoxic, but effectively antipsychotic agents. REFERENCES Aghajanian, A. K., and Bunney, B. S. (1974). Biochem. Phannacol. 43, Suppl., 523-528. Agid, Y., Javoy, F., and Glowinski, J. (1973). Nature (London), New Biol. 245, 150-151. Alpert, M.,and Friedhoff, A. J. (1976). Clin. Phurmacol. Thm. 19, 103. Ambani, L.H.,Van Woert, and Bowers, M. K. (1973).Arch. Neurol (Chicago) 49,444-446. American College of Neuropsychopharmacolagy-Food and Drug Administration (ACNP-FDA) Task Force (1973). New Engl. J. Med. 289, 20-23. Ananth, J., and Costin, A. (1977). Am. J. Psychiatv 134, 689-690. Anden, N.-E., Rubenson, A., Fuxe, K., and Hokfelt, T.(1967).J. Phann. Phannacol. 19, 627-629.
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SOVIET LITERATURE ON THE NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
.
By Theodore H Bullock and Vladimir
S. Gumvich
Noumbbkgy Unit
krippr Institution of Oaranogmphy and e k p a h n t of Nounaciencos School of W i c i n Univrniiy of California. San Diego Lo Jolla. California and H u b s s.0 World Reswrch Institute h n Diago. California
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Neuroanatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.Genera1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Ontogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Peripheral Nervous System . . . . . . . . . . . . . . . . . . . . D . Spinal Cord. Brain Stem. and Cerebellum . . . . . . . . . . . . . E . Chemoreceptors and Behavioral Chemoreception . . . . . . . . . . F. Auditory System . . . . . . . . . . . . . . . . . . . . . . . . . G . Visual System . . . . . . . . . . . . . . . . . . . . . . . . . . . H . Cerebral Cortex . . . . . . . . . . . . . . . . . . . . . . . . . 111. Neurophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . A.Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Reflex Control of Air Movement . . . . . . . . . . . . . . . . . . C. Acoustic Signals and Behavioral Audition . . . . . . . . . . . . . . D . Echolocation . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Physiology of Acoustic Sense Organs and Centers . . . . . . . . . . F . Cerebral Cortex . . . . . . . . . . . . . . . . . . . . . . . . . G . Brain Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . H . Sleep and Wakefulness . . . . . . . . . . . . . . . . . . . . . . LBehavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J . Higher Nervous Activity . . . . . . . . . . . . . . . . . . . . . . IV . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
49 52 52 52 53 56 57 58 64 64 70 70 73 74 84 88 89 94 95 97 98 103 107
The original idea of an annotated guide to the findings and opinions of Soviet authors on the brain of cetaceans had to be broadened because of the strong Russian interest in “higher nervous functions” and in acoustic communication and echolocation. These latter topics are not treated exhaustively here. but a selection of the most pertinent papers is deemed essential. Such a broad topical and restricted geographic scope 47 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL 21
.
Copyright @ 1979 by Academic Press. Inc . All rights of reproduction in any form reserved . ISBN 0-12-366821-2
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prohibits the usual type of review. We cannot place each Soviet finding in the contextual background of world literature nor evaluate the degree of originality. The result cannot be a coherent statement of the status of knowledge. We have tried to go beyond a mere annotated bibliography, but in the nature of the subjects and the papers this cannot be accomplished evenly. We have tried to be as balanced as possible; we believe the views herein fairly represent the more scholarly Soviet opinion. Our sampling of the literature is no doubt incomplete, but it is a large sample, probabIy much more than 90% of the papers that meet our criteria of relevance and scholarship. The search was intensive, prolonged, and without screening until after we had read. Then we excluded a good many citations as irrelevant or trivial. A few of the 1978 conference abstracts are in the Bibliography but not cited in the text. It has not been our intention to inject our own interpretations of the available data or to take issue with the interpretations reported. We could not help evaluating how persuasive a case each author makes. However, our evaluations are mainly implicit in the treatment: occasionally, it was necessary to be explicit. Non-Soviet literature is only rarely mentioned, but in a few instances it was necessary because of the direct relevance or because the point of interest is the Soviet opinion about it. The reader will share our frustration with the impossibility of reviewing the background of each claim, indicating what is new, what was already known, what is in disagreement with non-Soviet literature, and arguing the merits of propositions-in short, reviewing the world literature. To offer a modicum of subjective opinion we venture the following overall impressions. The enormous Soviet effort in this field, far beyond that of any other country, has grown rapidly and now contributes scores of papers every year. Among these, one can be sure to find very striking and interesting results. Without passing any judgment on the value of the results in terms of the cost in animals, we are clearly far richer in knowledge of these marvelous creatures as a consequence of the work of this veritable army of investigators. The literature in the area of our title is far from a literature: i.e., although it is substantial and in spite of numerous conferences on marine mammals, there is less integration, less of a continuum from anatomy and physiology to behavior, less cross-referencing and bridging between disciplines than in the literature on other mammals. Although the work comes from institutes that for the most part conduct modern, interdisciplinary research on other animal groups, the cetacean research often seems curiously antediluvian, with significant exceptions. This applies to the techniques employed, the questions asked, the formulation
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of interpretations, and the evidence of integration with work from other approaches or on other animal groups. The literature we have reviewed shows a doctrinaire tendency and relatively little concern with convincing the reader. One would like more details on techniques, controls, and data, and more evidence of close attention by referees. There is, nevertheless, a large corpus of work and great activity on many fronts, much of it far ahead of the rest of the world. This deserves to be better known. Unfortunately, it will not be, under present conditions. The journal literature is only infrequently translated, monographs even more rarely. Whatever ought to be true, the great majority of scientists cannot read the original articles. It is regrettable that so small a fraction of the Russian work is submitted to international journals, especially synthetic reviews. We hope that this compilation, although not properly a review, will call attention to the substantial Soviet contributions to the study of cetaceans.
1. Introduction
Rather suddenly in the late 1960s a profusion of papers on the cetacean nervous system and behavior, including echolocation, began to appear in the USSR. Since most of the findings of Soviet workers are little known in the West, even in their general thrust, a review is overdue. We have undertaken to provide at least an annotated guide. The following previous reviews have dealt with the same subject, but in an even broader scope. Tomilin (1957) published a book in Russian reviewing all the literature, both Soviet and foreign, on Cetacea of the USSR and neighboring areas. Although essentially a faunistic account, it included information on behavior and ecology. Sokolov ( 1971) published a comprehensive review in English of cetacean research in the USSR covering the Soviet literature since 1950; the nervous system and behavior received about three pages of treatment. Voronin and Sokolov (1971) reviewed in Russian the entire Soviet and foreign literature on dolphin biology in 22 pages, particularly the hydrodynamics of locomotion, echolocation, and the physiological and anatomical bases of behavior. Yablokov, Bel’kovich, and Borisov (1972) published a 528-page book in Russian (translated into English in 1974)covering the world literature on all aspects of the biology of whales and dophins, with simplified reviews of the more behavioral aspects. New findings from Soviet authors in our area were still meager. Linebaugh (1976) provided a brief overview in English of all kinds of Soviet dolphin research from 1900; neuroscience was only touched upon.
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A book by Bel’kovich and Dubrovskiy (1976) on the sensory basis of cetacean orientation reviews, in Russian, both Soviet and foreign literature on the skin, chemoreception, vision, and hearing (both passive and active echolocation), as well as aspects of the environment impinging on these several modalities, and some special problems involved with navigation and social behavior. This should become a standard starting reference for investigations of such matters as the visual fields, the accuracy of lateralization of sound sources (alone and in the presence of a second source), and other performance measures. A small monograph on electrophysiological investigations of the dolphin brain (Supin et al., 1978) has just appeared, reviewing a field in which Soviet workers have, in recent years, taken the lead. Although cetacean research on a significant scale got a late start in the USSR, it received new recognition in the middle 1960s and special facilities for work on dolphins began to appear along the Black Sea coast. An interest in “bionics” was one of the early influences, and the new wave of research emphasized, in particular, the areas of behavior, communication, echolocation, and the hydrodynamics of swimming. Half a dozen institutes, in Moscow, Leningrad, Kiev, and elsewhere, took u p the subject and, in the early 1970s, facilities became available at several points along the Black Sea coast, at Pitsunda, Batumi, Karadag, and some others not openly named. Because of this concentration of interest, the present review, which focuses on the nervous system, finds that most of the relevant literature is in the areas of behavior, acoustics, and anatomy. Even within these fields, a substantial number of workers have tended to concentrate their efforts, so that one finds very few, o r no, publications on areas such as ethology (in the rigorous sense), cognitive capacity (in the sense of batteries of tests of graded difficulty), neuropharmacology or neurochemistry, experimental anatomy (by either degeneration or axoplasmic transport methods), electron microscopy, ontogeny, studies of the cerebellum, the motor systems, the hippocampus, the hypothalamus, the thalamus, or the physiology of the ear. Nevertheless, as the bibliography shows, Soviet scientists have been busy with cetacean brain and behavior. T h e number of references is increased by a high percentage of two o r three page notes and of abstracts of presentations at meetings. These workers are capable of detailed and quantitative papers with adequate description of methods, but in many cases never go beyond the preliminary note. Methods are typically classical or, if modern, are usually several years behind the western literature. However, many workers have exploited their methods in new ways.
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Soviet scientists have a virtual monopoly at present on the physiology of the dolphin brain, a subject where at least United States scientists are inhibited by both cost and social milieu. In that country it is less acceptable to undertake invasive research upon dolphins than upon apes, although the former are, in general, far less threatened and their mental faculties are not known to be more advanced. Soviet scientists reflect the latter view, yet it cannot be said on the evidence that they have been generally cavalier or careless in the use of these animals. The principal institutes and groups may be enumerated here for convenience. A good many publications show workers collaborating from different groups, sometimes based on working together at Black Sea stations. In Moscow, some 15 workers that we know of at the Brain Institute of the Academy of Medical Sciences have authored papers in the area of this review, by an approximate count, through 1977. Among the branches of the Academy of Sciences of the USSR, eight workers at the Acoustical Institute who work closely with the group at Karadag (see below), eight at the Laboratory of Evolution of Sensory Systems plus eight others in the Institute of Evolutionary Morphology and Animal Ecology of which that laboratory is a part, and twelve at the Institute of Developmental Biology have authored papers. About ten at the Biology Department of Moscow State University, and a few each at the All-Union Agricultural Institute and the Institute of Automatics and Hydraulics of the Ministry of Defense have authored papers in our area. In Leningrad, the Academy of Sciences’ Sechenov Institute of Evolutionary Physiology and Biochemistry has had some 13 authors in this area; Leningrad State University’s Cathedra of Comparative Physiology has had nine, the Ukhtomsky Physiological Institute, one, the Cathedra of Higher Nervous Activity, six, the Zoological Institute, three, and the Leningrad Institute of Aviation Instrumentation has had eight. In Kiev, one author came from the Institute of Cybernetics, 18 from the Institute of Zoology of the Academy of Sciences of the Ukrainian SSR and one from the Ukrainian Institute of Hydrodynamics of the same Academy. A few authors each credit the Minsk State University, the Atlantic Institute of Oceanography and Fishery in Kaliningrad, the Pacific Institute of the same name in Vladivostok, and the Gorkiy State University. Some 26 authors are known to have worked at unnamed facilities in the Sevastopol’ area and eight at the Karadag Biological Station of the Academy of Sciences of the Ukrainian SSR Institute of the Southern Seas, in the Crimea. These last collaborate especially with the group at the Acoustical Institute in Moscow. This survey is reasonably comprehensive for the topics listed back to
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about 1960, before which there was almost no original literature in this area from the USSR.’ Some of the findings reported here may have been reported earlier in non-Soviet literature; we could not undertake a distribution of credit for priority of observation. We have tried to emphasize the concepts, claims, and methodological approaches so that readers, according to their areas of interest, will know what Soviet literature overlaps with those areas. II. Neuroanatomy
A. GENERAL An underlying position of a number of Soviet authors is that the cetacean brain developed in a different direction from that of familiar terrestrial mammals. We will see later, especially under “Cerebral Cortex,” some evidence for this. This means that the size of the brain does not have the same significance it would have in a primate or other mammalian order. For example, Tomilin (1968b), one of the generalists and long-term students of the cetaceans, regards the huge dolphin brain, with its unparalleled degree of convolution, exceeding that in the human, to be somehow a special adaptation to the living conditions and habits of life. Echolocation and acoustic analysis play a large role in this adaptation but in his view dolphins do not have real language or speech (see also Blinkov and Glezer, 1964; Krushinskaya, 1974; Simkin, 1974; Tomilin, 1975). The book of Blinkov and Glezer has a good deal of quantitative information about the brain size, area of the cortex, glial index, number of fibers in the optic nerve and the like, in several species of cetaceans. In agreement with the general view above, Kesarev (1970a, 1975) points to the relative paucity of paleocortex, hippocampus, and hypothalamus in dolphins as well as the relative monotony and simplicity of the structure of the neocortex. This in turn may be related to the proportionately small corpus callosum compared to that of the human brain. B. ONTOCENY
A series of short papers deals with the embryonic development of a few morphological and reflex characteristics of cetaceans. The formal The published proceedings of the Seventh All-Union Conference on Marine Mammals held at Simpheropol in 1977 arrived very recently and only some of the 44 relevant papers therein could be included here. All are cited in the Bibliography, however. The same is true of an important new book by Supin et al. (1978b).
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
53
tion of the optic nerve in early embryos of the sperm whale (Physeter catodon) from a hollow optic stalk to a compact trunk, including the nonsimultaneous development of its sheaths, are described by Petrova (1975). Khomenko (197513) described a sequence of myelinization in the three main branches of the dolphin’s facial nerve, and related this to the development of the respiratory and suckling reflexes. T h e branches innervating the muscles of the blowhole and system of nasal sacs become myelinated much faster than those for suckling. T h e last to be myelinated are the branches of the facial nerve that innervate the muscles of the bed of the melon; this is attributed to the immaturity of the young animals’ echolocation system (see also Agarkov and Sokolov, 1975). Leontyuk et al. (1975a,b) dealt with the development of the phrenic nerve and diaphragm, the trigeminal and spinal ganglia and some nuclei of the brain stem and ventral horn of the spinal cord. Yasyunas et al. (1975) described the early development of the hypophysis in the dolphin and sperm whale. It is emphasized that much is in common with the ontogeny in terrestrial mammals. A few facts about the fetal cerebral cortex, given by Kesarev et al. (1978), are presented in Section 11, H. C. PERIPHERAL NERVOUS SYSTEM
Agarkov et al. (1974) have brought together in a 167-page book, the results of a series of papers on some aspects of the morphology of the family Delphinidae, including gross anatomy of some of the peripheral nerves, the receptor and effector endings in muscles, and the structure and innervation of the integument (see also Agarkov et al., 1971, 1972a, 1973, 1975a; Khomenko, 1973a, 1974, 1975a). Special aspects of the anatomy of marine mammals relevant to “hydrobionics,” engineering principles in animals specialized for life in the aquatic environment, are brought together by Agarkov (1969; see also Karandeyeva et al., 1970). T h e skin of dolphins has long been supposed to play some special role in lowering hydrodynamic resistance (Karandeyeva et al., 1970). One of these suppositions is the presence of reflexly controlled movements of the skin such as to improve the laminar flow of water. This depends on drastic and primarily conjectural propositions concerning both direct responses within the skin to mechanical perturbations in the boundary layer (between skin and external fluid) and reflex changes. Superficial nerves are thought to sense the perturbations and generate impulses that, besides being transmitted to the CNS, also spread “through the axoplasm” to other branches of fibers terminating in the papillae and underlying adventitial vessels, so that the size of the lumen of these vessels and the blood volume in the papillae will change in
54
THEODORE H. BULLOCK AND VLADIMIR S . GUREVICH
response (Khomenko and Khadzinskiy, 1974). These statements are clearly conjectures. T h e postulated changes in blood volume are supposed to have “damping properties” that occur “almost in synchrony with change in the hydrodynamic conditions of the boundary layer.” If the compensatory mechanisms of the skin are inadequate, then a reflex arc is thought to be triggered, providing additional antiturbulence mechanisms. For example, the cutaneous musculature would contract, blood would be redistributed in vessels in the deeper skin layers, and the density of the fatty tissues would change. T h e damping properties of the skin are not elaborated but presumably are the supposed properties that laminarize the water flow over the skin. There appears to be no consideration of the probable time constants of movements in the skin which would have any influence on laminar flow, in order to compare them with the reasonable time constants of continuous muscle action. These authors and others (see Agarkov and Ferents, 1967; Agarkov et al., 1974; Agarkov and Valiulina, 1974; Gilevich and Khomenko, 1975; Il’ich and Khomenko, 1975; Manger and Khomenko, 1975) described a rich innervation of free and of complex encapsulated and unencapsulated receptors in the skin. Khomenko (1969a, 1970, 1975a) described some features of the innervation and histology of the melon in Tursiops truncatus by branches of the trigeminal and facial nerves and regarded the melon as an acoustical lens that has various types of sensory terminals acting as a “retina” that can image acoustical space! Receptor structures of varying complexity are classified into three main groups. (1) Preterminals end in dermal papillae and in the epidermis. (2) A second group of receptors appear as compact clusters in the fatty tissue around the air sac walls. (3) T h e third group consists of Krause’s corpuscles, unencapsulated basketlike structures and polyaxonic glomerular receptors in the dermis and hypodermis. The author suggests that the first and second groups may serve as sensors of stimuli connected with air movement during respiration, vibration of the air sac walls and septa in sound generation and displacement of fatty tissue of the melon during echolocation beaming adjustments. The third group possibly functions as “mechanoreceptors receiving stimuli connected with pressure oscillations in the air sacs, vascular wall tone stress, pressure changes in the fluid filling the peripheral sheaths, and possibly sonic and ultrasonic” acoustic stimuli. Kozak (1973, 1974a, 1975) called attention to the very numerous papillae or rugae on the posterior wall of the frontal air sac in the sperm whale (Physeter catodon). He stained the nerve endings in these papillae with reduced silver and methylene blue and estimated the number of
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
55
neural units. In a whale of 15 m length, weighing 34 tons (ca. 27,000kg), the papilla field is about 1 X 0.5 m and includes ca. 3000 papillae. T h e density of nerve endings in the upper portion of the papilla approaches 37 “units”/mm2.T h e total number of receptors in the papilla field of 0.7 m2 is well over 2 x lo7 units. T h e author interprets this structure as a specialized receptor for acoustic stimuli representing the equivalent of an acoustic retina and forming an image of the surrounding space in the water, corresponding to the absorption and reflection of acoustic energy. This appears to be a gratuitous interpretation, without supporting evidence of the acoustic function. T h e same idea had been proposed and criticized in marine mammal conferences in the United States. The possible specialization of trigeminal nerve endings in cetaceans as in other aquatic mammals is well worth physiologic investigation and it seems quite likely that unfamiliar mechanoreception is involved but it also seems premature to regard these endings as acoustic. Morozov (1976)devotes a paper to a critical evaluation of Kozak’s hypothesis, concluding that the presumed function as an image-forming acoustic receptor is insufficiently substantiated; Kozak‘s findings, he believes, can be interpreted as compatible with the principles typical of all echolocating animals. Kolchin and Bel’kovich (1973)studied tactile sensitivity in Delphinus delphis. They report the threshold of touch allows dolphins to discriminate differences of 1C-40 mm of water pressure. They consider this a high degree of discrimination and suggest the participation of cutaneous receptors in the perception of mechanical disturbance in the aquatic medium and, in particular, acoustic waves. Surkina (1971, 1975)described the structure and function of the skin muscles in dolphins of several species. Their extent and complexity permit the formation of localized skin folds on the body of Tursiops such as were described by Essapian (1955)and photographed in underwater motion pictures. The relation of the terminal branches of nerves that spiral and curl in association with thick networks of capillaries and arterioles close to the skin surface and unprotected by fatty tissue suggests a role in thermal regulation (Agarkov et al., 1974). Nechayeva et al. (1973)reported a systematic survey of the surface of Tursiops and Phocoena, measuring the electric potential of points on the skin relative to a reference electrode on the fluke. T h e animals were out of water and the portion of the skin under measurement was carefully dried. A silver/silver chloride electrode with a salt bridge was brought in contact with the surface and potentials recorded which are stated to be within the range of 135-180 mV. These are extraordinary values com-
56
THEODORE H. BULLOCK AND VLADIMIR S. GUREVICH
pared to skin potentials in other animals where they are typically 100 to 1000 times less. There is no internal evidence in the publication to confirm that the units reported are millivolts rather than microvolts or that the authors are aware of the reported values being extraordinarily high. Local peaks and left-right asymmetries are noted. Measurements over time vary slowly, perhaps due to changes in the general state of the animal. Malyshev (1969), Manger (1972), and Il'ich et al. (1973) studied the innervation of the airways, pharynx, and larynx, following up earlier work of Khomenko (1969b) and Khomenko and Nechayeva (1971). Solntseva ( 1972) described sensory innervation of the external acoustic canal. Rodionov (1974) detailed the muscles of the dolphin head, including those related to the supracranial respiratory passages; he hypothesized that these muscles, besides protecting from incoming water, function in sound generation. Agarkov and Veselovskiy (1975) described the innervation of the adrenal and thyroid glands, Khadzinskiy ( 1975)that of the myocardium, Vronskiy (1975) the innervation of the pharynx, and Vronskiy and Manger (19'15) and Vronskiy (1975) the branches of the vagus nerve in the pharynx and larynx of the Black Sea dolphins.
D. SPINAL CORD,BRAINSTEM,AND CEREBELLUM Zhukova and Kesarev ( 1972) described some structural peculiarities of the spinal cord. Accounts of the gross anatomy of the brain, including the cranial nerves, are given by Kulikov (1974), who regards the differences between an odontocete (harbor porpoise) and a mysticete (sei whale) as confirming the diphyletic origin of cetaceans, by Agarkov et al. (1972a, 1973, 1975a), and by Khomenko (197313). Amunts (1975a,b) estimated the cell density in brain stem nuclei of a dolphin (Tursiops truncatus), comparing reticular formation nuclei with those of the cranial nerves. She divided nuclei into three groups: highdensity nuclei with 100-173 cells per 0.01 mm3, nuclei of moderate (30-100) and low density (20-30). In the reticular nuclei it is claimed that cell density is inversely proportional to the number of afferent connections. Based on correlations among some 20 nuclei, it is said that those with a high cell density perform an autonomic function or play an important part in coordinating motor and sensory influences. In contrast, reticular nuclei with a moderate cell density are those of the lateral receptor or associated parts of the reticular formation and their density is similar to that of the sensory nucleus of the trigeminal nerve. Nuclei with low cell density are situated primarily in the medial part of the
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
57
reticular formation which is regarded as efferent and as characterized by particularly long and extensive dendrites receiving input from cerebral cortex and many other sources. The total volume of brain stem reticular nuclei is low in proportion to the rest of the brain compared to land mammals, and some particular nuclei are remarkable. Zvorykin (1975) and Trykova and Verbitskaya (1976) found that the lateral vestibular nucleus is much larger than the other three nuclei of the vestibular complex in Tursiops and Delphinus, whereas it is the smallest of the four in man. Although large in dolphins (9 to 16 times that of man), the lateral is the nucleus with the lowest cell density. Adanina (1969) studied the cytology of some of the cell types in the cerebellar cortex, by Nissl and Golgi methods. Aside from a generally “high specialization,” nothing peculiar is claimed for the cetacean.
E. CHEMORECEPTORS AND BEHAVIORAL CHEMORECEPTION Yablokov (1961a,b) and Bel’kovich and Yablokov (1963) pointed to “fossulae”in the tongue of toothed whales, but not baleen whales, which are presumably gustatory, though called olfactory by these authors, without explanation. In baleen whales, however, at the ends of their snouts are peculiar paired hollows which these authors identify as Jacobsen’s organs. They state that whales travel routes which are perpendicular to isohaline lines on the map and propose that they do so by very accurately perceiving the concentration of salts in the water, although this is varying by hundredths of one percent! Without more reason than is offered, this proposal appears naive. Sukhovskaya (1972) described probably the same sense organs in the fossae of the root of the dolphin’s tongue, and appears to be the first to call them taste buds; she saw typical nerve endings (see also Shvyrev, 1975). Tomilin and Valiulina (1972) and Valiulina and Khomenko (1976) stained the tongue of Tursiops with a silver method for nerve fibers and saw two nervous plexuses, one superficial and one deep. Receptors include free endings with compact and ,with diffuse branching and encapsulated bodies. Without giving reasons, they state that these findings suggest a gustatory function, whereas it would seem more likely that these descriptions apply to receptors of ordinary cutaneous modalities. Kuznetsov (1974) reported that a bottlenosed dolphin solved a complex problem requiring the detection of indole or camphor (0.01%). Indole is said to have no gustatory effect and the concentrations are believed to be too low for gustatory perception so that these authors concluded functional olfaction must be assumed. Without better evi-
58
THEODORE H. BULLOCK AND VLADIMIR S. CUREVICH
dence this hardly seems justified, but neither should we categorically reject the possibility of cetacean olfaction. Kuznetsov (1974) undertook to test dolphins' discrimination on the basis of chemical composition of solutions placed in the mouth using food-rewarded, lever-press conditioning. Two bottlenosed dolphins were trained to swim to a platform after a sonic cue and to hold the rostrum out of the water with the mouth open to receive a solution. The stimuli used were salt water and solutions of valeric acid in salt water (0.1 and 0.05%), oxalic acid (2.5 and 0.7%), and P-phenylethyl alcohol (0.04%).Water or chemical solutions were delivered in random order at 3-min intervals. The behavior indicated that the two animals made correct discriminations between each of the substances and salt water, in each of the concentrations used. In 1978 Kuznetsov reported that Tursiops responds to a dilution in sea water of a 0.9% ethanol solution of the prostate gland secretion and 0.005% solution of the secretion of the excretory gland. He concludes that there is chemical communication between dolphins. F. AUDITORY SYSTEM Solntseva (1975b) distinguished four hypotheses on the reception of acoustic reflections from the echolocation signals emitted by dolphins. These are (1) that sonic signals can enter through the obliterated external auditory meatus, (2) that sound enters through the mandible and conducts through a lipid-filled channel, (3) that the echo signal is picked up by a multichannel system of mechanoreceptors distributed over the head and imaged in the brain from these widely distributed inputs (Agarkov et al., 1971), and (4) that the melon constitutes an analog of an optical system while the pericranial air sacs are analogs of the retina of the eye (Reznikov, 1970). The last two would involve mainly the trigeminal nerve and presumably high intensity acoustic stimuli. No evidence is offered for these or for the first alternative. The second has been physiologically demonstrated by Western authors (Bullock et al., 1968). Bel'kovich and Solntseva (1970) and Solntseva (1975a) described the ear, from the meatus to the cochlea, with only a few statements about how dolphins differ from other mammals. Bogoslovskaya (1975) provided a note on the electron microscopy of the spiral ganglion and acoustic nerve in dolphins. The cytology of the large cells of the spiral ganglion and their sheaths is detailed. The parts of the acoustic nerve examined contained only myelinated fibers, usually of 5-7 pm diameter compared to the 2-4 pm usual in other animals. In longitudinal section, significant changes in diameter seem to occur, for
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
59
example, local constrictions of 9- to 16-fold were common. Small processes could be the start of internal collaterals of the acoustic nerve, thought perhaps to form an acoustical-vestibular anastomosis. Bogoslovskaya (1974) reviewed previous work on the structure of the cochlear nuclear complex and added new points from Nissl and Golgi stains. As already known the exceptionally large complex is chiefly the result of the high development of the ventral cochlear nucleus (Table I), which in common laboratory mammals includes a tonotopic organization. In contrast, the dorsal cochlear nucleus is relatively, though not absolutely, reduced in cetaceans. In Phocoenu phocoena Bogoslovskaya ( 1974) confirmed three important features of the dolphin’s dorsal cochlear nuclear complex. (1) A sharp decrease in the number of granule cells compared to laboratory mammals goes with (2)a virtual disappearance of lamination. (3) There is a parallel increase in the number of “reticular” cells and forms derived from them. Since the granule cells, in species where they are well developed, can form a “substantial or even a major portion” of the dorsal cochlear nucleus, providing the short axon cell linkages and much of the laminated cortical structure, their virtual absence in the Cetacea as in some other groups of mammals, such as the higher apes, must have some significance. Four features of the dolphin’s ventral cochlear nucleus were emphasized by Bogoslovskaya (1974). (1) The main increase in the number of neurons in the cochlear nuclear complex is attributable to particular cell types and derivatives of them in the ventral nuclei; these are involved in both intranuclear and internuclear interactions as well as connections between cochlear nuclei and other ventral nuclei. (2) There is a considerable number of large multipolar neurons with a complex, dendritic system in the anterior and posterior ventral nuclei. These she believes to be integrative cells that project axons to motor structures of the medulla. (3) Four main types of neurons and transitional forms can be recognized in the anterior ventral nucleus, the main sensory center of the acoustic nerve, including the types mentioned in (2),above, and (4), below. Some patterns of proportion of the different forms have been demonstrated in areas presumably receiving low-, medium-, and highfrequency sounds. The most complex portion of the anterior ventral nucleus with respect to composition of cell types and connections is the superficial area that is said to coordinate the function of the projection areas for different frequencies. (4) The tufted neurons, receiving cochlear projections in the anterior ventral nucleus, are more complex in dolphins than in bats, carnivores, and primates. Petelina (1973) examined the superior olivary complex in Tursiops tmncatus and Delphinus deZphis. Using the light microscope and the Nissl
Dolphin Bat Man
1
2
3
nuc. cochl. dors.
nuc. cochl. vent.
oliva nuc. super. trapez.
7.0 0.02 1.2
160. 0.23 10.8
150. 0.16 0.96
4
85.
I
Visual
Auditory
5
6
7
8
9
10
11
12
13
nuc. lem. lat. vent.
nuc. lem. lat. interm.
nuc. lem. lat. dors.
collic. inf.
corp. genicul. med.
sum, 1 to 9
collic. sup.
corp. genicul. lat.
Sum Ratio 11 + 12 10:13
217. 0.04 1.10
160 0.08 0
40 0.02 0.43
470 1.21 38.0
340 0.22 48.5
1629 1.98 101
143 0.10 92
251 0.60 194
108 0.50 102
14
15
Brain weight
( P I 6.5
3.3 0.52
780 0.5" 1250
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
61
stain, she was able to extend somewhat the previously published information (Fig. 1). The whole complex is strongly developed, formed by the closely packed nuclei of the trapezoid body and the superior olive. Within the former it is possible to recognize two subdivisions, the medial and the lateral nucleus of the trapezoid body. ( 1 ) T h e medial nucleus can be subdivided into the medial nucleus proper and the medial ventral segment. The former is much enlarged. In contrast to that of other animals, in the dolphin the medial nucleus is separated from the midline by a pronounced medial ventral segment. This segment approaches the raphe and forms a lamina bent into nearly a right angle. In this angle lies the medial nucleus proper. Four types of cells are distinguished in each of the trapezoid nuclei (see also Man’kovskaya and Petelina, 1973; Zharskaya, 1978). (2) The lateral nucleus of the trapezoid body is
ml
0
2 m3
FIG. 1. The superior olivary complex of the brain of the bottlenosed dolphin, Turstops truncatus; schematic outlines in a series of transverse sections of the medulla of one side. The midline is the right-hand margin. Symbols: 1 = medioventral segment of the medial nucleus of the trapezoid body; 2 = the medial nucleus proper; 3 = lateral nucleus of the trapezoid body; Iso = lateral segment; m o = medial segment of the superior olive; n = VIIth nerve (facialis) nucleus; n. cochl. = cochlear nucleus: nll = nucleus of the lateral lemniscus; f i r = pyramid. (From Petelina, 1973.)
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THEODORE H. BULLOCK A N D VLADIMIR S. CUREVICH
situated under the olive and consists of average and small cells rather widely and randomly spaced. Rostrally, the nucleus extends without a noticeable boundary into the nucleus of the lateral lemniscus. T h e superior olive in the dolphin is substantial in size. It is “somewhat more impoverished” in cells than in some other mammals such as cats, rats, and bats. Presumably this means the packing density is low although the total cell number is still high. The medial segment of the olive is only a narrow column of cells. In the whole complex, however, it is not so much the superior olive as the trapezoid body that is responsible for the great development of the complex in dolphins, exceeding in this respect other mammals, even bats. The lateral lemniscus of dolphins is not only prodigiously large (4 17 mms vs 1.53 mm3 in man; see Table I), with a major nucleus unknown in the human (nucleus intermedius), but its cell density is much higher. T h e inferior colliculus is 12 times larger in a dolphin than in man (Table I) and, though the cell density is somewhat less, it is more differentiated. The inferior colliculus of dolphins is the subject of two short papers by Sukhovskaya (1968, 1975). The author begins with the curious conclusion based on a review of the literature that the ability of animals to perceive high frequency sound depends upon size and complexity of the subcortical auditory centers, without proposing what aspect of perception requires or makes use of this size and complexity once the peripheral end organ is adapted to detect it. Based on studies of Tursiops truncutus and Delphinus delphis and Golgi as well as standard histological methods, four groups of cells are described. While the form of the neurons in two of these groups corresponds with humans, they are more complex in dolphins. They are characterized by extremely welldeveloped synaptic apparatus of both axosomatic and axodendritic types; the total number of synapses on the neurons on one of the groups in the dolphin considerably exceeds that of the cells on the same group in the inferior colliculus of the bat. In several species of insect-eating bats as well as in the dog and cat there is said to be a prevalence of small afferent neurons in the lateral group, whereas the large multipolar neurons of the central group are thought to be efferent to the reticular formation of the mesencephalon and medulla connecting to motor centers and centers of other sensory modalities. The medial geniculate is seven times larger in a dolphin than in man and, moreover, the packing density of neurons is about twice as high (Zvorykin, 1963). The auditory cortex has been repeatedly sought histologically in the temporal lobe. A series of papers by Ladygina and Supin (1970,1975, 1977) and Sokolov et al. (1972) provided the first maps of the auditory area of
NERVOUS SYSTEM A N D PSYCHOBIOLOGY OF CETACEA
63
the cortex, determined by the evoked potential technique. These papers are reviewed more fully in Section 111, F. As shown in Fig. 5 (p. go), the auditory area is large and is in a remarkable position-high on the parietal lobe, adjacent to the visual area. Its center is only 15-25 mm lateral to the midline suture of the skull. They distinguished secondary response areas adjacent to the primary projection zone on physiological grounds. Thus the topographic location of the auditory areas in the cortex seems to be significantly displaced from that in terrestrial mammals. Comparison of the size of the auditory area with that of other mammals depends on the assumption that the area recognized so far by the evoked potential technique corresponds to auditory areas 1 and 2 of other mammals. If this is correct, the auditory area is indeed large in these cetaceans. Avksent’yeva ( 1974) reported that the auditory cortex, like the visual, is unspecialized. In the sensory cortex, as in the cortex generally, the fourth layer is not differentiated from the third and fifth. Kesarev et al. (1975) did not disagree with this but claimed to distinguish primary and secondary cortical acoustic areas on cytoarchitectonic criteria, particularly the density of cells in the third layer and the size of cells in the fifth layer. They remarked that the difference between primary and secondary acoustic areas is less than that in carnivores, “which may be indicative of less specialization of sensory functions, in particular auditory ones, in the cetacean cerebral cortex than in terrestrial mammals”-a remarkable conclusion if it holds up. Further details from these authors are given in Section 11, H. Figurina (1978) raised the question of whether perhaps not only the parietal areas revealed by evoked potentials but also some temporal cortex may be auditory. She applied the Nauta-Gygax technique-the first use of modern experimental anatomical techniques, on cetacean+ seven days after lesions in the posterior sylvian temporal cortex and suprasylvian parietal cortex. She stated that both regions send fibers down to the ipsilateral medial geniculate body, nucleus of the lateral lemniscus, superior olivary complex, and ventral cochlear nucleus. On this ground she concluded that the auditory cortex includes both parietal and temporal regions. It will be important to see the evidence in full; at this writing only an abstract is available. Krasnoshchekova (1978) also used the method of lesioning and staining degenerate axons and terminals (Fink-Heimer), but this time for the projections from medial geniculate to cortex. A large area of both parietal and temporal cortex receives projection, perhaps differing according to the part of the medial geniculate coagulated. In some regions, the degenerating axons ascend partly to layer 111, where degenerating terminals can be seen, and partly to the upper third of layer I, where they turn horizontally. In the middle of
64
THEODORE H . BULLOCK AND VLADIMIR S. GUREVICH
gyrus suprasylvius, degenerating axons and terminals are found in all layers, including I. G. VISUALSYSTEM Andreyev (1975a) reported that among eight species of delphinids and eight species of pinnipeds, accommodation of the eye is by change in the cornea-retina distance, not by alteration of lens shape. L. M. Herman (personal communication) cites W. Dawson to the contrary. Andreyev (1975b) reviewed the distinctive features of the light transmitting system of the eye in a number of marine mammals, both cetacean and pinniped. More details on these features for dolphins were given in a 1974 paper together with some facts about the retina. There is a negligible number of cones compared to rods (10,000:200,000 per mm2 in the center of the retina). He counts ca. 200 ganglion cells per mm2, or more than 1000 photoreceptors per ganglion cell. L. M. Herman (personal communication) warns that higher ganglion cell counts and lower ratios are soon to be published. Photosensitivity of the dolphin eye was measured by a conditioned response technique and the threshold was given as 0.009 lux. Acuity was also measured and found to be poor (contra Herman et al., 1975). Padalkin ( 1975) briefly described peculiarities of the blood supply to the eye in Delphinus. As in the brain and elsewhere, there are very unusual features in the disposition of networks, loops, spiral vessels, circular venous sinuses, venules, arterioles, and anastomoses. Malofeyeva (1978) has studied the lateral geniculate body in three species, in respect to cytoarchitectonics, but provided little beyond stating that the structure is “nuclear” and the distribution of cells is “monotonous.” Information on the visual cortex is treated in the next section. H. CEREBRAL CORTEX
Kesarev (1968, 1969, 1970a,b, 1971, 1975), Kesarev and Malofeyeva (1969), Kesarev et al. (1975, 1977), and Malofeyeva et al. (1975) have employed standard stains as well as an original modification of the Golgi impregnation and a good deal of quantitative estimation of areas in a series of papers on the structural organization of the cortex of dolphins. They confirmed the main features already known: that the dolphin has a larger area of cortex for its brain size than other mammals and that a uniquely high percentage of the cortex is neocortex (97.8% in dolphin compared to 95.9% in man), that the cortex is very thin (ca. 60% of the
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
65
human) with a virtual absence of the granular layer, especially in the posterior parts of the cerebrum, and relatively thick layers 1 and 6 (Fig. 2). They add several other characteristics, including an almost complete absence of vertical striation and only a slight variation of cytoarchitectonic characteristics that can distinguish different areas, even as between limbic, motor, and sensory cortex. T h e horizontal fibers of the cortex, to which an association function is ascribed, in the dolphin lie mainly in the deepest layers, said to be the oldest and functionally most primitive, whereas in man they are more developed in the superficial, phylogenetically newer levels. Neurons of pyramidal shape are relatively more dominant than in other animals. Stellate cells are relatively scarce, whereas so-called transitional forms, triangular, quadrangular, clublike, and others, are numerous. N o quantitative comparison with other orders of mammals is made in this respect; however, they regard the dolphin cortex as low in the degree of differentiation of neurons. Pyramidal cell size does not range as widely as in man (Table 11). These authors believe that the neocortex plays a more important role
FIG. 2. Cytoarchitectonicsof the cerebral cortex of a dolphin (right) compared with that of a human (left) and a dog (center): sections cut normal to the surface of the motor area. Cresyl violet; all x 30. The layers are numbered I-VI. (From Kesarev, 1971.)
66
THEODORE H. BULLOCK AND VLADIMIR S. GUREVICH
TABLE I1 VOLUME OF CORTICAL PYRAMIDAL NEURONS IN CUBIC MICROMETERS” Layer
Dolphin (Tursiops) (Mean 2 )
Human (Homo) (Mean 2 )
980 & 34
735 f 35
~
I1 (smallest pyramidal cells)
V 17,780 & 794 (largest pyramidal cells, including giant cells of Betz in human)
41,780 f 1308
Modified from Kesarev et al. (1977).
in the regulation of visceral mechanisms and correlations between visceral and somatic mechanisms in the dolphin than in terrestrial animals. In part this appears to be due to the relative paucity of limbic cortex. They cite as one explanation for this fact the work of Filimonoff (1949, 1965), who related the quantitative deficiency of dolphin archicortex and mesocortex to the absence of olfactory input. This correlation is hardly a sufficient explanation but we may note the further correlation with a poorly developed hippocampus and hypothalamus. Nuclear formation in the hypothalamus of the dolphin is almost completely lacking; the ventromedial and dorsomedial nuclei cannot be detected. In spite of the relatively “monotonous” character and the absence of sharp transitions, Kesarev and co-workers (1975, 1977) have made cytoarchitectonic distinctions and divided the cortex into a small number of fields on criteria which are necessarily different from those used in familiar mammals, particularly of density of cells in the third layer and the size of cells in the fifth layer (Fig. 3). They emphasized that this requires determination of the epicenters where the morphological characteristics are the most distinct and estimation of transitional regions since there are not the sharp boundaries that are found in more familiar terrestrial mammals. In the parietal cortex they distinguished superior, central, inferior, and transitional cytoarchitectonic fields. In the temporal region, which is considerably smaller than the parietal in dolphins, only two fields were distinguished, external and internal temporal, both of which are transitional in structural organization. In an early study, Zvorykin (1963) considered the temporal cortex to be relatively more developed than other areas since layer I11 can be subdivided; he believed he was looking at auditory cortex but now it appears that at least the primary and secondary auditory cortex is not temporal. With the benefit of the physiological studies of Ladygina, Supin, and Sokolov referred to below, Kesarev had physiological evidence defining the primary and secondary sensory areas of visual and
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
67
-...."_..". Prn
FIG. 3. Cytoarchitectonic map of dolphin cerebral cortex. (A) anterior aspect; (B) posterior aspect; (C) lateral aspect; (D) medial aspect. a, anterior; c, central; e, external; f, frontal; i, inferior; 1, lateral; L, limbic; Lla, anterior borderline limbic; Up, posterior borderline limbic; Ls, anterior subgenual limbic; m, medial; 0, occipital; p, posterior; P, parietal; s, superior: T, temporal. (From Kesarev el al., 1977).
acoustic cortex and could compare these histologically. The finding was that primary and secondary zones are less different than would be true in carnivores, for example. Limbic cortex is also less distinct from isocortex than is usual in mammals. From all these findings Kesarev and his co-workers conclude that the cetacean cortex is less specialized in sensory functions and, in particular, in auditory functions than is the case in terrestrial mammals. Malofeyeva et al. (1975) quantitatively compared the frontal and occipital regions with respect to the absolute and relative width of the layers and of the entire cortex, the neuron density in the different layers, the volumetric fraction of neuronal bodies in different layers, the mean neuron volume in different layers, and the distribution of pyramidal cells, horizontally in layer 5. They found no appreciable differences in overall thickness between the occipital and frontal samples. The narrowest and most stable layers are layers 2 and 5; layer 3 is the most variable and is better developed in the occipital region. Layers 1 and 6 together occupy over half the thickness, relatively more in the frontal
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cortex. Neuron density is greater in all areas and the frontal and occipital cortex of the dolphin are more similar than would be true in terrestrial mammals, with the greatest divergences in the well-developed layer 3, which they regard as phylogenetically new. Bogolepova and Kesarev (1975) developed the idea that overlapping of transitional zones between neocortex and archicortex, referring to the wedgelike tapering of the respective laminae into one another, is in most mammals an ontogenetic stage that disappears earlier in groups that are higher on the evolutionary scale. Since in this respect the cortex of dolphins shows marked overlapping even in the adult, these animals are regarded, to this extent, as relatively lower. The view expressed in the book by Yablokov et al. (1972) that the neocortex of cetaceans has a high degree of differentiation among placental mammals, based on the supposed large variety of nerve cell types in Golgi impregnations, is apparently unique in Soviet literature. This chapter in the book of Yablokov et al., a general compendium on whales and porpoises, reviewing the world literature, was written by Professor G. Pilleri and Dr. M. Gihr of the University of Bern, Switzerland. These authors agree on the thinness of the cortex, the absence of a granular layer, and the relatively small corpus callosum, and they do not claim a regional differentiation comparable to that of primates or even of carnivores, especially the sharp boundaries between cytoarchitectonic regions in familiar mammals. The number of nerve cell types has not been quantitatively compared with that in other mammals using modern criteria (Szentagothai, 1978). Kesarev (1970a, 1971, 1975) and other authors already cited (see Section 11, A) concluded that homologies of cortical structures (macroand micro-) are so difficult as between cetaceans and other mammals, the former being so peculiar in their neocortex that one must recognize a new type of cortex, “preneocortex” (Kesarev et al., 1977), found only in this group and never observed in terrestrial mammals (see also Ladygina and Supin, 1978). Entin (1973) described Golgi and Nissl material from the pole and from the medial surface of the occipital lobe in dolphins. Previous authors, he said, advocated contradictory views of the level of organization of the dolphin cortex, from the denial of the possibility of comparing it with the brain of other animals to the assertion that the dolphin brain reaches the primate level, although by other means. Entin concludes that the cetacean neocortex is of a significantly distinct type from the primate but cannot be considered to be primitive. He confirms the features listed above and states his impression that dendrite spine distribution, length, and density are not essentially different from those
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features in the human. Considering the great variety of cell types and the differences in relative abundance of them, this impression should be quantitatively checked. T h e occipital cortex is unusual in that the layer he designates as 4 splits into three sublayers. Other authors have described, with respect to other regions of the cortex, layer 3 as subdivided into three sublayers. Pyramidal cells of layer 5 are not in groups of only three to four neurons as in the cat but up to seven or eight. Bundles of apical dendrites are conspicuous, extending up to layer 1 where they splay out into overlapping fans. Ladygina and Supin (1970, 1974, 1975, 1977), Ladygina etal. (1978), and Sokolov et al. (1972) have mapped the sensory cortex by the evoked potential technique (Fig. 5, p. go),as already mentioned and as will be more fully treated in Section 111, F. The visual area is not at the occipital pole but in the superior portion of the parietal cortex, as in nonprimate orders generally. The auditory area is also high in the parietal cortex, occupying a rather extensive region just rostral and adjacent to the visual area. The somatosensory and motor areas overlap nearly completely just in front of this, with the rostral region of the body represented in the lower anterior part of the gyrus and caudal regions in the upper parts of the gyrus. O n the basis of latency, they distinguished a primary and a secondary auditory area. These authors regarded the sensory projection areas of different orders of mammals to be homologous, whereas the nonprojection areas presumably developed in cetaceans from other parts of the cortex of primitive mammals than did the analogous areas of carnivores and primates. There seems to be a logical inconsistency here unless more is asserted about the definitions of the entities compared. However, the authors are properly emphasizing that the visual and auditory areas in particular are not located topographically where one might expect from a comparison with primates. They could not establish retinotopic or tonotopic projection for the visual and auditory cortical areas or define the boundaries of primary and secondary sensory cortices because of the difficulties posed by the closely spaced and very deep sulci of inconstant pattern. Zvorykin (197 1) made the same point as E. D. Adrian did in the 1930s, namely that sensory modalities apparently important to the species occupy relatively large areas of the cortex as well as of subcortical and brain stem structures. This is essentially based on the comparison of auditory and visual areas in dolphins, bats, and humans (see Table I). T h e first cytoarchitectonic study based on this physiological work and therefore dealing with cortex which may be justifiably labeled auditory or visual is that of Avksent’yeva (1974). She compared the auditory and visual cortices of Phocoena, Tursiops, and Delphinus and, by way of con-
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trast, the auditory cortex of some seals (Pinnipedia). Apparently differing at least in emphasis from Entin, she described layer 4 as not differentiated separately, its neurons being scattered in the lower part of layer 3 and the borderline of layer 5. The auditory and visual cortices are said to present no structural differences. The seals examined are typical of carnivores in these respects, and they contrast with the dolphins, in the greater thickness of cortex, the more conspicuous horizontal lamination and vertical striation, the well-developed layer 4 and the granule cells, the columnar arrangement of cells in layer 6 and the more “correct” orientation of the apical dendrites of cells in layers 3,5, and 6. Taking all features together, the seal cortex is regarded as more complex in cytoarchitecture than the dolphin cortex. Kesarev et ul. (1977) provided some details about the cortex of a fetal dolphin (Tursiops truncutus) whose brain weight (552 gm) was about one third of the adult’s. The sulci and gyri are already well developed, but the cytoarchitectonic features differ considerably from those of the adult. The lack of a granular layer appears to be a primary character, true in both fetus and adult, whereas agranular cortex in other mammals is a secondary loss of a fetal granular layer. Differences between anterior and posterior regions show up more vividly in the fetus. Layer 5 pyramids are nearly half the adult volume in the posterior parietal and occipital regions, but only one-eighth in anterior frontal and central regions. Nerve cells are abundant in the white matter, and especially noteworthy are large clusters of neurons appearing as islands. In anterior regions the border between cortex and white matter is less definite than in posterior regions.
111. Neurophyriology
A. METHODS In this section we call attention to papers that deal mainly with methodology. Other papers that include considerations of technique but report findings are dealt with in the corresponding sections. Akhutin et al. (1972) dealt at some length with the methodology of training marine animals by instrumental conditioning. While most of the article is devoted to the theoretical aspects of such conditioning, some paragraphs deal with the particulars of a system that is attached to the dolphin by straps and suction cups and provides remote control of signalling to the animal and of delivery of negative reinforcement by electric shock o r loud sound. There are at least 20 papers on the practi-
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cal aspects of training dolphins; we have excluded these from the present review. Agarkov et al. (1972b, 1975b) described implanted telemetering devices. Two models of sensors are mentioned, without specifying the parameters sensed. T h e sensors were glued to the skin with “cyacrin.” Electronic telemetering devices were installed within the dorsal fin. It is not stated how successful the system proved to be, or the duration or the distance of its useful signalling. Soft Teflon and a plastic called Titan are recommended for implants. Gurevich and Korol’kov ( 1973) described a roentgenological method for the study of the mechanics of respiration in order to estimate to what extent the nasal air sacs may fill with air and take part in ventilation. Another method applied to the study of function in the respiratory system is implantation of miniature hydrophones for recording sounds in various places. Romanenko ( 1975a) described the placement of piezoceramic hydrophones, 2 x 3 mm at the end of a 1.5-mm-diameter shielded wire, through the blowhole at the time of inspiration to a depth of about 6 cm. Similar hydrophones were attached to the dolphin’s head near the rostrum and a hydrophone of 15-mm diameter was attached near the external auditory meatus. The three hydrophones were connected by wires to a three-channel tape recorder attached to the dorsal fin and operated by radio. In this way evidence could be obtained that the larynx is evidently not the source of echolocating sounds; in fact, no sound pulses were recorded in the nasal passages during active echolocation as recorded by the external hydrophones, whereas the internal hydrophone recorded sound accompanying expiration and inspiration. Romanenko et al. (1974) used a similar method to record the echolocating sound pulses emitted while the dolphin swam freely in a pool. Two small hydrophones were attached anteriorly by suction cups to the blowhole; two radio transmitters were attached to the dorsal fin; two radiating aerials extended along the body of the dolphin in the form of segments of flexible wire. Carrier frequencies of 1.55 and 1.87 mHz were used. Further details on the radio telemetric methods, as well as other instrumentation and techniques for the investigation of bioacoustics in relation to dolphins, are given in several chapters of the book by Romanenko (1974~);many of the methods, as acknowledged, are taken or adapted from those used by Western authors. A compact threechannel magnetic recorder, designed to be attached to the dorsal fin, operated by radio, and carried by the free-ranging animal, is described, as well as miniaturized piezoelectric wide-band hydrophones. Several arrangements of three hydrophones attached on and about the head are
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described, permitting conclusions about the origin of the sounds and some explanations for the interesting fact that the high-frequency pulses emitted in lateral directions are shorter than the pulses propagated in a forward direction. A method is described for modelling certain hypotheses of the mechanism of sound generation and shaping. Mathematical approaches are given to the solution of problems of characteristic oscillation in air volumes with stiff but not absolutely rigid walls such as are believed to play a strong role in the characteristics of the low-frequency pulses and whistles. The methods of studying the directionality of the radiation of sounds from dolphins are described, including the insertion of a sound source into the head of a dead dolphin. The large role of the skull and the lesser role of the soft tissue in narrowing the beam of emitted sound can be evaluated. Describing a method of evaluating the hypothesis that the dolphin protects its own hearing from the overload of its strong emitted acoustic signal by shielding the port of entry of the sound to the auditory system, the author attempted to measure the latter with a hydrophone fixed to the head in the position of the principal portal, on the mandible. Agizin et al. (1974) described a sound generator for stimulating aquatic animals with controlled bursts of AM and FM tones. Bel'kovich and Gurevich (1971) reviewed aspects of veterinary care, holding, and capture of dolphins. Supin et al. (1975a) mentioned in a short note some experiences with a variety of tranquilizers, muscle relaxants, analgesics, and anesthetics. The tranquilizers Elenium and Trioxazaine are given to dolphins with fish and depress defense reactions so that the animals become calmer and more approachable. The short-acting muscle relaxant, Ditilene, can be used while performing operations. Large doses of Hexenal and sodium thiopental impair respiration but small doses are useful in combination with the local anesthetic, Novocain. Hexenal, in a dosage of 5-7 mg/kg, and intravenous thiopental, in a dosage of 5 mg/kg, combined with local anesthesia, are recommended for operations. In a review of electrophysiological recording techniques, Gapich et al. (1971; see also Popov and Supin, 1976b)described briefly their method of intubating for artificial respiration of smaller animals that cannot be intubated via the mouth, because the pharynx is too small for the hand. They conduct a tube through the blowhole and advance it past the sphincters during the moments the animal exhales and inhales. With artificial respiration they use deep Nembutal or light Hexobarbital plus local anesthetic infusion. Electrodes are sometimes led through a 4-mm guide tube that penetrates the skin and soft tissues and through which a drill penetrates the skull; this method requires X-ray control to reach a
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chosen target but is used for chronic implantation. For acute experiments, the Soviet authors expose an area of skull large enough to find landmarks and then drill holes 1-1.5 mm in diameter for electrodes aimed at chosen cortical targets. Voronov et al. (1977) gave some details on the stereotaxic procedures used and a table of the coordinates for some of the main auditory centers. Units are recorded in the cortex with glass micropipettes, driven hydraulically, from a manipulator cemented to the skull. Supin et al. (197513) described a method of chronic implantation of electrodes in the dolphin brain involving tungsten electrodes fastened to the skull under local anesthesia. Animals may survive up to 2 or 3 years after implantation. Postmortem examination reveals that the electrodes were securely fixed “without major destruction of the brain.” Cherepanov et al. (1975) reviewed electrophysiological methods applied to unrestricted dolphins, including stimulating electrodes connected by direct wire to the stimulating source, or radio-controlled. One of the methods involved spiral electrodes inserted in the brain inside a guide tube that was subsequently removed; implanting with a specially designed stereotaxic apparatus is done under local anesthesia. B. REFLEXCONTROL OF AIR MOVEMENT Kleinenberg ( 1956) reviewed the specializations in cetacean respiration, both morphological and physiological, as far as was known at that time (see also Yablokov, 1961a). He pointed out the relative insensitivity of the respiratory center to accumulation of carbon dioxide and the specialized vascular reflexes that change the blood circulation during the apneustic plateau or respiratory pause. Kolchinskaya et al. (1971, 1975) and Karandeyeva et al. (1975) contributed substantial papers on the respiration of porpoises. They employed a new breathing mask, an electronic device for the automatic collection of samples of alveolar air and techniques favoring the retention of the natural breathing regime. Besides a body of normative data on the parameters of ventilation, they studied the reflex alterations of these and of minute volume of blood in relation to oxygen capacity and heart rate. T h e reflexes caused by decreased oxygen or increased carbon dioxide content of the inspired air due to oxygen consumption and active movement are evaluated quantitatively. Two species are compared with each other and with the human. Shapunov et al. (1975) reviewed the responses to hypoxia and hypercapnia. Pershin and Sokolov (1975) compared a number of species in respect to duration of dive, motor activity, and maximum swimming speed.
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Kozak and Tomilin (1975) discussed several adaptations for deep diving, including the lack of a cough reflex. Cetaceans tolerate the introduction of fluid into the respiratory tract. The alveolar walls include muscle fibers that aid in the process of evacuating fluids at the time of forced expirations after the dive. C. ACOUSTIC SIGNALS A N D BEHAVIORAL AUDITION
1. Mechanism of Producing Sounds, Including Echolocating Clicks A series of papers concerned with the mechanics of the production of sounds will not be properly reviewed here; only a brief indication of the topics dealt with, to suggest the ways in which receptors and neural control may be involved, will be given. I n connection with the role of the airways in influencing sound generation, Kleinenberg and Yablokov ( 1958) compared the supracranial nasal canal, the larynx and pharynx in Odontoceti and Mystacoceti, and found these structures to be quite different in the two suborders of Cetacea (see also Kozak, 1977). Gurevich (1972) studied the nasal air sacs in Delphinus de1phi.s by filling with an X-ray-opaque substance. Some sacs are larger on the right and others on the left side of the head. Air sacs are usually larger in the male. Muscles which surround the supracranial respiratory passages and are associated with the nasal air sacs are described. They are arranged so that contraction could modify the shape of the sacs and presumably modify sound production. Romanenko (1972, 1973b, 1974b,c, 1978) considered the contributions of direct muscular contraction on the one hand and of rapid inflation of air sacs or nasal passages on the other. He believed the echolocation clicks are nonresonant in origin and that the air sacs do not participate in generating them. Low-frequency pulses and whistles, on the other hand, may be influenced by resonance and by the air sacs, o r nasal passages which may contribute to the left-right phase difference and asymmetry of sound pressure. Khomenko (1973a,b, 1974) considered the topography of the muscle of the melon and the mobility of its soft tissues to be important factors permitting the dolphin to vary the emitted sound as though varying an acoustic lens. He also called attention to the muscular apparatus of the nasal sacs, indicating that these are not passive organs but capable of regulating the volume of air within the sacs. He believed that the air sacs can participate in the generation of echolocation sounds, in apparent contradiction to the conclusion of Romanenko. He reported some experiments in which the nerves regulating the supracranial respiratory passages were blocked unilaterally, from the results of which he concluded that the respiratory passage is one of the possible mechanisms by which acoustic signals acquire differ-
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ent frequencies and intensities. He pointed to histological complexity and a large number of layers in the walls of the nasal sacs associated with their ability to regulate the volume of air. He found that the form of the nerve endings and receptors in the walls of the sacs differs depending on the functional role of that part of the passage. Thus the wall of the blowhole vestibule, which is highly sensitive, and the tubular and premaxillary sacs are equipped with various types of free and encapsulated nerve endings. The upper segment of the respiratory passage, which is an air tube, differs in that it has limited mobility and sensitivity and contains simpler unitypic nerve endings. Most receptors of the nasal sacs can be called mechanoreceptors in a broad sense and are presumed to detect various stimuli connected with the passage of air, fluctuations of pressure within the nasal cavity, vibration of the sac walls, and the like. Giro and Dubrovskiy (1973,1974)and Dubrovskiy and Zaslavskiy (1975) considered the origin of different components of the acoustic pulse, especially in relation to the reflecting properties of the skull and of the soft tissues lining the air cavities; the former is considered to play a major role. It contributes in particular to the directivity of the emitted sound (see also Romanenko et al., 1965),but additional factors must be operating since the directionality of the actual sounds emitted is much narrower than would be explained by the properties of the skull. The origin of the low-frequency component that follows the echolocation pulse in Delphinus is assumed to be an action of the main impulse of ultrasonic sound upon the nasal air sacs. Testing with a model nasal air sac, a rubberlike sphere with mechanical properties close to the parameters of muscular tissue, the agreement between theoretical expectation and experimental data is said to be satisfactory. Markov and Tarchevskaya (1978)emphasized that two or three independent sound sources in the head can act simultaneously or sequentially and that dolphins produce sounds during exhalation and inhalation, not only during the apneustic plateau (see Section 111, C, 2). Bel'kovich and Nesterenko (1975)attributed to Reznikov the claim that dolphins can, quite apart from head scanning, rapidly rotate the highly directional ultrasonic beam at 3.5 x lo5 deg/sec during each click, presumably by small muscular distortions of the acoustical lens in the melon. This means there is a significant shift of the direction during the single echolocating pulse, and a small target is irradiated for only a very short time. The factual basis for this interesting claim is unfortunately not available.
2, Characteristics .f Sounds (Nonecholocating) Several papers dealt with the acoustic analysis of the sounds emitted by dolphins. Those concerned with echolocating sounds are treated in
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the next section. Zlatoustova and Nizova (197 1) studied the variation in a single type of whistle with a dynamic spectrograph making spectral sections (frequency-amplitude graphs) every 10 msec. The variants within this single whistle type are only slightly different. Markov et al. (1974; Markov and Ostrovskaya, 1973, 1975a, 1978; Markov and Prokhorov, 1978; Markov and Tarchevskaya, 1978) studied the combinatory nature of dolphin whistles from the point of view of the organization of communication signals. They found that dolphins can independently manipulate five or six different parameters in producing pulsatile and whistling signals. Presumably each parameter can significantly affect the meaning of the acoustic signal as a form of communication. T h e number of independent parameters and the number of possible variations in structure increase still further in the complex signals; these may involve two or more generators of sound acting simultaneously. They distinguished five classes of whistles in Tursiops and pointed to further complexity in the superposition of other kinds of sound upon the whistles, both simultaneously and in patterns of alternation. Reznik and Chupakov (1975) made a quantitative comparison of 1700 whistles classified into eight types and grouped as either during communication between two animals or without communication. Influenced by the paper of Lang and Smith (1965), they tabulated the shape of the sound spectrogram, the duration and frequency range of each type. They could not find any difference between the signals used under the two conditions. None of the sounds studied extended above 15 kHz or below 6 kHz. In the most extensive study, Markov et al. (1974) distinguished seven types of “initial” elements (clicks, ringing smacks, long smacks, crackles, roars, wails, and whistles) which may appear as independent signals or as components of complex combinations at three primary levels. The permutations of elements and levels of combinations are given alphabetical symbols, of which 3 1 are needed. Most complex signals consist of two to five elements, but they may contain up to 24. The construction of complex signals involves five linking operations (“shift,” “connection,” insertion,” “superposition,” and “break”). T h e superposition operation 64.
means not only the simultaneous working of two o r more sound generators but the simultaneous nervous command to two separate control systems for these generators, each in its own structural and temporal pattern. The authors were properly cautious about the communicative significance of this rich potential for complexity. They emphasized that it is still necessary to prove the semantic value, to demonstrate coding rules, and to study the syntax. “For this reason, accepting on the basis of the data submitted above, that a formally ‘open’ communication system does exist in the dolphin, we abstain, for the time
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being, from any categorical evaluations of its semantic complexity and effectiveness.” Kreychi et al. (1975), in a short note, described a variety of complex signals in terms of the superposition and succession of noise, pulsed and tonal sounds, recognizing in this way nine categories. They concluded that the emission of complex acoustic signals warrants the assumptions that they “bear a certain meaning-related load in communication,” apparently going beyond Markov’s more cautious position (see also Romanenko, 1974c; Bel’kovich et al., 1977).
3 . Parameters Suggestive of Communication Function of Sounds Tomilin (1969c, 197 1) classified the whistle sounds as communicative and characterized them as “rather monotonous and stereotyped.” “The extreme monotony of communicative signals cannot support an idea that dolphins possess a language.” He considers the development of acoustic signals that can be utilized for communication as “moderate.” “Terrestrial carnivores such as the fox, have a much higher number of communicative signals than dolphins.” He also evaluates the intelligence of dolphins at approximately the same level as that of the dog o r the monkey. T h e large size of dolphin brains must have different bases than that of the human; Tomilin gives a long list of them. What is known about dolphin behavior in the wild does not support the interpretation of logical abstract thinking ability, in his view. Titov (1971; and co-workers, 1971), classified a large sample of sounds from three species into echolocating clicks, whistles, mixed whistles with clicks, and other complex sounds. This fourth group was further divided; some of the subclasses were (1) quacking, (2) croaking, (3) screaming, (4) explosions, (5) barking, (6) howling, (7) roaring, and (8) chirping and trilling. They regard quacking as prevailing when dolphins are hungry o r overexcited and in Tursiops it is apparently a sound of pleasure. Clearly this and some other sounds express emotional state and probably have some signalling value. Croaking was noted during “conversation,” when two dolphins are connected only by an electroacoustic channel. Screaming occurred during collecting, sexual behavior, and play with a ball, but seldom during feeding. Barking is mainly produced by recently collected animals when excited, during feeding, or on the appearance of people in the pool. In contrast to the dolphins Tursiops and Delphinus, the harbor porpoise Phocoena shows a much smaller repertoire of sounds, and these are almost only produced during collecting or other stressful states. Under normal conditions, the porpoise almost never makes any emotional signals and when it does they are very low in frequency (0.4-4 kHz) and in duration (0.02-1.0 sec). The most extended search for evidence of communication was that
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of Burdin et al. (1974) on Tursiops truncatus. Their method was basically to separate animals into pools linked by a wideband, two-way, electroacoustic line and to analyze the occurrence during periods when the link is functional, as compared with control periods, of various classes of sounds. These were in the first place divided into whistles, clicks, and gurgles. The clicks were grouped into low, medium, and high repetition rates. Whistles were subdivided into eight types. It was found that the same kinds of emissions occur during communication as occur in isolation, that the frequency of one type of sound or another is typically characteristic for the individual in either situation, and that some of the types change their relative occurrence markedly as between the two situations. The authors agreed that certain types of whistles serve as identification signals but believed that the role of these signals is not limited solely to identification. (The periods identified as “with communication’’ are apparently not random samples but selected by unstated criteria, from the tape recordings made during the time the link was available; it is not clear whether the periods marked “without communication” were similarly selected or were random samples from recordings when the two-way link was not available.) I t is suggested that the trains of clicks may be used in two-way communication. T h e emphasis is placed on the need for further investigation to determine the true role of clicks. It is proposed that most of the information content in whistles consists of short, high-frequency pulses superimposed on the base frequency and consisting of harmonics up to the fifth with a significant fine structure. These high-frequency pulses are called “inclusions” and vary from 0.5 to over 200 msec. Unlike the broadband clicks, the inclusions are narrow-band and apparently not as abrupt in risetime. Assuming that inclusions d o not exceed 50/sec and that the frequency of each inclusion may correspond to one of four harmonics, the maximum throughput of the communication channel would be 100 bitdsec, which is close to the estimates of the rate of perception of information in the human. Considering the need for some redundancy to implement reliable transmission, the authors expected that the actual rate of exchange of information would not exceed 20 bitshec in dolphins. They viewed the low-frequency components of the whistles, which are more powerful and less attenuating as well as more widely beamed, as serving to establish contact. T h e two dolphins emit a chain of partially overlapping whistles that serve as a communication bridge over which the short, high-frequency inclusions with fine structure carry the main information load, more narrowly beamed and rapidly attenuating. Since these are harmonics of the base frequency, the bridge enables the dolphins to design the optimal filter, both with respect to direction and frequency, to detect the fine structure in the noisy environment of the sea.
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Kaznadzey et al. (1975), using a dynamic spectrum analyzer, analyzed the signals emitted by a single specimen of Tursiops truncatus in a closed tank in nine different situations. They distinguished 59 types of signals but do not propose how they are used in communication. From the same group of workers and apparently the same body of recorded bottlenosed dolphin sounds, some 2000 signals, in nine different situations classified into 59 types, were analyzed (Khakhalkina, 1975) with the idea of searching for types of signals that are repeated characteristically in specific behavioral situations relating to presumed emotional states. It was assumed that if this criterion were met, a sound could be designated as an emotional signal. If the whole repertoire of signals was found to consist of a constant number of types related to specific emotional states, the communicative system of dolphins could be classified as the “closed” type. However, the authors emphasized that the question of the dolphin’s communication system remains debatable for lack of adequate data. Of all the 59 types of signals, only two have satisfied this criterion and these were extremely similar. One was produced throughout an hour of great excitement and dashing about by an isolated dolphin in a small tank during a severe thunderstorm. T h e other was similarly emitted throughout a long period of play with a rope stretched in the water, causing excited, rapid swimming and signs of pleasure. T h e circumstances are believed to exclude the possibility of informative or semantic content. From experience with other individuals in similar situations, the authors believe that long whistles are emotional in most cases and, furthermore, that they may be strictly individual. Titov and Nikolenko (1975) classified all the sounds produced in 1O-min sample periods, in Tursiops, Delphinus, and Phocoena, into clicks, whistles, and complex sounds. They counted the number of each during various states such as free swimming, feeding, sex play, ball play, orientation, solving an echolocation problem, and the like. The counts were also grouped into the broad state classes: satiated, hungry, quiet, and excited. First, it is notable that the picture resulting is quite different among the three species. Speaking only of Tursiops, clicks are increased only in the hungry state,’but are similar in the other three broad categories; they are increased while solving echolocation problems and during ball play, among the specific activities. Whistles are only increased in the excited state, especially during feeding but not during ball play, sex play, solving an echolocation problem, o r being caught o r transported. Complex sounds are only increased, among the lumped categories, in the hungry state, among the specific activities both during feeding and sex play. The “identification signals” (IS) of 11 bottlenosed dolphins were reported by Markov and Ostrovskaya (1975b) based on recordings in
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many situations: alone, in groups, and in electroacoustically connected tanks. In all, 64 phonograms, each of 10-min duration, of about 27,000 whistles were analyzed. Among the wide repertoire of signals used by these animals in groups, the authors could distinguish, in each animal, one, occasionally more, particularly preferred signal (PS) which, however, did not necessarily coincide with its IS. On different occasions an animal could use very different types of signals including the IS as its PS. T h e IS is identified as that signal which is repeated almost to the exclusion of other signals when communication between animals is interrupted, and which is different in each animal with only rare apparent coincidences. T h e repeated IS’Sfrequently show a common element and a superimposed variable component, occasionally quite complex. Markov and Ostrovskaya (1975a) analyzed the sounds emitted by three bottlenosed dolphins used in pairs in separate tanks connected by twoway, wide-band, electroacoustic lines, comparing 1O-min or 2-min periods with and without the communication lines. The material aggregated 50 phonograms covering a period of 8 hr and 20 min. The signals from the same pair of dolphins were found to be very unstable; the differences between 10-min runs were quite significant, regardless of whether these runs were recorded a few days o r a few minutes apart. T h e authors conclude that there is a complex syntactic organization in the communications. T h e following kinds of evidence are offered for this hypothesis. (1) Isolated signals are rare; usually signals are combined in groups with stable intersignal intervals easily distinguished from the intergroup intervals. (2) Signalling is typically richer and more diverse during periods of communication; when these were disrupted, in all of the pairs without exception, the signals became simpler, and the grouping of signals was impaired. (3) Apparently the nature and the sequence of signals and the duration of intersignal intervals within specific groups are meaningful to the animals. (4) Usually the exchange of signals is in the nature of a dialogue, such that the beginning of one animal’s signal coincides with the end of the other animal’s signal. Markov and Ostrovskaya ( 1978) reported further evidence for syntactical organization in communications that obey the Zipf-Mandelbrot law. Shevalev and Flerov (1975) mainly reported evidence that sounds produced by a dolphin encountering a sign of food may attract another dolphin. In general, the considerable body of results may be said to leave the question of communication poised. It is compatible with a view that dolphins are not vastly different from other nonhuman mammals in having a limited variety of signals with distinct information content, probably much less than 40, among which some are complex but may
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
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not carry much information beyond identifying the individual and a crude indication of its state. There is no compelling suggestion of syntax or vocabulary at a level beyond that common in nonprimate orders.
4. Perception .f Sound; Psychophysical Studies Several studies dealt with auditory perception as a function of signal characteristics or background sounds. Sukhoruchenko (1971, 1973) and Supin and Sukhoruchenko (1970, 1974) made a logical contribution in the introduction of the technique of classical conditioning and recording of autonomic responses in order to permit efficient plotting of audiograms of reasonable numbers of animals. Heretofore, single individuals have been studied by much more laborious conditioning methods. These authors, taking advantage of the cooperativeness of the harbor porpoise, Phocoena phocoena, nearly immobilized the animal with straps and recorded respiration, heart beat, and the galvanic skin reflex. They paired the conditioned stimulus, which is a tone, with the unconditioned electric shock to the skin. The animal was in a small tank lined with rubber which is said to be so sound absorbing that they are not troubled by interference due to reflections! Since the frequencies used extend from 4 to 180 kHz, this is a remarkable statement. Usually 10-15 pairings were enough to develop a conditioned reflex and when every fifth to tenth conditioned stimulus was reinforced the response was stable and did not extinguish. T h e complete audiogram could be made in a single experiment or within two or three experiments (Fig. 4). A minimum threshold N/m2, which for audibility at the best frequency is of the order of corresponds to about 1 O-I8 W/cm2. Repeated experiments yield wellreproduced results. Morosov et al. (1971) gave a similar audiogram for Tursiops with a minimum threshold (80 kHz) of 1.7 X lo-'' W/cm2,which is close to the value for the human at our best frequency (2-3 kHz). Differential frequency thresholds were measured (Supin and Sukhoruchenko, 1974) by 2-4 Hz frequency modulation at an intensity about 40 dB higher than the threshold of absolute perception at the frequency band under test. After developing the conditioned reaction to frequency-modulated tones as distinct from unmodulated tones, the depth of the frequency modulation was reduced to the threshold. This was found to be below 0.5%for all frequencies from 4 to 190 kHz, and in a considerable part of the range, 0.1%-0.2%.The low values of 0.2%0.3% at the highest frequencies (128-190 kHz) are noteworthy (but dubious in view of Thompson and Herman, 1975) because they should control for the possibility that hearing at these high frequencies may be analogous to ultrasonic hearing in man. It is known that if the stimulus is delivered to the cochlea, bypassing the middle ear, we can hear u p to
82
THEODORE H. BULLOCK A N D VLADIMIR S. CUREVICH
A
SOUND.
\
/
C
Bud.
Vis.
Somot.
,
V
FIG. 4. Evoked potentials in cerebral cortex of Tursiops truncatus. (A) Dorsal view of the brain, pictorially (right) and semischematically(left) to show the nomenclature used for the principal sulci and gyri. Rostral, above. (B) Diagram of an electrode track obliquely from the suprasylvian gyms medialward and the average electrical activity evoked at the correspondingly numbered loci with clicks and light flashes. Negativity upwards. (C) Diagram of the cortex with the sensory areas where evoked potentials occur to the indicated stimulus modality. (From Ladygina and Supin, 1978.)
dozens of kilohertz, but such perception does not permit frequency discrimination. This possibility is important in marine animals since direct conduction of the sound through body tissues is more important than in air. Such “false” ultrasonic hearing has been found in seals, which apparently perceive up to 160 kHz, although there is no frequency discrimination above 60 kHz. Dolphins clearly have normal frequency analysis in the entire range of perceived frequencies. Chilingiris (1977, 1978) showed the dependency of the frequency-difference limen upon the duration of the tone burst when it is below 1 msec. Vel’min et al. (1975b) and Titov and Yurkevich (1975) found a lowering
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
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of audibility thresholds for 15-msec tone bursts with increased recurrence frequency, in the range of 1-6 bursts. Vel’min et al. (1975a) measured the differential intensity threshold for short pulses in bottlenosed dolphins by the method of conditioned reflexes, obtaining values ofi 1-2 dB over a wide range of intensity levels: this is the same order of magnitude as the differential intensity threshold for tone and noise signals of longer duration and indicates the exceptionally high resolving power and adaptation of the auditory system of this animal for shortpulse echo signals. Actually, the pulses used in these experiments were very long compared to most dolphin echolocating pulses, i.e., 15 msec. Vel’min and Titov ( 1975) measured the discrimination of interpulse intervals by the same methods, and using again the long 15-msec duration pulses, with mean intervals of 50 psec, the difference threshold was 11.5%. Zanin and Zaslavskiy (1977) compared man and dolphin with respect to the dependence of the discrimination threshold of paired sound upon the interval between them. Both species show two regions in this relation, one at shorter intervals (50-250 psec) and another above 350 psec. Bel’kovich and Dubrovskiy ( 1976), in their monograph reviewing the sensory abilities of cetaceans, provided tables from the work of Korolev et al. and of Morozov that summarize the accuracy of the lateralization of a sound source, both when alone and when in the presence of another sound. Lateralization is less accurate when the source is close to the projection of the longitudinal axis, but appears to be significant even at angles as small as 1 deg; it is better at 20 kHz than at 3.8 kHz and worse in the presence of a second sound. Lashkaradze (1977), Saprykin et al. (1978), and Zaytseva (1978) added further to the same subject. Zaslavskiy (1978) argues that dolphins cannot be using binaural differences to an important degree. Levin et al. (1978) reported on the dolphin’s estimation of the speed of a sound source. A series of studies have dealt with the perception of signals against a background of noise. Burdin et al. (197 la) found that the lowest signalto-noise ratio at which a dolphin still recognized the signal to a certain criterion was nearly the same for tones from 1 to 42 kHz frequency, each against a background of band-limited noise of approximately proportional width and approximately centered on the tone frequency. Comparison of the threshold signal-to-noise ratio with that of a human showed the dolphin to be three to six times more resistant to interference in the range of 1-10 kHz: i.e., the dolphin can recognize a signal in much more intense noise. Burdin et al. ( 1971b) used a left-right choice instead of a go-no-go paradigm to measure the just noticeable intensity difference for band-limited white noise by mixing 3-Hz intensity modulated noise, of a fixed depth of modulation, with unmodulated noise.
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THEODORE H. BULLOCK AND VLADIMIR S. GUREVICH
T h e dolphin was trained with food rewards to go to the right for unmodulated noise and to the left for an intensity-modulated noise. T h e one animal studied fairly successfully distinguished modulated from unmodulated noise down to a value of modulation equal to only about 3%. Even a slight decrease of modulation depth below this level greatly reduced the probability of correct choice, increased the number of errors and, as the experiment proceeded, increased the number of refusals to choose. Although the reinforcement value is the same for an error and a refusal, namely, the dolphin is denied a fish, it apparently prefers not to make a mistake. The results agree rather well with cited measurements on humans. Romanenko (1975b) performed variants of this experiment. Saprykin et al. (1975d) examined the same questions with more elaborate procedures and the forced-choice method. For the highest probability of correct response, there is a clear optimum duration of the signal which, under the stated conditions, was 4.25 msec. T h e form of the “efficiency” curve is compared with that of correlated or optimal filters which are much more peaked. T h e authors concluded that the “mechanism for receiving acoustic signals in the dolphin achieves a self-tuning as a function of the impulse signal during information processing.” Zaytseva et al. (1975, 1978) and Morozov et al. (1975) found a sharp drop in the masking effect of noise as the angle of the noise direction increased. The high degree of directionality is one of the most important mechanisms promoting the high resistance to noise in the dolphin echolocation system. Within the range 40-120 kHz, the higher the frequency, the better the directionality. Saprykin et al. (1975c, 1976) investigated the differential sensitivity of the auditory system in the dolphin, relative to Doppler-signal transformations. T h e expectation was confirmed that the probability characteristics of the auditory system for the frequencies investigated are invariant relative to a parameter called alpha of Doppler transformations. D. ECHOLOCATION Although late in starting, the Soviet research in this field has accelerated rapidly and there is at least some literature on each of many aspects, including the mechanisms of production of sounds (Section 11, C, l), the characteristics of the sounds emitted and their variations with the problem confronting the dolphin, the use of the echolocating system and the quantitative assessment of its performance capabilities, without and with interference, the physiology of the auditory systems, at least at its highest levels (Sections 111, E and F), and the theoretical analysis of echolocation
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
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in terms of optimal filter theory. We can only indicate briefly the directions of some of the studies. General reviews were provided by Romanenko ( 1964) and especially Ayrapet’yants and Konstantinov ( 1 970), whose book on echolocation in animals brought to light a new Soviet effort, including behavioral studies on dolphins and comparisons with bats. These authors and others (Zaslavskiy, 1972; Romanenko, 1973a,b, 1974c, 1975b, 1978) analyzed the physical characteristics of the brief echolocating sound pulses, including the shape of the sound field and its rapid changes duringa train of pulses, apparently adapting to the problem at hand. Agarkovet al. (1971) briefly reviewed hypotheses of echolocation and the experiments that should be done. Dubrovskiy et al. (1970a) corrected a previous report to the effect that the harbor porpoise Phocoena phocoena does not use ultrasonic echolocating signals by showing that they do, although the intensity of the echolocating pulses at 1 m from the animal’s head is more than 100 times weaker than that for Tursiops o r Delphinus. Phocoena does not exhibit the same degree of independence of the ultrasonic and lowfrequency sound emissions as the other two genera, but instead the ultrasonic pulses are fairly rigidly tied in time to emission of a lowfrequency sound which they “always” precede. Morozov et al. (1972) tested the hypothesis that the repetition rate of the echolocating pulses is limited by the consideration that the animal emits successive pulses only after reception and appropriate analysis of the echo from the preceding pulse. Measuring every successive interval between echoranging pulses during approach runs of trained Tursiops truncatus starting at distances up to 30 m from the target, they showed that the minimum interval almost never drops below the time required at that distance for the echo to return. Averaging a number of runs, the intervals plotted as a function of distance to the target are remarkably parallel to the slope determined by the time for the echo to return at that distance. Above 4 m, the average interval is about 20 msec longer than this echo-arrival time. At the closer range, it goes down to 3 msec or less. When individual runs are examined, without averaging the intervals, emitted pulses are seen to fluctuate smoothly around the mean, with a period of roughly 1 sec and an excursion of roughly 50% of the mean. Saprykin et al. (1974) measured the fall-off in probability of correct identification of targets when the sound pulse emitted by the dolphin had to pass through an aperture in a screen, as a function of the diameter of the spatial filter. The evidence indicates that all signal frequencies participate in identification, but the most informative frequencies are those above 5 kHz (see also Saprykin et al., 1975b,c).
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THEODORE H. BULLOCK AND VLADIMIR S. GUREVICH
The largest group of papers on echolocation dealt with its achievements measured by discriminations made by trained dolphins (Tursiops truncatus, unless otherwise noted, see, e.g., Golubkov et al., 1969a,b). Of particular interest are numbers given for distance discrimination and azimuth discrimination of objects by echo, since these are the first substantial claims of a difference between dolphins and bats. The achievements of dolphins (Table 111) are remarkable, and if these comparisons are correct and fair, they may help in accounting for the vast difference in absolute (though not relative) size of the inferior colliculi and other auditory structures of the brain (Table I). These important measurements should be repeated. Based on a target 5-15 cm in length, Ayrapet'yants et al. (1969) estimated the range of useful echolocation to be 5 m, whereas the range of social communication is 5-10 km. One series of papers by the group under Bel'kovich (Bel'kovich et al., 1969c; Bagdonas et al., 1970; Bel'kovich and Borisov, 1971; Bel'kovich and Reznikov, 1971; see also Gurevich, 1968a,b) dealt with the discrimination of geometric figures by Delphinus de1phi.s. In passing, it is noted TABLE 111 ECHOLOCATION PERFORMANCE (COMPARISON OF DOLPHINS A N D BATS) Dolphin
Bat
Echo target size threshold: (Diameter of metal wire detectable by pulse rate increase criterion, distance 1 m for bat; unstated for dolphin, which is in water, using much longer wavelength, shorter pulses)
Phocoena 0.22 mm*
Myotis 0.12 mmb
Discrimination of difference in distance: (Threshold for 75% correct response; simultaneous presentation; distance = 1 m = 3 msec echo time for bat; unstated for dolphin) difference in echo time =
Tursiopsb 1.5-4 mm 2-5 psec
Myoh* 25 mm 160 psec
Tursi@s* 14' 48' Delphinrrs' 1' 4 0 I' 40"
Rhinolophw 4'30' -
-
Minimum angular separation of targets: (Cylinders: 70% correct response) horizontal vertical horizontal vertical ~
~~
~
~
~~~
Busnel (1967) and Busnel et al. (1965a,b). Ayrapet'yants and Konstantinov (1970). Bel'kovich (1970).
