International Review of Neurobiology Volume 25

INTERNATIONAL REVIEW OF Neurobiology VOLUME 25 Editorial Board W.Ross ADEY JULIUS AXELROD KETY SEYMOUR KEITH KILL...

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INTERNATIONAL REVIEW OF

Neurobiology VOLUME 25

Editorial Board

W.Ross ADEY JULIUS

AXELROD

KETY SEYMOUR KEITH KILLAM

Ross BALDESSARINI

CONANKORNETSKY

SIR ROGERBANNISTER

ABELLAJTHA

FLOYDBLOOM

BORISLEBEDEV

DANIELBOVET

PAULMANDELL

PHILLIPBRADLEY

H UMPHRY OSMON D

JOSE

DELGADO

RODOLFOPAOLETT~

SIR JOHN ECCLES

SOLOMONSNYDER

JOEL ELKES

STEPHEN SZARA

H. J. EYSENCK

SIR JOHN VANE

KJELL FUXE

MARAT VARTANIAN

B o HOLMSTEDT

RICHARD WYATT

PAULJANSSEN

OLIVER ZANGWILL

INTERNATIONAL REVIEW OF

Neurobiology Edited by JOHN R. SMYTHIES Department of Psychiotry and The Neurosciences P rogrom University of Alobomo Medical Center Birmingham, Alabama

RONALD J. BRADLEY The Neurosciences Program University of Alabama Medical Center Birmingham, Alabomo

VOLUME 25

1984

ACADEMIC PRESS, INC. (Harcourt Brace Jovonovich, Publishers)

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COPYRIGHT @ 1984, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS,INC.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/20 Oval Road. London N W l IDX

LIBWRYOF CONGRESS CATALOG CARDNUMBER: 59 - 1 3 8 2 2 I S B N 0-12-366825-5 PRINTED IN THE UNITED STATES OF AMERICA

04 05 06 01

98 1 65 4 3 2 1

CONTENTS CONTRIBUTORS

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

iX

Guanethidine-Induced Destruction of Sympathetic Neurons

EUGENEM. JOHNSON. JK..A N D

PAMELA TOY

MANNING

I . Introduction I l l . Discovery of Guanethidine-Induced Sympathectomy V. Structure-Activity Relationships

VI. Mechanism of Destruction of Sympa

eurons .......................

13

VIII. Assessment of the Degree of Sympathectomy Produced by Guanethicline . . . 23 IX. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 References ...............................

Dental Sensory Receptors

MAKGAREI-R. BYEKS 1. Introduction

Ill. IV. V. VI. VI1.

Location of Sensory Nerve Endings Ultrastructure of Sensory Nerve Endings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neural Relationship to Other Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensory Transduction Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ........................ .....

68

74 80 85 87

Cerebrospinal Fluid Proteins in Neurology

A. LOWENTHAL, R. CROLS,E. DE SCHUTI'EK, J . GHEL'ENS. D. KARCHER,M. NOPPE,A N D A. T.4S"IER 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Quantitative Determinations of Proteins . . . . 111. Qualitative Studies ...............................

V. Enzymatic Determinations

V

95

vi

CONTENTS Muscarinic Receptors in the Central Nervous System

MORDECHAI SOKOLOVSKY I. Introduction

........

111.

IV. V. VI. VII. VIII. IX.

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

139

... Binding of Antagonists ................................................. 147 . . . . . . . . . . . . . . . 156 Binding of Agonists .............. ................................. 161 Receptor-Receptor Interactions ...

11. Direct Receptor Chara

zation: Kadioligand-Binding Studies

Localization of Muscarinic Receptor Solubilization and Isolation of the Muscarini Structure and Function Relationship of Muscarinic Receptors . . . . . . . . . . . . . . 174 Concluding Remarks ......................................... I78 References ............................................................ Peptides and Nociception

DANIEL LUTTINGER, DANIEL E. HERNANDEZ, CIIARLES B. NEMEROFF, A N D A R T H U R J . PRANCE, JR.

I. Introduction . . . . . . . . . . . . . . . ............................ 11. Algesic Pgptides . . . . . . . . . . . . ............................ Ill. Opioid Peptides ................................... IV. Naloxone-Sensitive Nonopioid Peptides .................................. ................................ V. Naloxone-Insensitive Peptides ......