~~~
-
NERVOUS SYSTEM A N D PSYCHOBIOLOGY OF CETACEA
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that the percentage of correct responses is somewhat lower in the morning hours, upon presentation of difficult tasks. A series of papers (Dubrovskiy et al., 1970b, 1971; Dubrovskiy and Titov, 1975; Titov, 1975) on the discrimination of metal spheres of different materials and diameters led to the hypothesis that discrimination is based on analysis of the intensity and spectrum of a so-called secondary echo. However, in situations in which the spectral clue is not available, dolphins find additional signs (see also Abramov et al., 1972). Titov provided numbers for the sound level at which the probability of target recognition drops to 75% as a function of interference. Maximum masking occurs when the interference overlaps the spectral maxima of the echolocating pulses of the dolphin. An increase in the tolerable interference comes with experience. Vel’min (1975) and Vel’min and Dubrovskiy (1975, 1978) reported further on the detection of targets in the presence of interference, in this case artificial “reverberations” similar to echolocating pulses in shape and duration, placed at fixed time intervals after the emission of the echolocating pulse. With a strong interfering reverberation, the percentage of correct detections by the animal remains close to 100% until the time interval between echolocating pulse and reverberation is less than 200 psec. Another test, based on detection of a low-amplitude acoustic pulse in the background of a lagging and relatively strong interference pulse, gave similar values for the high-resolution temporal window. T h e threshold of signal audibility becomes twice as high when there is a 300-psec lag in the interference pulse in relation to the signal. Ayrapet’yants et al. (1973) found adaptation of the signals to the parameters of the objects to be discriminated (see also Golubkov et al., 1969b; Markov and Prokhorov, 1978). Ayrapet’yants et al. (1969) described some features of the strategy used in solving a near-threshold discrimination: shorter echolocating pulses (30-150 psec), changes in the pulse envelope, in the frequency composition, in the number of principal sinusoids in the pulse, and in the modulating sinusoids in the pulse, and scanning head movements which began 7-8 m away from the target. They emphasize that the directionality of the beam can be varied widely and its direction can be rapidly changed without head movement. As we have already mentioned, Bel’kovich and Nesterenko (1975) cited Reznikov as finding that the directivity, of the club-shaped sonic beam is rotated at high speed during each pulse independent of head scanning: they give the figure of 3.5 x lo5 deglsec but without supplying enough evidence in methods or results to make it acceptable as an
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established datum. This means a small target is irradiated for only a very short time and, taken together with the known steepness of the acoustical wavefront, they argued that this provides for an extraordinarily accurate orientation ability, all the more because collision excitation of the target can produce spectral harmonics that can convey to the dolphin a good impression of the nature of the reflecting object. Livshits (1974, 1975)proposed a physical model of the fast changes in the directionality diagram of the radiated echolocation pulse due to scanning. He assumed that the dolphin performs a cross-correlation between the pulse and the reflected signal from the target and provided a methodology for calculating the potential accuracy and angular resolving power. Golubkov et al. ( 1975) compared dolphin echolocation signals with theoretically optimum signals. They conclude that “the agreement between the theoretical and actual signals of detection and resolution is evidence of the lability of the hydro-acoustic apparatus of the dolphin” (see also Koshevoy and Mikhaylovskiy, 1972; Abramov et al., 1978). We have alluded to the proposition of an acoustical imaging of the world on sensory surfaces distributed on and in the head. This idea, particularly advocated by Reznikov (1970), Kozak (1973, 1974a, 1975), and Khomenko (1969a, 1970, 1975a), depends on the assumption of extracochlear receptors in the skin, air sacs, melon, or elsewhere that are sufficiently sensitive to ultrasonic stimuli to detect faint echoes. The arguments put forward are compatible with the view that such echoes are detected only by the cochlea.
E. PHYSIOLOGY OF ACOUSTICSENSE ORGANS AND CENTERS There are few studies on this topic and the most important are treated under the next heading, “Cerebral Cortex.” Solntseva (1974) gave a survey of the peripheral organs of hearing in aquatic animals in general, including Cetacea. In particular, specializations of the ossicles and of the gross form of the cochlea are emphasized. Cetacea are very different from pinnipeds in the relative sizes, forms, and angles of rotation of the ossicles. In relation to earlier claims (Yanagisawa et al., 1966; Bullock et al., 1968) that the principal port of entry of ultrasonic frequencies is the mandible, then with somewhat lower sensitivity the melon, and with still lower sensitivity the region of the external auditory meatus, Valiulina (1975) implanted a miniaturized acoustic receiver, 1.5 x 3 mm in size, in the proximal part of the external auditory meatus of the bottlenosed dolphin, and stimulated with a spherical piezoelectric source 15 mm i n diameter pressed actually against the skin in various parts of the dol-
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
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phin’s head. She found the sensitivity maximal over the mandible and somewhat less over the maxilla, the melon, and the region of the blowhole. Curiously, she did not report results over the region of the external auditory meatus. It should also be pointed out that the locus of the implanted receiver might not adequately sample the special pathway for sound and discovered by Norris (1964). Lipatov (1978a,b) claimed that a measurable effect of air in the external canal means, under certain conditions, that sound may enter the dolphin head via the external acoustic meatus, when the tympanum is in contact with air. He also argued that “calculations” (not given) based on directivity diagrams support the external acoustic canal instead of the mandible (Norris theory) as the sound path. A few recent publications deal with subcortical structures. Voronov and Stosman (1977, 1978) reported evoked potentials from cochlear nuclei, trapezoid body, superior olive (sic), lateral lemniscus, inferior colliculus, and medial geniculate body. T h e most effective frequency under their conditions was about 40 kHz, close to the value determined behaviorally. A few peculiarities are noted among these structures, but the yield of physiological findings about the properties of responses, given all these unique recording loci, was very meager. Stosman ( 1978) compared the potentials evoked at different levels by ipsilateral, contralateral, and binaural acoustic stimuli. At the trapezoid, ipsilateral sound is most effective, binaural next, and contralateral least. In the lateral lemniscus, contralateral sound is most effective. Supin et al. (1978b) summarize new experiments on the medial geniculate and lower brain stem structures. Papers on responses to acoustic stimuli in the cerebral cortex are treated immediately below. F. CEREBRAL CORTEX
A series of papers, beginning in 1970, from the laboratory of A. Ya. Supin, is based on recording evoked potentials from the cortex through small holes in the skull in unanesthetized dolphins. Apparently the first scientific paper reporting the results of electrical recording from the cortex in Cetacea is that of Ladygina and Supin (1970), whereas the papers of a Japanese-United States group (Yanagisawa et al., 1966; Bullock et al., 1968) which were the first reports of recording from the brain in this order, concentrated primarily on recording evoked potentials from the mesencephalon (see also Bullock and Ridgway, 1972a,b; Bullock et al., 1971). T h e Russian authors used clicks, light flashes, electrical stimulation of the skin and of the brain to map the cortex for auditory, visual,
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THEODORE H . BULLOCK AND VLADIMIR S. GUREVICH
somatosensory, and motor areas (Fig. 4).The precision of the map depends on the number of points recorded in each animal; based on the data reported, this is moderate, not high, especially in relation to the large extent of the responsive cortex. An inherent problem with the cetacea is the richness of convolution, the most elaborate of all animals, with a profusion of gyri and deep sulci that are for the most part not consistent among specimens. Those sulci that may be consistent are not readily distinguished from others, and a very large portion of the whole cortex is buried within the sulci, which are typically deeper than in other mammals including primates, commonly reaching 20 mm. In these first papers the authors have attempted only in a preliminary way to distinguish primary and secondary sensory areas. They did not attempt to map the retina or cochlea upon the cortex, that is, areas of visual field and frequencies of sound. They reported areas of "rapid
A
B
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100
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kHz FIG. 5. Frequency threshold curves for behavioral response, averaged from four P h o c o m phocom. (A) Absolute threshold of perception for pure tones, by conditioning a visceral response. d B are referred to 1-pbar sound pressure. (B) For octave-wide bands of interference noise; dB in relation to spectral density of 0.5 X lo-'' W/cmP/Hz.(C) Same as (A) in high-frequency range, for two individuals, to show the variation. (D) Differential thresholds plotted in Hertz (closed circles, left ordinate) and as a fraction of the carrier frequency (open circles, right ordinate). (From Supin and Sukhoruchenko, 1974.)
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negative response” and others of “slower positive response.” In the former, the minimal latency to click stimuli is 6 msec; the main peak is a negativity that crests at 15 msec (we will use the system of notation that calls this N15). The surface negativity of the primary response is in apparent contrast to other mammals, where the primary response is surface positive, with peaks at ca. P28 and at P50. The “slower positive response” in other cortical loci has a latency of 15-20 msec, and its peak is P50. Zanin et al. (1978) confirmed some of these results and added a few details. The rapid negative peak in the dolphin does not diminish and reverse in sign within the thickness of the cortex as in other mammals but grows as the electrode goes deeper to between 4 and 10 mm, without signs of crossing the deep sulci but gradually increasing in a monotonic fashion. As the electrode continues deep to the folded cortical mantle, the deflection disappears. In addition to the rapid negative and slower positive types of response, these authors found a spikey response of very short latency (less than 1 msec), of unidentified subcortical origin. In a second paper, Sokolov et al. (1972) gave some additional details on responses to light, sound, and somatosensory stimuli. Evoked responses to photic stimuli are multiphasic, with the first important peak a negativity at 35-55 msec. The suggestion of secondary responses in associative regions surrounding a primary projection zone is based on longer latencies. In the somatosensory area, which is at the rostral pole, primary surface negative responses have a peak at ca. 12 msec, measured from electrical stimulation of the skin. The rostral region of the body is represented in the lower anterior part of the exposed gyri, the caudal region of the body in the upper part. The acoustical area is large; the areas responding to photic and to somatosensory stimuli are relatively small. Ladygina ( 1974) improved the spatial resolution of the recording method by using a five-electrode Laplacian array. Comparison of monopolar and Laplacian potentials showed the greater spread of activity in the former case, where the recorded potential results from currents flowing from regions several millimeters distant. Specific results with this method have not been reported, and the newer methods of current source density analysis have not been used. Popov and Supin (1975, 1976a,b) used the method of implanted electrodes on Tursiops truncatus. This permits recording the evoked potential chronically in the behaving animal, as shown by Bullock and Ridgway (1972a,b). Later, using TursaOps trmncatus and the technique of averaging evoked potentials, Popov and Supin (1978) did an admirably quantitative study (see also Supin et al., 1978a,b). They found the threshold for the appear-
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THEODORE H. BULLOCK AND VLADIMIR S. CUREVICH
ance of evoked potentials in the auditory cortex (minimum 1 X Pa) to be close to the behavioral threshold for this species; it is lowest for long (20 msec) noise bursts, higher for the optimal tone and highest for clicks. Evoked responses occur not only to the on and off of a sound but also to sudden changes in intensity of about 0.8 dB and in frequency of as little as 0.25%, saturating as low as 1%. These values are said to be close to the differential thresholds measured behaviorally. Noise bursts are more duration-dependent than optimal tones near threshold. A maximal evoked potential may occur to intensities only 20 dB above the threshold. Although the authors did not discuss the differences between cortex and inferior colliculus, a major point of Bullock and co-workers, their results add several properties to the list of differences, including the long summation time and, perhaps, less dependence on a very short rise time. Attention was not focused on the contrast in responses to echo-type and social communicating sounds. Much of the latter are below 5 kHz, which was the lower limit of tones used. Ladygina and Supin (1978) and Ladygina et al. (1978) also studied the bottlenosed dolphin Tursiops truncatus, using implanted electrodes and recording without anesthesia; they showed that the cortical areas responding to sound, light, and skin stimulation are similar to those previously reported for Phocoena. Supin et al. (1978a) examined about 100 points in the auditory cortex with tones of different frequency. The optimum was often a very narrow band, but not always; different components of the evoked potential depend differently upon frequency. A large region is very sensitive to the octave from 50 to 100 kHz. Supin et al., in a new monograph (1978b), add substantially to the quantitative description of properties. For example, many curves are given for the effect upon amplitude of evoked potential of sound, of a step change in intensity, of duration, composition, rise time, tone frequency, size of sudden small change in frequency upwards, the same downwards, rare of FM, interval since a conditioning click, the same with a long series of clicks, the same with a brief FM stimulus, and intensity of a masking noise with a test click. Several of these effects were studied parametrically, at different values of some other variable. Some were studied separately upon the amplitude of the short-latency (ca. 17 msec) peak of the cortical evoked potential and that of a longer-latency (ca. 26 msec) peak, and a late (ca. 150 msec) wave. Besides clicks, tone bursts, FM tones, and noise bursts, some use was made of pairs of brief (ca. 10 psec) clicks separated by intervals in the 30-1000 psec range. Often the character of a dependency of evoked potential upon some of these parameters, e.g., rise time, is drastically different at separate recording
NERVOUS SYSTEM AND PSYCHOBIOLOGY OF CETACEA
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sites. This monograph arrived too Iate for a full translation. These are simply indications of the wealth of detail presented. Glass capillary microelectrodes recording extracellularly from single units were successfully used for the first time by Gapich and Supin (1974) in the area of cortex previously defined as acoustic (see also Supin et al., 1978b). They were introduced through a hole in the skull, after opening the dura mater, and advanced by a hydraulic micromanipulator. The authors examined 45 neurons in the acoustic cortex and, for comparison, 31 additional neurons in the “frontal occipital region” 2-3 cm rostrad of the acoustic zone (“central occipital region”) (Fig. 6). Background activity was usually 6-9 impulses/sec in the acoustic zone and 1-6 impulses/sec in the frontal occipital region. Three-fourths of the neurons in the central occipital zone responded well to acoustic clicks either by intense excitation or inhibition, whereas response to clicks was extremely rare in the frontal occipital region. Excitatory responses in the acoustic region were sometimes phasic, sometimes tonic, and sometimes both phasic and tonic (Fig. 6). There may be a postexcitatory rebound silence. T h e latency judged from poststimulus time histograms was 7- 14 msec, but sometimes very long latencies were observed, from 70-250 msec. Some forms of stimuli repeated after 1-2
lo 50/ ,
I
Ii 10
m 0
200 400
m
0
200 400
m
0
200 400
FIG. 6. Extracellular single unit responses to acoustic stimuli, recorded by a capillary microelectrode in the central occipital region of the cortex of Phcoena phocoenu. Upper row: Poststimulus histograms of different types of neurons to a click stimulus: 1, fast phasic; 2, phasic-tonic; 3. tonic; 4, long inhibitory pause. The moment of the click is indicated by the arrow. Lower row: Stimulation by pure tones of long duration: 1,30 kHz; 2, 40 kHz; 3, 60 kHz. Stimuli delivered by a piezoelectric ceramic emitter in contact with the skin of the lower jaw; rise times of tones are not given. Ordinates: impulses per second; abscissa: time in milliseconds. (From Gapich and Supin, 1974.)
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sec caused a gradual change in the response, enhancing the inhibitory pause following initial excitation. Long tones evoked reactions in fewer neurons; out of 20 examined, 7 were found to be excited, 1 to be inhibited, and 12 did not react significantly. Ultrasonic frequencies (dozens of kilohertz) were the most effective, but characteristic curves were not plotted for individual neurons, nor was the effective rise time of the stimulus studied. The authors believe that neurons have selectivity for particular frequencies. N o neurons of the frontal occipital region were observed to react to tones. G. BRAINSTIMULATION The papers reporting evoked potential recording may be considered relevant to this topic insofar as they report the techniques for short-term and long-term implantation of electrodes in chosen areas of the brain. We cite here only Gapich et al. (197 l), but other papers treated in the preceding section are equally relevant. Belyayev et al. (1975) investigated the use of electrical stimulation of motivational regions of the brain in the bottlenosed dolphin as reinforcement in conditioned reflex activity. With the stereotactic methods available, they could not with sufficient precision aim at the septum pellucidum, hypothalamus, or medial forebrain bundle and, therefore, studied primarily the striopallidal system and cingulate gyrus. They used spiral electrodes and a remote-controlled stimulator (3 msec pulses at 200 Hz up to 15 V for 0.5 sec) under quasi-free behavior conditions. T h e animal was first trained to press a lever with its rostrum for food reinforcement. This was then combined with electrical stimulation, and finally the food reinforcement was completely replaced by electrical stimulation. They compared extinction of the conditioned reflex following electrical stimulation with that following only food reinforcement. Self-stimulation of the dolphin was continued for up to eight consecutive hours. There was a change in the nature of lever depressions after switching from food to electrical reinforcement. While there were isolated lever presses with the food reinforcement, after which the animal usually circled the tank, with electrical reinforcement there was even more distinct differentiation between the two types of lever presses. T h e dolphin “demanded” fish through abrupt and distinct isolated lever presses, whereas with electrical stimulation these were grouped in series. Self-stimulation was also observed when the animal was completely satiated. The authors cite the early work of Lilly (1962), who was the first to induce self-stimulation in dolphins, but remarked that the sparse information submitted by that author made it impossible to repeat his
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experiment. Another equally short note by an overlapping group of authors (Bogdanova et al., 1975) adds a little information. There were no overt vegetative changes in dolphins with electrical stimulation of the brain, unlike terrestrial animals (rats and dogs) which they studied. They mentioned, without giving results, that these stimulus parameters are effective also in motor areas of the brain. Bogdanova and Fedenko ( 1975) reported some experiences with the development of conditioned reflexes using implanted electrodes. Besides lever pressing, they trained the animals to perform several other tasks. They are concerned with the rate of reflex development and extinction and questions of dolphin memory, but few data are provided. H. SLEEPA N D WAKEFULNESS Mukhametov and Supin (1975a,b) (see also Mukhametov et al., 1977; Mukhametov, 1978) implanted electrodes in a series of nine bottlenosed dolphins to record electroencephalograms, electromyograms of oculomotor and cervical muscles, electrocardiograms and pneumograms. T h e waking state and “slow” sleep were identified according to EEG criteria; the former is characterized by high frequency and low amplitude of electrical activity, while slow sleep was characterized by low-frequency, high-amplitude rhythms. Stages of drowsiness (alphalike rhythm), light sleep (sleep spindles), and deep sleep (theta and delta activity) could be distinguished. N o “rapid eye movement” sleep stage was identifiable because the usual physiological criteria for this stage are not applicable to dolphins; if rapid eye movement sleep does exist in dolphins, it is differently organized than in terrestrial mammals. There was no prolonged and complete relaxation of skeletal muscles in any phase of sleep in dolphins, since the animal must continuously maintain its position for normal respiration. Sleeping and waking states cannot be determined in dolphins on the basis of visual observation of the animal’s mobility alone, since it can be almost completely motionless while awake, and while it is asleep, slight movements of the fluke and head are normal, Synchronization as well as desynchronization of the EEG may be observed when the eyes are open. T h e entire set of movements required for expiration and inspiration can normally occur during slow sleep on a background of bilateral, synchronized, large-amplitude, slow waves of the EEG. Hence, the respiratory act does not require that the dolphin be awake, as has been repeatedly assumed. Mukhametov et al. (1976) reported interhemispheric asymmetry during certain functional states (Fig. 7). Whereas, in the waking state and in slow wave sleep there was bilateral symmetry of the electrocorticogram,
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A
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FIG. 7. EEG,sleep, and hemisphere asymmetry. Recordings with implanted electrodes in Tursiops truncatus. (A) Location of intracranial electrodes, numbered to correspond to the records. (B) and (C) During sleep, 1 hr apart; unipolar recording. (D) and (E)Stages of sleep (1, desynchronized; 2, intermediate synchronization: 3, maximal synchronization) in left (L) and right (R)hemispheres during two different nights. Bipolar recording; parietal cortex; time scale in hours. The beginning of (E)corresponds to the end of a 60-hr period of total sleep deprivation. (From Mukhametov et al., 1977.)
there can be distinct synchronization in the sense of large-amplitude slow waves at three widely separated points in one hemisphere and desynchronization in the sense of low-amplitude fast activity at corresponding points in the other hemisphere. The sides alternate in state approximately on a 3-hr cycle during the night. Such asymmetry is said to be unknown in other animals and man. Koval'zon (1978) confirmed the presence of epochs of asymmetry in Tursiops and found, with implanted thermometers, that temperature is also asymmetrical at these times. The synchronized hemisphere has a more fluctuating thermograph and is slightly cooler; the desynchronized side has a flat thermograph and is usually warmer. Stage 3, with at least two-thirds of each scoring epoch occupied by delta waves of maximum amplitude, was only recorded unilaterally; bilateral delta waves were only observed during barbiturate anesthesia, when spontaneous breathing stopped. Bilateral
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light sleep is compatible with respiration, but bilateral deep slow sleep appears to be incompatible with respiratory excursions in the dolphin. Supin et al. (1978b) summarize the previous work and add new data on sleep and asymmetry of brain waves. I. BEHAVIOR This section is intended to cover the literature on descriptive and analytical ethology or on characterization of the free behavior of cetaceans, either in the wild or in captivity, that have the opportunity to express a considerable choice in their behavioral repertoire. It is brief because of the omission of several groups of papers. Those dealing solely with the acoustic communication aspects of behavior were treated in Section 111, C; those on echolocation activity in Section 111, D; those on sleep and wakefulness in Section 111, H; those in which the focus of interest is on assessment of higher nervous activity are in Section 111,J. A considerable body of papers has been omitted from this review that deal with methods and accomplishments in training dolphins to perform at the will of the trainer, i.e., papers not basically concerned with a study of the learning process, let alone its substratum, which is the main focus of this review. The residuum is a small group of papers, mostly notes of two to four pages and chiefly anecdotal observations on individual dolphins, almost entirely those kept in captivity. To avoid disappointment, it should be said that rigorous ethology has not infected Soviet cetaceologists, whereas, on the contrary, they find essentially unrefereed outlets for casual or narrative accounts. In the nontechnical paper of Bel’kovichet al. (1969a), observations of two species of dolphin in captivity, the common and the bottlenosed, were recounted under such headings as grouping in the school, games, sexual behavior, mutual assistance, attitude towards sick relatives, and behavior in isolation. Social structure and interindividual relationships are moderately complex, perhaps on a par with many nonhuman primates. Play is well developed, vigorous, inventive, imitative, and frequently involves objects. Their sexual behavior can hardly be called complex but is diversified and not confined to the opposite sex or to the same species. Mutual assistance is frequent but selective, some sick individuals, formerly members of the group, are allowed to die, unaided. Isolation frequently causes signs of deep emotional distress. The observations behind such conclusions are, for the most part, not new or more quantitatively or elegantly detailed. Similar remarks may be made about the natural history observations of Morozov ( 1 970), Tomilin ( 1 97 l), Romanenko (1974a), Kozarovitskiy et al. (1971), and Shurepova (1971),
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who described several levels of aggressive behavior of a captive, trained Turszops toward its trainer. An exceptional publication, with respect to dolphins and porpoises, in that it deals with observations of behavior in the wild, is that of Bel’kovich et al. (1975), who observed groups of two to four “scout” dolphins (Tursiops trmncatzcs) from a herd, seeking food along the coast and apparently maintaining acoustic communication with the main herd. A stereotyped searching behavior was described with respect to single large fish as well as schools of fish. They believe that under normal lighting and water transparency conditions the echolocating system is used only in the last phases of capturing the fish as well as periodically for orientation with respect to the immediate vicinity. Somewhat more interpretive is the paper by Krushinskaya and Dmitriyeva (1975), who compared such interindividual behavior as mutual aid and aggression in cetaceans and pinnipeds in relation to their biological significance for the social system of the species. Karandeyeva and Shurepova (1975) elaborated the conclusion that, as compared to easily tamed and trained terrestrial animals, it is quite difficult to establish emotional contact between a trainer and a dolphin, in relation to the dominance-subordination relationship between them. Saprykin et al. (1975a) stated that they have a model making it possible to formalize quantitatively the capacity for classification of values or significance of events and of probabilities of occurrence thereof in the training of bottlenosed dolphins; the model apparently involves peripheral and central analyzers and an integrative center. Stochastic processes are introduced at several stages. J. HIGHER NERVOUSACTIVITY
This heading is frequently used by Soviet scientists, and the intention here is to treat papers whose main focus is the assessment of evidence for complex, presumably conscious, behavior involving cognition, communication, or the more derived forms of learning. We have already touched upon some such evidence in Sections 111, C and 111, I. Voronin and Kozarovitskiy (1969) described the responses of bottlenosed dolphins to difficult tasks; they dealt with “sharply pronounced inertness of nervous processes,” “extensive excitation irradiation,” imitation, orientation, investigation and play activity, a tendency towards developing unachieved reactions, and ritual motions. In these respects, dolphins resemble other higher animals, notably apes. Kreyn ( 1970a) reviewed American papers that purport to investigate the language and intellect of dolphins. She concluded that the approach of Dreher (1966), “although of definite interest, cannot be used as a key
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to decoding the language of dolphins,” that the approach of Lilly (1962, 1966) to teach the dolphins English “has so far been fruitless-which is completely understandable,” and that both of these approaches “have not produced any significant results.” On the other hand, the work of Bastian (1967) and of Pryor et al. (1967) “are the most interesting of all the work done so far in the United States in the area of language and intellect of dolphins, since they indicate a new approach to this problem.” (Bastian experimented with the ability of dolphins to transmit arbitrary types of information and Pryor et al. attempted an experiment to estimate the capacity of dolphins to make logical generalizations.) Voronin (1970) provided a broader review of both Soviet and foreign literature. Otherwise sympathetic or neutral to the contributions of most workers, when he came to the proposition of Lilly (1967); e.g., that dolphins have a language comparable in complexity to humans, he reviewed the classes of argument Lilly implied and concluded that “not one of these arguments significantly indicates a capacity for abstract conceptual thinking in the dolphin. . . . as far as the capacity of the dolphin to adopt the words of an experimenter by imitation and to learn them by conditioned reflex is concerned, this capacity is not evidence of language and conceptual thinking in dolphins . . .” He found the reported responses to be not essentially different from conditioned responses in other animals, combined with extraordinary acoustic and sound generating abilities. “Thus the question of dolphin language cannot be answered in such a way as to favor the hypothesis of its existence.” Turning to Lilly’s conclusion that “. . . although dolphins are not similar to us (they are even far apart from us), they are probably not inferior to us in adaptability, learning ability, and wisdom . . .”, Voronin discussed the nature of the argument and concluded that “it appears to us that all of Lilly’s considerations are extremely preconceived and that the sole ‘argument’ (concerning brain weight) in favor of cetacean intelligence is, gently speaking, incorrectly interpreted by the author.”
* Whereas, in general, we cannot give the background or the relevant non-Soviet literature, since a review of world literature is beyond our present scope, the Soviet views on Lilly’s writings are a special case. The following comments are quoted, with permission, from a recent letter by Dr. Lilly: “. . . I find that my separation of hypothesis and theory from experimental fact is not followed in the critiques of the work; suggestive experimental results are misquoted as if they were absolute solutions or failure of solution of major problems. . . He is apparently confusing hypothesis in the current state of the art of that time with conclusions which I am not willing to make as yet. . . . The hypothesis (1967, Mind of the Dolphin) and its tests are both derived from computer science (“biocomputers, dolphin and human”), and for the last two years, have been under detailed experimental development in both hardware and software in realizable forms (the JANUS project).”
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Voronin (1970) regarded as fully justified the views of McBride and Hebb (1948) that the emotional and motivational lability in dolphins can be ranked between the dog and the chimpanzee. Voronin noted that the passive attitude of dolphins, which do not manifest aggression during capture and during experiments, has evoked great interest but no scientific explanation in the literature. “Lilly’s attempts at explaining this by saying that the dolphin does not attack man because he knows how dangerous he is, can hardly be considered seriously.” Voronin pointed out that there are a number of reports in the literature of occasions when dolphins become aggressive in relation to each other and to other species, including humans (see Shurepova, 1971). He went on to discuss some special features of the dolphin’s nervous system, including the pronounced passive defense reaction, well known in some other species, and “inertia” with respect to some forms of restructuring of responses, also well known elsewhere. Citing Starodubtsev (1968), he pointed out that if a monkey is offered eight pedals which it has learned to press to receive food, reinforcement of just a certain sequence of depression, e.g., the second, fifth, and seventh pedals, irrespective of actions related to the other pedals in the intervals between the correct pedals, leads to development of a chain of motion. Such a task was found to be impossible for the bottlenosed dolphin, which, failing to receive food upon pressing any particular pedal, continued to act upon it repeatedly in different ways (from above, from the side, from below, from the front) and with different parts of the body. It did not sample the other pedals as did the monkeys. Although the species was said to be “inventive” in respect to play and shows irradiation in respect to the motor systems that are evoked, it shows some kind of generalization that is represented by the term “inertia.” “The anthropomorphic interpretation differs sharply from the physiological one. For example, Morgan (1968) interprets the general movements of a dolphin that had made an error as an expression of its anger. Thus, in her words, ‘one of the dolphins was so angered that it destroyed the experimental apparatus’.” Voronin noted the extensive knowledge of the special characteristics of higher nervous functions in chimpanzees and other anthropoids. These include such properties as rapid “reanimation” of formerly developed temporal associations and their combination with conditioned associations being developed at the moment-the basis for the so-called “inspiration” reaction (“ah-ha”),the presence of elementary counting, and the broad use of “active” signalization, manifested in the form of the pointing gesture, and other features. He regretfully concluded that “presently the results of excessively specialized dolphin experiments and training, which in addition, have not been subjected to adequate physiological analysis,
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preclude our possibility for fully comparing their behavior with that of anthropoid monkeys.” Voronin et al. (1971) contributed a paper based on simultaneous training of two bottlenosed dolphins under conditions permitting either one or both to give the correct reaction, which then released fish available to both so that either one or both could get the reinforcement. This permits three variants of “conformity”: dolphin 1 shows the correct response and takes the fish, or dolphin 2 shows the response and takes the fish, or both dolphins show the response and both take fish. But six variants are possible of “incongruity”: dolphin 2 shows the response but dolphin 1 eats the fish or vice versa; dolphin 2 shows the response, but both eat the fish; or dolphin 1 shows the response and both eat fish; or both show the response and only dolphin 1 or only dolphin 2 eats fish. In three series under somewhat different conditions, the authors studied the relative occurrence of conformities and incongruities, as well as the frequency of correct responses in the two animals. In the first series, a single stimulus, a single response manipulator, and a single feeding trough were approximately in the center of the pool. In the second, a duplicate setup was at each end and only responses at the manipulator corresponding to the end where the stimulus was presented were reinforced. In the third, stimuli were presented in the center but when a red ball was presented, the animal’s response at the left manipulator was reinforced and when the blue ball was presented in the center, responses at the right manipulator were reinforced. The results cannot be summarized in a simple way but significant trends were noted in the successive series. The dolphins “sorted out” at first the manipulators, then the sides, and only then, to a small extent, the stimuli. There was apparent interaction in that the trends of behavior in one animal were accompanied by changes in the behavior of the other. But the design of the experiment does not permit readily interpretable statements about the higher nervous functions thus manifested. Krushinskiy et al. (1972a,b) examined an apparently more complex form of behavior in the same species, a behavior indicating what the authors consider to be “elementary mental activity.” The ability to solve problems requiring recognition of two-dimensional versus threedimensional figures was used as an indicator of “elementary mental activity.” The experiment was based on the capacity of volumetric figures to hold objects whereas flat figures cannot; the method was based on the play activity of dolphins. The animals had to choose one of two figures, volumetric or flat, in which a volumetric bait, namely a ball, was placed behind an opaque screen. It was found, at a high level of significance, that these two dolphins were able, without preliminary learn-
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ing, to devise a program of behavior based on their capacity to operate with empirical dimensions of figures. One looks in vain for a series of studies based on modern ideas for assessing the development of concept formation at different levels of sophistication. A sufficient set of such tests and, likewise a set of graded tests of learning-set such as reversal learning and delayed response, oddity nonoddity, and the like, would go far toward permitting significant comparative statements of the level of intellectual capacity in these animals compared to other species. It is a most striking fact that such a battery of tests has not been applied, considering the number of patiently conducted experiments involving training of dolphins that have been performed in the aggregate, both in the Soviet Union and elsewhere. Krushinskiy et al. (1972a,b) devised an even more complex group experiment with three animals. They believe the results revealed that in a group situation, the behavior of each dolphin was biologically purposeful and allowed them to obtain fish. For example, the passive male made complex adaptations, one after the other, to changes in conditions or behavior of the other dolphins, permitting it to switch from tactics of waiting and grabbing another dolphin’s fish in the first series to “selfservice” and “static” monopoly of one of the levers. Thereafter, increased activity of the female caused a change in his tactics, and, after a period of antagonism, when peaceful relations with the other male were reestablished, it became possible to return to the initial tactic of depending on that animal instead of working for itself. The authors felt that their observations confirmed the strong tendency toward inertia of nervous processes in dolphins but at the same time showed that some individuals can display considerable lability. The dominant male showed a period of aggressive behavior, scaring off the other male, and even pouncing on the female, rapidly giving way to neutral and then markedly friendly behavior. In two short notes from the same group (Protasov and Sergeyev, 1975; Saga1 et al., 1975), the characteristics of conditioned responses were analyzed, The first noted that the slowest and most difficult of the conditioned food-searching responses to develop in dolphins are responses involving differentiation of several positive conditioned stimuli and those requiring simultaneous development of positive and inhibitory conditioning, as well as certain forms of conditioned chain responses. Several forms of conditioned response are easily and rapidly developed in dolphins as in most higher terrestrial animals: inhibitory differentiation, primary and secondary conditioned inhibition, secondary reflexes, several dynamic stereotypes, as well as conditioned re-
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flexes with “switching over.” Dolphin behavior is characterized by specific inhibition of orientation and exploratory activity. They related these characteristics to the ecology of hunting in groups upon schools of fish. In the second communication an effort was made to use the methods and procedures of general communication theory to describe the forms of higher nervous activity. The development of a conditioned response was considered to be a reflection of a simple homogeneous confluent Markov chain with a small number of states. Kovtun (1975) argued that as yet little is known about the types of unconditioned reactions of dolphins and those that are known have been little studied with respect to their properties and physiological mechanisms. These are regarded as first-order needs in order to progress toward understanding higher nervous activity. When conditioning animals, one must use methods, he argued, that take into consideration the evolutionary and ecological status of the species, including their sensory and motor apparatuses. The author felt that the evidence available, considering the adaptations to specific conditions of their habitat, does not yet enable us to maintain that the level of higher nervous activity in dolphins is higher than that of familiar terrestrial animals, let alone that of the human. Shurepova et al. (1975) assessed long-term memory in five bottlenosed dolphins trained to perform a series of tasks on command. About four basic types of tasks were listed: approaching the trainer, towing the trainer, delivering different objects from the surface to the trainer, and delivering different objects from the trainer at the bottom to the source of a signal on the surface. Variations on several of these were also built in, such as towing the trainer in different directions. Subsequently, at different intervals without training, 3 months to 2 years, the animals were tested for retention and it was found that there is a well-developed long-term memory. Even after dolphins had been out in the open sea for up to 2 years without contact with man, they retained a degree of skill in performing previously developed tasks which was completely restored after issuing a few commands. Previously, Kreyn (1970b) had taught a dolphin a “rule” and retested it without intervening training 1 year later; Gurevich (1968a,b) and Bel’kovich et al. (1969a) had similar experiences. Such ability, of course, is not unusual and does not measure intellectual capacity (cf. Thompson and Herman, 1977). IV. Summary
Over 360 Soviet papers have been reviewed in the areas of cetacean neurobiology and psychobiology. This is probably nearly the whole of
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the scientific literature on these subjects. We signal the main contributions in something a little more than an annotated bibliography, but with a few exceptions we cannot properly evaluate the contributions in perspective, since that would require reviewing the world literature. This is primarily a summary of what Soviet authors have studied, claimed, and opined. A list of the main laboratories is given. Areas in which activity has been especially strong include anatomy, histology, sound production, echolocation, behavioral audition, the electrophysiology of the sensory cortex, and social behavior exemplifying higher nervous function. Areas in which Soviet authors have not been particularly or at all active include ethology in the rigorous sense, cognitive capacity, neuropharmacology, neurochemistry, and, with exceptions noted, ontogeny, electron microscopy, experimental anatomy, and even classical studies on the cerebellum, lower motor systems, hippocampus, hypothalamus, thalamus, the ear, and some other structures. The anatomy of peripheral nerves and the microscopy of innervation of skin, melon, air sacs, and certain viscera have occupied a number of authors. Some studies appear uncritical, even naive, or give no evidence for unlikely claims. A few papers deal with the proportions of nuclei in the brain stem, and the cell types in the cerebellum. Some claims of olfactory endings are made, on weak evidence. A few experiments on trained discrimination of chemicals give values for detectable concentrations of such solutions. The lowest value for a known substance is 0.0 l%(indole) and for an unknown biological product (gland secretion), 0.005%. One author speculates that there is chemical communication between dolphins. A number of studies focus on the peripheral and central anatomy of the auditory system, the simplicity of the dorsal cochlear nucleus, the elaboration of the ventral cochlear and higher acoustic nuclei, and the unspecialized character of the very large auditory cortex, which is high in the parietal lobe. A few studies deal with the visual system, from receptors to cortex. The cerebral cortex has attracted repeated attention because of its strange combination of great extent, thinness, less marked lamination and regional differentiation, and relative paucity of limbic structures compared to primates, carnivores, and other mammals. A common view among Soviet authors is that the cetacean brain has developed in a different direction from that in terrestrial mammals, so that size does not have the same significance it would have in other groups. Homology of areas becomes a serious question, since the auditory cortex is so far from its position in primates; the visual area is contiguous caudally but much smaller, the somatosensory is adjacent rostrally and overlaps nearly
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completely with the motor, as far as that has been mapped. Authors who address this question consider these areas homologous to their analogs in other mammals but that association cortex has developed quite differently. Several studies of reflex control of air movement provide empirical results but do not address the question of central control. Behavioral studies of audition and of the production of acoustic signals are numerous. There is still major uncertainty about the roles of various structures in producing sounds; several independent sources, under nervous control, seem likely. At least five parameters of the acoustic emission can be independently manipulated. In addition, its direction (axis of maximal emission) can be changed, perhaps at high rates; a value of 3.5 X lo5deg/sec is given, although supporting evidence is not. This amounts to scanning without head movement. Some authors have classified large numbers of recorded dolphin sounds according to elements and combinations, inferring lower and higher order neural operations in generating the patterns. In spite of considerable complexity, the authors cautiously “abstain for the time being from any categorical evaluations of [the] semantic complexity or effectiveness” of sounds as an “open” communication system. Others assume that they “bear a certain meaning-related load in communication,” but do not claim a high level of language. Some explicitly conclude that the evidence does not support a language in dolphins. Very modest progress is reported in associating sounds with situations, emotional states, activities, or communication between individuals in separate pools linked by wire. There are proposals that dolphins choose sounds to maximize the audibility of fine structure in a noisy environment according to optimal filter theory. Among a wide repertoire, animals are said to have an identification signal and a preferred signal, which may be the same or different. The considerable and heterogeneous body of results leaves open the question of how elaborate is the communication system. It is compatible with a view that dolphins are not greatly different in complexity from other nonhuman mammals, use a limited variety of signals, among which a few may be very long and involved, though little more than signature or identification calls. Psychophysical measurements of sound perception thresholds as functions of frequency produce some interesting new values, including differential frequency and differential intensity thresholds. Discrimination of intervals, accuracy of lateralization of a sound source, estimation of the speed of a sound source, of the azimuth of an echoing target, and of the relative distance of a target are reported. Threshold values for the last two are 14‘ of arc (cf. 4’30’ for a bat) and 1.5- to 4-mm difference in
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distance (equal to 2-5 psec difference in echo time; cf. 160 psec for a bat). The effect of masking noise decreases as the angle of the noise direction from the signal direction increases. Echolocation is the subject of a large number of papers, including, besides the topics mentioned above, the discrimination of objects, including geometric figures, the duration of the period of susceptibility to interfering false echoes (300 psec), the strategies invoked in solving near-threshold discriminations, and theoretical studies of the dolphin’s potential accuracy. The physiology of the cerebral cortex has been studied by recording evoked potentials in the unanesthetized dolphin. The main effort has been to map areas responsive to clicks, light flashes, skin stimuli, and direct electrical stimulation of the motor cortex. Some of the physiological properties of responses and limited evidence of primary and secondary sensory projection areas are reported. With glass capillary microelectrodes, single units have been recorded, some of which were responsive to sound with phasic or tonic, excitatory or inhibitory changes in spike firing. Self-stimulationof motivational regions (striopallidal system and cingulate gyrus) has been studied. Electroencephalographic characteristics of the waking and the sleeping states are described; several stages of sleep are distinguished, but no paradoxical or “rapid eye movement” sleep stage has been recognized because the usual criteria for this stage are not applicable. In certain stages of sleep the hemispheres can be asymmetrical, one having large, slow waves and a slightly lower temperature, the other with low voltage, fast waves, and higher temperature. These relations may alternate, left and right, on a 3 hr cycle. A few quasi-ethological reports of the behavior of dolphins in captivity are reviewed. Some of these and a long series of special tests are believed to involve “higher nervous activity.” While these are often ingenious and indeed suggest conscious mental processes, they are not designed to permit comparisons with other orders of mammals. The dominant opinion among these authors seems to be that dolphins are not superior to apes in cognitive abilities and may be considerably inferior. ACKNOWLEDGMENTS This work was aided by grants to T.H.B. from the National Science Foundation, the National Institutes of Health, and the National Aeronautics and Space Administrationand by support of V.S.G. from the Naval Ocean Systems Center, San Diego, and the Office of Naval Research.
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Valiulina, F. G. The role of the mandible in conduction of sonic oscillations in man and dolphins. In: Morskiye Mlekopitayushchiye, G. B. Agarkov (ed.), Izd. Naukova Dumka, Kiev, Part I, pp. 68-71, 1975. Valiulina, F. G. and B. G. Kohmenko. Micromorphology of the upper tongue receptor system in Tursiops truncatus. Zool. Zh., 55, 467-470, 1976. Vasilevskaya, G. I. On innervation of the extraocular muscles in dolphins. VII-aya Vses. K q f . Morsk. Mlekopitaywhchim, Simpheropol‘, pp. 6 1-62, 1978. Vel’min, V. A. Detection of targets by bottlenosed dolphins (Tursiops truncabus) in the presence of artificial reverberations. In: Morskiye Mlekopitayushchiye, G. B. Agarkov (ed.), Izd. Naukova Dumka, Kiev, Part I, pp. 75-77, 1975. Vel’min, V. A. and N. A. Dubrovskiy. Auditory analysis of sound pulses in dolphins. Dokl. Akad. Nauk SSSR, Ser. Biol., 225, 470-473, 1975. (Also in English, Neurosc. Behm. Physiol., 7 , 562-565. 1977.) Vermin, V. A. and N. A. Dubrovskiy. Auditory perception of the impulse signals by Tursiops truncatw. In: Morskiye Mlekopttayushchiye. Resul’taty i Metody Issledovaniya, V. Ye. Sokolov (ed.), Izd. Nauka, Moscow, pp. 90-99, 1978. Vermin, V. A. and A. A. Titov. Auditory discrimination of interpulse intervals by bottlenosed dolphins (Tursiops truncatus). In: Morskiye Mlekopitayushchiye, G. B. Agarkov (ed.), Izd. Naukova Dumka, Kiev, Part I, pp. 77-78, 1975. Vel’min, V. A., A. A. Titov and I,. I. Yurkevich. Differential intensity thresholds for short-pulsed signals in bottlenosed dolphins (Tursiops truncatus). In: Morskaye Mlekopitayushchiye, G. B. Agarkov (ed.), Izd. Naukova Dumka, Kiev, Part I, pp. 73-74, 1975a. Vel’min, V. A., A. A. Titov and L. 1. Yurkevich. Temporal pulse summation in bottlenosed dolphins (Tursiops truncatus). In: Morskiye Mlekopitayushchiye, G. B. Agarkov (ed.), Izd. Naukova Dumka, Kiev, Part I, pp. 78-80, 1975b. Vinnikov, Ya. A. and L. K. Titova. Organ OfCorti. Izd. Nauka, Moscow-Leningrad, 234 pp., 1961. Vomnin, L. G. Behavior of the “primate” of the sea-the bottlenosed dolphin (Turnups truncatus Montagu). Usp. Sovrem. Biol., 2, 191-207, 1970. Voronin, L. G. and L. B. Kozarovitskiy. Some peculiarities of high nervous activity of the Black Sea dolphin (Tursiops truncatus). Zh. Vyssh. Neru. Dqat., 19, 233-242, 1969. Voronin, L. G. and V. Ye. Sokolov. Survey of data on dolphins. In: Morfologiya i Ekologiya Morskikh Mlekofitaywhchikh, V. Ye. Sokolov (ed.), Izd. Nauka, Moscow, pp. 3-25, 1971. Voronin, L. G., L. B. Kozarovitskiy and Yu. D. Starodubtsev. Group behavior of Black Sea bottlenosed dolphins ( T u r y f ~ shuncalu~ Montagu). In: Mmjdagija i Ekologrya Monkikh Mlekopitayushchikh, V. Ye. Sokolov (ed.), Izd. Nauka, Moscow, pp. 88-93, 1971. Voronin, L. G., L. B. Kozarovitskiy and Yu. D. Starodubtsev. On the behavior of the Black Sea dolphin (Tursiops truncatus, Family Delphinidae) in a group experiment. Morfologiya, Fiziologiya, i Akuctika Morskikh Mlekopitayushchikh, V. Ye. Sokolov (ed.), Izd. Nauka, Moscow, pp. 99-108, 1974a. Voronin, L. G., Yu. D. Starodubtsav, L. B. Kozarovitskiy, V. A. Gorshkov and M. Ye. Ivshin. Some data dealing with taming Black Sea dolphins. In: Morfologiya, Fiziologiya i Akwtika Morskikh Mlekopitayushchikh, V. Ye. Sokolov (ed.), Izd. Nauka, Moscow, pp. 108-122, 1974b. Voronov, V. A. and I. M. Stosman. Frequency threshold characteristics of subcortical elements of the auditory analyzer of Phocoenu phocoena. Zh. Evol. Biokh, Fiziol., 6, 719-723, 1977. Voronov, V. A. and I. M. Stosman. Frequency range of the sounds of dolphins. Vll-oye Nauchn. Soveshch. Evol. Fiziol., Leningrad, pp. 57-59, 1978. Voronov, V. A., E. I. Krasnoshchekova, T. M. Mal’tseva and V. A. Protasov. Method of
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BINDING AND IONTOPHORETIC STUDIES ON CENTRALLY ACTIVE AMINO ACIDS-A SEARCH FOR PHYSIOLOGICAL RECEPTORS*
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By F V Dehudir Cntn & N.umchimk du CNRS. et InFacult6 & &ine.
& Chimb Bbbdqw.
Strarboug odex, France
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. y-Aminobutyric Acid (GABA) . . . . . . . . . . . . . . . . . . . B. Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.Taurine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Glutamate and Aspartate . . . . . . . . . . . . . . . . . . . . . I1. Physiologic-Pharmacologic Studies in Vertebrates . . . . . . . . . . . A.GABA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.Taurine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Glutamate and Aspartate . . . . . . . . . . . . . . . . . . . . . I11. Physiologic-Pharmacologic Studies in Invertebrates . . . . . . . . . . . A.GABA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.Glycine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.Taurine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Glutamate and Aspartate . . . . . . . . . . . . . . . . . . . . . IV . Biochemical Studies with Vertebrate Preparations . . . . . . . . . . . A.GABA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.Taurine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Glutamate and Aspartate . . . . . . . . . . . . . . . . . . . . . V. Biochemical Studies with Invertebrate Preparations . . . . . . . . . . . A.GABA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Glutamate and Aspartate . . . . . . . . . . . . . . . . . . . . . VI . Relevant Studies on Glial Cells . . . . . . . . . . . . . . . . . . . . VII . Relevant Studies with Tissue Cultures . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Autoradiographic Studies on the Uptake of “Active” Amino Aids . . . C . Electrophysiologic-Pharmacologic Studies . . . . . . . . . . . . . . D . Other Biochemical Studies . . . . . . . . . . . . . . . . . . . . . E . Criticism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130 131 131 132 132 133 133 144 147 147 150 150 151 151 152 154 154 177 185 187 189 189 192 192 198 198 199 200 202 202 202 204
* This review was essentially completed in November. 1976 while the author was Professor of Physiology in the Facultad de Medicina. Universidad Aut6noma. Madrid. Spain . 129 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 21
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Copyright @ 1979 by Academic Press. Inc All rights of reproduction in any form reserved
ISBN 0-12-366821-2
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1. Introduction
Certain pharmacologically active amino acids may, on a quantitative basis, be the major neurotransmitters of the vertebrate CNS (see for review Roberts, 1956, 1968; Elliott and Jasper, 1959; Curtis and Crawford, 1969; McLennan, 1970a, 1975; Johnson, 1972; Baldessarini and Karobath, 1973; Krnjevik, 1974; Curtis and Johnston, 1974a; DeFeudis, 1975a; Ryall, 1975; Snyder and Bennett, 1976; Roberts et al., 1976). Neutral amino acids [e.g., y-aminobutyric acid (GABA), glycine, and taurine] are leading candidates for roles as postsynaptic inhibitory transmitters. Acidic amino acids (e.g., glutamate and aspartate) may function as postsynaptic excitatory transmitters. Since these “active” amino acids are present in all regions of the vertebrate CNS and since they can modify the excitability of most central neurons, they may serve as transmitters in a variety of central pathways involved in the control of vital functions and behavior (see, e.g., Davidson, 1976; Roberts et al., 1976). Studies with tissue slices or subcellular particles have revealed that most regions of the mammalian CNS possess “high-affinity” uptake or binding mechanisms for these amino acids (see, e.g., reviews by DeFeudis, 1975a; Martin, 1976). I n vivo and in vitro studies on the central “release” of these amino acids have further supported their proposed transmitter roles (e.g., Crowshaw et al., 1967; Obata and Takeda, 1969; Jasper and Koyama, 1969; Mitchell and Srinivasan, 1969; Bradford et al., 1973; Pasantes-Morales et al., 1973a,b; Osborne et al., 1973; Okada and Hassler, 1973; Collins, 1974; Mulder and Snyder, 1974; Kaczmarek and Adey, 1974; DeFeudis, 1974a; Davies et al., 1975; L6pez-Colome et al., 1976). Some of these amino acids (e.g., GABA, glutamate) have been collected in newly synthesized form from certain brain structures of anesthetized monkeys (DeFeudis et al., 1969, 1970), and more recently a K+-induced release of newly synthesized GABA from synaptosomal fractions of rat brain has been demonstrated (Ryan and Roskoski, 1975). Neuronal membrane hyperpolarizations produced by applications of GABA, glycine, or other inhibitory amino acids appear to involve increases in C1- permeability (e.g., Krnjevik, 1974; Curtis and Johnston, 1974a), whereas increased neuronal influxes of both Na+ and Ca2+ appear to be involved in the depolarizing actions of glutamate and aspartate (Harvey and McIlwain, 1968; Ramsey and McIlwain, 1970; Bernardi et al., 1972; Curtis et al., 1972a; Zieglgansberger and Puil, 1973; HBsli et al., 1973a, 1976; Onodera and Takeuchi, 1976). “Binding” and iontophoretic studies may be considered to represent, respectively, the biochemical and electrophysiological approaches for studying mechanisms of receptor interaction and inactivation of released
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transmitters. Recently, added emphasis has been placed on in vitro studies of the “binding” of these amino acids to subcellular particles of nervous tissues. In particular, a search is being made for receptor antagonists. However, it has not yet been possible to separate processes of uptake-related “binding” from receptor binding of these amino acids. This pitfall is difficult to overcome since both “receptor binding” and “uptake binding” would be expected to occur at 0°C. As a working hypothesis, “uptake binding” might be expected to be strongly Na+dependent and to occur in glial, as well as in neuronal, membranes, whereas specific “receptor binding” may not be so strongly Na+dependent and might be confined to postsynaptic membranous structures. Many pitfalls are also inherent in the use of the iontophoretic method. In addition to the general problems related to the solubilities and other characteristics of the substances used and the inability to accurately quantitate the amount of substance applied, many other problems exist when employing presumed amino acid antagonists (e.g., nonspecific effects; nonreversibility of action; intrinsic actions of substances; local anesthetic actions). This review will focus on recent advances made using iontophoretic and biochemical approaches. Supporting evidence derived from studies on the active uptakes of “active” amino acids, though inextricably related to the subject at hand, will be discussed only when necessary for the development of particular concepts.
A. y-AMINOBUTYRIC ACID(GABA) In mammals, GABA appears to be localized mainly in the CNS; it is also found in the peripheral nervous systems of various inverte-
brates. GABA is not a substrate for protein synthesis, though it is a constituent of certain small peptides; e.g., y-aminobutyrylhistidine; a-(y-aminobutyry1)-lysine(Nakajima et al., 1969; van Regemorter et al., 1972). GABA is likely to be a major postsynaptic inhibitory transmitter throughout the vertebrate neuraxis, and may also be involved in presynaptic inhibition in the spinal cord and brain stem. All established criteria for characterizing central neurotransmitters have been met by GABA (see, e.g., Curtis and Johnston, 1974a; Krnjevit, 1974; DeFeudis, 1975a; Roberts et al., 1976).
B. GLYCINE Unlike GABA, glycine is present in most mammalian tissues, and is involved in many metabolic reactions (both central and peripheral), including protein synthesis. Hence, the separation of transmitter from
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F. V. DEFEUDIS
metabolic pools of glycine may be more difficult than for GABA. Physiologic-pharmacologic evidence has indicated that glycine possesses actions which mimic that of a postsynaptic inhibitory transmitter in several regions of the vertebrate CNS, e.g., in the spinal cord, particularly at inhibitory synapses on motoneurons, and in some brain stem areas (e.g., Curtis and Johnston, 1974a; Ryall, 1975). Almost all criteria for characterizing CNS transmitters are met by glycine in these CNS regions. Curtis et al. (1968a, 1971a) found that strychnine, a convulsant long known to potently antagonize spinal inhibitory mechanisms (Owen and Sherrington, 1911; Dusser de Barenne, 1933),also antagonized the depressant action of glycine, but not that of GABA, on spinal motoneurons. Other significant physiologic-pharmacologic and biochemical advances indicating a transmitter role for glycine have been recently reviewed (Krnjevii., 1974; Curtis and Johnston, 1974a; DeFeudis, 1975a; Johnston, 1977; Davidson, 1976). C. TAURINE The sulfonic amino acid, taurine, is present in high concentration in the vertebrate CNS (e.g., Tallan et al., 1954; Shaw and Heine, 1965; Kandera et al., 1968; Battistin et al., 1969; Yoshino et al., 1970; Guidotti et al., 1972) and is enriched in synaptosomal fractions of rat brain homogenates (Agrawal et al., 1971). Like GABA, taurine is not a substrate for protein synthesis, but unlike GABA it is present in many non-nervous tissues. Its release in uiuo from cat cerebral cortex (Jasper and Koyama, 1969) and in uitro from rat cerebral cortex slices (Kaczmarek and Davison 1972) can be evoked by electrical stimulation, and its release from isolated chicken retina can be stimulated by light or high K+ concentrations (Pasantes-Moraleset al., 1973a; Lopez-ColomC et al., 1976). Like GABA and glycine, it is rapidly taken up by slices of rat cerebral cortex (Kaczmarek and Davison, 1972; Lahdesmaki and Oja, 1973; Starr, 1973). Although the taurine uptake system appears to be distinct from that for GABA (Iversen and Johnston, 1971), both amino acids appear to be accumulated by synaptosomal particles of rather identical size, volume, and density (Sieghart and Karobath, 1974). Its potent depressant action on spinal neurons was first shown by Curtis and Watkins (1960a). Many of the criteria for characterizing CNS inhibitory transmitters are met by taurine. AND ASPARTATE D. GLUTAMATE
The dicarboxylic amino acids L-glutamate and L-aspartate are present in high concentration in the vertebrate CNS and, like glycine, they
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are involved in many metabolic reactions (both central and peripheral), including protein synthesis. These substances exert potent excitatory actions on neurons of most areas of the mammalian CNS (see, e.g., Curtis et al., 1960a,b; Krnjevit and Phillis, 1963; see also reviews by Johnson, 1972; KrnjeviS, 1974; Curtis and Johnston, 1974a; DeFeudis, 1975a). Many of the criteria for CNS excitatory transmitters are met by glutamate and aspartate.
11. Physiotogic-Pharmacologic Studies in Vertebrates
A. GABA Studies concerning GABA receptors, conducted primarily with possible GABA antagonists, have been recently reviewed (Krnjevit, 1974; Curtis and Johnston, 1974a; DeFeudis, 1975a; Johnston, 1976, 1977). The convulsant agents, picrotoxin (PIC) and bicuculline (BIC), which block the depressant actions of GABA on vertebrate central neurons (e.g., Galindo, 1969; Nicoll, 1970, 1971; Obata and Highstein, 1970; Engberg and Thaller, 1970; Curtis et al., 1970a,b; ten Bruggencate and Engberg, 1971; Duggan and McLennan, 1971; Hill et al., 1973a; Altmann et al., 1976),along with strychnine, which predominantly blocks depressions produced by “glycine-like” amino acids (e.g., Curtis et al., 1968a, 1971a),are the most widely used agents for examining inhibitory processes of the vertebrate CNS (Fig. 1).These agents also block synaptically mediated postsynaptic inhibitions (e.g., Kellerth and Szumski, 1966; Curtis et al., 1970a,b; Ito et al., 1970; Nicoll, 1970; Obata and Highstein, 1970; Obata et al., 1970; Altmann et al., 1976), and in the cases of PIC and BIC, presynaptic inhibitions (e.g., Curtis et al., 19’71b; Davidoff, 1972a,b) in the vertebrate CNS. BIC-sensitive, strychnineinsensitive synaptic inhibitions have been found in most regions of the vertebrate CNS, whereas strychnine-sensitive, BIC-insensitive inhibitions are more restricted to spinal cord and brain stem. 1. Picrotoxin (PZC) as a GABA Antagonist The first demonstration of a PIC-sensitive GABA-mediated inhibition in the vertebrate CNS was made on neurons of the feline cuneate nucleus (Galindo, 1969). In this region, PIC could be a specific, competitive, GABA antagonist (see also Kelly and Renaud, 1973). PIC also blocked both stellate and basket cell inhibitions of Purkinje cells and Golgi cell inhibitions of granule cells when administered intravenously (2-5 mg/kg) or by iontophoresis (Bisti et al., 1971; Woodward et al.,
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F. V. DEFEUDIS
2H N, no ,!,
GLYCINE
noL
N
H
2
GABA
e0 ‘
0
OJ STRYCHNINE
BICVCULLINE
FIG. 1. Structures of the agonist-antagonist pairs, glycine and strychnine, GABA and bicuculline. (Reproduced with permission from Johnston, 1976.)
1971), and granule cell synaptic inhibition of mitral cells in the olfactory bulb (Nicoll, 1971), all of which may be mediated by GABA. PIC also antagonizes the action of GABA and blocks neurally evoked inhibitions at several other central sites (see, e.g., references in Krnjevit, 1974; DeFeudis, 1975a; see also Altmann et al., 1976). However, PIC does not appear to block specifically the neuronal depressant action of GABA in cat cerebral cortex (Krnjevii: et al., 1966), or in rat caudate nucleus (Bernardi et al., 1976). The report by Davidoff and Aprison (1969) that iontophoresed PIC consistently blocked the depressant action of glycine on spinal interneurons could not be confirmed (e.g., Curtis et al., 1969; Engberg and Thaller, 1970; Felpel, 1972). Even though PIC has been helpful in attempts to show GABA-mediated inhibitions in various regions of the CNS, its usefulness is limited, as it has low aqueous solubility, it may not be a competitive antagonist specific to GABA, and it may exert direct excitatory actions on neuronal membranes (see, e.g., Krnjevit, 1974). The actions of picrotoxin analogues have been recently reviewed (Curtis and Johnston, 1974b; Johnston, 1977). Picrotoxinin appears to be the active principle of picrotoxin; picrotin is a much less potent convulsant. Iontophoretically applied dendrobine blocked the inhibitory action of glycine, but not that of GABA, on spinal neurons. Tutin can serve as a GABA antagonist on spinal neurons, but it also reduces considerably the depressant action of glycine (Curtis et al., 1973a). Shikimin, administered intravenously (1- 1.3 mg/kg) to cats, reduced prolonged (presynaptic) inhibition of spinal reflexes, while not affecting direct inhibition (Curtis et al., 1973a).
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135
2. Bicuculline (BZC) as a GABA Antagonist Curtis et al. (1970a,b) first showed that bicuculline (BIC) can antagonize GABA-induced depressions and certain evoked central inhibitions in the mammalian CNS. Studies on the central actions of this substance have been extensively reviewed (e.g., Curtis and Johnston, 1974a; Krnjevik, 1974; Davidson, 1976; Johnston, 1977). Like PIC, BIC does not produce an effective and specific blockade of GABA-induced depression or of synaptic inhibition in the mammalian cerebral cortex, and it may exert direct excitatory effects on neuronal membranes (see, e.g., Straughan et al., 1971; Krnjevii, 1974). On rat caudate neurons, BIC also did not behave as a specific GABA antagonist (Bernardi et al., 1976). Also, BIC antagonizes the actions of some “glycine-like” amino acids (e.g., p-alanine and taurine) on spinal neurons (see, e.g., Curtis et al., 1971b), as well as the depressant action of droperidol on feline Purkinje cells (Maruyama and Kawasaki, 1975).
3 . Other Possible GABA Antagonists Intravenous benzylpenicillin antagonized BIC-sensitive prolonged inhibition, but not strychnine-sensitive direct inhibition in the spinal cord, and its iontophoretic application blocked GABA-, but not glycineinduced, spinal depressions (Davidoff, 1972a,c; Curtis et al., 1972b). Iontophoretically administered benzylpenicillin is a weaker GABA antagonist than BIC (Curtis and Johnston, 1974b; Johnston, 1977). d-Tubocurarine exerts a relatively potent GABA-antagonistic action on neurons of the feline cerebral cortex and cuneate nucleus, but also affects glycine action (Hill et al., 1972, 1973a,b), and, of course, is the classic antagonist of nicotinic acetylcholine receptors. Systemic administration of tetramethylenedisulfotetramine (TMDST) antagonized prolonged spinal inhibition (Curtis and Johnston, 1974a) and the action of GABA on rat superior cervical ganglion (Bowery et al., 1975a). Since TMDST antagonized the actions of both GABA and glycine in parallel on neurons of the rat cuneate nucleus (Collins et al., 1975), mesencephalic reticular formation, and medulla (Dray, 1975a),this agent could interact with an ionophore that is common to GABA and glycine (Dray, 1975a). However, it should be realized that TMDST, though effective as a GABA antagonist, is virtually insoluble in aqueous solutions (Kerr et al., 1976), as well as being rather nonspecific. Electroosmotically applied cuaniol reduced the depressant effect of GABA, relative to that of glycine, on feline spinal neurons (Curtis and Johnston, 1974b). As with PIC (Precht and Yoshida, 197l),systemic administration of caprolactams to rabbits blocked positive field potentials evoked in the substantia nigra
136
F. V. DEFEUDIS
by strio-pallidal stimulation (Kerr et al., 1976). Bowery et al. (1976a) have shown that some bicyclic organophosphorus esters, that are potent convulsants, antagonized the depolarizing action of GABA on rat superior cervical ganglion by a mechanism that appeared to be rather specific. Thus far, none of these compounds appears to be superior to PIC or BIC as GABA antagonists. 4. StructurelActivity Studies Curtis et al. (1970a) noted that structural similarities exist between GABA and BIC, indicating that both agents might interact with the same membrane receptor. The phthalide isoquinoline alkaloids, BIC-HCl, BIC-methochloride or methiodide, and corlumine-HC1, along with the derivative, bicucine methyl ester-HC1, are the only compounds investigated which appeared to behave as selective GABA antagonists on feline spinal neurons (Johnston, 1976). Corlumine has the same absolute configuration as BIC (Johnston, 1976, 1977). Interesting studies performed on neurons of rat cerebral cortex and cuneate nucleus by Collins and Hill (1974) have revealed that (-)BIC-methochloride, the optical antipode of (+)BIC-methochloride (the one used in iontophoretic studies), was ineffective as a GABA antagonist, although it produced neuronal excitation similar to that of the (+) isomer. Also, (+)BICmethiodide antagonized both the action of iontophoretically applied GABA and synaptic inhibition in the cat cuneate nucleus, ivhereas the (-) isomer possessed neither action (Hill et al., 1974). Further evidence supporting the similarity of GABA and BIC receptors comes from the findings that most other substances that exert BIC-sensitive depressions (e.g., y-amino-P-hydroxybutyric acid; imidazoleacetic acid) are structurally related to GABA (Johnston, 1977). Since both GABA and BIC are flexible molecules, the problems of the “active conformation(s)” of GABA have been approached indirectly using structurelactivity correlations of GABA analogues that possess restricted conformations. The pitfalls of using this approach have been discussed by Johnston (1977). Several analogues have been tested on BIC-sensitive, strychnine-insensitiveinhibitions in feline spinal neurons, and five compounds have been found to act as GABA agonists (see Fig. 2). The most potent analogue was trans-S-aminocyclopentane-l-carboxylic acid (about twice as potent as GABA) and the least potent is 4-aminotetrolic acid (about 20%-50% as potent as GABA). As these compounds possess three isosteric atoms that correspond to the charged atoms of the GABA zwitterion, GABA might act on its BIC-sensitive receptors in extended conformation (Johnston, 1977). Both BIC and PIC affect the actions of depressants other than
137
AMINO ACID RECEPTORS
MUSC’MoL
T R A N S 4 AMINO CROTONIC ACID
4
AMINOTETROLIC ACID
FIG. 2. Structures of bicuculline methochloride, GABA, and related bicucullinesensitive depressants drawn from Dreiding stereomodels to emphasize structural similarities. The three stars in each compound denote atoms which are isosteric with those marked in the other compounds. (Reproduced with permission from Johnston, 1976.)
GABA (e.g., glycine, imidazoleacetic acid; taurine, 5-HT) in various CNS regions (see, e.g., Straughan and Watson, 1972; see also reviews by Krnjevib, 1974; Curtis and Johnston, 1974a; DeFeudis, 1975a). These findings led Dray (1975b) to compare the actions of PIC, BIC, and BIC-methochloride against the depressant effects of various transmitter candidates on neurons of the rat brain stem. On spontaneously active cells of the medulla-pons of urethane-anesthetized rats, BICmethochloride was a more potent and more selective GABA antagonist than PIC or BIC. Also, all three compounds reduced or abolished imidazoleacetic acid-induced inhibitions before those of GABA, and were more potent antagonists of imidazoleacetic acid than of GABA; these compounds exerted no significant effects on norepinephrine- or 5-HT-induced depressions (see Table I). Such differences between BIC and BIC-methochloride are perhaps best explained by differences in their solubility. If one considers that BIC and BIC-methochloride are relatively specific GABA antagonists, then imidazoleacetic acid might also interact with GABA receptors, as had been previously indicated (Curtis et al., 1971b; Haas et al., 1973). However, Dray (1975b) has suggested that the more potent antagonism by these agents of the action of imidazoleacetic acid could indicate that its receptor might differ from the GABA receptor.
TABLE I COLLECTIVE DATAFOR THE EFFECTSOF BICUCULLINE METHOCHLORIDE, BICUCULLINE, AND PICROTOXIN ON DEPRESSION OF NEURONS OF RATBRAINSTEM PRODUCED BY GABA, GLYCINE, IM~DAZOLEACETIC ACID(IMA), NOREPINEPHRINE (NE), OR 5-HYDROXYTRYPTAMINE (5-HT)' Bicuculline methochloride Antagonismb GABA Glycine IMA NE 5-HT
0 $5 fQ
18 l%
Mean shift ratioc
Mean expellimg charge (C x 10+y
1.8 0.2 3.2 0 0
1.1 1.7 0.4 2.5 1.4
Bicuculline Antagonisml
Q & A
18
Q
Picrotoxin
Mean shift ratioc
Mean expelling charge ( c x 10-5y
1 .o 0.4 2.4 0.1 0
3.6 3.8 2.9 3.2 3.2
Antagonismb
Mean shift ratio'
Mean expelling charge (C x lo-s)c
H
0.7
A
0.1 2.9 0 0.1
4.2 3.9 3.9 3.9 5.2
* a %
Reproduced by permission of Dray (1975b). T h e times for which constant ejecting currents were maintained to provide 50%depression (TS)of firing were used as measures of the drug effects. For each antagonist, the numerator in the first column refers to the number of cells antagonized and the denominator to the number studied. of agonist response in the presence of antagonist - TS control agonist Antagonism is expressed as the shift ratio,calculated from the relationship (Tm response)mS control agonist response. T h e control responses of most cells selected for study varied by 2 10% so that a change in the agonist response of four times this variation during the concurrent application of an antagonist was considered to be significant and is indicated by a shift ratio of 0.4. The mean shift ratios for all the cells tested are shown. T h e mean charge (current x time) passed through the antagonist barrel when antagonism was measured is given in the third column.
AMINO ACID RECEPTORS
139
In other iontophoretic studies, Krogsgaard-Larsen et al. (1975) attempted to evaluate conformationally restricted GABA analogues. For separating receptor- from uptake- (“carrier”-) binding activities, these workers used muscimol(3-hydroxy-5-aminomethylisoxazole) and its derivatives, since muscimol exerts a potent “GABA-like” depressant action (Johnston et al., 1968), while being a weak inhibitor of GABA uptake (Johnston, 1971). Results from studies of the effects of these agents on spinal interneurons of cats and from studies with the same compounds on GABA uptake (Krogsgaard-Larsen and Johnston, 1975) led to the conclusion that different conformations of GABA are involved in its interaction with its receptor and uptake system. 5. Drugs Affecting Bicuculline (BZC)-Znsensitive GABA Receptors
The action of iontophoretically applied cis-4-aminocrotonic acid, unlike that of trans-4-aminocrotonic acid, was not antagonized by BIC or strychnine. Trans-2- and trans-3-aminocyclohexane carboxylic acid also produced BIC-insensitive, strychnine-insensitive depressions on feline spinal neurons (Johnston et al., 1975; Johnston, 1977). Hence, BICinsensitive GABA receptors appear to exist in the mammalian CNS (Ryall, 1975; Johnston, 1977). GABA might act with BIC-sensitive receptors in an “extended” conformation and with BIC-insensitive receptors in a “folded” conformation, since cis-4-aminocrotonic acid is a folded analogue of GABA (Johnston et al., 1975; Johnston, 1977; see also Kerr et az., 1976). Lioresal (Baclofen, or P-(p-chloropheny1)-GABA), when administered iontophoretically to central neurons, also exerted a BIC-insensitive depressant action (Davies and Watkins, 1974; Curtis et al., 1974). 6. Possible “Activators”of GABA-ergic Synapses a. Barbiturates. Regarding postsynaptic effects of barbiturates, Nicoll ( 1975a) found that pentobarbital ( 10-4 M, or more) hyperpolarized frog motoneurons by an action that was reversibly blocked by PIC and BIC but not by strychnine. Pentobarbital, at lower doses (> 2 X M ) , also selectively and reversibly depressed glutamate- and aspartate-induced depolarizations of these motoneurons, and at 2 X M to lop4M, it also increased the amplitude and duration of the GABA response (Nicoll, 1975a). Thus, three actions of pentobarbital have been shown in spinal cord, two of which (those on pre- and postsynaptic inhibitions) could involve an activation of GABA receptors. Further support for a role of barbiturates in mediating postsynaptic inhibition derives from the study of Nicoll et al. (1975), which revealed that intravenous pentobarbital (30 mg/kg) hyperpolarized feline hippocampal neurons and
140
F. V. DEFEUDIS
markedly prolonged their IPSPs by a direct, dose-dependent action on inhibitory synapses. These findings extended the proposal, based on an analysis of field potentials of olfactory bulb (Nicoll, 1972), that barbiturates prolong synaptic inhibition. Nicoll et al. (1975) suggested that barbiturates could produce such an effect by prolonging the release of inhibitory transmitter, or by their possible direct action on the GABA conductance mechanism, or by a delayed removal of GABA from postsynaptic receptor sites. Prolonged GABA release does not appear to be the sole basis for the effect, since pentobarbital can prolong the action of directly applied GABA (Nicoll, 1975b; Ransom and Barker, 1975). As pentobarbital competitively inhibits GABA uptake (Cutler et al., 1974), such a prolongation might well be caused by delayed removal of GABA from postsynaptic receptor sites (Nicoll et al., 1975). b. Benzodiatqbines. Benzodiazepines facilitated GABA function in cerebellum without altering the tissue content of GABA (Ma0 et ad., 1975; Costa et al., 1975a,b, 1976) and decreased the firing rates of Purkinje cells (Haefely et al., 1975) and the cyclic-GMP content of cerebellar cortex (Costa et al., 1976). It has also been claimed that benzodiazepines preferentially reduce the increase in cerebellar cyclic-GMP content caused by climbing fiber activation and preferentially antagonize convulsions that have been correlated with a deficit in brain GABA mechanisms (Costaet al., 1975a, 1976). Such findings have led Costaet al. ( 1975b) to suggest that benzodiazepines might stimulate GABA receptors indirectly by causing a release of GABA from cells of the cerebellum (see also Haefely et al., 1975). To test the hypothesis that benzodiazepines might act by facilitating “GABA-ergic”transmission, Steiner and Felix (1976) studied the effects of GABA and benzodiazepines on vestibular and cerebellar neurons. In N20-anesthetized cats, Deiters’ neurons were activated by antidromic stimulation of the vestibulospinal tract, and this response was inhibited by the preceding (orthodromic) stimulation of Purkinje cell axons. Iontophoretic application of GABA to Deiters’ neurons mimicked the effect of Purkinje fiber stimulation, and antidromically evoked action potentials were reduced by GABA. Intravenous diazepam suppressed the GABA-induced inhibition of the antidromically evoked potential. The effect of increased cerebellar stimulation was antagonized by local application of BIC (see also Curtis et al., 1970a). Further experiments on urethane-anesthetized rats revealed that a preceding injection of bromazepam strikingly decreased GABA-induced depressions of spontaneously firing Purkinje cells (see Fig. 3). Furthermore, inhibition of Purkinje cells by basket cells, as well as the direct inhibitory effects of
141
AMINO ACID RECEPTORS
7
‘
I
I
Y‘”’ ( , \ I %1 1\1 1 1 \
-
12 in\ I)
1 %
FIG. 3. Single unit activity of a rat Purkinje cell recorded through the central barrel of a five-barreled micropipette. GABA was delivered from a lateral barrel as a 0.5-1 M solution at pH 3.0-3.5. Upper:Delayed depressant effect of CABA (10 nA) 47 min after injection of bromazepam (2 mg/kg, iv.); action potentials persisting during the second two-thirds of GABA iontophoresis represent nonsuppressible climbing fiber responses. Lower: Further attenuation of the GABA (10 nA) effect 2 min after a second injection of bromazepam (4 mg/kg, i.v.). (Reproduced with permission from Steiner and Felix, 1976.)
GABA on Purkinje cells, were abolished by direct iontophoretic application of chlordiazepoxide. These results do not support the notion that benzodiazepines act by facilitating “GABA-ergic” transmission, since GABA-mediated inhibition was clearly antagonized by these drugs. Recent studies by Gahwiler (1976a) on cultured cerebellar explants also revealed that benzodiazepines antagonized the depressant effect of GABA (see Section VI1,C). Indirect in vivo experiments and observations using isolated CNS receptor preparations have linked the actions of benzodiazepines to GABA (Polc et al., 1974; Costa et al., 1975b; Suria and Costa, 1975; Haefely et al., 1975) and glycine (Young et al., 1974a; Snyder, 1975). To test this hypothesis, the effects of chlordiazepoxide and flurazepam were compared with those of GABA and glycine administered to the same neurons by iontophoresis; BIC-methochloride and strychnine were used to determine whether flurazepam acted on GABA or glycine receptors (Dray and Straughan, 1976). On spontaneously active cells of the medullas of urethane-anesthetized rats, both flurazepam and chlordiazepoxide generally reduced cell firing, being about half as potent as GABA or glycine, but no selective actions could be shown on the depressions produced by GABA or glycine. However, other experiments revealed that both GABA- and flurazepam-induced depressions were consistently antagonized by BIC-methochloride, when glycine depressions were not affected, and that strychnine selectively reduced or abolished glycine depressions when GABA and flurazepam
142
F. V. DEFEUDIS
depressions were not affected. These results revealed an involvement of benzodiazepines with GABA-, rather than with glycine-mediated, processes (see also Section 11,B). 7. Presynaptic Inhibition; Possible Involvement of GABA a. Introduction. Presynaptic inhibition regulates sensory input and has been demonstrated in the vertebrate spinal cord and brain stem (Schmidt, 19’71). Its mediation appears to involve the effects of interneurons that synapse axo-axonally upon the terminals of sensory afferents. The transmitter involved is believed to depolarize nerve terminals of sensory afferents, thereby decreasing the amount of transmitter released. In the innervation of crustacean muscle, presynaptic as well as postsynaptic inhibition have long been known to exist (e.g., Marmont and Wiersma, 1938); more recent evidence has indicated that GABA serves as the neurotransmitter for both of these mechanisms (Dudel, 1962; Potter, 1968). A role for GABA as a presynaptic inhibitory transmitter was first proposed when its topical appplication was shown to depolarize spinal dorsal roots in the cat (Eccles et al., 1963). Some evidence now exists to support such a role for GABA in primary afferent depolarization (PAD) and in presynaptic inhibition, especially in the amphibian spinal cord (see, e. g., references in Davidoff et al., 1973; Davidoff and Adair, 1974; Barker et al., 1975a,b; see also for review Davidson, 1976). However, a role for GABA as the mediator of this process remains uncertain (see, e.g., Curtis et al., 1971b; Krnjevii, 1974; McLennan, 1976). Both BIC and PIC block PAD, long-duration inhibitions, and the effects of GABA on primary afferent terminals (e.g., Curtis et al., 1971b; Davidson and Southwick, 1971; Barker and Nicoll, 1972; Davidoff, 1972a,b; Levy and Anderson, 1972; Barker et al., 1975a). Also, experimentally produced decreases in spinal GABA content decrease PAD (Banna and Jabbur, 1971; Bell and Anderson, 1972; Miyata and Otsuka, 1972), whereas inhibition of GABA-a-oxoglutarate transaminase (the enzyme involved in GABA catabolism) increases PAD and facilitates presynaptic inhibition (Davidoff et al., 1973). However, recent studies have indicated that at least three different types of neutral amino acid receptors exist on primary afferent terminals in frog spinal cord; a “GABA-like” receptor, a “taurinelp-alanine” receptor, and a “glycinelike” receptor (Barker et al., 1975a,b). Barker et al. (1975a) showed that PIC and BIC both blocked depolarizing responses elicited by GABA, P-alanine, and taurine with equal specificity, whereas glycine responses were unaffected. Also, strychnine blocked p-alanine and taurine responses, whereas it had no effect on those produced by glycine (or
AMINO ACID RECEPTORS
143
GABA). This lack of specificity of PIC, BIC, and strychnine was shown by their antagonism, to varying degrees, of the actions of many of the neutral amino acids tested. It was perhaps best demonstrated with imidazoleacetic acid, which was the most potent depolarizing agent tested and whose action was effectively blocked by both PIC and strychnine. 6. Barbiturates. Besides having postsynaptic actions (see Section II,A,6,a), barbiturates exert presynaptic actions in the vertebrate CNS (e.g., Ldyning et al., 1964; Weakly, 1969; Nicoll, 1975b). Recent studies on isolated frog spinal cord have revealed that pentobarbital depolarized primary afferents and greatly prolonged presynaptic inhibition (Nicoll, 1975b).Since this depolarization was reversibly blocked by PIC and BIC, but not by glycine antagonists or M$+, it appeared to involve an activation of GABA receptors (possibly by a direct action of pentobarbital). These findings confirmed previous observations that indicated that presynaptic inhibition can be prolonged by anesthetics (Eccles and Malcolm, 1946; Eccles et al., 1963; Schmidt, 1963; Miyahara et al., 1966; Banna and Jabbur, 1969)and supported a role for GABA in presynaptic inhibition. c. Benzodiazepines. Recent studies have led to the notions that benzodiazepines might exert their sedative-hypnotic effects by a facilitatory action on GABA-mediated presynaptic inhibition (Costa et al., 1975b), and that their muscle relaxant effects might involve glycine-operated synapses (Snyder, 1975). In support of the liaison with GABA, very low doses of benzodiazepines facilitate PAD in the spinal cord (e.g., Schmidt, 1963; Schmidt et al., 1967), and GABA could be involved in presynaptic inhibition (see above). Also, enhanced presynaptic inhibition can be reduced by both PIC (Stratten and Barnes, 1971) and BIC (Polc et al., 1974). However, the recently demonstrated GABA-antagonizingactions of benzodiazepines should be considered before accepting this view (see Section II,A,6,6 and Section VI1,C). d . y-Aminobutyrylcholine (GABA-Ch). Bowery and Brown ( 1972a) showed that GABA-Ch exerted little “acetylcholine-like” activity, but strong “GABA-like”activity on sympathetic ganglia. GABA-Ch also depolarized primary afferents of frog spinal cord (Nicoll, 1975c) by an action which, unlike those of acetylcholine (ACh) and carbachol, was not blocked by the presence of Mg2+or tetrodotoxin. Experiments with ACh and amino acid antagonists revealed that GABA-Ch produced depolarization by activating GABA receptors. Both PIC and BIC antagonized the action of GABA-Ch, whereas strychnine (at a concentration that blocked p-alanine responses) was ineffective. Although this “GABA-like”action of GABA-Ch indicated that it might interact with GABA receptors, its low potency (about 1/20th that of GABA) on primary afferents makes it
144
F. V. DEFEUDIS
an unlikely candidate for mediation of BIC-sensitive PAD (see also Davidoff, 1972a,b; Barker and Nicoll, 1972). e. Lioresal (Baclofen). Lioresal [P-(p-chloropheny1)-GABA], an antispastic agent in man (see references in Davidoff and Sears, 1974), is structurally related to both GABA and phenylethylamine. Lioresal might activate presynaptic inhibition (Burke et al., 1971; Knutsson et al., 1973). However, its iontophoretic application to central neurons produced a depression that was resistant to both BIC and strychnine (Curtis et al., 1974; Davies and Watkins, 1974), and, unlike GABA, Lioresal hyperpolarized (rather than depolarized) primary afferent terminals in frog spinal cord (Davidoff and Sears, 1974). These findings indicated that this agent does not act by enhancing the action of GABA during presynaptic inhibition. Lioresal could act on aminergic, rather than on “GABA-ergic,”receptors (Curtis et al., 1974; Davies and Watkins, 1974). However, recent results have indicated that Lioresal can exert a quite potent direct postsynaptic inhibitory action on feline spinal motoneurons (Puil et al., 1976).
B. GLYCINE In their pioneering studies, Curtis and Watkins (1960a,b) found iontophoretically applied glycine to be less potent at depressing feline spinal neurons than GABA, P-alanine, and taurine. However, further studies (Curtis et al., 1968a,b; Werman et al., 1968) have revealed that glycine, in low concentrations, is at least as potent a spinal depressant as GABA and that it produces a rapid, reversible, hyperpolarization of motoneurons that has a reversal potential similar to that of the neurally evoked IPSP. These, and other findings (see, e.g., for review Krnjevii, 1974; Curtis and Johnston, 1974a; DeFeudis, 1975a) have made glycine a leading candidate for a role as postsynaptic inhibitory transmitter in the spinal cord and brain stem. The potency of the neuronal depressant action of glycine appears to decrease as the neuraxis is ascended, a change which correlates roughly with changes in its endogenous tissue concentration. Glycine may not be an inhibitory transmitter in the cerebral cortex (Kelly and Krnjevit, 1969). Iontophoretically applied strychnine reversibly blocked the depressant effects of glycine and b-alanine (but not those of GABA) as well as evoked IPSPs and the hyperpolarizing action of glycine on spinal motoneurons, findings which indicated that strychnine acted similarly at receptors for glycine and at those for the inhibitory transmitter (e.g., Curtis et al., 1968a, 1969; Engberg and Thaller, 1970; Felpel, 1972). In the medullary reticular formation, strychnine blocked both the de-
AMINO ACID RECEPTORS
145
pressant action of glycine and evoked inhibition, but not the depressant action of GABA (Tebecis and DiMaria, 1972; TebEcis and Ishikawa, 1973). By using both strychnine and PIC, Galindo (1969) was able to separate GABA- from glycine-mediated depressions of postsynaptic activity in cuneate neurons. More recently, GABA- and glycine-mediated depressions of neurons of the rat substantia nigra were separated by using BIC-methochloride and strychnine (Dray et al., 1976). Other studies by Curtis et al. (1976a), using strychnine, BIC, and tetanus toxin, have revealed that both glycine and GABA appear to be inhibitory transmitters released on Renshaw cells and that glycine appears to be the inhibitory transmitter released by Renshaw cells of the feline spinal cord (see also Belcher et al., 1976). In other studies on brain stem neurons of unanesthetized decerebrate cats, Haas and Hosli (1973) found that though glycine, taurine, and GABA all caused depressant effects (see also Denavit-Saubie and Champagnat, 1975), iontophoretically administered strychnine reversibly reduced the depressant actions of glycine and taurine, but not that of GABA; BIC did not affect the actions of glycine and taurine. These results indicated that taurine receptors might be similar to glycine receptors in the brain stem. Iontophoretic dose-response studies are in accord with the notion that strychnine competitively antagonizes glycine-induced neuronal depressions (Johnson et al., 1970; Curtis et al., 197 la). Tests by Curtis et al. (1968a) have revealed a series of “glycine-like” amino acids (e.g., L-aalanine; L- and D-serine; various isomers of cystathionine; p-alanine; P-aminoisobutyric acid; taurine), whose depressant effects on spinal neurons are antagonized by strychnine but are relatively insensitive to BIC. However, strychnine should not be considered to be a specific agent, though it may be the best amino acid antagonist presently available. Some actions of strychnine which indicate its lack of specificity include: its anti-acetylcholinesterase activity (Alid et al., 1974); its suppression of carbachol uptake by brain slices (Creese and Taylor, 1967); its direct effects on axonal or other membranes (e.g., see references in Pollen and Ajmone Marsan, 1965; Freeman, 1973); its antagonism of 5-HTinduced depression of some neurons of the cerebral cortex (Stone, 1973a); its lack of effect on the depolarizing response elicited by glycine on primary afferents of frog spinal cord (Barker el al., 1975a); and its effect of decreasing Na+-dependent GABA binding to synaptosomal fractions (DeFeudis et al., 1976a). T h e above-mentioned iontophoretic studies led Young and Snyder (1973) to study the glycine receptor using rH]-strychnine (see Section
146
F. V. DEFEUDIS
IV,B, 1). However, recent iontophoretic experiments by Curtis et al. (1976b) have failed to provide direct support for the proposal that benzodiazepines interact with spinal glycine receptors, which was based on such studies of [3H]-strychnine binding (Young et al., 1974a; Snyder, 1975). The main findings were: (1) Diazepam (minimum dose of 3.0 mg/kg; i.v.) did not diminish the reduction by strychnine of the inhibitory action of iontophoretic glycine on feline dorsal horn neurons. (2) In mice, diazepam (2.5 mg/kg; i.p.) did not appreciably affect the potency of strychnine as a convulsant, whereas it did provide some protection against BIC. (3) In cats, iontophoretically administered chlordiazepoxide failed to either inhibit the firing of spinal interneurons o r to reduce the antagonism between strychnine and glycine. Several indole alkaloids and their derivatives, as well as morphine and structurally related alkaloids (e.g., thebaine, codeine) can also antagonize the effects of glycine, but not those of GABA, on mammalian spinal neurons (Curtis and Johnston, 1974b; Johnston, 1977). Other compounds that appear to selectively antagonize the depressant action of glycine, with respect to that of GABA, have been discussed by Johnston (1977). Recent in vitro studies on cerebellar spontaneous action potentials have indicated that glycine and other inhibitory amino acids (GABA, taurine, and P-alanine) caused inhibition by increasing C1- (and perhaps K+) permeability, and that glycine, taurine, and p-alanine also interacted with strychnine-sensitive receptors (Okamoto et al., 1976; see also Section IV,A,6). Other recent studies revealed that strychnine could block hyperpolarizing responses elicited by glycine, p-alanine, and taurine, but not those induced by GABA on motoneurons of isolated frog spinal cord (Nicoll et al., 1976). However, strychnine did not affect depolarizing components of glycine and p-alanine responses. PIC and BIC did not affect glycine responses but did antagonize both GABA- and p-alanineinduced hyperpolarizations. Although large amounts of amino acids (up to M, except for glycine) were required to elicit these actions, such results revealed that frog motoneurons possess receptors for both hyperpolarizing and depolarizing actions of neutral amino acids. For depolarizing responses, the order of potency was: glycine > 8-alanine = taurine > GABA. Although depolarizing components always required higher concentrations than those producing hyperpolarizing components for GABA, p-alanine, and taurine, the glycine threshold concentration for depolarization ( M) was often the same as that required for hyperpolarization. The resistance of all depolarizations to the actions of both strychnine and PIC indicated that all four amino acids de-
AMINO ACID RECEPTORS
147
polarized motoneurons through entirely different receptor mechanisms from those associated with their hyperpolarizations. C. TAURINE Iontophoretically administered taurine produces neuronal depression in most vertebrate CNS areas tested, being especially potent on neurons of the spinal cord and lower brain stem (e.g., Curtis and Watkins, 1960a, 1965; Krnjevit, 1964, 1974; Denavit-Saubie and Champagnat, 1975; Krnjevik and Puil, 1976). Like p-alanine, taurine has been classified as a “glycine-like” amino acid (Curtis et al., 1968a, 1971a), and this has been supported by studies on spinal and medullary neurons which revealed that its depressant action was largely abolished by strychnine, but hardly affected by BIC (Curtis et al., 1971a; Haas and Hosli, 1973). In the cerebral and cerebellar cortices, taurine is a much weaker depressant than GABA, but perhaps stronger than glycine (Krnjevit and Phillis, 1963; Crawford and Curtis, 1964; Krnjevik and Puil, 1976). Like P-alanine and GABA, taurine can depolarize feline sympathetic ganglia (Bowery and Brown, 1974) and amphibian dorsal root fibers (e.g., Nicoll and Barker, 1973; Nishi et al., 1974). This latter action of taurine can be blocked by both strychnine and PIC (Barker et al., 1975a; see also Section 11, A, 7, a). Studies by Barker et al. 1975a,b) and Sonnhof et al. (1975) on isolated frog spinal cord have indicated that taurine could be a transmitter involved in the dorsal root potential elicited by antidromic stimulation of the ventral root, and in postsynaptic segmental inhibition. Taurine appears to act by increasing membrane C1- permeability (Hosli et al., 1973b), as do GABA and glycine. D. GLUTAMATE A N D ASPARTATE
Upon iontophoretic application, the L-isomers of glutamate and aspartate appear to be only slightly more potent excitants of feline spinal neurons than their D-isomers, whereas the D-isomers of N-methylaspartate and homocysteate are much more potent excitants than the corresponding L-isomers (Curtis and Watkins, 1963; Krnjevik, 1974). T h e excitatory action of L-aspartate is similar to, but often weaker than, that of L-glutamate (e.g., Curtis et al., 1960a; Krnjevik and Phillis, 1963). Some spinal interneurons are more sensitive to glutamate than to aspartate (Duggan, 1974; Johnston et al., 1974). Note that such differences in activity may be related to differences in both receptor interaction and inactivation (“re-uptake”) of these substances. Since intracellularly injected glutamate does not produce excitation (Coombs et al.,
148
F. V. DEFEUDIS
1955), the actions of glutamate (and aspartate) are likely mediated by their interactions with surface membrane receptors. However, as glutamate and aspartate excite almost all mammalian CNS neurons, their receptors could be nonspecific constituents of all neuronal membranes (Roberts and Hammerschlag, 1972). Using intracellular methods, Curtis et ad. (1960a) showed that a clear depolarizing effect accompanies the increase in membrane excitability caused by glutamate. Intracellular studies on cortical neurones (Krnjevit and Schwartz, 1967) revealed that this effect was associated with a marked fall in membrane resistance. Zieglgansberger and Puil (1973) found the reversal level for the action of glutamate to be about -30 to 0 mV, which indicated a simultaneous increase in membrane permeability to both Naf and K+. However, the depolarizing actions of acidic amino acids do not appear to be due to Na+ movements that are associated with the action potential since DL-homocysteate-induced depolarizations were not antagonized by tetrodotoxin (Curtis et al., 1972a). Kainate, domoic acid, quisqualic acid, P-N-oxalyl-L-a,P-diaminopropionic acid, N-methyl-D-aspartate, and ibotenic acid exert potent excitatory actions on vertebrate central neurons (Watkins et al., 1966; Johnston et al., 1968, 1974; Shinozaki and Konishi, 1970; McCulloch et al., 1974; Biscoe et al., 1975).Some of these agonists, which can be regarded as conformationally restricted analogues of glutamate and aspartate, have been useful in separating their receptors. Studies with kainate, ibotenic acid, and synthetic “cycloglutamate” (cis-laminocyclohexane- 1,3-dicarboxylic acid) have indicated that at least two types of receptor exist for excitant amino acids on feline spinal interneurons (Johnston et al., 1974). Glutamate might interact with “glutamate-preferring” receptors in partially extended conformations and with “aspartate-preferring” receptors in partially folded conformations, though aspartate might interact only poorly with “glutamate-preferring” receptors due to its shorter carbon chain. Regarding glutamate/aspartate antagonists, no available substance has been very useful for distinguishing between the excitations induced by glutamate and aspartate, but some separation of excitation induced by these amino acids and other excitants (e.g., ACh) has been achieved. Lysergic acid diethylamide (LSD) antagonized glutamate-induced excitation of brain stem neurons (Boakes et aA, 1970; Bramwell and Gonye, 1973), but not DL-homocysteate-induced excitation (Boakes et al., 1970). The glutamate analogues, glutamate diethyl ester (GDEE), and a-methyl-DL-glutamate appeared to exert more effective antagonisms of glutamate- than of aspartate-induced excitation on thalamic neurons (McLennan et al., 1971; Haldeman et al., 1972a), but this separation was
A M I N O ACID RECEPTORS
149
not evident on spinal neurons (Curtis et al., 1972a). GDEE can reversibly antagonize iontophoretically applied glutamate, aspartate, and DLhomocysteate actions, and reversibly blocks synaptic activations of cat thalamic relay neurons, cuneate neurons and spinal interneurons, while not affecting excitatory responses of these neurons to ACh (Haldeman and McLennan, 1972; Haldeman et al., 1972a,b; McLennan, 1974, 1975). Iontophoretic application of GDEE (50-125 nA) in the vicinity of rat striatal neurons excited by cortical stimulation almost totally suppressed excitation in 90% of cells, by a fully reversible action, and also suppressed neuronal excitation elicited by iontophoretically applied aspartate, glutamate, and DL-homocysteate (Spencer, 1976). GDEE reversibly antagonized excitations produced by glutamate and aspartate, but not those produced by ACh when applied iontophoretically to rat CA1 hippocampal neurons in methoxyfluorane-anesthetized cats and to CA 1 neurons in in vitro slice preparations, but did not appear to differentiate glutamate from aspartate responses (Spencer et al., 1976). l-Hydroxy-3-aminopyrrolid-2-one(HA-966) does not distinguish between glutamate- and aspartate-induced excitations of spinal neurons (Curtis et al., 197313). However, upon iontophoretic application, HA-966 did reduce glutamate- or aspartate-induced excitation of cells of the feline cortex while affecting ACh-induced excitation to a lesser extent (Davies and Watkins, 1972). This differential potency of HA-966 on amino acid- and ACh-induced excitation of cortical neurons was greater than that of GDEE and L-methionine-DL-sulfoximine.Also, Stone (1973b) showed that rat cerebral cortical neurons activated by stimulation of the pyramidal tract appeared to be particularly sensitive to iontophoretically applied L-glutamate; synaptic excitation of neurons by pyramidal tract stimulation could be blocked by GDEE, but only 5 out of 20 cells were blocked. In a more comprehensive study, Stone (1976) examined excitation of cells in the cuneate nucleus of the rat caused by pyramidal tract stimulation and used HA-966 as well as GDEE. Both agents (iontophoretically applied) reduced glutamate excitation of neurons; HA-966 was effective on more cells and was more potent and did not affect excitatory responses to ACh. Curtis et al. (1972a) have shown that 9-methoxyaporphine antagonizes acidic amino acid-induced excitations of cholinoceptive neurons in the spinal cord, ventrobasal thalamus, and pyramidal tract without affecting ACh-induced excitation. A blocking action of L-nuciferine ( 1,2-dimethyloxyaporphine)has also been described (Duggan et al., 1973). These analogues may all act as neuronal depressants in the spinal cord (e.g., Zieglgansberger and Puil, 1973), and although GDEE appeared to preferentially and reversibly antagonize glutamate-induced
150
F. V. DEFEUDIS
responses (see above references), this agent is neither highly specific nor potent, at least in striatum o r cortex (Spencer, 1976). L-Methionine-DLsulfoximine may also act in a similar nonspecific manner on the actions of glutamate and aspartate (Curtis et al., 1972a). Also, it is not yet possible to specify a single amino acid, since no antagonist examined thus far, including HA-966, distinguishes between glutamate- and aspartate-induced excitations (Davies and Watkins, 1972, 1973; Curtis et al., 197313). T h e actions of glutamate antagonists on the vertebrate CNS have recently been reviewed (McLennan, 1975; Usherwood, 1976).
111. Physiologic-Pharrnacologic Studies in Invertebrates
A. GABA Invertebrate studies actually initiated the search for the GABA receptor. Florey (1954) described an inhibition of impulse generation in the crayfish stretch receptor neuron by extracts of mammalian CNS and termed the responsible agent(s) “Factor I.” Bazemore et al. (1957) then isolated GABA from a beef brain extract by using a method for assaying “Factor I.” It was considered that extracts containing “Factor I” might have produced inhibition by means of their contents of GABA. Other studies have revealed that the effects of both GABA and synaptic inhibition were reversibly blocked by picrotoxin (PIC; 10-6M) in several crustacean preparations (Elliott and Florey, 1956; Florey, 1957; Robbins and Van der Kloot, 1958; Grundfest et al., 1959; Kuffler, 1960; Takeuchi and Takeuchi, 1969; Epstein and Grundfest, 1970; Earl and Large, 1972). Now it is believed that such inhibitory synapses are operated by GABA (see, e.g., for review Gerschenfeld, 1973; Takeuchi, 1976). PIC also antagonized synaptic inhibition and the inhibitory effects of GABA in certain insects (e.g., Usherwood and Grundfest, 1965). Takeuchi and Takeuchi (1969) analyzed the effects of PIC on the crayfish (Cambarus clarkii) neuromuscular junction (NMJ) and showed that its antagonism of GABA was noncompetitive; one molecule of PIC was required to decrease the conductance increase caused by two molecules of GABA. This action of PIC could be caused by its irreversible binding to the GABA receptor or by its interference with the GABA ionophore (Earl and Large, 1974). The action of bicuculline (BIC) on this same inhibitory NMJ was also shown to be noncompetitive, BIC being about 100 times less effective than PIC (Takeuchi and Onodera, 1972). However, McLennan (1970b) had earlier found that BIC (1-3 x lod4M) suppressed the inhibitory actions of both GABA
A M I N O ACID RECEPTORS
15 1
and impulses on inhibitory nerve fibers of the crayfish (Eustacus amatus) stretch receptor and that it was more potent than PIC. Most recently, Dude1 and Hatt (1976) have shown, with respect to desensitization and the effect of a specific GABA antagonist (P-guanidinopropionic acid), that four types of GABA receptors exist in crayfish (Astacusftuviatilis) leg muscles. M) was ineffective at the NMJ of the hermit crab BIC (5 X Eupagarus bernhardus (Earl and Large, 1972). Walker et al. (1971) found that BIC (0.7-2.7 X lop6M) reversibly blocked the effects of GABA in exciting and depolarizing the membrane potentials of certain snail (Helix aspersa) neurons and the effect of GABA in inhibiting and hyperpolarizing the membrane potentials of neurons of the isolated segmental ganglia of cockroach (Periplaneta americana). However, BIC also exerted blocking effects on the excitatory action of acetylcholine on some snail neurons and on the excitatory action of carbachol on some cockroach neurons. PIC was about ten times more potent than BIC as a GABA antagonist in the snail preparation, but about five times less potent in the cockroach (Walker et al., 197 1). d-Tubocurarine did not antagonize the inhibitory effect of GABA at the crab NMJ (Earl and Large, 1973a), whereas tetramethylenedisulfotetramine did show some antagonism (Large, 1975). Like BIC, benzylpenicillin did not affect GABA action at the crab NMJ (Earl and Large, 1973b). Cholinoceptive muscle fibers of isolated tube feet of several species of sea urchin were found to contract in response to GABA, perhaps by an excitatory action of GABA on cholinergic motoneurons, and this effect was blocked by BIC or PIC (Florey et al., 1975). Hence, GABA receptors differ markedly among various invertebrates and they are relatively insensitive to BIC.
B. GLYCINE Glycine, like GABA and glutamate, appears to interact with receptors on molluscan neurons (but see Takeuchi et al., 1976). Indirect evidence indicating an action of glycine in invertebrates involves the depolarizing effects on lobster (Homarus americanus) giant axon produced by strychnine (Freeman, 1973). However, strychnine does not appear to exert specific postsynaptic actions at several peripheral inhibitory junctions in crustacea (see review by Curtis and Johnston, 1974b). C. TAURINE Taurine exerts a “GABA-like” action on a variety of crustacean preparations (Edwards and Kuffler, 1959; McGeer et al., 1961; Koidl
152
F. V. DEFEUDIS
and Florey, 1975). Taurine also produced a C1--mediated reversible increase in membrane conductance of single muscle fibers of lobster, a change that was only partially blocked by PIC (Nistri and Constanti, 1976). Although taurine (8 x M) exerted no effect on the electrical activity of an identified neurone of the subesophageal ganglion of the African giant snail (Achatinafulica Firussac), inhibitory effects were produced by three sulfur-containing amino acids present in the urines of homocystinuric and cystathioninuric patients (Takeuchi et al., 1974). D. GLUTAMATE AND ASPARTATE L-Glutamate is a good excitatory transmitter candidate at neuromuscular junctions (NMJ) in crustacea and other arthropods (e.g., Robbins, 1959; Takeuchi and Takeuchi, 1964; Usherwood and Machilli, 1968; see also reviews by Florey, 1967; Gerschenfeld, 1973; Kehoe and Marder, 1976). Recent observations on lobster preparations (e.g., Kravitz et al., 1970; Shank and Freeman, 1975) have indicated that L-aspartate might also function in this process. Takeuchi and Takeuchi ( 1964) showed that iontophoretically applied glutamate depolarized the crayfish NMJ, and that L-glutamate receptors were stereospecific. Both stimulation of excitatory nerves and glutamate application depolarized the muscle membrane toward the same equilibrium potential, estimated to be at 8-10 mV (Ber6nek and Miller, 1968). Also, the same reversal potential for the neural excitatory transmitter and for L-glutamate has been observed at the crayfish NMJ (Taraskevich, 1971; Takeuchi and Onodera, 1973; Dudel, 1974; Onodera and Takeuchi, 1975). At the crayfish NMJ, glutamate depolarization and the excitatory junction potential appear to depend mainly on Na+ and slightly on Ca2+(Takeuchi and Onodera, 1973; Onodera and Takeuchi, 1976). Experiments by Kerkut et al. (1965) and Kravitz et al. (1970) have revealed a stimulus-induced release of glutamate, and more recently, Traubatch et al. (1973) observed that end-plate potentials were abolished by glutamate, indicating a desensitization of glutamate receptors. Insect striated muscle also appears to have excitatory innervation(s) operated by glutamate (e.g., Usherwood, 1972). Glutamate action appears to involve at least two distinct types of receptor in insect muscle (e.g., Cull-Candy and Usherwood, 1973; Lea and Usherwood, 1973a,b; Clements and May, 1974; Usherwood and Cull-Candy, 1974; Kerkut et al., 1975), in striated muscle of the crayfish vas deferens (Florey and Murdock, 1974), and in the crayfish NMJ (e.g., Shinozaki and Shibuya, 1974a). A glutamate-induced change in C1permeability in crayfish vas deferens appears to be mediated by extra-
AMINO ACID RECEPTORS
153
junctional glutamate receptors that can be blocked by PIC but that are insensitive to GABA (Florey and Murdock, 1974; see also review by Kehoe and Marder, 1976). In insect striated muscle, Cull-Candy and Usherwood (1973;Usherwood and Cull-Candy, 1974)showed that iontophoretically applied glutamate mimicked the depolarizing action of the transmitter (presumably glutamate) at the junctional membrane, but that its application to the extrajunctional membrane induced either a biphasic (depolarizing, followed by hyperpolarizing) o r a purely hyperpolarizing response, the latter being caused by an increase in membrane C1- permeability. Cull-Candy (1976)applied L-glutamate and the glutamate analogue DL-ibotenate iontophoretically to locust muscle membrane and was able to distinguish t w o pharmacologically distinct populations of extrajunctional receptors. This dissection was possible since an “H-receptor” was sensitive to glutamate and ibotenate, whereas another receptor (“D-receptor”) was sensitive to glutamate but insensitive to ibotenate. This finding provided an explanation for the biphasic response which is caused by glutamate (see above), i.e., via simultaneous activation of both types of receptor. As the D-response to glutamate remained after the H-component of the biphasic response had been desensitized by ibotenate, it was concluded that the depolarizing and hyperpolarizing phases of the extrajunctional response resulted from activation of two types of receptors (see also Cull-Candy and Usherwood, 1973). Interestingly, more than one type of glutamate receptor also appear to exist on central mammalian neurons (Johnston et al., 1974). Cull-Candy et al. (1976)have also reported that iontophoretic application of the glutamate analogue, DL-2-amino-4-phosphonobutyric acid (DL-APB), antagonized the excitatory action of glutamate (also iontophoretically applied) on junctional receptors of the locust muscle membrane. Dose-response relationships revealed that a competitive antagonism might exist between DL-APBand glutamate. With further regard to different types of glutamate receptors, Shinozaki and Shibuya (1974a)showed that L-glutamate-evoked depolarization of crayfish muscle was enhanced by the presence of kainate, whereas kainate did not affect extrajunctional potentials, which presumably involve synaptically released glutamate. Also, when glutamate was applied to the junctional membrane, kainate did not enhance the response, but kainate did increase both the amplitude and duration of the response when glutamate was applied over a wider area that included nonjunctional membrane (Takeuchi and Onodera, 1975).Thus, glutamate could interact with a kainate-insensitive extrajunctional receptor, which differs from the junctional receptor. Quisqualic acid is several hundred times more potent than glutamate at the crayfish NMJ
154
F. V. DEFEUDIS
(Shinozaki and Shibuya, 197413). Although quisqualic acid causes effects similar to those of glutamate on the crayfish opener muscle, its action was depressed, whereas that of glutamate was facilitated by domoate or kainate, indicating a difference between the pharmacological properties of glutamate and quisqualic acid (Shinozaki and Ishida, 1976). Domoate and kainate receptors also appear to differ from that of quisqualate. Gerschenfeld and Lasansky ( 1964) demonstrated both excitatory and inhibitory effects of glutamate, and Szczepaniak and Cottrell (1973) reported biphasic response to glutamate in molluscan neurons. Other studies by Oomura et al. (1974) have indicated that glutamate-induced hyperpolarization of mulluscan neurons occurs by a mechanism that involves positive cooperativity (i.e., more than one molecule of glutamate interacts with its receptor). Regarding possible glutamate antagonists, L-glutamate diethyl ester, a-methyl-DL-glutamate, L-methionine-DLsulfoximine, and 1-hydroxy-3-aminopyrrolid-2-one did not affect molluscan glutamate receptors, whereas a-aminopimelic acid (a weak excitant of feline spinal neurones; Curtis and Watkins, 1960a) antagonized glutamate-induced inhibition, but not excitation (Kerkut et al., 1975). Most recently, Takeuchi et al. (1976) showed that both L- and D-glutamate, as well as L-aspartate, glycine, and /3-alanine, did not exert any effects on two types of neurons of the subesophageal ganglion of an African giant snail (Achatina fulica Firussac), but that bath-applied /3-hydroxyglutamate (BHG) remarkably inhibited one type of neuron (periodically oscillating neuron). Among the BHG stereoisomers, erythro-L-BHG had the strongest effect. Earlier studies with mammals had revealed that BHG possessed convulsive properties when topically applied to motor cortex (Hayashi, 1959) and that it caused excitation of spinal neurons when given by iontophoresis (Curtis and Watkins, 1960a).
IV. Biochemical Studies with Vertebrate Preparations
A. GABA Several types of in vitro preparations have been used to study the properties of binding sites associated with the GABA uptake and GABA receptor systems of vertebrates. 1. Introduction to “Binding” Studies A major aim of these studies is the separation of GABA “binding” into components related to uptake (inactivation) and receptor interac-
AMINO ACID RECEPTORS
155
tion. Undoubtedly, there exist many types of binding sites for amino acids in CNS structures. Even after the tissue has been fragmented, some of these binding sites may be identical to, o r similar to those which exist in viuo (e.g., uptake sites, postsynaptic receptor sites, enzyme sites), T h e “binding” of GABA and other “active” amino acids to subcellular particles of the CNS appear to occur by energy-independent, nonenzymatic, physicochemical processes (e.g., Sano and Roberts, 1963; Elliott et al., 1965; DeFeudis, 1973a,b). In their classic work, Sano and Roberts (1963) showed that the “binding” of GABA to particulate fractions of mouse brain was Na+-dependent and had a pH optimum of about 7.4. Unlabeled glycine ( lop3M), added simultaneously with labeled GABA M), did not affect GABA “binding,” but preincubation of the particles with p-hydroxymercuribenzoate (5 x M ) did prevent this “binding.” Further studies on this Na+-dependent “binding” process indicated that a “carrier-mediated” mechanism might be involved in the transfer and accumulation of GABA by subcellular particles (e.g., Varon et al., 1965; Weinstein et al., 1965; Kuriyama et al., 1968a,b), and thus that GABA might be inactivated by a “re-uptake” mechanism. A recent study on mouse brain revealed that changes in neither the constitution of particulate fractions nor in their rates of sedimentation could account for the increase in GABA “binding” produced by Na+ (DeFeudis, 1973b). T h e lower Na+ requirement (about 40 mEq/liter) for maximal GABA “binding” found in some studies (e.g., Elliott et al., 1965; DeFeudis, 1973b), as compared with those of Roberts and co-workers (about 130-200 mEq/liter; see above references), is presumably due to the use of Tris buffer by the latter workers, since Tris interferes with Na+-dependent mechanisms (Bittar et al., 1972). Recent reviews (DeFeudis, 1975a; Martin, 1976) have indicated that it will be difficult to show that any amino acid is a transmitter substance using the Na+ dependency of its energy-mediated uptake as the criterion. Studies of Na+-dependent and Na+-independent “binding” processes may provide better clues about the identity of amino acids as CNS transmitters. Kuriyama et al. (1968b) showed that some GABA “binding” occurred to a mouse brain synaptic vesicle fraction in the presence of Na+. Rassin (1972) found that substantial amounts of GABA, taurine, aspartate, glutamate, and glycine were bound to synaptic vesicle fractions of guinea pig cortex, but as these vesicular amino acids were not released by hypoosmotic conditions, it was concluded that “transmitter” amino acids may not be stored in synaptic vesicles. In other studies, with synaptic vesicles of rat cerebral cortex, taurine and glutamate were bound in most significant amounts of the amino acids measured, and these vesicular pools were also not sensitive to osmotic shock (DeBelleroche and Brad-
156
F. V. DEFEUDIS
ford, 1973). More recently, Philippu and Matthaei (1975) have shown that pig caudate nucleus possesses GABA-containing synaptic vesicles that accumulated [l4C1-GABA by an ATP-M$+-dependent process which was abolished by osmotic lysis, followed by sonication. Mangan and Whittaker (1966), using guinea pig brain, found that amino acids, including GABA and glutamate, were not more highly localized to synaptosomes than K+ or lactate dehydrogenase (markers for occluded cytoplasm). A criticism which might be applied to all of these studies with subcellular particles is that “gliasomes,” which may be present in subcellular preparations, may accumulate GABA and other amino acids. Although Whittaker (1965) suggested that most glial elements would sediment in crude nuclear fractions with media of low ionic strength, no conclusive data were given for this, and even if this is the case, studies performed with crude homogenates (e.g., those of Elliottet al., 1965) would be heavily subject to criticism based on glial contamination. If glia are involved in the “clumping” effect of Na+, then, since GABA “binding” followed “clumping” (e.g., DeFeudis, 1972a, 1973b), glia could well be a major GABA-binding species (see also Section VI). A recent study concerning glial contamination of synaptosomal fractions has been published (Henn et al., 1976). Until about 1971, Na+-dependent GABA “binding” was considered to be a component of the GABA uptake (active transport) system. No studies had yet focused on receptor binding, but drugs which affect active transport processes (e.g., chlorpromazine (CPZ), imipramine) had been shown to inhibit Na+-dependent GABA “binding” to cerebral subcellular particles (e.g., Sanb and Roberts, 1963; Weinstein et al., 1971). 2. Bicuculline (BIC)-Sensitive GABA ‘%inding” The initial studies on BIC-sensitive GABA binding were carried out with a synaptosomal fraction of rat cerebellar cortex using a millipore filtration method (Peck et al., 1973; see also Peck et al., 1976). In an attempt to differentiate postsynaptic binding of GABA from binding associated with Na+-dependent GABA uptake, synaptosomal fractions were preincubated in the presence of various concentrations of CPZ for 10 min at 4”C, followed by a 20-min incubation in the presence of CPZ and 2 x M [3H]-GABA, with or without a 1000-fold excess of unlabeled GABA. CPZ-insensitive (“specific”)uptake of r3H]-GABA by the particles (10%-55% of total uptake) was saturable and was inhibited by BIC but not by L-diaminobutyrate (an inhibitor of “high-affinity” GABA transport; Sutton and Simmonds, 1974), picrotoxin (PIC), penicillin, or glycine. To examine “specific” GABA binding to a synaptic membrane
157
AMINO ACID RECEPTORS
fraction of rat cerebellar cortex, Peck et al. (1976) used [3H]-GABA M) in 10 mM “HEPES” buffer at 4°C for 20 min. In the (5-200 x absence of added Na+, binding was saturable, with a KB = 2.3 X M, and was competitively inhibited by BIC, theK, for BIC being about 5.7 X lop5M. An attempt was made to solubilize the GABA binding site by treating synaptic plasma membranes with various concentrations of Triton X-100 (Peck et al., 1976). Using hydroxyapatite to absorb rH]GABA-receptor complexes, a solubilized GABA-binding component M and a B,, of 3.4 pmole was isolated which had a K B 2.3 X GABNpg protein; BIC also competitively inhibited this binding with a K , = 5.6 X M. T h e optimal p H for GABA “binding” to these Triton-solubilized particles was 7.1-7.3. T h e destructive actions of various degradative enzymes on this GABA-binding component indicated that it was either a lipoprotein or a protein dependent on lipid for its structural integrity and/or binding activity (Table 11; see also Sano and Roberts, 1963). In other studies, Zukin et al. (1974) examined r3H]-GABA (3.2 x low8 M) binding to a synaptic membrane fraction of rat brain incubated at 4°C for 5 min in 50 mM Tris-citric acid buffer. “Specific” GABA binding was obtained by subtracting from the total bound radioactivity the amount not displaced by high concentrations of BIC ( M) or GABA ( M). This crude synaptic membrane fraction possessed about ten times as much GABA binding activity as the “synaptosomalTABLE I1 EFFECTOF VARIOUS DEGRADATIVE ENZYMES ON THE SOLUBILIZED GABA RECEPTOR“ Enzyme pretreatment (Pgw None Trypsin (20) Trypsin (2) Trypsin (0.4) Bacterial protease (20) Bacterial protease (2) Phospholipase A (35) Phospholipase C (200)
Tryptic activity A440
GABA binding % control
0 0.733 0.116 0.024 0.334 0.037 0.0 18 0.027
100 47 59 110 36 97 54 14
a Triton-solubilized membrane components were exposed to varying concentrations of trypsin, bacterial protease, or phospholipase A or C for 20 min at 22°C. t3H]-GABA binding capacity was then measured by the HAP assay, as described by Peck et al. (1976), and maximal binding capacity expressed as percent of a control not exposed to degradative enzymes. The degradative enzymes were assayed for tryptic activity by the azoalbumin method of Tomarelli et al. (1949). Results are presented as the change in absorption at 440 nm per 20 min. Reproduced by permission from Peck et al. (1976).
158
F. V. DEFEUDIS
mitochondrial” (P2)fraction from which it was prepared. “Specific” PHI-GABA binding was saturable, with a K B = 1 x lo-’ M, whereas “nonspecific” GABA binding (that portion of [3H]-GABA not displaced M GABA or lob4 M BIC) was not saturable. Half-maximal by displacement of “specific” GABA binding was produced by about 5 X M BIC, and maximal displacement by about 1 X M BIC, i.e., BIG had about 1/50th the affinity of GABA for these sites. The potencies of various amino acids (e.g., 3-aminopropanesulfonic acid; imidazoleacetic acid) in inhibiting “specific” GABA binding paralleled their potencies as neuronal depressants, but differed markedly from their relative affinities for synaptosomal GABA uptake (Zukin et al., TABLE 111 SUBSTRATE SPECIFICITY OF THE GABA RECEPTOR^
Compound GABA 3-Aminopropanesulfonic acid Imidazoleacetic acid 1-Methylimidazoleacetic acid 3-Hydroxy GABA B- Alanine 2,4-Diaminobutyric acid p -Aminophenylmercuric acetate Chlorpromazine d-Tubocurarine Bicuculline Strychnine
SodiumSodiumdependent Synaptosomal independent GABA GABA GABA binding uptake binding
Percent GABA-like neurophysiologic activity
10
0.37
100
1,400
0.25 0.24
130-150 90- 100
1.2 160 100
35 540 11
21 860 130
-
> 1,000 > 1,000
> 1,000
100 55,000 260
1.o 80 > 1,000
0 50-70 30-50 5-10
2.6 12 7,500 > 1,000 100
> 1,000
0 0 0 0 0
160 38 4 100
a Inhibition of “specific”GABA binding by various tompounds was determined by the methods described by Zukin et al. (1974) and Enna and Snyder (1975). Na+-independent binding was determined using previously frozen membranes incubated in Na+-free buffer. Na+-dependent binding was studied using freshly prepared synaptic membranes incubated in the presence of 100 mM NaCI. For experiments on synaptosomal GABA uptake, an aIiquot of a nuclei-free sucrose hornogenate was incubated for 5 min at 25°C in Krebs-Ringer-Tris (pH 7.4) containing 50 nM pH]-GABA (Enna and Snyder, 1975). ID,, values were calculated by log-probit analysis. Data for imjdazole compounds are from Godfraind et al. (1973); other data are from Curtis and Watkins (1960a,b, 1965) and from Curtis et al. (1968a,b). Reproduced by permission from Young el al. (1976).
AMINO ACID RECEPTORS
159
1974; Enna and Snyder, 1975; see Table 111). Glycine, p-aminoisobutyric acid, p-alanine, and taurine had very little affinity for these GABA-binding sites. It was concluded that the postsynaptic GABA receptor is best shown in the absence of added Na+, using frozen and thawed synaptic membranes. Na+-dependent GABA binding was hardly affected by BIC, imidazoleacetic acid, and 3-aminopropanesulfonic acid, which exerted considerable effects on Na+-independent binding (Table 111). Regional binding assays revealed no close correlation with endogenous GABA content (but, see Section IV,A,4). Perhaps a better correlation would have been shown if BmaXvalues, rather than those obtained with a given concentration of 13H]-GABAhad been compared. Scatchard plots of GABA binding to synaptic membrane fractions of rat brain indicated single populations of binding sites for both Na+dependent and Na+-independent mechanisms (Enna and Snyder, 1975). The KB for Na+-dependent GABA binding was about 1.2 x lo-' M, whereas that for Na+-independent binding was about 4 X lO-'M; B,,, was about 30 pmole/mg protein for Na+-dependent binding and about 0.7 pmole/mg protein for Na+-independent binding. Hill plots were linear for both types of GABA binding, with coefficients of about 0.9, indicating neither positive nor negative cooperativity. T h e Na+independent process had a broad pH optimum with a maximum at about pH 7; the Na+-dependent process had a much sharper pH optimum with a maximum at about p H 7.7. This result, that agents inhibiting synaptosomal GABA uptake exerted parallel effects on Na+dependent GABA binding was, of course, expected, since Na+-d@endent GABA binding might represent the initial phase of Na+-dqbendent GABA uptake (see Section IV,A,l). T h e suggestion of Enna and Snyder (1975) that Na+-dependent GABA binding might be associated with glial rather than neuronal uptake sites is unwarranted in light of the short incubation periods that were used (see Section VI). Rate constants for the association and dissociation of Na+-independent GABA binding were so rapid, even at 4"C, that they could not be measured with the methods of Enna and Snyder (1975); however, for Na+-dependent GABA binding, association was complete after about 100 sec, half-maximal binding occurred at about 30 sec, and the rate of dissociation of bound r3H]GABA had a half-life of about 115 sec. Using millipore filtration techniques, Fiszer de Plazas and DeRobertis (1975) showed that the binding of [I4C]-GABAto a fraction enriched in nerve-ending membranes had a KB 3 x M and Bmax 0.4 pmole GABAJpg protein, and was inhibited by about 60% by CPZ (5 X M) M) in the presence of Na+. A and by about 40% by BIC (4 x
160
F. V. DEFEUDIS
hydrophobic protein fraction, separated from the total lipid extract of nerve-ending membranes, bound [I4C]-GABA,in the presence of Na+, 10.5 pmole GABNpg protein and an apparent K B = 3 x with a M. Binding curves for both the membrane fraction and the protein fraction indicated the presence of a single type of GABA-binding site. BIC competitively inhibited GABA binding to this protein, its& = 2.7 x 10“‘ M. Since CPZ and BIC together inhibited 100% of the total [14C]GABA binding, it was suggested that these drugs act through different mechanisms (see also Peck et al., 1973, 1976).Some separation of the GABA-binding sites from L-glutamate-binding sites was achieved. This GABA-binding species differed kinetically from that isolated from crustacean muscle by DeRobertis and Fiszer de Plazas (1974)(see Section V). The above-mentioned studies can lead one to believe that GABAreceptor binding is best shown in the absence of added Na+ (see also DeFeudis, 1975a),as the uptake of GABA is blocked under this condition. However, a BIC-sensitive component of GABA binding can also be demonstrated under more physiological conditions (Peck et al., 1973; DeFeudis et al., 1975; Fiszer de Plazas and DeRobertis, 1975). The interaction between GABA and BIC-methiodide has been studied at 0°C in a synaptosome-enriched fraction of rat cerebral cortex in an isosmotic medium containing physiological concentrations of Na+ and other ions (DeFeudis et al., 1975).BIC-methiodide ( 10-7-10-3M) significantly decreased the binding of r3H]-GABA (4.8X lo-*M) to the particles (see Fig. 4).At M, the drug decreased GABA binding by about 23%. The binding of r3H]-GABA(4.8X 1 O+M) to synaptosomal particles of six regions of feline brain in physiological medium was also inhibited by M); at M BIC-methiodide, GABA binding to BIC-methiodide ( particles of cerebral and cerebellar cortices and medulla was decreased, and at M it was decreased only to particles of cerebral cortex (de Sagarra et al., 1975).BIC-methiodide decreased GABA binding to particles of rat cerebral cortex (DeFeudis et al., 1975)more than to those of feline cerebral cortex (de Sagarra et al., 1975). Other studies conducted with a crude mitochondria1 fraction of mouse brain at 0°C revealed that [I4C]-GABA uptake (“binding”) was saturable and had an apparent K , E 2.8 X M (Olsen et al., 1975a). This process was strictly Na+-dependent in synaptosomal fractions and synaptosomal membranes from whole brain, cerebral cortex, and cerebellar cortex, as shown by using 100 mM NaCl in 10 mM Tris-HC1 buffer (pH 7.8)vs. Na+-free buffer. Chlorpromazine and imipramine M ) inhibited GABA uptake noncompetitively by as (both at 5 x much as 100%; PIC (3 x lo4 M)had no effect, whereas high concen-
Concentratratm of N-methyl-bicuculline (M) FIG. 4. Displacement of r3H1-GABA(4.8 X 10-aM) “binding” to synaptosome-enriched fractions of rat cerebral cortex by bicuculline methiodide. T o p and bottom plots indicate data of individual sets of experiments ( 1 1 or 12 samples per point); middle plot contains the combined data from both sets of experiments (22-24 samples per point). Mean values f S.E.M.; asterisks indicate that these values differed significantly ( p < 0.05 top < 0.001) from their respective control values which were obtained in the absence of the drug (Student’s t-test; two-tailed). (Reproduced with permission from DeFeudis et al., 1975.)
162
F. V. DEFEUDIS
trations (3 X M) of BIC-like compounds did inhibit the process. Giambalvo (1975) showed that GABA was bound with positive cooperativity (Hill coefficient of 2.17) to junctional complexes of rat cerebellum at 25°C; BIC-methiodide and PIC inhibited this binding, whereas d- tubocurarine and strychnine had no effect. Phospholipids competed with GABA for binding sites. The above studies revealed a wide range of apparent dissociation constants for GABA binding (about 8 x 10-8M to 3 x 10-5M), depending on the preparation and techniques used. Also, a rather wide range of BIC o r BIC-like substances were needed for half-maximal displacement of the BIC-sensitive component of bound GABA. T h e active concentrations of BIC-like agents at their site(s) of action have been estimated to be about 1-1.5 x lo-' M for vertebrate central neurons (Curtis and Johnston, 1974a; Johnston, 1977). Young and Snyder (1974a) found an apparent binding constant of strychnine to rat spinal synaptic membranes of 2.6-4 X 10-0 M (see Fig. 9). Since strychnine and BIC are of similar potency as convulsants, it might be expected that BIC would show an affinity for the GABA receptor which is similar to that of strychnine for the glycine receptor (Curtis et al., 1974). In any case, BIC perhaps does not act directly on GABA receptors. Like PIC, BIC might block ionophores (see Section 111, A). Furthermore, besides being poorly soluble in aqueous medium and hydrolyzed to its less active derivative (bicucine) under physiological conditions (see e.g., Olsen et al., 1976), several effects of BIC-like compounds have been noted that are not related to a.GABA-receptor action, i.e., its inhibition of acetylcholinesterase (Svenneby and Roberts, 1973; Olsen et al., 1976), its potentiation of acetylcholine action (Miller and McLennan, 1974), its direct effect on axonal (Freeman, 1973) or muscle membrane conductance (Shank et al., 1974). 3. Other Drugs with Possible Actions on the GABA Receptor; Substrate Specijicity of the GABA Receptor Several types of in vitro studies have been performed in an effort to elucidate the substrate specificity of postulated GABA receptors, e.g., those on the displacement of [3H]-GABA binding to membrane particles by GABA analogues and drugs, with emphasis on comparing Na+dependent and Na+-independent GABA binding (e.g., Zukin et al., 1974; Enna and Snyder, 1975; see also above); others using agents in the presence of physiological concentrations of Na+ (Peck et al., 1973, 1976; DeFeudis et al., 1975; Olsen, 1976); and others with purified membrane particles (e.g., Pecket al., 1976) or protein fractions (e.g., Fiszer de Plazas
A M I N O ACID RECEPTORS
163
and DeRobertis, 1975). These results have been compared with the relative potencies of the agents as inhibitors of synaptosomal GABA uptake and as neuronal depressants. Snyder’s group has found bicuculline (BIC) to be useful for distinguishing between Na+-independent and Na+-dependent GABA “binding” (see above). Of the other possible GABA antagonists, picrotoxin (PIC) had no effect on GABA-receptor binding, whereas d- tubocurarine did inhibit Na+-independent GABA binding with a half-maximal inhibition constant (I&) of about 3.8 X 10-5M (Enna and Snyder, 1975). d-Tubocurarine, though having only about 1/100th the potency of GABA for inhibiting Na+-independent GABA binding, had over 200 times more affinity for Na+-independent than for Na+dependent GABA binding (Young et al., 1976). These results are summarized in Table 111, which also reveals the expected result that substances that are most effective at inhibiting Na+-dependent binding are also potent inhibitors of synaptosomal GABA uptake. In general, the potency of agents at inhibiting Na+-independent GABA binding paralleled their neuropharmacologic effects on “GABA-ergic” synapses much better than did Na+-dependent binding of GABA or synaptosomal GABA uptake. Interestingly, strychnine had some effect on Na+independent GABA binding (Zukin et al., 1974; Young et al., 1976), and has also been shown to inhibit the binding of both GABA and glycine to synaptosome-enriched fractions in the presence of Na+ (DeFeudis et al., 1976a, 1977). Recent studies have revealed that BIC can also inhibit the binding of both glycine and GABA (G. Svenneby and E. Roberts, personal communication to R. W. Olsen, 1976). These findings revealed that binding studies do not necessarily provide results that reflect in uivo drug actions, and indicated further that both BIC and strychnine are not highly specific agents. Filtration or equilibrium dialysis assays of the binding of [l4C1-CABA to crude mitochondria1 fractions of mouse brain were carried out at 0°C in Ringer’s solution or in Tris-buffered 0.1 M NaCl (Olsen et al., 1975a; Olsen, 1976). These workers considered that uptake was being measured at 0°C since results indicated an entry of GABA into the membrane-bound space of the particles. Even though no corrections for the uptake of GABA into the membrane-bound space (e.g., with inulin or sucrose) were applied to these data, the potencies of GABA analogues and other substances at inhibiting this GABA uptake corelated well with their neuropharmacological potencies, indicating that some of the GABA-binding sites shown in the presence of Na+ might have been GABA receptors. Values obtained for the potencies of inhibitors of the GABA “uptake” system in mouse brain, GABA binding to crayfish
164
F. V. DEFEUDIS
muscle membranes, crustacean transport, and invertebrate GABA synapses are provided in Table IV. The binding of GABA to crayfish muscle membranes and to mouse brain particles was similar. These studies, together with others (see above), have revealed that “GABA receptors” can be studied in the presence of Na+. Although some properties of these receptors may be shown in Na+-free medium, one cannot avoid considering that the properties of a physiological process, receptor interaction, should depend upon a normal physiological environment.
4. Regional Distribution of GABA Binding If GABA is a central neurotransmitter, one might expect that the regional distribution of its “binding” mechanisms should exhibit some correlation with regional variations in its endogenous content and/or iontophoretic potency. T h e present discussion will center on comparing regional variations in both Na+-dependent and Na+-independent GABA binding with regional variations in GABA content and uptake. In the first study on regional dependency of Nu+-dependent GABA binding, rat cerebral cortex, hippocampus, whole cerebellum, and ponsmedulla were homogenized at 0°C in an isosmotic sucrose solution containing 40 mM NaCl plus [14C]-GABA(and [3H]-acetylcholine),and then PI (“crude nuclear”) and P2 (“synaptosomal-mitohondrial”) fractions were separated by differential centrifugation (DeFeudis and Black, 1973). Results indicated that Na+-dependent GABA “binding” occurred to a greater extent in PI fractions prepared from cerebral cortex and hippocampus than in those of whole cerebellum or pons-medulla, under the conditions employed. In another study, the binding of [3H]-GABA and [14C]-glycineto subcellular particles of rat cerebral cortex and spinal cord was examined both by the above-described method (homogenization of tissue in an isosmotic sucrose solution containing 40 mM NaCl plus the radioactive ligands) and by a method in which P2particles were first separated in the absence of added salt (to prevent “clumping”; see Gray and Whittaker, 1962) and then exposed to the labeled amino acids Results revealed that in the presence of 32 mM NaCl (DeFeudis, 1973~). “regional dependency” existed, as GABA was bound to a greater extent to particles of the cerebral cortex than to those of the spinal cord, and that “regional specificity” also existed, as GABA was bound to a greater extent than glycine to particles of the cerebral cortex and as glycine was bound to a greater extent than GABA to particles of the spinal cord. Thus, the concept of pfefeerentiul binding of amino acids emerged (DeFeudis, 1973~). In further studies, the binding activities of GABA and glycine over
TABLE IV POTENCIES OF GABA INHIBITORS'
Compound GABA ~~-2,4-Diaminobutyrate DL-y-Amino-&hydroxybutyrate 4-Amin0, &urn-crotonate 8-Guanidinopropionate 4-Aminopentanoate Imidazoleacetic acid D-Glutamate Homohypotaurine Homotaurine @-Alanine L-Glutamate Chlorpromazine Imipramine Picrotoxin Bicuculline Bicucine Bicucine methyl ester N-Methyl bicuculline
'All
GABA uptake in mouse brainb 28 f 5' 26 f 4 45 f 10 60 f 20 135 f 20 200 f 50 600 f 100 >500 150 f 30
500 f 100 500 f 100 >500 90 f 20
120 f 40 >500 500 f 50 >500 100 f 30 >500
GABA binding to crayfish muscle membranesC 1.3 f 0.5' 16 f 10 11 2 4 8 2 5 12 f 10 300 f 50 9 0 2 10 250 f 50 500 f 100
>500 1000 500 f 100 40 f 20 200 100 >500 350 f 100
*
Crustacean transportd 60@,22
20-200
500
20-200
> 1000 >500
50-500 ?
> 10,000
1000
? 1000
300
> 10,000 > 1000
>500
references to these data are in Olsen (1976). Reproduced by permission from Olsen (1976). K , in /AM.
* Crude mitochondria1 fraction at OOC, filter assays.
Equilibrium dialysis assays at 4°C. Recalculated values, estimating the concentration of inhibitor blocking GABA transport by 50%. Concentration of inhibitor blocking 50% of the tissue response to inhibitory nerve or to 50 /AM GABA application. 'KD app. Concentration giving 50% of maximal response, recalculated from original data.
10-100" 1000-10,000;< 100
1000
>200 250 f 50
SK,.
Invertebrate GABA synapsese
3,300 200, 50-100, 1, 10" >10 5 1,100
166
F. V. DEFEUDIS
wide concentration ranges were examined in a medium containing 32 mM NaCl with preisolated P2 fractions (DeFeudis, 1974b, 1975b; DeFeudis and Schiff, 1975). T h e order of potency of binding at 0°C was as follows: GABA, cortex > GABA, cord = glycine, cord > glycine, cortex. GABA binding to particles of the cerebral cortex occurred with a KB 1.8 X M and a Bmax=65 nmole/gm cortex, whereas glycine was M and a Bmax 43 bound to spinal particles with a K B zs 3.3 X nmole/gm cord (DeFeudis, 1974b). Calculations revealed that, at saturation, the capacity of the GABA-binding mechanism at 0°C in cerebral cortex (at about 0.16 x lo-’’ mole GABA per nerve ending) was great enough to account for both the depressant effect produced by iontophoretic application of GABA and for inactivation of the amount of GABA required to cause cortical inhibition. These “binding” data were significantly correlated with the iontophoretic potencies of these amino acids (see below). At O”C, the binding of both GABA and glycine occurred by tripleafjnity processes to particles of cerebral cortex and by double-affinity processes to particles of spinal cord (DeFeudis and Schiff, 1974, 1975). Respective binding affinities (reciprocals of KB values) of GABA for particles of the cortex were .generally greater than those for glycine, whereas B,,, values for GABA were about twice those for glycine (Fig. 5a). In spinal cord fractions, although the binding affinities for GABA were about twice those for glycine, BmaXvalues for GABA were smaller than those for glycine (Fig. 5b). Differences between Bmax values for GABA and glycine in particles of cerebral cortex and spinal cord reflected their different endogenous concentrations. With the increased resolution provided by using a wide range of amino acid concentrations, two additional (one of higher and one of lower affinity) components became apparent for both GABA and glycine in cortex, and one additional (low-affinity) component was shown for both GABA and glycine in spinal cord. Note that the “high-affinity” processes of DeFeudis (1974b) and the “intermediate-affinity” processes of DeFeudis and Schiff ( 1975) for cerebral cortex (see Fig. 5a) possessed rather identical KB values, which were also rather identical to the K , values reported for the “high-affinity” uptake of GABA and glycine by small slices or homogenates of cortex at 22” or 37°C (e.g., Iversen and Neal, 1968; Balcar and Johnston, 1973; Peterson and Raghupathy, 19’73). Hence, “highaffinity” GABA uptake and Na+-dependent GABA binding involve similar processes (DeFeudis, 1973~).“Triple-affinity” processes for the binding of GABA to a membrane fraction of rat cerebral cortex in the presence of Na+ (C. Estrada, 1976 unpublished observations) and for the binding of glutamate and aspartate to hydrophobic protein fractions
167
AMINO ACID RECEPTORS
CEREBRAL CORTEX, O'c .a7
a3
150
-0
10
25
50
75
100
125
I.n m o h r amino acid in
FIG. 5. Hofstee plots for the binding of ['HI-GABA and [%I-glycine to synaptosome-enriched fractions of rat cerebral cortex (a) and spinal cord (b) at OT, 10 min in the presence of 32 mM NaCI. Each point represents the mean value from three to eight experiments; values for half-maximal binding constants (KB) and maximal binding capacities (Emax)for these "triple-affinity" or "double-affinity" processes are indicated. (Reproduced with permission from DeFeudis and Schiff, 1975.)
168
F. V. DEFEUDIS
isolated from rat cerebral cortex in the absence of Na+ (DeRobertis and Fiszer de Plazas, 1976a; Fiszer de Plazas and DeRobertis, 1976) have recently been demonstrated (see below). A further study on Na+dependent GABA binding to 17 regions of the feline CNS (Balfagon et al., 1975; see also de Sagarra et al., 1975) indicated a greater binding of GABA to synaptosome-enriched fractions of cerebral and cerebellar cortices than to some other CNS regions studied, providing support for pharmacological studies which have indicated that GABA exerts its most pronounced depressant effects in these regions (e.g., Curtis and Watkins, 1965; KrnjeviS, 1974; Curtis and Crawford, 1969; DeFeudis, 1975a). Regional dependency of GABA binding has also been studied by Zukin et al. (1974) with crude membrane fractions of rat brain. In the absence of added Na+, “specific,” BIC-displaceable, [3H]-GABA (3.2 X M) binding at 4°C was greatest in cerebellum, least in spinal cord and medulla oblongata-pons, and of intermediate potency in hippocampus, cerebral cortex, and several other regions. These values, obtained with a single concentration of [3H]-GABA,did not correlate closely with regional variations in endogenous GABA content. [Note that this correlation can best be shown by using B,,, values, rather than KB or single binding values (see DeFeudis and Schiff, 1975).] In a further study on rat CNS, Na+-independent GABA binding was also found to be greatest in membrane preparations of cerebellum and lowest in those of spinal cord, and marked differences existed between Na+-dependent and Na+-independent processes among the eight CNS regions examined (Enna and Snyder, 1975). It seems noteworthy that in the presence of physiological concentrations of Na+ and other ions, the binding of [3H]-GABAto synaptosomal-mitochondria1 fractions of cerebellar cortex was also found to be as great (Balfagon et al., 1975) or greater (de Sagarra et al., 1975) than its binding to other CNS regions. Regional variations in synaptosomal GABA uptake correlated neither with Na+dependent nor with Na+-independent GABA binding, but, again, B m a , values were not compared; only 2.5 x lo-* M [SH]-GABA was used (Enna and Snyder, 1975). However, a further study with crude membrane fractions of 31 regions of rhesus monkey (Macaca mulatta) CNS did reveal a rather close correlation between Na+-independent GABA binding, GAD activity, and synaptosomal GABA uptake (Enna et al., 1975; see also Table V). Na+-dependent GABA binding did not correlate well with Na+-independent GABA binding or with the other parameters (see Table V). Enna et al. (1975) realized that the concentrations of labeled GABA that they used were considerably lower than theKB for GABA binding in
AMINO ACID RECEPTORS
169
both Na+-dependent and Na+-independent systems, and that the regional variations they observed could have been due to variations in affinity for GABA rather than to differences in total number of tissue sites. Therefore, they estimated the number of sites and affinities for Na+-dependent and Na+-independent GABA binding in cerebellar cortex, frontal cerebral cortex, and thalamus or putamen. No regional differences among the KB values for Na+-dependent (see also Bond, 1973) and among those for Na+-independent GABA binding were found. However, the total number of binding sites (Bmax)for both Na+-dependent and Na+-independent GABA binding did differ among these regions, but these differences were similar to variations in binding obtained with single concentrations of GABA (Table V). Interestingly, this conclusion of Enna et al. (1975), that variations in GABA binding appeared to reflect differences in number of sites rather than in affinity, based on studies of both Na+-independent and Na+-dependent GABA binding to frozen and thawed membrane fractions, did not differ from that obtained by studying Na+-dependent GABA binding to freshly prepared synaptosomal fractions (DeFeudis and Schiff, 1975). Recent reviews of the regional distributions of GABA, glutamate-adecarboxylase (GAD) activity (Fahn, 1976; Okada, 1976), and GABA uptake (Martin, 1976) allow more valued judgments to be made about these results on regional dependency of GABA binding. Data presented by Fahn (1976) revealed that GABA content and the activity of its synthesizing enzyme (GAD) do correlate remarkably well among various CNS regions of several species, thus lending validity to the use of GAD activity as an indication of endogenous GABA concentration. However, this analysis of Fahn (1976) revealed also that the GABA contents of CNS regions of monkey and rat are also strikingly similar (see Table VI), leading one to suspect that regional GABA binding in these two species should also exhibit a similar trend. However, close correlations were shown among Na+-independent GABA binding, GAD activity, and synaptosomal GABA uptake among regions of monkey CNS (Enna et al., 1975),but not between this binding and GABA uptake among regions of rat CNS (Zukin et al., 1974; Enna and Snyder, 1975). Also, the finding that the average KB values for both Na+-dependent and Na+independent GABA binding in monkey brain (Enna et al., 1975) were only about one-eighth those of rat brain (Enna and Snyder, 1975) cannot be explained on the basis of the very similar concentrations of GABA that exist among CNS regions of these two species (see Table VI). Also, the use of regional synaptosomal GABA uptake (or active transport) to represent the endogenous GABA contents of CNS regions (Enna and Snyder, 1975; Enna et al., 1975) is not entirely valid since the positive
TABLE V REGIONAL DISTRIBUTION OF SODIUM-DEPENDENT AND SODIUM-INDEPENDENT GABA BINDING, GABA UPTAKE, AND GLUTAMIC ACIDDECARBOXYLASE (GAD) IN MONKEYCNS"
Binding (fmole/mg)
-
4 0
Region Olfactory bulb Cerebrum Frontal cortex Frontal pole Occipital pole Temporal pole Precentral gyrus Postcentral gyrus . Superior temporal gyrus Medial temporal gyrus Cingulate cortex White matter areas Corpus callosum Corona radiata Optic chiasm
Na+dependent
Na+independent
Uptake (fmole/mg)
5.1 f 0.2
0.04 f 0.03
232
38
11.06 & 1.20
17.1 f 1.9 15.8 f 1.9 12.8 f 1.8 9.4 f 0.4 8.6 f 1.0 10.5 -t 0.6 18.8 f 0.7 13.1 f 1.0 6.0 f 0.5
1.53 f 0.19 1.82 & 0.20 2.03 & 0.33 0.67 f 0.12 0.40 f 0.03 1.33 2 0.13 2.18 f 0.26 1.58 5 0.24 1.52 f 0.27
607 21 641 f 34 533 & 25 739 & 51 535 & 39 563 2 26 815 f 69 746 2 34 764 t 61
9.07 f 0.72 8.50 2 0.99 14.60 & 1.60 7.87 2 0.77 11.23 f 0.66 11.40 -t 0.57 8.70 0.87 9.53 f 0.89 9.93 f 1.47
3.4 f 0.4 2.6 +- 0.2 3.2 & 0.2
0.23 f 0.13 0.11 "0.04 0.13 f 0.08
97 f 3 6 8 5 11 5 5 2 11
0.43 f 0.09 0.30 2 0.12 0.47 f 0.34
&
GAD (nmole/mg/hr)
-2
Limbic cortex Am ygdala Hi ppocam pus Septum Hypothalamus Thalamus Extrapyramidal areas Caudate Putamen Globus pallidus Substantia nigra Midbrain Cerebellum--lower brain stem Cortex Nodule and uvula Cerebellar deep nuclei Pons Medulla oblongata Spinal cord Cervical Thoracic Lumbar Data are the meandmg wet weight
4.7 f 0.5 7.3 f 0.6 5.4 f 0.8 12.8 f 1.2 43.8 f 2.7
1.40 f 0.11 0.74 f 0.1 1 1.21 f 0.11 0.41 f 0.07 1.02 f 0.08
847 f 49 390 f 38 491 f 44 787 f 75 224 f 30
9.10 2 0.86 4.87 f 0.77 8.60 -t0.37 20.00 f 1.43 7.50 ? 0.37
11.7 f 0.8 15.3 f 1.0 4.8 f 0.3 7.9 f 0.9 31.6 f 3.5
2.52 f 0.49 2.37 f 0.40 2.00 2 0.11 1.14 f 0.16 0.70 f 0.11
339f 16 299 f 8 852 f 68 587 f 75 405 f 25
13.23 2 1.33 13.00 f 1.23 47.87 f 2.16 27.20 f 3.97 14.40 t 1.99
23.9 f 1.9 4.7 f 1.0 12.4 f 0.99 15.8 t 1.9 21.4 2 0.8
1.72 f 0.28 0.55 f 0.03 0.30 f 0.13 0.20 t 0.07 0.06 f 0.02
229 f 26 286 f 46 148 f 14 158 12 270 f 35
8.07 & 1.20 8.67 t 0.96 8.67 t 0.63 1.17 f 0.17 4.67 f 0.30
9.8 f 0.5 4.4 f 1.1 4.1 f 0.2
0.07 f 0.02 0.09 f 0.05 0.08 f 0.04
154 f 21 102 2 9 196% 13
1.17 f 0.17 0.83 f 0.02 2.60 f 0.80
f S.E.M.
*
of seven determinations. Reproduced by permission from Enna et al. (1975).
172
F. V. DEFEUDIS
TABLE VI REGIONALDISTRIBUTION OF GABA’ Rhesus monkey
Rabbit
Rat
Guinea Pig
Region of CNS
(4)
(5)
(5)
(5)
(2)
(5)
Substantia nigra Globus pallidus Hypothalamus Inferior colliculus Dentate nucleus Superior colliculus Periaqueductal gray Oculomotor nucleus Putamen Pontine tegmentum Caudate nucleus Medial thalamus Hypoglossal nucleus Amygdala Hippocampus Lateral thalamus Occipital cortex Anterior thalamus Medullary tegmentum Inferior olivary nucleus Temporal cortex Frontal cortex Motor cortex Cerebellar cortex Spinal cord gray Red nucleus White matter
9.70 9.54 6.19 4.70 4.30 4.19 4.02
8.50 6.43 5.33 3.06 4.09 4.93
10.07 7.67 7.68 5.06
9.69 8.17 5.76 4.73
9.63 8.85 4.48 5.49 4.80
5.3 1 5.6gd 3.72
7.67
4.59
-
-
3.58b 3.34 3.58b 4.19 3.58
2.91b 2.95 2.9Ib
4.08
3.03 2.52c
-
3.62 3.34 3.20 3.00
-
-
2.68 2.68 2.50 2.27 2.25
-
2.10 2.09 2.03
-
0.3 1
-
4.52 3.48 1.82 -
-
3.25 2.39 2.30
-
-
-
-
2.67
-
-
2.76
2.11
2.29
2.10
2.86
1.80
3.01 1.92
2.61 2.48
-
-
1.91 0.75
-
0.96
-
-
Baboon Human
-
3.53
-
2.77
-
-
-
2.47
-
2.32 -
-
2.39
2.14 2.09
2.14
2.33
-
1.86 0.26
-
-
-
-
’Mean values; numbers in parentheses under each species indicate the numbers of animals studied. Data for monkey are from Fahn and CbtC (1968); those for rabbit, rat, and guinea pig are from Okada et al. (1971); those for baboon are from Okada (1976); those for human are from Perry et al. (1971). Reproduced by permission from Fahn (1976).
Putamen and caudate nucleus were combined. Whole thalamus was assayed. Globus pallidus and putamen were combined.
correlation shown in most studies between energy-mediated GABA uptake and GABA content has been general, but not perfect (Martin, 1976).
5 . Studies with Sympathetic Ganglia GABA “receptors” in mammals are not unique to the CNS. Application of GABA can depolarize cat superior cervical, vagal sensory, and
AMINO ACID RECEPTORS
173
dorsal root ganglia in vivo (DeGroat, 1970, 1972; DeGroat et al., 1972) and rat superior cervical ganglion in vitro (Bowery and Brown, 1974), and can elicit a release of catecholamines from isolated bovine adrenal gland (Sangiah et al., 1974). In isolated desheathed superior cervical ganglia of the rat, studied in Krebs-Ringer solution, half-maximal depolarization was elicited by about 1.3 X M GABA, and was antagonized by bicuculline (BIC), half-maximal inhibition occurring at about 1.4 X M (Bowery and Brown, 1974). Conformationally restricted analogues of GABA (4-aminotetrolic acid, trans-4-aminocrotonic acid, and imidazoleacetic acid) produced depolarizations similar to that of GABA, and their actions were prevented by BIC, methyl-BIC, and tetramethylenedisulfotetramine (Bowery et al., 1975a; Bowery and Jones, 1976).
6. Relevant Studies with Tissue Slices In superfused slices of guinea pig cerebellar cortex, both PIC and BIC suppressed GABA-induced depression of spontaneous activity in high-Cl- medium, and abolished GABA-induced excitation in low-CImedium (Okamoto et al., 1976). Interestingly, BIC blocked the depressant effects of glycine, taurine, and P-alanine on these cells in highC1- medium, but not their excitatory effects in low-cl- medium. All results obtained with this preparation, which likely involved mainly Purkinje cells, were consistent with the notion that GABA, glycine, taurine, and P-alanine cause inhibition by increasing C1- (and perhaps K+) permeability (see also Section 11,B). In other studies with this preparation, Okamoto and Quastel (1976) showed, by kinetic analyses of dose-response relationships for amino acids in the presence or absence of PIC or strychnine, that the numbers of amino acid molecules which combined with their respective receptor sites to produce inhibition or excitation were: 3 for GABA, 2 for glycine, 3 for taurine, and 4 for /I-alanine. One molecule of either PIC or strychnine combined with its receptor site (see also Takeuchi and Takeuchi, 1969). These findings provided good evidence that the GABA receptor in cerebellar cortex differs from the receptor(s) for “glycine-like” amino acids. All four of these amino acids depressed the spontaneous firing of neurons of the feline cerebellum in situ (e.g., Kawamura and Provini, 1970). 7. Effects of Maturation and Environment on GABA Binding a. Maturation. Stimulated by the pioneering work of Roberts and colleagues (Roberts, 1962), several developmental studies on the GABA system have been performed. Kuriyama et al. ( 1 9 6 8 ~ )examined the developments of GABA-a-oxoglutarate transaminase (GABA-T) activity, GAD activity, and GABA content in the cerebella of chick embryos at
174
F. V. DEFEUDIS
tissue and subcellular levels. “Synapse-like’’structures were observed as early as 11 days of incubation, synaptogenesis increasing markedly thereafter, The time course of development of the whole GABA system correlated better with the development and increase in synaptic structures than with the accretion of the total cerebellar mass. At all ages of development studied, GAD activity was higher in presynaptic nerve ending fractions than in other fractions, and GABA-T activity was especially high in free mitochondria. Regarding GABA uptake by chick cerebrum, Levi (1970) showed the presence of a “double-affinity” system in 8-, 14-, and 19-day-old embryos, whereas in 15-day-old chicks only a “single-affinity’’ system was present. K , values for GABA transport for the high-concentration limbs of the plots obtained for embryos and for the 15-day-old chick “singleM and constant, whereas correaffinity” system were about 8 x sponding V,,, values increased from 0.15 to 1.4 pmole/ml intracellular water/min from 8-day-old embryos to 15-day-old chicks. Johnston and Davies (1974) found that the V,,, for “high-affinity” GABA uptake by small rat brain slices increased dramatically as animals developed from the neonatal to adult stage, a result that is not in accord with those of Levi (1970, 1972), which showed that the “high-affinity” GABA transport system of chick brain tended to decrease in activity during development. T h e studies of Levi (1970, 1972) may possess a geometric artifact related to the thick (0.4 mm) slices that were used (Martin, 1976). In any case, the study of Levi (1970) introduced not only the effects of maturation on GABA uptake mechanisms, but also the “double-affinity’’ system for GABA uptake. Changes in the Na+-dependent “binding” of GABA to subcellular particles of rat brain also occur during maturation (DeFeudis, 1972b, 1973a,d). At O’C, [3H]-GABA “binding” occurred to a greater extent to “synaptosomal-mitochondrial” (P2) fractions prepared from the brains of 11- or 15-day-old rats than to those prepared from younger or older rats (Fig. 6). GABA binding to “crude nuclear” fractions increased until 21-22 days of age, after which it remained rather constant. Maximal Na+-dependent GABA binding to P2 fractions coincided with the period during which brain growth rate (Davison and Dobbing, 1968), synaptic development (Aghajanian and Bloom, 1967), and cerebral excitability (Timiras et al., 1968) are also maximal. Electrophysiological studies that have revealed that inhibitory neural influences predominate in the immature nervous system (Crain and Bornstein, 19’74)appear to support the functional significance of these observations, but it should be noted that similar age-related changes in binding have been shown for the excitatory amino acid, glutamate, and for glycine and arginine (De-
175
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FIG. 6. Na+-dependent binding of [3H]-GABA and retention of [‘4C]-sucrose by “synaptosomal-mitochondrial” (P2)fractions of the brains of rats during development. (Reproduced with permission from DeFeudis, 1973a.)
Feudis, 1973a). The result with arginine indicated that this phenomenon is not specific to transmitter-candidate amino acids. Enna et al. (1976) studied the development of Na+-dependent and Na+-independent GABA binding and GAD activity in chick embryo brain. Both GAD activity and Na+-independent GABA binding were barely detectable up to 9 days of conceptual age, and thereafter both systems increased rapidly, but no appreciable time difference existed between the development of GAD activity (reflecting presynaptic nerve terminals) and Na+-independent GABA binding. The greatest increases in GAD activity, Na+-independent GABA binding, and protein content occurred between 12 and 20 days of development. These effects correlated temporally with the appearance of morphologically distinct synapses throughout the chick embryo brain and with mature cerebral
176
F. V. DEFEUDIS
electrical activity and motor coordination (Rogers et al., 1960; Oppenheim and Foelix, 1972; Foelix and Oppenheim, 1973). Coyle and Enna (1976) showed that the development of “highaffinity”, Na+-dependent GABA uptake by particulate fractions of whole rat brain differed markedly from that of GAD activity, uptake being near adult levels at birth, peaking considerably above adult levels at 1 to 2 weeks after birth, and declining toward adult activity by 4 weeks after birth. Also, as had been shown previously by Levi (1970) in slices of chick cerebrum, Coyle and Enna (1976) found that the development of GABA uptake was reflected by differences in Vmax,while K , remained constant. Development of “high-affinity” Na+-dependent GABA uptake by P, fractions of brain regions (Coyle and Enna, 1976) was remarkably similar to that of Na+-dependent binding of GABA to P, particles at 0°C (DeFeudis, 1972b, 1973a,d). In all brain regions studied, Na+independent GABA binding increased dramatically after 8 days following birth, approximated adult levels by 4 weeks of age, and correlated best with the increase in GAD activity (Coyle and Enna, 1976). Hence, development of the “GABA receptor” might correlate best with that of presynaptic “GABA-ergic” elements. Since so many differentiating processes occur concurrently in the brain, it is very difficult to correlate physiological events with the development of any one specific neurochemical element. A further criticism that could be applied to these studies is that such experiments are particularly difficult to interpret in terms of GABA transport or receptor binding because they were not carried out with morphologically characterized particles and because the composition of the fractions obtained could vary greatly with age (Martin, 1976). In defense against this criticism, the writer suggests that these studies have revealed that the developmental changes which occur in GABA binding and uptake do indeed have a morphological basis. b. Environment. Less GABA was bound, in the presence of Na+, to synaptosome-enriched (P2)fractions prepared from the brains of “isolated” (singly housed) male C-57 Black mice than to those of their “aggregated” (group housed) littermates after 5 weeks of differential housing (DeFeudis, 1972a). Further studies revealed that the binding of both GABA and glycine to P, fractions of the brains of male Swiss albino mice was lower in mice that had been isolated for 8 to 10 weeks than in their “aggregated” counterparts (DeFeudis et al., 1976a). Centrifugation of P, fractions on discontinuous sucrose gradients revealed that both GABA and glycine were bound to a lesser extent to a “heavy synaptosomal fraction” of the brains of “isolated” mice when the data were expressed as mole per fraction (DeFeudis et al., 1976b; see also Fig. 7a). However, since the protein contents of fractions from “isolated” mice
AMINO ACID RECEPTORS
177
were also lower than those of their “aggregated” counterparts, no difference existed between the two groups of mice when these data were expressed on a protein basis (Fig. 7b). It seemed apparent that an environmentally induced morphological change had been produced in the brain; the brains of “isolated” mice contained fewer subcellular binding sites for GABA and glycine. Thus, the binding of GABA and glycine may provide a measure of the number (or development) of cerebral nerve endings that can be affected by long-term changes in external environment. T h e brains of animals exposed to individual housing, by having a decreased capacity to bind GABA, could have less inhibitory nerve endings, a change which could be related to the behavioral changes (e.g., increased aggressiveness) caused by environmental impoverishment. These results support the view that the brain exhibits structural plasticity when an animal is adapting to conditions that produce dramatic changes in its behavior. A change in internal environment, produced by intracerebral injection of rat parvovirus into the left hemisphere of newborn hamsters, was paralleled by a decrease (about 99%)in cerebellar granule cells, and by a selective reduction (60%-75%) of Na+-independent GABA binding to synaptic membranes when these animals were killed at 2 1 and 40 days of age (Simantov et al., 1976; see also Fig. 8). Such findings support the view that excitatory granule cells of the cerebellar cortex might possess GABA receptors on their dendrites. B. GLYCINE Glycine is one of the amino acids that, like GABA, are bound to cerebral subcellular particles by Na+-dependent mechanisms (DeFeudis, 1973a; Valdes and Orrego, 1975). However, the Na+-dependency of glycine binding does not appear to be as pronounced as that for GABA (DeFeudis, 1973a). Studies on the binding of glycine in the presence of Na+ and on the comparison of this binding with that of GABA in synaptosome-enriched fractions of rat cerebral cortex and spinal cord (DeFeudis, 1973c, 1974b; DeFeudis and Schiff, 1975) have revealed a regional dependency of glycine binding (see Section A,4), and also that theKBfor glycine binding at 0°C (2-3.3 x M; see Fig. 5b) approximated the K , for “highaffinity” glycine uptake (3.1 x lop5M; Neal, 1971) in rat spinal cord. 1. Glycine-Sensitive Strychnine Binding-An Approach toward the Glycine Receptor
Studies on the “binding” of [3H]-strychnine and its displacement by glycine as a measure of glycine-receptor activity in subcellular fractions
F. V. DEFEUDIS
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were initiated by Snyder and colleagues (1973). First of all, it should be noted that these studies on the “glycine receptor” are not comparable to those performed on the “GABA receptor” (see above). For studying the GABA receptor, [3H]-GABA binding was displaced by its antagonist, bicuculline (e.g., Zukin et al., 1974), whereas in studies on the glycine receptor, displacement by glycine of [3H]-strychnine binding has been examined. Perhaps the most important difference in experimental design was that the medium used to demonstrate the interaction of GABA with its postulated receptor was free of added Na+, whereas in the studies
AMINO ACID RECEPTORS
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on the glycine receptor ([3H]-strychninebinding) the medium contained Na+ (0.05 M Na+, K+-phosphate buffer -+ 200 mM NaCI). Such a difference in experimental design can influence the binding (and related KBvalues) of both GABA and glycine as well as the binding (and related Ki values) of their antagonists. T h e rationale in support of the use of Na+ in studies on r3H]-strychnine binding involves the idea that Na+dependent binding of glycine, itself, was not being studied, whereas in
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studies with [3H]-GABA,its Na+-dependent binding (related to its uptake mechanism) would have been involved. However, it should be noted that the amount of glycine required to displace [3H]-strychninewould likely be increased by the presence of Na+ in the medium, since, like GABA, glycine might be bound mainly to its uptake sites under these conditions (see, e.g., DeFeudis, 1973a). An additional reason for using [3H]-strychninerather than labeled glycine as the ligand in studying the glycine receptor is that strychnine appears to have a greater affinity for the glycine receptor than glycine itself (see referenc.es in Young and Snyder, 1973). The writer tends to believe, however, that the reverse type of study, i.e., the effect of strychnine on [3Hl-glycinebinding, would have provided different results. Some evidence for this contention derives from recent studies on strychnine displacement of glycine binding
AMINO ACID RECEPTORS
181
(ValdCs and Orrego, 1970, 1975; DeFeudis et al., 1976a, 1977; see also Section IV,B,S). Using a centrifugation method, [3H]-strychnine binding to crude synaptic membrane preparations of spinal cord and brain stem of rat and monkey was demonstrated (Young and Snyder, 1973, 1974a,b; Snyder et al., 1973; Snyder, 1975). In studies with rats, Young and Snyder (1973) found that “specific” [3H]-strychnine binding (i.e., that M glycine) to synaptic membranes was enriched threedisplaced by and eightfold, respectively, over the dialyzed and undialyzed whole homogenate. In 0.05 M Na+, K+-phosphate buffer, “specific” [3H]strychnine binding was saturable, with half-maximal binding occurring M, whereas nonspecific [3H]-strychnine binding (i.e., rH]at 3 X M nonstrychnine binding in the presence of 10” M glycine, or radioactive strychnine) was not saturable and increased linearly with increasing [3H]-strychnineconcentration. Displacement of bound [3H]strychnine by glycine and other amino acids paralleled their “glycinelike” neurophysiologic activities, and the regional distribution of rH]strychnine binding sites in the CNS correlated closely with variations in endogenous glycine concentration. The affinity of glycine for these sites was three or four orders of magnitude less than that of strychnine, which is consistent with neurophysiological findings (see above; see also Young and Snyder, 1974a; Snyder, 1975) (Fig. 9). Displacement curves of [3H]-strychnine by glycine indicated cooperative interactions with a Hill coefficient of about 1.7, whereas the Hill coefficient for displacement of [3H]-strychnine by unlabeled strychnine was about 1.O (Young and Snyder, 1974a). [Note: When a binding curve is expressed as a Hill plot, i.e., as log occupancy/(1-occupancy) vs. log of concentration, its gradient (the Hill coefficient) should be about 1.0 for antagonists (see, e.g., Birdsall and Hulme, 1976).] Hence, glycine and strychnine appeared to bind to distinct sites which interact in a cooperative fashion. Strychnine appeared to bind to a single population of receptor sites, the amount of which was about 39 pmole/gm of rat spinal cord (Young and Snyder, 1974a). The potencies of a series of anions at inhibiting [3Hl-strychnine binding to synaptic membranes of spinal cord correlated closely with their neurophysiologic capacities to reverse IPSPs in mammalian spinal neurons (Young and Snyder, 197413). C1- displaced strychnine binding in a noncompetitive fashion with Hill coefficients of about 2.3 to 2.7. This noncooperative inhibition of strychnine binding by C1- indicated that strychnine and C1- do not bind to the same site or that more than one C1- ion is needed to displace each strychnine molecule. These results indicated that strychnine binding is associated with the glycine ionophore. Studies of the actions of benzodiazepines on [3H]-strychninebinding
182
F. V. DEFEUDIS
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LOG,, [LIGAND,M] FIG. 9. Displacement of ['HI-strychnine binding by unlabeled glycine and by unlabeled strychnine. Synaptic membrane suspensions (1 .O mg proteinltube) were incubated with 2 nM [3H]-strychnineand increasing amounts of glycine or strychnine at 4OC for 10 min. Nonspecific binding, obtained in the presence of 1.0 mM glycine or 0.1 mM strychnine, has been subtracted from all experimental points. Mean values of triplicate determinations. (Reproduced with permission from Young and Snyder, 1974a.)
have led to the proposal that these drugs might act by mimicking the action of glycine at CNS receptor sites (Young et al., 1974a). In crude synaptic membrane fractions of rat spinal cord and brain stem, the rank-order of potency for 2 1 benzodiazepines at displacing [3H]strychnine binding correlated with their rank-order of potency in various pharmacological and behavioral tests in humans and other animals. A 50-fold variation in potency was evident with the benzodiazepines used; diazepam (Valium)and chlordiazepoxide (Librium) had respective ICso values of 2.6 X M and 2 X M; ICJOfor glycine was 2.5 x M. Hill coefficients for the displacement of 13H]-strychnine by unlabeled strychnine and by benzodiazepines were both about 1.0, and that for glycine displacement of [3H]-strychninewas about 1.7, indicating that benzodiazepines might interact with the strychnine site of the receptor. Comparison of the ICJOof unlabeled strychnine on rH]strychnine binding of about 4 X 10-'M (Young and Snyder, 1974a; see also Fig. 9) with that for diazepam (which was comparable to that for glycine at 2.6 x M) indicated that diazepam could be regarded as a rather weak strychnine antagonist, or agonist, rather than as a glycine antagonist (see also Johnston, 1977).
AMINO ACID RECEPTORS
183
2 . Studies on Glycine Binding As mentioned above, C3H]-strychnine binding was studied in the presence of Na+, but generally not in balanced physiological media. Other experiments, also conducted in the presence of Na+, have been aimed at studying the binding of radioactive glycine. Although Na+ would be expected to enhance the binding of glycine to its uptake carrier, such studies provide a more direct assessment of the physiological properties of glycine-binding sites as well as information about glycine binding to its uptake carrier. T h e first study of this type was conducted by Valdes and Orrego (1970), who showed, at 37°C in an artificial CSF medium containing 123 mEq/liter Na+, that strychnine (5 x lo4 M) decreased the binding of [I4CJ-glycine(5 x M) by about 25% to a membrane fraction of rat cortex, and that 1 X lo-’ M strychnine decreased this binding of glycine by about 75%. Strychnine inhibited glycine binding by a strictly competitive mechanism. An action of strychnine on this mechanism was also revealed by the study of DeFeudis et al. (1976a), in which strychnine-SO4 and M) decreased the binding of glycine and GABA (both at 1.2 x M) to synaptosomal fractions of mouse brain. In a further study, strychnineSO4 ( and M) inhibited the binding of both GABA and glycine (both at 6 x M) to synaptosomal fractions of several regions of feline brain: at M, this drug inhibited the binding of GABA and glycine only to particles of cerebral cortex (DeFeudis et al., 1977). Such findings have indicated that, with as little as one order of magnitude difference in concentration between the amino acid and strychnine, a clear interaction can be shown in physiological media. That such an effect of strychnine could include some postsynaptic receptor sites for these amino acids seems unlikely if only 39 pmole/gm of tissue of glycine-receptor sites exist (see Section IV,B, 1); more likely, these results indicate an effect of strychnine mainly on the binding mechanisms associated with the transport of glycine and GABA. The binding of [14C]-glycineto a membrane fraction of rat cerebral cortex incubated in artificial CSF occurred with a “high-affinity” ( K B =4 X M) and a “lower-affinity’’component at 37°C and was strongly Na+- and temperature-dependent (Va1di.s and Orrego, 1975). The affinity pattern for the binding of glycine and other amino acids indicated that the receptor might be similar to that involved in the transport of small neutral amino acids in brain slices. It was suggested that in cerebral cortex only transport receptors, as opposed to synaptic receptors, can be demonstrated for glycine and that this interaction of glycine with brain cortex membrane fragments represented its transport into
184
F. V. DEFEUDIS
resealed membrane vesicles. Further studies by Valdes et al. (1977) indicated that “high-affinity”Na+-dependent glycine-binding sites of rat cerebral cortex were located in myelin proper and possibly also in some other glial plasma membranes but not in nuclei, mitochondria, endoplasmic reticulum, or synaptosomes. The above-mentioned results provided further evidence for the existence of a “high-affinity”transport, or binding, mechanism for glycine in cerebral cortex preparations (see also Valdes and Orrego, 1970; Peterson and Raghupathy, 1973; DeFeudis, 1974b; DeFeudis and Schiff, 1975); other workers (e.g., Johnston and Iversen, 1971; Logan and Snyder, 1972; Bennett et al., 1974) have not observed this process in slices or homogenates of cerebral cortex. ValdCs et al. (1977) suggested that this inability to observe the “high-affinity” process for glycine in cerebral cortex preparations might have been due to the presence of substantial amounts of endogenous amino acids in the homogenates or slice preparations that were used (see also DeFeudis, 1975a). According to Valdes et al. (1977), the more conspicuous “high-affinity” glycine transport of spinal cord preparations might reflect the much higher myelin content of this CNS region, as compared with cerebral cortex. 3. Regional Distribution of Glycine Binding The regional distribution of “specific” [3H]-strychnine binding (a possible indicator of glycine receptors) within the mammalian CNS and the regional distribution of glycine binding to six regions of feline CNS, both in the presence of Na+, have already been discussed. Regional distributions of glycine and strychnine binding in the absence of added Na+ have not yet been reported.
4. Effects of Maturation and Environment on Glycine Binding a. Maturation. Na+-dependent binding of glycine, like the binding of GABA, glutamate, and arginine, to synaptosome-enriched fractions of rat brain was maximal in 11- and 14-day-old animals and declined thereafter (DeFeudis, 1973a). These results are in accord with those of the earlier developmental study of Piccoli et al. (1971), which showed that the uptakes (influxes) of high concentrations (e.g., 5 x lob4M or 2 x M) of glycine, like those of taurine, glutamate, and GABA, increased rapidly during the fetal and early neonatal periods, reached a maximum between 2 and 3 weeks of postnatal age, and then declined to adult levels. Such results indicated that the Na+-dependent binding of glycine to subcellular fractions represents the initial phase of the active uptake process (i.e,, binding of amino acid to its “carrier” site). Zukin et al. (1975) showed, in chick embryo spinal cord, that both glycine uptake and “glycine-receptor” binding (measured with VHI-
A M I N O ACID RECEPTORS
185
strychnine) increased dramatically during development, glycine uptake tending to precede the development of receptor binding by about a day (see Fig. 10). As was evident from studies on GABA binding (Enna et al., 1976), a rapid increase in potency of the glycine uptake mechanism occurred between 11 and 17 days after embryo incubation. T h e developmentally linked increase of “glycine-receptor” binding displayed at least two components. Half-maximal reduction of [3H]-strychnine binding to spinal cord preparatians of 14-day embryos occurred at about 2 x 10-0 M strychnine and at about 2 X lop5M glycine, values similar to those obtained in adult rat spinal cord (Young and Snyder, 1973, 1974a). After 10 days of embryonic development, receptor binding increased markedly in spinal cord and brain stem, but not in cerebral hemispheres (Zukin et al., 1975). b. Environment. Plots of the binding of glycine (and GABA) to synaptic particles of the brains of differentially housed mice are shown in Fig. 6; see Section IV,A,7, b. T h e lower glycine binding that occurred in the region of discontinuous sucrose gradients containing “heavy synaptosomal elements” of the brains of mice subjected to prolonged individual housing provided evidence that environmental modification can produce morphological changes that affect glycine binding sites. Studies on the binding of [3H]-strychnine in relation to environment have not yet been performed. C. TAURINE Although saturable “high-affinity” (Kaczmarek and Davison, 1972; Lahdesmaki and Oja, 1973; Schmid et a/., 1975) and “low-affinity” (e.g., Pasantes-Morales et al., 1972; Honegger et al., 1973) taurine uptake mechanisms have been shown in mammalian cerebrum and retina, a more recent investigation by Davidoff and Adair (1976) failed to reveal a Na+-dependent, saturable, “high-affinity” system for taurine in slices of frog spinal cord. These latter results corroborated the earlier findings of Gadea-Ciria et al. (1975) that synaptosomal fractions of 17 regions of the feline CNS exhibited either slight binding or no binding of taurine (7.5 x M). Thus, “high-affinity” Na+-dependent mechanisms perhaps play a minor role in the inactivation of taurine, if it is released as an inhibitory neurotransmitter. Since taurine appears to be metabolized slowly by neural tissue (Peck and Awapara, 1967), a metabolically linked mechanism for its inactivation also does not appear to exist. These negative results concerning taurine inactivation should not be taken to mean that taurine should not be further studied as a possible transmitter. Other studies (see, e.g., Sections I,C and I1,C) certainly indicate a neurophysiological role for taurine in vertebrates.
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FIG. 10. Specific [3H]-strychnine binding associated with the glycine receptor and [3H]-glycine uptake in embryonic chick spinal cords expressed per spinal cord and per milligram of protein, as percentages of values obtained for 1-day chicks. For binding experiments, tissue homogenates were incubated at 4°C for 20 min with 4 nM [3H]strychnine, with M glycine added to half the tubes serving to displace “specific” [SH]-strychninebinding. For uptake studies, crude synaptosomal preparations were incubated at 37°C for 4 min with [SH]-glycinewith unlabeled glycine (lO-sM) added to half the tubes. Values represent means of five (binding) or two (uptake) experiments, each performed in triplicate, varying less than 20%. (Reproduced with permission from Zukin etal., 1975.)
A M I N O ACID RECEPTORS
187
D. GLUTAMATE A N D ASPARTATE Biochemical studies on the subcellular distributions (Wofsey et al., 1971) and kinetic properties of the transport systems for glutamate and aspartate (e.g., Logan and Snyder, 1972; Bennett et al., 1972; Neal and White, 1973; Balcar and Johnston, 1973) with mammalian nervous tissues have led to the beliefs that a “unique synaptosomal fraction” selectively accumulates both glutamate and aspartate and that these amino acids may be transported by the same system. However, more recent studies on the binding of these amino acids to subcellular and protein fractions of CNS tissues have enabled some separation of these amino acids. I n rat brain homogenates, two types of glutamate binding, a “highaffinity” (KB s 2 X lo-’ M) and a “low-affinity” (KB = 4.4 x lo4 M) type, have been described (Michaelis et al., 1974). “High-affinity” binding was associated primarily with plasma membrane fractions, and, in particular with a synaptic membrane fraction, was strongly stereospecific for L-glutamate and was almost totally reversible. This glutamate binding activity did not depend on Na+, thus distinguishing it from the glutamate uptake system (see above), and was partially prevented by excitatory and inhibitory amino acids, but not by amino acids devoid of neuropharmacologic activity. L-Aspartate competitively inhibited ( K , = 1 x M) this binding of glutamate, in agreement with observations made on the locust neuromuscular junction (Dowson and Usherwood, 1972; see also below). GABA and glycine blocked glutamate binding noncompetitively, possibly by allosteric interactions with its binding site, M and 4.5 x their respective K , values being about 1 x M. A further study revealed that this glutamate binding species is a lowmolecular-weight ( 13,800) acidic glycoprotein (Michaelis, 1975). Inhibitory amino acids (e.g., GABA) did not affect the binding of L-glutamate to this glycoprotein, whereas excitatory amino acids and glutamate diethyl ester, as with the synaptic membrane preparation (Michaelis et al., 1974), were effective inhibitors. In a related study, Roberts (1974) showed a K Bvalue of about 8 x 1OPfiMfor the Na+-independent binding of L-glutamate to a synaptic membrane fraction of rat brain; this binding was weakly inhibited by o-glutamate but strongly inhibited by L-aspartate, DL-homocysteate, and L-glutamate diethyl ester. Fiszer de Plazas and DeRobertis ( 1975) have isolated a hydrophobic protein fraction from rat cerebral cortex that binds glutamate and GABA to separate sites, the binding of L-glutamate not being affected by GABA. However, in contrast to shrimp muscle (Fiszer de Plazas and DeRobertis, 1973; see also Section V), the protein fraction binding glutamate could not be separated from that binding GABA. In a further
188
F. V. DEFEUDIS
study, a hydrophobic protein fraction binding ~-[‘~C]-glutamate was separated from the total lipid extract of nerve-ending membranes or homogenate of rat cerebral cortex (DeRobertis and Fiszer de Plazas, 1976a), and although the glutamate binding species could only be partially separated from that binding GABA, these two amino acids appeared to bind to different sites. High (KB 3 X lO-’M), medium (KB 5 X M), and low (KB 5.5 x M) affinity binding sites were demonstrated for glutamate. The “high-affinity” binding site had a capacity of 0.53 nmole/mg protein and was highly stereoselective for L-glutamate. T h e binding of ~-[‘~C]-glutarnate was not inhibited by GABA, was slightly inhibited by glycine and glutamine, and strongly inhibited by DL-a-methylglutamate, L-nuciferine, L-aspartate, and L-glutamate diethyl ester. Use of a wide concentration range of glutato 2.5 x M) led to the demonstration of this mate (6 x “triple-affinity” mechanism; such wide ranges were not employed by Roberts (1974) o r by Michaelis et al. (1974) (see above). The glutamatebinding protein isolated previously from shrimp muscle (Fiszer de Plazas and DeRobertis, 1973, 1974) possessed only a single type of binding site M, but it should be noted that a narrow with a KB = 1.3 X concentration range of L-glutamate (1.8 X to 1 X 10-4M) was used in these experiments. The effect of L-aspartate on the binding of L-glutamate reminds one of the report of Curtis and Watkins (1960a) that both these amino acids could interact with a common receptor. Aspartate also potently inhibits glutamate binding to membranes (see above) and the Na+-dependent uptake of glutamate (e.g., Logan and Snyder, 1972). Thus, glutamate-receptor sites and glutamate-uptake sites may have some common properties. Fiszer de Plazas and DeRobertis (1976) also showed that a hydrophobic protein fraction from rat cerebral cortex possessed three saturable binding sites for ~-[‘~C]-aspartate with apparent KB values of 2 X lo-’, 1 X lod5,and 5 X M ;respective binding capacities of these three sites were 2.8, 132, and 617 nmole/mg protein. There were about 8 nmole of “high-affinity” binding sites for L-aspartate and about 1.5 nmole for L-glutamate per gram of fresh rat cerebral cortex. Differentiation between the binding of L-aspartate and L-glutamate was clearly shown by cross-binding and competition experiments with agonists and antagonists. These results indicated that there might exist separate aspartate and glutamate receptors in rat cerebral cortex (see also DeRobertis and Fiszer de Plazas, 1976b). However, it has not been determined whether these binding sites reside on a single or on different proteins. Since these studies were carried out in the absence of Na+, and since several of the compounds used to antagonize the binding (e.g.,
AMINO ACID RECEPTORS
189
N-methybaspartate, kainate; see Fig. 11) are not substrates for the transport systems (e.g., Balcar and Johnston, 1972a,b),such binding sites could be related to synaptic receptors. Simon et al. (1976) found that [3H]-kainatewas bound specifically to a M. synaptic membrane fraction of rat brain with a KB = 6 x Quisqualic acid was about one-third as potent, and L-glutamate about 1/25th as potent as kainate at displacing bound [3H]-kainate (Fig. 12). “Specific” kainate binding was localized to cerebral gray matter, was most enriched in crude synaptosomal membrane preparations, and varied by five- to sixfold in various brain regions. Interestingly, aspartate possessed relatively low affinity for kainate binding sites (Simon and Kuhar, 1975; Simon et al., 1976), indicating that separate glutamate and aspartate receptors might exist in the brain. Kainate, although possessing great potency for glutamate receptors, apparently lacks affinity for glutamate “high-affinity” uptake sites. Other studies have revealed, by indirect means, a difference between glutamate and aspartate receptors. Young et al. (1974b) showed that virus-induced destruction of cerebellar granule cells in hamsters caused a 40% decrease in endogenous glutamate concentration with no change in any other amino acid; “high-affinity,’’Na+-dependent glutamate and aspartate uptakes were, however, both reduced by 85%, whereas the uptakes of other amino acids were not lowered. Another recent study revealed, in the presence of Na+, that glutamate was bound to a greater coi I
L-GLUTAMATE
L-ASPARTATE
KAINATE
N ME THYL- D- ASPARTATE
-
FIG. 11. Structural formulas of L-glutamate and L-aspartate and their respective analogues, kainate and N-methybaspartate. (Reproduced with permission from DeRobertis and Fiszer de Plazas, 1976b.)
190
F. V. DEFEUDIS
z
z
20
MOLARITY OF DISPLACER
FIG. 12. Displacement of specific ['HI-kainic acid binding by unlabeled kainic acid and L-glutamate. Increasing concentrations (lo-'' to M ) of unlabeled kainic acid and L-glutamate were added to the tubes for displacement of specific ['HI-kainic acid binding. (Reproduced with permission from Simon et al., 1976.)
extent than aspartate to synaptosome-enriched fractions of several regions of the feline brain, except for cerebral cortex in which the binding of both amino acids was about equal (DeFeudis et aZ., 1976~).Another partial separation of glutamate from aspartate has been achieved with rat synaptosomal fractions; in Na+-free media, Li+ supported the uptake of glutamate to a lesser extent than that of aspartate (Peterson and Raghupathy, 1974). With regard to maturation, Na+-dependent binding of glutamate to synaptosome-enriched fractions of rat brain was similar to that of GABA and glycine (DeFeudis, 1973a). V. Biochemical Studies with Invertebrate Preparations
A. GABA DeRobertis and Fiszer de Plazas (1974) showed that a "high-affinity'' binding of [14C]-GABA(KB= 8 X M) occurred to a hydrophobic protein fraction (free of lipid phosphorus) isolated from shrimp (Arternisiu Zonginaris) muscle (see also Fiszer de Plazas and DeRobertis, 1973). GABA binding to this protein fraction tended to saturate between 2-3 X
AMINO ACID RECEPTORS
191
10-5M;at saturation, 1 mole of GABA was bound to about 80,000 gm of protein. A Hill coefficient of about 1.O and the hyperbolic nature of the saturation curve indicated a single type of binding site with no cooperativity. BIC (2.1 x M) produced about 50% inhibition, and at 5 X M it caused about 80% inhibition of GABA binding. PIC inhibited GABA binding by about 23% at M and by about 33% at lop4M. It is noteworthy that BIC was more potent than PIC at blocking GABA binding to this protein, since BIC appears to exert a weak, noncompetitive GABA-antagonistic action in crustacea (Takeuchi and Onodera, 1972; Earl and Large, 1972; Swagelet al., 1973). Muscimol was about as potent as PIC at inhibiting GABA binding, 3-(pchloropheny1)-GABA produced slight inhibition, and glutamate did not bind to this protein. Fiszer de Plazas and DeRobertis (1975) have compared the properties of a GABA-binding protein of rat cerebral cortex (see Section IV,A,2) with those of this crustacean protein. Besides having different chromatographic properties, the KB value of the crustacean protein was about 3.7 times smaller than that of the cortical protein. However, the major difference concerned the K i value of BIC; for the crustacean protein this was about 0.9- 1.4 x 10-5M, or only about 1/20th that of the cerebral cortex protein (2.7 X lo4 M). The idea that GABA receptors may differ among various species is therefore emphasized. This demonstration of a single type of binding site lacking cooperative interaction does not agree with the Hill coefficient of about 2 calculated by Takeuchi and Takeuchi ( 1967) based on electrophysiological studies of membrane conductance. However, as suggested by DeRobertis and Fiszer de Plazas (1974), measurement of the binding of GABA to an isolated receptor should not necessarily be expected to give the same result as measurements of the receptor in situ. Equilibrium dialysis was used to measure the binding of [l4C]-GABA to particulate fractions of tail or claw muscle of crayfish in Trisbuffered Van Harreveld's solution at 0°C (Olsen et al., 1975b). GABA binding saturated at about 7 pmole/mg protein (about 2 X lo-'' mole/ gm wet tissue) and had a KB = 1.3 x lops M (see also Olsen, 1976). In accord with the results of DeRobertis and Fiszer de Plazas (1974), a single type of binding site with no apparent cooperativity appeared to be involved. PIC (3 x lop4M) did not affect GABA binding, but BIC was a weak inhibitor ( K i GZ 3.5 X lop4M), as was chlorpromazine (see Table IV). Experiments with various agents revealed that binding, as measured, agreed well with that of synaptic receptors and differed significantly from that of GABA transport, leading these workers to suggest that their binding system might be useful for measuring GABA-receptor sites. Binding sites of crayfish muscle differed from those of rat brain
192
F. V. DEFEUDIS
(e.g., Peck et al., 1973, 1976); Zukin et al., 1974) and shrimp muscle (DeRobertis and Fiszer de Plazas, 1974).
B. GLUTAMATE AND ASPARTATE Lunt (1973) extracted and solubilized a crude protein fraction from locust (Schistocerca pegaria) muscle which showed “high-affinity”binding for glutamate but practically no binding of aspartate or glutamine. Using organic solvent extraction followed by chromatography, two protein fractions were isolated which exhibited specific binding for glutamate. It was concluded that these hydrophobic proteins could constitute part of the glutamate receptor at the insect NMJ. The glutamate analogue, ~~-2-amino-4-phosphonobutyric acid, competitively inhibited the binding of glutamate to these receptor-like hydrophobic proteolipids with a KB 6.6 X lo4 M (James et al., 1974; Cull-Candy et al., 1976). Fiszer de Plazas and DeRobertis (1974) isolated from shrimp muscle two hydrophobic protein fractions, one of which possessed specific binding activity for glutamate. This shrimp protein bound glutamate with aKB 1.3 x lou5M, had a single type of binding site, and saturation was achieved at about 1 mole of ~-glutamate/320,000gm protein. This glutamate-binding protein did not bind GABA, aspartate, or glutamine, and the binding of ~-[‘~C]-glutamatewas inhibited by DL-Qmethylglutamate and L-glutamate diethyl ester. VI. Relevant Studies on Glial Cells
Many studies have focused on the relationships between glia and neuronal function (see, e.g., DeRobertis, 1965; Sotelo, 1967; HydCn, 1973). Lugaro (1907) suggested that glia might inactivate products of neuronal activity and thereby exert a controlling influence on the extraneuronal ionic milieu. This concept has been strengthened by findings that revealed the close investment of synapses by glial membranes (e.g., Peters and Palay, 1965). Studies with microelectrodes (e.g., Kuffler and Nicholls, 1966), with hand-dissected samples (e.g., HydCn, 1967, 1973), and with glia- and neuron-enriched subcellular fractions (e.g., Rose, 1969) have contributed greatly to our understanding of glial function. Glia possess potent uptake systems for the “active”amino acids, and therefore, they might be involved in the inactivation of released synaptic transmitters. KrnjeviC and Schwartz ( 1967)observed that electrically unresponsive
AMINO ACID RECEPTORS
193
cells of the feline cortex were depolarized by GABA, and they suggested that this depolarization might represent an active transport of GABA coupled to the electrogenic Na+ pump and that glial cells might be involved. Indirect evidence was thus provided for a role of glia in the inactivation of GABA. An accumulation of GABA by glial cells was demonstrated (Hokfelt and Ljungdahl, 1970; see also below). Henn and Hamberger (1971) then showed that in 30-40 min a glial fraction concentrated GABA (6 X lO-’M) over 100-fold from the medium, but that a neuronal fraction concentrated GABA only about fourfold; GABA uptake by glial cells was about 30%-50% that of synaptosomal fractions. The presence of a “high-affinity”GABA uptake system in glia (Km= 2.7 X M) supported the notion that glia are involved in transmitter inactivation. Another significant contribution was made by Bowery and Brown (1972b), who showed that “high-affinity’’ mechanisms for the uptake of GABA, /3-alanine, and taurine (all at 5 x lo-’ M) exist in sympathetic ganglia and other nervous tissues that are considered to be devoid of “GABA-ergic” neurons. It seems likely that glial cells may be involved in this uptake mechanism, since depolarization of sympathetic ganglion cells by carbachol M) or by stimulation of pre- or postganglionic trunks did not affect the efflux rate of previously accumulated GABA, whereas this GABA was released from the ganglia by high K+ concentrations or by direct electrical stimulation. Studies with tissue cultures have also revealed that GABA and other “active” amino acids are taken up extensively by glial elements (see Section VII). Hokfelt and Ljungdahl (1970) used light microscopic autoradiography to examine the cellular localization of [3H]-GABA (4 x M) that was taken up by slices of rat cerebellar cortex. After 45 min, glia (e.g., oligodendroglia; pericytes of blood vessels; Bergmann glia) were labeled by [3H]-GABA (see also Hokfelt and Ljundahl, 1972a,b,c). T h e glial reaction was most pronounced after administration of M in vitro; 40 mg/kg, i.p. in rat, in aminooxyacetic acid (AOAA) viuo), which prevents GABA catabolism (Wallach, 1961). Ljungdahl and Hokfelt (1973) also showed a marked accumulation of GABA by glia of spinal cord both in vivo and in vitro, and CABA was accumulated mainly into Muller (glial) cells of rat retina incubated in vitro (Neal and Iversen, 1972). Other studies revealed a glial uptake of GABA by sympathetic ganglia (Bowery and Brown, 1972b; Young et al., 1973), sensory ganglia (Schon and Kelly, 1974a,b, 1975), and brain tumors (Snodgrass and Iversen, 1974). Hokfelt and Ljungdahl (1972b) noted also that glutamate uptake by
194
F. V. DEFEUDIS
rat cerebellar slices was strictly confined to glia. In rat, rabbit, and frog retinas, accumulated glutamate was localized predominantly in glial cells (Ehinger and Falck, 1971; Ehinger, 1972; Kennedy et al., 1974; White and Neal, 1976). [3H]-Glycinealso produced an autoradiographic reaction over glia in slices of spinal cord (Hokfelt and Ljungdahl, 1971a,b; Ljungdahl and Hokfelt, 1973). P-Alanine was taken up by amacrine cells of rabbit retina, whereas aspartate and taurine (like glutamate) were found in glial elements (Ehinger and Falck, 1971; Ehinger, 1972, 1973; Brunn et al., 1974). Other autoradiographic studies revealed that in rat brain slices p-alanine is taken u p mainly by glia, whereas GABA is taken up mainly by nerve terminals (Iversen and Bloom, 1972; Schon and Kelly, 1975). Although both uptake systems showed relatively high affinity and Na+ dependency (see below), the V,,, of /3-alanine uptake by brain slices was about an order of magnitude lower than that for GABA (Iversen and Neal, 1968; Iversen and Kelly, 1975; Schon and Kelly, 1975). Schon and Kelly ( 1974a,b), using autoradiographic methods, showed that the uptakes of both [3H]-GABA and [3H]-glutamate into rat sensory ganglia (which are devoid of any synaptic innervation) were exclusively localized to satellite glial cells. GABA was also accumulated by glial cells surrounding unmyelinated axons and by the Schwann cell bodies of the large myelinated fibers. Glycine and a-alanine were taken u p to much lesser extents than GABA or glutamate and were localized equally within neuronal and glial cell bodies. Schon and Kelly (1974b) found a K , SE 1x M for the Na+-dependent, “high-affinity” uptake of pH]-GABA into satellite glial cells, and this mechanism was not inhibited by bicuculline, picrotoxin, glutamate, glycine, or glutamine. Using this glial uptake model it was shown that ~ - 2 , 4 diaminobutyrate was about 20 times more potent as an inhibitor of GABA uptake in cortical slices than in sensory ganglia, whereas p-alanine was about 200 times more potent at inhibiting GABA uptake by sensory ganglia (Schon and Kelly, 1974b). [3H]-fi-alanine was rather exclusively accumulated by glial sites in both sensory ganglia and slices of rat cerebral cortex, and a virtually identical “high-affinity” process for [3H]-/3-alaninewas demonstrated at both these sites. For further information about these autoradiographic studies, the reader is referred to the review by Hokfelt and Ljungdahl (1975). The uptake of GABA by glia is far slower than its neuronal uptake by tissue slices or synaptosomal fractions of rat brain (Bowery and Brown, 1972b; Schon and Kelly, 1974b, 1975). Since the slow nature of l3H1-palanine (6.7 x M) uptake by synaptosomal fractions, brain slices, and isolated sensory ganglia and the relatively low tissue: medium ratios
AMINO ACID RECEPTORS
195
at 25°C resembled characteristics of the GABA uptake process into sensory ganglia, Schon and Kelly (1975) proposed that p-alanine was taken up by the glial GABA transport system. Further indirect support that glial GABA uptake in cerebral cortex is the same as that in sensory ganglia stemmed from their findings of nearly identical ICBOvalues for GABA inhibition of [3Hl-/3-alanine (6.7 x M) uptake into synapM), and cortical slices tosomal fractions ( 1.8 X 1O+ M), ganglia (2 X (3.2 x 10-5 M ) . Even though such a glial uptake system could contribute significantly to the total GABA uptake by various tissue preparations, especially at room temperature or at 37°C for rather long (>20 min) incubation periods, it appears likely that it plays no significant role in “binding” studies carried out at low temperatures for 5-15 min. Some evidence for this contention has recently been provided by results obtained in a study of the binding of p-alanine and GABA to synaptosome-enriched fractions of rat brain. After 10-min incubations at 0°C with [3H]-GABA and [14C]-/3-alanine( - 10-4M), a marked binding of GABA occurred but the binding of P-alanine was slight or negligible; this was especially well demonstrated by comparing distribution ratios for these two amino acids (see Fig. 13). Hence, the suggestion of Enna and Snyder (1975) that Na+-dependent GABA binding at 0°C might occur at glial rather than at neuronal sites may not be valid. Also, Schon and Kelly (1975) have pointed out that even in the case of active uptakes of GABA with 10-min incubations the ratio of “nerve-termina1:glial” uptake is about 30: 1. Johnston and Stephanson (1976) have suggested that it may be misleading to consider that the same glial system mediates the uptake of both p-alanine and GABA, since GABA uptake by this system has not been demonstrated in rat brain slices, and that the finding that GABA is a competitive inhibitor of p-alanine uptake (Schon and Kelly, 1975) does not provide direct evidence that GABA serves as a substrate for this system. Also, a recent study by Roberts (1976), based on the proposal of Levi and Raiteri (1974) that homoexchange (of intra- and extracellular amino acid) might account for apparent “high-affinity” uptake by synaptosomes, has revealed that the presumed “uptake” of [3H]-GABA by rat sensory ganglia might largely reflect its homoexchange with endogenous (intracellular) GABA (see also Simon et al., 1974; DeFeudis, 1975a; Martin, 1976). In any case, the findings of Schon and Kelly (1975) led Johnston and Stephanson (1976) to search for substances that are more potent inhibitors of p-alanine uptake than of GABA uptake so that these might be used to test the role of glial uptake in the inactivation of GABA (or p-alanine) by iontophoretic methods. p-Alanine, itself, cannot be used
F. V. DEFEUDIS
io 20
do
tbo
0
(GABA,y&l?)
FIG. 13. Distribution ratios for the “binding” of GABA (a) and 8-alanine (b) to a synaptosome-enriched fraction of rat cerebral cortex in physiological medium. Corrected distribution ratios were calculated using the equation: D , - S.Sp. 1 - s.sp. in which D, = corrected distribution ratio, D , = measured distribution ratio, and S.Sp. = distribution ratio of sucrose, which provided an estimate of the amount of supernatant fluid trapped in the pellets. The value of S.Sp. used was 0.71 f 0.02 (326)(mean f S.E.M.; number of determinations in parentheses; DeFeudiset al., 1975). Means +. S.E.M.; nine determinations in all cases; S.E.M. not indicated were within the symbols, themselves. (Unpublished graphs; data in Somoza et al., 1977.)
D, =
since it exerts a potent direct depressant action on central neurons (Curtis and Watkins, 1960a; Biscoe et al., 1972). Using small slices of rat cerebral gray matter, the uptakes of [3H]-&alanine (3 X lo-’’ M) or [14C]-GABA(4 x lo-’ M) were monitored for 10 min at 37°C after 15-min preincubations of the slices with the possible inhibitors (at M). Several substances selectively inhibited p-alanine uptake, and of these, 2,2-dimethyl-P-alanine, ~-2,3-diaminopropionate, O-phosphorylethanolamine, and AOAA might be useful in iontophoretic experiments on spinal neurons, since they d o not directly affect the firing of these neurons (Curtis and Watkins, 1960a; Curtis et al., 1968a; Johnston et al., 1975).
AMINO ACID RECEPTORS
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Schon and Kelly (1975) further suggested, based on their finding that p-alanine was taken up exclusively by glial cells, that this and the previous finding that endogenous p-alanine is present in minute amounts in mammalian brain (about 3-8 x mole/gm wet weight; Tallan et al., 1954; Yoshino et al., 1970) virtually precluded its consideration as a neurotransmitter. However, much other evidence indicates a possible transmitter role for p-alanine (see, e.g., references in Johnston and Stephanson, 1976), and furthermore, Brunn et al., (1974) have recently demonstrated its uptake into rabbit retinal neurons (see also Brunn and Ehinger, 1974). In any case, a low concentration of p-alanine in the CNS (i.e., about the same as, or greater than, those of norepinephrine, dopamine, and ACh in some CNS areas) certainly does not preclude its possible role as a neurotransmitter, and neither does the possibility that its inactivation could involve a purely glial "re-uptake" mechanism. Other studies, performed on isolated desheathed rat superior cervical ganglia in the presence of AOAA ( 10-5M) at 25"C, have revealed that glia might be involved in the indirect GABA-mimetic effect of p-alanine and P-amino-n-butyric acid (BABA) (Bowery et al., 197613). Preincubation of ganglia with 1 X loF3M GABA (60 min) potentiated neuronal depolarizations produced by p-alanine or BABA but not those produced by GABA or 3-aminopropanesulfonic acid (a potent receptor agonist with low affinity for the GABA uptake system). Bowery et al. (1976b) concluded that P-alanine and BABA elicit a release of GABA from glia into interstitial spaces; thus, the depolarization of ganglion cells that they produce (in low doses, at least) might be caused by their release of GABA rather than by their direct actions on GABA receptors. Invertebrate studies have also indicated that glia might be involved in the inactivation of transmitters. The uptake of [3H]-GABAby a lobster nerve-muscle preparation (Iversen and Kravitz, 1968) has been localized exclusively to Schwann cells that surround inhibitory synapses at the NMJ (Orkand and Kravitz, 1971). Also glutamate is localized in Schwann cells at the insect NMJ (Salpeter and Faeder, 1971; see also Usherwood, 1972). Many criticisms apply to the studies discussed in this section; some have already been mentioned. In general, except for cases in which GABA uptake was measured in the presence of AOAA, metabolism of the amino acids and their incorporation into protein (except for GABA and taurine) would be expected to influence their accumulation (or that of their metabolites) by glia, especially with prolonged incubations (>30 min) at 22"-37°C. This metabolism would affect both autoradiographic and biochemical analyses.
198
F. V. DEFEUDIS
Recent approaches employed by Benjamin and Quastel (1972-76), in which the effects of metabolic inhibitors and other drugs on the metabolic compartmentation of amino acids in slices of rat cerebral cortex were monitored, offer a means for overcoming this latter criticism. By using tetrodotoxin to suppress the release of amino acids from neurons caused by the combined action of protoveratrine and ouabain, Benjamin and Quastel (1972) found that the major pools of glutamate, aspartate, serine, and probably GABA, are located in neurons, whereas the major pool of glutamine is in glia. These findings, together with others obtained with slices incubated in the presence of fluoroacetate and malonate, revealed that glutamate which is released from neurons is taken up, in part, by glia, converted to glutamine, and then returned to neurons where it is converted to glutamate and GABA. Slices were also shown to contain two pools of glutamate, one of which is neuronal and is derived from glucose and exchanges only poorly with extracellular glutamate, the other of which is glial and is derived from extracellular glutamate and is freely exchangeable with extracellular glutamate (Okamoto and Quastel, 1972). These results have revealed the existence of a cycle of events in which neurons and glia are coupled. Thus, both neurons and glia appear to be involved in terminating the excitatory activity of extracellular glutamate (see also Benjamin and Quastel, 1974). Further studies which revealed a constancy of the glutamate-ammonia system under a variety of metabolic conditions led Benjamin and Quastel (1975) to suggest that, like their action on extraneuronal K+ (Okamoto and Quastel, 1970), glia appear to be involved in “buffering” the extracellular content of glutamate and perhaps other physiologically active amino acids that may be released from neurons during synaptic activation (see also Benjamin and Quastel, 1976). These studies have contributed much to our knowledge about the possible roles that glia may play in the regulation of functionally active amino acids of the brain. VII. Relevant Studies with Tissue Cultures
A. INTRODUCTION Tissue culture methods offer several advantages for electrophysiological, pharmacological, and biochemical analyses of the actions of “active” amino acids. Tissue explants or cultured cell lines can be studied under direct visual control, substances can be applied in controlled amounts, and the extracellular (ionic) environment can be controlled (e.g., Hosli et al., 1973c, 1974; Hokfelt and Ljungdahl, 1975). Such studies have confirmed some in vivo observations concerning changes in
AMINO ACID RECEPTORS
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membrane potential produced by “active” amino acids and actions of possible amino acid antagonists, as well as some mechanisms of in vitro amino acid uptake by other preparations. B. AUTORADIOGRAPHIC STUDIES ON ACIDS
THE
UPTAKE OF “ACTIVE” AMINO
These studies have recently been aimed at separating glial from neuronal uptake systems; the “binding” involved is related to Na+dependent uptake, except when using Na+-free media, in which cases the binding could involve synaptic receptor sites. A recent electron microscopic autoradiographic study with dispersed cell cultures of postnatal rat cerebellum revealed that GABA (0.3 or 1.0 X lov6M) was taken up by both neuronal and glial cells; grain density over GABA synapses was 92% greater than over glial cells (Burry and Lasher, 1975; see also Lasher, 1975). This GABA uptake by glia and neurons in culture was consistent with the “high-affinity” uptake of GABA reported for cultured spinal cord (Hosli et al., 1972) and glioma cell lines (Schrier and Thompson, 1972, 1974), as well as with that of satellite glia of dorsal root ganglia (Schon and Kelly, 1974a,b) and retinal Muller cells (Neal and Iversen, 1972; see also Section VI). Also, the results of Burry and Lasher (1975) and those of McLaughlin et al. (1974) indicated that presumed “GABA-ergic” nerve terminals were both asymmetrical and symmetrical in developing cerebral cortex, as opposed to being only symmetrical in the adult. This evidence for morphological changes in GABA synapses during development was in accord with other results which indicated that changes in the “binding” of GABA (and other amino acids) occur during maturation (see Sections IV,A,7,a and IV,B,4,u. Using intraocular transplants of cerebellar tissue, GABA uptake was shown to occur into Purkinje, stellate, Golgi, and possibly glial cells (Ljungdahl et al., 1973). A Na+-dependent uptake of glycine by glia and neurons of cultured rat spinal cord and medulla oblongata has also been shown by autoradiography (e.g., Hosli and Hosli, 1972; E. Hosli et al., 1972; L. Hosli et al., 1974; 1975b). [3H]-Glycine was also taken up by neurons and glia of explant cultures of fetal human spinal cord and brain stem (Hosli et al., 1974). Other studies have revealed that [3Hl-glutamate and [3H]-aspartate are taken up by both glia and neurons of cultured explants of human fetal and rat spinal cord (Hosli et al., 1973a, 1974; Hosli and Hosli, 1976), and that [3H]-glutamate is taken u p by neurons and glia of cultured human fetal brain stem (Hosli et al., 1974). These uptake
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F. V. DEFEUDIS
processes were temperature- and Na+-dependent, but less Na+dependent than the uptakes of GABA and glycine (L. Hosli and Hosli, 1972; E. Hosli and Hosli, 1976; Hosli et al., 1975a). C. ELECTROPHYSIOLOCIC-PHARMACOLOCIC STUDIES Tissue culture methods offer a direct approach for characterizing amino acid receptors and associated changes in membrane ionic permeability. Bicuculline (lo-' M) totally blocked the hyperpolarizing effect of M) on disaggregated neurons of 7-day cultures of neonatal GABA ( mouse brain (Bonkowski and Dryden, 1976). In explants of rat cerebellum, GABA ( M) depressed spontaneous firing of Purkinje cells by an action that was antagonized by a low concentration (10" M) of BIC M PIC (Gahwiler, 1975). Gahwiler (1976a) has shown also and by that benzodiazepines exert potent GABA-antagonistic actions on Purkinje cells of rat cerebellar explants. With regard to antagonism of the effect of GABA ( M) of blocking spontaneous activity of these cells, diazepam had an EC5,-,= 2.4 x 10-lOMand chlordiazepoxide had an ECm = 1.5 X 1O-O M; GABA and drugs were bath-applied. Preliminary experiments revealed further that bath-applied diazepam ( 1O-O M) partially blocked the action of iontophoretically applied GABA (Gahwiler, 1976a). Iontophoretically applied glycine hyperpolarized membranes of cultured rat spinal and medullary neurons (e.g., Hosli et al., 1971, 1973c), changes that were associated with increases in membrane conductance (see Fig. 14). Hosli et al. (1973c, 1974, 1975a) showed also that glycine ( M) alters the C1- permeability of rat and human spinal neurons in culture. Thus, cultured neurons appear to possess glycine receptors that are similar to those of spinal motoneurons in situ. Glycine exerted no effect on glial cells (Hosli et al., 1975b). Regarding antagonists, studies by Crain (1966) and by Crain and Peterson (1967) showed that strychnine enhanced bioelectric responses in cerebral and spinal cord cultures. Strychnine also exerted an excitatory influence on cultured rat Purkinje M), but higher cells when applied at low concentrations (lo-* concentrations of strychnine (>1Od5 M) inhibited spontaneous activity M glycine was needed to pro(Gahwiler, 1976b). However, since duce a sustained depression of firing and since this effect was insensitive to strychnine, it was considered to be nonspecific (see also Schlapfer, 1969; Geller and Woodward, 1974). However, Crain (1972, 1974) had shown earlier in spinal cord explants of mouse, that glycine depressed synaptically mediated discharges by an action that could be prevented by
AMINO ACID RECEPTORS
20 1
A mV
-- 35 05
i
-
20 sec
B Glut
-- 46 0
f
C
- 64
c
~ o - ~ M
-
20 sec
-
20 sec
FIG. 14. Depolarization by glutamate (Glut M) and hyperpolarization by glycine (Glyc lO-'M) of spinal neurons in tissue culture. A, Neuron of a human fetus (1 1 weeks in ulero; 12 days in vitro).B, Rat spinal neuron (16 days in vztro). C, Human spinal neuron (15 days in vitro; fetus 1 1 weeks in utero). Durations of perfusions with solutions containing amino acids are indicated by horizontal bars above the tracings. Ordinate: membrane potential in millivolts. Time bar represents 20 sec. (Reproduced with permission from Hosli et al., 1974.)
concomitant addition of strychnine. Application of taurine to cultured spinal neurons of rat and human produced an action similar to that of glycine (i.e., membrane hyperpolarization and increased C1- permeability), but taurine was frequently less potent (Hosli et al., 1975a,b). Glutamate and aspartate ( or 10-3M) depolarized cells of rat and human spinal cord cultures by actions that were reversed in Na+-free media (Hosli et al., 1973a, 1974, 1975a,b; see also Fig. 14). Spinal cord cells, cultured from fetal mice, generally responded to iontophoretically applied L-glutamate and L-aspartate with depolarizing potentials, one group of glutamate-induced depolarizations having an equilibrium potential between -30 and 0 mV (Ransom and Nelson, 1975). Hence, in accord with in vivo studies, the depolarizing actions of these acidic amino acids appear to depend mainly upon an increase in Na+ permeability of the membrane. Glutamate M), like glycine, had no effect on cul-
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tured glial cells (human and rat astrocytes), indicating that glia may not have specific receptors for these amino acids (Hosli et al., 1975b;see also Wardell, 1966). D. OTHERBIOCHEMICAL STUDIES
Hutchison et al. (1974)showed that clonal astrocytoma and neuroblastoma cells lines take up GABA by Na+-dependent “high-affinity” mechanisms ( K , = 2 x M), thus providing further evidence that glia might be involved in the inactivation of GABA. Lasher (1975) showed, in dispersed cell cultures of rat cerebellum, that “GABA-ergic” neurons had an average velocity of uptake that was several orders of magnitude greater than that for nonneuronal cells, but that the K, values were both about 3 x lo-’ M. Interestingly, no “low-affinity” system was detected in nonneuronal cells, whereas both “high-” and “lowaffinity” systems were evident in neuronal cells. Calculations revealed that at concentrations of 5 x 10-9Mto > 10-3M,over 99% of the GABA should be accumulated by “GABA-ergic” neurons, given equal access of all cells to the label. About 75%-85% of the total uptake of [3Hl-GABA (4.4x M) by both neurons and nonneuronal cells was Na+- and temperature-dependent.
E. CRITICISM Some advantages of culture methods have already been mentioned (see Section VI1,A). However, it should be realized that there are several distinct disadvantages to these methods (see, e.g., Ransom and Nelson, 1975; Hokfelt and Ljungdahl, 1975). For example, GABA (or other substances) can be lost from cells during preparation for autoradiography (see, e.g., Lasher, 1974,1975).Also, the morphology of cultures only partially resembles in situ morphology, and a preferential loss of cellular elements can occur (Hokfelt and Ljungdahl, 1975),but note that Lasher and Zagon (1972)have taken advantage of such a preferential cellular loss. Tissue transplants offer certain advantages over tissue culture (see, e.g., Olson and Seiger, 1972; Hoffer et al., 1974).
VIII. Concluding Remarks
Binding and iontophoretic studies have contributed greatly toward the elucidation of mechanisms involved in receptor interaction and synaptic inactivation of “active” amino acids. Some properties of amino
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acid receptors and the possible role of glia in inactivation processes have been realized. However, approaches to amino acid receptors remain limited due to a lack of specific antagonists. Both vertebrate and invertebrate findings have revealed that the actions of the most potent and reliable GABA antagonists, PIC and BIC, act indirectly, perhaps by influencing GABA ionophores, and that these agents and the glycine antagonist, strychnine, exert many nonspecific actions. Thus far, glutamate/aspartate antagonists (e.g., HA-966, GDEE) have not been very useful for distinguishing between the excitations elicited by these amino acids. In general, proposed amino acid antagonists appear to modify receptor properties rather than interacting specifically with receptors. Regarding binding studies, strict comparisons of results obtained in various laboratories are usually not possible because of the use of different experimental procedures. However, regardless of the neural tissue preparation (e.g., synaptosomal fractions; small slices of CNS tissue; isolated hydrophobic protein) or the techniques used, a “high-affinity” system for GABA “binding” or uptake has been demonstrated that has a M. Such a “high-affinity’’ half-maximal constant of about 2 x GABA uptake by certain preparations (e.g., glia of sensory ganglia) could be largely due to homoexchange of added GABA with GABA of the intracellular pool (Section V1; see also Roberts, 1976). Most recently, “triple-affinity” mechanisms for the binding of GABA and glycine to synaptosome-enriched fractions of rat cerebral cortex (DeFeudis and Schiff, 1975) and for the binding of glutamate and aspartate to hydrophobic protein fractions of rat cerebral cortex (DeRobertis and Fiszer de Plazas, 1976b) have been demonstrated (Section IV). In such cases, “highest-affinity’’ processes possessed K B values of about 2 X lo-’ M. Results obtained in several recent studies, at both physiological and biochemical levels, have indicated that a separation of glutamate from aspartate receptors can be achieved (Section IV, D; see, e.g., DeRobertis and Fiszer de Plazas, 1976b). However, other evidence indicates a considerable overlap of these two systems. Structure/activity studies performed in vitro and in vivo have indicated that GABA may act with BIC-sensitive receptors in an “extended” conformation and with BIC-insensitive receptors in a “folded” conformation, and that different conformations of GABA are involved in its interaction with its receptor and uptake system (Johnston, 1977). I n other studies, BIC inhibited GABA binding to cerebral synaptosomal and synaptic membrane fractions in the absence of added Na+ (e.g., Zukin et al., 1974; Peck et al., 1976). The potencies of agents to inhibit this Na+-independent GABA binding, which could be associated with
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GABA receptors, paralleled their neuropharmacological actions at synapses much better than did Na+-dependent binding o r synaptosomal GABA uptake (Section IV,A). GABA binding to synaptosome-enriched fractions was also inhibited by BIC-methiodide in the presence of physiological concentrations of Na+ and other ions (DeFeudis et al., 1975). Two interesting model systems have emerged from recent studies. Sympathetic ganglia can serve as a model for the study of CNS GABA receptors (e.g., Bowery et al., 1975a,b), and an in vitro system of superfused brain tissue can be used to simultaneously examine amino acid receptors and ionophores (Okamoto et al., 1976). Developmentally linked changes in Na+-dependent and Na+independent binding of GABA and glycine have been shown (Sections IV,A,7 and IV, B,4L Other findings have revealed that the binding of GABA and glycine to cerebral synaptosomal particles can be affected by long-term changes in environment (e.g., Section IV,A,7). These results add support to the view that the brain exhibits structural plasticity when an animal is adapting to conditions that produce dramatic changes in its behavior, It seems likely that further attempts to characterize, identify, and isolate receptors for “active” amino acids in both vertebrate and invertebrate preparations will be fruitful. In addition to continuing the search for specific amino acid receptors, perhaps more emphasis should now be placed on variables that affect both these receptors and the behavior of the organisms. Such studies might be conducted by analyzing receptor properties in relation to development, environmental modification, and genetic differences, all of which are major determinants of animal behavior. Only by correlating the data obtained thus far with these variables will the physiological roles of these amino acids and the significance of their receptors be fully understood. REFERENCES Aghajanian, G. K., and Bloom, F. E. (1967). Brain Res. 6, 710-727. Agrawal, H. C., Davison, A. N., and Kaczmarek, L. K. (1971).Biochem.J. 122, 759-763. Alid, G., Valdes, L. F., and Orrego, F. J. (1974). Experientia 3 0 , 2 6 6 2 6 8 . Altmann, H., ten Bruggencate, G., Pickelmann, P., and Steinberg, R. (1976). Brain Res. 111,337-345. Balcar, V. J.. and Johnston, G. A. R. (1972a).J. Neurobiol. 3, 295-301. Balcar, V. J., and Johnston, G. A. R. (1972b). J. Neurochem. 19, 2657-2666. Balcar, V. J., and Johnston, G. A. R. (1973).J . Neurochem. 20, 529-539. Baldessarini, R. J.. and Karobath, M. (1973). Annu. Rev. Physiol. 35, 273-304. Balfagon, G., Gervas-Camacho, J., Gadea-Ciria, M., Somoza, G., and DeFeudis, F. V. (1975). Exp. Neurol. 48, 383-386. Banna, N. R., and Jabbur, S. J. (1969).Int. J. Neuraphrmacol. 8, 299-307.
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PRESYNAPTIC INHIBITION: TRANSMITTER AND IONIC MECHANISMS By R A. Nicoll and B. E. Alger
Dqmhnentr of Phamw~olcgyand Phyriokgy Univrnity of California Son Fmncirco, California
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Presynaptic Inhibition in Invertebrates . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Electrophysiological Mechanism of Presynaptic lnhibition . . . . . . C. Anatomical Considerations of Presynaptic Inhibition . . . . . . . . . D. Pharmacology of Presynaptic Inhibition . . . . . . . . . . . . . . . E. Electrical Transmission and Presynaptic Inhibition . . . . . . . . . . 111. Presynaptic Inhibition in Vertebrates . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Demonstrating the Presence and Location of PAD . . . . . . . . . . C. The Origin and Nature of PAD . . . . . . . . . . . . . . . . . . D. Evidence that CABA Is a Transmitter of PAD . . . . . . . . . . . E. Properties of CABA Responses . . . . . . . . . . . . . . . . . . . F. Effects of PAD on Synaptic Transmission . . . . . . . . . . . . . . IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Although the existence of a presynaptic form of inhibition was originally inferred from studies in the 1950s in the mammalian spinal cord, our current understanding of this type of inhibition is derived to a large extent from work done on the crustacean neuromuscular junction. However, recent studies have greatly advanced our knowledge of presynaptic inhibition in vertebrates and indicate that there is a remarkable similarity between the findings. In this review we will compare the properties of presynaptic inhibition in vertebrates and in invertebrates, and will concentrate on the pharmacology and ionic mechanisms. Various aspects of this review have recently appeared in other reviews (Atwood, 1976; Burke and Rudomin, 1977; Levy, 1977). Other aspects of presynaptic inhibition such as its organization are covered in earlier reviews (Eccles, 1964; Schmidt, 1971). No attempt will be made to cover in general the rapidly growing area of presynaptic receptor pharmacology. There is considerable evi-
INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 21
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dence based largely on pharmacological and biochemical studies that the release of transmitter can be modified by the action of neurotransmitters on a variety of presynaptic receptors (Usdin and Bunney, 1975; Starke and Endo, 1976). However, in most cases there is little evidence for a physiological role for these presynaptic receptors. 11. Presynaptic Inhibition in Invertebrates
A. INTRODUCTION At the conclusion of their study on postsynaptic inhibition at the crab neuromuscular junction, Fatt and Katz (1953) noted that the conductance increase mechanism they described could not fully account for the excitatory junction potential (e.j.p.) depression which followed inhibitory nerve stimulation. This observation led to the classical demonstration by Dudel and Kuffler (1961) that the additional inhibition was presynaptic. Crustacean claw opener and closer muscles are innervated by one or two excitatory axons and one inhibitory axon. Dudel and Kuffler examined cases in which the inhibitory junction potential (i.j.p.) reversal potential was more positive than the amplitude of an e4.p. In this case a single i.j.p. occurring simultaneously with an e4.p. could only increase the size of the e.j.p. Hence the finding of Dudel and Kuffler that superimposing an e.j.p. on an i.j.p. caused a substantial decrease in e.j.p. amplitude provided good evidence that some process other than a postsynaptic conductance increase was at work. Quanta1 analysis revealed that the effect involved a decrease in quantal content with no change in quantal size, i.e., that it was presynaptic in nature. We will examine evidence regarding the physiological, anatomical and pharmacological characteristics of presynaptic inhibition in invertebrates. Because the most complete body of evidence has come from work done on the crustacean neuromuscular junction (NMJ), we will focus primarily on this preparation. Atwood (1976) has recently provided a comprehensive overview of the literature through 1974 on the organization and physiology of the crustacean NMJ, and the reader is referred to that article for a broader approach.
B. ELECTROPHYSIOLOGICAL MECHANISM OF PRESYNAPTIC INHIBITION There are several distinct issues involved in this discussion: (1) The nature of the potentials which invade excitatory nerve terminals; (2) the action of presynaptic inhibitory transmitter on these potentials; and (3)
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the relationship of the potentials to transmitter release. T o date, interest has focused on the first two issues for technical reasons; however, the resolution of the third is of central importance. 1. Excitatoly Nerve Terminal Potentials There are two opposing interpretations of extracellularly recorded excitatory nerve terminal potentials (e.n.t.p.s), i.e., whether or not the responses indicate the nerve terminal region is invaded by propagated action potentials. In attempting to explain the various forms taken by e.n.t.p.s as they approach the “ultimate end” of the nerve, Dudel (1963) proposed that a major part of the terminal region is activated solely by passive currents. Locations from which large tri- and diphasic potentials can be recorded represent regions of active invasion. The e.n.t.p.s there have a prominent negative phase due to the large currents flowing into the nerve at the point of action potential generation. There is no disagreement that large e.n.t.p.s represent active propagation. Difficulties arise in interpreting the small e.n.t.p.s, which are compatible with either active or passive conduction models. T h e amplitudes of extracellularly recorded potentials are proportional to transmembrane current density, positive and negative potentials corresponding to outward and inward currents respectively (Eccles, 1964). Dudel (1963) has emphasized that, because passive currents have a capacitive component (proportional to dVldt, where V is the membrane potential) a negative extracellular potential can be produced during the falling phase of an action potential, even if propagation does not continue to the point of recording (Eccles, 1964). Purely resistive currents (proportional to V ) account for the small monophasic e.n.t.p. recorded at the nerve tip. Dudel (1963, 1965a,c) showed that small e.n.t.p.s tend to be associated with large unit postsynaptic potentials and interpreted this to mean that the smaller potentials are recorded closer to the synapses. He therefore focused much attention on changes occurring to small e.n.t.p.s. In contrast, Katz and Miledi (1965) showed that in frog motoneuron terminals the metamorphosis from tri- to di- and monophasic forms of e.n.t.p. may be a natural consequence of action potential propagation to the “closed end” of a nerve fiber. Since extracellular potentials are proportional to transmembrane current density, changes in this current will affect e.n.t.p. shape. Along a fiber transmembrane current is proportional to the first derivative of the longitudinal intracellular current and (since the longitudinal current itself is proportional to the first derivative of the intracellular potential gradient) to the second derivative of the intracellular potential (Katz, 1966). At the end of a fiber all current becomes transmembrane current. Hence, at the end, the ex-
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tracellular potential is proportional to the first derivative of the intracellular potential, and will be predominantly diphasic (positive-negative). Essentially monophasic potentials might come about via the cancellation of negative and positive currents at the nerve tip. In sum, the shapes of e.n.t.p.s may be qualitatively explained by either active or passive conduction processes. Dudel (1963, 1965a,c) considered three other pieces of evidence as supporting the idea of passive conduction. He was not able to set up antidromic action potentials by electrotonic stimulation in the terminal region, suggesting that region is electrically inexcitable. He also found that while small di- and monophasic e.n.t.p.s are invariably reduced during presynaptic inhibition, some e.n.t.p.s are increased in size. Cable theory explains that such an increase can occur near a point of conduction block. That is, owing to the decrease in length constant (caused by the presynaptic transmitter) more current crosses the membrane near the point of block and extracellular potentials become larger. (At a distance from the block, less current is available and extracellular potentials are smaller.) Nerve terminal potentials which increased in size tended to be associated with smaller unit postsynaptic potentials, indicating their greater distance from release sites. Dudel considers the existence of increased e.n.t.p.s as especially significant for his interpretation since increases are not obviously predicted by other models. While investigating mechanisms of facilitation, Zucker (1 974a,b) reexamined some of these findings. He discovered that it was often possible to produce antidromic impulses in the excitatory nerve with stimulation in the terminal region, thus demonstrating that much of this region can support active propagation. Zucker also provided new information regarding the nature of monophasic e.n.t.p.s by showing that such potentials may be contaminated by nonspecific muscle responses and probably by activity in neighboring nerve branches. In addition, small muscle movements, which may be entirely undetectable visually, were shown to have considerable effects on recorded e.n.t.p.s. It appears from Zucker’s results that many synaptic sites do experience a full depolarization. [Some of Dudel’s (1963) records, e.g., Fig. 6, could also be interpreted in this way.] However, it does not appear possible to decide if the ultimate tip of the nerve is actively invaded. Placing an electrode near the tip, which cannot be seen, is difficult, and even in preparations in which the entire branch is visible, as in the frog, stimulation of the tip may not set up antidromic potentials (Katz and Miledi, 1965). Similarly, although many changes in e.n.t.p.s may be due to nonspecific factors, as Zucker’s findings imply, lack of changes in i.n.t.p.s (Dudel, 1963; Takeuchi and Takeuchi, 1966a) might indicate
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that presynaptic inhibition does alter e.n.t.p.s in a specific way. It will be difficult to establish that all release points see a full action potential. Despite the possibility that axonal conduction block plays a role in presynaptic inhibition, there is little direct evidence regarding the details of conduction block during inhibition. What is known has come from studies on the conduction failure which follows repetitive stimulation. Parnas (1972) and Grossman et al. (1973) took advantage of the readily accessible branching pattern of a single axon which innervates two distinct sets of deep abdominal muscles in lobster. Recordings made both proximal and distal to bifurcations revealed that there was differential invasion of the two branches at different frequencies of stimulation. One branch reliably followed stimulation up to 40-50 Hz while the other showed good frequency following to 80-100 Hz. Grossman et al. (1973) provided a detailed picture of this block by recording intraaxonally in the main axon and larger branch. Activity in the smaller branch was monitored extracellularly. Intraaxonal records showed that after a few seconds of relatively high frequency activation the distal response in the large branch was reduced in amplitude, showed delayed latency and intermittent failure, followed by complete block. However, the simultaneously recorded activity in the smaller branch showed no change until much higher frequencies were used. The relevance of these experiments, and this form of conduction block to the discussion of presynaptic inhibition is not certain. From the observation of an increase in the normal spike after-depolarization following repetitive stimulation in these axons, Grossman et al. (1973) were led to propose that extracellular potassium, accumulating as a result of activity, played a role. Potassium accumulation would produce depolarization, decrease in input resistance, and increase in sodium inactivation. Subsequent action potentials would be decreased in amplitude, and the load resistance imposed by the branches would increase, as would the action potential threshold. Some of these effects might occur during presynaptic inhibition as a result of decreased membrane resistance. One problem with inferring this similarity is that, with repetitive stimulation, block of conduction into the larger of two branches occurs first. This seems to be contrary to the expected effect of conduction block in presynaptic inhibition, in which activation of small branches would be preferentially eliminated (Atwood, 1976; Dudel, 1965~).A further difficulty is that the mechanism of block produced by repetitive stimulation is not understood, Explanations based on increases in extracellular ionic concentrations predict that these effects will be greatest when the surface/volume ratio of the membrane is largest, i.e., near smallerdiameter fibers (Grossman etal., 1973; Hatt and Smith, 1976a), whereas
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the block occurs first into larger fibers. I n the absence of resolution of these questions, any inferences of similarity between this type of conduction block and that occurring with presynaptic inhibition must remain tentative. Nevertheless, the phenomenon of axonal conduction block itself is of interest as another means by which transmitter output may be limited. Hatt and Smith (1976a,b) studied the effects of conduction block on synaptic transmission at crayfish NMJ. They reported that prolonged stimulation of the excitatory fiber for several minutes resulted in a decrease in ej.p.s that could be attributed to branch point failure. Using simultaneous intracellular and extracellular focal recordings, it was possible in some cases to observe sporadic disappearance of e.n.t.p.s and focal e.j.p.s, with little concomitant change in intracellular potentials (suggesting that the block affected release from only a small subset of the synapses). This selectivity is similar to the case of presynaptic inhibition. Hatt and Smith (1976a,b) performed a quantal analysis of release after several minutes of repetitive stimulation and found depression to be due to a decrease in the binomial release parameters n a n d p . However, at short times, transmitter release was not well described by binomial statistics and Hatt and Smith attributed the deviation to nonuniformities of quantal release (i.e., differing probabilities of release among releasable quanta; Brown et al., 1976) at these times. A reexamination of release statistics during presynaptic inhibition would be interesting.
2. Effect of Inhibitory Transmitter
in Altm'ng Nerve Terminal Potentials
There is general support for the hypothesis that the presynaptic inhibitory transmitter, after combining with presynaptic receptors, causes an increase in conductance to one o r more small ion species whose combined equilibrium potential is near the terminal resting membrane potential (Dudel and Kuffler, 1961; Dudel, 1965~). Interpretation of this effect in producing inhibition of release depends on the issue of passive vs. active terminal invasion. For the passive conduction model, the transmitter action is twofold: (1) T h e conductance increase causes a decrease in the length constant, which in turn limits the spread of electrotonic currents. A smaller depolarization occurs at the synapse, and less transmitter is released. (2) A greatly decreased length constant, occurring at points of low safety factor for propagation, causes a shunting of action currents sufficient to cause conduction failure. Hence the presynaptic inhibitory transmitter can also shift points of conduction block proximally and further reduce electrotonic current available to cause transmitter release. If it is supposed that the synaptic region is normally invaded by action potentials, and that depolarization of that order of magnitude is
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necessary for transmitter release, then the major effect of the presynaptic transmitter might be to block action potential propagation (Atwood, 1976). However, the inhibitory effect may be simply to reduce the action potential and thus limit the depolarization present at the release points (Takeuchi and Takeuchi, 1966a). T h e intracellularly recorded action potential is reduced during the PAD (primary afferent depolarization) associated with presynaptic inhibition at crayfish tactile afferents (Kennedy et al., 1974). Although the distinction between the active and passive models appears clear, the evidence does not always support a sharp dichotomy, and the different interpretations may be due to differences in emphasis. For example, despite the fact that Dudel stresses cases in which small e.n.t.p.s are associated with large quantal sizes, he reports data (e.g., Dudel, 1965c, Fig. 6) in which large diphasic e.n.t.p.s are associated with large quantal sizes. As Zucker (1974a) mentions, synapses are known to be made all along nerve branches (Atwood and Morin, 1970) and there appears to be no reason to concentrate solely on the synapses made at nerve tips. On the other hand, Atwood contrasts his view of active conduction block with Dudel’s view, but some of Dudel’s records (e.g., Dudel, 1965c, Fig. 4) illustrate the mechanism clearly, and Dudel considers block of conduction to be one effect of presynaptic inhibition. Finally, the suggestion of Takeuchi and Takeuchi (1966a) appears reasonable and is not obviously at variance with aspects of either Dudel’s or Atwood’s interpretations. Lack of major effects on spontaneous transmitter release during the application of inhibitory drugs has been cited as evidence (Dudel, 1965d; Takeuchi and Takeuchi, 1966a) against the possibility that large potential changes take place in the excitatory terminal during presynaptic inhibition. Recently, Kawai and Niwa ( 1977) have recorded intracellularly from inhibitory and excitatory axons of the lobster walking leg. Action potentials could be recorded from both axons. However, a small hyperpolarization was recorded only in the excitatory axon during repetitive stimulation of the inhibitory nerve. T h e hyperpolarization developed slowly and was not clearly visible without many seconds of stimulation, indicating that the site of recording was distant from the synapses, where the potentials would therefore be larger. Hyperpolarizing potentials have also been reported to occur in crayfish excitatory axons following inhibitory nerve stimulation (Fuchs, 1977). In the crayfish, inhibition is found to reduce depolarizing after-potentials, which normally accompany action potentials in the excitatory axons. The importance of small hyperpolarizations for the mechanism of presynaptic inhibition is not yet clear. Kennedy et al. (1974) have investigated intracellular potentials in
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crayfish primary afferent terminals during presynaptic inhibition. Either natural or electrical stimulation of sensory nerves produced a primary afferent depolarization (PAD) which was mediated interneuronally. The PAD involved a conductance increase which could reduce the action potential in the terminal region. It was suggested that the conductance increase, rather than the depolarization per se, was responsible for the presynaptic inhibitory effects. Understanding the significance of the PAD, though, is complicated by some unresolved discrepancies. As Kennedy et al. (1974) point out, the peak PAD is well correlated in time with maximal presynaptic inhibition, but the latter may outlast the former (Kennedy et ad., 1974, Fig. 3). Another question arises from the work of Bryan and Krasne (1977), who tested the excitability of the tactile afferents during presynaptic inhibition by setting up antidromic impulses in the terminal region. They found a decrease in terminal excitability, which they attributed to the conductance increase occurring near the terminals of the afferents. Measured in this way, the conductance increase appears to last substantially longer (at least 100 msec; Bryan and Krasne, 1977) than the PAD (about 55 msec; Kennedy et al., 1974) and has a time similar to the observed inhibition. The explanation of this discrepancy in PAD and excitability is not certain. However, this discrepancy might be explained if the site at which PAD was recorded by Kennedy et al. (1974) were remote from the site of generation, so that the entire time course was not accurately measured. 3. Transfer Function .f the Excitatory Synapses
A key problem in these investigations is the lack of information on the transfer function of the synapses, i.e., the relationship of presynaptic potential to transmitter release. A qualitative approach to the problem was made by Dude1 (1971, 1973), who used elegant methods for polarizing nerve terminals. Hyperpolarizing current increases the amplitude of axonal action potentials and reduces miniature e.j.p. frequency, while depolarizing current has opposite effects. These changes are in the direction expected from work on other synapses (e.g., Katz and Miledi, 1967) and may imply other similarities as well. Katz and Miledi (1968) showed that, in the frog, local blockade of motor nerve terminals with iontophoretically applied T T X leads to abolition of transmitter release evoked by motor nerve stimulation. This, they argue, indicates the necessity of action potentials in producing release. However, they point out that their analysis does not preclude possible effects of electrotonic action on terminal branches much shorter than a length constant. Since crustacean motor nerves ramify extensively, with many fine branches being quite short (Atwood, 1976), results from the frog may not pre-
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clude the importance of decrementing potentials in the crayfish. Unfortunately, the effects of local T T X blockade do not appear to have been examined at crayfish junctions showing small e.n.t.p.s and large quanta1 size. Quantitative data will be required to resolve this issue. Determination of the transfer function at other synapses has shown that there is a definite threshold for release. At the squid giant synapse (Katz and Miledi, 1967, 1969) and the lamprey Muller axon-lateral interneuron synapse (Martin and Ringham, 1975), where two electrodes can be inserted into the presynaptic element, depolarizations less than 30-40 mV are ineffective in causing transmitter release. Duration of polarizing pulses is also an important variable. T h e slopes of the transfer functions are steep, with a tenfold change in postsynaptic response resulting from 10 and 24 mV changes in prepotential at squid and lamprey synapses, respectively. However, it is important to consider that the slopes apply only to the linear portion of the curves, approximately 50-80 mV prepotential, and that above 80 mV the slopes decrease. In lamprey, the postsynaptic response saturates at presynaptic depolarizations of about 100 mV. This indicates a relative insensitivity of release to reduction in action potential amplitudes in this range (Martin and Ringham, 1975). The same conclusion can be drawn from preterminal voltage clamp data, showing that the inward calcium current which triggers transmitter release is maximal at 60-mV depolarization and declines above this level (Llinas and Steinberg, 1977). In the absence of data pertaining to cable properties and transfer function, it is not possible to reject the notion of transmitter release by reduced action potentials or electrotonic potentials at the crustacean NMJ. Finally, it is interesting that Katz and Miledi (1968) found a lack of correlation in e.n.t.p. size and transmitter release. Specifically, the e.n.t.p. could decline substantially in size before the end plate response was noticeably diminished. Katz and Miledi suggest that this means that transmitter release, depending as it does on action potential amplitude and duration, is not sensitive to membrane current density, which is what is measured by the focal e.n.t.p. Another interpretation might be that a reduced action potential, or even an electrotonic potential, is capable of releasing transmitter until depressed below a critical level. In any event, this finding does cast doubt on the validity of interpretations of presynaptic inhibitory mechanisms based on changes in extracellularly recorded fiber potential size. 4. Conclusion While the phenomenon of presynaptic inhibition is well established in crustacea, the physiological mechanisms involved are not fully understood. T h e question regarding the electrical activity in the terminal
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regions is still outstanding. Full action potentials can evidently propagate to many synapses, but the question of the importance of electrotonic conduction in fine terminal branches remains open. T h e actual effect of the inhibitory transmitter in reducing release will depend on the resolution of this question. It is likely that more than one effect is at work (Atwood, 1976). The possibilities include blocking action potential conduction, reducing action potential amplitude and perhaps its duration, and limiting the spread of decrementing potentials in fine branches. T h e alternatives are not mutually exclusive; conductance increase is probably the major contributor to these effects. A crucial issue concerns the transfer function of the excitatory synapse. T h e minimal requirements for transmitter release are not known. C. ANATOMICAL CONSIDERATIONS OF PRESYNAPTIC INHIBITION
The anatomy of the crustacean NMJ has been extensively reviewed (Atwood, 1976) and a brief overview will suffice here. The most commonly used preparation, the opener muscle of crayfish walking legs, receives one excitatory and one inhibitory axon (Atwood and Morin, 1970). Axonal branching over the muscle surface continues down to a very fine level, with some branches extending only a few microns from the main branch. The axon terminals, which are situated singly, in pairs, or in clusters, are associated with a Schwann cell element and are typically embedded in the muscle (Atwood and Morin, 1970). Inhibitory axons are typically larger, as much as twice the size of excitatory axons. T h e anatomical questions involved in the investigation of presynaptic inhibition concern: ( 1 ) whether or not it is possible to discover a morphological substrate of the process, and (2) if so, what a study of this substrate can contribute to the understanding of the physiological mechanisms of presynaptic inhibition.
1. Anatomical Substrate for Presynaptic Inhibition The first question has apparently been answered in the affirmative based on a series of experiments by Atwood and his collaborators (Atwood, 1976). Inhibitory synapses in general and axo-axonic synapses with the appropriate inhibitory-excitatory profiles in particular have been extensively described. Nakajima et al. (1973) have reported finding axo-axonic synapses in which the postsynaptic axon also appears to be inhibitory, at the crayfish stretch receptor. (No physiological function has yet been ascribed to these synapses.) Axo-axonic synapses at the crayfish NMJ are relatively rare (Atwood and Morin, 1970; Jahromi and Atwood, 1974), occurring once or twice for every 3-4 p m of terminal axon Uahromi and Atwood, 1974).
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2. Implications f o r Mechanisms of Presynaptac Inhibition Given the likelihood that axo-axonic synapses mediate presynaptic inhibition, it is important to consider the implications of the morphological picture for the mechanisms of presynaptic inhibition. Atwood and his co-workers (Atwood, 1976) find these synapses located many times on narrow constrictions connecting excitatory axon branches with the parent axon and, hence, ideally situated for controlling conduction into fine excitatory branches. These findings are consistent with the model proposed by Atwood in which inhibitory synapses, located at points of low safety factor for transmission, such as constrictions, can stop propagation of the action potential along the branch. The anatomical picture is not inconsistent with other physiological models, however. T h e anatomy does provide a ready explanation for the finding of all-or-none blockade of propagation which can occur during presynaptic inhibition (Dudel, 1965~).In addition, serial reconstruction of the terminal regions Uahromi and Atwood, 1974) shows that many excitatory neuromuscular synapses are made with the main axon branch, and it seems probable that these experience a full action potential. However, in some instances, axo-axonic terminals are found within a few microns of an excitatory NMJ (Atwood and Jones, 1967, Fig. 2). It is not clear that axo-axonic synapses positioned so close to the neuromuscular synapses would have the same switching function as those positioned elsewhere. On the other hand, they could function to reduce further the level of depolarization at the release point. T h e finding that a series of presynaptic inhibitory terminals can occur along a single stretch of axon (Atwood, 1976) implies that each axo-axonic synapse does not act to block action potential conduction in an all-or-none way.
D. PHARMACOLOGY OF PRESYNAPTIC INHIBITION 1. Identity of Presynaptac Transmitter In their original investigation of presynaptic inhibition, Dudel and Kuffler (1961) provided evidence that GABA might be the presynaptic transmitter. They noted that the GABA equilibrium potential is the same as the postsynaptic inhibitory equilibrium potential, i.e., depolarized with respect to the resting potential. Therefore, in an experiment analogous to their prior physiological demonstration, they showed that small e.j.p.s could be reduced by GABA concentrations which produced no apparent postsynaptic depolarization. Since the total depolarization of e.j.p. plus GABA response would have been below the GABA equilibrium potential, this reduction suggests that the action of GABA was to reduce the amount of excitatory transmitter released.
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It has been well established that presynaptic inhibition at the crayfish NMJ is chemically mediated and that the chemical is GABA. Presynaptic inhibition involves a delay of about 1 msec (Dudel and Kuffler, 1961), facilitates (Dudel and Kuffler, 1961), and is dependent upon, external calcium (Parnas et al., 1975) and chloride (Takeuchi and Takeuchi, 1966b; Kawai and Niwa, 1977). Its effects are mimicked by GABA (Dudel and Kuffler, 1961; Dudel, 1965b,d; Takeuchi and Takeuchi, 1966a) and blocked by GABA antagonists (Takeuchi and Takeuchi, 1969). GABA and its synthetic and degradative enzymes (Kravitz et al., 1963; Otsuka et al., 1967; Hall et al., 1970) are present exclusively in inhibitory nerves. GABA is released in a calcium-dependent way with electrical stimulation (Otsuka et al. , 1966), and a specific, high-affinity, sodium-dependent GABA uptake system has been described (Orkand and Kravitz, 1971).
2 . Ionic Conductance Mechanism As it does postsynaptically (Boistel and Fatt, 1958; Takeuchi and Takeuchi, 1967),GABA is found to increase chloride (Cl-) permeability presynaptically at crustacean NMJ (Takeuchi and Takeuchi, 1966b; Kawai and Niwa, 1977). Takeuchi and Takeuchi (1966b) found that replacement of C1- with large impermeant anions reduced presynaptic inhibition and the postsynaptic conductance increase produced either by stimulation of the inhibitory axon or by application of GABA. When GABA was applied to a neuromuscular junction washed briefly in C1-deficient saline, there was a transient increase in the frequency of spontaneous miniature potentials, an effect attributed to C1- leaving the excitatory axon, thus depolarizing it, through a GABA-activated conductance. No increase in frequency was seen after prolonged washing in Cl--free medium or following repeated GABA treatments, both of which would abolish the reversed C1- gradient and the depolarization. Finally, they found that terminal invasion and neuromuscular transmitter release were facilitated in low-C1- saline. These facilitatory effects could be explained by a reduction in either a resting C1- conductance or one due to a tonic GABA release. Recently, Parnas et al. (1975) have shown that tonic GABA release does occur both pre- and postsynaptically. The postsynaptic effect was seen in the increase in membrane resistance that occurred with GABA-induced desensitization (see below), picrotoxin, and low temperature, all procedures that limit transmitter release or postsynaptic effectiveness. In fibers known to be subject to both pre- and postsynaptic inhibition (R fibers), picrotoxin increased amplitudes of ej.p.s above that predicted on the basis of the postsynaptic increase in membrane resistance alone. R fibers also showed a decreased
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dependence of e.j.p. amplitudes on external calcium. This supported the conclusion that GABA was tonically released presynaptically, since tonic release would also be influenced by external calcium and would partially counteract the direct calcium effect on e.j.p. amplitude. In fibers not subject to presynaptic inhibition (S fibers), the reduction in membrane resistance was the same as the e.j.p. reduction and the dependence of e.j.p. amplitude on calcium was greater than in R fibers. This indicates that axons that do not receive inhibition do not have presynaptic recep tors. Chloride-dependent hyperpolarizations have been recorded by Kawai and Niwa (1977), using intracellular electrodes, in the excitatory axons of lobster opener muscles during inhibitory nerve stimulation and GABA application. At other synapses, Cl--dependent hyperpolarizations during inhibition have been associated with an outward C1- pump which maintains the C1- gradient (e.g., Lux, 1971; Meyer and Lux, 1974). Ammonium ion has been found to block the C1- pump and thus to eliminate the hyperpolarization, with no effect on the underlying C1conductance change. Evidence for the role of the conductance increase per se, against that of the hyperpolarization, in mediating presynaptic inhibition would entail showing a resistance of presynaptic inhibition to ammonium chloride. Intracellular recordings from crayfish primary afferents (Kennedy et al., 1974) indicated an approximate reversal potential for the PAD of 12-20 mV depolarized with respect to the resting potential although the distance of the electrode from the synaptic site limited the precision of this measurement. Recent evidence indicates that GABA may be the transmitter at these synapses (R. Fricke, 1978 personal communication). It is interesting that at primary afferents of both vertebrates and invertebrates the GABA response is markedly depolarizing, whereas, at other pre- and postsynaptic sites, the potential change is either very slight or markedly hyperpolarizing.
3. Dgfmences between Pre- and Postsynaptic GABA Receptors Despite similarities in transmitter, C1- requirement, tonic release, and susceptibility to block by picrotoxin, there is evidence that pre- and postsynaptic GABA receptors may not be identical. In comparing the action of various drugs on presynaptic and postsynaptic responses of the crayfish claw opener muscle, Dude1 (1965b) was able to differentiate two classes. One, the P-guanidinopropionic acid (PGP)group, produced no apparent alteration in postsynaptic membrane conductance, although drugs of this class did cause neural inhibition. The second group, the GABA group, produced a postsynaptic conductance increase and inhibi-
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tion. Both classes produced effects that were similar to presynaptic inhibition, as determined by quanta1 analysis as well as by their exclusive action on excitatory nerve terminal potentials (Dudel, 1965b). PGP did act competitively to block the postsynaptic action of GABA (Dudel, 1965b; Feltz, 1971), implying that presynaptic receptors may differ from postsynaptic receptors in their conductance mechanisms rather than in their receptor functions. Balashov et al. (1975) found that while GABA was equally effective in stimulating pre- and postsynaptic receptors in the hermit crab, the former were ten times more sensitive to PGP than the latter. Picrotoxin blocks both pre- and postsynaptic inhibition (Takeuchi and Takeuchi, 1969; Earl and Large, 1974; Balashov et al., 1975; Parnas et al., 1975). It also depresses GABA-induced conductance increases in a C1--dependent fashion (Takeuchi and Takeuchi, 1969). Picrotoxin acts noncompetitively in blocking postsynaptic receptors of crab (Balashov et al., 1975) and crayfish (Takeuchi and Takeuchi, 1969). [Competitive antagonism of neural inhibition and GABA has been reported at the lobster NMJ (Grundfest et al., 1959; Shank et al., 1974).] I n contrast to its noncompetitive action postsynaptically in crab, picrotoxin blocked presynaptic inhibition, as well as the presynaptic effects of GABA and PGP, competitively (Balashov et al., 1975). Bicuculline is less potent than picrotoxin at these synapses, although it is reported to block the postsynaptic GABA-induced conductance increases noncompetitively at crayfish (Takeuchi and Onodera, 1972; Earl and Large, 1974) and lobster (Shank et al., 1974) NMJs. Pre- and postsynaptic GABA receptors differ in their susceptibilities to desensitization, as was first made clear by Epstein and Grundfest ( 1970). Crab postsynaptic GABA receptors desensitize so rapidly (within about 2 min) that early experiments had indicated that there was no postsynaptic GABA sensitivity. Nevertheless, Epstein and Grundfest found that inhibition of e.j.p.s was still strong after the postsynaptic conductance increase had been greatly reduced, suggesting resistance of presynaptic receptors to densensitization. A similar conclusion was reached by Parnas et al. (1975) in studying crab NMJ. Dudel and Hatt (1976) have exploited this finding in showing that, in various crayfish muscles, four types of GABA receptors can be distinguished largely on the basis of their susceptibility to desensitization: ( 1) postsynaptic; inhibition with GABA, no desensitization, no effect of PGP; (2) postsynaptic; inhibition and desensitization with GABA and PGP; (3) presynaptic; inhibition with both GABA and PGP, but no desensitization; and (4) presynaptic; inhibition with GABA and PGP, desensitization only to GABA. Results from crab and lobster were found to be consistent with the hypothesis of four receptor types.
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However, it is worth considering the problems inherent in distinguishing among receptor “types” on the basis of an observed physiological response. It is conceivable, as Atwood (1976) suggested, that the mechanism of presynaptic inhibition is sufficiently potent that substantial desensitization of GABA receptors does not abolish its effectiveness. The finding of Balashov et al. (1975) that high concentrations of picrotoxin did not block full attenuation of e.j.p.s by GABA indicated to them that the presynaptic inhibitory mechanism is a very effective one. A further problem that occurs when using desensitization to distinguish among receptors is that the result depends on the mechanism of the desensitization process itself. Sarne (1976) reported that in the crab closer muscle desensitization and recovery from desensitization are calcium-dependent processes. Local influences acting on calcium availability might account for apparent distinctions among receptors in different muscles. Although Dudel and Hatt ( 1976) evidently considered such a possibility, they found no effects of increasing calcium on desensitization. Reasons for the discrepancy are not entirely clear; however, Sarne (1976) compared the effects of GABA in high-calcium (34 mM, standard saline) to those in calcium-free saline (with EDTA added), whereas Dudel and Hatt used a moderate level of calcium (13.5 mM normal saline) and looked for effects of high calcium (40mM). As suggested above, GABA receptors may also differ according to their manner of antagonism by picrotoxin. These characteristics d o not obviously fit into Dudel and Hatt’s (1976) scheme of four receptor types (Dudel and Hatt, 1976, Table 1) and it appears there may be a great variety of GABA receptors. It will be interesting to learn whether they can be assigned functionally distinct roles. 4 . Pharmacology of Presynaptic Inhibition in Aplysia
Tauc (1965) reported a form of depression of a monosynaptic potential caused by stimulation of a heterosynaptic connective in the mollusc Aplysia californica. The depression was not accompanied by changes in postsynaptic membrane conductance or excitability. T h e inhibition, which was extremely long lasting, u p to 20 min, was not affected by blockade of known inhibitory synapses. Recent reports have supported Tauc’s proposal that the effect takes place at the presynaptic terminal of a monosynaptic junction (Woodson et al., 1976; Tremblay et al., 1976). These authors find that the inhibitory action can affect some of the presynaptic response plasticities, e.g., synaptic depression, frequency facilitation, and posttetanic potentiation (PTP) at the synapse RC1-R15 in Aplysia. Biogenic amines, dopamine, serotonin, and related compounds were also found to act on the synaptic plasticities. Haloperidol and propanolol antagonized the effects of heterosynaptic stimulation
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and dopamine (but not serotonin). Both amine and stimulation actions were sodium dependent, The amines reduced the size of an evoked EPSP, synaptic depression, and PTP, whereas they increased frequency facilitation. The reduction of PTP was considered especially interesting, since it was longer lasting than other heterosynaptic effects. RCl-R15 is presumably a cholinergic synapse, yet the ACh potential was not affected by the amines, and their action was not blocked by high divalent cation concentrations. Postsynaptic changes in membrane conductance .and potential were shorter in duration than EPSP duration. These results were interpreted as indicating the actions of the amines were on presynaptic terminals and that no interneurons were involved. Some questions remain regarding the actual nature of these effects. As Woodson et al. (1976) pointed out, the possibility of the involvement of interneurons is not entirely eliminated. In fact, there does not appear to be good evidence that the effects are synaptic, rather than “neurohormonal.” Apparently, the presynaptic cells have not been identified, nor have axo-axonic synapses, thought likely to mediate the response, been described. While the findings of Woodson et al. (1976) demonstrated a phenomenon that is not as long lasting as that reported by Tauc (1965) (approx 1 min vs 20-40 min), it is considerably longer than the ordinary presynaptic inhibition seen at crayfish NMJ, which lasts no more than 100 msec. [Rathmayer and Florey (1974) described a long-lasting form of presynaptic inhibition at the slow muscle system of the crab. Following a brief stimulus train delivered to the inhibitory nerve, there was a prolonged depression of muscle contractions and ej.p.s which outlasted the postsynaptic conductance increase by many minutes.] It is not clear whether these long-lasting influences are accompanied by overt physiological signs (e.g., presynaptic potential changes) or if they represent a series of intracellular reactions that are merely triggered by the action of the presynaptic transmitter. It may be significant that the inhibitory phenomenon itself is very susceptible to modification. The effect weakens considerably if elicited more often than once per hour (Tauc, 1965; Tremblay et al., 1976). Tremblay and Plourde (1977) have described similar results at the RCl-R15 synapse which implicate GABA in a heterosynaptic modulatory role. Again, the effects were attributed to a presynaptic site and acted in the same way as the amines to modify the presynaptic response plasticities. The GABA action appeared partially C1--dependent but, surprisingly, was not antagonized by picrotoxin, GABA or bicuculline. Metabolic inhibitors potentiated GABA actions to some extent. Since these effects were not sodium dependent they appeared to comprise a
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system different from the dopaminergic system discovered earlier (Woodson et ul., 1976; Tremblay et ul., 1976). N o anatomical substrate for this GABA influence has been described. E. ELECTRICAL TRANSMISSION A N D PRESYNAPTIC INHIBITION This review has focused on instances of presynaptic inhibition involving chemical transmissions, but presynaptic inhibition need not be chemically mediated. Waziri (1977) has demonstrated a form of interaction between two neurons in the abdominal ganglion of Aplysiu that may be an example of electrically mediated presynaptic inhibition. (It has not been rigorously demonstrated that these effects are presynaptic.) When the cell L,, is polarized intracellularly, the chemical transmission of its electrically coupled partner cell L,, is modified. Hyperpolarization of Lz0 reduced the output of Ll0, and depolarization had the opposite effect. These effects are contrary to those expected for polarization of nerve terminals directly (e.g., Dudel, 1971, 1973). The reason for this difference may be related to the anomalous rectification seen in these cells. Hyperpolarization would decrease, and depolarization would increase, the space constant, and hence the spread of electrotonic potential to the terminal (Waziri, 1977). In the preceding case the effects of electrical coupling on chemical transmission are considered as a possible form of presynaptic inhibition. The converse, that of the effects of chemical transmission on electrical coupling, may be seen as another form. Carew and Kandel(l976) report that increased conductance excitatory postsynaptic potentials (EPSPs) decrease electrical coupling in Aplysiu. (Decreased conductance EPSPs increased coupling.) It is not known how common such effects are, but they could conceivably be significant in any case in which electrically and chemically transmitting cells interact.
111. Prerynaptic Inhibition in Vertebrates
A. INTRODUCTION Our current understanding of presynaptic inhibition in vertebrates comes from the synthesis of data collected over a period of nearly half a century. The stage was set when Gasser and his colleagues (Gasser and Graham, 1933; Hughes and Gasser, 1934) found that, following an afferent volley to the spinal cord, a prolonged positive wave (p-wave) could be recorded from the cord dorsum and that this p-wave had the
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same time course as the inhibition of flexor reflexes, which had been described earlier (Gerard and Forbes, 1928; Eccles and Sherrington, 1931). This was followed by the discovery of Barron and Matthews (1938) that an afferent volley also elicited a depolarization of the intramedullary portion of adjacent primary afferents, which could be recorded as an electrotonic potential from the dorsal root. These authors noted that the time course for this dorsal root potential (DRP) was similar to that of the p-wave and concluded that the two events were indices of the same process and that this process was responsible for the central inhibition. T h e nature of this inhibition remained obscure until the late 1950s and early 1960s. Frank and Fuortes (1957) reported that monosynaptic excitatory postsynaptic potentials (EPSPs) recorded from motoneurons could be depressed by conditioning afferent volleys that produced no change in the membrane properties of the motoneurons. They therefore referred to this inhibition as “presynaptic,” but did not speculate on the mechanism underlying this inhibition. Later, Frank ( 1959) changed the terminology from “presynaptic” to “remote,” thus leaving open the possibility that it could be occurring postsynaptically but far out in the motoneuron dendrites where electrical changes in the motoneuron membrane might go undetected by a recording electrode located in the soma. The coupling of this finding of Frank and Fuortes on inhibition to the previous work on DRPs was made by Eccles et al. (1961a), who established that these two processes had the same time course and that the falling phase of the monosynaptic EPSP was unaltered during the inhibition. Thus they reinstated the term “presynaptic” for this form of inhibition. The purpose of this section is to consider the possible mechanisms and pharmacology of presynaptic inhibition in the vertebrate. First, the process responsible for depolarization of synaptic terminals will be discussed and then the effects which this depolarization has on synaptic transmission will be discussed. It is important to keep in mind that whether the depolarization causes an inhibition in transmitter release may depend on how the depolarization is mediated. Thus, demonstrating a depolarization of the presynaptic terminals in a particular pathway is not sufficient for concluding that this depolarization results in a presynaptic inhibition.
B. DEMONSTRATING THE PRESENCE AND LOCATION OF PAD As already mentioned, Barron and Matthews ( 1938) demonstrated that impulses coming into the spinal cord set up a depolarization both in the same and neighboring groups of afferent fibers. This depolarization was recorded from the dorsal root with bipolar electrodes, the electrode near the spinal cord becoming negative relative to the peripheral lead. Since this depolarization spread decrementally along the dorsal root,
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they concluded that it was generated in the spinal cord and spread electrotonically out along the dorsal root. When the primary afferent depolarization (PAD) is recorded from the dorsal root, the depolarization is referred to as the dorsal root potential (DRP). Another way of demonstrating this primary afferent depolarization is to measure the change in excitability by directly stimulating the afferents in the spinal cord with a microelectrode and recording the size of the antidromic volley in the dorsal root (Wall, 1958). During PAD the excitability increases; thus more afferents are activated by the stimulus from the microelectrode, which produces a larger antidromic response. T h e advantage of excitability testing is that one can identify the type of fiber receiving PAD and, in addition, one can determine approximately where the fiber is being maximally depolarized. Thus it has been found that afferent fibers of various types are maximally depolarized in the region where these fibers are thought to terminate. For instance, Group I afferents are maximally depolarized in the motoneuronal nucleus (Eccles et al., 1962b, 1963a,b), whereas Group I1 cutaneous afferent fibers are maximally depolarized more superficially (Wall, 1958, 1962; Eccles et al., 1962a, 1963~).Similarly, in the cuneate nucleus the largest excitability increase occurs in the region where the afferents terminate (Andersen et al., 1964b). Systematic plotting of the size and polarity of the field potentials occurring during PAD can also give an indication of where the maximal depolarization is taking place. It has been found that the zones of maximum extracellular negativity which signify the site of depolarization correspond very well to the results obtained with excitability testing (Eccles et al., 1962b, 1963a,b,d; Andersen et al., 1964a). The conclusion reached from these physiological experiments, that it is the terminals which receive PAD, is in accordance with the anatomical evidence showing that the terminals receive axo-axonic synapses (see Section C,2). Finally, PAD can be recorded by directly inserting a fine microelectrode into single fibers (Koketsu, 1956a,b; Eccles and Krnjevii, 1959a,b). Such recordings unequivocally demonstrate that primary afferents are subjected to depolarization and that they also permit identification of the type of fiber receiving PAD. However, the site of penetration is undoubtedly in the large unbranched portion of the fibers, some distance from the site of PAD generation. Thus, intrafiber recording is of no value in determining the location of the depolarizations. OF PAD C. THEORIGINAND NATURE
Three explanations have been advanced to account for the mechanism involved in the generation of PAD. These include electrical, chemi-
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cal, and ionic. Each of these proposals can be further divided into direct interactions among afferents or indirect actions that are mediated by interneurons. 1. Interactions Mediated by Electrical Fields An early hypothesis was that PAD occurred as a result of extracellular currents activated by primary afferents. Indeed, this mechanism may explain the small, short-latency dorsal root reflexes in cats that are sometimes observed in cold preparations (Eccles et al., 1961b; Van Harreveld and Niechaj, 1970). Similar short-latency, presumably electrical, interactions have been reported to occur between motoneurons and between motoneurons and primary afferents (Washizu, 1960; Grinnell, 1966, 1970; Nelson, 1966; Decima and Goldberg, 1970; Gutnik et al., 1975). However, the long latency and long duration of PAD and its blockade by magnesium ions suggests that interneurons are involved in the pathway and that a chemical synapse is interposed somewhere in the pathway. If the interneurons generate PAD by electrical fields it should be possible to record these fields in the spinal cord and they should be unaffected by chronic deafferentation, since the primary afferents would only be responding passively to the extracellular fields. In the frog spinal cord, ventral root stimulation evokes PAD and a slow field potential of similar time course having its maximum size in the region where the primary afferents terminate (Glusman and Rudomin, 1974). In addition, synaptic potentials evoked by dorsal root stimulation are inhibited with a similar time course (Glusman and Rudomin, 1974; Czeh, 1977). Degeneration of the primary afferents results in a disappearance of this field, suggesting that the primary afferents themselves are generating the extracellular currents associated with PAD and not responding passively to extrinsic currents (Glusman and Rudomin, 1974).
2 . Interactions Mediated by Neurotransmitters Fatt (1954) was the first to propose that PAD might be due to the prolonged depolarizing action of a transmitter. He suggested that the transmitter might be released from one group of presynaptic terminals and diffuse to adjacent terminals. There is some anatomical support for this possibility from Ralston's (1965, 1968) studies, which show that, following dorsal root section, presynaptic elements of axo-axonic synapses in lamina I11 degenerate. Koketsu (1956a) considered the involvement of a chemical transmitter and additionally raised the possibility of its release from interneurons. As already mentioned, a number of observations favor the existence of interneurons in the pathway, including spatial and temporal summation, the very wide segmental distribu-
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tion, and the fact that volleys in one dorsal root produce PAD of similar size in itself and in adjacent roots. a. Anatomical Evidence. Eccles (1961) extended the chemical hypothesis by boldly suggesting that PAD resulted from the release of a depolarizing transmitter from synapses located on the presynaptic terminals of the primary afferents. Subsequently, Gray ( 1962) discovered axo-axonic synapses in the spinal cord. These results have been confirmed, and axo-axonic synapses have been described in the spinal cord in Clarke’s column (Rethelyi, 1970; Saito, 1974), substantia gelatinosa (Ralston, 1965; Rethelyi and Szentagothai, 1969), and the motor nuclei (Conradi, 1969), although, as to the last site, there has not been unanimous agreement (Szentagothai, 1968; McLaughlin, 1972a,b; but see McLaughlin et al., 1975). Axo-axonic synapses have also been found in the cuneate nucleus (Walberg, 1965; Rosenstein and LeureDuPree, 1977) and trigeminal nucleus (Gobel, 1974). In a few cases it has been possible to identify the postsynaptic element of the axo-axonic synapses as being of dorsal root origin. In the cuneate (Walberg, 1965) and rat spinal cord (Barber et al., 1978), dorsal root rhizotomy leads to degenerative changes in the postsynaptic element. In the frog spinal cord, dorsal root fibers have been labeled with cobaltous chloride iontophoresis and found to be present in the postsynaptic component of the axo-axonic synapse (Szekely and Kosaras, 1977). Typically, the axoaxonic synapse has flattened vesicles and a symmetrical synaptic contact, while the presumed primary afferent contains round vesicles and forms an asymmetrical contact (Uchizono, 1973). These synaptic features are generally associated with inhibitory and excitatory functions, respectively (Uchizono, 1965). b. Physiological Studies. If PAD is produced by a chemical transmitter released from axo-axonic synapses, one would expect PAD to be associated with an increase in conductance and to have a reversal potential more positive than the resting potential. A major difficulty in analyzing the ionic mechanism of PAD is that it is generated in the fine axon terminals, thus preventing direct recording at the site of origin. Early attempts at polarizing the terminals with extrinsic current applied across the spinal cord produced inconclusive results, presumably because the current also affected the interneurons in the pathway (Eccles et al., 1963~). Thus both hyperpolarizing and depolarizing currents decreased the size of DRPs, and no reversal could be seen with depolarization. On the other hand, when the afferent fibers are hyperpolarized, either by tetanic stimulation (Eccles and Krnjevib, 1959b)or by extrinsic current to the dorsal root (Padjen et al., 1973 and unpublished observations), the size of the DRP and PAD recorded from single fibers increases up to
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fivefold, whereas depolarizing the dorsal root decreases the size of the DRP and PAD. A major problem in interpreting these results is that the rising phase of these potentials is altered very little by the polarization, which indicates that there has been little change in the driving potential for this presumed synaptic event. Possibly, much of the change in size is a result of changes in membrane resistance, which would greatly affect the electrotonic propagation of the potentials. Such nonlinearity of the afferent membrane properties also causes extrapolation of reversal potentials to be of limited value. However, the slow field potential generated in the dorsal horn by ventral root stimulation in the frog, which is a reflection of the current generated during PAD (Glusman and Rudomin, 1974), can be modified by dorsal root polarization, indicating that dorsal root polarization can, to some extent, alter the driving potential for PAD (Nicoll, unpublished observations). In summary, the physiological results are inconclusive concerning the ionic mechanism of PAD, although the effects of dorsal root polarization on the amplitude of the field potentials generated by PAD are consistent with a transmitter-coupled, ionic conductance increase mechanism. The rather large volume of data specifically suggesting that GABA is the transmitter will be presented in a later section.
3 . Interactions Mediated by Potassium In their original description of DRPs, Barron and Matthew (1938) suggested that these potentials might arise from a transient shift in the ionic environment surrounding the primary afferents. This hypothesis has recently been revived by the introduction of potassium-specific microelectrodes to neurophysiological investigations (KTii' et al., 1974, 1975; ten Bruggencate et al., 1974; Krnjevit and Morris, 1972, 1975; Lothman and Somjen, 1975; Sykova et al., 1976). Specifically, it is proposed that increases in extracellular potassium during neuronal activity depolarize afferent fibers. A direct interaction among primary afferents due to A[K], could not contribute more than a few percent to the DRP evoked by single volleys, because a block of chemical transmission by magnesium ions almost completely blocks the DRP. However, with high-frequency stimulation, a sustained depolarization appears which is approximately 10%-20% of the response seen with synaptic transmission intact, and this may be due to A[Kl, (Sykova and Vyklickjr, 1977; Nicoll, 1979). There are a number of obstacles to accepting the hypothesis that the release of K+ during interneuronal activity is responsible for generating the DRP to single or short trains of stimuli to afferent fibers. First, the magnitude of the A[K], to a single dorsal root stimulus is very small, and
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the time course is much slower than the DRP. It has been argued (KPZ et al., 1974, 1975) that these discrepancies might arise from the dead space which undoubtedly exists around the tip of the K-selective microelectrode. However, the use of glial cell recordings to monitor changes in [K], should overcome this technical problem, since glial cell membranes are as close to primary afferent fibers as primary afferent fibers are to each other. However, the results with glial cell recording are similar to those obtained with K-selective microelectrodes (Lothman and Somjen, 1975; Nicoll, 1979), supporting the evidence that the A[K], in the immediate vicinity of primary afferents is much slower than the DRP. Second, low-frequency dorsal root stimulation results in a decremental summation of the change in [K],, while the DR-DRP remains of constant size or decreases slightly (Nicoll, 1979). Third, in the frog spinal cord, ventral root stimulation fails to alter [K], and yet elicits a DRP (Nicoll, 1976, 1979). Fourth, pharmacological manipulations produce opposite effects on the DRP and [K],. For instance, barbiturates reduce A[K], and yet prolong DRPs (ten Bruggencate et al., 1974). Fifth, conditioning volleys in the flexor reflex afferents, which block group I evoked DRPs, have no effect on the simultaneously recorded K signals (ten Bruggencate et al., 1974). Sixth, conditioning volleys in flexor group I afferents, which depress l a EPSPs, have no effect on descending monosynaptic EPSPs, even though the two inputs are in electrotonic proximity (Rudomin et al., 1975). It is difficult to imagine such specificity of action occurring by a K mechanism. All of these findings suggest that A[K], does not contribute substantially to generating DRPs evoked by single stimuli. However, with high-frequency sustained stimulation of dorsal roots, [K], might contribute to the depolarization of adjacent dorsal roots. Under these conditions [K], can reach 6-8 mM (KPZ et al., 1974, 1975; Lothman and Somjen, 1975; Sykova et al., 1976; Nicoll, 1979). In the frog spinal cord (Nicoll, 1979), an 8 mM increase in K depolarizes the dorsal root about 15 mV. Although the single DR-DRP averages about 1I mV, during high-frequency stimulation, the summated DR-DRPs can build with a time course similar to A[K], to a depolarized level of approximately 15 mV. Thus, a substantial portion of the sustained depolarization may be due to A[K], and a small percentage (ca. 10%) of the A[K], may originate directly from primary afferents, as suggested by experiments in which synaptic transmission is blocked by high magnesium (Sykova and Vyklickjr, 1977; Nicoll, 1979). Another observation suggesting that high-frequency neuronal activity can release sufficient K to depolarize primary afferents comes from experiments with the convulsant picrotoxin. Addition of picrotoxin to the Ringer’s solution results
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in the depression of the DR-DRP, but a later depolarization of slow time course appears and can reach depolarizations similar in size to the DR-DRP observed in control conditions (Barker et al., 1975b). Simultaneous measurement of [K], indicates that the time of appearance and the magnitude of this picrotoxin-resistant depolarization correlate well with the observed A[K], (Lothman and Somjen, 1976; Sykovi and Vyklickf, 1978; Nicoll, 1979). In summary, it appears that changes in [K], play a minor role in the genesis of DRPs evoked by single afferent volleys. However, during high-frequency sustained neuronal activity, a substantial portion of the depolarization of the dorsal root may be due to A[K],.
D. EVIDENCE THAT GABA Is
A
TRANSMITTER OF PAD
1. Pharmacological Evidence In their pioneering study on the pharmacology of PAD and presynaptic inhibition, Eccles et al. (1963e) demonstrated that pre- and postsynaptic inhibition had distinct pharmacological properties. Strychnine, which blocked postsynaptic inhibition, actually increased and prolonged DRPs in the cat. On the other hand, picrotoxin blocked DRPs and presynaptic inhibition but did not block postsynaptic inhibition. Since picrotoxin was known to block GABA-mediated inhibition in crustacean peripheral nervous system (Robbins and Van der Kloot, 1958), this suggested that GABA might be the transmitter. To test this possibility, GABA was applied topically to the spinal cord and was found to have an excitatory effect, since it increased the size of the dorsal root reflex. Furthermore, it had been shown previously that GABA applied iontophoretically to motoneurons decreased the monosynaptic EPSP to a greater extent than would have been predicted by the change in electrical properties of the motoneurons (Curtis et al., 1959). However, there were two reservations in accepting GABA as the transmitter released from axo-axonic synapses. First, the effects of topically applied GABA could have been indirect, and second, GABA was known to decrease excitability of postsynaptic neurons (Curtis et al., 1959), and thus if it were the transmitter of PAD, its effects would be opposite to those found on cell bodies of neurons in the CNS. Studies on the frog spinal cord also revealed that GABA increased the excitability (Schmidt, 1963) and depolarized the primary afferents (Tebecis and Phillis, 1969) and picrotoxin reduced the DRPs. In addition, Tebecis and Phillis (1969) reported that picrotoxin antagonized the GABA responses, but these early studies emphasized that (1) high doses of GABA were required, (2) GABA
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might well be acting indirectly on interneurons, and (3) picrotoxin was a nonspecific antagonist and did not invariably antagonize the DRPs. In addition, Curtis and Ryall (1966) reported that GABA applied iontophoretically to cat primary afferent terminals actually decreased excitability of the terminals. However, topical application experiments in the rat cuneate nucleus (Davidson and Southwick, 1971) demonstrated quite clearly that GABA increased excitability and that the GABA antagonists (picrotoxin and bicuculline) selectively blocked the response. One possible explanation for the discrepancy between the iontophoretic and topical experiments was that GABA was indeed acting indirectly when applied topically. This possibility was effectively ruled out by two types of experiments. First, in the frog spinal cord, blockade of synaptic transmission, either with tetrodotoxin or magnesium ions, did not alter the depolarizing action of GABA on primary afferents, and the depolarization was blocked by picrotoxin and bicuculline (Davidoff, 1972a,b; Barker and Nicoll, 1972, 1973). Similar experiments with tetrodotoxin and intravenous injections of GABA have been done in the cat (Levy, 1975). Second, GABA application to mammalian and frog dorsal root ganglia, which contain no synapses, resulted in a depolarization (DeGroat et al., 1972; Obata, 1974; Nishi et al., 1974; Deschenes et al., 1976; Lawson et al., 1976; Gallagher et al., 1978). Curtis et al. (1977) have recently reexamined the effects of iontophoretic GABA on the excitability of single afferent fibers and confirmed similar experiments by Gmelin and Cerletti (1976) that GABA has an excitatory action on primary afferents. Thus, the pharmacology of DRPs and GABA fit very well with the notion that GABA is released from axo-axonic synapses and depolarizes primary afferent terminals. In the frog spinal cord, both picrotoxin and strychnine entirely block the DRP evoked by ventral root stimulation (VR-DRP) while only picrotoxin antagonizes the DR-DRP (Barker et al., 1975b). Although it cannot be entirely excluded that these two drugs act at different sites in the VR-DRP pathway, e.g., strychnine possibly blocking the cholinergic step by its known curariform action (Alving, 1961), it is possible that these two antagonists both exert their action at the final step in this pathway. Indeed, both taurine and p-alanine depolarize primary afferents, and their actions are entirely blocked by both picrotoxin and strychnine (Barker et al., 1975a; Nistri and Constanti, 1976), whereas only picrotoxin antagonizes the action of GABA. Thus, from the pharmacological results, the DR-DRP may be mediated by GABA, while the VR-DRP might be mediated either by p-alanine o r taurine. Pharmacological manipulation of the levels of GABA in the spinal cord has also been shown to alter the size of PAD. Depleting GABA by blocking the enzyme glutamate decarboxylase with either semicarbazide
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or thiosemicarbazide reduces the size of PAD and sometimes abolishes it altogether in the cat (Banna and Jabbur, 1971; Bell and Anderson, 1972; Banna, 1973; Polc et al., 1974; Polc and Haefely, 1976). On the other hand, inhibition of GABA transaminase, the degradative enzyme for GABA, by aminooxyacetic acid (AOAA) or hydroxylamine, results in increased GABA levels, and has been reported to increase PAD in the frog spinal cord (Davidoff et al., 1973). In the cat, AOAA has little (Polc and Haefely, 1976) or no (Bell and Anderson, 1972; Polc et al., 1974) enhancing effect on PAD or DRPs, although an increase in the longduration inhibition of monosynaptic reflexes has been reported (Polc et al., 1974).
2. Biochemical and Hbtochemical Evidence Early neurochemical studies reported that both GABA (Graham et al., 1967) and glutamate decarboxylase (Albers and Brady, 1959) are present in mammalian spinal cord and the highest concentrations are in the dorsal horn. Taurine is also present in high amounts in the mammalian and frog spinal cord (Collins, 1974), whereas @-alanineis present in extremely low amounts in mammalian CNS (Yoshimo et al., 1970) but has not been measured in frog. Miyata and Otsuka (1975) did a detailed study on the distribution of GABA in the cat spinal cord with a resolution of 200 pm. They found the highest concentrations in the dorsal part of the dorsal horn. A similar distribution was found in the rabbit spinal cord (Berger et al., 1977). In addition, cauterization of the blood vessels supplying the dorsal horn results in a marked depression of the DRP, a loss of interneurons in the dorsal horn, and a depletion of GABA in this area. This supports the earlier suggestion of Wall (1962) concerning the location of the interneurons in the pathway responsible for PAD. There is also a high-affinity, sodium-dependent uptake system for GABA in spinal cord tissue, and sucrose density gradients of mammal (Iversen and Johnston, 1971) and frog (Davidoff and Adair, 1975, 197613) suggest that accumulated GABA is present in nerve terminals (Iversen and Johnston, 1971). Glusman (1975) examined the regional distribution of this uptake process for GABA in the frog and compared this distribution to the depth profile of the PAD-related slow field potential evoked by ventral root stimulation. He found that the region of highest GABA uptake was in the dorsal horn, where the field potential was largest. Both @-alanine(Johnston, 1977) and taurine (Kaczmarek and Davidson, 1972) are taken u p into mammalian CNS. In the frog spinal cord, @-alanineis also taken up, but there is evidence that this uptake may be into glial cells (Adair and Davidoff, 1977). Interestingly, there does not
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appear to be an active uptake process for taurine in frog spinal tissue (Davidoff and Adair, 1976a; Muller and Snyder, 1978). Autoradiographic studies have been done to determine what types of cells accumulate these amino acids. Both [3H] GABA and [3H] P-alanine are taken up into glial cells in the dorsal root ganglia (Schon and Kelly, 1974, 1975), and [3H]GABA has been localized in glial cells in the spinal cord (Ljungdahl and Hokfelt, 1973). However, Ljungdahl and Hokfelt ( 1973) also localized [3H] GABA in axo-axonic synapses. More recently, detailed studies have been performed on the localization of glutamic acid decarboxylase, the rate-limiting enzyme in the synthesis of GABA. This was accomplished by immunohistochemistry and therefore required isolating the enzyme to homogeneity. Horseradish peroxidase labeling was used, and results with light microscopy showed that the label was concentrated in the dorsal horn. In electron micrographs, heavy peroxidase labeling occurred over axo-axonic synapses, and the profiles that were postsynaptic had the appearance of primary afferent terminals. In addition, axo-dendritic synapses were labeled (McLaughlin et al., 1975). In a later study, immunohistochemistry was combined with degeneration of the dorsal roots and confirmed that primary afferent terminals were postsynaptic to axo-axonic synapses (Barber et al., 1978). The release of radioactive or endogenous amino acids have been examined in a number of studies. Depolarizing concentrations of potassium cause a large increase in the efflux of [3H] GABA (Mulder and Snyder, 1974; Johnston, 1977) and p-alanine (Johnston, 1977) from mammalian spinal cord tissue preparations. This release is dependent on external calcium ions. Roberts (1974) has obtained similar results from the rat dorsal column nuclei in vivo and, in addition, has shown that endogenous GABA is released during stimulation of dorsal column tract fibers. In the isolated spinal cord of the frog, stimulation of the descending tracts results in a calcium-dependent release of [3Hl GABA, but dorsal root stimulation has no effect (Roberts and Mitchell, 1972; Collins, 1974). High potassium also releases both r3H] GABA and [3H] p-alanine from frog spinal tissue, but, since veratridine, which selectively depolarizes neuronal membranes, only releases [3H] GABA, it has been concluded that p-alanine is being released from glial cells by high potassium (Adair and Davidoff, 1977). Recently, it has been shown that a number of putative neurotransmitters exhibit high-affinity, saturable, stereospecific binding to membrane fragments of CNS tissue. This binding, which is independent of uptake sites, is likely to reflect binding to postsynaptic receptors (Snyder and
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Bennett, 1976). GABA binding sites have been found in both mammalian (Zukin et al., 1974; Enna and Snyder, 1975) and frog (Enna and Snyder, 1977) spinal cord. Although the levels are quite low in the spinal cord, compared to other regions of the CNS, the regional distribution of this binding in spinal tissue has not been investigated. The evidence summarized above presents a very convincing case that GABA is a depolarizing transmitter released from axo-axonic synapses onto primary afferents. T h e only results which might question the role of GABA in PAD are the negative experiments in which dorsal root stimulation in the frog failed to release GABA. The basis for these negative results is unclear. T h e conclusion that GABA is released from axo-axonic synapses resolves a paradox raised by the anatomical studies, i.e., that the morphology of the axo-axonic synapses is inhibitory and yet the function is excitatory. T h e morphological correlation was initially made in cerebellum (Uchizono, 1965), where the inhibitory terminals are thought to release GABA (Curtis and Johnston, 1974). Since the vesicle shape is presumably related in some way to the transmitter released from the terminal, it is to be expected that at axo-axonic synapses, where GABA has an excitatory action, the presynaptic morphology should be the same as at synapses where GABA has an inhibitory action. A neurotransmitter role for p-alanine or taurine is much less certain. Although the pharmacological results are suggestive of a role in the generation of the VR-DRP in the frog, the neurochemical data, which show that there is no uptake system for taurine and that p-alanine is probably taken up and released from glial cells, provide little support for such a role. E. PROPERTIES OF GABA RESPONSES 1. Ionic Mechanism of GABA Action and PAD
The two most likely explanations for the ionic mechanism involved in the GABA depolarization of primary afferents are an increase in sodium conductance or an increase in chloride conductance. For an increase in chloride conductance to produce a depolarization, the chloride equilibrium potential would have to be at a level more positive than the resting membrane potential. This in turn would imply the existence of an inwardly directed chloride pump. Initial experiments in which the concentrations of extracellular sodium and chloride were altered and the responses recorded extracellularly from the dorsal root suggested that sodium ions are involved in the response (Barker and Nicoll, 1972, 1973). This was based largely on the observation that a sodium-free
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Ringer’s solution abolished the GABA response and that there was no transient increase and often only a decrease in the size of the response in a chloride-free Ringer’s solution. A transient increase in the response would be expected if the response were due to an increase in chloride permeability because the gradient for chloride exit would transiently be greatly increased. Others have also seen a depression of the GABA response in low-chloride solutions (Nishi et al., 1974; Kudo et al., 1975; Otsuka and Konishi, 1976) and sodium-free solutions (Nishi et al., 1974; Constanti and Nistri, 1976; Otsuka and Konishi, 1976). Interestingly, PAD is augmented by superfusing the cuneate with a low-chloride Ringer’s solution (Davidson and Simpson, 1976). A marked prolongation of the DRP evoked by dorsal root stimulation is seen in the frog in a lowchloride Ringer’s solution (Katz and Miledi, 1963; Barker and Nicoll, 1972, 1973, and unpublished observations), despite the fact that the response to exogenous GABA is depressed. Picrotoxin produces the same effects (Barker et al., 1975b; see also Section III,C,3). Both of these conditions lead to a marked increase in neuronal excitability as revealed by the increase in polysynaptic activity in motoneurons. This increased excitability, which presumably results from a block of inhibition on spinal interneurons, brings out a non-GABA component to the DR-DRP which may be due to increases in [Kl, (see Section III,C,3). On the other hand, the VR-DRP is entirely blocked by low-chloride Ringer’s and picrotoxin. Further experiments with sodium substitution have revealed that only when the concentration of sodium is below 10% of control does the GABA response disappear and that at intermediate concentrations the response can actually be larger than control (Nishi et al., 1974; Otsuka and Konishi, 1976; Nicoll and Padjen, unpublished observations). Although the loss of GABA responses in sodium-free Ringer’s remains a mystery, these findings are difficult to explain in terms of sodium being the major ion involved in the GABA response. Since the primary afferent terminals are too small for intracellular recording, advantage has been taken of the presence of GABA receptors on dorsal root ganglion cells (DeGroat, 1972). Nishi et al. (1974) found that the GABA response was associated with an increase in membrane conductance and had a reversal potential of 33 mV. T h e reversal potential was found to be related to the log of the extracellular chloride concentration, but to be unaffected by changing extracellular sodium or potassium. Similar conclusions have been reached from experiments on mammalian dorsal root ganglion cells (Deschenes et al., 1976; Gallagher et al., 1978) and sympathetic ganglion cells (Adams and Brown, 1975). Furthermore, Gallagher et al. (1978) investigated the effect of intracellular injection of 20 different species of anions of varying hydrated size
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and found that GABA increased permeability only to those anions with a hydrated radius smaller than Br03-. Except for SH- and HC02-, which are permeant in ganglion cells but not in motoneurons, these results are very similar to the results obtained for IPSPs in motoneurons (Araki et al., 1961), which appear to be generated by the transmitter glycine (Curtis and Johnston, 1974) and to the IPSPs in hippocampal pyramidal cells (Eccles et al., 1977), which appear to be generated by GABA (Curtis and Johnston, 1974). Gallagher et al. (1978) also found that large foreign anions in the Ringer’s solution depressed the GABA response in a noncompetitive manner. Such an effect would explain the depression of the neutral amino acid responses on primary afferents and the block of the VR-DRP in chloride-free Ringer’s solution. Although these results clearly establish that the GABA response on dorsal root ganglion cells involves a selective increase in permeability to chloride ions, it is uncertain whether the same mechanism also occurs on the terminals of primary afferents. Experiments with substances that are known to block chloride transport in other systems have been examined for their effects on GABA responses. Although a number of agents, including ammonium ions, which are thought to block an outwardly directed chloride pump in motoneurons (Lux, 1971; Llinas et al., 1974) had little effect on the depolarization, furosemide was found to block both the GABA responses and the DRPs (Nicoll, 1978b). Since this compound is known to block chloride fluxes in a number of systems (Burg et al., 1973; Brazy and Gunn, 1976), a similar action might well explain these results. However, it is apparent that furosemide is blocking the increase in conductance to chloride and not the presumed inward chloride pump, because there is little change in the reversal potential of the GABA response.
2 . Pharmacological Properties Structure activity studies have been performed on both the intramedullary portion of the primary afferent (Barker et al., 1975a) and on dorsal root ganglion cells (Nishi et al., 1974; Gallagher et al., 1978), and two interesting differences were observed. First, the dicarboxylic amino acid glutamate is devoid of activity on the cell bodies but depolarizes the terminals. Second, whereas glycine has only weak activity at both sites, the amino acid with an intermediate chain length, p-alanine, has little activity on the cell bodies, but on the intramedullary fibers has a potency only slightly less than GABA. T h e difference in activity for P-alanine at the two sites is also seen with taurine. T h e findings on dorsal root ganglion cells are remarkably similar to the depolarizing responses recorded from the superior cervical ganglion (Bowery and Brown, 1974)
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and the evidence suggests that one class of amino acid receptor that favors GABA is present on these membranes. T h e potent effects of p-alanine on the intramedullary portion of frog primary afferents might be explained by the presence of specific receptors for this amino acid, and this is supported by the finding that strychnine selectively blocks the action of p-alanine (Barker et al., 1975a). T h e use of conformationally restricted amino acid analogues also supports this conclusion (Nicoll, 1977). A variety of drugs has been found to antagonize GABA responses on primary afferents, including picrotoxin (Tebecis and Phillis, 1969; Davidson and Southwick, 1971; Barker and Nicoll, 1972; Barker et al., 1975a; Constanti and Nistri, 1976), bicuculline (Davidoff, 1972a,b; Barker et al., 1975a; Levy, 1975), pentylenetetrazole (Nicoll and Padjen, 1976; Simmonds, 1978), and bemegride (Simmonds, 1978; Nicoll, unpublished observations). Comparison of the pharmacological properties of GABA responses on primary afferents to those obtained from neurons within the CNS reveals a marked similarity. However, the relative sensitivity of these responses to antagonists does vary considerably. For instance, in the frog spinal cord, the concentration of picrotoxin and bicuculline needed to antagonize motoneuron responses is 10 to 100 times higher than that needed to produce a similar antagonism of primary afferent responses (Nicoll et al., 1975b). Pentylenetetrazole has been reported to block GABA responses on primary afferents but to have little effect on frog motoneuron hyperpolarizing responses (Nicoll and Padjen, 1976), although others have seen antagonism of GABA responses on central neurons (MacDonald and Barker, 1977). Pentobarbital, which has long been known to prolong presynaptic inhibition (cf. Nicoll, 1978a), interacts with GABA responses in a number of ways. In low doses, it enhances the action of GABA (Nicoll, 1975a, 1976). It also depolarizes the dorsal root with a potency similar to GABA (Nicoll, 1975b). T h e pharmacological properties of this response are identical to those of GABA. It is unclear at present whether this effect of pentobarbital is exerted at the level of the receptor or the iontophore. T h e observation that GABA-mediated postsynaptic inhibition is prolonged by barbiturates to an extent similar to that seen with PAD (Nicoll et al., 1975a) provides further pharmacological evidence that GABA is the transmitter mediating PAD.
3 . Distribution of GABA Receptors As mentioned previously, GABA receptors are not confined to regions of membrane that receive synapses. GABA receptors are present
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on dorsal root ganglia (DeGroat et al., 1972), sympathetic ganglia (Bowery and Brown, 1974), and adrenal medulla (Sangiah et al., 1974). In addition, GABA applied to the sciatic nerve has been reported to block action potentials in sensory, but not motor, fibers (Sabelli et al., 1974), and sensory C-fibers in the vagus are depolarized by GABA (Brown and Marsh, 1978). It might be postulated that the existence of these depolarizing GABA responses (and, therefore, possibly an inward chloride pump) is related to the common embryological origin of these neurons, which are all derived from the neural crest. However, there are two sites which d o not fit into this generalization. First, the preganglionic sympathetic fibers have depolarizing GABA receptors (Koketsu et al., 1974), and, second, the terminals of mitral axons in the lateral olfactory tract are depolarized by GABA (Simmonds and Pickles, 1978). This finding is particularly interesting since it is known that GABA has an inhibitory action and is thought to mediate the chloride-dependent IPSP in the soma of mitral cells (Nicoll, 1969, 1971). Thus, if this depolarizing response is due to a chloride conductance mechanism, as occurs in ganglion cells (Adams and Brown, 1975; Gallagher et al., 1978), an inward chloride pump in the terminal region would create a large chloride gradient between the terminal and the cell body. 4. Are Axo-axonic Synapses Needed for PAD?
The possibility that an elevation in extracellular potassium during high-frequency stimulation might contribute to PAD has already been discussed. In the lateral geniculate nucleus, the optic nerve terminals are depolarized by a number of inputs to this nucleus and yet axo-axonic synapses have not been found in this structure. Singer and Lux (1973) have found that activation of these inputs also elevates extracellular potassium in the vicinity of the optic nerve terminals. The idea that GABA might be involved in neurotransmission in systems that d o not involve clearly defined anatomical synapses has been advanced by Ramon-Moliner (1977). In this context, recent studies on the olfactory cortex slice (Pickles and Simmonds, 1976, 1978) are of particular interest. It has been found that, at low stimulus frequencies to the lateral olfactory tract (LOT), antidromic action potentials, analogous to dorsal root reflexes, can be recorded from the LOT. This reflex is blocked by bicuculline, suggesting that LOT stimulation releases GABA, which interacts with the depolarizing GABA receptors on LOT fibers. However, electron microscopic studies (Westrum, 1969) have failed to find any axo-axonic synapses on LOT axon terminals. Presumably, the GABA is released from neighboring synapses and diffuses to the terminals of LOT axons.
PRESYNAPTIC INHIBITION
OF PAD F. EFFECTS
ON
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SYNAPTIC TRANSMISSION
Up to this point, emphasis has been placed on the pharmacology and ionic mechanism of PAD. It was suggested in the Introduction that PAD resulted in a presynaptic inhibition. However, the evidence for this is not as conclusive as that for the crustacean neuromuscular junction, which is based on quanta1 analysis. T h e reason for believing that PAD is associated with presynaptic inhibition is that the time course for PAD and the depression of monosynaptic EPSPs in spinal motoneurons is similar to the duration of PAD, and there is no change in the properties of the motoneuron membrane detected by a microelectrode located in the soma. This has been found in cat (Frank and Fuortes, 1957; Eccles et al., 1961a; Eide et al., 1968) and frog motoneurons (Grinnell, 1966; Czeh, 1977). In addition, the falling phase of the EPSP is unchanged during the inhibition (Eccles et al., 1961a; Eide et al., 1968). Since the falling phase of the EPSP, which represents EPSP components from distal dendrites, is not altered, this finding favors a presynaptic over a remote postsynaptic mechanism. However, in later studies, which used more sensitive methods for detecting subtle postsynaptic changes, it was recognized that some postsynaptic inhibition (Granit et al., 1964; Kellerth, 1968; Cook and Cangiano, 1972), with the same pharmacology and time course as presynaptic inhibition (Kellerth, 1965; Kellerth and Szumski, 1966a,b), can occur with the conditioning stimuli that are used to generate presynaptic inhibition. That the two types of inhibition might occur together should not be surprising since axon terminals which form axo-axonic contacts can also synapse with the postsynaptic element (e.g., Walberg, 1965),just as occurs at the crustacean NMJ (see Section 11, C). In addition, the presence of GAD-containing synapses on motoneurons (McLaughlin et al., 1975) supports the idea that GABA-mediated postsynaptic inhibition does occur in motoneurons. Cook and Cangiano’s (1972) findings also support the concept that pre- and postsynaptic inhibition can both be evoked by the standard conditioning stimuli used to generate presynaptic inhibition. With stimuli that produced no evidence for postsynaptic inhibition there was no change in the falling phase of the EPSP, whereas, when there was evidence for a concomitant postsynaptic inhibition, the falling phase of the EPSP was accelerated. Bergmans et al. (1974) have also observed long-duration pre- and postsynaptic inhibition occurring after flexor reflex afferent stimulation. Other evidence that strongly suggests the existence of a presynaptic form of inhibition is the finding that EPSPs evoked in motoneurons by stimulating descending pathways are unaffected by conditioning volleys
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in flexor group 1 afferents which at the same time depress l a EPSPs (Eide et al., 1968). Rudomin st al. (1975) have extended these findings by showing that, even when these two types of EPSPs are in electrotonic proximity, only the l a EPSP is depressed by the conditioning stimulus. The precise mechanism by which PAD reduces transmitter release is not clear. It is thought that in the crustacean (see Section 11, B) the conductance increase primarily acts to shunt and reduce the size of the action potential or electrotonic potential in the nerve terminal. Since the number of quanta released from a nerve terminal is dependent on the magnitude of the depolarization in the terminal, the number of quanta reIeased would be reduced during presynaptic inhibition. A blocking of impulse invasion into the terminal might also contribute to the inhibition. Eccles (1964) offered a similar mechanism to explain presynaptic inhibition in the vertebrate CNS, but, in addition, proposed that the depolarization of the terminal during PAD would also decrease the size of the action potential. However, the mechanism underlying the release of transmitter from l a afferents on motoneurons is, at present, far from clear. Kuno (1964a) reported that stimulation of single l a afferent fibers elicited in motoneurons an EPSP composed of smaller unitary components, which he treated as quanta, in analogy with the NMJ. However, Edwards et al. (1976a,b) provided fairly strong evidence that the fluctuations in release of transmitter do not imply a quanta1 release process, and they provided some evidence that the fluctuations represent the all-ornone activation of separate boutons, possibly due to a variable branch point failure. It is unclear whether the single-bouton EPSP is composed of a great many quanta or only a single quantum. If there is a large number of quanta, then reduction in the size of the action potential would cause a graded decrease in release from terminals that receive axo-axonic synapses, as Eccles (1964) proposed. T h e results of Kuno (1964b), although not extensive, imply that during presynaptic inhibition there is a decreased probability of occurrence of unitary EPSPs (probably single-bouton EPSPs), suggesting that PAD has increased the probability of branch point failure. These two mechanisms, i.e., a graded reduction of release from single boutons and failure of invasion of boutons, are not mutually exclusive, and a similar situation probably exists at the crustacean NMJ. The major problem is to explain how PAD, which is associated with an increase in fiber excitability, increases, rather than decreases, the probability of failure at branch points. It is generally agreed that when testing fiber excitability, the large, parent, myelinated portion of the afferent is being stimulated, and yet, on the basis of the location of axo-axonic synapses, PAD is generated in the terminals. It is
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possible that in the fine terminals the conductance increase produced by the presynaptic inhibitory transmitter predominates over the depolarization, so that the shunting would cause the excitability of the terminal to decrease, whereas for the PAD seen in the large myelinated portion of the afferent, which has spread electrotonically from the terminal, the depolarization would predominate over the conductance increase. A similar argument has been made to account for the effects that PAD exerts in the crustacean CNS. In this preparation, PAD is often associated with a decrease in excitability, indicating that the conductance increase is of greater importance than the depolarization and that the stimulating electrode may be close to the terminals of the primary afferents (Bryan and Krasne, 1977). One additional point should be made. Presumably, there is a threshold for transmitter release at the afferent terminal, because there is no evidence that PAD by itself induces the release of transmitter from afferents. As discussed in Section II,D, there is considerable evidence that GABA is the transmitter at axo-axonic synapses. It is also likely that, during high-frequency stimulation, potassium can contribute to the depolarization of primary afferents. T h e effects of these two substances on transmitter release in the spinal cord are unknown, but the overall effect of potassium on the spinal cord is to facilitate reflex transmission (Nicoll, 1979). In the lateral geniculate nucleus there is some evidence that potassium may mediate the depolarization of optic nerve terminals, and yet the EPSPs generated by the optic nerve fibers are not reduced when evoked during this depolarization (Singer and Lux, 1973). T h e sympathetic ganglion provides a good model for comparing the presynaptic action of GABA and potassium, since in the sympathetic ganglion GABA depolarizes the preganglionic fibers and quantal analysis can be accurately carried out at this site. In this preparation, GABA reduces quantal content by half (Kato et al., 1978). On the other hand, potassium actually increases quantal content, and the average size of the EPSP is larger and more likely to fire an action potential in the presence of potassium (Nicoll, unpublished observations). Thus, one cannot automatically conclude that, if the fibers in a particular pathway receive a presynaptic depolarization, transmission in the pathway will be inhibited. IV. Conclusion
We have discussed the pharmacology and ionic mechanism of presynaptic inhibition in both vertebrates and invertebrates. For invertebrates, we have concentrated mainly on the crustacean nervous system.
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The presynaptic inhibitions observed in these two groups have many aspects in common. Pharmacological and anatomical studies suggest that the inhibition results from the release of GABA from axo-axonic synapses. There is evidence that in mammalian CNS presynaptic fibers may have GABA receptors even though they do not receive axo-axonic synapses. In addition, electrical stimulation of these presynaptic fibers can result in the activation of these receptors, presumably due to the diffusion of GABA from remote synapses. This raises the possibility that presynaptic inhibition might occur in regions which do not contain axo-axonic synapses. At the crustacean NMJ there is good evidence that GABA increases chloride permeability in the motor nerve terminal, which in turn results in a slight hyperpolarization. In vertebrates there is also evidence that GABA, at least on the cell bodies of primary afferents, increases chloride permeability. At the crustacean NMJ the increased conductance of the motor nerve terminal results in a reduction in spike amplitude and/or a block in impulse invasion into the terminal that reduces the number of quanta released per stimulus. The major difference in the presynaptic action of GABA in vertebrate primary afferents and in the crustacean NMJ is that, in the former, the increased chloride conductance results in a large depolarization. This in turn suggests that chloride ions are not passively distributed across the primary afferent membrane, but, rather, that there is an inwardly directed chloride pump. It is interesting that the primary afferents of crustacea receive a depolarizing synaptic input just as do vertebrates. Although the pharmacology and ionic mechanism of this has not been studied, the results suggest that the presence of an inward chloride pump may be a general feature of primary afferent neurons. The advantage of having a depolarization of the synaptic terminal during presynaptic inhibition is not clear. Partly, this is due to our ignorance of events that normally are involved in transmitter release from primary afferents. Eccles ( 1964) originally proposed that the depolarization would reduce the size of the terminal action potential and thus reduce the number of quanta released. However, only if (1) the action potential normally fully invades the terminals of primary afferents and (2) the quantal content of release from the terminal is large, could the depolarization contribute to the presynaptic inhibition. Yet, recent experiments have suggested variable invasion of terminals, and the quantal nature of transmitter release from primary afferent terminals is unclear. Particularly confusing is the old finding that the depolarization can, by itself, bring the fiber whose release is being inhibited, to threshold. These fiber discharges can be recorded as antidromic discharges from the dorsal
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root and are called dorsal root reflexes. The fact that the presynaptic inhibition of a particular fiber can result in its activation would appear to represent a breakdown in the inhibitory process. Other areas for future research include whether all primary afferent depolarization (PAD) is mediated by GABA. Although potassium appears to play only a minor role in PAD evoked by single spinal afferent stimuli it may contribute substantially during high-frequency afferent stimulation. The possibility of other transmitters being involved in PAD should be considered. Further experiments on other presynaptic fibers should be done to pursue the question of how widespread presynaptic GABA receptors are and whether these receptors have any physiological role. Finally, the mechanism of presynaptic inhibition in the vertebrate is still poorly understood, and there is little direct evidence that transmitter release is reduced during PAD. This lack of. clarity will persist until advances are made in our understanding of transmitter release from synapses of the central nervous system. ACKNOWLEDGMENTS T h e authors’ research is supported by Grants GM-23478 and NS00287 to R.A.N. and 9F32 NS-05744-02 to B.E.A.
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Simmonds, M. A. (1978).Br. J.Phurmacol. 63,495-502. Simmonds, M. A., and Pickles, H. G . (1978). In “Iontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System’’ (R. Ryall and J. Kelly, eds.), pp. 279-281. ElseviedNorth-Holland Biomed. Press, Amsterdam. Singer, W., and Lux, H. D. (1973). Bruin Res. 64, 17-23. Snyder, S. H., and Bennett, J. P., Jr. (1976). Annu. Rev. Physiol. 38, 153-176. Starke, K., and Endo, T. (1976). Gen. Phurmacol. 7, 307-312. Sykovb, E., and Vyklickf, L. (1977). Nmrosci. Lett. 4, 161-165. Sykova, E., and Vyklickf, L. (1978). Neuroscience 3, 1061-1067. Sykova, E., Shirayev, B., Kriz, N., and Vyklickf, L. (1976). Brain Res. 106, 413-417. Szekely, C., and Kosaras, B. (1977). E x f . Bruin Res. 29, 531-539. Szentagothai, J. (1968). In “Structure and Function of Inhibitory Neuronal Mechanisms” (C. von Euler, S. Skoglund, and U. Soderberg, eds.) pp. 15-32. Pergamon, New York. Takeuchi, A., and Onodera, K. (1972). Nature New Bwl. 236,55-56. Takeuchi, A., and Takeuchi, N. (1966a).J. Physiol. (London) 185, 418-432. Takeuchi, A., and Takeuchi, N. (1966b).J. Physiol. (London) 183, 433-449. Takeuchi, A., and Takeuchi, N. (1967).J. Physiol. (London) 191, 575-590. Takeuchi, A., and Takeuchi, N. (1969).J. Physiol. (London) 205, 377-391. Tauc, L. (1965).J . Physiol. (London) 181, 282-307. Tebecis, A. K., and Phillis, J. W. (1969). Comp. Biochem. Physiol. 28, 1303-1315. ten Bruggencate, G., Lux, H. D., and Lieble, L. (1974). PJluegers Arch. 349, 301-317. Tremblay, J. P., and Plourde, G . (1977). Can. J . Physiol. Phurmucol. 55, 1286-1301. Tremblay, J. P., Woodson, P. B. J,, Schlapfer, W. T., and Barondes, S. H. (1976).Bruin Res. 109, 61-81. Uchizono, K. (1965). Nature (London) 207, 642-643. Uchizono, K. (1973). Proc. Jpn. A d . 49, 569-574. Usdin, E., and Bunney, W. E., eds. (1975). “Pre- and Postsynaptic Receptors.” Dekker, New York. Van Harreveld, A., and Niechaj, A. (1970). Bruin Res. 19, 105-116. Walberg, F. (1965). Exp. Neurol. 13, 218-231. Wall, P. (1958).]. Physiol. (London) 142, 1-21. Wall, P. (1962).J. Physiol. (Ladmq) 164, 508-526. Washizu, Y. (1960)
[email protected]. Physiol. 10, 121-131. Waziri, R. (1977). Science 195, 190-192. Westrum, L. E. (1969). Z . ZelEforsch. Mikrosk. Anat. 98, 157-187. Woodson, P. B. J., Tremblay, J. P., Schlapfer, W. T., and Barondes, S. H. (1976).Bruin Res. 109, 83-95. Yoshimo, Y., De Feudis, F. V., and Elliott, K. A. C. (1970). Cun.J. Biochm. 48, 147-148. Zucker, R. S. (1974a).J. Physiol. (London) 241, 69-89. Zucker, R. S. (1974b).J. Physiol. (Lon&) 241, 111-126. Zukin, S. R., Young, A. B., and Snyder, S. H. (1974). Proc. Natl. Acud. Sci. U.S.A. 71, 4802-4807.
MICROQUANTITATION OF NEUROTRANSMITTERS IN SPECIFIC AREAS OF THE CENTRAL NERVOUS SYSTEM By Juan M. Saavedm
Section on Pharmacokgy, Labomby of Clinical Scmnce, Natbnal Institute of Mental Health, Bethesda, Marybnd
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Biochemical Techniques . . . . . . . . . . . . . . . . . . . . . . B. Microdissection of Brain Areas . . . . . . . . . . . . . . . . . . 111. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Distribution of Biogenic Amines in Selected Areas of the Brain . . . B. Interaction o f Catecholamines and Serotonin in Specific Brain Areas C. Effects of Hypertension and Stress on Catecholamine Levels in Localized Brain Stem Areas . . . . . . . . . . . . . . . . . . . . IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
.
.
259 261 26 1 265 270 270 270 271 273 273
1. Introduction
In recent years, a number of sensitive enzymatic-isotopic methods have been developed to measure biogenic amines and other compounds in small amounts of tissues and body fluids (Saavedra, 1974). These methods are based on the incubation of the biological material with specific methyltransferase enzymes, together with a donor of methyl groups, S-adenosyl-L-methionine (SAME). These enzymes catalyze, in the test tube as well as normally in tissues, the transfer of the methyl group from SAME to the 0 or N terminal positions of the amines, resulting in the formation of the corresponding 0 or N methyl derivatives (Axelrod and Tomchick, 1958; Axelrod, 1977; Saavedra, 1976). The use of a donor of radioactive methyl groups ([3H]methyl-SAME) of high specific activity, and the utilization of selective organic and chromatographic procedures to separate and quantitate the radioactive 0 or N derivatives formed, resulted in a number of specific micromethods, most of them with a sensitivity at the picogram level (Saavedra et al., 1973; Henry et al., 1975; Da Prada and Zurcher, 1976; Paul and Axelrod, 1977; Peuler and Johnson, 1977). Table I presents the general procedure for the measurement of 259 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 21
Copyright @ 1979 l by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-366821-2
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TABLE I GENERAL PROCEDURE FOR THE ENZYMATIC-ISOTOPIC ASSAYOF BIOCENIC AMINESAND RELATEDSUBSTANCES 1. Extract the amine from the tissue or body fluid (acid or buffer extraction). 2. Incubate with the corresponding N- or 0-methyltransferase and radioactive methyl donor (t3H]methyl-SAME). 3. Extract the radioactive N- or 0-methylated products formed in the reaction with specific organic solvents. 4. Eliminate the radioactive contaminants by selective drying or chromatographic procedures. 5. Count the radioactivity by liquid scintillation spectroscopy.
biogenic amines in tissues by the enzymatic-isotopic method. Table I1 lists some of the methods currently available. Microdissecting techniques to specifically isolate discrete brain areas have been available for a number of years (Eik-nes and Brizzee, 1965). Recently, these techniques, coupled with the new sensitive micromethods, have been widely used to study the distribution, metabolism, TABLE I1 SENSITIVITY OF ENZYMATIC-ISOTOPIC METHODSFOR THE DETERMINATION OF BIOCENIC AMINESAND RELATEDCOMPOUNDS ~~~
~~
~
~
~~
Compound A. Indoles 1. Tryptamine" 2. N-acetykerotonin* 3. SerotoninP B. Catechols 1. Norepinephrined 2. Norepinephrinee 3. Epinephrinee 4. Dopamine' 5 . Catechol estrogens'
" Saavedra and Axelrod
Enzyme
Sensitivity (picograms)
NMTP HIOMT NAT/HIOMT
250 25 25
PNMT COMT COMT COMT COMT
10 5 5 10 25
(1972).
Brownstein et al. (1973). Saavedra et al. (1973). Henry et al. (1975). Da Prada and Zurcher (1976) and Peuler and Johnson (1977). 'Paul and Axelrod (1977). Abbreviations used: NMT, Nonspecific N-methyltransferase from rabbit lung; HIOMT, Hydroxyindole-0-methyltransferase (EC 2.1.1.4) from bovine pineal gland; NAT, N-acetyltransferase (EC 2.3.1.5) from rat liver; PNMT, Phenylethanolamine N-methyltransferase from bovine adrenal; COMT, Catechola-rnethyltransferase (EC 2.1.1.a) from rat liver.
MICROQUANTITATION OF NEUROTRANSMITTERS
261
and response to physiological manipulations of the putative neurotransmitters in brain (Palkovits et al., 1974; Saavedra et al., 1979). The results obtained by the newly developed techniques in the last three years cover a wide area, relating to neuroanatomy, physiology, pharmacology, endocrinology, and biological psychiatry. We will restrict ourselves here to the presentation of a few examples regarding the specific localization, interaction, and response to some physiological manipulations of brain serotonin and catecholamines.
II. Methods
A. BIOCHEMICAL TECHNIQUES 1. Serotonin The essay is based on the conversion of serotonin to [3H]melatonin by a two-step reaction involving the N-acetylation of serotonin to form N-acetylserotonin, followed by the O-methylation of N-acetylserotonin to form melatonin, utilizing [3H]methylS-adenosyl-~-methionine (r3H1 SAME) as the methyl donor (Fig. 1). [3H]Melatonin is isolated from the rH]SAME by a simple organic extraction. Under the conditions of the essay, [3]melatonin is the only radioactive product isolated in detectable amounts. Other compounds and enzymes in the serotonin pathway can also be measured by this principle (Fig. 1). Tissues are dissected as described below and homogenized in 25 p1 of 0.1 N HCl at 4°C. Five p1 of the homogenate is removed for protein determination (Lowry et al., 1951)and the homogenates are centrifuged at 5000 rpm for 20 min. Ten p1 of the supernatant is transferred to 15-ml glass-stopped tubes combining 10 pl of a solution made with 10 parts of 0.2 M sodium phosphate buffer, pH 7.9, and 1.1 parts of 1 N NaOH (final pH 7.9) (solution A). a. Formation of N-Acetykerotonin. The reaction is carried out at 37”C, and it is started by the addition of 5 p1 of a mixture containing equal proportions of partially purified rat liver N-acetyltransferase and acetylcoenzyme A (AcCoA) (4 mg/ml in 0.1 mM HC1). b. Conversion of N-Acetykerotonin to [3HJklelatonin. After 30 min, 5 p.1 of a mixture containing 1.5 parts of partially purified hydroxyindoleO-methyitransferase (HIOMT), 1 part of [3H]SAME,and 2.5 parts of 0.2 M sodium phosphate buffer, pH 7.9 is, added. The reaction is allowed to proceed for 10 min and then stopped by the addition of 0.5 pl of 0.5 M
262
JUAN M. SAAVEDRA
0 7
CHz--fH--COOH NHZ
TRYPTOPHAN
1
TRYPTOPHAN HYDROXY LASE (6- methylpterin)
H O O T -
CHZ-iH-CWH NHZ
-
-
5 HY DROXY TRY PTOPHAN
L-AMINO ACID DECARBOXYLASE (Pyridoxal phosphate)
SEROTONIN I
N -ACETY L TRANSFERASE (Ac~I-COA)
N-ACETYL SEROTONIN HY DROXY INDOLE 0 - M E T H Y L TRANSFERASE (CH3 *-same)
MELATONIN
FIG. 1. Enzymatic-isotopic assays for serotonin and related enzymes.
borate buffer, pH 10, and 10 pl of a 1 mg/ml solution of melatonin in 25% ethanol. The radioactive product is extracted into 6 ml of toluene by mixing for 30 sec on a Vortex mixer and the aqueous and organic phases are separated by centrifugation. Five ml of the organic phase are transferred to counting vials containing 2 ml of toluene and are dried in a chromatography oven at 80°C overnight. After drying, 1 ml of ethanol plus 10 ml
MICROQUANTITATION OF N E U R O T R A N S M I T T E R S
263
of phosphor containing 40 ml of Liquifluor per liter of toluene are added to each counting vial, and the radioactivity of the samples is determined by liquid scintillation spectroscopy. Internal standards are prepared by adding 1 ng of authentic serotonin, dissolved in 10 p1 of solution A, to 10-pl aliquots of supernatant from cortical tissues. Tissue blanks are obtained by replacing the AcCoA by 0.1 mM HCI. Figure 2 represents a flow sheet for the serotonin assay.
2 . Catecholamines (Dopamine, Norepinephrine,
and Epinephrine)
This assay is based on the use of the partially purified enzyme catechol-0-methyltransferase to transfer a tritium-labeled methyl group from S-adenosyl-L-methionine to the catecholamines to form a radioactive 0-methylcatecholamine derivative (Da Prada and Zurcher, 1976; Peuler and Johnson, 1977). T h e radioactive products are then extracted into an organic solvent and separated by thin layer chromatography. Brain tissue from one rat is homogenized in 50 p1 of 0.1 N perchloric acid, and 5 pl is removed for protein determination. After centrifugation at 50,000 g for 20 min, a 30-p1 aliquot of the supernatant is transHOMOGENIZE TISSUES ( 0 . 1 N HCl )
+
SEPARATE ALIQUOT FOR PROTEIN DETERMINATION
+
CENTRIFUGE
+
SEPARATE WPLICATE ALIQUOTS OF SUPERNATANT f
ADO SEROTONIN INTERNAL STANDARD f
INCUBATE WITH LIVER NAT. AND ACETYL COENZYME A
+
INCUBATE WITH PINEAL HIOMT AND 'H-METHYL
+
S A k
STOP WITH BORATE BUFFER AND MELATONIN
+
EXTRACT INTO TOLUENE
+
EVAPORATE TOLUENE AT 80°C
+
ADD PHOSPHOR
+
COUNT
FOR 'H-MELATONIN FORMED
FIG. 2. Flow sheet of the radioenzymatic assay for serotonin (see text for details).
264
JUAN M. SAAVEDRA
ferred to incubation tubes. Ten pl of 0.1 N perchloric acid is added, followed by the additon of 40 p1 of incubation mixture, containing: (a) 0.1 mg of dithiothreitol; (b) 0.4 pl of 1 M MgCl,; (c) 35.6 p1 of 2 M Tris-HC1 buffer, pH 9.6, containing 4 mM EGTA; (d) 3 p1 of rH]Sadenosyl-L-methionine (1.5 pCi); and (e) 3 pl of partially purified rat liver catechol4-methyltransferase. After incubation at 37°C for 30 min, the reaction is stopped by the addition of 100 p1 of a freshly prepared mixture containing 80 p1 of 1 M borate buffer, pH 8.6, and 20 p1 of carrier (0.5 mg each of 3methoxytyramine, normetanephrine, and metanephrine in 0.0 1N HCI). After adding 50 p1 of 1.5% sodium tetraphenylborate, the O-methylated radioactive products are extracted into 2.5 ml of an organic solvent (3 parts of toluene and 2 parts of isoamyl alcohol, v/v). After shaking for 10 min in a mechanical shaker, and centrifuging to separate the phases, the aqueous phase is frozen in an acetone-Dry Ice bath, the organic phase is decanted into another tube containing 100 p1 of 0.1 N HC1, and the methoxylated catecholamines are back extracted into the acid phase. After shaking and centrifuging, the aqueous phase is frozen in an acetone-Dry Ice bath, and the organic phase is aspirated and discarded. The acid phase is washed once with 1 ml of the tolueneisoamyl mixture, and the organic phase is discarded. Methanol (100 pl) is added to the acid phase, and the total amount of liquid (200 p1) is spotted on silica-gel TLC plates (LQDF, Quanta Gram). The development of the thin layer chromatography plate is accomplished by the use of a solvent system containing ch1oroform:ethanol: 70% ethylamine (80:15: 10) (v/v). The methylated catecholamines are localized under UV light and extracted as follows: a. Methoxytyramine. For the assay of dopamine, the methoxytyramine product is scraped into disposable tubes containing 1 ml of 0.05 M ammonium hydroxide. After shaking for 15 sec, 10 ml of toluene:isoamyl alcohol (3:2)are added, and the product is extracted into the organic phase by shaking for 10 min in a mechanical shaker. After centrifugation to separate the phases, the aqueous phase is frozen in an acetone-Dry Ice bath and the organic phase is decanted into counting vials. After adding 10 ml of Aquasol (New England Nuclear), the radioactivity is counted in a liquid scintillation counter. b. Nomnetanephrine and Metanephrine. For the assay of norepinephrine and epinephrine, the radioactive normetanephrine and metanephrine formed are scraped into separated counting vials containing 1 ml of 0.05 M ammonium hydroxide. After shaking for 15 sec, 50 pl of 4% sodium periodate solution is added, and the cleavage reaction is stopped 5 min later by the addition of 50 pl of 10% glycerol.
MICROQUANTITATION OF NEUROTRANSMITTERS
265
The content of the vials is acidified with 100 p1 of 1 N acetic acid, the radioactive products extracted into 10 ml of toluene containing 400 p1 of Liquifluor (New England Nuclear) by shaking for 15 sec. After separation of the phases, the radioactivity is counted in a liquid scintillation counter. Internal standards consist of 30-p1 aliquots of brain tissue homogenate plus 10 pl of 0.1 N perchloric acid containing 0.1 to 1 ng of each catecholamine (dopamine, noradrenaline, and adrenaline). Blanks consist of 40 pl of 0.1 N perchloric acid (nontissue blanks) o r brain tissue homogenates and the incubation mixture which are incubated separately and combined after addition of the borate buffer. Figure 3 depicts a flow sheet for the catecholamine assay.
OF BRAINAREAS B. MICRODISSECTION
1. Preparation of Tissues Animals are killed by decapitation between 1 1 A.M. and 12 noon. T h e brains are removed and rapidly frozen on microtome specimen holders on Dry Ice. Serial sections of 300 pm thickness are cut in a cryostat at a temperature of - 10°C. Specific brain stem areas and nuclei are located under a dissecting microscope and dissected by the use of needles with an internal diameter of 0.5 mm or 0.3 mm, as described elsewhere (Schlumpf, 1973; Schlumpf et al., 1974; Eik-nes and Brizzee, 1965; Koning and Klippel, 1963). 2. Dissection of Specific Nuclei Specific brain stem areas are designated A 1, NCO, A2, NTS, and LC, as follows (Saavedra et al., 1979): The area designated as A1 corresponds to the catecholamine cell group A 1 of Dahlstrom and Fuxe (1964) and includes the PNMT-rich C1 region of Hokfelt et al. (1974). It is located in the rostral and lateral part of the nucleus reticularis lateralis of the medulla oblongata, between the pyramidal tract and the tractus spinalis nucleus trigemini, lateral to the olivary complex and just under the ventral surface of the medulla oblongata. Samples from this area are dissected from four serial brain sections, at a level 6.0-7.4 mm caudal to the interauricular line and 0.6-2.0 mm rostral to the spinal cord-medulla oblongata border. Areas containing parts of the nucleus tractus solitarii are dissected at three different levels in the brain stem, and include neurons from other vagal nuclei.
266
JUAN M. SAAVEDRA H W G E N I Z E TISSUES (0.1 N PU)
t SEPARATE M I Q W T FOR PROTEIN DETERMINATION 4
CENTRIFUGE t SEPARATE DUPLICATE ALIQUOTS OF SUPERMTANT t ADO CATECHOLAMINE INTERNAL STANDARDS t INCUBATE WITH OTT, EGTA. MgOlz. COMT AN0 3H-METHYL SAME t STOP WITH BORATE BUFFER, METHOXYCATECHOLAnINE CARRIERS, AN0 TPB t EXTRACT INTO TOLUENE-ISOAMYL ALCOHOL (3:Z) .I
BACK-EXTRACT INTO 0.1 N HCL t WASH WITH TOLUENE-ISOAMYL ALCOHOL 4
ADO METHANOL t SPOT I N SILICA-GEL TLC PLATE t DEVELOP I N CHLOROFORn/ET~ML/ETHrLAMINE 70X ( 1 2:3:2) t VISUALIZE S W T S BY U.V. LIGHT
t
M
'
t
NE OR E
t
SCRAP INTO DISPOSABLE NBES
t SCRAP S I L I C A INTO COUNTING VIALS
t EXTRACT WITH AmoNIUM HYDROXIDE
t EXTRACT PROWCTS WITH A m O N I U I HYMIOXIDE t EXTRACT INTO TOLUENE-IUMIIYL ALCHOHOL + (3:Z) CLEAVE WITH Iu PERIODATE .I
DECANT O R W I C PHASE TO COUNTING VIALS
t AOO AQUASOL COUNT
t
t STOP WITH GLYCEROL t ACIDIFY WITH ACETIC ACID t EXTRACT INTO TOLUENE t ADO PHOSPHOR t COUNT
FIG. 3. Flow sheet of the radioenzymaticassay for catecholamines (see text for details).
267
MICROQUANTITATION OF NEUROTRANSMITTERS
TS
FIG. 4. Frontal sections at different levels of the rat brain stem. Distances a r e in millimeters from the spinal cord-medulla oblongata border. Circles correspond to the tissue pellets removed. a: Area A l , 2: (NCO) nucleus tractus commissuralis, 3: Area A2.4: nucleus tractus solitarii, anterior part (NTS). DP: decussato pyramidis, rl: nucleus reticularis lateralis, tsV: tractus spinalis nervi trigemini, nic: nucleus intercalatus, cc: central canal, P: tractus corticospinalis, PCI: pedunculus cerebellaris inferior, nXII: nucleus origini nervi hypoglossi, AP: area postrema, on: olivary complex, nX: nucleus originis dorsalis vagi, NTS: nucleus tractus solitarii.
268
J U A N M . SAAVEDRA
-7.0
-5.0
FIG. 5. Localization and dissection of the raphe nuclei in the rat. The circles represent the size of the needle used to punch the area studied. 1: n. raphe pallidus; 2: n. raphe obscurus; 3: n. raphe magnus; 4: n. raphe pontis; 5: n. raphe dorsalis; 6: n. centralis superior; 7: n. raphe lineares; P: tractus cortico spinalis; io, nucleus olivaris inferior; nrp,
MICROQUANTITATION OF NEUROTRANSMITTERS
269
T h e caudal part of the nucleus tractus solitarii, or nucleus commissuralis (NCO),located in the midline just dorsal to the central canal, is dissected from two serial sections, at a level 7.4-8.2mm caudal to the interauricular line and 0.0-0.6 mm rostral to the spinal cord-medulla oblongata border (Fig. 4). The medial part of the nucleus tractus solitarii corresponds to the A2 area, although this area also includes neurons from another vagal nucleus, the nucleus intercalatus. The A2 area is located lateral to the central canal and ventrolateral to the area postrema, as well as close to the bottom of the fourth ventricle. This area is dissected from three sections, 7.4-6.5mm caudal to the interauricular line, and 0.6-1.5 mm rostral to the spinal cord-medulla oblongata border (Fig. 4). T h e anterior part of the nucleus tractus solitarii (NTS) also includes neurons from the nucleus originis dorsalis vagi. It is located lateral to the bottom of the fourth ventricle, and is dissected from two sections, at a level 5.5-5.0 mm caudal to the interauricular line and 2.0-3.0 mm rostral to the spinal cord-medulla oblongata border (Fig. 4). Both the A2 area and the NCO,as dissected here, are parts of dense catecholamine-fluorescent cell bodies of the A2 area, as described by Dahlstrom and Fuxe (1964),and of the C2 group, as described by Hokfelt el al. (1974). The locus coeruleus (LC)is located rostral to the facial genu, between the medial edge of the pedunculus cerebellaris superioris and the fourth ventricle. It is dissected from three sections, at a level 2.8-1.5 mm caudal to the interauricular line and 4.8-6.4mm rostral to the spinal cordmedulla oblongata border (Fig. 4). T h e raphe nuclei are all located in the midline and are dissected as follows (Palkovits et al., 1974) (Fig. 5): T h e nucleus raphe pallidus is located between the inferior olives and the pyramidal tract, 0.6-1.5 mm from the pyramidal decussation. The nucleus raphe obscurus, dorsal and rostral to the raphe pallidus, is situated in the midline between the paramedian reticular nuclei, and halfway between the ventral and dorsal surfaces of the medulla. It is dissected between 1.5 and 0.2 mm from the pyramidal descussation. T h e nucleus raphe magnus is removed from sections 3.0-4.0 mm nucleus reticularis paramedianus; XII, nucleus originis n. hypoglossi; FLM, fasciculus longitudinalis medialis; nrv, nucleus reticularis medullae oblongatae pars ventralis; lc, locus coeruleus; rpoc, nucleus reticularis pontis caudalis; LM, lemniscus medialis; PCS, pedunculus cerebellaris superior; ntv, nucleus tegmenti ventralis Gudden; rtp, nucleus reticularis tegmenti pontis; nl, nucleus linearis; ip, nucleus interpeduncularis. Frontal sections are numbered with the coordinatesrostral (+) and caudal (-) to the interauricular line.
270
JUAN M. SAAVEDRA
from the pyramidal decussation in the midline and just above the pyramidal tract. The nucleus raphe pontis is removed with a small needle (0.3 mm internal diameter), in the midline of the pons, at a distance of 4.5-6.0 mm from the pyramidal decussation. T h e center of this nucleus is halfway from the basal surface of the brain and the bottom of the fourth ventricle. The most caudal portion of the nucleus raphe dorsalis is located at 6.0 mm from the pyramidal decussation, and is removed from 6.3 mm to 7.2 mm from the decussation. T h e nucleus raphe medianus is continuous rostrally with the nucleus centralis superior, and both nuclei are removed together. The pedunculus cerebellaris superior (PCS) is used as a point of reference so that the nuclei are removed at the level where the PCS is horizontal and its fibers are beginning to decussate (Fig. 5).
111. Results
A. DISTRIBUTION OF BIOCENIC AMINES IN SELECTED AREASOF THE BRAIN Table I11 shows the distribution of the catecholamines dopamine, norepinephrine, and epinephrine, and of serotonin, in specific areas of the brain stem and in the raphe nuclei. All amines are present in all areas studied. Serotonin levels are higher in the raphe areas, where the cell bodies of the serotonin-forming neurons are located, but are also high in the A1 and A2 areas, where catecholamine-forming neurons are concentrated. Conversely, although the catecholamines are concentrated in catecholamine-rich areas such as areas A1 and A2 and the locus coeruleus, they are also present in the serotonin-rich raphe nuclei. These anatomical correlations strongly suggested the possibility of a physiologic interaction between the two aminergic systems (Saavedra et al., 1976b).
B. INTERACTIONOF CATECHOLAMINES AND SEROTONIN IN SPECIFIC BRAINAREAS Specific brain areas of the rat were examined for their content of serotonin after treatment of the animals with an inhibitor of the synthesis of norepinephrine, the dopamine-P-hydroxylase inhibitor
27 1
MICROQUANTITATION OF NEUROTRANSMITTERS
TABLE 111 DISTRIBUTION OF BIOCENIC AHINESIN SELECTED AREASOF THE RAT BRAIN Nuclei N. raphe pallidus N. raphe obscurus N. raphe magnus N . raphe pontis N. raphe dorsalis N. raphe centralis superior N. raphe lineares A1 area NCO A2 area NTS LC
Serotonin (5-HT)
Dopamine @A)
Norepinephrine (NU
Epinephrine (E)
18.3 f 3.6' 25.9 f 9.8 17.7 f 2.4 18.3 f 3.9 31.1 f 5.2 19.5 f 2.9 26.2 f 2.2 18.2 f 1.1
1.4 f 0.0 0.9 f 0.3 1.5 f 0.4 1.0 f 0.2 4.0 f 0.7 2.4 & 0.8 2.0 f 0.4 1.5 f 0.2 5.1 ? 1.6 2.2 f 0.2 1.6 f 0.3 3.0 2 0.4
2.8 f 0.4 2.7 f 0.6 6.2 f 1.3 2.0 2 0.1 9.9 -c 2.2 3.2 f 0.5 3.0 f 0.1 13.0 f 2.0 36.1 f 5.0 34.2 f 3.6 19.6 f 1.2 41.4 f 8.1
NAb NA NA NA NA NA NA
NAb 20.1 f 3.2 9.7 f 0.6 10.6 f 0.7
' Results are expressed as n g h g protein, determinations. NA = not assayed.
2*
0.8 ? 0.1 1.7 f 0.3 1.2 f 0.2 0.6 f 0.1 0.8 f 0.3
SEM for groups of eight individual
l-phenyl-3-(2-thiazolyl)-2-thiourea (Saavedra et al., 197613). This treatment resulted in a more than 80% depletion of brain norepinephrine, and in selective increases in serotonin concentrations in the nucleus raphe magnus and in the nucleus centralis superioris (Table IV). On the other hand, no change in serotonin levels was detected in the nucleus raphe dorsalis. Thus, alterations in norepinephrine metabolism could result in rapid modifications in serotonin levels, these changes occurring only in specific brain areas.
C. EFFECTSOF HYPERTENSION AND STRESS ON CATECHOLAMINE LEVELS I N LOCALIZED BRAINSTEMAREAS Catecholamines have long been implicated in the central regulation of the stress reaction and in some forms of hypertension (Van Zwieten, 1975). T h e specific localization of catecholamines in selected brain stem areas, which formed part of recognized circuits for the central cardiovascular control (Van Zwieten, 1975), prompted us to study the changes in steady state level of catecholamines in those areas after acute immobilization stress (Kvetnansky et al., 1977) and in spontaneous hypertensive (SH) rats (Saavedra et al., 1976a, 1977). Table V summarizes our recent findings. Decreases in norepinephrine and epineph-
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JUAN M. SAAVEDRA
TABLE IV EFFECTOF INHIBITION OF DOPAMINE-/~-HYDROXYLASE ON THE LEVEL OF NOREPINEPHRINE AND SEROTONIN IN SPECIFIC AREASOF THE RAT BRAIN’ NE (nglmg protein)
5-HT (ng/mg protein)
Area A1 A2 Raphe magnus Locus coeruleus Raphe dorsalis N. centralis superior
Control
DBHI
% Change
Control
DBHI
% Change
12.6 f 1.0 12.7 f 1.9 16.9 2 1.3 16.7 f 1.9
+8.7 -1.2
9.6 f 0.7 0.2 2 O.lb 18.6 f 2.8 1.4 f 0.3’
-90
18.5 f 4.0 28.0 & 3.1c
+51.7
16.1 k 3.7 0.3 f 0.2b
-98
15.82 2.4 15.8 f 0.6
0
29.2 2 6.6 6.0 f 0.8’
-79 -98
43.6 2 4.0 45.3 -C 1.5
+3.8
17.1 2 1.6 0.2 f 0.2’
25.4 k 2.6
+42.5
12.1 f 2.8
36.2 2 2.3b
-92
NDd
’Data from Saavedra et al. (1976b). l-Phenyl-3-(2-thiazolyl)-2-thiourea was administered intraperitoneally 18 hr (200mg/kg) and 4 hr (25mg/kg) before sacrifice. Results are expressed as means f SEM. N = 8 in all regions. * Statistically significant, P < 0.01. c P < 0.05. ND = not detectable (< 0.2 ng/mg protein) (Student’s t test).
TABLE V OF CATECHOLAMINES I N SPECIFIC BRAIN STEM AREAS CHANGES IN STEADY STATE LEVELS IN STRESS AND IN HYPERTENSION’ Spontaneous hypertensive (SH) rats
Stress Area
DA
NE
E
NE
E
A1
-1 +4 +25 - 10 -30
-2 -15 -10
- 32b
+9 NA‘
-47’
-3
-7
NCO A2 NTS LC
- 5ob - 16
-63b -37’ -4ob
-33’
NA‘
NA
NA
-18
-47b
a Results are expressed as percent change over controls. Stress was produced by immobilizing the rats for 4 hr. Controls were nonimmobilized rats (Kvetnansky et al., 1977).Spontaneously hypertensive rats (SHR) and Wistar-Kyoto controls were killed at 4 weeks of age (Saavedra et al., 1977).Results modified from Saavedra et al. (1978)and Saavedra et al. (1979). Statistically significant, P < 0.05;Student’s t test. NA = not assayed.
MICROQUANTITATION OF NEUROTRANSMITTERS
273
rine levels, possibly indicating increased release and metabolism of these amines after neuronal activation, are noted in some brain stem areas. The response is more generalized for epinephrine, which is found to be decreased in almost all the areas analyzed. Norepinephrine is specifically decreased in a few brain stem areas, and only significantly in the NTS area, after acute stress (Table V). These results strongly suggest that catecholamines, and specifically epinephrine, are implicated in the central regulation by brain stem areas of the acute stress reaction, and in the development of spontaneous hypertension in the rat. Since the stress reaction results in significant changes in peripheral catecholamine metabolism (Kvetnansky et al., 1977), and changes in the peripheral sympathetic system occur also in hypertension (Grobecker et al., 1975; Nagatsu et a!. , 1976), it is tempting to speculate that central catecholaminergic areas could control the catecholamine metabolism in the periphery, or that central catecholamines are able to respond to corresponding changes in peripheral amines. Since serotonin is also concentrated in those brain areas, and this amine has been implicated in the central co-ntrol of hypertension as well as in the central regulation of the stress reaction (Van Zwieten, 1975), it would be interesting to study the metabolism of this amine under similar conditions.
IV. Conclusion
Newly developed microdissecting and microanalytical techniques have opened new perspectives for the study of central biogenic amines and related substances in a number of physiological and pathological conditions. Interactions between neurotransmitter systems in the brain may very well be restricted to a few key areas. These changes can only be studied by the dissection of restricted brain areas, and by the use of specific microquantitative techniques with sensitivity at the picogram level. REFERENCES
Axelrod, J. (1977). In “The Biochemistry of Adenosylmethionine” (F. Salvatore, E. Borek, V. Zappia, H. G. Williams-Ashman, and F. Schlenk, eds.), pp. 539-554. Columbia Univ. Press, New York. Axelrod, J.. and Tomchick, R. (1958).J. Eiol. Chem. 23, 702-705. Brownstein, M.J., Saavedra, J. M., and Axelrod, J. (1973). Mol. Pharmacol. 9,605-61 1. Dahlstrom, A., and Fuxe, K. (1964). Actu Physiol. Scund., SupPl. No. 232, 1-55.
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Da Prada, M., and Zurcher, G. (1976). LiJe Sci. 19, 1161-1 174. Eik-nes, K. B., and Brizzee, K. R. (1965). Biochim. Biophys. Acta 97, 320-333. Grobecker, H., Roizen, M., Weise, V., Saavedra, J . M., and Kopin, I. (1975). Nature (London) 258, 267-268. Henry, D. P., Starman, B. J,, Johnson, D. G., and Williams, R. H. (1975). Life Sci. 16, 375-384. Hokfelt, T., Fuxe, K., Goldstein, M., and Johansson, 0. (1974). Brain Res. 66, 235-251. Konig, J. F. R., and Klippel, R. A. (1963). “The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem.” Williams & Wilkins, Baltimore, Maryland. Kvetnansky, R., Sun, C. L., Torda, T., and Kopin, I. J. (1977). Pharmacologist 19, 241. Lowry, 0. H., Rosebrough, N. J., Fan, A. L., and Randall, R. J. (1951).J. Biol. Chem. 193, 265-275. Nagatsu, T., Ikuta, K., Numata (Sudo), Y., Kato, T., Sano, M., Nagatsu, I., Umezawa, H., Matsuzaki, M., and Takeuchi, T. (1976). Science 191, 290-291. Palkovits, M., Brownstein, M. J.. and Saavedra, J. M. (1974). Brain Res. 80, 237-249. Paul, S. M., and Axelrod, J. (1977). Lfe Sci. 21, 493-502. Peuler, J. D., and Johnson, G . A. (1977). L f e Sci. 41, 625-636. Saavedra, J. M. (1974). Prog. Anal. Chem. 7, 33-44. Saavedra, J. M. (1976). In “Essays in Neurochemistry and Neuropharmacology” (M. Yaudim and W. Lovenberg, eds.), Vol. 1, pp. 1-41. Wiley, New York. Saavedra, J. M., and Axelrod, J. (1972).J. Pharmacol. Exp. Ther. 182, 363-369. Saavedra, J. M., Brownstein, M. J., and Axelrod, J. (1973).J. Pharmacol. Exp. Ther. 186, 508-515. Saavedra, J. M., Grobecker, H., and Axelrod, J. (1976a).Science 191,483-484. Saavedra, J. M., Grobecker, H., and Zivin, J. (197613).Brain Res. 114, 339-345. Saavedra, J. M., Grobecker, H., and Axelrod, J. (1977).Mayo Clin. Proc. 54, 391-394. Saavedra, J. M., Grobecker, H., and Axelrod, J. (1978). Circ. Res. 42, 529-534. Saavedra, J. M., Kvetnansky, R., and Kopin, I. J. (1979).Brain Res. 160, 271-280. Schlumpf, M. (1973). Doctoral Thesis, Eidgenoessichem Techniashen Hochschule Zurich, Zurich. Schlumpf, M., Waser, P. G., Lichtensteiger, W., Langemann, H., and Schlup, P. (1974). Biochem. Phannacol. 23, 2447-2449. Van Zwieten, P. A. (1975). Prog. Pharmacol. 1, 1-63.
PHYSIOLOGY OF GLIA: GLIAL-NEURONAL INTERACTIONS By R. Malcolm S t e w * and Roger N. Rosenbeg Depattment of Nmumlogy, Southwerhrn Medical School, The University of Texas Health Science Center at Dallas, Texas
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Classification of Glial Cells . . . 111. Role of Glia in CNS Development
. . . . . . . . . . . . . . . . . . . . . . . A. Neuronal Migration and Connectivity . . . . . . . . B. Myelination of Neurons . . . . . . . . . . . . . . . C. Glial-Neuronal Differentiation . . . . . . . . . . . IV. Potassium and Neuroglial Function . . . . . . . . . . A. Glial Membrane Potential . . . . . . . . . . . . . .
. . . . . . . B. GIiaI Cells and Regulation of Extracellular Potassium .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Depolarization of Glia and Extracellular Potential Shifts . . . . . . . D. Spreading Depression and Seizures . . . . . . . . . . . . . . . . . V. Putative Neurotransmitters and Glia . . . . . . . . . . . . . . . . . . A. Amino Acid Transport . . . . . . . . . . . . . . . . . . . . . . B. Release of Amino Acids . . . . . . . . . . . . . . . . . . . . . . C. Glutamine-Glutamate Neuronal-Glial Cycle . . . . . . . . . . . . . D. Cyclic Nucleotide Responses and Glial Cell Receptors . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
275 276 278 278 281 283 291 291 293 294 296 297 297 300 301 301 304 304
1. Introduction
T h e purpose of this paper is to provide an overview of the physiology of neuroglia and to discuss some of the recent advances in this field. T h e central task we have set for ourselves is the development of a general awareness of the close interaction of glia with neurons. This glialneuronal relationship begins during the embryonic development of the organism and continues throughout its life. Pertinent aspects of glial pathophysiology and its consequences for neural function and dysfunction will be discussed in the hope of better understanding the normal physiology. T h e physiology of microglia has been discussed by Cammermeyer (1970) and will not be discussed here. T h e blood-brain barrier appears to be more a function of vascular endothelial cells than glial cells and will not be reviewed here (Kuffler and Nicholls, 1966). This review will emphasize functional glial-neuronal interactions and suggest our view that glia are active rather than passive partners in the ongoing 275 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 2 1
Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-366821-2
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glial-neuronal relationship and are critical for sustained and efficient neuronal functioning. For completeness, previous reviews on neuroglia should be consulted (Galambos, 1964; Kuffler and Nicholls, 1966, 1976; Bunge, 1968; Lasansky, 1971; Sidman and Rakic, 1973; Watson, 1974; Varon, 1975; fomjen, 1975; Somjen et al., 1976). II. Classification of Glial Cells
Glial cells were first distinguished from neurons by Virchow (1871), who conceived of them as a form of connective tissue within the CNS and gave them the name neuroglia, or nerve glue. T h e major types of glia include astrocytes, oligodendroglia, ependymal cells, and microglia. T h e first three cell types are generally thought to derive from neuroectoderm, whereas microglia are primarily of mesenchymal origin (histocytes) (see Table I). Neuroepithelial cells that line the primitive neural tube differentiate into neuroblasts and astroblasts. The primitive astroblast has the potential to become either an astrocyte or an oligodendrocyte, both of which subsequently develop special relationships with neurons. Early in embryogenesis, the astrocytes develop radial glial guides, and, later, oligodendrocytes myelinate axons (Bunge, 1968; Sidman and Rakic, 1973). Astrocytes are classfied histologically into fibrous astrocytes and protoplasmic astrocytes (see Fig. 1). Fibrous astrocytes are recognizable by the presence of fibers running in their processes. T h e processes are longer than those of the protoplasmic astrocytes, in which branching is more frequent. As a rule, fibrous astrocytes are found predominantly in white matter, whereas protoplasmic astrocytes are more prevalent in gray matter. T h e Bergmann glia in the cerebellum are a specialized form of astrocyte. Distinguishing oligodendroglia from other glia was accomplished by del Rio Hortega (1919). Although oligodendroglia may be present in gray matter and are known as perineuronal satellites, they are many TABLE I
ONTOCENY OF NEUROCLIAL CELLS ~~~~~
Type Astroglia Oligodendroglia Ependymal cells Microglia
~~
~
Origin Neuroectoderm Neutoectoderm Neuroectoderm Mesoderm
Presence Early Late Early Rare
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PHYSIOLOGY OF GLIA
GLIAL CELL TYPES
PROTOPLASMIC ASTROCYTE
' \
FIBROUS ASTROCYTE
OLIGODENDROCYTE ROD MICROGLIACYTE
MlCROGLlAL PHAGOCYTE
EPENDYMAL CELL
FIG. 1. Glial cells in the CNS. Astrocytes, oligodendrocytes, and ependymal cells are considered to be of neuroepithelial origin, whereas microglia are predominantly, if not exclusively, of mesenchymal origin.
times more common in the white matter and may be referred to as the intrafascicular glia. Occasionally, oligodendroglia may appear as perivascular satellites (Sen Sharma and Singh, 1973). Oligodendroglia are recognizable by a small rounded nucleus with dense cytoplasm in a small irregular cell with few processes. They have abundant microtubules. Oligodendroglia are closely applied to myelinated fibers in white matter and are the synthetic cell for CNS myelin. They are considered to be functionally analogous to the Schwann cell of the peripheral nervous system, which synthesizes peripheral nervous system myelin (Bunge, 1968). Ependymal cells line the ventricular system within the brain and central canal of the cord. They may be ciliated and are aligned in a palisade formation. T h e nuclei are dark and the cell bodies are cylindrical. Little is known about their function other than possible participation in the circulation and secretion of cerebrospinal fluid (Fleishhauer, 1973). Microglia are dense, dark, small cells of mesenchymal origin and can be confused with endoplasmic reticulum. When found in gray matter they are generally bipolar or multipolar free cells, whereas in white matter they are found predominantly around blood vessels. T h e microglial nucleus is dense and elongated in bipolar cells and more irregular
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when processes are present. Microglia may lengthen and enlarge and are recognized as rod microglial cells. Importantly, microglial cells may phagocytize lipid, as may be observed during breakdown of myelin, at which time the cells enlarge and become more rounded and the nucleus becomes more compact. Microglia can migrate and, in fact, are predominantly of extracranial origin. A small number of microglia arise from neuroectoderm (Cammermeyer, 1970).
111. Role of Glia in CNS Development
A. NEURONALMIGRATIONA N D CONNECTIVITY 1. Cerebral Cortex The exact origin and earliest ontogeny of glia in the CNS have not been completely elucidated, but the development of neuroglia during later embryonic stages has been well characterized (Sidman and Rakic, 19’73). An early and active interaction between astroglia and neurons seems evident, however, as is shown by the critical relationship of glia in neuronal migration to the cerebral cortex. Following the formation of the neural tube, the CNS wall is occupied by a ventricular zone of proliferating cells and an outer marginal cell sparse zone, separated by an intermediate zone consisting of postmitotic cells (Sidman and Rakic, 1973). A subventricular zone consisting of radially aligned cells develops between the intermediate and the ventricular zones. At present, there is no general agreement concerning whether o r not the dividing cells in the subventricular zone are common stem cells for both glia and neurons or have differentiated into separate committed populations of glial and neuronal-cell precursors. At somewhat later stages of fetal development, this subventricular zone contains radially aligned cells whose processes traverse the entire thickness of the developing CNS (Rakic, 1971, 1972). These radially oriented glia have been identified in the fetus as embryonal astrocytes and are first clearly recognizable by about midgestation. These radial glia are modified astrocytes and serve as guide cells for the postmitotic neurons migrating from the subventricular zone to form the successive layers of the cerebral cortex in an inside-out pattern (Sidman and Rakic, 1973). Using the glial guide, the migrating neuron can traverse the multitude of cells and processes and arrive at its proper destination. As these glial cells are not recognizable in the adult CNS, they are presumed to either degenerate or perhaps transform into astrocytes after neuronal migration is complete (Sidman and Rakic, 1973) (Fig. 2).
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279
Myelin Formation by Oligodendrocyte
Cell Migration on Radial Glial Guide
FIG. 2. Glial-neuronal relationships. On the left, two neurons are migrating from the subventricular zone to the cortex and are in contact with the radial glial guide cells (modified astrocytes). On the right, an oligodendrocyte envelops a neuron with myelin.
Dysfunction relating to the radial glial guides in the cerebral cortex has been proposed as being responsible for producing the human cerebral malformation, lissencephaly (agyria-pachygyria), which, pathologically, is considered to be a disorder of neuronal cell migration. Clinically, microcephaly, seizures, blindness, deafness, and mental retardation are present (Stewart et al., 1975). This congenital malformation is characterized by a smooth cerebral cortex instead of the normal convolutional pattern. Neuropathological examination reveals heterotopic (misplaced) neurons that are located below the cerebral cortex in the area normally occupied by the white matter. Histologically, those neurons that have reached the cortex as well as those that have failed to reach their assigned destination appear relatively normal and are arranged in a radial pattern. A cell-sparse zone separates these two groups of neurons. The pathological picture of this human malformation is consistent with the formulation that the dysfunction in the neuronal migration process relates to this radial glial guide. Interruption of this radial guide would leave stranded those neurons migrating after a pathological intrauterine event. Although an experimental model with histopathological evidence of glial guide dysfunction in cerebral cortex is forthcoming, an analogous model is present for the cerebellum.
2. Cerebellum A similar glial guide-neuronal relationship apparently holds for the migrating granule cells in the developing cerebellum. The processes of
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R. MALCOLM STEWART AND ROGER N . ROSENBERG
the Bergmann glia (modified astrocytes) stretch from the outer surface of the external granule layer, across the molecular layer, to the Purkinje cell layer (Das, 1976; Del Cerro and Swarz, 1976; Basco et al., 1977). This glial guide allows the migrating granule cell to cross the highly complex field of cellular processes in the molecular layer and to reach its destination in the internal granular layer. Several granule cell neurons may migrate in sequence on the same glial guide. Not all cells in the CNS, however, migrate by means of glial guides and may depend on other mechanisms, such as the recognition of cell surface properties of other cells, to provide contact guidance (Das et al., 1974). A dysfunction of the glial cell guide (Bergmann glia) can affect the migration of granule cells in the cerebellum from the external to the internal granule cell layer (Rakic and Sidman, 1973a,b,c). For instance, in the homozygous weaver mouse mutant, most granule cells degenerate in the external granule layer of the cerebellum before they migrate, whereas in the heterozygous mutant an intermediate disorder of granule cell migration is evident. Electron microscopic analysis demonstrates that the Bergmann glia show degenerative changes and are reduced in number in the heterozygous form and are almost totally absent in the homozygous weaver mutant (Rakic and Sidman, 1973a). Those granule cells that do migrate in the heterozygous mutant do so more slowly than those of controls (Rakic and Sidman, 1973a). The migration failure leads to a cerebellum slightly reduced in size in the heterozygote and markedly reduced in the homozygote (Rakic and Sidman, 1973b). The glial changes are recognized at 3 days of postnatal life, using electron microscopic analysis, by their enlarged irregular size and dense bodies and vacuolation in the heterozygous mice (Rakic and Sidman, 1973b), whereas in the homozygous mice glia appear reduced in number. Heterozygous weaver and control mice, however, could not be distinguished using a glial fibrillary acidic (GFA) protein immunofluorescence technique. Although the Bergmann glia of homozygous strains were not decreased in number by this technique, they did demonstrate a shortening and an irregularity in shape and spacing compared to controls (Bignami and Dahl, 1974). The in uztro maintenance of granule cells from the weaver and controls shows equal survival of both cells in tissue culture (Messer and Smith, 1977). These results support the premise of a primary defect in the glia, which, in turn, leads to an abnormality of glial-neural interaction and failure of migration. The granule cells apparently die after failing to achieve the proper synaptic connectivity from Purkinje cells, although Sotelo and Changeux (1974) reported a few degenerating granule cells that had already migrated, thereby suggesting an intrinsic granuIe cell defect. The dendritic spines taken
PHYSIOLOGY OF GLIA
28 1
from these mutant Purkinje cells, which normally make contact with granule cells, show normal postsynaptic development (Hirano and Dembitzer, 1973) but are inappropriately surrounded by abnormal glial processes, as shown by electron microscopic (Rakic and Sidman, 1973b) and freeze fracture studies (Hanna et al., 1976).
3 . Spinal Cord T h e radial glial guides may also be important for neuronal connectivity in the developing spinal cord. In the human cervical spinal cord, glioblasts synthesizing DNA, studied in embryos, are first detected at the 8-week stage in the mantle and marginal zones (Fujita, 1973). As the dendrites grow from the motor neurons, they begin to course along the processes of the radial glial guides in the direction of the marginal zone. As they begin receiving synapses, the dendrites may turn away from the glial guide (Henrikson and Vaughn, 1974). Although neuronal cell bodies themselves are not migrating, the glial cells seem important in providing contact guidance of the neural processes to their correct connections. 4. Retina
The development of the retina also depends on cell proliferation, migration, and differentiation. Recent studies of chick retina, using the scanning electron microscope, have demonstrated that migrating ganglion cells are oriented in columns radial to the vitreal surface. These migrating ganglion cells appear to have cell-to-cell contact. It has been proposed that the organization of the Muller cell (a glial cell) in the retina may function in a manner analogous to the radial glial guides of the cerebral cortex (Meller and Tetzlaff, 1976).
B. MYELINATION OF NEURONS The role of oligodendroglia in myelin formation has been extensively discussed by Bunge (1968). A knowledge of the physiology of oligodendroglia is important, however, for understanding the process of normal myelination as well as for a consideration of remyelination occurring, for instance, after demyelination (breakdown of normally formed myelin) in multiple sclerosis or allergic encephalomyelitis. Although the role of the Schwann cell in the peripheral nervous system clearly relates to increasing nerve conduction velocity, the analogous role of the oligodendroglia in the CNS is less clear. It is generally thought that myelin may help isolate and insulate neurons. Certainly after demyelination, dysfunctions such as blindness, ataxia, and spasticity attest to the importance of CNS
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myelin. The origin of the oligodendrocyte also remains unclear. Although an origin from a common stem cell for astroglia and oligodendroglia has been proposed (Vaughn and Peters, 197 l ) , oligodendroglia are recognized rather late in brain development, usually following neuronal migration and the development of astrocytes and just prior to my elination. Most myelination occurs postnatally. In the cat myelination occurs postnatally in cerebellar cortex up to 60 days (Lange, 1978). In the rat it is usually complete by 60 days, whereas in the human it continues past the first year of development and into adulthood (Yakovlevand Lecours, 1967). This myelination of the CNS, which begins in the brainstem prior to birth and progressively involves the spinal cord and cerebral hemispheres, correlates generally with the successive milestones of development (Yakovlev and Lecours, 1967). Intrauterine or postnatal insults, however, may alter the development or function of these oligodendroglia. For example, rats made hypothyroid at birth show poor development and demonstrate sluggish behavior. Pathological examination of the brains from these animals shows oligodendroglia that apparently fail to migrate to the cortex and to areas of white matter development, leading to a failure of myelination (Bass and Young, 1973). In the rat cerebellum, hypothyroidism results in a doubling of Bergmann glia as well as of the total number of astrocytes, yet the disappearance of the external granular layer is retarded (Clos et al., 1973). A genetic form of dysmyelination (failure to form normal myelin) is seen in the sex-linked recessive mutant mouse strain “jimpy.” A decrease in the maturation and proliferation of oligodendroglia is associated with the failure of myelination (FarkasBargeton et al., 1972; Meier and Bischoff, 1975) and with an accumulation of lipid droplets in differentiated cells and oligodendroglia. This genetic disorder may be similar to the inherited human neurological condition sudanophilic leukodystrophy (Phillips, 1954), which is associated with a failure to achieve myelination. Phenylketonuria, a disorder of phenylalanine metabolism, may, in the untreated state, result in, among other defects, a dysmyelination with subsequent mental-retardation and neurological dysfunction (Knox, 1976). A turnover of oligodendroglia cells has been demonstrated (KrausRuppert et al., 1973), with the number of oligodendroglia cells depending on a dynamic balance. In the rat, subependymal cells arise after birth, migrate into the corpus callosum and other areas, and can be identified as oligodendroglia (Paterson et al., 1973). T h e cycle time for proliferating glial cells outside the subependymal layer has been calculated to be about 20 hr in brains of adult mice for both astrocytes and
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283
oligodendroglia (Korr et al., 1973). These oligodendrocytes develop through a light phase (4-7 days) with prominent organelles, followed by a medium phase lasting about 2 weeks, and then a dark phase of fairly long duration (weeks to months) (Vaughn, 1969; Imamoto et al., 1978; Sturrock, 1974). Myelination probably occurs during the active light and medium stages, whereas the dark phase may be only for maintenance of the myelin sheath (Imamoto et al., 1978). In the jimpy mouse mutant the medium and dark oligodendroglia are absent (Privat, 1975). Early growth of the axonal sheath occurs in a random fashion, with a helical extension around the axon like the thread of a screw rather than concentric spiraling of cytoplasm (Knobler et al., 1976; Caley and Maxwell, 1968). In the white matter of the spinal cord, a rapid sixfold increase in the number of neuroglia occurs in neonatal and postnatal rats up to 6 weeks, after which time the number of neuroglia appears stable (Gilmore, 1971). Oligodendroglia in optic nerve begin final division a day o r two before the onset of myelination, but most are produced during myelination. Few are produced after myelination (Skoff et al., 1976a,b). Myelination is most rapid in rat optic nerve in the first 3 weeks of postnatal life and is synchronous with proliferation of oligodendroglia (Hirose and Bass, 1973). Oligodendroglia do continue to proliferate even at an advanced age (Dalton et al., 1968), although the turnover is slow, with a renewal of only two to three cells per 1000 each day (Nadler, 1978). Some decrease in the density of astrocytes and oligodendrocytes has been noted before 108 days of age in rat occipital cortex but not between 108 and 650 days (Diamond et al., 1977). Similarly, the number of glia in white matter, as studied in the anterior and posterior commisures of mice, decreased to about 60% at 18 months compared to 5 months, although no change in myelination occurred (Sturrock, 1976). Part of this decrease may relate to selective cell death in the early postnatal period of development and may play a role in internodal elongation, with a possible increase in conduction velocity, as illustrated in the study of the cat spinal cord white matter (Hildebrand, 1971).
C. GLIAL-NEURONAL DIFFERENTIATION 1. Environmental and Metabolic Influences Ramon y Cajal(l928)speculated on a metabolic relationship between neurons and glia. He spoke of a “mutually serviceable” metabolic relationship and a symbiotic interaction between neurons and glia in the
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mammalian brain. He was led to this conclusion on anatomic grounds, because of the relationship that glia have to neurons and blood vessels. H e could trace both protoplasmic and fibrous astrocytes having processes on blood vessels and the same astrocyte having another process on a neuron. Further, glia were juxtaposed between neurons, and still others could be traced between neurons and the ependymal surface of the lateral ventricles. This intermediate position of glia suggested a buffering or regulatory role between blood vessels and the neuronal compartment. Subsequent research, however, suggests that endothelial cells, rather than glial cells, are primarily responsible for the blood-brain barrier (Kuffler and Nicholls, 1966). Galambos (1961, 1971) suggested a close neuronal-glial metabolic relationship, comprising a single unit made of two cell types, glia and neurons. Communication within this postulated unit would occur at synapses o r specialized junctions between neurons but also between glia at gliapses and between neurons and glia via the extracellular space. Effects of neurons on glial cells have been investigated in several in vivo studies. In rats raised in enriched environments the number of glial cells is said to increase, resulting in a thicker cortex compared to littermate controls raised without such stimulation (Altman and Das, 1964; Diamond et al., 1966). The increased ratio of glia to neurons of 16% was seen in 12 out of 17 pairs of rats. In another study, dehydration in the rat, substituting saline for water, apparently stimulated glial proliferation specifically in the supraoptic nucleus and posterior pituitary, as measured by radioautographic analysis of [3H]thymidine uptake (Murray, 1968; Paterson and Leblond, 1977). No change in [3H]thymidine uptake in optic tract or trigeminal nerve was noted. Another study, however, using similar techniques (Watson, 1965), did not report changes in glial DNA synthesis in the supraoptic nucleus. Although these studies may suggest some glial response to environment or stress, they do not support the inference of a closely linked o r direct interaction between neurons and glial cells. It is also difficult to ascribe reciprocal differentiating events between neurons and glia studied from a homogenate of brain. Such a homogenate contains elements of both neurons and glia as well as other brain cell types, allowing for the steady-state level of activity of a protein or enzyme to be achieved by cell mixing within the homogenate. T h e question of specific genetic regulation between neurons and glia, however, can be more directly approached by using clonal cell lines of neurons and glia. Utilization of neuoblastoma and glioblastoma cells can be most effective in examining cell interaction and regulation. Newburgh and Rosenberg (1972) studied the effect of catecholamines on
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regulating glucose metabolism in glioblastoma cells. Norepinephrine induced glioblastoma cell adenylcyclase, which in turn activated glycogen phosphorylase, resulting in glycolysis and formation of glucose 1-phosphate. This is an example of a putative neurotransmitter associated with the induction of a glial enzyme. One could conceive of signaling by an adrenergic neuron releasing neurotransmitter for the postsynaptic cell and simultaneously signaling to adjacent glia the need for glucose. In similar experiments, Newburgh and Rosenberg (1973) cocultured neuroblastoma and glioma cells and demonstrated that the coculture resulted in a reduction in glycolysis and in I4CO2production that was not produced by individual cultures of glia o r neurons or the coculture of a neuronal cell line or glial cell line with HeLa cells. It is suggested that a soluble factor may be present that maintains and regulates glycolysis at an efficient level within the neuronal-glial metabolic unit.
2. Growth Factors T h e C-6 rat astrocytoma cell line used in the above-mentioned studies represents a cell line that expresses differentiated glial properties. T h e presence of glial acidic filamentous protein, the s-100 protein, and the synthesis, high-affinity uptake, and efflux of glutamate, y-aminobutyric acid, and taurine are among the differentiated properties (Rosenberg et al., 1978a). It is noteworthy that this cell line contains a factor described by Monard et al. (1973), probably a soluble lipoprotein, which results in the morphologic differentiation of neuroblastoma cells in vitro (see Table 11). Another factor, described by Lim and Mitsunobu (1974), results in the morphologic differentiation of glioblastoma cells. TABLE I1 NEURAL-GLIAL GROWTH FACTORS ~~~~
~
Glial factor (GF) Neuroblastoma differentiation Glioblast differentiation Maintains dissociated cultures Source Biochemistry
~
Glial maturation (MF)
Nerve growth factor (NGF)
Yes
No
No
Yes
Murine No Human Yes No
No
No
Yes
Glial rat cortex Lipoprotein
Glial rat cortex Protein .MW 350,000
-
Glial fibroblasts Protein (dimer) -MW 26,500
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Recent in vitro studies have suggested the existence of a glial maturation factor: glioblasts in monolayer culture can be induced to undergo morphological transformation and differentiation, with retraction of the cell body and extension of processes. This maturation factor (MF), which has been isolated from 17-day-old fetal rats and added to the monolayer culture (Lim et al., 1977b), is a protein of about 350,000 molecular weight (Lim and Mitsunobu, 1974, 1975). Electron microscopic studies demonstrated a change in the cells from a predominance of 50 A microfilaments to a predominance of 100 A microfilaments. Microtubules changed from a random arrangement to one parallel to the axis of the glial process. Nevertheless, glial cells in culture can undergo maturation without addition of this factor, suggesting that glial cells may synthesize this factor during normal development (Lim et al., 1977a). This protein factor also increases the amounts of cyclic 3'5'-adenosine monophosphate (CAMP)and of the S-100 protein in the glial cells. These biochemical changes follow, rather than precede, the morphological differentiation (Lim et al., 1977b). Nerve growth factor (NGF) is inert in this system. Addition of dibutyryl cyclic AMP causes a retraction of the glial soma (Steinbach and Schubert, 1975), whereas transforming (maturation) factor causes on outgrowth of processes (Lim et al., 1976). Established glial cell lines also release a macromolecular factor (GF) that can induce morphological differentiation in neuroblastoma cells (Monard et al., 1973). This factor could be involved in the development and neural arborization that occur postnatally. This factor has also been detected in media conditioned by primary cultures of rat brain and it varies with the brain region and age of the animal (Schurch-Rathgeb and Monard 1978). Glial factor (GF) is apparently distinct from nerve growth factor (NGF) (Levi-Montalcini and Hamberger, 1953), which does fiot induce morphological differentiation in murine neuroblastoma C 1300derived cells (Monard et d., 1975). Furthermore, anti-NGF antibodies can prevent formation of NGF-induced process formation in chick dorsal root ganglia. Anti-NGF antibody added to glia-conditioned media blocks neurite production in mouse dorsal root ganglia (Varon et al., 1974; Varon, 1975). These antibodies, however, fail to block glial factor ("GF" of Monard) differentiation of neuroblastoma (Monard et al., 1975). In chick embryo dissociated dorsal root ganglia cultures, neurons can differentiate in the absence of satellite glial cells, but their presence accelerates this phenomenon (Lodin et ad., 1973). Use of glioma cell extracts has also produced neurite formation in neuroblastoma (Longo and Penhoet, 1974) as well as in embryonic dorsal root ganglia explants. Gel electrophoresis experiments suggested an overlap of this purified glioma extract and submaxillary NGF anti-
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gen. Glial production of NGF is certainly suggested by these experiments, but is not proved. For instance, the NGF shown by the glioma cell may reflect a neoplastic property rather than a normal glial property. This effect might also result from “contamination” by glial factor (Monard et al., 1975). NGF, however, is necessary for the survival of most neurons in dissociated embryonic dorsal root ganglia cultures (Varon, 1975) or postnatal mouse dorsal root ganglia cultures. Homologous nonneuronal cells from the same source (glial or fibroblasts) can supplant the requirement for NGF in these cultures. Nonneuronal cells from areas other than the dorsal root ganglia, however, are not able to replace NGF. Nonneuronal cells from xenogeneic species can replace NGF only with regard to homologous neuronal cells. Similarly, sympathetic ganglia neurons also require NGF when cultured in vitro. Further information about glial-neuronal interaction has come from coculture of specific clones of neuronal and glial cells. For instance, nonneuronal cells can enhance the differentiation of dissociated neurons. In both a neuroblast and a hamster astroblast line an increase of ecto-ATPase activity was seen from a relatively low level in either line prior to coculture. This activity persisted for more than 20 replications after reseparation of the neuroblastoma and glial lines. This phenomenon can be attributed either to a cell-to-cell interaction followed by cell differentiation o r to selection of cells with a higher ATPase activity. Somatic cell hybridization appears to be ruled out by a stable karyotype analysis (Stefanovic et al., 1976, 1977); ecto-5’-nucleotidase activity, however, did not change. Glial cells can influence the amount of neurotransmitter synthesized by sympathetically derived neurons either by coculture or by conditioned media (Patterson et al., 1977). Sympathetic satellite cells and the C-6 glial cell line, but not 3T3 fibroblasts, influenced the growth and development of cholinergic synapses and acetylcholine synthesis (Patter?on and Chun, 1974). Neuronal cells may also influence the differentiation of glial cells. Organ cultures of chick spinal cord explanted at an early neural tube stage were studied by electron microscopy (Lyser, 1972). Oligodendroglia and astroglia were noted predominantly in regions of welldifferentiated neurons. Although both glial types were not always noted in the same location or culture, these results suggest that the neuronal environment may affect glial differentiation. In an electron microscopic study of differentiation in chick embryo neural tube, utilizing reaggregation cultures, neuronal cells differentiated during the first 2 weeks, whereas glial cells differentiated during the third week (Suburo and
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Adler, 1977). It is not clear, however, whether each cell type proceeded to develop on an independent time scale o r whether the later development of the glial cell may have been influenced by neuronal cell differentiation. Steroid hormones may influence neural growth and function through their action on the glial cell (Vernadakis et al., 1978). For example, steroid hormones induce the activity of butyrylcholinesterase, an enzyme localized in glial cells (Giacobini, 1964). Steroids also increase the migration of glial cells in culture (Vernadakis, 1971), and steroids accumulate within glial cells. Cortisol also reduces the uptake of r H ] norepinephrine into C-6 cells (Vernadakis and Nidess, 1976) and thus may affect the microenvironment of the neuron and result in hyperexcitability in the CNS (Vernadakis et al., 1978).
3. Somatic Cell Hybrids and Possible Genetic Complementation Somatic cell hybrids of neuroblastoma and glioma cells have been used to investigate the genetic regulation that may exist between their genomes (Minna et al., 1971). Thus, hybrid cells serve as a model system for exploring neuronal-glial genetic regulation, which, analogously, may be in effect in the intact brain. These hybrids resemble parent neuroblastoma cells in many of their properties, including acetylcholinesterase activity (Amano et al., 1974), dopamine-/3-hydroxylase activity (Hamprecht et al., 1974), clear- and dense-core vesicle formation (Hamprecht, 1974), the ability to extend long neurites (Daniels and Hamprecht, 1974; Schubert et al., 1971; Seeds et al., 1970), and possession of electrically excitable membranes (Hamprecht, 1974). It is noteworthy that some hybrid clones possess activity of choline acetyltransferase that is 500 times greater than the parent neuroblastoma or glioblastoma in individual cultures (Amano et al., 1974). This argues for genetic complementation between the genomes of the neuroblastoma and glioblastoma cells to induce high steady-state levels of choline acetyltransferase in the hybrid cell and may be the mechanism for enzymic induction in the intact mammalian brain. Klee and Nirenberg (1974) have found a high density of opiate receptors on hybrid cells, which again argues for genetic complementation. Christian et al. (1978) found a soluble factor produced by neuroblastoma-glioblast hybrid cells that results in the aggregation of acetylcholine receptor molecules to form mature aggregates capable of generating miniature end-plate potentials in vitro. This finding suggests that genetic complementation between neuroblasts and glioblasts may be required for the production of a
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soluble factor, which may in part be responsible for the aggregation of specific proteins to form an active acetylcholine receptor and thus maintenance of neuromuscular transmission. The glial genome may also be important in forming acetylcholine receptor aggregates, thus providing a better target for synapse formation during embryogenesis and after denervation. Similar genetic cooperativity exists between peripheral nerve Schwann cells and motor axons, as described by Aguayo et al. (1976), who showed that Schwann cells of the superior cervical ganglion do not receive the appropriate signal from associated axons to induce myelination. However, these Schwann cells, when transplanted into a nerve where axons are normally rnyelinated, result in myelination of regrowing axons by grafted superior cervical ganglion Schwann cells. Thus, the Schwann cells of this sympathetic ganglion have the potential to cause myelination of motor axons but depend on an appropriate neural signal for this to occur. Rosenberget al. (1978a) examined the species of proteins synthesized by parent individual cultures of neuroblastoma and glioblastoma cells. The patterns for proteins synthesized by neuroblastoma or glioblastoma cells, as identified on two-dimensional polyacrylamide gels, were very different. Proteins were identified that were dominant for one cell type and not expressed in the other. A hybrid cell line, NG 108-15, composed of parent mouse neuroblastoma and rat glioblastoma genomes, expressed many of the neuroblastoma types of proteins and relatively fewer of the glioma type of proteins. A specific protein species, referred to as Z protein, was identified in hybrid cells, but was not present in either parental neuroblastoma or glioma cultures. T h e Z protein (53,000 molecular weight) was expressed, however, by the coculturing of neuroblastoma and glioma cells, suggesting that its induction is dependent on a possible soluble factor. T h e Z protein in hybrid cells was demonstrated on both stained gels and by autoradiography. Chrornosome analysis of hybrid cells confirmed the presence of both rat and mouse chromosomes. The expression of the Z protein induced in hybrid cells or by coculture of parents, but not by the individual culture of parent alone, is good evidence in favor of the concept of reciprocal genetic regulation between neuroblastorna and glioma genomes. The reexpression of the Z protein in hybrid cells is presumably a glioma-induced function of a repressed neuroblastoma character, as Z protein is present in the original parent N18 clone, which in turn is the parent of the cell line N18TG2, one of the parents of the hybrid cell line. Z protein was also found in homogenates of intact Ajax mouse brain, the strain of mouse from which the
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parent neuroblastomas were obtained. Thus, it is possible that the Z protein synthesized by Ajax mouse brain may be due to genetic complementation between glia and neurons (Rosenberg et al., 1978b). In view of these data, the occurrence of trophic interaction between neurons and glia in the intact brain must also be considered as a possible mechanism for differentiation. The dominance of neuroblast genetic expression in the hybrid cell suggests the possibility of similar regulation and inhibition of glial function by neurons in vivo. Neuronal-glial interaction may be necessary for the genetic expression of differentiated functions that are absent from, or weakly expressed by, either cell type alone, as, for example, the presence of opiate receptors (Klee and Nirenberg, 1974) and increased choline acetyltransferase activity in hybrid cells (Amano et al., 1974), and the showing of a unique protein species in hybrid cells, the Z protein (Rosenberg et al., 1978a). Could a trophic interaction also be relevant to the control of postnatal gliogenesis, which persists through life, in contrast to the neuronal population, which is postmitotic? This ability of glial cells to proliferate allows the brain to respond to injury with the potential for repair and cellular reaction. Nevertheless, the ability to proliferate carries with it the potential for uncontrolled growth such as is seen in a brain tumor. The various types of tumors appear to correspond to the various glial cell precursors (see Table 111). The role of neurons in maintaining glial differentiation, however, remains speculative. TABLE I11 BRAIN TUMORS ARISING FROM GLIAL Glial cell type Astrocyte Oligodendrocyte Ependymal cell Microgliacyte
DERIVATIVES
Brain tumor Astrocytorna, glioblastornas, optic gliomas Oligodendrogliomas Ependymomas, choroid plexus papillomas, colloid cysts, subependymomas Microgliomas
Frequency Common Rare Rare Rare
The studies reviewed emphasize the point that the determination of enzyme activity or protein concentration in a homogenate of brain is in actuality the combined expression of neurons and glia, mutually monitoring and regulating their genetic expression. It may be that a similar modulation and reciprocal inhibition of the genetic material of the opposite cell type, as seen experimentally, may be an essential mechanism of differentiation within the mammalian brain.
PHYSIOLOGY OF GLIA
29 1
IV. Potassium and Neuroglial Function
A. GLIALMEMBRANEPOTENTIAL Much of the available information concerning the physiology of glia has been contributed by Kuffler and his associates, and has been reviewed by Kuffler (1967), Kuffler and Nicholls (1966, 1976), and Somjen (1975). Glial cells in the CNS have a higher resting potential (-75 mV to -86 mV) than do the neurons (-50 mV) (Dennis and Gerschenfeld, 1969). This resting potential appears to be a logarithmic function of the intracellular and extracellular potassium concentrations, and appears to follow the Nernst equation:
where E is the membrane potential, R is the gas constant, T is the absolute temperature, F is the Faraday constant, [KO]is the outside concentration of potassium, and [KJ is the inside concentration of potassium (see also Lebovitz, 1970, for theoretical considerations). It is in this sense that glial cells act much like a potassium electrode (Kuffler and Nicholls, 1966; Cohen et al., 1968). This relationship holds more for nonmammalian glia and for the mammalian spinal cord (Lothman and Somjen, 1975). Glial cells, like neurons, have a high intracellular potassium concentration. However, glia seem to be more permeable to potassium than other ions such as Na+ and C1- (Kuffler and Nicholls, 1966). The response of the optic nerve of necturus to varying potassium concentrations gives an experimental slope of 59 mV, which is in agreement with the Nernst equation (Kuffler and Nicholls, 1966). Furthermore, the potential varies with temperature (Bracho et al., 1975; Somjen, 1973). In the cerebral cortex of the cat, however, the glia do not conform so precisely to this equation (Pape and Katzman, 1972; Somjen, 1973). Ransom and Goldring (1973a) showed that, at lower concentrations of extracellular potassium (<3 mM K+), the slope was less than 38 mV. For intermediate concentrations (3-4 mM K+), the slope was 38 mV for a tenfold change in K+, whereas, at high (100- 125 mM K+) concentration, the slope was approximately 60 mV. Nevertheless, it was thought that potassium was the primary determinant of the resting membrane potential and that chloride or calcium did not affect this potential. In response to extracellular potassium, rat optic nerve had a slope of 42 mV (Dennis and Gerschenfeld, 1969). The slope for the Nernst equation was approached in a human glioblastoma line only after addition of ouabain (Manuelidis et al., 1975). These results may suggest that the membrane
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potential for mammalian species could be bi-ionic and be influenced to some degree by the external sodium concentration. This relationship is described by the Goldman-Hodgkin-Katz equation:
where the symbols are as for the Nernst equation. In addition, however, “a], describes the extracellular concentration of sodium, andp the ratio of Naf to K+ permeability, p(Na+/K+).An equation can be made that would fit the experimental points but is discrepant when sodium is lowered while maintaining a constant extracellular potassium concentration (Ransom and Goldring, 1973a). In further studies, the average resting membrane potential in a rat glioma line in tissue culture was -36 mV (Kukes etal., 1976a). T h e slope was 3 1 mV in response to changes in external potassium, also suggesting that while the membrane is predominantly influenced by the internal and external K+ ratio, it may be permeable to other ions besides potassium. A modification of the Constant Field Equation (3) may even more accurately describe the effect of the potassium and sodium concentrations on the resting membrane potential of glia (Kukes et al., 1976b; Woodbury, 1965):
where the symbols are as before but, in addition, the internal sodium concentration [Nal] is considered. In experimental substitution of choline for sodium to lower the “a,] from 120 to 20 mM, the potential increased from -36 to -49 mV. This differs from the theoretical prediction of an increase of -25 mV. This discrepancy may have been due to loss of potassium in a low-sodium medium. In another study, the resting potential of cultured human glial cells was -7.7 mV, but these cells were studied at 26°C and the WNa ratio was reduced in the media (Trachtenberg et al., 1972). Furthermore, in studies of cultures, the resting potential of oligodendroglia (Vernadakis and Berni, 1973) from 15-day-old chick embryos varied, with 2-day cultures showing 34% of the cells in the range of -35 to -40 mV, whereas at 90 days most cells showed membrane potentials of - 15 to 20 mV; and in another study, the glial cells showed a membrane potential of - 10 to -20 mV after 16 days. These studies suggest that glial cells in organotypic cultures vary the resting potential with age.
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B. GLIALCELLSAND REGULATION OF EXTRACELLULAR POTASSIUM Neuronal activity is associated with an extracellular release of potassium. For instance, stimulation of the cortex or thalamus causes an increase in extracellular potassium. Following this stimulation, the potassium level rapidly returns to normal or even slightly below normal (Krnjevii: and Morris, 1972). Lugaro (1907) suggested that glia may protect the neurons from increases in the extracellular potassium. Interestingly, intracellular injection of Na+ o r Li+ into cortical glia causes glial depolarization and discharge of surrounding neurons, apparently on the basis of reduced uptake of extracellular Kf by glia (Grossman and Seregin, 1977). Glia might accomplish the extracellular clearance of potassium by either a passive or an active mechanism (Somjen, 1975). Glia are highly permeable to potassium, so that increased potassium release from neurons would result in an increased flux of potassium into the glial cell (Kuffler and Nicholls, 1966). T h e glial cell would be depolarized and the potassium would move from the depolarized cell to other nondepolarized cells at a distance (see Table IV). On the other hand, potassium may be cleared by an active transport system, utilizing the Na+, K+-ATPase system (Embree et al., 1971; Henn et al., 1972); this would agree with the high intracellular potassium concentrations. Glial cells apparently increase their oxygen consumption with increased extracellular potassium concentration (Hertz et al., 1973). On the other hand, oxygen consumption did not increase in the NN line of hamster astroglia following added potassium (Ciesielski-Treska et al., 1976). This line, however, has low Na+, K+-ATPase activity (Embree et al., 197 1). Addition of high potassium concentrations to incubations of TABLE IV POTASSIUMAND ELECTROPHYSIOLOGY OF GLIA
Functional state
Glial cell membrane potential
Extracellular potassium (d)
Oxidative metabolism
Quiescent
Normal
Extracellular potential shift
Depolarized
-5-6
+ ++
Spreading depression
Depolarized
40-80
++++
Seizure
Depolarization followed by hyperpolarization
13-20
++++
2-4
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leech glial cells, however, did not increase oxygen consumption, in contrast to experiments with rat brain slices and astrocytes, and this suggests that the glial cells play a passive role in the leech but a more active role in the mammalian system (Hertz and Nissen, 1976). Increased potassium in the extracellular fluid of glia prepared from optic nerve of necturus resulted in a reversible decreased fluorescence of NADH (Orkand et al., 1973). Barbiturates slow the rate of potassium uptake, along with a decrease in oxidative metabolism (Hertz et al., 1978). A similar, though less marked, effect has been seen for phenytoin. One study has shown that the uptake of potassium in mammalian glial (C-6) cells is associated with an outward sodium transport (Kukes et al., 1976a,b).Also noted is a decrease in ATP content and an uptake of potassium along with chloride. This leads to a swelling of the glia. The uptake of chloride appears to be mediated by a K+-dependent enzymic process (Gill et al., 1974). On the other hand, a study involving measurement of extracellular potassium [KJ and the membrane potential V , showed little relationship between AV, and A[KJ for single events (Futamachi and Pedley, 1976) but a closer agreement to the Nernst equation, with sustained elevation of extracellular potassium concentration [K,,].
C. DEPOLARIZATION OF GLIAAND EXTRACELLULAR POTENTIAL SHIFTS
Glial cells, unlike neurons, are inexcitable when stimulated by electrical currents. They do, however, depolarize with a slow decline whenever the extracellular potassium level is increased (Kuffler and Nicholls, 1966).These slow depolarizations can summate to produce a substantial negative potential shift. Various studies have tried to determine whether these shifts may play a role in the electrophysiology of the mammalian brain (Orkand et al., 1966). The glial depolarization appears to be related to the increased number of potassium ions that accumulate in the intercellular cleft following neuronal activity (Grossman et al., 1969). Neuronal activity itself, however, is not as closely linked as glia to this voltage shift (Castellucci and Goldring, 1970; Somjen, 1969, 1970, 1975). Slow electrical stimulation of the thalamus (6-10 Hz) in the adult cat showed the configuration of the steady potential shift presumed to be related to glial slow depolarization, as neurons d o not show sustained depolarization at this frequency. With direct slow cortical stimulation, intracellular recordings also show a gradient of slow depolarization and the electrocorticogram shows a cortical potential shift in the same distribution. At higher frequency, the slow depolarization still occurs but
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the cortical potential shifts appear to be more unpredictable and suggest a neuronal contribution (Castellucci and Goldring, 1970). Ransom and Goldring (1973a,b) showed that repeated stimulation of cat cerebral cortex resulted in a decreasing step of depolarization that had a logarithmic relationship. Monitoring of the slow depolarizing potential while altering the external potassium concentrations demonstrated an inverse relationship between the slow depolarizing potential and the potassium concentration. These authors suggest that K+ released from active neurons accumulates in the intercellular clefts, depolarizes glia, and leads to the cortical potential shifts (see also Orkand et al., 1966; Baylor and Nicholls, 1969). Oxidative metabolism also increases in this area, as measured by changes in fluorescence in intramitochondrial NADH (Orkand et al., 1973). This steady potential shift may be augmented by barbiturate and reduced by procaine (Ransom et al., 1977). However, since no effect on glial slow depolarization or potassium concentration was noted, this argues against glial slow depolarization as the only source of cortical steady potential shift during barbiturate anesthesia. Studies of sustained potential shifts in cat spinal cords using micropipettes within presumed glial cells and extracellular space also showed that glial depolarization is the major contributor to the sustained potential shifts in spinal cord (Strittmatter and Somjen, 1973). Administration of thiopental, pentobarbital, or phenytoin decreased both the slow depolarization of membrane and the extracellular sustained potential shifts and relates to decreased neuronal activity. T h e mechanism of the extracellular voltage gradient generated by the glial cells is of interest. A cell can generate a current if different portions of its membrane are at different electrical potentials. Although spatial configuration and geometry might permit this effect, it is likely that the glial cells are coupled electrically and in this way act as a syncytium. Current flow between glial cells may be facilitated by the presence of gap junctions, features not seen between neurons (Tani et al., 1977; Sipe, 1976; Sipe and Moore, 1976; Quigley, 1977). Gap junctions between glial cells were first described in the leech and in necturus, and have more recently been described in fetal rat neocortex and adult rat hypothalamus and optic nerve. A computer model of this electrical syncytium has been evaluated (Joyner and Somjen, 1972; Somjen, 1975). Lowering the membrane resistance enhances the intracellular and extracellular voltage shifts within the depolarized area. Decreasing the coupling resistance between cells increases the extracellular voltage shifts.
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D. SPREADING DEPRESSION AND SEIZURES 1. Seizures
The glial cell may play a role in seizure activity in the CNS. Glial cells apparently depolarize during seizure activity (Sugaya et al. , 1964, 197 1, 1975). This is related to increased extracellular potassium. Failure to remove potassium from the extracellular space has been suggested as a cause of further neuronal excitement and continuing seizures. On the other hand, one study of glial cells with an epileptogenic focus indicated that the glial cells repolarized faster than normal so that elevated extracellular potassium probably results from the seizures, rather than causing them (GIotzner, 1973). Glial cells from rat cerebral cortex (Ransom, 1974) studied during seizure activity have large resting membrane potentials (- 105 mV). With tonic paroxysms, the glial cells showed a slow depolarization that was accompanied by steady potential shifts. It is estimated that K+ concentration increases up to a level of 13 mM during a seizure. On the other hand, the K+ concentration can be raised to 40 mM and not cause a seizure. An increase of K+ concentration to a certain level apparently is not sufficient to cause a seizure. A slow hyperpolarization of glial cells can occur following a seizure. This hyperpolarization may result from the activation of an electrogenic sodium pump (Grossman and Rosman, 1971) that is caused by the increased intracellular neuronal sodium following neuronal activity. The increased inward potassium flux, coupled with the outward pumping of the sodium, may result in a temporary decreased external potassium concentration (Ransom and Goldring, 1973b). How this activity affects the brain surface is complex, and the final effects of glia and neurons may add or cancel. For instance, the neurons may be hyperpolarized by the electrogenic pump, while the glia may be depolarized by the increased extracellular potassium (Baylor and Nicholls, 1969; Sypert and Ward, 1971).
2. Spreading Depression Grafstein ( 1956) has suggested that spreading cortical depression is related to the increased K+ released by excessive activity of neurons. The increased K+ leads to depolarization of the neurons and increased recruitment of involved neurons. Similarly, glial cells undergo depolarization during spreading depression (Higashida et al., 1971, 1974). The exact mechanisms involved in spreading depression are not clearly understood. Studies indicate that glial depolarization is concurrent with spreading depression, but the DC polarity was opposite to the glial
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intracellular potential. Neuronal burst discharges did not show a close relationship with spreading depression and frequently followed the slow wave by 10-30 sec. Spreading depression could be noted in the cortex even when neuronal activity was abolished with tetrodotoxin (TTX). KCl application, but not electrical stimulation, could produce spreading depression after TTX. T h e spreading depression could pass through the T T X area and into normal cortex. T h e above finding suggests that spreading depression and glial depolarization can occur without neuronal discharge. The extracellular potassium measured by a microelectrode showed a [KO]of 40-50 nM at the peak of the spreading depression and it may reach 80 mM. In contrast, during seizure activity the [KO]did not exceed 20 mM. The [KO] with spreading depression elicited after T T X follows the same course as the extracellular potential. These data suggest that spreading depression is linked to dysfunction of the intercellular modulation of K+ by the glia (Sugaya et al., 1971; Higashida et al., 1971, 1974). Perhaps both glial and neurons may contribute to spreading depression, but both probably work through increased potassium in the extracellular space (Higashida et al., 1971). In other studies, spreading depression has been seen in tissue cultures of glial cells without neurons. In studies of cat neocortex, spreading depression and seizures appeared to be associated with increased oxidation of NADH (Lothman and Somjen, 1975).
V. Putative Neurotransmitters and Glia
A. AMINOACID TRANSPORT It is increasingly thought that glial cells are important in maintaining and regulating the ionic and metabolic environment of the extracellular space in the nervous system. In addition, the uptake, release, and metabolism of amino acids, including those, such as glutamate, that have been proposed as neurotransmitters at central neuronal synapses (Henn and Hamberger, 1971), may be regulated in part by glial cells. Glial cells have been shown to take u p many of the substances that have been proposed as putative neurotransmitters o r their precursors, including the amino acids glutamic acid, y-amino butyric acid (GABA), glycine, taurine, aspartate, the serotonin precursor tryptophan, as well as the nonamino acids, such as choline (reviewed by Hamberger et al., 1976). In fact, the glial uptake of some of these substances may be greater than in neuronal perikarya (Hamberger, 1971). The uptake of the transmitter amines, however, seems to be lower in glial cells than in
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neurons. Synaptosomes (pinched-off nerve endings) seem to concentrate all these substances better than glial cells (Henn and Hamberger, 1971; Henn et al., 1974). The nature of the uptake by the glial cells varies with the substance. The uptake may be accomplished by an energy- and sodium-dependent high-affinity system, which is characteristic of isolated nerve-ending fractions, in combination with a low-affinity system, or by a low-affinity system alone. Uptake studies are usually performed in tissue obtained from bulk isolation with separation of neuronal and glial elements or glia obtained through tissue culture. A comprehensive general review on the uptake and utilization of amino acids by cell cultures has been published by Patterson (1972). The use of tissue culture for glia offers the advantage of obtaining a specific response by avoiding contaminating neurons found in bulk isolation or in explant cultures. A possible disadvantage, however, is that pathological rather than normal function may result when tumor cell lines are employed, and results must be interpreted cautiously. The bulk isolated glial cells have the advantage of using normal tissue but have the disadvantage of possible contamination of the fractions (Henn, 1975). 1. Glutamic Acid
A high-affinity uptake system for glutamic acid has been demonstrated in partially isolated glial cells from cerebral cortex, from glial cells, or cell fractions obtained from CNS tissue, dorsal root ganglia, and from relatively complex slices or explants of some human glial tumors (Nagata et al., 1974; Henn et a)., 1974; Roberts and Keen, 1974; Snodgrass and Iversen, 1974). In addition, high-affinity uptake has been demonstrated for glial cells in tissue culture from both human and nonhuman tumors (Henn et al., 1974; Schrier and Thompson, 1974; Stewart et al., 1976). For instance, glutamate uptake studied in eight human astrocytoma lines grown in tissue culture demonstrated high affinity in seven of the eight lines. This property appeared to be generally independent of grade of malignancy or anatomical site, although the absence of high-affinity uptake in one of the more malignant tumors remains unexplained. High-affinity uptake of glutamate appeared during the confluent state of growth, which presumably more closely resembles the natural condition of cells in the intact nervous system, and was absent during the proliferating phase of growth. T h e absence of highaffinity uptake in three optic gliomas, however, suggests that not all glial cells share the same biochemical properties (Stewart et al., 1976). Highaffinity uptake of glutamate was also noted in a nontumorous astroblast line (Balcar et al., 1977).
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Although the physiological significance of high-affinity uptake by glial cells is not currently understood, there are several possible explanations (Henn, 1976). Perhaps the astrocytes help to inactivate extraneural neuroactive amino acids after release at CNS synapses. In addition, glial cells may have specific roles for uptake of amino acids to modulate neuronal activity (Henn and Hamberger, 1971). High-affinity uptake may be rate limiting for enzymic processes saturated at substrate concentrations higher than those of the transport process (Stewart et al., 1976). The extrapolation of results from in vitro studies to normal brain function in vivo should be considered cautiously. 2. GABA Similarly, the high-affinity uptake of y-aminobutyric acid (GABA) has been demonstrated for glia. Henn and Hamberger (197 1) investigated the uptake of several neurotransmitters, including GABA, in cell fractions derived from rabbit brain. These studies showed that glial cells and neuronal perikarya accumulate GABA in a linear fashion for almost 20 min, giving rise to tissue-to-medium GABA ratios of eight for neuronal perikarya, 53 for glia, and 165 for synaptosomes. A highaffinity uptake of GABA by glial tumors was demonstrated by Haber et al. (1973), Hutchison et al. (1974), and Schrier and Thompson (1974). In a study involving the uptake of GABA in slices of surgically removed human brain tumors, Snodgrass and Iversen (1974) found that GABA was lower than in rat brain slices, although other amino acids were taken up as in normal brain slices. Autoradiographic studies of GABA show uptake by astrocytes. [3H]GABA uptake into the satellite glial cells and Schwann cells of rat superior cervical sympathetic ganglia has been demonstrated (Bowery and Brown, 1972), as well as into the satellite cells of rat dorsal root ganglia (Schon and Kelly, 1974), the glial Muller cells of the retina (Neal and Iversen, 1972), and the gliocyte cells of the rat pineal gland (Schon et al., 1975).
3 . Other Transmitters Uptake of other substances into glial cells has been studied. Taurine was found to have both a high- and low-affinity uptake into C-6 glioma cells (Schrier and Thompson, 1974) and into astrocytes cultured from dissociated mouse cerebral cortex (Schrier and Thompson, 1974; Borg et al., 1976; Schousboe et al., 1976). Subcellular fractions of C-6 glioma cells also accumulated taurine (Sieghart and Karobath, 1976). Choline uptake was studied in cultures of dissociated chick cerebral cortex (Massarelli et al., 1974a). A sodium-dependent uptake was noted, and ouabain inhibited uptake in dissociated cultures with a higher proportion of glial cells.
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In a separate study, however, the high-affinity system was not demonstrated in mature glial cells from these mixed cultures. A high-affinity uptake system for choline has been demonstrated in the C-6 and NN glioma cell lines (Massarelli et al., 1974b). L-Tryptophan, studied in C-6 glial cells, showed a high- and low-affinity component. However, this did not appear to be sodium dependent (Bauman et al., 1974). Glycine also had a high-affinity uptake in bulk isolated glial preparations and appeared to be concentrated in the same areas where glycine is thought to function as a neurotransmitter, i.e., in spinal cord more than brain (Henn, 1976). Aspartate also showed high-affinity uptake in a glial culture (Balcar et al., 1977). Some substances studied had only a lowaffinity uptake. For instance, glutamine, a metabolite of glutamate, had a low-affinity uptake in glial cells or neurons (Hamberger et al., 1976), although one study of dorsal root ganglia apparently did show highaffinity uptake (Roberts and Keen, 1974). 4. Uptake Inhibitors Various substances have been investigated in order to selectively inhibit the uptake of the amino acids into the glial cells (Iversen and Kelly, 1975; Hamberger et al., 1976). For instance, P-alanine was reported to inhibit GABA uptake into dorsal root ganglia but not into slices of cerebral cortex. /I-Alanine appeared to be a more potent inhibitor for glia than ~-2,4-diaminobutyricacid (DABA), which. is an inhibitor of GABA uptake in neuronal systems (Schon and Kelly, 1974; Sutton and Simmonds, 1974). In one study of bulk isolated glial cells and neurons, however, the inhibitory effects of DABA were equipotent for either glial or neuronal uptake of GABA (Sellstrom and Hamberger, 1977). Autoradiographic studies of [3H]/I-alanine did, however, show an accumulation into satellite glial cells, whereas [3H]DABA was not accumulated selectively by these cells (Iversen and Kelly, 1975). These inhibitors may be acting as selective substrates for the two uptake systems (Iversen and Kelly, 1975). Folate appears to inhibit the high-affinity glial uptake of glutamate in rat dorsal root ganglia (Roberts, 1974). OF AMINOACIDS B. RELEASE
The release of amino acids and transmitters has been demonstrated in uitro in tissue slices and has been considered to be a primary function of the presynaptic neuron. However, there is evidence for the release of GABA from glial cells from the dorsal root ganglia in the rat by depolarizing (64 mM) concentrations of potassium (Minchin, 1975; Mil:chin and Iversen, 1974). This was Caz+dependent. Glutamate, glycine,
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alanine, and leucine were not released by potassium depolarization. Potassium-stimulated GABA release was also noted in bulk isolated neuronal and glial cells (Sellstrom and Hamberger, 1977). Substances that inhibited uptake also stimulated release, and this would be consistent with a bidirectional carrier system for GABA (Kuriyama et al., 1969; Levi and Raiteri, 1974; Simon et al., 1974). T h e Kf stimulation of GABA release may have some physiological significance in regulation of the synapse following neuronal activity, although sufficient information for a clear understanding of this phenomenon is still lacking. T h e release of GABA from synaptosomes appeared to be Ca2+dependent, whereas that from glial cells was Ca2+independent (Sellstrom and Hamberger, 1977; Lazarewicz et al., 1977). Conversely, GABA causes release of Ca2+from perfused C-6 rat glioma cells on the order of moles of Ca2+ released per cell in response to 5 mM GABA (Henn, 1976). These findings suggest a possible role in stabilization of extracellular Ca2+by glia (Henn, 1976) in a manner suggested for extracellular K+ by Kuffler and Nicholls (1966). Glial S-100 protein may play a role in binding of intraceliular Ca2+in glial cells and thereby be important in the GABAstimulated release of Ca2+ from glial cells (Henn, 1976). C. GLUTAMINE-GLUTAMATE NEURONAL-GLIAL CYCLE
Glial cells may play an important role in maintenance of the neuronal glutamate metabolism in cells that utilize glutamate as a neurotransmitter. Using an immunohistochemical technique, it was found that, in rat brain, glutamine synthetase, the enzyme that converts glutamate to glutamine, is predominantly found in glial cells (Martinez-Hernandez et aE., 1977). Glutaminase is found predominantly in nerve cell preparations rather than in glial cells (Svenneby, 1970). A cycle has been proposed in which glutamate released by the neuron is taken u p by a high-affinity uptake system into glial cells (Hamberger et al., 1976) (see Fig. 3). Glutamate is converted to glutamine by glutamine synthetase in the glial cell. Glutamine is released from the glial cell and is taken up by a low-affinity process into the neuron. Within the neuron, glutamine is reconverted by glutaminase into glutamate and thereby appears to be available for recycling. These studies suggest that glial cells act as modulators of neuronal activity (Krnjevit, 1974; Henn and Hamberger, 1971; Roberts, 1976).
D. CYCLICNUCLEOTIDERESPONSES A N D GLIALCELLRECEPTORS Cyclic nucleotides may increase in glial cells following exposure to neurotransmitters (Gilman, 1972). Norepinephrine or N-isopropyl-
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GLUTAMATE-GLUTAMINECYCLE
7 K -
PRESYWlC NEURON
POSTSYNAPTIC
Glutamate
Glutamlm syntbtaso GLIAL CELL
FIG. 3. Possible glial-neuronal interaction for the compartmentation and metabolism of glutamate and glutamine. The glutamate is taken up by a high-affinity system into the glial cell. Glutamine is taken up by a low-affinity system.
norepinephrine increased the intracellular concentration of cAMP more than 250-fold in three clonal lines of glial tumor cells (including a human line and the C-6 rat glioma induced by N-nitrosourea). This response was not blocked by phentolamine, an a-adrenergic blocker (Gilman and Nirenberg, 1971). A cultured human astrocytoma line responded with an increased intracellular cAMP concentration after stimulation with norepinephrine, epinephrine, or histamine (Clark and Perkins, 1971). This response to norepinephrine was blocked by propranolol, a @-adrenergicantagonist, whereas histamine did not block the response, suggesting that different receptors may be present in this line of astrocytoma. Adenylate cyclase activity measured in homogenates of a rat glial tumor line showed stimulation by catecholamines in the order: isoproterenol > epinephrine > norepinephrine (Schimmer, 1971; Jard et al., 1972). Histamine and serotonin were without effect. This effect was blocked by propranolol but not by phentolamine, suggesting that a P-adrenergic receptor is present on the glial cell. Dopamine also stimulated adenylate cyclase, whereas serotonin, acetylcholine, tryptamine, and tyramine were inactive (Jard et al. , 1972).Although serotonin, tryptamine, and tyramine apparently do not stimulate glial CAMP,they can be metabolized by glial monoamine oxidase, type A (Murphy et al., 1976; Hazama et al., 1976). Dopamine stimulated an increase in intracellular cAMP levels in several lines of glia (Schubert et al. , 1976),and this response was blocked by propranolol, but not by phentolamine, an a blocking agent. Moreover, many of the CNS dopamine binding sites, as measured in
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neuronal and glial fractions prepared from rabbit cerebral cortex by haloperidol binding, appear to be found on glial membranes and to be associated with the adenylate cyclase present on these membranes, which is also four times higher in the glial than in the neuronal fraction (Henn et al., 1977). Binding of ['2511iodohydr~xybenzylpindololto putative P-adrenergic receptors in rat glioma cells appears to be associated with adenylate cyclase in a stoichiometric manner (Maguire et al., 1976). In other studies, the dopamine-increased cAMP in glia was blocked by propranolol to a greater degree than by the neuroleptic drugs and thus appears to be modulated through a P-receptor (Schubert et al., 1976), and in this regard differs from the response found for the dopamine receptor located on neurons. Repeated exposure of tissues with the P-receptor-associated cAMP response to P-adrenergic agonists results in a decreased cAMP response to a subsequent P-adrenergic challenge (Franklin and Twose, 1976). T h e reason for this decreased sensitivity is uncertain, but may involve changes in receptor mechanisms or intracellular processes. Other studies of C-6 rat glioma in culture show that the epinephrine-sensitive increase in cAMP has a sixfold increase in confluent cultures over those of low density, suggesting that differentiation may be important in this effect (Morris and Makman, 1976). a-Adrenergic agents may also modulate the cAMP response to P-adrenergic stimulation of glia. Simultaneous activation of the glial cells with phenylephrine (an (Y agonist) reduced the cAMP response to P-adrenergic agonists, including norepinephrine, adenosine, and prostaglandin El. a-Adrenergic receptors may thus modulate the response of glia to agents that increase cAMP levels (McCarthy and deVellis, 1978). The physiological meaning of this increase in glial intracellular cAMP is unclear. Perhaps this glial cAMP response to neuronal activity might be related to a feedback control system in regulating neuronal activity. In addition, the glial location of dopamine receptors may be important in mediating the effects of the neuroleptic antipsychotic agents (Henn et al., 1977). In addition to the more immediate effects of catecholamines on glial cells, some more delayed and long-term effects occur. deVellis and Inglish (1969) noted an increase in levels of lactate dehydrogenase in cultures of C-6 glioma 24 hr after the addition of epinephrine. Another enzyme whose activity was increased in glial cells by the presence of catecholamines was ornithine decarboxylase (Bachrach, 1975). Within 2-4 hr after the addition of norepinephrine to cultured glioma cells, the ornithine decarboxylase activity was observed to increase 500- to 1000fold, an effect Bachrach attributed to the production of CAMP, which regulates the transcription of the enzyme.
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VI. Conclusions
Neuroglia have important physiological functions in support of neuronal activity. The glial-neuronal relationship begins early in the developing nervous system when modified astrocytes guide migrating neurons or their processes so that proper connectivity of the nervous system can be achieved. This is followed by the myelination of neuronal processes by oligodendroglia. Neurons and glial cells may interact by a reciprocal influencing of differentiation. The level of potassium in the extracellular space surrounding the neuron appears to be regulated by glia. The membrane potential of glia is primarily dependent on the extracellular and intracellular distribution of potassium. Perturbations in this ratio lead to depolarization, as well as to potential shifts that can be recorded electrocortically. Glial cells contribute to neuronal functioning by extraneuronal regulation of neurotransmitters in the CNS and, possibly, more directly, as in the glutamate-glutamine cycle. It is clear that glia and neurons are true partners in the CNS. The efforts of future research will expand our current knowledge so that we can appreciate this relationship more fully. The prophecy of Cajal made 50 years ago about a possible symbiotic relationship between neurons and glia is being documented and fulfilled. ACKNOWLEDGMENTS This work was supported in part by the Southern Medical Association and BRSG Grant No. 5-507-RR5426-16 to the University of Texas Health Science Center at Dallas. We thank Leslie Sheaffer for her skillful secretarial assistance and Bob Heckmann for his help in this endeavor.
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Privat, A. (1975). Int. Rev. Cytol. 40, 281-323. Quigley, H. A. (1977). Invest. Ophthal. Visual Sci. 16, 582-585. Rakic, P. (1971). Brain Res. 33, 471-476. Rakic, P. (1972).J. Comp. Neurol. 145, 61-84. Rakic, P., and Sidman, R. L. (1973a).J . Comp. Neurol. 152, 103-132. Rakic, P., and Sidman, R. L. (1973b).J. Comp. Neurol. 152, 133-162. Rakic, P., and Sidman, R. L. (1973~).Proc. Natl. Acad. Sci. U.S.A. 70, 240-244. Ramon y Cajal, S . (1928). “Degeneration and Regeneration of the Nervous System.” Oxford Univ. Press, London. Ransom, B. R. (1974). Brain Res. 69, 83-99. Ransom, B. R., and Goldring, S . (1973a).J. Neurophyswl. 36, 855-866. Ransom, B. R., and Goldring, S. (l973b).J. Neurophysiol. 36, 879-892. Ransom, B. R., Greenwood, R. S., Goldring, S., and Letcher, F. S. (1977).Brain Res. 134, 479-499. Roberts, E. (1976).In “Nervous System Function” (E. Roberts, T. N. Chase, and D. B. Tower, eds.), pp. 515-544. Raven, New York. Roberts, P. J. (1974). Nature (London) 250, 429-430. Roberts, P. J.. and Keen, P. (1974).J. Neurochem. 23, 201-209. Rosenberg, R. M., Vance, C. K., Morrison, M., Prashad, N., Meyne, J., and Baskin, F. (1978a).J. Neurocha. SO, 1343-1355. Rosenberg, R. N., Vance, C. K., Morrison, M., Prashad, N., Meyne, J., and Baskin, F. (1978b).Proc. SOL. Neurosci., Abstr. 4, 323. Schimmer, B. P. (1971). Biochim. Biophys. Acta 252, 567-573. Schon, F., and Kelly, J. S . (1974). Brain Res. 66, 289-350. Schon, F., Beart, P. M., Chapman, D., and Kelly, J. S. (1975). Brain Res. 85, 479-490. Schousboe, A., Fosmark, H., and Svennegy, G. (1976).Brain Res. 116, 158-164. Schrier, B. K., and Thompson, E. J. (1974).J. Biol. Chem. 249, 1769-1780. Schubert, D., Humphreys, S., DeVitry, F., and Jacob, I. (1971).Dev. Biol. 25, 514-546. Schubert, D., Tarikas, H., and LaCorbiere, M. (1976). Science 192, 471-472. Schurch-Rathgeb, Y., and Monard, D. (1978). Nature (London) 273, 308-309. Seeds, N. W., Gilman, A. G., Amano, T., and Nirenberg, M. (1970).Proc. Natl. Acad. Sci. U.S.A. 66, 160-167. Sellstrom, A., and Hamberger, A. (1977). Brain Res. 119, 189-198. Sen Sharma, G. C., and Singh, S. (1973).IndianJ. Med. Sci. 27, 12-21. Sidman, R. L., and Rakic, R. (1973).Brain Res. 62, 1-35. Sieghart, W.,and Karobath, M. (1976).J. Neurochem. 26, 981-986. Simon, J. R., Martin, D. L., and Kroll, M. (1974).J. Neurochem. 431, 981-993. Sipe, J. C. (1976). Cell Tissue Res. 170, 485-490. Sipe, J. C., and Moore, R. Y. (1976). Anat. Rec. 185, 247-252. Skoff, R. P., Price, D. L., and Stocks, A. (1976a).J. Comp. Neurol. 169, 291-312. Skoff, R. P., Price, D. L., and Stocks, A. (1976b).J. Comp. Neurol. 169, 313-334. Snodgrass, S. R., and Iversen, L. L. (1974). Brain Res. 76, 95-107. Somjen, G. G. (1969). Brain Res. 12, 268-272. Somjen, G. G. (1970).J. Neurophysiol. 33, 562-582. Somjen, G. G. (1973).Prog. Neurobiol. (Oxford) 1, 199-237. Somjen, G. G. (1975).Annu. Rev. Fhysiol. 37, 163-190. Somjen, G. G., Rosenthal, J., Coordingley, G., LaManna,J., and Lothman, E. (1976).Fed. Proc., Fed. Am. SOL. ESP. Biol. 55, 1266-1271. Sotelo, C., and Changeux, J. P. (1974).Brain Res. 77, 484-491. Stefanovic, V., Ciesielski-Treska,J., Ebel, A., and Mandel, P. (1976). Ex@. Cell Res. 98, 191-203.
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Stefanovic, V., Ciesielski-Treska, J., and Mandel, P. (1977). Brain Res. 125, 313-323. Steinbach, J. H., and Schubert, D. (1975). Exp. Cell Res. 91, 449-453. Stewart, R. M., Richman, D. P., and Caviness, V. S., Jr. (1975).ActaNeuropathol. 31, 1-12. Stewart, R. M., Martuza, R. L., Baldessarini, R. J., and Kornblith, P. L. (1976).Bruin Res. 118, 441-452. Strittmatter, W. J., and Somjen, G. G. (1973). Brain Res. 55, 333-342. Sturrock, R. R. (1974).J . Anal. 117, 27-35. Sturrock, R. R. (1976).J . Gerontol. 31, 513-522. Suburo, A. M., and Adler, R. (1977). Cell Tissue Res. 176, 407-416. Sugaya, E., Goldring, S., and OLeary, J. L. (1964).Electroencephlogr. Clin. Neurophysiol. 17, 661-669. Sugaya, E., Takato, M., and Noda, Y. (1971).J . Physiol. SOC.Jpn. 33, 654-655. Sugagy, E., Takato, M., and Noda, Y. (1975).J . Neurophysiol. 38, 822-841. Sutton, I., and Simmonds, M. A. (1974).J. Neurochem. 23, 273-274. Svenneby, G. (1970).J . Neurocha. 17, 1591-1599. Sypert, G. W., and Ward, A. A. (1971). Exp. Neurol. 33, 239-255. Tani, E., Itagaki, R., and Nakano, M. (1977). Cell Tissue Res. 184, 139-142. Trachtenberg, M. C., Kornblith, P. L., and Haupili, J. (1972). Brain Res. 38, 279-298. Varon, S. (1975). Exp. Neurol. 48, 93-134. Varon, S., Raiborn, C. H., and Norr, S. (1974). Exp. Cell Res. 88, 247-256. Vaughn, J. E. (1969). Z. Zellforsch. Mikrosk. Anat. 94, 293-324. Vaughn, J. E., and Peters, A. (1971). In “Cellular Aspects of Neural Growth and Differentiation” (D. C. Pease, ed.), UCLA Forum in Medical Sciences, No. 14, pp. 103-140. Univ. of California Press, Los Angeles. Vernadakis, A. (1971). In “Influences of Hormones on the Nervous System” (D. H. Ford, ed.), pp. 42-55. Karger, Basel. Vernadakis, A., and Berni, A. (1973). Brain Res. 57, 223-228. Vernadakis, A., and Nidess, R. (1976). Neurochem. Res. 1, 385-402. Vernadakis, A., Culver, B., and Nidess, R. (1978). Psychoneuroadocrinology 3, 47-64. Vichow, R. (187 1). “Cellularpathologie in ihre Begrundung auf Physiologische und Pathologische Gewebelehre,” 4th Ed. Hirschwald, Berlin. Watson, W. E. (1965).J . Physiol. (London) 180, 754-765. Watson, W. E. (1974). Physiol. Rev. 54, 245-271. Woodbury, J. W. (1965). In “Physiology and Biophysics” (T. C. Ruch and H. D. Patton, eds.), pp. 1-25. Saunders, Philadelphia, Pennsylvania. Yakovlev, P. I., and Lecours, A. (1967). In “Regional Development of the Brain in Early Life” (A. Minkowski, ed.), pp. 3-70. Blackwell, Oxford.
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MOLECULAR PERSPECTIVES OF MONOVALENT CATION SELECTIVE TRANSMEMBRANE CHANNELS By Dan W. Urry hborrrtory of Mabcuhr Biophyricr and Cardiovascular bsemrch and Training Center,
Uniwnity of Alabama M.dical Gnter, Birmingham, Alabama
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Channel Models . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and Relevance o f the Gramicidin A Channel . . . . . . . . . A Molecular Theory of Electric-Field-Dependent Channel Formation . . Perspective of Ion Selectivity Derived from Gramicidin A and Poly-AAG . A. Anion vs. Cation Selectivity . . . . . . . . . . . . . . . . . . . . B. Monovalent vs. Divalent Cation Selectivity . . . . . . . . . . . . . . C. Selectivity among Monovalent Cations . . . . . . . . . . . . . . . VI. Polymorphism of Channel-Forming Peptides . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. 11. 111. IV. V.
311 312 3 14 317 324 324 327 329 329 332
1. Introduction
Cell membranes, as protein-containing, lipid bilayer structures, demarcate aqueous domains of different chemical composition. T h e differing chemical compositions are achieved by proteins variously incorporated within, or attached to, the lipid membrane. One such class of proteins, adenosine triphosphate, as the name implies, utilizes adenosine triphosphate as a source of energy to pump chemical species against concentration gradients (Skou, 1965; Dahl and Hokin, 1974). Ionic gradients set up in this manner result in a separation of charge across the membrane, i.e., a transmembrane potential, which, in excitable cells such as nerve and muscle, can rapidly be dissipated locally and for short periods of time to give an action potential. T h e entire process of depolarization and repolarization, which is the action potential, is complete within a few milliseconds (Hodgkin, 1961). On the basis of the Eate at which ions traverse the membrane during the action potential, it can be concluded that the mechanism of translocation is too fast for selective complexation and movement of the complex from one side of the membrane to the other (Armstrong, 1975a; Lauger, 1972; Conti et al., 1976a; Bamberg et al., 1976). Rather, the evidence is that transmembrane structures exist that can rapidly undergo a structural transition to an open, selective, cation-conducting structural state and then almost as 31 1 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 21
Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form rererved. ISBN 0-12-966821-2
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D A N W. URRY
rapidly close. In the case of the transmembrane structure that selectively conducts sodium-the sodium channel-closing is not the reverse process of opening, but, rather, a third structural state occurs for a component of the transmembrane protein (Armstrong, 1975b; Conti et ad., 1976b). Also, the evidence is compelling that sodium and potassium conductances occur by means of separate and distinct channels (Armstrong, 197513) and that the sodium channel has a permeability for Na+ that is about ten times greater than for K+ (Ebert and Goldman, 1976; Hille, 1976).
II. Channel Models
There are now three basic structural models for transmembrane channels. One is a barrel stave or parallel alignment of repeating units (see Fig. la), in which the number of subunits determines the diameter of the channel. A classical helix, such as an a- or 310-helix,forming the walls of a pore, would be a conformational model. Alamethicin, an example of this model (Baumann and Mueller, 1974; Boheim, 1974), exhibits an interesting voltage dependence that, with sufficient membrane fluidity, can be fast enough to be a model for the sodium channel Transmembrane Stru
a. porollel alignment alamethicin (radial symmetry)
b. series alignment
grarnicidin A (single stranded P-helices) c. twist alignment
grarnicidin A (double stranded P -he I ices)
FIG. 1 . Transmembrane structures (channels)of repeating units. (From Urry, 1977.)
MOLECULAR STRUCTURE OF CHANNELS
313
(Mueller, 1975). While it exhibits very little ion selectivity on its own, as both monovalent and divalent cations and anions can pass through (Eisenberg et al., 1973), binding of cationic protamine or spermidine, e.g., can give it an anionic selectivity (Mueller, 1975). A second model is that of a series alignment of repeating units, wherein each repeat completely envelops a linear segment of the channel and determines the channel diameter, and the number of subunits determines the length of the channel (see Fig. lb). The evidence is that gramicidin A forms this type of channel by amino-end-to-amino-end (head-to-head) hydrogen bonding of two single-stranded @-helices (see Fig. 2; see also Urry, 1972a; Urry et al., 1971). A third model, a combination of the first two, is a twist alignment of repeating units wherein each repeating unit spirals around the channel but more than one unit is required to complete the circumference (see Fig. lc). Gramicidin A is capable of forming this type of structure (Veatch et al., 1974; Veatch and Blout, 1974; Fossel et al., 1974), specifically, a double-stranded @-helix,at high concentrations and temperatures, as shown in Fig. 3 (Urry et al., 1975). Although there is no evidence as yet that this structure forms ionconducting transmembrane channels in lipid bilayer membranes (see Table I below), it remains an intriguing structural possibility. The invaluable and physiologically relevant method whereby these model compounds are evaluated was first developed by Mueller and Rudin (1967). Lipid dissolved in a hydrocarbon such as decane is
FIG. 2. The gramicidinA single-stranded@-helicalchannel. (a) Channel view showing 4-A-diam channel. (b) Side view showing head-to-head hydrogen-bonded dimer with formyl protons juxtaposed. (c) Side view wire model showing backbone and Pcarbon atoms. (From Urry et al., 1975.)
314
DAN W. URRY
FIG. 3. The gramicidin A antiparallel, double-stranded P-helical structure. (a) Helix axis view showing channel. (b) Side view. (c) Side view in wire model. While there is evidence that this structure occurs at elevated concentrationsand temperatures,there is no evidence as yet that it forms conducting channels in planar lipid bilayer membranes. (Frnm Urry et al., 1975.)
brushed onto, or extruded into, a small hole in a septum separating two aqueous chambers, each containing an electrode. The lipid in the hole naturally thins to a lipid bilayer. When the thinning process is viewed through a light microscope, a rainbow pattern develops that finally gives way centrally to a black-appearing membrane that signals that the lipid bilayer state has been reached. For this reason, the method has been called black lipid membranes. The molecular species to be evaluated for its ion transport properties is added directly to the water solution on one or both sides of the membrane, or it is added directly to the lipid solution and its conduction properties are evaluated by means of the electrodes. The value of this approach, and its subsequent refinements, to the development of our understanding of mechanisms of selective ion transport across lipid bilayer membranes cannot be overstated.
111. Structure and Relevance of the Gramicidin A Channel
The evidence that gramicidin A, H C 0 - ~ - V a l ~ - G l y ~ - ~ - A l a ~ - ~ - L e u , L-A1a5-D-Val,-L- Val,-D- Vals-~-Trp9-~-LeuIo-~-Trp, I-~-LeU12-~-Trp13-DLeu14-~-Trp,,-NHCH2-CH20H(Sarges and Witkop, 1964), forms a continuous channel across the membrane is compelling: conductance increases by fixed increments (Hladky and Haydon, 1970); freezing of the lipid membrane stops carrier conductance but not gramicidin A conductance (Krasne et al., 197 1); and proton conductance indicates
MOLECULAR STRUCTURE OF CHANNELS
3 15
diffusion rates greater than that of ions in bulk solution, suggesting a continuous file of water and a proton jump mechanism (Myers and Haydon, 1972). That the channel is a dimer is also established: T h e initial hypothesis that the channel was a dimer, based on an approximate second-order concentration dependence for both equilibrium conductance and conductance development (Goodall, 1970, 1971), was confirmed by the first-order concentration dependence and kinetics of the covalent dimers (Urry et al., 197 1; Goodall, 1973), by the kinetics studies employing temperature, voltage step relaxation, and correlation analysis methods (Bamberg and Lauger, 1973, 1974; Kolb et al., 1975), by concentration studies on the O-pyromellityl, water-soluble derivative (Apell et al., 1977), and finally by fluorescence studies on the incorporation of a fluorescent-labeled gramicidin A into planar lipid bilayers (Veatch et al., 1975). That the functional dimeric channel is formed by head-to-head (amino-end-to-amino-end) hydrogen bonding of single-stranded /3helices, as originally proposed (Urry, 197 1, 1972a; Urry et al., 197 l), is also compelling, as summarized in Table I. T h e desformyl gramicidin A (I) was inactive (Urry, 1971); the amino-end-to-amino-end covalent dimers (111, IV, and V) are very active, with first-order concentration dependence and very long channel lifetimes (Urry et al., 1971; Goodall, 1973; Bamberg and Janko, 1977); the O-pyromellityl gramicidin A (VII) is active, with properties similar to those of gramicidin A only when added to both sides (Apell et al., 1977); and theN-pyromellityl derivative (VIII) is inactive whether added to one or both sides and whether added to one side with VII on the other side (Bamberg et al., 1976). An interesting derivative is 11, where the formyl proton is replaced by a methyl. This derivative has no discernible effect on the association in ethanol solution to form the double-stranded P-helix (Veatch and Blout, 1974), but its efficacy as a functional channel is reduced to 1% of that of gramicidin A (see Urry, 1971) due to an almost two order of magnitude reduction in channel lifetime (Szabo and Urry, 1979), as shown in Fig. 4. T h e conclusion is that the methyl causes a steric crowding on head-to-head dimerization (see Fig. 2b), which destabilizes the dimeric state. Any modification of the amino end of the molecule either completely destroys the capacity to form the channels (VIII and XII) or dramatically changes the channel lifetimes (11, 111, IV, V, VI, XI), whereas modification of the carboxyl (or ethanolamine OH) end has much more limited effects (VII and X, Harris et al., 1976) on channel activity. These effects are all explicable in terms of the head-to-head dimerized channel structure shown in Fig. 2 (Urry et al., 1971; Urry, 197213).
TABLE I: SUMMARY OF BILAYER ACTIVITIESOF GRAMICIDIN A' DERIVATIVES" Structural implications
HCO
Gmicidin A' derivative NHCHCH,OH
1. IkrfoAyl: H.N 11. N-Acetyl:CH,CO 111. Malonyl:
CO
Bilayer activity
NHCHCH,OH NHCH.CH.OH
Inactive (a) 1% Activity (b) r = 60 mwc (c) Exceptionally (4 active (4 (first order) (f,
NHCH&H.OH
I CHz I I
co 1V. Succinyl: CO
v. oxalyl:
co
I I co
NHCH,CH.OH NHCH.CH.OH
T
NHCH&H.OH N HCH&HsOH
Single stranded (head to head)
Double svanded
J J
? ?
J
Parallel
very long
Active (first order) (')
Pardel
Active (first Order) (c)
Parallel
NHCHzCH.OH 0.1% Active (b) r<
VI. Ni-Butyloxycarbonyl (CH&=-NHCH.CHPH
?
0 COOVII. 0-Pyrumellityl: HCO
N HCH,CI*d+COO-
COO
VIII. N-pyromellityldesformyl: -0OC
COOIX. 0-Pyromellityl (one side) + N-pyromcllityl (other side) X. 0-Succinyl: HCO NHCHICHz-OCOCHtCHzCOOXI. N-succinyldesformyl: methylester: CH,ooCCH.CHH,CO-NHCH.CHIOH acid: HOOCCHICHICO X11. N,O-bissuccinyldesformyl: -OOCCH.CHtCONHCH&HzOCOCH*CHzCOO-
(1) One side: inactive (2) Both sides: active )'
J
Antiparallel
(I) One side: inactive (2) Both sides: inactive (')
J
X
Inactive (i) Active (sidednw) (i) + = I 3 sec Active (sidedness) r=2mmmpc
Inactive 0)
0)
J
X
J
Antipadel
J
?
J
X
From Urry (1978). (a)Urry (1971): (6) Urryctd. (1975); (c) Szaboand Urry (1978); (d) Urryct al. (1971); (4 Goodall (1973): (f, Bamberg and Janko (1977): (g) Sraboand Urry (unpublished data); (h)Apelletol. (1977): ( i ) Bamberg cf al. (1977); (i) Bradley ef al. (1978). r = Channel mean lifetime. ? = Doubtful structure. J = Acceptable structure. x = Highly unlikely structure. a
317
MOLECULAR STRUCTURE OF CHANNELS
1 N-Acetyl-Desformyl Gramicidin A
n
A
1
e
Lhiz
0
\
V
I
/
B
\../
FIG. 4. Single-channel conductance events in monoolein planar lipid bilayer membranes due to (a) N-acetyldesformyl gramicidin A and (b) gramicidin A. Note, in particular, the difference in time scales, where the mean channel lifetime is much shorter for the derivative than for gramicidin A. The replacement of the formyl proton by a methyl results in a steric crowding (see Fig. 2) that destabilizes the dimeric structure required for channel formation. The conditions are 1 M RbCl at an applied voltage of 100 mV. (From Szabo and Urry, 1979.)
The great interest in the gramicidin A channel is due to its property of monovalent cation selectivity (Myers and Haydon, 1972; Sandbloom et al., 1977; Bamberg and Lauger, 1977) and the similarity of unit channel conductances to those of monovalent cation selective physiological channels, as shown in Table 11. Yet gramicidin A is limited as a model for physiological channels, because it contains D-amino acids as an important structural feature, it lacks a comparable voltage dependence (Bamberg and Benz, 1976), its on-off kinetics are slow, with channel lifetimes on the order of several seconds, and it exhibits selectivity of K+ over Na+ by only a factor of three (Myers and Haydon, 1972), whereas the selectivity of the Na channel for Na+ over K+ is a factor of 13 (Ebert and Goldman, 1976). IV. A Molecular Theory of Electric-Field-Dependent Channel Formation
As a means of overcoming some of the aforementioned limitations while retaining the favorable structural features giving rise to monova-
318
DAN W. URRY
TABLE 11" CONDUCTANCES, DENSITY, AND GATING CHARGES OF CHANNELS Source Physiological channels Node of Ranvier
Squid giant axon
Neuromuscular junction (Ach channel) Extrajunctional (muscle) (Ach receptor)
Glutamate receptor
Channel conductance (pS = mhos)
yNe= 8 pS Conti et al.
(1976a,b) yn = A 4 pS Begenisich and Stevens (1975) YNa = 4 p s Y K = 12 p s Wanke et al. (1974); DeFelice et al. (1975); Conti et al. (1975) y = 32 pS Anderson and Stevens (1973); Stevens (1975) y = 15,23,39 pS Neher and Sakrnann (1976a,b); Sachs and Lecar (1973, 1977) y = 231 pS at 23°C y = 39 pS at 8.5"C Anderson et a[. (1976)
Photoreceptors
y = lops Simon et al. (1975); Lamb and Simon (1976a,b); Simon and Lamb (1979)
Mechanoreceptors (hair cells)
y = lops DeFelice and Alkon (1977)
Density of channels
Gating charge
2000/p* DeFelice (1977)
330/p2 60/p2 Wanke et al. (1974); DeFelice et al. (1975); Conti el al. (1975) 10' Channels at end plate DeFelice (1977)
4e ( 19 DIA) Keynes (1976)
50 DIA Stevens (1975)
Sarcopfasmic reticulum 4.8 DIA Almers et al. (1975) Opposite polarity to neuromuscular junction Anderson et al. (1976)
Polypeptide models Alamethicin
y = 20-200 p s Boheim'( 1974)
14 DIA (for a 6-helix channel) (continued)
319
MOLECULAR STRUCTURE OF CHANNELS
TABLE I1 (Continued) Gramicidin A
yNa = 6-30 pS
(Ala-Ala-Gly)S
Bamberg el al. (1976) YNa = 14 p s YK = 25 pS Goodall ( 1973)
2.6-4 DIA Urry et al. (1978)
Conformational models Syn-vz-spiral = ss-a?&4-helix Two parallel &'-helices = p,
[email protected] Urry (1972a. 1973)
3.2 DIA 5 D/A Venkatachalam and Urry ( 1979) ~
a
~
~
_
_
_
~
~
Adapted from Urry (1977).
lent cation selectivity, a molecular theory of electric-field-dependent channel formation was developed (Urry, 1972b, 1973) after the necessary derivation of a more generally applicable conformational concept of cyclic conformations with linear conformational correlates. T h e concept is best exemplified by the structures of the enniatin B-Kf complex and the single-stranded @!;&helix of gramicidin A, as shown in Fig. 5a and b. Both structures have the general features of alternating C-0 moieties pointing parallel and antiparallel to the symmetry axis. It is apparent that rupture of the cyclic structure, with but minor and acceptable changes in dihedral angles b, and Jl, can convert to the linear P-helical structure. Thus the gramicidin A @-helixmay be thought of as the linear conformational correlate of the cyclic enniatin B structure. This concept allows us to go from well-known cyclic conformations to linear conformations which had not, at the time (Urry, 1973), been described. Figure 6a shows a well-known backbone conformation for a cyclic hexapeptide which is two @-turns,i.e., two 1O-atom hydrogen-bonded rings, related by a twofold symmetry axis. With small and acceptable changes in the dihedral angles, 4 and Jl, of the cyclic structure, the linear structure shown in Fig. 6c can be constructed, which is called a syn-@$spiral. The superscript 6 indicates the number of residues in the cyclic analog; the subscript 2 means two p-turns per turn of spiral; syn indicates that the C-0 moieties of the two end peptide units are pointing in the same direction, which is up in Fig. 6a and c. A schematic cyclic representation of this @-spiral is given in Fig. 7b. Whereas the end peptide
320
DAN W. URRY
FIG. 5. Space-filling molecular models of the enniatin B-K+ complex (a) and gramicidin A (b) in the single-stranded @;%-helicalconformation. Enniatin B is a cyclohexadepsipeptide with an alternating L-D sequence. Of the six carbonyls that coordinate the cation alternately three are directed up out of the plane and three are directed down. This is a cyclic conformation for which the gramicidin A structure is the linear conformational correlate achieved by rupture of the cyclic structure and only small variations in r#~and $ dihedral angles. Note that the linear gramicidin A structure has 6.3 residues per turn and the carbonyls alternately point parallel and antiparallel to the helix axis. This exemplifies the concept of cyclic conformations with linear con formational correlates.
doiety C - 4 directions are directed up out of the plane of the paper, the remaining peptide moieties are in the plane. Rotation of those peptide moieties, such that their C-0 directions are down, as shown in the CPK model in Fig.Gb, results in an opening of the structure to a state analogous to that of enniatin B when complexed. The linear conformational correlate of the structure in Fig. 6b is that of Fig. 6d, which results in a new type of P-helix, with structural similarity to that of gramicidin A but with a net two C-0 moieties directed downward. Structures shown in Fig. 6c and d are related by a rotation of four of the six peptide moieties per turn, resulting in a transition from a structure with no channel to a channel-containing structure. As the peptide moiety contains a substantial dipole moment, the t w o structures, on maintaining the same helix axis orientation, would have a dipole moment difference of about 3 D/W, say from + 1.5 D/I$ to - 1.5 D/I$. This is sufficient, on changing the lipid bilayer transmembrane potential by 100 mV, to shift the equilibrium between the two states by a factor of more than10 (Urry, 1975). The structures shown as Corey-Pauling-Koltun models in Fig. 6c and d may be considered to be idealized structures. Recent con-
MOLECULAR STRUCTURE OF CHANNELS
321
FIG. 6. Cyclic conformations (a) and (b) and linear conformational correlates (c) and (d), respectively, of the repeating sequence L-Ala-L-Ala-Gly. (a) The cyclohexapeptide, L(L-Ala-L-Ala-Gly),~in a conformation with two p-turns related by twofold symmetry. (b) The same cyclohexapeptide as in (a) but with the four peptide moieties involved in the &turn hydrogen bond rotated such that the four C-0 directions all point down.(c) The linear conformational correlate of (a). (d) The linear conformational correlate of (b). The structure in (c). a syn-&spiral, can convert to the channel structure in (d), a singlestranded @,,-helix, by appropriate rotation of the &turn hydrogen-bonded peptides of (c). (From Urry, 1978.) formational energy calculations (Venkatachalam and Urry, 1979) on poly-(~-Ala-~-Ala-G1y), abbreviated as poly-AAG, have shown these two conformational states to be of similar energy and competitive with the a-helix. A corresponding structure, derived from detailed theoretical calculations, is shown in helix-axis perspective in Fig. 8a and in side view in Fig. 8b. Note that the slightly oval channel of the P-helix is lined with peptide carbonyl oxygens, with four carbonyls pointing down and two pointing up per turn. As indicated previously (Urry, 1972b), the nomenclature for this structure is @;:-helix, where the superscript is the number of residues per turn and the subscript indicates the above-
322 0
DAN W. URRY
onti
-4-
spiral c-0
= fl36.3- helix
c - 0 up
UD
L-Roa 4 - N
Gly
-0-
f--]
1
L-R88=
f
N-H-0-
\
-o-i%_i- L-Raa
L-Roa
GIY
C - 0 down
C - 0 down
a'
- splrol
Gly
a
L:x7.
b synfl:
6 == 4,,helix
c - 0 up
c - 0 up
L-Rea
L-RIs
c-0 up
b'
c-0
UP
b
FIG. 7. Schematic cyclic representations of linear structures. In (a) the end G O moieties point in opposite directions (anti) such that their dipole moments cancel. Similarly, in (a') the peptide dipole moments approximately cancel in the linear structure. This is a cyclic representation of the gramicidin A backbone, with Gly in place of D-residues. In (b') the end peptide C-0 moieties point in the same direction (syn), such that in the linear correlate they add. In (b) rotation of the four cross-hydrogen-bonded peptide moieties, such that the four C-0 groups are directed downward, results again in a net dipole moment but with the opposite sign. The linear correlates of these cyclic structures, therefore, have substantially different dipole moments along their axes, and one is nonconducting and the other is a conducting-channel-containing structure. (b') corresponds to the structure in Fig. 6c and (b) to the channel structure in Fig. 6d. (From Urry, 1972a.)
MOLECULAR STRUCTURE OF CHANNELS
323
Chamel Vew FIG. 8. Right-handed @$-helix of (L-Ala-L-Ala-Gly)., calculated structure using the partitioned potential energy method. Note the orientation of peptide moieties. (From Venkatachalam and Urry, 1979.)
noted orientation of carbonyls. The nomenclature is P&helix for gramicidin A. This different relative orientation of peptide carbonyls has implications with respect to end-to-end dimerization. T h e gramicidin A &helix, with three C-0 and three N-H moieties directed outward at each end, makes possible, from this aspect alone, any combination of end-to-end hydrogen-bonded dimerization, i.e., head-to-head, head-to-tail, and tail-to-tail. It has already been noted that the formylated amino (head) end, as well as other factors, makes head-to-head association preferable. In the case of the poly-(L-Ala-L-Ala-Gly)right-handed @2,4-helix,there are four carbonyls and two NH moieties directed outward at the amino end (see Fig. 8) such that six intermolecular hydrogen bonds could not form with head-to-head dimerization. With head-to-tail hydrogenbonded association, six hydrogen bonds could form, making head-to-tail association likely. This has important implications with respect to voltage dependence because, with head-to-tail dimerization, the dipole moments of the associating species will add (i.e., + d), whereas the dipole moments of the head-to-head dimer (t+) will cancel. Recalling that gramicidin A is a pentadecapeptide, a structure such as HCO-(L-Ala-LAla-Gly),-OMe might be expected to form head-to-tail hydrogenbonded dimers similar to those of gramicidin A but with a resultant
324
DAN W . URRY
dipole moment across the membrane that could couple to the transmembrane electric field. As the structure is similar to that of gramicidin A, there remains the possibility that it wauld retain the favorable feature of monovalent cation selectivity with a comparable conductance. Following the development of the above molecular theory for electric-field-dependentchannel formation (Urry, 1972b), the polypeptides HCO-(L-Ala-L-Ala-Gly),-OMe, with n = 4 and 5, were synthesized and found to be channel-forming peptides that turned on and off with voltage sweeping (Goodall and Urry, 1973; Goodall, 1973). Recently, HCO-(L-AIa-L-Ala-Gly),-OMe was examined in more stable diphytanyl-L-a-lecithin membranes (Urry et al., 1978) and was found to form monovalent cation selective channels (being impermeable to Caz+ and Cl-), with a conductance of about 50 pS for Cs+ (see Fig. 9a), and to respond to increasing voltage with a nonlinear, exponential rise in current (see Fig. 10). A calculation of the number of channels at 50 mV and 100 mV for the + and - voltage sweeps in Fig. 9b and c allows four approximations to the dipole moment change, with values ranging from 2.6 to 4 D/A. This experimental estimate compares favorably with the theoretical value of 3.2 D/A calculated for the conversion from the right-handed @-spiral,with - 1.8 D/A, to the right-handed @-helix,with 1.4 D/A. This is equivalent to a gating charge of 0.6 and is similar to that of the acetylcholine receptor channel of neuromuscular junction (Anderson and Stevens, 1973; Stevens, 1975). Another feature of the synthetic channel is a mean lifetime of about 30 msec (Urry et al., 1978; see also Fig. 9b), which is again similar to that of the neuromuscular junction channel (Neher and Sakmann, 1976a). Thus, this synthetic channel, while retaining the favorable unit channel conductance and monovalent cation selectivity of gramicidin A, exhibits, in addition, a channel mean lifetime and a voltage dependence that are similar to those of physiological channels. V. Perspective of Ion Selectivity Derived from Gramicidin A and Poly-AAG
A. ANIONvs. CATIONSELECTIVITY
The experimental Gibbs free energy of hydration for ions of about 1 .O-A radius is approximately - 100 kcal/mole for a monovalent cation, -400 kcaI/mole for a divalent cation, and -900 kcal/mole for a trivalent cation (Rosseinsky, 1965; Stokes, 1964; Noyes, 1962). For monovalent cations, nearly half of the energy comes from the first two waters of
MOLECULAR STRUCTURE OF CHANNELS
325
FIG. 9. Planar lipid bilayer studies on HCO-(L-Ala-L-Ala-Gly)5-OMe.(a) Activity of a single channel turning on and off with a conductance of 50 pS at 50 mV, 25"C, and 1 M CsCI. (b) Power spectral density of bilayer current due to 100 pM concentration of peptide in the aqueous bath. The spectrum gives a mean channel lifetime of 30 msec. (From Urryet al., 1978.)
hydration and nearly all of the energy comes from the first coordination shell (Dzidic and Kebarle, 1970; Arshadi et al., 1970; Kistenmacher et al., 1973, 1974). Also, energies of hydration for the chloride anion are of the same magnitude as for the monovalent cations (Arshadi et al., 1970; Kistenmacher et al., 1974). If a channel diameter is too small for the ion to pass through with its first hydration shell (which is not an unreason-
326
DAN W. U R R Y
FIG. 10. Planar lipid bilayer membrane conductance in the presence of HCO-(LAla-L-Ala-Gly),-OMe as a function of voltage achieved by 0.1 Hz voltage sweeps between + 100 mV and - 100 mV. In (a) a single channel is seen to turn on and off. In (b) several hundred channels in the membrane turn on and off as a function of voltage. The membrane current is seen to increase exponentially with increased voltage. In (c) with a higher concentration of channels in the membrane the nonlinear current vs. voltage curve is also apparent. Note the changes in scale for (a), (b). and (c) and that the capacitance step in (a) that causes the rise at + 100 mV and the drop at - 100 mV is lost in the scale change for (b) and (c). A voltage dependence of from 2.6 D//l to 4 D/A can be calculated from the four nonlinear curves in (b) and (c). (From Urry et al., 1978.)
MOLECULAR STRUCTURE OF CHANNELS
327
able prerequisite, in general, for high selectivity), and if one coordinated water molecule can precede and one follow the ion through the channel, then it follows that some 50 kcal/mole of energy must be provided by interaction with the walls of the channel. Because of the distance dependence of liganding energies (Urry, 1978), this requires that the van der Waals radius of the interacting atom approximately make contact with the ionic radius. In gramicidin A and poly-AAG channels, with 4-A diameters, this requires that there be a flexibility in the wall that is provided by the peptide libration. As depicted in Fig. 11, libration of the peptide moiety to decrease the diameter of the channel lines the wall of the channel with the negative oxygen of the peptide moiety, providing an adequate interaction energy for a monovalent cation but making the channel repulsive to anions. Thus, anions are excluded from the channel. vs. DIVALENT CATIONSELECTIVITY B. MONOVALENT Because the energy of hydration is four times greater for a divalent cation than for monovalent cations, it is reasonable to expect that substantial hydration energy is gained from molecules in the second coordination shell and beyond. Whereas enniatin B-like linear hexapeptides (Bradley et al., 1977) selectively bind Ca2+in polar organic solvents with little interaction with monovalent cations, calcium ions inhibit the potassium carrier activity of these synthetic peptides in planar lipid bilayer studies (Bradley et al., 1977). Presumably, the hexapeptide interacts with Ca2+but does not cross the lipid layer due to insufficient decreases in the ion self-energy, which result in a repulsive image force. In other words, the lipid does not provide a second coordination shell sufficient to lower the energy. In trifluoroethanol solution gramicidin A readily complexes Ca2+and shows little interaction with Na+ (Urry, 1978), yet Ca2+neither passes through the channel nor does it bind and block the channel (Bamberg and Lauger, 1977). Divalent cations at molar concentrations d o decrease the unit channel conductance of gramicidin A channels, apparently by being able to very rapidly approach the mouth of the channel, but are unable to pass through the channel (Bamberg and Lauger, 1977). It appears that the lateral single coordination shell (i.e., the single thickness of polypeptide backbone) provided by the gramicidin A channel is insufficient in lowering the energy of the divalent cation to compete with water binding. In other words, the close proximity of the lipid represents a repulsive, positive image force. These concepts have been developed in greater detail elsewhere (Urry, 1978). Thus, the gramicidin A and poly-AAG structures, being simple, single-walled,
2 I- 3.00 )
Zfl.38)
H
a. Top View
b. Side View
FIG. 11. Peptide libration mechanism for the interaction of the walls of the channel with a permeant monovalent cation. The negative dipole of the peptide carbonyl oxygen makes the channel impermeable to anions. T h e energy for the rotation of the peptide oxygen inward could contribute to selectivity based on ionic radius. Divalent cations are thought to be repulsed due to an inadequate shielding by a single thickness of polypeptide backbone from the surrounding lipid environment. (From Urry, 1973.)
MOLECULAR STRUCTURE OF CHANNELS
329
&helical structures, provide a rationale for monovalent vs. divalent cation selectivity.
C. SELECTIVITY AMONG MONOVALENT CATIONS Utilizing erythrocyte lipid plus n-decane membranes with cation concentrations of 0.1 M at 20"C, Myers and Haydon (1972) reported the permeability ratios with respect to Na+ to be H+ (150) > NH,+ (8.9) > Cs+ (5.8) > Rb+ (5.5) > K+ (3.9) > Na+ (1.0) > Li+ (0.33) for gramicidin A. Whereas this is poor selectivity when compared to a carrier such as valinomycin, whereP(K+)/P(Na+)> lo3,it is not so poor when compared to physiological channels, where for the sodium channel P(Na+)/P(K+) is reported to be 13 (Ebert and Goldman, 1976). As P(K+)/P(Na+)is 3.9, one need only find a means of achieving a small increase in the energy for discrimination (0.5 kcaymole) in order to achieve the same relative selectivity. One way of accomplishing this is to change the energetics for peptide libration (see Fig. 11). Any structural change which would sufficiently alter the energy profile for peptide libration should, in this proposed mechanism, be able to effect changes in cation selectivity. This could be achieved most simply by changing the R group, i.e., change the amino acid residue. More stringent means would be to N-methylate available peptide NH moieties, to replace selected residues with proline, or to use a residue such as a,P-dehydrotryptophan. VI. Polymorphism of Channel-Forming Peptides
Fundamental features of gramicidin A and poly-AAG are the numbers of conformational states available to the peptide. The conformation of gramicidin A is dependent on concentration, solvent, and temperature, as shown in Figs. 12 and 13 by the variable circular dichroism spectra of hydrogenated gramicidin A (Urry et al., 1975). As the sidechain chromophores are removed by hydrogenation, this reflects only backbone conformations. Furthermore, the analog of gramicidin A, poly-y-benzyl-D,L-ghtamate, has been shown by fiber diffraction studies to occur in the a-helical, single-stranded &helical (Heitz et al., 1975), and double-stranded &helical (Lotz et al., 1976) conformational states. The conversion from one con formational state to another is achieved by successively elevating temperatures in a manner previously shown to be effective for gramicidin A in solution (Urry et al., 1975), i.e., elevating the temperature favors the double-stranded conformational
B
Hydrogenoled Gromicidin A' in EtOH 0 1 25%
Concentration Effecl 0 . 0.01 mg/ml b. 0.10 mglml c. 1.0 mg/ml d. 10.0 mg/ml e. 100 mg/ml
200
X
C
D Hydrog~nalrdGromicidln A'
in Methanol ot 25' C Concentration Effect
a. b. c. d. e.
.b
0.01 mg/ml 0.10mg/ml 1.0 mg/ml 10.0 mg/ml 100 mg/ml
in E10H:H~O(3:l) at 25-
0.5
I
Concentration E f f ~ c t
a. b.
0.01 mp/ml
O.lOmp/ml 1.0 mg/ml d. 10.0mg/ml e. 100mg/ml
c.
0
x c
x o c I
240
(nm)
Hydrogenoled Gramicidin A'
0.5
220 A (nm)
.3
s
I
-01
-0.5
- 1.1
- 1.c 200
220 A (nm)
240
200
220 A (nm)
240
FIG. 12. Concentration dependence of the, circular dichroism patterns of hydrogenated gramicidin A' in four different solvent systems. This demonstrates that the conformation of the polypeptide backbone varies with concentration and solvent. (From Urry ct al., 1975.) 330
-R
A 6
Hydrogenated Gramtcidin A' I mglrnl in Absolute Ethanol
4
Temperature Study
A
a. b. c. d.
2
-a 0
y
-20.C IO'C 24.C 57 *C
I?
o
E -2 -4
-2
-
-4
-
-6
-
-8
-
Hydrogenated Gromictdon A'
-6
I mglml in Trifluoroethanol Temperature Study
-8 -1.0
200
220
240
A lnm)
- 1.2
I,,
-2 0
,
-2.2
200
220
,
240
A Inm)
D Hydrogenated Gramicidin A'
Hydropanoted Gramicidin A'
I mg/ml in Ethanol:Waler 9:l
I mg/ml in Methanol
Temperature Study
4-
-26.C b. 8.C c. 24.C d. S8.C 0.
2-
2-
Temperature Sludy a. -1O.C b. I0.C c. 24-12 d. 57.C
.)
s, 0 x
b a
o
x o
-2
-
-4
-
-2
-
-4
-
d
-6
" U 200
220 A lnm)
240
200
220
240
A lnm)
FIG. 13. Temperature dependence of the circular dichroism patterns of hydrogenated gramicidin A' at 1 mg/ml in four different solvent systems. The state with negative ellipticity near 220 nm is favored by increased temperature and, as seen in Fig. 12, by increased concentration. In addition to providing an interesting paradox wherein an ordered state is favored by increased temperature it emphasizes the polymorphic nature of gramicidin A'. (From Urry et al., 1975.) 33 1
332
DAN W. URRY
state, Because of its limited solubility, poly-AAG cannot be studied in a variety of solvents with varied concentration and temperature, as has been done for gramicidin A, but, in a single solvent of 5% trifluoroacetic acid in trifluoroethanol, poly-AAG gives an a-helix-type circular dichroism (CD) spectrum when n is very large and a CD spectrum when n = 5 which we expect to be that of a @spiral. When incorporated into liposomes, poly-AAG gives the latter CD spectrum. What this polymorphism means is that any structural determination that is not tied experimental4 to the lipid membrane state cannot be concluded to be relevant, Therefore, the conformational states within the liposome and planar bilayer membranes necessarily become the target states. Interestingly, conformational mobility is just what is required for voltage-dependent channel formation and particularly for those voltage-dependent channels that exhibit a separate inactivation step. ACKNOWLEDGMENT This work was supported in part by the National Institutes of Health, Grant No. HL-11310.
REFERENCES Almers, W., Adrian, R. H., and Levinson, S. R. (1975). Ann. N.Y. Acad. Sci. 264, 278. Anderson, C. R., and Stevens, C. F. (1973).J. Physiol (London) 235, 655. Anderson, C. R., Cull-Candy, S. G., and Miledi, R. (1976). Nature (London) 261, 151. Apell, H.-J., Bamberg, E., Alpes, H., and Liuger, P. (1977).J. Membr. B i d . 31, 171. Armstrong, C. M. (1975a). Biophys. J . 15, 932. Armstrong, C. M. (1975b). Q. Rev. Biophys. 7 , 179. Arshadi, M., Yamdagni, R., and Kebarle, P. (1970).J. Phys. Chem. 74, 1475. Bamberg, E., and Benz, R. (1976). Biochim. Biophys. Acta 426, 570. Bamberg, E., and Janko, K. (1977). Biochim. Biophys. Acta 465,486. Bamberg, E., and Liuger, P. (1973).J. Membr. Bid. 11, 177. Bamberg, E., and Lauger, P. (1974). Biochim. Biophys. Acta 367, 127. Bamberg, E., and Lauger, P. (1977).J. Membr. Biol. 35, 351. Bamberg, E., Kolb, H.-A., and Lauger, P. (1976). In “The Structural Basis of Membrane Function” (Y. Hatefi and L. Djavadi-Ohaniance, eds.), pp. 143- 157. Academic Press, New York. Bamberg, E., Apell, H. J., and Alpes, H. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 2402. Baumann, G., and Mueller, P. (1974). J. Supramol. Struct. 2, 538. Begenisich, T., and Stevens, C. F. (1975). Biophys. J. 15, 843. Boheim, G. (1974).J. Membr. B i d . 19, 277. Bradley, R. J., Romine, W. O., Long, M. M., Ohnishi, T., Jacobs, M. A., and Urry, D. W. (1977). Arch. Biochm. Biophys. 178, 468. Bradley, R. J., Urry, D. W., Okamoto, K., and Rapaka, R. (1978). Science 200, 435. Conti, F., DeFelice, L. J.. and Wanke, E. (1975).J. Physiol. (Londrm) 248, 45. Conti, F., Hille, B., Neumcke, B., Nonner, W., and Stampfli, R. (1976a)J. Physwl. (London) 262, 699.
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MOLECULAR STRUCTURE OF CHANNELS
Conti, F., Hille, B., Neumcke, B., Nonner, W., and Stampfli, R. (1976b).J. Physiol. (London) 262, 729. Dahl, J. L., and Hokin, L. E. (1974). Annu. Rev.Eiochem. 43, 327. DeFelice, L. J. (1977). Int. Rev. Neurobiol. 20, 169. DeFelice, L. J., and Alkon, D. S. (1977). Eiophys. Soc. Annu. Meet. Abstr. 17, 19a. DeFelice, L. J., Wanke, E., and Conti, F. (1975).Fed. Proc., Fed. Am. SOC.Exp. Eiol. 34, 1338. Dzidic, I., and Kebarle, P. (1970).J. Phys. Chem. 74, 1466. Ebert, G. A., and Goldman, L. (1976).J. Gen. Physiol. 68, 327. Eisenberg, M., Hall, J. E., and Mead, C. A. (1973).J. Membr. Eiol. 14, 143. Fossel, E. T., Veatch, W. R., Ovchinnikov, Y. A., and Blout, E. R. (1974). Biochemisby 13, 5264. Goodall, M. C. (1970). Eiochim. Eiophys. Acta 519, 471. Goodall, M. C. (1971). Arch. Eiochem. Biophys. 147, 129. Goodall, M. C. (1973). Arch. Eiochem. Eiophys. 157, 514. Goodall, M. C., and Urry, D. W. (1973). Eiochim. Biophys. Acta 291, 317. Harris, R. C., Jacobs, M. A., Long, M. M., and Urry, D. W. (1976). Anal. B i o c h a . 73,363. Heitz, F., Lotz, B., and Spach, G. (1975).J. Mol. Eiol. 92, 1. Hille, B. (1976). In “Dynamic Properties of Lipid Bilayers and Biological Membranes” (G. Eisenman, ed.), Membranes-A Series of Advances, Vol. 3, pp. 255-323. Dekker, New York. Hladky, S. B., and Haydon, D. A. (1970). Nature (London) 225, 451. Hodgkin, A. L. (1961). “The Conduction of the Nervous Impulse,” Sherrington Lectures, VII. Thomas, Springfield, Illinois. Keynes, R. D. (1976). In “The Structural Basis of Membrane Function” (Y. Hatefi and L. Djavadi-Ohaniance, eds.), pp. 33 1-338. Academic Press, New York. Kistenmacher, H., Popkie, H., and Clementi, E. (1973).J. Chem. Phys. 58, 1689. Kistenmacher, H., Popkie, H., and Clementi, E. (1974).J. Chem. Phys. 61, 799. Kolb, H.-A., Lauger, P., and Bamberg, E. (1975).5. Membr. Biol. 20, 133. Krasne, S., Eisenman, G., and Szabo, G. (1971). Science 174, 412. Lamb. T. D., and Simon, E. J. (1976a). J . Physiol. (London) 263, 257. Lamb, T. D., and Simon, E. J. (1976b). Proc. Physiol. SOC.July, 5-7. Liuger, P. (1972). Science 178, 24. Lotz, B., Colonna-Cesari, F., Heitz, F., and Spach, G . (1976).J. Mol. Eiol. 106, 915. Mueller, P. (1975). Ann. N.Y. Acad. Sci. 264, 247. Mueller, P., and Rudin, D. 0. (1967). Biochem. Eiophys. Res. Commun. 26, 398. Myers, V. B., and Haydon, D. A. (1972). Eiochim. Eiophys. Acta 274, 313. Neher, E., and Sakmann, B. (1976a).J. Physiol. (London) 258, 705. Neher, E., and Sakmann, B. (1976b). Nature (London) 260, 799. Noyes, R. M. (1962).J. Am. Chem. SOC.84, 513. Rosseinsky, D. R. (1965). C h a . Rev. 65, 467. Sachs, F., and Lecar, H. (1973). Nature (London), New Eiol. 546, 214. Sachs, F., and Lecar, H. (1977). Eiophys.J. 17, 169. Sandbloom, J., Eisenman, G . , and Neher, E. (1977).J. Membr. Eiol. 31, 383. Sarges, R., and Witkop, B. (1964).J. Am. Chem. SOC. 86, 1862. Simon, E. J., and Lamb, T. D. (1979).1n “Vertebrate Photoreception” (H. B. Barlow and P. Fatt, eds.), pp. 291-304. Academic Press, New York. Simon, E. J., Lamb, T. D., and Hodgkin, A. L. (1975). Nature (London) 256, 661. Skou, J. C. (1965). Physiol. Rev. 45, 596. Stevens, C. F. (1975). Cold S p ‘ n g Harbor Symp. Qwlnt. B i d . 40, 169-173. Stokes, R. H. (1964).J. Am. Chem. SOC. 86, 879. ~~
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NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR By Albert Wouquier Depahant of Pharmacology. k n r s e n Pharmaceutico.
8-1340kern. &lgium
.
I Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . General Methods . . . . . . . . . . . . . . . . . . . . . . . . . . A. Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Test Procedures and Control Results . . . . . . . . . . . . . . . . . A . Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . B. Control Results: Analysis of Variance . . . . . . . . . . . . . . . . IV . The Influence of Neuroleptics on Brain Self-stimulation Behavior . . . . A. General Description . . . . . . . . . . . . . . . . . . . . . . . . B. Dose-Effect Relationship . . . . . . . . . . . . . . . . . . . . . C. Potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D Control Base-line . . . . . . . . . . . . . . . . . . . . . . . . . E. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Implantation Site and Species Differences . . . . . . . . . . . . . . . A . Implantation Site . . . . . . . . . . . . . . . . . . . . . . . . . B. Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Self-stimulation and Psychotropic Assays . . . . . . . . . . . . . . . . A. Classification of Neuroleptics . . . . . . . . . . . . . . . . . . . . B. Positioning of Psychotropic Assays . . . . . . . . . . . . . . . . . VII . Studies on Drug Interaction . . . . . . . . . . . . . . . . . . . . . A . Literature on Neuroleptic-Anticholinergic Interaction . . . . . . . . B. Reversal of the Specific Inhibition Obtained with Anticholinergics . . C. Differential Antagonism . . . . . . . . . . . . . . . . . . . . . . D. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . A . Neuroleptic Impairment of Self-stimulation . . . . . . . . . . . . . B . Clinical Implications . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
335 337 337 337 338 339 339 340 349 351 358 358 359 362 365 365 367 372 373 377 379 379 380 384 388 391 391 395 398
.
1 Introduction
In 1954. Olds and Milner discovered that rats rapidly learned to press a lever which triggered electrical brain stimulation through a chronically implanted electrode . 335 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL . 21
Copyright 0 1979 by Academic Press. Inc. All rights o f reproduction in any form reserved. ISBN 0-12-966821-2
336
ALBERT WAUQUIER
The questions of which neuronal pathways are involved in selfstimulation behavior and what role they play in the different aspects of this behavior have been the subject of a large number of studies (Wauquier and Rolls, 1976). T h e reinforcing properties of electrical stimulation of particular neuronal pathways must in some way be related to the activation of these pathways, caused by the electrical stimulation. Pharmacological studies on self-stimulation and neurochemical identification of the pathways involved gave rise to the so-called catecholamine hypothesis of self-stimulation behavior (Crow, 1972; Dresse, 1966; Stein, 1968). According to this hypothesis, the effects of electrical self-stimulation are mediated by noradrenergic and dopaminergic neuronal pathways. It has been shown, however, that self-stimulation behavior can also be induced by applying electrical stimulation to non-aminergic structures (e.g., Liebman and Butcher, 1974). Moreover, there are controversies with regard to the functional role of the different aminergic structures in self-stimulation behavior. There are a variety of neuroleptics belonging to different chemical classes: the rauwolfia alkaloids (reserpine-like), the phenothiazines (chlorpromazine-like), the butyrophenones (haloperidol-like), and the diphenylbutylamines (pimozide-like). Particular attention has been directed towards neuroleptics that effectively control psychotic symptoms, reduce relapse, and enable patients to be reintegrated into society. Some neuroleptics are of benefit in neurotic or psychosomatic disorders; most of the neuroleptics, however, lack any beneficial effect on emotional disorders. Shortly after the discovery of self-stimulation, Olds and co-workers tested the influence of chlorpromazine and reserpine (e.g., Olds et al., 1957). These drugs were found to be powerful inhibitors of selfstimulation behavior. However, site-dependent effects were apparent and self-stimulation in the posterior hypothalamic area was more susceptible to the disruptive effects of these agents than the other sites tested. Dresse (1966, 1967) described the effects of larger groups of phenothiazines and butyrophenones and related the effects obtained on self-stimulation to a biochemical substrate. In recent years, some butyrophenones (haloperidol, spiroperidol) and the diphenylbutylamine pimozide have been tested more extensively, but the number of neuroleptics tested still remains small. In addition, no comprehensive data on the characteristics of the inhibition obtained with neuroleptics and, consequently, the nature of the inhibition, have been formulated.
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
337
II. General Methods
A.
SUBJECTS
Adult male Wistar rats from the Janssen breeding laboratory, weighing 250 & 10 gm at the time of surgery were used. They were transferred to the laboratory, in which environmental conditions were kept constant, i.e., temperature of 21" -+ 1"C, relative humidity of 65 5 5%. A normal light (12 hr)-dark (12 hr) cycle was used, and rats were tested during daytime (light period). The rats were kept in individual cages and provided with food and water ad libitum,except during the experimental sessions. When the oral route of drug administration was used, rats were deprived of food for a period of 24 hr prior to the experimental session. B. SURGERY Rats were anesthetized with Thalamonal' (0.5 mVkg s.c.) and positioned in a David Kopf Stereotaxic instrument with rat adaptor. The tooth bar was placed 5 mm above the ear bars. A sagittal incision was made on the skin of the skull, the membranes covering the skull were cut away, and the skull surface was made clean, so that the external landmarks became clearly visible. The bregma was taken as reference point and with the use of coordinates adapted from the stereotaxic atlas of Konig and Klippel(l963) the site at which the electrode had to be placed was marked. Four holes were then drilled in the skull using a dental drill: one where the electrode entered the brain and three others into which jeweller's screws were driven. One of the screws served as an indifferent electrode and was placed anterior to the bregma, the other two served as anchor points for the dental cement covering the contact points after the electrode had been put into position. Coordinates for electrode placement in the lateral hypothalamus of the MFB were: anterior 4.0 mm, lateral 1.4 mm and 3.0 mm above the zero stereotaxic point, according to the atlas; and actual coordinates: 1.2 mm posterior to the bregma, 1.4 mm lateral and 8.8 mm beneath the surface of the skull. All studies involved stimulation via a monopolar electrode. However, a bipolar electrode was implanted in such a way that two monopolar electrodes would be used in the same rat. The electrodes consisted of 0.254-mm nichrome wires twisted together, insulated at all points except for the cross section of the tip. Thalamonal? droperidol 2.5 mg and fentanyi citrate 0.0785 mg per ml.
338
ALBERT WAUQUIER
After implantation, rats were given 3 mg of nalorphine HCl i.v. in order to accelerate recovery from anesthesia. Thereafter, they were transferred to their home cage. The rats were allowed to recover for at least 1 week before experiments were begun. C. MATERIALS
Figure 1 is a schematic diagram of the general setup.
Slimulalor
P:
'
L=l
Stimulator
m
[11I1II17
m m m m unlcul
000 000 000
000 000 000
0 0 0 000 000
El Stimulator
N
w
w Recorder
Recorder
Recorder
FIG. 1. General setup.
Recorder
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
339
1. Cage The experimental compartment was a 20-cm wide, 25-cm long, and 33-cm deep PVC (polyvinylchloride) cage with a 6 x 3 cm stainless steel lever mounted in the back wall of the cage, 6 cm above the floor, that was made of stainless steel grid. Underneath was a funnel that collected feces and urine separately. T h e front wall of the cage was made of transparent Perspex, allowing direct observation of the rat. T o prevent twisting of the electrode leads, a modification of Berkley and Kling’s mercury contact swivel device with vertical movement compensation (Berkley and Kling, 1967) was constructed. 2. Stimulator and Recording Electrical stimulation was given by a stimulator of our own construction (Geivers et al., 1973, 1975) with constant current output from an integrated circuit. T h e rats could obtain electrical stimulation by pressing on a lever. The trains of biphasic rectangular pulses elicited were adjustable for constant current intensity, pulse frequency, pulse width, and train duration. Fixed ratio schedules (ratio of the number of stimulations to the number of responses) could be programmed; an inhibition circuit prevented the responses given during the stimulation train from being rewarded. T h e output of the stimulators was monitored on an oscilloscope, and the amplitude of the constant current measured across 1 Q. T h e voltage from oscilloscope readings over the current input provided the impedance. The number of responses (number of leverpressings) and the number of stimulations received were visualized by seven segment displays. The numeric indicators were driven via Binary Decimal Code (BDC) and converted in the interface unit to American Standard Code for Information Interchange (ASCII code). T h e information from the stimulators was changed in interface units from parallel to serial. The interface unit drove the teletype, which collected every minute the number of lever-pressings given and the number of stimuli applied. The numbers of responses and stimuli from each stimulator were translated via a digital converter to an analog voltage that controlled a pen recorder. The responses and stimuli were recorded separately. 111. Test Procedures and Control Results
A. TESTPROCEDURES In a quantitative approach to a particular type of behavior, drug effects are defined as facilitatory or inhibitory according to whether they
340
ALBERT WAUQUIER
increase or decrease base-line behavior. It is clear that the assessment of drug effects is partly dependent upon base-line response rate. For instance, high levels of base-line responding are not suitable for the investigation of facilitatory effects of drugs, because of the physical limits to the frequency of response. As we have previously demonstrated, electrical stimulation parameters also play an essential role in base-line responding. Accordingly, different base-lines resulting from different stimulus parameter combinations (SPCs) given during the same session, within the same rats, were studied. After self-stimulation had been established during pretraining, rats were selected and further trained on the schedule used for the drug experiments. The criterion for selection was: at least 300 responses per half hour during pretraining. The schedule used in training and during drug experiments was as follows: daily 1-hr sessions were held, 5 days a week. Each session consisted of six 10-min periods during which rats could obtain brain stimulation by pressing on a lever. The periods were separated by a 1-min “reset period” during which no brain stimulation was available. During each period, a different SPC was selected (based on the study of Wauquier et al., 1972).Of the six SPCs chosen, two elicited low response rates, two high response rates, and two intermediate response rates. A fixed-ratio schedule 2: 1 was used, i.e., two lever-pressings were needed to obtain one stimulation.*Lever-pressings during the stimulation were not rewarded. Table I depicts the different SPCs used. Two stimulators, differing slightly with respect to the available SPCs, were e m p l ~ y e dThe . ~ SPCs were given to groups of three rats in a partially randomized sequential order. The sequence of the SPCs is also given in Table I. The particular sequence given to a rat remained constant during the whole experiment. Training was continued until the rats had adapted to the schedule. Criteria for adaptation were: indications of differential responding on the SPCs and a total of approximately 900 or more responses on the highest SPCs. After training, which required 2 to 3 weeks, drug experiments were started.
B. CONTROL RESULTS: ANALYSIS OF VARIANCE The response rates obtained during the control session were determined by various factors. These factors, such as stimulus parameter This schedule was used in order to minimize the possibility of seizures occurring when rats get continuous brain stimulation. The use of two different types of stimulators is purely incidental: the second type of stimulator was developed at a later stage of the research.
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
341
TABLE I
(SPC)U S E D IN PRE-DRUG TRAINING EXPERIMENTAL SESSIONS A N D THE SEQUENCE I N WHICHTHEY WEREGIVEN
SIX SELECTED
AND
STIMULUS PARAMITERS
SPC stimulator 1 a Quantity of charge Intensity Frequency Pulse width Train duration Sequence C 1 c2 c3
(&)
(PA) (PPS) (msec) (msec)
COMBINATIONS
1 LIC
2 L2
3 MI
MI
5 H1
HI
12.5 250 20 5 500 4 6 5
16.5 66 5 500 1 3 2
20.0 250 32 5 500 5 4 6
24.8 150 66 5 500 2 1 3
31.8 250 50 5 500 6 5 4
40.0 200 80 5 500 3 2 1
8 LZ
9
10
11
12
MI
M2
HI
H2
12 100 60 4 500 1 3 2
18 150 60 4 500 2 1 3
20 250 40 4 500 5 4 6
30 250 60 4 500 6 5 4
32 200 80 4 500 3 2 1
7 SPC stimulator 2* Quantity of charge Intensity Frequency Pulse width Train duration Sequence C 1 c2 c3
(&)
(PA) (PPS) (msec) (msec)
Ll 10
250 20 4 500 4 6 5
100
4
6
a This schedule was used in order to minimize the Occurrence of seizures when rats get continuous brain stimulation. The use of two different types of stimulators is purely incidental: the second type of stimulator was developed at a later stage of the research. L: low, M: intermediate, H: high.
combinations, subjects, and time of experiment, were manipulated during control sessions. An adequate analysis of the control data therefore required a factorial approach. An analysis of variance was used to evaluate the data.
1 . Dejinition of the Variables Table I1 depicts schematically the different factors involved. A first variable is the stimulator, further symbolized as treatment A. This treatment has two levels (A, and A*),i.e., two different stimulators were used. A second variable is the various stimulus parameter combinations (SPCs) (Table I), and is further symbolized as treatment B. T h e effects of this treatment are restricted to a single level of A, i.e., B is nested within A (B IIA). T h e reason for this is that the two stimulators differed slightly as regards the possibilities for selecting SPCs (see Table I). In Table 11, stimulator 1 corresponds to Al and the SPCs 1 to 6 to B1 to B,; stimulator
TABLE I1 SCHEMATIC REPRESENTATION OF THE DIFFERENT TREATMENTS AND THE REPARTITION OF
THE SUBJECTS WITHIN THE
A,
TREATMENT COMBINATIONS
Az
CI
b,.
. . b j . . . b.
.
b i . . b j . . .be
b,.
. .b l . . .b.
b 7 . . . b,.
. , b,
b7.
. . bj . . . biz
b,.
. . b j . . . b,
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
343
2 corresponds to A, and the corresponding SPCs to B, to BIZ.For both stimulators, two low SPCs (L, and L2), two intermediate SPCs (MI and M2), and two high SPCs (HI and H,) can be distinguished (see Table I). A third variable is the sequence of the SPCs, further symbolized as treatment C (Table I) (C," A). The fourth variable is the time factor, further symbolized as treatment D. D has five levels, i.e., the first 2 weeks (D1 and D,) after termination of the training period, 1 week in the middle of the experimental period (D3),and the last 2 weeks of the experimental period (D., and D5). Thus, the selected rats were tested for at least 5 weeks. The fifth variable is the subjects, and is further symbolized by S. The subjects are nested within a treatment combination of A and C (S n AC). For each AC combination, 14 subjects were used, i.e., a total of 84 rats.
2 . Design This is a description of the structural model of the design, the expected values of mean squares, and the choice of an adequate error term. The treatments A, B, C, and D have fixed effects, i.e., all treatment levels about which inferences had to be drawn were included in the experiment. The subjects (S) were randomly selected from the same population and after implantation randomly assigned to the different treatments (C). It follows that the factorial design used is a mixed model (Model 111) (see Kirk, 1968; Huitson, 1966), with both crossed and nested treatments. The following notation rules are used: (1) Fixed effects are designated by Greek letters.
(2)Random effects are designated by Roman letters. (3) Nested effects are indicated by adding the subscripts between brackets to the symbols referring to the particular level of the treatment(s) within which they are nested. The effects of the levels of the different treatments are designated in the accompanying tabulation.
A: B:
c: D:
s:
a,
ai
aP
PIC0
Prca
Pdi)
YlW
YkW
YHi)
6,
6,
6,
Sl(ik)
Sm(ik)
Ss(ik)
(p = 2) (q = 6 ) (r = 3 ) (t = 5 ) (s = 14)
(fixed crossed effects) (fixed nested effects) (fixed nested effects) (fixed crossed effects) (random nested effects)
LetXijkumQ be a measure for a randomly selected observation ( ( 6 a )), in a treatment population ABCDSUbm. Under the mixed-effects model, it is assumed that measurement XijkumQ is equal to the following terms:
344
ALBERT WAUQUIER
-k ai -k Pj + Y k ( f ) -k a u -k smlik) -k a a i u -k P Y j k ( f ) -k PajjuCf) -k @jrn(fk) -k -k PYSJkM) -k P h u m t i k )
XUkuma =
yaku(f)
-k
&um(fk)
+ Ea(wm) where p is the grand mean for treatment populations at,
PJ, Y k ( f h and Sm(ik):
e f f e c t s of the different treatments; aSfU,
h J k ( i h PaJu(i), f i J m ( f k ) , Y a k u ( i ) , &um(fk)t
and
P Y a J k u ( f ) tP&Jum(fk)
-effects that represent nonadditivity of the stated effects (respectively, first- and second-order interactions); &(Ukum)
-experimental error, which is normally and independently distributed with mean = 0 and variance = (within cell error term). The expected values of the mean squares [E(MS)I are determined according to the general rules described by Kirk (1968) and are given in Table 111. However, in this design only one observation is available for each treatment combination (1 . . . a . . . n :N = 1). For such an experiment, a mean square within-cell (ea(ifkum)) term cannot be computed. There~
TABLE I11 THEEXPECTEDVALUE^ OF MEANSQUARES [E(MS)], D ~ R M I N ACCORDING ED TO THE GENERAL RULESDESCRIBED BY KIRK(1968)
Effects
E(MS)
Degrees of freedom
Error term n>l
Error term n = l
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
345
fore, the pooled highest-order interaction($, instead of the within-cell error term, is used as an estimate of the experimental error. 3. Fundamental Assumptions
Cochran and Cox (1957) stated that failure to meet the fundamental assumptions effects both the significance level (in both directions) and the sensitivity of the test. However, the F-distribution is very robust with respect to violation of these assumptions. Cochran (1947) stated that it is impossible to be certain that all required assumptions are satisfied. Analysis of variance must therefore be regarded as approximate rather than exact (Kirk, 1968). One of the requirements for a ratio of variances in order to obey the F-distribution is that the numerator and denominator of the ratio are independent. If scores are randomly sampled from a normal population, this requirement is satisfied. The errors also have to be normally distributed for each treatment population. Because the only source of variation within a treatment population are the errors, the above-mentioned assumption is equivalent to the assumption of normally distributed scores. Fortunately, the F-distribution is relatively unaffected by lack of symmetry or by kurtosis, provided the populations are homogeneous in form (Lindquist, 1953). The F-distribution is robust with respect to violation of the assumptions of homogeneity of population-error variances, provided the number of observations in the samples is equal (Cochran, 1947; Lindquist, 1953). A further basic assumption of the statistical model used to analyze the present data is that a score is thesum of the effects of the linear model. A particular situation occurs when the number of within-cell observations is 1, as is the case in the present model. As pointed out, the higher-order interactions are considered to be zero. Thus the corresponding E(MS) of these terms yields an estimate of experimental error. This means that a " p r s and cr2py8 are supposed to be zero. If this is true, the E(MS) for effects (12) and (13) (see Table 111) are both estimates of aZe. The mean square for the experimental error term is calculated by pooling the SS of (12) and (13), i.e., MS (12 13) = "l2 ss13 = MS residual
+
+
y12
+ y-I3
The basic question to be answered is: Are the higher-order interactions equal to zero? Some a posteriori points concerning this assumption are discussed in the next section. 4. Results
The results of the analysis of variance are shown in Table IV. The following factors were significant: stimulus parameter combinations (B)
346
ALBERT WAUQUIER
TABLE IV ANALYSIS OF VARIANCE TABLE Source of variation
Sum of squares
(1) A (2) B (within A) (3) C (within A)
632,922.210 3 10,011,501.391 8,191,105.027 2,038,725.140 68,246,111.355 254,704.844 8,538,556.925 1,444,287.263 92,707,544.317 1,051,850.902 29,808,017.846 48,075.63 1.605 57 1.000.958.825
(4) D
(5) S (within AC) (6) A X D (7) B X C (within A) (8) B x D (within A) (9) B X S (within AC) (10) C x D (within A) (11) D X S (within AC) (12) Residual Total
df
F
Mean squares
1 632.922.210 10 31,001,150.139 4 2,047,776.257 4 509,681.285 78 874,950.146 4 63,676.21 1 20 426,927.846 40 36,107.182 390 237,711.652 16 65,740.681 312 95,538.519 1640 29,314.410 25 19 -
(115) 0.723 (219) 130.415" (315) 2.340 (411 1) 5.335" (5112) 29.847" (6111) 0.667 (7/9) 1.796b (8112) 1.232 (9112) 8.109' (10111) 0.688 (11/12) 3.259"
'p f 0.01. bp G 0.05.
(p < O.Ol),time (D)(p < 0.01), and subjects (S) (p < 0.01).The following interactions were significant: stimulus parameter combinations x sequence (B x C) (p < 0.05), stimulus parameter combinations x subjects (B X S) ( p < 0.01), and time X subjects (D X S) (p < 0.01). In the present table of variance, the MS residual was found by pooling the variance of the second-order interactions B X C X D (within A) and B X D X S (within AC) (respectively, effects 12 and 13 in Table 111). If the MSs are calculated for these interactions, we then find: 27382.6488 for BCD (within A) (effect 12) and, 29413.4742 for BDS (within AC) (effect 13). The E(MS) for these effects are, respectively, (12) a:
(13)
+ nf3pss + S n d g y 8 + nu2@a8
If the interaction term 02p8s is equal to zero, the F-ratio of effect 12 to effect 13 should be significant. By contrast, the F-ratio is even slightly smaller than 1, proving that the assumption should not be rejected. From the present design, no such exact test can be derived for the assumption that cr2p& is equal to zero. If uppcis not equal to zero, the error variance will be overestimated in the denominator will be negatively biased. and an F-test involving u2pss On the other hand, if a significant F-ratio is obtained, one can be confident that there are real treatment differences. This is exactly the
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
347
case in the present analysis of variance. All effects tested against MS residual are significant atp < 0.01. There is only one exception, i.e., the B X D (within A) interaction. However, in this test, no negative bias can appears both in the numerator and in the denominator of occur, as uzpSs the F-ratio [see Table 111: E(MS)]. 5 . Discussion
The factor B, effects of the SPCs, was found significant (p < 0.01). Figure 2 shows the mean response rates per SPC for stimulator 1 and 2. In general, the higher the quantity of charge of the SPC, the higher the response rates (see also Wauquier el al., 1972). The response rates obtained on stimulator 1 are slightly higher than those obtained on stimulator 2. Although not significant (see factor A), this difference could depend on the higher quantity of charge of the SPCs of the first stimulator (see Table I). There is a tendency to obtain higher total response rates with sequences starting with a low SPC (C, and C,) and the lowest total response rate with those starting with high SPCs (C3 and c6) (see Fig. 3). However, the difference is not significant. The interaction B x C was significant ( p < 0.05) and is illustrated in Fig. 4. This clearly shows that the effects of factor B (SPCs) will differ according to the sequence in which they are tested. The most marked differences occurring with both stimulators are the higher response rates for B2 and BB with sequence C, and C4, respectively, and for B5 and B1, with sequence C3 and (26, respectively. Higher or lower responding caused by reward shifts are described in the literature (Panksepp and Trowill, 1969). The reinforcing value of the different SPCs may depend on the sequence in which they are tested and actually produce higher or lower responding. However, the present shifts cannot clearly be interpreted because all possible sequences of the SPCs were not tested. The time factor (D) was significant (p < 0.01). As seen in Fig. 5 , the further response rates increased slightly from the first to the second week of testing, and subsequently decreased over the following weeks. It is of interest to note that the analysis was done on rats that had been tested for 5 weeks, as well as on rats that had been tested for longer periods. The latter increases the possibility of response decline, so that the actual results may underestimate the response decrease as a function of time. Two explanations are possible for the progressive decrease in responding as a function of time. The first explanation refers to the fact that the rats received drug treatment between control sessions. Drug treatment can cause shifts in control values, which outlast direct drug effects. For example, chlordiazepoxide markedly enhanced responding
348
ALBERT WAUQUIER
1000
900
800
700
60C VI
50[ 0
n VI
u K
L 00
3 00
200
100
0
FIG. 2. Mean response rates per SPC (L: low;M: intermediate; H: high; see details in Table I) for stimulators 1 and 2.
in tests for some days following drug injection (Wauquier, 1974). A second explanation for the decline in responding as a function of time may be that prolonged electrical stimulation induces lesions in the brain structures stimulated. The subjects factor (S) was found significant ( p < 0.01). The total response rates emitted by the subjects varied over a large scale. The variability in sensitivity to brain stimulation has been described previously (see, e.g., Wauquier e6 al., 1972).
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
349
ml
g2000
2! 1000
OJ
1
I
1
2
3
Sequence
FIG. 3. Total response rates (sum of six SPCs) as a function of the sequence of the SPCs: Sequence 1: low SPC (Cl, C4);sequence 2: intermediate SPC (C2, C3); sequence 3: high SPC (Cs, C8), given at the start of the session.
The significant interaction B x S (SPCs x subjects) (p < 0.01) pointed to the fact that, for individual rats, there are qualitatively different relationships between the quantities of charge (SPCs) and the response rates. For instance, some rats exhibited no further increase in, or even decreased, responding at the highest SPCs either because the limits of performance were reached or because the stimulation became aversive. Furthermore, large differences with respect to the evolution of responsiveness as a function of time were observed for individual rats. Consequently, the interaction D x S (time x subjects) was found significant ( p < 0.01). In conclusion, because of the significant effects of the factors B (SPCs), D (time), and S (subjects), and the significant interactions in which these factors are involved, it is absolutely necessary to relate data involving drug effects to control data obtained (a) in the same rats, (b) for the same SPCs, and (c) during the same week.
IV. The Influence of Neuroleptics on Brain Self-stimulation Behavior
In all, 20 neuroleptics belonging to several chemical groups (Fig. 6) were investigated with respect to their effects on self-stimulation behavior. Nineteen neuroleptics were tested on groups of six rats and one neuroleptic (droperidol) on a group of three rats. Four doses of each compound were given, the lowest during the first week and progressively higher doses in subsequent weeks. The neuroleptics were administered either subcutaneously or orally, with one exception: pimozide was administered orally to a group of six rats and subcutaneously to another group of six rats. Saline was given on Tuesdays and drugs on Thursdays.
ALBERT WAUQUIER 1100-
1000-
900-
800-
700-
;600n
c 0
a
:500-
K
400-
300-
200-
100-
0-
FIG. 4. Interaction B (SPC) X C (sequence). Mean response rate as a function of stimulator (Al, A*), SPCs (B, . , B6 and B, , B12),and sequence (C, . , , c6).
.
..
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
35 1
40007
f
0’
r D1 first
I
02 second
I
I
1
03
Dl
D5
medium
forelast
FIG. 5. Total response rates as a function of time (D,
last week
. . . . . Ds).
The long-acting neuroleptics clopimozide, penfluridol, and pimozide (orally), however, were given on Monday sessions. Saline sessions preceded successive drug treatments, and recovery to control levels occurred within 1 or 2 weeks after each dose of drug. The figures represent the response rates obtained with all rats at each SPC and after each dose of a neuroleptic during the drug session (fourth day of the week, except for the long-acting neuroleptics clopimozide, penfluridol, and pimozide, given orally, for which the first day was taken). They are expressed as percentages of the response rates obtained during the control session (second day of the week, and fifth day for clopimozide, penfluridol, and pimozide). Significant ( p d 0.05) differences are indicated by an asterisk (Wilcoxon matched-pairs, signedranks test, one-tailed probability). Additional statistical tests are described in subsequent sections. The effects of the neuroleptics described have been reported very briefly by Wauquier (1976),and particular neuroleptics have been described more extensively by Janssen et al. ( 1 975), Wauquier and Niemegeers (1972, 1975, 1976a,b).Because the profiles of the neuroleptics tested were quite similar, it was not considered necessary to discuss each one separately.
A. GENERAL DESCRIPTION Figure 6(a)-(f) shows the percentage of lever-pressing as compared with the control response rates, obtained with each of four doses of the 20 neuroleptics and at each of the six SPCs. All neuroleptics caused pronounced response inhibition; however, a slight response enhancement was seen with a few drugs at some doses.
I TRICYCLIC NEUROLE?TlCS
.u
10
a PHINOTRAZING CHLORPROHAZIN~
in1 foul. llm. 01 In1 rals
n
m Dl6
sc
- Ih, 6
m 031
THlORIOAZINE
~~
@ 6.7-6
RlNO SYSTEMS CLOTHIAPINE
m a04
m am
CLOZAPINE
m a6a
m as
FIG. 6(a) FIG. 6. (a)-(f) Self-stimulation response rates in relation to control response rates, obtained with each of the four doses of 20 neuroleptics on each of the six SPCs. Asterisks indicate significantdifference (p 0.05) as compared to control (for details see Section 11). 352
1 BUIVRO~HENONES
0 1-PHENVL-~lPEltlOlNO HALOPERlOOL IR 16151
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is"
-50-
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_
_
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I23156 0.01
1 2 1 4 5 6 0.02
1 2 3 1 5 6 0.01
src DOSE
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0-10-
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-30-10-
-50-
-60
-
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1 2 3 1 5 6 0.06
0.01
FIG. 6(b). See legend on p. 352. 353
src
now
m
DIPHENVLBUIVLAMINES
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357
358
ALBERT WAUQUIER
The total response inhibition (effects on the 1-hr session, i.e., sum of the six (SPCs) ranged from 24% (thioridazine) to 65% (pimozide, given orally). The inhibition was dose-related, ranging from a dose which was almost ineffective, to a dose causing almost complete suppression of self-stimulation. It appears that the response inhibition is inversely related to the base-line of responding.
B. DOSE-EFFECT RELATIONSHIP Figure 6(a)-(f) also shows the dose-effect relationship. The Wilcoxon matched-pairs, signed-ranks test, one-tailed probability, was used as a test for the significance of differences between drug and control response rates. Significant effects (p s 0.05) are indicated by an asterisk. All neuroleptics tested inhibited self-stimulation in a dose-dependent way. The dose-dependent decrease of self-stimulation obtained after some of the neuroleptics described here has been observed in different situations and with different electrode localizations (Dresse, 1967; Fibiger et al., 1975, 1976; Kadzielawa, 1973; Olds and Travis, 1959, 1960; Phillips et al., 1975, 1976; RolIs et al., 1974b; Stark et al., 1969). The inhibition, however, is clearly base-line dependent.
C. POTENCY Although there are great differences between the neuroleptics with respect to chemical structure, as well as biochemical, behavioral, and clinical effects, they all decrease self-stimulation in a dose-dependent way. There are, however, considerable differences with respect to potency. In order to estimate quantitative differences between neuroleptics, EDm values with confidence limits and potency ratios were calculated, as described in the following paragraphs. The median effective dose, often referred to as EDs0,is a term used to characterize the potency of a treatment by reference to the amount (dose of a drug) that produces a response in 50% of the cases. In practice: after defining a criterion (for our data: =Z 79%, which corresponds to the 0.05 probability level of distribution of the control values), one obtains data of the all-or-none type. Therefore, it is possible to solve a dose-percent curve, by which doses are converted to logarithms and percent effect to probits (Logarithmic-probability paper enables one to plot the data in original units, but leaves one with the problem of converting log-probit equations to their arithmetic equivalent). After the data have been plotted, a straight line is fitted through these points (for instance, percentage lever-pressing rate as compared with control vs.
NEUROLEPTICS A N D BRAIN SELF-STIMULATION BEHAVIOR
359
doses of the neuroleptic). A goodness-of-fit test is applied by performing a chi-square test (expected against observed values). If the test is acceptable, the EDB0value can be read off directly on the intersection of the plotted line and the 50% line on the ordinate. The confidence limits represent the values between which the EDBois supposed to lie. The width of these limits is influenced by the heterogeneity of the data besides the slope of the curves. If this heterogeneity proves to be significant, a correction for significant heterogeneous data is performed. Additional tests can be carried out if it proves necessary to compare two or more drugs, i.e., a test for parallelism (slopes) and the estimate of the relative potency. If no significant deviation of parallelism can be found, tests to detect significant differences in potency can be carried out and potency ratios with confidence limits may be calculated. For a detailed description of the procedures just described, see Litchfield and Wilcoxon (1949). The results of the tests are given in Table V. Table V gives the EDB0 values of the neuroleptics, slopes, slope function, potencies, and potency ratios of the neuroleptics as compared with those of the most potent compound, fluspiperone. The ED50 of pimozide (orally given) could not be calculated, since three out of six rats were already found positive at the lowest dose tested. Comparison with fluspiperone was made since no significant differences from parallelism were found with the most potent neuroleptic. As seen, thioridazine, the weakest neuroleptic, is 659 times less potent than fluspiperone. The inhibition obtained with fluspiperone, spiperone, benperidol, bromperidol, haloperidol, or droperidol does not differ significantly from one substance to another.
D. CONTROL BASE-LINE It appears from Fig. 6(a)-(f) that there is an inverse relationship between base-line responding (response, rates increase from SPC 1 to SPC 6) and neuroleptic-induced inhibition, i.e., response inhibition is more pronounced at the low SPCs than at the high SPCs. This relationship was statistically substantiated by the Friedman two-way analysis of variance (see detailed description in Siegel, 1956, pp. 166-172) (Fig. 7). Basically, the test applied used the rank-order of the percentage inhibition obtained at the 6 SPCs with the four doses of the 20 neuroleptics. The analysis was made as follows: The percentage inhibition obtained with the first dose of a neuroleptic, at SPC 1, SPC 2 . . . SPC 6, is given a rank-order (column); this is repeated for each of the 20 neuroleptics tested (rows). For example, see the tabulation at the bottom of p. 361.
TABLE V EDso VALUES WITH CONFIDENCE LIMITS,SLOPES,SLOPERATIOS,AND POTENCY RATIOS OBTAINED WITH 20 NEUROLEPTICS TESTED O N BRAINSELF-STIMULATION ~
Compound Fluspiperone Spiperone Benperidol Bromperidol Haloperidol Droperidol Moperone Oxiperomide Clothiapine Fluanisone Pimozide or. Pimozide S.C. Butaclamol Clopimozide Chlorpromazine Metoclopramide Azaperone Penfluridol Pipamperone Clozapine Thioridazine
EDm (limits)
Slope
Slope ratio (limits)
Potency ratio (limits)
0.0182 (0.0111-0.0299) 0.0185 (0.0106-0.0323) 0.0200 (0.0148-0.0270) 0.0200 (0.0124-0.0322) 0.02 18 (0.0125-0.0380) 0.0285 (0.0193-0.0422) 0.0330 (0.0238-0.0457) 0.0569 (0.0374-0.0867) 0.0970 (0.066-0.143) 0.133 (0.073-0.243) (-) s0.160 0.160 (0.101-0.253) 0.270 (0.144-0.505) 0.340 (0.207-0.560) 0.5 13 (0.340-0.774) 0.520 (0.180-1.50) 0.534 (0.193-1.48) 1.36 (0.806-2.30) 6.35 (2.85-14.1) 7.84 (2.32-26.5) 12.0 (4.17-34.5)
1.859 2.008 1.305 2.075 1.635 1.414 1.334 1.692 1.405 2.523
1.o 1.08 (0.53-2.20) 1.42 (0.90-2.25) 1.12 (0.57-2.17) 1.14 (0.63-2.05) 1.31 (0.82-2.1 1) 1.39 (0.88-2.21) 1.10 (0.64-1.90) 1.32 (0.82-2.14) 1.36 (0.54-3.42) 1.08 (0.57-2.05) 1.18 (0.51-2.71) 1.00 (0.53-1.88) 1.11 (0.65-1.90) 2.72 (0.73-10.1) 1.32 (0.60-2.93) 1.03 (0.53-2.00) 1.46 (0.71-3.02) 2.47 (0.61-10.0) 1.37 (0.59-3.15)
1.o 1.02 (0.48-2.14) 1.10 (0.62-1.96) 1.10 (0.55-2.19) 1.20 (0.57-2.52) 1.57 (0.83-2.95) 1.81 (1.OO-3.28)' 3.13 (1.63-5.99)' 5.33 (2.84-9.99)" 7.31 (3.34-16.0)"
~
~~
'Significantly different from fluspiperone ( p < 0.05).
-
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361
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
I Ranks 120100-
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3rd dose
SPCl
SPC2
SPC3
SPC4
SPC5
SPC6
fluspiperone (7% inhibition): (rank-order):
-84.7 1
-33.2 2
-
8.5 6
-28.3 3
-19.5 4
-
9.4 5
spiperone (% inhibition): (rank-order):
-81.6 1
-51.9 2
- 5.9 5
-26.5 3
- 8.1 4
-
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-50.5
-18.8 4
-27.9 3
-48.2 2
- 9.6 6
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... thioridazine (% inhibition): (rank-order):
1
362
ALBERT WAUQUIER
Then, the sum of ranks is made and, finally, x? is calculated according to the formula described by Siege1 (1956, p. 168). T h e same is repeated
for the second, third, and fourth dose and for the sum of all doses. If there is no preference for one of the conditions (SPC effect), then one finds a nearly equal sum of ranks; if not, one can expect a systematic effect. It was found that, with each of the doses tested, there is a systematic relationship between SPC and percentage inhibition, i.e., high inhibition (low sum of ranks) for low SPCs and vice versa (p < 0.001). L
E. DISCUSSION All neuroleptics tested induced a dose-related inhibition of selfstimulation. T h e dose-response curves were quite similar, but the neuroleptics differed largely with respect to potency. However, as described in Section VI, neuroleptics also vary with respect to their effects on DA and NA. Although self-stimulation was nearly completely suppressed by the highest dose of the neuroleptics tested, rats tended to self-stimulate at normal or higher rates than control animals during the first minutes of the session. Figure 8 illustrates such an observation (similar observations were reported by Liebman and Butcher, 1974; Rolls et al., 197413). This was confirmed by Fouriezos and Wise (1976) and discussed as follows. T h e temporal pattern of responding suggests that cessation of responding occurs in a manner similar to extinction. If neuroleptics block the motor system, one would not expect the rats to start self-stimulating. The latter observation not only suggests that the ability to press a lever is intact, but also that the anticipation of brain-stimulation reward may be unaffected. T h e cues associated with self-stimulation (conditioned motivation, i.e., smell, sight of the lever, etc,) are still present and induce the rats to start lever-pressing. T h e lack of sustained responding is not related to drug onset, because lever-pressing occurs whenever the rats are put in the self-stimulation cage after drug injection (Fouriezos and Wise, 1976). This observation suggested that the reinforcing value of brain-stimulation is lowered or eliminated after high doses, just as it is in extinction. The extinction effect can alternatively be interpreted in terms of increased motor fatigue (Fibiger, 1977): neuroleptic-treated rats are able to start responding but are unable to maintain the behavior, except when sufficiently aroused. Lowering of the reinforcing value of brain stimulation is suggested by the fact that chlorpromazine increased the threshold for brain stimulation (Stein, 1962), but this may be conceived as an indirect effect.
NEUROLEPTICS A N D BRAIN SELF-STIMULATION BEHAVIOR
363
g LOO-
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FIG. 8. Self-stimulationresponse rates of six rats obtained after the the S.C. injection of saline (0-0) or haloperidol (0.08 nig/kg ( 0 3 )and during extinction ( 0 4 )(no brain stimulation available).
364
ALBERT WAUQUIER
Colpaert et al. (1977) showed that haloperidol blocked the discriminative stimulus properties produced by electrical stimulation of the lateral hypothalamus. An interruption of the response-stimulus contingency predicts an extinction pattern as described above. Further to this, Colpaert ( 1977) reported that haloperidol increased the detection threshold of this electrical stimulation. T h e ultimate effect of altering the stimulus control of behavior is a decrease in the reinforcing value of the stimulus. It is expected that the inhibition obtained with neuroleptics is inversely related to the strength of the stimulation. Indeed, response inhibition was more pronounced at the low SPCs than at the high ones. Similar observations, using a different method, were reported for pimozide by Liebman and Butcher (1973), who showed that pimozide (0.35 and 0.5 mg/kg, given i.p., 3 hr before the test) reduced self-stimulation for current intensities yielding from 50 to 75% of maximal rate. When the current was doubled, lever-pressing increased to the base-line level. That base-line rates of responding determine the sensitivity of the animal to the suppressive effects of neuroleptics was also demonstrated in an experiment by Fibiger et al. (1976). Lever-pressing for food or brain stimulation was affected to a similar extent by haloperidol, provided that the rates of responding did not differ significantly for the two reinforcers. The differential inhibition suggests that the neuroleptic inhibitory property depends on the strength of the stimulation. Within a given structure, different response rates are obtained by varying the SPCs and possibly reflect a different rewarding or discriminating level. Rats do not work continuously for a low-intensity or a low-frequency stimulation, and the response rates are often erratic. Consequently, the expectancy is low and the habit strength less than when rats work for higher intensities or frequencies of stimulation. In the latter case, the expectancy of reward is stronger. In order to answer the question of whether neuroleptics also affect reward, some authors used a “rate-free” test of self-stimulation, which does not require lever-pressing (Valenstein and Meyers, 1964). In short, the apparatus used is a ,shuttle box with a tilt platform. Moving the animal to one part of the cage closes a microswitch that activates programming equipment, so that the rat automatically obtains brain stimulation. The time spent in the “active part” of the cage gives an indication of “self-stimulation.” The programming is such that the rat has to move only a few times during an experimental session. In the rate-free situation used by Liebman and Butcher (1974), the gross locomotor activity was also measured. An increase in motor activity does not necessarily increase the time of stimulation. The authors
NEUROLEPTICS A N D BRAIN SELF-STIMULATION
BEHAVIOR
365
showed, conversely, that a decrease in activity does not necessarily result in a reduction of brain stimulation. Apomorphine, for instance, increased self-stimulation, despite a decreased locomotor activity. Liebman and Butcher (1974) also showed that pimozide (0.35 mg/kg and 0.5 mg/kg) reduced self-stimulation as measured in this rate-free situation, with rats bearing electrodes in the substantia nigra and lateral hypothalamus. Because this rate-free test probably constitutes a more valid measure of the rewarding value of the stimulation, the authors concluded that pimozide reduced self-stimulation by interfering with the reward. However, the fact that pimozide also significantly decreased the number of crossings and locomotor activity does suggest that the inhibition might be due to a failure to maintain behavioral output. Moreover, Atrens et al. (1976) reported that haloperidol increased the latency to initiate self-stimulation and escape for brain stimulation, both in a ratefree situation. This suggested to them a “non-specific decrement in performance.” In addition, neuroleptics not only inhibit positively motivated behavior but have the same effect on negatively motivated behavior. Doses almost identical to those inhibiting self-stimulation also inhibit Sidman shock-avoidance (see Table VI). In conclusion, in the self-stimulation paradigm where response rates are the dependent variable, neuroleptics inhibit the behavior by affecting both the response output (motor fatigue, maintenance of behavior) and the stimulus control produced by electrical stimulation. This results in a disruption of the response-stimulus contingency through which the stimulation itself loses its capability to reinforce behavior.
V. Implantation Site and Species Differences
A. IMPLANTATION SITE Most studies have concentrated on self-stimulation in the lateral hypothalamic region. One would expect that a specific dopaminergic blocking neuroleptic would have little or no effect on self-stimulation maintained by an electrode implanted in purely noradrenergic cell bodies, the underlying assumption being that a purely dopaminergic neuroleptic selectively disrupts DA-mediated systems. Such an assumption was apparently validated by Ritter, and Stein (1973), who injected 1 mg/kg pimozide S.C. as an aqueous suspension and found a differential inhibition of self-stimulation in the locus coeruleus (89.7%of control) and the medial forebrain bundle (69.8% of control). Liebman and Butcher (1974), also working with pimozide, stated that 0.5 mg/kg inhib-
366
ALBERT WAUQUIER
TABLE VI OF VARIOUS NEUROLEPTICSTO EDw VALUES (mglkg) AFTER ADMINISTRATION BRAINSELF-STIMULATING (BS) RATS,AND AFTER THE FOLLOWING TESTS: AMPHETAMINE ANTAGONISM (AM), NOREPINEPHRINE ANTAGONISM (NE), CATALEPSY (CA), PALPEBRAL (AMB), A N D REARING (REA) PTOSIS(PP), SIDMAN SHOCKAVOIDANCE (SA), AMBULATION Tests
Compound Generic name
BS
AM
NE
CA
PP
SA
1 Azaperone 2 Benperidol 3 Bromperidol 4 Butaclamol" 5 Chlorpromazine 6 Clopimozidead 7 Clothiapine 8 Clozapine" 9 Droperidol 10 Fluanisone 11 Fluspiperone 12 Haloperidol 13 Metoclopramide 14 Moperone 15 Oxiperomide 16 PenfluridoP" 17 Pimozide" 18 Pipamperone 19 Spiperone 20 Thioridazine
0.534 0.020 0.020 0.270 0.5 13 0.340 0.097 7.84 0.029 0.133 0.018 0.022 0.520 0.033 0.057 1.36 0.160 6.35 0.019 12.0
2.5 0.012 0.053 0.31 0.60 0.085 0.20 20 0.023 0.20 0.020 0.038 5.0 0.03 0.03 0.3 1 0.1 2.5 0.02 9.12
0.33 0.30 7.24 >10 0.60 5.0 0.89 4.0 0.1 0.1
8.0 0.18 0.12
1.5 1.2 1.7
0.015 0.045
2.3 0.50
2.3 5.0
1.2
-
0.2
-
2.1
>I60 1.5 5 10 40 3.0 1.2 1.o
-
0.38 2.0 0.18 19 1.2 >40 1.2 0.18 16.5 0.036 14
-
0.38 0.65
-
-
-
0.025 0.12
-
0.03 1.0 32 2.3 0.04 2.5 36 >0.63 4.5 0.16 2.7 5 0.27 0.015 2.7 5
-
" Given orally. T h e EDw values of the long-acting neuroleptics penfluridol and clopimozide were obtained 4 hr after injection.
ited self-stimulation in the substantia nigra slightly more than in the lateral hypothalamus. Site-related effects have also been described with other neuroleptics. Rolls et al., ( 1974a) examined the inhibitory effects of spiroperidol on stimulation in the nucleus accumbens, the septa1 region, the hippocampus, the anterior hypothalamus, the lateral hypothalamus, and the ventral tegmental area. Although the inhibition was related to base-line, further site-related differences were apparent. For example, the response rates obtained with spiroperidol, 0.05 mg/kg, were approximately 40%, 20%, 0%, 60%, 50%, and 70% of control values for the afore-mentioned structures. Similarly, Mora et al. ( 1976), also working with spiroperidol, reported a site-related effect on self-stimulation,after intracerebral injection in the monkey. In this work again the locus
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
367
coeruleus appeared less affected by spiroperidol than other areas. Mora et al. (1975) also stated that spiroperidol reduced responding more for lateral hypothalamic stimulation when animals were injected in the nucleus accumbens than in the globus pallidus. Stark et al. (1969), working with chlorpromazine, were able to show that posterior hypothalamic self-stimulation was more affected by a low dose than the anterior hypothalamic or septal areas. Olds and Travis (1959) and Olds et al. (1957) described anatomically related differences with respect to chlorpromazine. They noted that chlorpromazine strongly inhibited self-stimulation in the ventral posterior hypothalamus, was less effective in the septal region, and had negligible effect in the anterior hypothalamus. There are also a number of articles in which no site-related effects were found. Phillips et al. ( 1975) obtained a similar dose-related response inhibition with pimozide and haloperidol in rats self-stimulating in the dorsal noradrenergic bundle and the dopaminergic nucleus accumbens. Similarly, Liebman and Butcher (1973) found no difference in the effects of pimozide injected into rats self-stimulating in either the lateral hypothalamus or in the mesencephalic central gray area, especially as the latter area contains no dopaminergic neurons (Ungerstedt, 197la). Broekkamp and Van Rossum ( 1975) reported that haloperidol inhibited self-stimulation to a similar extent whether injected in the ipsilateral or contralateral neostriatum. The discrepancies among all these studies of site dependency may be more apparent than real in that methodological differences appear to be largely involved. Apart from differences related to central distribution of the drug with time, state-dependent effects, doses used, injection route, etc., an important factor overlooked in many studies is the base-line rate of responding (see Section IV, D). Furthermore, it is known that rates of responding d o not adequately reflect motivational strength (Valenstein, 1964). Thus, even if base-line rates are the same, the stimulation strength might differ for different structures. This suggests that other types of experiments, such as preference tests, would be more valuable tools in determining whether there are site-dependent effects. B. SPECIES 1. Introduction For obvious reasons, rats were the subjects of choice for the pharmacological self-stimulation experiments. Drug results obtained in one species may not be generalized to other species without restrictions. A
368
ALBERT WAUQUIER
good example of species-related effects are the acute effects of narcotic analgesics. Morphine, and related drugs, cause catatonia (rigidity and loss of righting reflex) in rats, excitation in mice, and sham rage in cats. There are only two reports of drug effects on self-stimulation in other species. Stark (1964) tested cholinergics, anticholinergics, serotonin-like and serotonin-antagonistic drugs in dogs. Horovitz et al. (1962) studied the effects of chlorpromazine in cats. They reported that chlorpromazine (2.5, 3.75, and 5.0 mg/kg) decreased self-stimulation in the lateral hypothalamus and equally in the caudate nucleus. In two out of seven cats, 0.5 and 1.0 mglkg of chlorpromazine did not affect responding. T h e effects of different doses of pimozide, haloperidol, and pipamperone on self-stimulation in dogs have been investigated (briefly reported in Wauquier, 1975). Dogs are extremely suitable for selfstimulation experiments because of their extensive behavioral repertoire. The aims of our studies were to investigate whether neuroleptics inhibited self-stimulation in the same way as in rats and whether neuroleptic-induced inhibition depended on the brain structure. 2. Methods Thirteen dogs (seven beagles and six Labrador retrievers) were implanted with bipolar stainless steel electrodes in the nucleus accumbens, the lateral preoptic region, the basolateral amygdala, the lateral hypothalamus, and the substantia nigra. A total of 5 1 out of the 76 implanted electrodes sustained self-stimulation. After the initial shaping, dogs were further trained to press a lever for brain stimulation during x times 10-min periods (x being the number of positive electrodes). After at least six sessions (three sessions a week), the dogs were treated S.C. with either pimozide (4 hr before the session), haloperidol (2 FIG. 9. Electrode placements of two dogs. D-Sh and LIM refer to the anterior position according to the atlas of Dua-Sharma et al. (1970) and Lim et al. (1960), respectively. Electrodes E l , E2, and E3: right side of the brain: E4, E5, and E6: left side of the brain. Dog left: position E 1 and E4 in nucleus accumbens; E2 and E5 in lateral preoptic region: E3 and E6 virtually outside amygdala (self-stimulation in El, E2, E4, and E5). Dog right: position E l and E2 ventral to the caudate nucleus and between internal capsula and nucleus accumbens; E4 and E5 in nucleus accumbens; E3 in basolateral amygdala; E6 in substantia nigra (self-stirnulation in E l , E2, E3, E4, and E6). Abbreui&’m: NC-caudate nucleus, CC-corpus callosum, Spt-septum, CI-internal capsule, Acc-nucleus accumbens, Put-putamen, Fx-fornix, CA-anterior commissure, Pal-globus pallidus, APr-preoptic area, m-medial, I-lateral, ChO-optic chiasm, TMT-tractus mammillothalamicus, Pyr-lobus pyriformis, Hipp-hippocampus, CM-corpora mammillaria, amygdala: NL-lateral nucleus, NB-basal nucleus (nomenclature after Lim), RH-rudimentum hippocampi, NO-optic nerve, Hab-habenula, CP-corticopontine tract, CB-corticobulbar tract, SN-substantia nigra, FMT-fasciculus mammillotegmentalis, SGC-substantia grisea centralis, Aqaqueductus cerebri, El . . . , -tip of the electrode.
DSh 25-5
E4 D-Sh 29
Acc put
Acc
Put
-
D S h 23.5
D S h 23
E2
E5
D-Sh 26
t5
NO LIM R18
E6
LIM R16
LIM R 1 5 / 1 6 v
Arnygdola
s 35/36 L I M Rlb
Arnygdola
370
ALBERT WAUQUIER
hr before the session), fluanisone (1 hr before the session), pipamperone (1 hr before the session), or azaperone (1 hr before the session). These time intervals were selected on the basis of pilot experiments in which different doses of these neuroleptics were given to Labradors and the approximate time of peak effect was determined by a series of 10-min self-stimulation periods 1 hr, 2 hr, 4 hr, and 6 hr after injection. Histology was carried out after celloidin embedding and staining of brain slices by the Weil method. Examples of electrode placements are given in Fig. 9.
3. Pre-drug Results In each dog, the stimulus parameters selected in the control sessions were kept constant during all drug experiments. Self-stimulation was supported by 23 out of 26 electrodes in the nucleus accumbens, 15 out of 24 in the lateral preoptic region, 5 out of 8 in the amygdala region, 4 out of 9 in the lateral hypothalamus, and 4 out of 9 in the substantia nigra region. Electrodes producing unstable response rates were, however, discarded for drug testing. Electrodes in the anterior forebrain regions (nucleus accumbens, lateral preoptic area, and basolateral amygdala) sustained self-stimulationslowly and mostly in bouts; delayed extinction was also apparent. This contrasted strongly with stimulation in the lateral hypothalamus or substantia nigra, which was characterized by continuous fast lever-pressing and instantaneous extinction.
4. Drug Effects Since only those electrodes eliciting stable response rates were selected it was possible to compare the response rates during test sessions with control sessions and express drug results as percentages of controls (see Fig. 10). The five neuroleptics inhibited self-stimulation obtained in various brain structures in the dog. In general, this inhibition was dose-related, but the sensitivity of brain structures and the potency of the neuroleptics differed from results obtained in rats. Further behavioral changes accompanied the self-stimulation inhibition induced by specific neuroleptics. The most sensitive brain sites were the amygdala with pimozide and pipamperone; the lateral preoptic with haloperidol; the lateral hypothalamus with fluanisone; and the substantia nigra with azaperone. There was no clear evidence that self-stimulation in dopaminergic, or noradrenergic-mediated sites was differentially affected by neuroleptics, which are selective dopaminergic, noradrenergic, or serotonergic recep-
NEUROLEPTICS A N D BRAIN SELF-STIMULATION BEHAVIOR La1 preoplic
Accumbens
Subs1 nlgra
La1 hypothalamus
37 1
b I Amygdala
--- n=6
008
031
n=!
n=2
004
016
-
_. -k
006
016
008
063
031
004
016
O63mglkg
PImoz ode
n-1 1
063
1
4
008
031
016
063
031
125
008
031
I
.
OOL
016
004
016
5
1
O63mglkg Haloperidol
LO 20 n: 2
08
031
"\' OJ 016
n:5 063
n=L 250
0 31
063
008
125
.
063
125
250
;:-\ci LO 20 0
016
031
L
n.1
n= L
n:3
OOL
OOL
016
063
O63mglkg Azaperone
FIG. 10. Self-stirnulation response rates in relation to control response rates (CRR = 100%)after the injection of five neuroleptics, obtained with 13 dogs, self-stimulating in different brain structures, (see text for details).
tor blockers. The fact that other authors (e.g., Ritter and Stein, 1973) have reported differential effects may be due to methodological differences (e.g., injection route, base-line rates, etc.) rather than reflecting the presence or absence of site-related effects. As compared to the rat: similar doses of pimozide inhibit ICS in both species, although with haloperidol and fluanisone almost ten times
372
ALBERT WAUQUIER
higher doses are required in the dog. However, dogs were inhibited by four to six times lower doses of pipamperone and azaperone. Thus, neuroleptics, which preferentially block noradrenergic and serotonergic receptors, are potent inhibitors of self-stimulation in the dog. These results partially confirm and extend the data reported by Stark (1964). T h e differences may reflect species differences, indicating that noradrenergic and serotonergic substrates play a role in self-stimulation in the dog. The doses of the specific neuroleptics (pimozide, haloperidol, fluanisone) producing inhibition were higher than those causing loss of avoidance (e.g., Janssen et al., 1965a), which suggests that motor incapacitation plays a role in inhibitory effects. However, dogs were still able to perform, since over short periods they pressed the lever at very high rates. However, the temporal pattern of responding was completely changed. Instead, of an almost constant rate of lever-pressing, prolonged periods of nonpressing occurred, interspersed with periods of rapid pressing. This change in pattern of response is the reason why inhibition was nonlinearly related to dose (see, e.g., the effects of pimozide on substantia nigra self-stimulation). During the period of nonpressing, dogs often showed a stereotyped scratching of the floor with both forepaws. 5. Conclusion There were no clear-cut site-related effects, and self-stimulation in most sites was dose-relatedly inhibited. Atypical dose-response curves were obtained with specific dopaminergic blocking neuroleptics apparently due to motor effects. Neuroleptics that preferentially block noradrenergic and serotonergic receptors appeared to be potent inhibitors of self-stimulation. The fact that these neuroleptics also block selfstimulation in sites in which dopamine might play an important role may confirm the suggestion of Franklin et al. (1976) and Stephenset al. (1976) that dopaminergic self-stimulation also requires a transsynaptic activation of noradrenergic structures. VI. Self-stimulation and Psychotropic Assays4
T h e preceding section described neuroleptic-induced inhibition on a quantitative basis. In spite of certain characteristics shared by all neuroleptics, these drugs are not a homogeneous group. T h e aim in this This section was written in collaboration with P. J. Lewi.
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
373
section is to compare the neuroleptics on the basis of different pharmacological tests. Also, appropriate methods make it possible to integrate the self-stimulation test within the results of other pharmacological experiments.
A. CLASSIFICATION OF NEUROLEPTICS Classificationsof neuroleptics can be made on different bases, such as chemical (e.g., Janssen, 1970, 1973), biochemical (e.g., Anden et al., 1970), pharmacological (e.g., Janssen et al., 1965a), and clinical (e.g., Bobon et al., 1972). Here we describe classifications of neuroleptics based on pharmacological and clinical activity. 1. Chs$cation Based on Potency Ratios Qualitative observations and comparative pharmacology of a large group of neuroleptics have been described extensively over the past years (Janssen, 1970, 1972;Janssen and Van Bever, 1975; Janssen et al., 1965a,b, 1966, 1967; Niemegeers, 1974).These allowed the construction of neuroleptic activity spectra based on potency and ratios between potencies. Four main pharmacological tests have been used to differentiate the neuroleptics: the induction of catalepsy (CA), palpebral ptosis (PP), the antagonism of amphetamine-induced stereotype behavior (AM), and the antagonism of a lethal dose of norepinephrine (NE). The procedures used and criteria applied to determine neuroleptic potency in these tests were described by Niemegeers (1974). The CA and AM tests are indicative of predominant dopamine-blocking activity, and the N E and PP tests are indicative of a more pronounced noradrenergic blocking activity. High doses of incisive neuroleptics cause catalepsy. The same neuroleptics antagonize stereotyped behavior induced by amphetamine. These neuroleptics have antipsychotic activity and often cause extrapyramidal side effects in the clinic. High doses of sedative neuroleptics cause sedation and palpebral ptosis. These neuroleptics antagonize a lethal dose of norepinephrine. They have less, or are devoid of, antipsychotic activity. The EDJovalues of the neuroleptics tested in self-stimulation and in the CA, PP, AM, and NE tests are given in Table VI. The relative adrenolytic vs. antipsychotic activity and the relative sedative vs. neurologic effects are indicated by the ratio of EDSo-basedactivities. A low ratio of NE/AM and PP/CA is found with a-adrenolytic neuroleptics, which preferentially block the noradrenergic receptors that cause sedation in rats and autonomic side effects in human subjects. A high ratio of
374
ALBERT WAUQUIER
NEIAM and PP/CA is found with incisive neuroleptics, which preferentially block the dopaminergic receptors that induce catalepsy in rats and cause extrapyramidal side effects in humans. The separation between incisive and sedative neuroleptics is evidently not an all-or-none phenomenon, but a continuum. The ED50 values of the neuroleptics tested in self-stimulation were correlated with the ED50 values obtained in the different tests mentioned (Janssen and Van Bever, 1975), i.e., CA, PP, NE, and AM (Spearman rank-order correlation) (Fig. 1 1). Significant correlations were found between the self-stimulation inhibition and the induction of catalepsy (r = 0.736,p < O.Ol),antagonism of amphetamine (r = 0.925,p < 0.01),and induction of palpebral ptosis
CA
100-
a a
a
a
am
4
10-
ma
a
m
a m a
I rs: 0.925 P C 0.01
W-
0.01 0 01 001
,
01
1
10
1W B S
NE 100-
r,:0.736 P < 0.01
0.01 0.01
10
100 B S
PP 100-
a
a . 10-
10-
a * .ma
a a
om 1.
a
l-'
a
0
a' 01-
1
0.1
n.19 rs = 0.208 P > 0.05
0.1-
e
m
n = 16 r s = 0.693 P C 0.01
FIG. 1 1 . Correlation (Spearman rank-order correlation r.) between the EDsovalues of the inhibition on brain self-stimulation (BS) and the EDSo values of antagonism of amphetamine-induced stereotypes (AM), catalepsy (CA), norepinephrine antagonism (NE), and palpebral ptosis (PP),obtained with various neuroleptics (n) (see Table 11).
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
375
( r = 0.693,p < 0.01). The self-stimulation inhibition was not significantly correlated with the antagonism of norepinephrine lethality (r = 0.208, p > 0.05). Apart from the classical differ'entiations among the neuroleptics, one recent extensive study (Niemegeers et al., 1977) demonstrated that neuroleptics might also be differentiated on the basis of their serotonergic blocking action (as shown in the antagonism of tryptamine-induced effects). Again, a significant (p < 0.01) correlation ( r = 0.650) was found between the self-stimulation inhibition and the antiserotonergic effects. It could be inferred that the dopaminergic receptor blocking activity is more specifically related to the self-stimulation inhibition than the noradrenergic receptor blocking effects. However, the doses of the sedative neuroleptics effectively inhibiting self-stimulation also block noradrenergic as well as dopaminergic receptors (see, e.g., A n d h el al., 1970; Table VI). The doses required to antagonize the tryptamine effects were much higher than those inhibiting self-stimulation, except for azaperone, pipamperone, clozapine, and thioridazine. Serotonergic blocking activity is thus, to a minor extent, involved in the inhibition of selfstimulation. It is important to realize, however, that the correlation may simply reflect the potency relationships. It is, therefore, appropriate to apply a method that separates the potency of neuroleptics from their spectral information. 2. Spectral Map Analysis A technique that separates the potency from the spectral information has been described by Lewi (1976a). In short, spectral map analysis is a mathemathical method that extracts from the pharmacological data (such as ED,,, values of neuroleptic activity in various tests) relevant dimensions that are related to the ratios between the assays. The compounds are projected on a (multidimensional) plane, and the dominant axes drawn through this plane can be identified with the principal components. T h e percentage contributions to the total variance of the spectra are calculated. T h e spectral mapping in a plane thus provides two-dimensional information on the relative positions of the compounds. Figure 12 shows the spectral maps (Lewi, 1975, 1976b) of 24 neuroleptics according to four pharmacological assays in rats Uanssen and Van Bever, 1975; see Section VI,A,l) and with respect to six clinical observations (Bobon et al., 1972). The pharmacological map shows the more incisive neuroleptics on
376
ALBERT WAUQUIER
a Ilusptrilrnr pimozidr
*pipamperone oxyprrline
FIG. 12. Spectral map of various neuroleptics based on pharmacological assays (upper figure) and clinical observations (lower figure) (with permission of North Publishing Co.).
the left, whereas more sedative compounds appear on the right. The incisive/sedative ratio of the compounds can be estimated from the relative position of the projections of their images on the map upon the horizontal axis. This axis accounts for about 89% of the total information contained in the spectra. The second principal axis contributes no more than 6% to the original information. The clinical map, on the other hand, shows a larger contribution of the minor principal axis, contributing 26%. This axis appears to be related to the antimanic/antiautistic differential score. It can be seen that the incisive/sedativeclassification derived from the horizontal axis of the clinical map agrees with the same classification based on the phar-
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
377
macological assays in animals. The Spearman rank-order correlation between these two Lambert-type (Lambert and Revol, 1960) classifications is 0.79.
B. POSITIONING OF PSYCHOTROPIC ASSAYS This method (spectral map analysis) was applied to the neuroleptics tested in self-stimulationand enabled the demonstration of the interrelationship between the self-stimulation test and other pharmacological assays, such as the tests discussed in the previous section, as well as other tests of operant behavior. The spectral map analysis was programmed and calculated on an interactive typewriter using APL, a computer language devised by Iverson (1962). The program listings are reported by Lewi (1976~). Spectral map analysis was carried out on 40 neuroleptics as tested in 12 assays in rats. Two principal components were found, of which the horizontal one accounted for most of the information (73%). This component reproduced the pharmacological classification described in the previous section, namely the bipolar incisive/sedative scale (Fig. 12). Many neuroleptics were tested in a variety of learned behavioral situations using different schedules in which responding was maintained by negative reinforcement: escape (Niemegeers et al., 1970b, 1972) and avoidance (Janssen and Niemegeers, 1961; Janssen et al., 1965a; Niemegeers, 1974; Niemegeers et al., 1969a,b, 1970a, 1972). The ED,,, values of the inhibition obtained with various neuroleptics on brain self-stimulation, on the one hand, and various escape or avoidance situations, on the other hand, are highly correlated (one example, Sidman shock-avoidance,is given in Table VI). In all these experiments (a) rats learned to press or to jump in order to escape, avoid, or obtain reward, and (b) well-trained rats and good performers were selected. The situations differed with respect to (a) acquisition rate, (b) training required before stable performance was achieved, and (c) motivation (positive vs. negative). The following operant behavior assays are positioned on the map of neuroleptics (Fig. 13): noise escape (effects on latency time and on number of responses), Sidman shock escape/avoidance, jumping-box shock avoidance, and intracranial self-stimulation. It is observed that all these operant behavior assays are located on a line oriented from apomorphine and amphetamine inhibition towards unrestrained locomotor activity (rearing, ambulation), conditioned feeding (weight gain), and catalepsy. Furthermore, typical escape inhibition is associated most closely with the inhibition of apomorphine and amphetamine,
378
FIG. 13. Spectral map of various neuroleptics and various operant behavior assays
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
379
whereas typical loss of avoidance is most correlated with conditioned and unrestrained activities. Mixed escape/avoidance and intracranial selfstimulation can be seen to occupy intermediate positions between these two extremes. As previously stated (Section VI,A, l), it appears that the neuroleptic effects on operant behavior are more related to dopaminergic receptor blocking activity than to the noradrenergic receptor blocking effects. T h e neuroleptic-induced inhibition of operant behavior is rather independent of the motivational sign, i.e., reward or aversion. T h e intermediate position of self-stimulation shows the relationship with typical neuroleptic effects and with neurologic side effects (motor impairment). Common to all these operant behavioral situations is the involvement of complex learned behavior and the occurrence of a reinforcement process.
VII. Studies on Drug Interaction
Self-stimulation behavior depends, at least partly, on catecholaminergic and cholinergic interacting functions (Olds and Ito, 1973; Stein, 1968). Olds (1972) showed that scopolamine (0.5 mg/kg) antagonized about 60% of the inhibition of self-stimulation induced by chlorpromazine (2.5 mg/kg). T h e aim of the following studies was to reveal the functional interaction between dopaminergic and cholinergic neurons. These studies have clinical relevance because of the treatment of Parkinson’s disease with drugs that also antagonize the neurolepticinduced Parkinson-like symptoms, and further, because of the routine treatment of psychotic patients with combined neuroleptic and antiparkinsonian agents. In all the studies reported in this article, we used rats implanted with electrodes in the lateral hypothalamic region of the medial forebrain bundle. Rats were trained in 15- or 30-min daily sessions, using a stimulus parameter combination that elicited high response rates (SPC 6). At least two control sessions separated each treatment. The test used to show significant differences between drug session and control session was the Wilcoxon matched-pairs, signed-ranks test with two-tailed probability. The level of significance selected was p C 0.05. Further details are reported in the different experiments. A. LITERATURE ON NEUROLEPTIC-ANTICHOLINERGIC INTERACTION Relatively few authors have studied the interaction between the pharmacological effects of neuroleptics and anticholinergics (see
380
ALBERT WAUQUIER
Wauquier et al., 1975, 1976). The effects studied were primarily concerned with changes in the extrapyramidal system, both at the behavioral level, e.g., catalepsy (Table VII), and at the biochemical level, e.g., modulation of the increased levels of homovanillic acid in subcortical brain regions (AndCn and BCdard, 1971; Bowers and Roth, 1972; Corrodi et al., 1972; O’Keeffe et al., 1970) and increased rate of dopamine depletion after synthesis inhibition (Puri and Lal, 1973; Puri et al., 1973). Partial blocking of the neuroleptic-induced inhibition of avoidance behavior has been previously reported (Table VIII). We have also reported on the antagonism of pimozide-induced inhibition of jumping in amphetamine-dopa treated mice (Colpaert et al., 1975). It follows that centrally acting anticholinergics are able to antagonize neuroleptic-induced effects. The doses required to antagonize catalepsy or inhibit avoidance are much higher than those producing central or peripheral anticholinergic activity, as measured in the anti-pilocarpine test in rats (Jamsen and Niemegeers, 1967) (Tables VII and VIII). Quantitative inter-drug comparisons are almost impossible to perform, because of the wide range in activity of the different compounds used, the variable doses, and the different injection schedules and routes of administration. It will be shown in the subsequently described experiments that self-stimulation is a reliable means of studying neurolepticantagonistic drug interaction. OF THE SPECIFIC INHIBITION OBTAINED WITH B. REVERSAL ANTICHOLINERGICS
It has been stated (see Section VI) that specific neuroleptics inhibit self-stimulation by interference with the nigrostriatal system. These dopaminergic neurons interact with striatal cholinergic interneurons. Since DA neural transmission appears to be involved in the neurolepticinduced inhibition of self-stimulation, one would expect anticholinergics to antagonize the inhibitory effects of specific DA-blocking neuroleptics. 1. D@?rentiation from Narcotic Anulgesacs
Haloperidol and morphine have a number of similarities: they increase the turnover of striatal DA (Puri and Lal, 1974); they release prolactin (Dickerman et al., 1972); they inhibit the release of luteinizing hormone (Dobrin and Mares, 1974);and they produce a state of immobility that can be reversed with apomorphine (Puri et al., 1973). They differ, however, in many respects: haloperidol lacks analgesic effects and tolerance (La1 and Puri, 1973) and neuroleptics cause catalepsy, whereas morphine-like drugs cause catatonia.
TABLE VII: ANTAGONISM OF NEUROLEPTIC-INDUCED CATALEPSY IN RATS Neuroleptics
Chlorpromazine
Perphenazine
Dose (mg/kg)
10.0
10.0
Route
S.C.
i.p.
Effect"
+++(+)
++++
Dose Anticholinergics (mg/kg) Ethybenzatropine Ethybenzatropine Scopolamine ,Trihexyphenidyl
1.00 3.00
S.C.
0.50 5.00
i.p. i.p.
2.00 5.00 10.0 2.00 2.00 0.10 0.50 2.00
i.p. i.p. f.p. f.p. 1.p. i.p. i.p. i.p.
2.00 5.00
i.p. i.p.
'Atropine 11.5 Piethazine >64.0 Orphenadrine 18.0 Procyclidine 14.0 Profenamine B64.0 Trihexyphenidyl 9.50 ,Scopolamine 1.20
i.p. i.p. 1.p. f.p. f.P. 1.p. i.p.
4.00 0.45
S.C. S.C.
5.00 12.5 J .OO
i.p. i.p. i.p.
0.50 5.00
i.p. i.p.
Atropine Atropine Atropine 'Benztropine Biperiden 1~co polarnine Scopolamine Scopolamine rrihexyphenidyl Trihexyphenidyl
Rochlorperazine
8.00
i.p.
++++
Thiopropazate
0.29
S.C.
++
Biperiden Chlorphencyclan
Thioproperazine
0.20
i.p.
++
Benztropine homethazine Scopolamine
++++
Scopolamine Trihexyphenidyl
Trifluoperazine a
10.0
i.p.
Route S.C.
Effect"
ED, = lb
Reference
+(+)
++ +++
von Taeschler ct al. ( 1962) Morpurgo and Theobald ( 1964)
+++ +(+)
+++ +++ ++ ++ +(+) +++(+)
++++ ++
++(+)
++
++ ++
8 X' 21 x 42 x 8x,
-
6 x 31 x
Morpurgo (1962) Morpurgo and Theobald ( 1964)
Morpurgo (1962) Morpurgo and Theobald ( 1964)
-
++ ++ ++++ +
+++ +++(+)
++++ ++++
++(+)
Malatray and Simon (1972)
Schauman and Kurbjuweit (1961) Leslie and Maxwell ( 1964) Morpurgo and Theobald ( 1964)
Agonistic effects obtained with neuroleptics and antagonistic effects with anticholinergics expressed as +25%, ++50%, +++75%,
+ + + + 100%.
ED, = Dose of central anticholinergic activity in 50% of the rats (according to Janssen and Niemegeers, 1967).
TABLE VIII ANTAGONISM OF NEUROLEPTIC-INDUCED INHIBITION OF AVOIDANCE I N RATS
Neuroleptics
Dose (mg/kg)
Route
Chlorpromazine
2.00
i.p.
+++
Perphenazine
0.20
i.p.
+++
S.C.
++
10.00
Trifluoperazine a
0.40
I I
Route
Effecta
Scopolamine Trihexyphenidyl
0.20 2.00
i.p. i.p.
++(+) ++(+)
Scopolamine Trihexyphenidyl
0.20 2.00
i.p. i.p.
++
0.37
S.C.
0.86 1.80 4.90 0.10
S.C.
0.50
S.C.
1.OO 0.20 2.00
S.C.
++
S.C.
0
S.C.
S.C.
+(+) +(+)
Biperiden Chlorphencyclan Biperiden Chlorphencyclan Ethybenzatropine Ethybenzatropine Ethybenzatropine
i.p.
++(+)
Scopolamine Trihexyphenidyl
Thiopropazate
Thioridazine
Dose Anticholinergics (mg/kg)
Effect'
++(+)
Morpurgo and Theobald (1964) 12 x 8X
+(+)
-
S.C.
Reference
EDm= I*
S.C.
+(+)
S.C.
0
-
Schaumann and Kurbjuweit (1961)
++++
+ + +(+)
Taeschler et al. (1962)
S.C.
0.20 1x 2x
i.p. i.p.
++ + ++ +
12 x 8x
Morpurgo and Theobald (1964)
Agonistic effects obtained with neuroleptics and antagonistic effects with anticholinergics expressed as +25%,
++ ++ 100%. * EDs
= Dose of central anticholinergic activity in
50% of the rats (according to Janssen and Niemegeers, 1967).
+ +50%, ++ +75%,
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
383
The inhibition of self-stimulation obtained with 0.16 mg/kg of fentanyl or with 40 mg/kg of morphine could not be reversed by the centrally acting anticholinergic dexetimide (2.5 mg/kg). T h e associated catatonia was not reversed either. Naloxone, on the other hand, reversed the self-stimulation inhibition and catatonia induced by the former drugs (Wauquier et al., 1974). These experiments showed that some actions induced by morphine-like agents are reversed by a specific antagonist. Since DA receptors appear to be involved in causing morphine- or neuroleptic-induced effects, one would expect different mechanisms of action (see also Broekkamp and Van Rossum, 1975).
2. Dose-Related Antagonism As shown previously (see Section IV,B), a dose of 0.08 mg/kg of haloperidol given S.C. 1 hr before the session caused a nearly complete suppression of self-stimulation. Dexetimide, a centrally acting anticholinergic Uanssen and Niemegeers, 1967; Janssen et al., 197 l), antagonized, in a dose-related manner, the suppression of self-stimulation induced by haloperidol (Wauquier et al., 1975). Eight rats were trained to press a lever for brain stimulation (SPC 6) in the lateral hypothalamus, during two daily 15-min sessions separated by a 4-hr interval. After training, they were injected S.C. with 0.08 mg/kg of haloperidol 1 hr before the first daily session, followed 30 min later with either 0.04, 0.16, 0.63, or 2.5 mg/kg of dexetimide S.C. Dexetimide gradually reinstated self-stimulation in a dose-related manner. Isopropamide, a peripherally acting anticholinergic drug, at a dose (10 mg/kg) 1000 times higher than those producing peripheral anticholinergic activity Uanssen and Niemegeers, 1967), did not antagonize the haloperidol-induced inhibition. T h e reversal of the inhibition by dexetimide suggests that the haloperidol effect was due to some altered relationship between dopaminergic and cholinergic activity in striatum (Klawans, 1973; Sigwald, 197 l), either by cholinergic inhibition or dopaminergic stimulation. In the following studies, we selected the dose of 0.63 mg/kg of dexetimide, because this was the lowest dose to reverse more than 50% of the haloperidol-induced inhibition.
3. Antiparkimonian Drugs The reversal of the haloperidol-induced inhibition of self-stimulation was not an exclusive action of dexetimide. Benztropine (10 mg/kg) likewise antagonized the inhibition brought about by haloperidol (Wauquier et al., 1974).
384
ALBERT WAUQUIER
In another study, we compared the antagonism of the penfluridoland clopimozide-induced inhibition by three antiparkinsonian agents; dexetimide, benztropine, and trihexyphenidyl. Seven rats were trained to self-stimulate for SPC 6 in daily 30-min sessions, 5 days a week, except for Monday, when they were run twice, with a 4-hr interval between the sessions. A dose of the long-acting neuroleptics penfluridol(5 mg/kg) (Janssenet al., 1970) and clopimozide (1.25 mg/kg) (Janssen et al., 1975), which, as described, suppressed self-stimulation virtually completely, was given orally 1 hr before the first session on Monday. During subsequent weeks, rats were treated with the neuroleptic followed 4 hr later, i.e., 1 hr before the second session on Monday, with either dexetimide (0.63 mg/kg), benztropine (10 mg/kg), or by trihexyphenidyl (10 mg/kg) S.C. Rats were only treated once per week. Figure 14 depicts the results obtained. At the doses used, penfluridol and clopimozide significantly inhibited self-stimulation 4 hr, 24 hr, and 48 hr after injection. The inhibition was most pronounced 4 hr after neuroleptic treatment, and selfstimulation gradually recovered during the following 4 days. The three antiparkinsonian drugs, which by themselves did not significantly affect self-stimulation, completely reversed the self-stimulation inhibition obtained 4 hr after neuroleptic treatment. Self-stimulation was completely normalized (not significantly different from controls), except for the combination of penfluridol with trihexyphenidyl. However, during the following days, self-stimulation rates did not differ from the rates obtained after neuroleptic treatment alone. This study showed that the self-stimulation inhibition induced by specific neuroleptics could be reversed by three different antiparkinsonian drugs at a time when maximum inhibition could be expected.
C. DIFFERENTIAL ANTAGONISM
Inasmuch as DA neural transmission is involved in the antagonism by antiparkinsonian drugs of neuroleptic-induced inhibition, one would expect anticholinergics to antagonize the inhibitory effects of specific DA-blocking neuroleptics, without affecting the inhibition brought about by NA-blocking neuroleptics. Further, different mechanisms can be advanced to account for the reversal of this inhibition: anticholinergic activity (noncompetitive antagonism); release or DA-uptake blocking activity (competitive antagonism). It was the aim of the following studies to elucidate the possible mechanism involved, by using various putative antagonists.
NEUROLEPTICS AND BRAIN SELF-STIMULATION BEHAVIOR
385
P E N F L U R I D O L * A N l A G O N l SlS 'I* 120, 100. 10.
60.
LO. 20. 0. hr
1 2 3 4 I
1 2 3 4
1 2 3 4 24
4
1 2 3 4
48
1 2 3 4 72
1 2 3 4 96
CLOP1 M O Z l D E * A N T A G O N l S l S
I3
3 4 hr
1 ;
1 24 3 1
1 2 3 4 24
1 2 3 4 LB
1 21 23 4
il:
1 2963 4
FIG. 14. Median self-stimulation response rates as a percentage of control values obtained with seven rats, 1 hr, 4 hr, 24 hr, 48 hr, 72 hr, and 96 hr after oral administration of 5 mg/kg of penfluridol (1) (upper) or 1.25 mg/kg of clopimozide (1) (lower) or, after combined treatment with either penfiuridol or clopimozide, plus 0.63 mg/kg of dexetimide (2), 10 mg/kg of benztropine (3) or 10 mg/kg benztropine (3) or 10 mg/kg of trihexyphenidyl(4). The latter antiparkinsonian drugs were given S.C. 1 h r before the 4-hr session.
1. D i f f e n t i a l Antagonism among the Neuroleptics
a. Three different neuroleptics were selected: pimozide, haloperidol, and pipamperone. Pimozide and haloperidol are both specific DAblocking neuroleptics; haloperidol, however, also blocks NA receptors at high dose levels. Pipamperone, on the other hand, blocks DA and NA receptors at approximately equal dose levels (AndCn et al., 1970) (see Section VI). Four doses of each neuroleptic were given, the second dose being approximately the EDSovalue for inhibition, the fourth dose being
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16 times higher. All rats were also given the combination of the neuroleptic and 0.63 mg/kg of dexetimide (Wauquier and Niemegeers, 1975). Figure 15 shows the results obtained. Dexetimide completely reversed the self-stimulation inhibition induced by pimozide, and self-stimulation was normalized to control levels. The haloperidol-induced inhibition was also significantly antagonized at all dose levels, but self-stimulation was not normalized to control levels with the combination of dexetimide with 0.16 mg/kg and 0.63 mg/kg of haloperidol. T h e pipamperone-induced inhibition was not antagonized by dexetimide. The inhibition of self-stimulation induced by pimozide and haloperidol is probably due to the DA-blocking activity, whereas the pipamperone-induced inhibition is related to the DA- and NA-blocking activity. It follows that dexetimide reversed the DA-blocking effect, whereas the NA-blocking effect would not be antagonized. This conclusion was further examined by using different neuroleptics, which preferentially block NA receptors. b. A group of rats were treated with various sedative neuroleptics (ratio NE/AM lower than 1; see Section V1,A) and a combination of these neuroleptics with 0.63 mg/kg of dexetimide. T h e neuroleptics were: azaperone (2.5 mg/kg), chlorpromazine (2.5 mg/kg), chlorprotixene (2.5 mg/kg), clozapine (40 mg/kg), haloanisone (2.5 mg/kg), oxypertine ( 10 mg/kg), piperazetacine (0.63 mg/kg), promazine (40 mg/kg), and thioridazine (40 mg/kg). The chosen dose of neuroleptic was the lowest dose of a geometrical series (0.04, 0.08, . . . 40 mg/kg of body weight) which produced complete inhibition of self-stimulation (based on Section IV,B and pilot experiments). The inhibition of self-stimulation induced by these neuroleptics was not reversed by dexetimide, except for thioridazine (see, further, Section VI1,D). T h e self-stimulation inhibition is probably due to its NA-blocking and DA-blocking activity. Dexetimide could reverse the DA-blocking effect completely (as was shown for pimozide), but, because of the NA-blocking effect, self-stimulation remained inhibited. 2. Competitive and Noncompetitive Antagonism
Sedative neuroleptics (ratio NA/DA < 1; see Section VI) could not be antagonized with the anticholinergic dexetimide, whereas specific (DAblocking) neuroleptics were. Most anticholinergics, apart from their anticholinergic activity, are also DA-uptake blockers. It is possible that the reversal of the effects of the specific neuroleptics requires an action on synaptic DA mechanisms, i.e., potentiation of the synaptic action of DA might overcome the receptor blockade. T h e reversal of the sedative neuroleptics (blocking DA and NA) probably requires an action on both NA and DA.
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P I P A M PE R 0 N E
FIG. 15. Self-stimulation in rats: median (eight rats) response rate expressed as a percentage of the preceding control (= 100%)obtained after neuroleptic (0)and combined neuroleptic-anticholinergic treatment (M)(with permission of Archives Intentationales & Phunnacodynamie et de Therapie).
To test this hypothesis, several compounds, i.e., piribedil and
apomorphine (DA agonists), dexetimide (anticholinergic and DA-uptake blocker), cocaine and nomifensine (DA- and NA-uptake blockers), and amphetamine (release of NA and DA), were tested for their ability to restore self-stimulation inhibited by pimozide or chlorpromazine. The results indicate that anticholinergics (noncompetitive an-
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tagonism) or drugs that enhance endogenous neurotransmission by increased release or uptake blockade (competitive antagonism) are able to reverse the neuroleptic-induced inhibition of self-stimulation more effectively than receptor agonists. (Part of the present results are reported in Wauquier and Niemegeers, 1976b.) The mean percentage (k SEM) of lever-pressing obtained after administration of pimozide, chlorpromazine, and a combination of these with potential antagonists is shown in Fig. 16 and compared to the respective preceding control session. Pimozide and chlorpromazine induced an almost complete suppression of self-stimulation, The antagonists were without significant effects on high base-line responding, except for apomorphine, which inhibited lever-pressing by approximately 40%. Although the pimozide-induced inhibition of self-stimulation was significantly antagonized by all compounds tested, marked quantitative differences were observed: cocaine, dexetimide, and nomifensine restored self-stimulation to more than 85% of the controls, amphetamine restored self-stimulation to about 50% of normal levels, whereas the two DA agonists, piribedil and apomorphine, showed very little antagonism. The chlorpromazine-induced inhibition of self-stimulation was not antagonized by dexetimide, but was completely reversed by amphetamine and nomifensine ( p > 0.05 as compared to the controls). Cocaine restored self-stimulation to about 50% of normal levels. Apomorphine had no effect on chlorpromazine-induced inhibition, whereas piribedil was slightly antagonistic. The differential antagonism reported here shows that reversal properties are not necessarily dependent upon the inherent facilitatory effects of the antagonists (Wauquier, 1976; Wauquier and Niemegeers, 1974). It appears that the DA-uptake blocking activity, rather than the central anticholinergic action of dexetimide, is responsible for the antagonism of specific DA-blocking activity, since the two DA-uptake blockers, cocaine (Ross and Renyi, 1967) and nomifensine (Hunt et al., 1974), completely restored self-stimulation inhibited by pimozide. In other experiments it was found that pretreatment with a-methylparatyrosine anesthylester (150 mg/kg, i.p., 5 hr before the session), which in itself reduced self-stimulation for more than 50%, prevented dexetimide from enhancing low base-line responding. D. DISCUSSION The results reported are evidence that anticholinergics and DAuptake blockers are able to overcome the inhibition induced by
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FIG. 16. Mean percentage (+ S.E.M.) of lever pressing for brain stimulation in the lateral hypothalamic region, after S.C. injection of saline (1 h r before the session), pimozide (PIM) (2 hr before the session), or chlorpromazine (CPZ) ( 1 hr before the session), or combined treatment with either pimozide or chlorpromazine with amphetamine (AMP), apomorphine (APO), cocaine (COC), dexetimide (DEX), nomifensine (NOM), or piribedil (PIR) given S.C. f hr before the session. Single asterisk indicates significant (p s 0.05) difference as compared to control. Double asterisk indicates significant (p s 0.05) difference as compared to pimozide or chlorpromazine. Circled asterisk indicates significant (p s 0.05 difference as compared to pimozide or chlorpromazine, but not significantly different from control.
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neuroleptics. Additional experiments are required to show that the antagonism obtained with dexetimide is competitive (at the DA-receptor sites) or a mixture of competitive and noncompetitive (at the site of the cholinergic neurones) mechanisms. Further, the serotonergic blocking activity of neuroleptics will have to be taken into account. Dexetimide did not restore self-stimulation inhibited by chlorpromazine, a neuroleptic that blocks NA at lower doses than DA. Nomifensine, which blocks DA and NA uptake (Schacht and Heptner, 1974), however, completely reversed the chlorpromazine-induced inhibition of self-stimulation. The partial antagonism of the pimozideinduced effects and the complete restoration of the chlorpromazineinduced effects achieved with amphetamine can be ascribed to its ability to increase the release of both DA and NA. Apomorphine is able to restore the inhibition of self-stimulation obtained after blockade of the synthesis of DA by a-methylparatyrosine or depletion by reserpine (Stinus et al., 1976). This points to a direct receptor activation. DA agonists have been used with varying success in the treatment of parkinsonism: both apomorphine (Cotzias et al., 1970, 1972) and piribedil (Sweet et al., 1974; Vakil et al., 1973) affected tremor and rigidity. These two direct receptor stimulating agents were not able to antagonize the neuroleptic-induced self-stimulation inhibition. These results point to the difference between the receptor blocking activity and parkinsonism, the latter being due to a degeneration of the nigrostriatal DA pathway (Hornykiewicz,1972), resulting in a lack of DA input to the striatum and a distorted balance between the transmitters operating in the caudate (Coolset al., 1975). One should not forget, however, that the action of apomorphine is far more complex than was originally supposed (Costal1and Naylor, 1973; also see survey by Colpaert et al., 1976). The results with nomifensine clearly indicate that the anticholinergic activity, in itself, is not a prerequisite for antagonism of specific DA inhibition. It seems, therefore, that drugs that enhance the efficacy of endogenous transmitter-substances in a physiological way (by increasing the release or blocking the uptake) but do not impinge directly on the receptor are able to restore self-stimulation inhibited by neuroleptics. The data may also suggest that neuroleptic-induced inhibition and its counteraction by the antagonists tested occur at a presynaptic level. The results of the interaction of pimozide with various antagonists favor the supposed connection between DA and ACh neurons. The interaction could be explained by an interference with inhibitory DA neurons. It has indeed been suggested that there may be two different
NEUROLEPTICS AND BRAIN SELF-STIMULATION
BEHAVIOR
39 1
types of DA neurons, the second being excitatory and operating in the same way as the cholinergic striatal neurons (Cools, 1973). Future experiments should, therefore, take this difference into account. Neuroleptic treatment increased homovanillic acid (HVA) content in the striatum and in the limbic structures, an effect suggesting an increased DA turnover. Neuroleptics also increase the release of ACh and decrease its content in the striatum (Cuyenet et al., 1975; McGeer et al., 1974; Stadler et al., 1973) but not in the limbic system (Anden, 1972; Bartholini et al., 1973; Lloyd et al., 1973) or other brain structures, such as the cortex and the hippocampus (Sethy and Van Woert, 1973). Furthermore, in the striatum, but not in other brain structures, the neuroleptic-induced increase of HVA content is effectively antagonized by anticholinergics (Consolo et al., 1974; Stadler et al., 1973), whereas cholinergic drugs have been found to increase striatal HVA concentrations (Anden, 1974). A functional interaction between DA and AcH is indicated both by this study and by biochemical experiments. Both these types of study point to an interaction in the basal ganglia; however, the precise site of this interaction has not been determined. One approach to this problem is the use of intracerebral microinjection techniques. In one such experiment, Herberg and co-workers (personal communication, 1976) found that scopolamine injected into the nucleus accumbens partially restored spiroperidol-inhibited selfstimulation. Scopolamine had no antagonistic effects, however, when injected into the caudate. In an unpublished study we were able to show that dexetimide injected into the substantia nigra partially restored self-stimulation inhibited by pimozide. The ventral midbrain, including the substantia nigra, is thus a putative site of ACh and DA interaction, as there are projections both to the caudate nucleus and the accumbens. Thus “autaptic” DA receptors may be located in there and cholinergic neurons may synapse at the nigra (Bartholini and Pletscher, 1971). T h e partial restoration of self-stimulation after injection of antagonists may thus depend on a high local systemic concentration of the antagonists, causing a “concerted action.”
VIII. General Discussion
A. NEUROLEPTIC IMPAIRMENT OF SELF-STIMULATION The many variables inherent in any study involving the effects of drugs on self-stimulation make it imperative that great attention be paid
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initially to the experimental design and secondly to a correct statistical analysis of control data obtained from that design. A careful analysis of control data allows more valid conclusions to be drawn regarding the effects of drugs. Regardless of the way data are analyzed, the selfstimulation paradigm itself does not allow a single firm conclusion to be made which explains the mechanisms of action of neuroleptics on selfstimulation. This study does allow the following general conclusions to be made. All neuroleptics dose-relatedly reduced self-stimulation; this inhibition was inversely related to the base-line rate of responding; this inhibition was seen in all brain areas that were stimulated. At dose levels inhibiting self-stimulation, the ability to perform locomotor activity was not affected. Although these conclusions apply to all neuroleptics, quantitative differences between them may also be concluded from this study. EDso values are used to measure these differences. Using a single or a small range of doses may lead to erroneous conclusions. Qualitatively, in terms of behavioral and clinical efficacy, the neuroleptics also differ. In general, at very high doses, incisive neuroleptics induce catalepsy whereas sedative neuroleptics induce sedation. This difference is apparent clinically: incisive neuroleptics are potent neuroleptics (especially the butyrophenones) and, at high dose levels, extrapyramidal side effects often become evident, whereas, although sedative neuroleptics may have antipsychotic effects, at the same dose levels the parasympathetic and noradrenergic brain systems are activated, resulting in such side effects as orthostatic hypotension. These qualitative phenomena caused by neuroleptics must be borne in mind when interpreting self-stimulation data. The fact that neuroleptics can pharmacologically induce catalepsy or sedation leads readily to the idea that inhibition of self-stimulation may be a motor effect. This is also suggested by spectral mapping, in which the self-stimulation test is positioned between catalepsy and amphetamine antagonism, suggesting that common factors are involved. In fact, at high doses of neuroleptics, locomotor activity (ambulation and rearing) is disrupted Uanssen et al., 1965a). Thus, at high doses, motor antagonism must have some function in neuroleptic-induced inhibition of self-stimulation. This motor debilitation can be overcome if the animals are sufficiently aroused or stressed in situations where selfpreservation is required or after strong sensory stimulation (e.g., Marshal et al., 1976). There are a number of observations that show that the ability to self-stimulate is still present in neuroleptic-treated animals. Such rats
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press at normal or at higher rates when first placed in the self-stimulation cage, and they will continue to self-stimulate if high SPCs are programmed. The extinction curve observed after neuroleptic treatment suggests that rats are increasingly less able to maintain responding. It is impossible to exclude motor fatigue or a subtle interference with the operant behavior from the explanation of this phenomenon. The experiments by Huston and Borbkly (1973), in which extensive brain lesioning was shown to interrupt “complex responding” (such as those responses requiring spatial orientation) but not simple operants, may be analogous to the motor debilitation aspect of the effects of neuroleptics on selfstimulation, in that neuroleptics may effectively inhibit complex behaviors. This explanation must, at present, remain only a possibility, as “complexity” is a blanket term, with many aspects. Further experiments using multiple discriminations, a series of operants, or comparisons between different operants, for instance, would be useful to further elucidate this idea. I n summary, motor debilitation is a factor that does operate in inhibition of self-stimulation by neuroleptics. The extent of this motor debilitation depends, for instance, on the paradigm chosen, the doses of the drug used, the stimulation strength (physical or sensory), and possibly the operant used. The neuroleptic-induced disruption of responding at relatively high doses indicates that rats are unable to maintain responding or even to initiate operant behavior (see also Ahlenius, 1973; Fibiger et al., 1975). The stimulus parameter combinations chosen have been shown to result in different base-line rates of responding. These different base-lines vary in their susceptibility to inhibition, in that the inhibition evoked by particular doses of neuroleptics is inversely proportional to the base-line. Although base-line rates do not adequately reflect motivation (Valenstein, 1964) in a single site, rates of responding may reflect motivational strength. Self-stimulation inhibition by neuroleptics could, thus, also represent a motivational deficit: low levels of motivation might be expected to be more sensitive to the suppressing influence of neuroleptics. The motivational sign, however, has been shown to be unimportant, in that negatively, as well as positively, motivated behavior is inhibited by similar dose levels of neuroleptics (Wauquier and Niemegeers, 1972). In addition, the cues associated with the reinforcing stimulation (the conditioned motivation, Trowill, 1976) are not affected. An alternative interpretation of the same data is that the inhibition due to neuroleptics depends inversely on the level of arousal associated with the stimulation strength, and thus not on the motivational strength.
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If this is true, then one would expect that brain sites in which the arousal level is low are more susceptible to the inhibitory effects of neuroleptics. It is known that self-stimulation in forebrain regions, such as septum and accumbens, for instance, is associated with quiet behavior (possibly parasympathetic in nature; see Sadowski et al., 1978). “Quiet” sites were inhibited by the same doses of neuroleptics that inhibited selfstimulation in brain sites where the arousal level is high, such as in the lateral hypothalamus. Further experiments are necessary to resolve this problem. Yokel and Wise (1975) showed that treatment with pimozide increased self-administration for amphetamine. These authors suggested that the perception of amphetamine-produced reward was altered by pimozide. In the same way, the neuroleptics may interfere with the way an animal perceives an electrical stimulation. Colpaert et al. (1977) found that haloperidol blocked the discriminative properties of electrical stimulation in the lateral hypothalamus. Further, Stein and Ray (1960) showed that chlorpromazine increased the threshold for self-stimulation. These results suggest that brain stimulation lost its capacity to act as a discriminative and reinforcing stimulation after neuroleptic treatment. Notwithstanding the different interpretations of the neuroleptic inhibition of self-stimulation presented here, neither one is capable by itself of explaining the inhibition. What is, therefore, required is a hypothesis that incorporates motor control, stimulus characteristics, perception of reward, and sensorimotor integration. It is well established that neuroleptics interfere with dopaminergic neural transmission. Three major dopaminergic pathways in the brain have been described. First, the nigrostriatal pathway (Dahlstrom and Fuxe, 1965; Broch and Marsden, 1972), which is the main input to basal ganglia structures (caudate putamen and, eventually, globus pallidus), which are important in motor behavior and sensorimotor integration. Second, the mesolimbic system, which inputs to limbic structures (tuberculum olfactorium, nucleus accumbens, lateral septum), which are involved in locomotor activity and processing of incentive stimulation. Third, the mesocortical system, which inputs to the cortex (limbic and basal part of the frontal cortex), which is implicated in sensory and motor aspects of behavior. In addition, according to Lindvall et al. (1974), there is a topographically ordered and restricted projection from all cell groups in the ventral tegmentum to the frontal cortex and from lateral parts of the substantia nigra to the anterior cingulate. All these dopaminergic pathways originate in the ventral mesencephalon and coalesce at cortical and basal ganglia structures concerned with sensory transformation, motor initiation, and control of motor behavior (see also
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Glickman and Schiff, 1967; Milner, 1976; Mogenson and Ciot., 1977). Since the dopamine pathways are mainly inhibitory and “intracortical connections are essentially, if not exclusively, inhibitory” (Creutzfeldt, 1977, p. 513), disinhibition would be the principle of activation of motor responses (disinhibition of programmed neural circuits, Roberts, 1974). Activation of dopaminergic pathways disinhibits response-inhibitory systems in cortex and striatum, resulting in repetition of behavior and maintenance of goal-directed behavior (Milner, 1975; Mogenson and Phillips, 1976). Since, in the concepts of Milner (1976), motivation is “the system that controls what gets into and through the motor system” (p. 546), motivation might largely be mediated by dopaminergic pathways. There is every reason to believe that a neuroleptic will interfere with all these different dopaminergic pathways. Specific experiments usually measure effects on the three pathways and interpret the resulting data in terms of one system, which leads to apparent discrepancies. Thus, motor disruption or decreased alertness induced by neuroleptics would occur by blockade of the nigrostriatal pathway (Wauquier and Niemegeers, 1972; Rolls et al., 197413; Fibiger, 1978), whereas changes in the discriminative properties (Colpaert et al., 1977) and perception of reward (Yokel and Wise, 1975) could be caused by blockade of dopamine receptors in mesolimbic and mesocortical systems. As all these dopamine pathways feed into neural structures concerned with initiation, execution, and maintenance of motor behavior, the final effect of neuroleptic treatment will be a reduction in the number of self-stimulations. This hypothesis is the key to an understanding of the effects of neuroleptics on self-stimulation. Manipulation of the variables within the experimental setup might show differential effects on the three systems and would explain the dispute between researchers as to which factors are predominant. What is now required are experiments in which discrimination is made between the different effects of neuroleptics on motor debilitation, perception of reward, and discriminative or reinforcing stimulus control. Whether this is possible using the self-stimulation paradigm remains an open question.
B. CLINICAL IMPLICATIONS Experimental studies on the interaction of neuroleptics with other drugs are few. One of the reasons for this could be the difficulty in obtaining reliable and quantitative data. There is, however, an obvious need for such studies. Firstly, neuroleptic-induced parkinsonian-like effects could be of heuristic value for a better understanding of the treatment of parkinsonism. The drawback to this model is the different etiology: parkinsonism is mainly related to a degeneration of
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dopaminergic neurons, whereas the neuroleptic-induced effects are due to a postsynaptic receptor blockade. Secondly, psychotic patients are routinely given antiparkinsonian drugs in conjunction with neuroleptics, in order to alleviate extrapyramidal side effects (associated with high doses of neuroleptics). The self-stimulation inhibition and its antagonism or reversal showed that self-stimulation was a reliable tool providing quantitative data. Drugs effective in the treatment of parkinsonism reversed the selfstimulation inhibition. It was shown that anticholinergic activity was not a prerequisite, but that drugs that block dopamine uptake were also potent antagonists. Apomorphine and piribedil were ineffective as antagonists. Yet, both compounds have been used in the treatment of parkinsonism. Nomifensine, according to Costall et al. ( 1975), resembles apomorphine in that its stereotypic action was still present after disruption of presynaptic events. It was concluded that nomifensine could be used as a potential antiparkinsonian agent (preliminary clinical data confirmed this action: B. Costall, personal communication). The antagonism of the neuroleptic-induced inhibition is not dependent upon its direct receptor-activating properties. Rather, it is the ability of nomifensine to inhibit reuptake processes (Hunt et al., 1974) that accounts for its reversal properties. If self-stimulation inhibition reflects more the neuroleptic effect and less the neurological deficit caused by neuroleptics, one would expect the antagonists effectively reversing the inhibition to reverse therapeutic effects too. This action is assumed to be localized at the level of the extrapyramidal structures. It was found that L-dopa treatment of Parkinson patients elicited psychotic side effects (Goodwin, 197 l), and L-dopa-treated schizophrenics were made worse, without improvement of the extrapyramidal side effects (Yaruyura-Tobias et al., 1970). Similarly, treatment with DA-receptor stimulants, such as piribedil, caused a deterioration of psychiatric status (Angrist el al., 1975). Some clinical studies report that antiparkinsonian drugs given in conjunction with neuroleptics induce a therapeutic rev6rsal (Haase, 1972; Haase and Janssen, 1965; Singh and DiScipio, 1972; Singh and Kay, 1974, 1975; Singh and Smith, 1973a,b). Singh and Kay (1975) found that the countertherapeutic effects were more extensive than originally described. The countertherapeutic reversal of the fundamental psychotic symptoms was not due to some kind of toxic effect but to an exacerbation of the disorder. Singh and Kay (1975) speculated that the countertherapeutic effects are due to an interference with a periventricular cholinergic system,
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reciprocally interacting with the facilitatory catecholaminergic system, in limbic forebrain structures described by Stein (1968). It is conceivable that anticholinergic drugs influence striatal, limbic, and cortical structures because in all these structures cholinesterase and acetylcholinesterase are present (Lewis and Shute, 1967; Shute and Lewis, 1967). This influence is of particular relevance when it can be shown that in these structures monoaminergic-containing systems produce effects contrary to those of the cholinergic system (Shute and Lewis, 1967). Many biochemical experiments substantiate the dopaminergic-cholinergic link in the caudate nucleus. Such a direct interaction in the limbic structures was, however, not evidenced. An interaction at the origin of the dopaminergic systems is not excluded and remains, therefore, a subject for further experimentation. Undoubtedly, anticholinergics influence the cholinergic neurons bypassing the dopaminergic links, whereas neuroleptics could affect dopamine functions in limbic structures, striatum, and cortex. The different psychotic symptoms are probably also related to a dysfunctioning of various brain structures, which is at variance with the hypothesis of Snyder et al. (1974) that dopaminergic receptors of the mesolimbic system are the exclusive site for antipsychotic activity. Stevens (1973) pointed to the striking parallelism between the “limbic striatum” and the neostriatum. Dopaminergic cell bodies located in the ventral midbrain project to the neostriatum and the limbic striatum and further, respectively, to the globus pallidus and the substantia inominata. Further, the cortex topographically projects into the caudate putamen, on the one hand, and the limbic striatum, on the other (Kemp and Powell, 1970). Both the neostriatum and the limbic striatum could act as central gating mechanisms for cortical output. It is thus quite feasible that both structures, when functioning pathologically, are substrates of psychosis. Stevens (1973, p. 187) wrote that “Although the circumstantial evidence for abnormal function in a limbic striatal gate in schizophrenia is heuristically attractive, direct anatomical evidence is sparse.” Anatomical evidence for an abnormal striatum was, however, already described in 1955 by Mettler. The self-stimulation inhibition and its reversal probably reflect a part of the extensive countertherapeutic effects found in the clinic. A failure of the cholinergic system in the striatum may be related to the social avoidance aspects described by Singh and Kay (1975). The practical consequence is that one should avoid the routine use of antiparkinsonian drugs in conjunction with neuroleptics. Because high doses of specific neuroleptics cause extrapyramidal side effects, the degree of parkinsonism has been used as an index of efficacy. The fact
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that both phenomena are related to a striatal dopaminergic receptor blockade leads to this erroneous conclusion and was experimentally contradicted by Bishop et al. (1965). ACKNOWLEDGMENTS I would like to thank Dr. E. T. Rolls of the University of Oxford, England, for reading a first version of this paper, Dr. B. Costall of the University of Bradford, England, for the
corrections she suggested, and Professor Dr. J. M. H. Vossen of the University of Nijmegen. The Netherlands, for many critical comments and suggestions which lead to a final version. I also would like to thank W. Melis and F. Fransen for their excellent technical assistance.
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enzymatic degradation, 157 maturation and, 174-177 pre- and postsynaptic, differences in, 229-23 1 regional distribution of, 164-172 substrate specificity, 158,163- 164 in sympathetic ganglia, 172 y-Aminobutyrylcholine, presynaptic inhibitionand, 143-144 truns-4-Aminocroronicacid, 137,139 3-Aminocyclopentane- 1-carboxylicacid, 137 4-Aminotetrolic acid, 137 Anion vs. cation transport selectivity, 324327 Anticholinergic chemicals, self-stimulation inhibition, 380-384 Antiparkinsonian drugs, 383-384 Antipsychotic drugs, see also Neuroleptic drugs action, 3-8 general characteristics, 2-3 Aspartate antagonists of, 148-150 biochemical studies with invertebrate preparations, 192- 193 with vertebrate preparations, 187-191 physiologic-pharmacologicstudies in invertebrates, 152-154 in vertebrates, 147-150 tissue localization, 132- 133 Astrocyte, see Astroglia Astroglia,seeafso Glial cell localization, 276 ontogeny, 276 Auditory cortex, of cetacea, 62-64 Auditory system, of cetacea, 58-64 Azaperone, self-stimulation behavior and, 355,360,366
A
N-Acetylserotonin, assay, 260-263 Acoustic sense organs, cetacean, physiology, 88-89 Acoustic signals, cetacean, 74-84 production mechanism, 74-75 Acute dystonia, clinical features, 9 Akathisia, clinical features, 9 PAlanine, 300 Alamethicin channel properties, 3 18 transport models and, 3 12-3 13 Amine, biogenic assay, 259-260 distribution in brain areas, 270 Amino acid neurotransmitters, 129-204, see also y-Aminobutyric acid; Aspartate; Glutamate; Glycine; Taurine transport into glial cells, 297-303 Amino acid receptors, tissue culture cells and, 198-202 /3-Amino-n-butyric acid, 198 y-Aminobutyric acid antagonistsof, 133-136,151,165 binding studies, 154-177.191- 192 biochemical studies with invertebrate preparations, 191-1 92 with vertebrate preparations, 154- 177 ionic mechanism of PAD action, 244246 ph ysiologic-pharmacologicstudies in invertebrates, 150-151 in vertebrates, 133-144 presynaptic inhibition and, 142- 144 as presynaptic transmitter, 227-228 as primary afferent depolarization transmitter, 240-248 structural formula, 134 structure/activity studies, 136- 139, 246-247 tissuelocalization, 131 transport in glial cells, 299 y- Aminobutyric add-ergic synapses, activators of, 139-142 y-Aminobutyric a d d receptors, 154- 177 bicuculline sensitivity, 156-163 distribution of, 247-248 environment and, 177
B
BABA, see @-Amino-n-butyricacid Balcofen,see Lioresal Barbiturate postsynaptic effects of, 139- 140 presynaptic inhibition and, 143 Bat auditory and visual nuclei, 60 echolocation, 86
405
406
SUBJECT INDEX
Behavior, of cetacea, 97-98 Behavioral chemoreception, of cetacean, 57-58
Benperidol, self-stimulation behavior and, 356,360,366
Benzodiazepines postsynaptic effects of, 140-142 presynaptic inhibition and, 143 Benzylpenicillin,antagonist, of GABA, 135 BIC,see Bicuculline Bicuculline, antagonist, of GABA, 134,135, 138,151,163,165
Bicuculline-insensitive GABA receptors, drugs affecting, 139 Bicuculline methochloride, 136, 137, 138 Brain microdissection techniques, 265-270 self-stimulation behavior, 335-395 Brain stem, of cetacean, 56-57 Brain stimulation, in cetacea, 94-95 Brain tumor, glial cell types and, 291 Bromperidol, self-stimulation behavior and, 353,360,366 Butaclamol, self-stimulation behavior and, 357,360,366
C Catecholamine assay, 260,263-265,266 hypertension and stress and, 27 1-273 interaction with serotonin, in brain, 270-271
Catecholamine hypothesis of selfstimulation behavior, 336 Cation, monovalent vs. divalent selectivity, 327-329
Cation, monovalent, membrane transport models, 3 11-332 Cerebellum of cetacean, 56-57 role of glia in, 279-28 1 Cerebral cortex of cetacea, 64-70.89-94 role of glia in, 278-279 Cetacea acoustic signals, 74-84 auditory system, 58-64 behavior, 97-98 behavioral audition, 74-84 behavioral chemoreception, 57-58
brain stem, 56-57 brain stimulation, 94-95 cerebellum ,56-5 7 cerebral cortex, 64-70,89-94 chemoreceptor, 57-58 cognition, 98- 103 communication, 77-8 I , 98- 103 echolocation, 74-75,84-88 learning, 98- 103 neural ontogeny, 52-53 neuroanatomy, 52-70 neurophysiology, 70-1 03 peripheral nervous system, 53-56 physiology of acoustic sense organs and brain centers, 88-89 psychobiology, 47- 107 reflex control of air movement, 73-74 sleep and wakefulness, 95-97 sound perception, 8 1-84 sound production mechanism, 74-75 sounds of, 75 Soviet research on, 49-52,103-106 spinal cord, 56-57 visual system, 64 Chemoreceptor, of cetacean, 57-58 Chlorpromazine, self-stimulation behavior and, 352,360,366,381,382,389 Clopimozide, self-stimulation behavior and, 354,360,366
Clothiapine, self-stimulation behavior and, 352,360,366
Clozapine, self-stimulation behavior and, 352,360,366
Cognition, in cetacea, 98- 103 Communication, in cetacea, 77-81,98-103 Cyclic nucleotides, glial cell receptors and, 301-303 D
DABA, see ~-2,4-Diaminobutyricacid Denervation supersensitivity, see Disuse supersensitivity L-2.4-Diaminobutyricacid, 300 Disuse supersensitivity, 12, 16-27 behavioral evidence for, 16-19 contradictory evidence, 19-22 evaluation of, in humans, 24-27 neurophysiological and biochemical evidence, 22-24
SUBJECT INDEX
Dog, self-stimulation behavior, 368-372 Dopamine assay, 260,263-265 metabolism in brain, 7 role in etiology of tardive dyskinesia, 15-30 Dopamine-p-hydroxylase, inhibition effects, 272 Dopamine neurons effect of neuroleptic drugs on, 6 in mammalian brain, 5 Dorsal cochlear nudeus, 59,60 Dorsal root potential, 235 potassium and, 238-240 Droperidol, self-stimulation behavior and, 356,360,366 DRF',see Dorsal root potential Dyskinesias,see Neuroleptic dyskinesias; Tardive dyskinesias E
Echolocation, in cetacea, 74-75,84-88 Electrical field, primary afferent depolarization and, 236 Ependymal cell, see also Glial cell localization, 277 ontogeny, 276 Epinephrine, assay, 260,263-265 Extrajunctional Ach receptor, channel properties, 3 18
F Factor I, 150 Fluanisone, self-stimulation behavior and, 355,360,366 Fluspiperone, self-stimulation behavior and, 356,360,361,366
G GABA,see y-Aminobutyric acid GF, see Glial factor Glial cell amino acid neurotransmitters and, 297303 amino acid release, 300-301 amino acid transport, 297-300 brain tumor types arising from, 291 classification,2 7 6 2 7 8
407
depolarization, 294-295 extracellular potential shifts, 294-295 function of, potassium and, 290-297 membrane potential, 290-292 neuronal migration and connectivity and, 278 ontogeny, 276 physiology of, 275-304 regulation of extracellular potassium and, 293-294 role in CNS development, 278-290 seizure activity and, 296 spreading cortical depression and, 296297 Glial cell receptors, cyclic nucleotide responses and, 30C303 Glial factor, 286-287 Glial-neural growth factors, 285-288 Glial-neuronal differentiation, 283-290 Glial-neuronal interaction, 193- 198,275304 Glutamate antagonists of, 148-150 biochemical studies with invertebrate preparations, 192-193 with vertebrate preparations, 187-191 physiologic-pharmacologic studies in invertebrates, 152- 154 in vertebrates, 147-150 tissue localization, 132-133 transport into glial cells, 298-299 Glutamine-glutamate neuronal-glial cycle, 301,302 Glutamate receptor, channel properties, 3 18 Glycine antagonists of, 144-147 binding studies, 183-187 biochemical studies with vertebrate preparations, 177-187 physiologic-pharmacologicstudies in invertebrates, 151 in vertebrates, 144-147 tissue localization, 131-132 uptake in glial cells, 300 Gramicidin A, transport selectivity models and, 324-329 Gramicidin A channel properties, 3 19,320 structure, 314-317
408
SUBJECT INDEX H
Hair cell mechanoreceptor, channel properties, 318 Haloperidol, self-stimulation behavior and, 353,360,363,366,387 5-HT,see 5-Hydroxytryptamine Human, auditory and visual nuclei, 61 bHydroxytryptamine, 138 I
IMA,see Imidazoleacetic acid Imidazoleacetic acid, 137,138 Inferior colliculus, 62 Ionic conductance mechanism, 228-229 Ion transport, selectivity theories, 324-329 K
Kainate, structural formula, 190 L
Lateral lemniscus, 62 Learning, in cetacea, 98-103 Lioresal, presynaptic inhibition and, 144 M
Medial nucleus, 6 1 Membrane channel electric-field-dependent formation theory, 317-324 models,312-314 monovalent cation selective,3 11-332 Membrane channel-forming peptides, polymorphism of, 329-332 Metanephrine, 264-265 Methoxytryptamine, 264 N-Methyl-maspartate, structural formula, 190 Metoclopramide, self-stimulation behavior and, 357,360,366 Microglia,see ako Glial cell localization, 277-278 ontogeny, 276 Moperone, self-stimulation behavior and, 353,360,366 Muscimol, 137
N
Narcotic analgesic, comparison to neuroleptic effects, 380-383 NE,see Norepinephrine Nerve growth factor, 286-287 Nerve terminal potential effect of inhibitory transmitter on, 2222 24 excitatory, 219-222 Neural ontogeny, of cetaceans, 52-53 Neuroanatomy ,of cetaceans, 52-70 Neuroglia,see Glial cell Neuroleptic-anticholinergicinteraction differential antagonism, 384-388 literature review, 379-380 Neuroleptic drugs, see a130Antipsychotic drugs action, 3-8 brain self-stimulation behavior and, 335-395 classification,373-377 control base-line, 359-362 dose-effect relationship, 358,383 effect on dopamine neurons, 6 impairment of self-stirnulation, 391-395 neurologic side effects, 8- 12 potency, 358-359,373-375 spectral map analysis, 375-378 Neuroleptic dyskinesia, experimental, 35-36 Neuromuscular junction Ach channel, channel properties, 318 Neuron function of, glial cells and, 193-198 migration and connectivity of, 278-28 1 myelination, 28 1-283 Neuronal-glial genetic regulation, 288-290 Neurophysiology of cetacea, 70- 103 methodology, in cetacean studies, 70-73 Neurotransmitter amino acids as,see Amino acid neurotransmitters autoradiographic studies, 199-200 localized, microquantitation, 259-274 primary afferent depolarization and, 236-238 research summary, 202-204 tardive dyskinesia and, 30-35
409
SUBJECT INDEX
Neurotransmitter, presynaptic, 227-228 NGF,see Nerve growth factor Node of Ranvier, channel properties, 3 18 Norepinephrine, 138 assay, 260,263-265 Normetanephrine, 264-265
0 Oligodendroglia, see aLFo Glial cell localization, 276-277 ontogeny, 276 role in myelin formation, 281-283 Oxiperomide, self-stimulation behavior and, 357,360,366 P
PAD,see Primary afferent depolarization Parkinsonism, clinical features, 9 Penfluridol, self-stimulation behavior and, 354,360,366 Peptide liberation mechanism, in channel model, 328 Perioral tremor, clinical features, 9 Peripheral nervous system, of cetaceans, 53-56 Perphenazine, self-stimulation behavior and, 38 1,382 Photoreceptor, channel properties, 3 18 PIC,see Picrotoxin Picrotoxin, antagonist, of GABA, 133-134, 142-143,145,152,165 Pimozide, self-stimulation behavior and, 354,360,366,387,389 Pipamperone, self-stimulation behavior and, 355,360,366,387 Poly-AAG channel properties, 319,321-323 transport selectivity models and, 324329 Postsynaptic inhibition, 139-142 Potassium dorsal root potential and, 238-240 neuroglial function and, 290-297 Presynaptic inhibition, 142- 144 anatomical considerations, 226-227 effects of PAD on, 249-251 electrical transmission and, 233
electrophysiological mechanism of, 218-226 in invertebrates, 218-233 mechanisms, 227 pharmacology of, 227-233 research summary, 251-253 transmitter and ionic mechanisms, 2 17253 in vertebrates, 233-251 Primary afferent depolarization axo-axonic synapses and, 248 effect on synaptic transmission, 249-25 1 electrical fields and, 236 GABA and, 240-248 neurotransmitters and, 236-238 origin and nature of, 235-240 potassium and, 238-240 presence and location, 234-235 Prochlorperazine, self-stimulation behavior and, 38 1 Psychotropic assay positioning of, 377-379 self-stimulation and, 372-379
R Rabbit syndrome, clinical features, 9 Rat, electrode implantation, surgical techniques, 337-338 Receptors, physiological,studies on, 129204 Respiration, of cetacea, 73-74 Retina, role of glia in, 28 1
s Seizure, glial cell and, 296 Self-stimulation behavior, 335-395 catecholamine hypothesis, 336 clinical implications, 395-398 data analysis, 340-349,361 experimental methods, 337-339 impairment of, 391-395 implantation site, 365-367 influence of neuroleptics on, 349-365 psychotropic assays, 372-379 species differences, 367-372 spectral map analysis, 375-378 test procedures, 339-340
410
SUBJECT INDEX
Serotonin assay, 260,261-263 interaction with catecholamines, in brain, 270-271
Sleep, in cetacea, 95-97 Somatic cell hybrids, neuronal-glial genetic regulation and, 288-290 Sound perception, in cetacea, 81-84 Spectral map analysis, 375-378 Spinal cord of cetaceans, 56-57 role of glia in, 28 1 Spiperone, self-stimulation behavior and, 356,360,361,366
Spreading cortical depression, glial cells and, 296-297 Squid giant axon, channel properties, 3 18 Strychnine, antagonist, of glycine, 134, 180-183
Superior olivary complex, 59-61,62 Synapse, excitatory, transfer function of, 224-225
Synaptic transmission, effects of PAD on, 249-251 T
Tardive dyskinesia, 1-37 clinical features, 9, 10 differential pharmacology of, 12, 13 disuse supersensitivity concept and, 12, 16-27 etiology, 12-36
experimental induction of, 35-36 neuroleptic drugs and, 8-12 pathophysiology, 12-36 presynaptic mechanisms for, 27-30
role of neurotransmitters in, 30-35 Taurine biochemical studies with vertebrate preparations, 151-152 physiologic-pharmacologicstudies in invertebrates, 15 1- 152 in vertebrates, 147 tissue localization, 132 uptake in glial cells, 299-300 Tetramethylenedisulfotetramine,antagonist, of GABA, 135 Thiopropazate, self-stimulation behavior and, 381,382 Thioproperazine, self-stimulation behavior and, 381 Thioridazine, self-stimulation response rates and, 352,360,361,366,381,382 Tissue culture cell, neurotransmitter studies in, 198-199 TMDST, see Tetramethylenedisulfotetramine Transport, of monovalent cations, 3 11-332 Trapezoid body, lateral nucleus, 61 Trifluoperazine, self-stirnulation behavior and, 381,382 Tryptamine, assay, 260-263 d-Tubocurarine, antagonist, of GABA, 135 V
Ventral cochlear nudeus, 59,60 Visual system, of cetacea, 64 W
Wakefulness, in cetacea, 95-97
CONTENTS OF PREVIOUS VOLUMES Volume 1
Hemicholiniums F. W. Schueler
Recent Studies of the Rhinencephalon in Relation to Temporal Lobe Epilepsy and Behavioral Disorders W. R. A&y Nature of Electrocortical Potentials and Synaptic Organizations in Cerebral and Cerebellar Cortex Dominick P. Purpura Chemical Agents of the,Nervous System Catherine 0.Hebb Parasympathetic Neurohumors: Possible Precursors and Effect on Behavior Carl C. Pfeqfer Psychophysiology of Vision G. W. Granger Physiological and Biochemical Studies in Schizophrenia with Particular Emphasis on Mind-Brain Relationships Rob& G. Heath Studies on the Role of Ceruloplasmin in Schizophrenia S. Martens, S. Vallbo, and B. Melander Investigations in Protein Metabolism in Nervous and Mental Diseases with Special Reference to the Metabolism of Amines F. Georgi, C. G. Honegger, D. Jordan, H . P. Rieder, and M.Rottenberg AUTHOR INDEX-SUBJECT
INDEX
Regeneration of the Optic Nerve in Amphibia R. M . Gaze Experimentally Induced Changes in the Free Selection of Ethanol Jorge Mardones Mechanism
Brain Neurohormones and Cortical Epinephrine Pressor Responses as Affected by Schizophrenic Serum Edward J . Walmzek The Role of Serotonin in Neurobiology Enninio Costa Drugs and the Conditioned Avoidance Response Albert Hertz Metabolic and Neurophysiological Roles of y-Aminobutyric Acid Eugene Roberts and Eduardo Eidelberg Objective Psychological Tests and the Assessment of Drug Effects H. J . Eysenck AUTHOR INDEX-SUBJECT
INDEX
Volume 3
Submicroscopic Morphology and Function of Glial Cells Eduardo De Robertis and H . M . GerschenfeeM Microelectrode Studies of the Cerebral Cortex Vahe E. Amassian
Volume 2
The
The Role of Phosphatidic Acid and Phosphoinositide in Transmembrane Transport Elicited by Acetylcholine and Other Humoral Agents Lowell E. Hokin and Mabel R . Hokin
of
Action
of
the
Epilepsy Arthur A. Ward, Jr. Functional Organization of Somatic Areas of the Cerebral Cortex Hiroshi Nakahumu Body Fluid Indoles in Mental Illness R. Rodnight
41 1
412
CONTENTS OF PREVIOUS VOLUMES
Some Aspects of Lipid Metabolism in Nervous Tissue G. R . Webster
The Electrical Activity of a Primary Sensory Cortex: Analysis of EEG Waves Walter J . Freeman
Convulsive Effect of Hydrazides: Relationship to Pyridoxine Hany L. Williams and James A. Bain
Mechanisms for the Transfer of Information along the Visual Pathways Koiti Motokawa
The Physiology of the Insect Nervous System D. M . Vowles
Ion Fluxes in the Central Nervous System F. J . Brinlqr, Jr.
AUTHOR INDEX-SUBJECT
INDEX
Volume 4
The Nature of Spreading Depression in Neural Networks Sidney Ochs Organizational Aspects of Some Subcortical Motor Areas Werner P . Koelh Biochemical and Neurophysiological Development of the Brain in the Neonatal Period Willbmina A. Himwich
lnterrelationships between the Endocrine System and Neuropsychiatry Richard P . Michael and James L. Gibbons Neurological Factors in the Control of the Appetite Andrt! Soukairac Some Biosynthetic Activities of Central Nervous Tissue R. V. Coxon Biological Aspects of Electroconvulsive Therapy Gunnar Holmberg AUTHOR INDEX-SUBJECT
INDEX
Substance P: A Polypeptide of Possible Physiological Significance, Especially within the Nervous System F. Lembeck and G. Zelter
Volume 6
Anticholinergic Psychotomimetic Agents L. G. Abood and J . H . Biel
Patterns of Muscular Innervation in the Lower Chordates Quentin Bone
Protein Metabolism of the Nervous System Abel Lajtha
Benzoquinolizine Derivatives: A New Class of Monamine Decreasing Drugs with Psychotropic Action A. Pletscher, A. Brossi, and K. F. Gqr
The Neural Organization of the Visual Pathways in the Cat Thomas H . M&l, Jr. and James M . S p a p
The Effect of Adrenochrome and Adrenolutin on the Behavior of Animals and the Psychology of Man A. Hoffer
Properties of Afferent Synapses and Sensory Neurons in the Lateral Geniculate Nucleus P . C. Bishop
AUTHOR INDEX-SUBJECT
Regeneration in the Vertebrate Central Nervous System Carmine D. Clemenle
INDEX
Volume 5
The Behavior of Adult Mammalian Brain Cells in Culture Ruth S . Geiger
Neurobiology of Phencyclidine (Sernyl), a Drug with an Unusual Spectrum of Pharmacological Activity Edward F. Domino
413
CONTENTS OF PREVIOUS VOLUMES
Free Behavior and Brain Stimulation Josi M. R . Delgado AUTHOR INDEX-SUBJECT
INDEX
Volume 7
Alteration and Pathology of Cerebral Protein Metabolism Abel Lajtha Micro-Iontophoretic Studies on Cortical Neurons K. Krnjevu Responses from the Visual Cortex of Unanesthetized Monkeys John R. Hughes Recent Development of the Blood-Brain Barrier Concept Ricardo E h t r h Monoamine Oxidase Inhibitors Gordun R. Pschkdt The Phenothiazine Tranquilizers: Biochemical and Biophysical Actions Paul S. Guth and Morris A. Spirtes Comments on the Selection and Use of Symptom Rating Scales for Research in Pharmacotherapy J . B. Wittenborn Multiple Molecular Forms of Brain Hydrolases Joseph Bemohn and Kevin D. Bawon AUTHOR INDEX-SUBJECT
INDEX
Effects of Drugs on Learning and Memory James L. McGaugh and Lewis F. Petrinovich Biogenic Amines in Mental Illness Grintrr G. Brune The Evolution of the Butyrophenones, Haloperidol and Trifluperidol, from Meperidine-like 4-Phenylpiperidines Paul A. J . Janssen Amplitude Analysis of the Electroencephalogram (Review of the Information Obtained with the Integrative Method) Leonide Goldrtein and Raynond A. Beck AUTHOR INDEX-SUBJECT
INDEX
Volume 9
Development of “Organotypic” Bioelectric Activities in Central Nervous Tissues during Maturation in Culture Stanley M . Crain The Unspecific Intralaminary Modulating System of the Thalamus P. KruH and M. Monnier The Pharmacology of Imipramine and Related Antidepressants Laszlo Gymnek Membrane Stabilization by Drugs: Tranquilizers, Steroids, and Anesthetics Philip M. Seeman Interrelationships between Phosphates and Calcium in Bioelectric Phenomena L. G.Abood
Volume 8
The Periventricular Stratum of the Hypothalamus Jerome Sutin
A Morphologic Concept of the Limbic Lobe Lowell E. White, Jr.
Neural Mechanisms of Facial Sensation I. Darion-Smith
The Anatomophysical Basis of Somatosensory Discrimination David Bowsher, with tht collaboration .f Denis Albe-Fessard Drug Action on the Electrical Activity of the Hippocampus Ch. Stumpf
AUTHOR INDEX-SUBJECT
INDEX
Volume 10
A Critique of Iontophoretic Studies of Central Nervous System Neurons G. C. Salmoiraghi and C. N. SlefanL
414
CONTENTS OF PREVIOUS VOLUMES
Extra-Blood-Brain-Barrier Brain Structures Werner P . Koella and Jerome Sutin
Endocrine and Neurochemical Aspects of Pineal Function Bila Mess
Cholinesterases of the Central Nervous System with Special Reference to the Cerebellum Ann Silver
The Biochemical Investigation of Schizophrenia in the USSR D. V. Lozovsky
Nonprimary Sensory Projections on the Cat Neocortex P . Bwer and K . E . Signal1 Drugs and Retrograde Amnesia Albert Weissmun Neurobiological Action of Some Pyrimidine Analogs Harold Koenig A Comparative Histochemical Mapping of the Distribution of Acetylcholinesterase and Nicotinamide Adenine Dinudeotide-Diaphorase Activities in the Human Brain T . Ishii and R. L. Frk&
Results and Trends of Conditioning Studies in Schizophrenia J . Saanna Carbohydrate Metabolism in Schizophrenia Per S. Lingiaerde The Study of Autoimmune Processes in a Psychiatric Clinic S.
F. S m o v
Physiological Foundations of Mental Activity N . P. Bechtereva and V. B . Gretchin AUTHOR INDEX-SUBJECT
INDEX
CUMULATIVE TOPICAL INDEX FOR VOLUMES
1-10
Behavioral Studies of Animal Vision and Drug Action Hugh Brown
Volume 12
The Biochemistry of Dyskinesias C. Curzon
Drugs and Body Temperature Peter Lomax
AUTHOR INDEX-SUBJECT
Pathobiology of Acute Triethyltin Intoxication R. Torack, J . Gordon, and J . Prokop
INDEX
Volume 11
Synaptic Transmission in the Central Nervous System and Its Relevance for Drug Action Philip B. Brad19 Exopepddases of the Nervous System Neville Marks Biochemical Responses to Narcotic Drugs in the Nervous System and in Other Tissues Dwis H . Clowt Periodic Psychoses in the Light of Biological Rhythm Research F. A . Jenner
Ascending Control of Thalamic and Cortical Responsiveness M . Steriade Theories of Biological Etiology of Affective Disorders John M . Davis Cerebral Protein Synthesis Inhibitors Block Long-Term Memory Samuel H . Barondes The Mechanism of Action of Hallucinogenic Drugs on a Possible Serotonin Receptor in the Brain J . R. Smythies, F. Beningtm, and R. D. Morin
415
CONTENTS OF PREVIOUS VOLUMES
Simple Peptides in Brain Isamu Sano
Protein Transport in Neurons Raymond J . Lasek
The Activating Effect of Histamine on the Central Nervous System M . Mmnier, R. Sauer, and A. M . Hatt
Neurochemical Correlates of Behavior M . H . Aprison and J . N. Hingtgen
Mode of Action of Psychomotor Stimulant Drugs Jacques M . van Rossum AUTHOR INDEX-SUBJECT
Some Guidelines from System Science for Studying Neural Information Processing Donald 0. Walter and Martin F. Gardiner AUTHOR INDEX-SUBJECT
INDEX
INDEX
Volume 13
Volume 14
Of Pattern and Place in Dendrites Madge E. Scheibel and Arnold B. Scheibel
T h e Pharmacology of Thalamic and Geniculate Neurons J . W . Phillis
The Fine Structural Localization of Biogenic Monoamines in Nervous Tissue Floyd E. Bloom Brain Lesions and Amine Metabolism Robert Y. Moore Morphological and Functional Aspects of Central Monoamine Neurons KjeU Fuxe, Tontc~sHokjXt, and Urban Ungerstedt Uptake and Subcellular Localization of Neurotransmitters in the Brain Solomon H. S n w , Muhuel J. Kuhur, Alan I. Green, Joseph T. Coyle, and Edward G. Sharkan Chemical Mechanisms of TransmitterReceptor Interaction John T. Garland and Jack Durell The Chemical Nature of the Receptor Site-A Study in the Stereochemistry of Synaptic Mechanisms J . R . Smythies Molecular Mechanisms in Information Processing Gewges Ungar The Effect of Increased Functional Activity on the Protein Metabolism of the Nervous System B. Jakoubek and B. Senaiginousky’
The Axon Reaction: A Review of the Principal Features of Perikaryal Responses to Axon Inquiry A. R . Lieberman CO, Fixation in the Nervous Tissue Sre-Chuh Cheng Reflections on the Role of Receptor Systems for Taste and Smell John G. Sinclair Central Cholinergic Mechanism and Behavior S. N . Pradhun and S. N. Dzlna The Chemical Anatomy of Synaptic Mechanisms: Receptors J . R. Smythies AUTHOR INDEX-SUBJECT
INDEX
Volume 15
Projection of Forelimb Group I Muscle Afferents to the Cat Cerebral Cortex Ingmar Rosin Physiological Paihways through the Vestibular Nuclei Victor J . Wilson
416
CONTENTS OF PREVIOUS VOLUMES
Tetrodotoxin, Saxitoxin, and Related Substances: Their Applications in Neurobiol-
Volume 17
ow
Epilepsy and y-Aminobutyric Mediated Inhibition B. S. Meldrum
Marh'n H. Evans
The Inhibitory Action of y-Aminobutync Acid, A Probable Synaptic Transmitter Kunihiko O h Some Aspects of Protein Metabolism of the Neuron M k Satahe Chemistry and Biology of Two Proteins, S-100 and 14-3-2, Specific to the Nervous
System
Blake W . Moore The Genesis of the EEG Rafml Elul Mathematical Identification of Brain States Applied to Classification of Drugs E. R. John, P. W a l k , D. Cawood, M. Rwh, and J . Gehrmann AUTHOR INDEX-SUBJECT
INDEX
Volume 16
Model of Molecular Mechanism Able to Generate a Depolarization-Hyperpolanzation Cyde Clara Tor& Antiacetylcholine Drugs: Chemistry, Stereochemistry, and Pharmacology T.D.Inch and R. W . Brimblecombe Kryptopyrrole and Other Monopyrroles in Molecular Neurobiology Donald G.I m h e RNA Metabolism in the Brain Victor E. Shmhowl A Comparison of Cortical Functions in Man and the Other Primates R. E. Passingham and G. Ettlinger Porphyria: Theories of Etiology and Treatment H.A. Peters, D. J . Cripps, and H. H. Reese SUBJECT INDEX
Acid-
Peptides and Behavior Georges Ungar Biochemical Transfer of Acquired Information S. R. Mitchell, J . M. Beaton, and R. J . Bradley Aminotransferase Activity in Brain M. Benuck and A. hjtha The Molecular Structure of Acetylcholine and Adrenergic Receptors: An All-Protein Model J . R. Smythies Structural Integration of Neuroprotease Activity Ekna Gabrielescu On Axoplasmic Flow Ldiana Lubihka Schizophrenia: Perchance a Dream? J . Christian Gillin and Richard J . Wyatt SUBJECT INDEX
Volume 18
Integrative Properties and Design Principles of Axons Stephen G. Waxman Biological Transmethylation Involving S-Adenosylmethionine: Development of Assay Methods and Implications for Neuropsychiatry Ross J . Baldessarini Synaptochemistry of Acetylcholine Metabolism in a Cholinergic Neuron Berialan Csillih Ion and Energy Metabolism of the Brain at the Cellular Level L e i Hertz and Arne Schowboe
CONTENTS OF PREVIOUS VOLUMES
Aggression and Central Neurotransmitters S. N. Pradhan
A Neural Model of Attention, Reinforcement and Discrimination Learning Stephen Grossberg Marihuana, Learning, and Memory Ernest L. Abel Neurochemical and Neuropharmacological Aspects of Depression B. E. L e m r d
417
Thymoleptic and Neuroleptic Drug Plasma Levels in Psychiatry: Current Status Thomus B. Cooper, George M . Simpson, and J . Hills? Lee SUBJECT INDEX
Volume 20 Functional Metabolism of Brain Phospholipids G. Brian Ansell and Sheila Spanner
SUBJECT INDEX
Isolation and Purification of the Nicotine Acetylcholine Receptor and Its Functional Reconstitution into a Membrane Environment Michael S. BrilS) and JeanrPiene Chungewr
Volume 19 Do Hippocampal Lesions Produce Amnesia in Animals? Susan D. Iversen Synaptosomal Transport Processes Giulio Leui and Maurizio Raitm' Glutathione Metabolism and Some Possible Functions of Glutathione in the Nervous System Marian Orlowski and Abrahum Karkowsky Neurochemical Consequences of Ethanol on the Nervous System Awn K. Rawat Octopamine and Some Related Noncatecholic Amines in Invertebrate Nervous Systems H. A. Robertson and A. V. Juorio Apomorphine: Chemistry, Pharmacology, Biochemistry F. C. Colpaert, W. F. M . Van Beuer, and J . E. M. F. Leysen
Biochemical Aspects of Neurotransmission in the Developing Brain Joseph T . Coyle The Formation, Degradation, and Function of Cyclic Nucleotides in the Nervous System John W . Daly Fluctuation Analysis in Neurobiology Louis J . DeFelice Lipotropin and the Central Nervous System W. H. Gispen, J . M . van Ree, and D. de Wied Tissue Fractionation in Neurobiochemistry: An Analytical Tool or a Source of Artifacts Pierre Lnduron Choline Acetyltransferase: A Review with Special Reference to Its Cellular and Subcellular Localization Jean Rossier SUBJECT INDEX
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