186 188 195 2 13 216

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

VII. Discussion . . ............................................ References ...............................

227

Opioid Actions on Mammalian Spinal Neurons

w. ZIEGLGANSBERGEK I. Introduction .......................................................... 11. Actions on Single Neurons .............................................

111. Concluding Remarks ................................................... References ............................................................

243 249 264 266

Psychobiology of Opioids A L B E R T 0 OLIVERIO, C L A U D 1 0 CASTELLANO, AND STEFANO PUGLISI-ALLECKA

I. Introduction

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

..........

11. Neurochemical Correlates o

111. IV. V. VI. VII.

Genetic Characterization of Endogenous Opioid Systems ... Opioids and Behavior . . . . . . Environmental Effects . . . . . . Brain Opiates and Mental Illness References . . . . . . . . . . . . . . .

............. 277 . . . . . . . . . . . . . . . . 278

..........

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

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

319

............. 322

vii Hippocampal Damage: Effects on Dopaminergic Systems of the Basal Ganglia ROBERT

L,. ISAACSON

In trodtiction . . . . . . . . . . . . . . . ................ 339 Behayioral and Anatoniical Ch The Basal Ganglia and the Hippocampal Formation . . . . . . . . . . . . . . . . . . . . . . 343 Biocheniical Changes in the Basal Ganglia after Hippocan Dopaminergic Intervention with Basal Ganglia Systems . . . . . . . . . . . . . 351 Hippocampal Lesions: Effects on Neuropeptide Actions . Dopaminergic Influences on Excessive Grooming ....... Dopaininergic Stimulation, Neuropeptides, and Hippocainpal 13estruction: Their Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Sunimary .......... ............................................... 3.37 References ............................................................ 3.58

I. 11. 111. IV. V. VI. VII. VIII.

Neurochemical Genetics

v. CSANYl I ntiwluction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potentials and Limitations of Genetic Analysis . . . . . . . . . . . . . . Natural and Experimenral Populations ................................... Heterogeneous Polylineal Populations . . . . . ........................... Honiogcneous Populations . . . . . . . . . . . . . . . . . . . . Regulated Heterogeneity ...................... MI. Conclusions .................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. 11. 111. IV. V. VI.

364 367

585

The Neurobiology of Some Dimensions of Personality b1AKVlN ZUCKEKMAN,JAMES

c. BALLENGEK. AKD KOBER'L k1. POS-I'

............................................... ......................... 111. Riocticmical Studies . . . . . . . . ..................... IV. C:onclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction

1NI)EX..

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

CON'lEN.I'S OF RECENT \'OLL:MES

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

392 998 428 432

437 442

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CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' conti-ihutions begin.

BAILENGER,~ Drpurtment (4 Behaiiiorul iZledzcinr and Py-hiatry, Lfiiiverhity of Vzrgnza Medzcal Center, CharlotteA.szdle, Vzrpriza 22901 (391)

J A M ~ C. S

MARGARE1' R. BYEKS,Departments of Anesthrhrology and Biological Slructute, Crnter fo? Reyrurch ington 98195 (39)

in

Orul Biology, U n i m yatj of Wushinglon, Senltle, W(i\h-

CLAUDIOC A S TELLANO, Institute of Psychobiology und Phycholhar~~iuroi,~, Nutzonal Research Counrzl of ltuly, 00198 Kome, Italy (277) R. CKOI.S,Luboratory of Nei(1ochYiiii\tiY, Boi ue-HiingP Foundulion, Uiizset \ilaire lnstellzng Antwerpen, B-2610 Antiiierp, Brlglurri (95) V. CSANYI. Depurtinerit of Behavior Genrlich, Loicind Edtoiis Univ~i.city,1~uc.apast, Hungary

(361)

K, Luhoratory ( f NeurocIiPrnistiy, Roi n-Bunge Foundutzon, Unrvrrsitaire Instelling Antwerpen, 8-2610 Anticlei p , Helpurn (95)

J . GHLUENS, Laboratory of Neurochemutry, Borri-Bungo Foundutzori, Univer\tlairr In stelltug Anlwerpen, B -2610 A ritiiirrl,, BPIgiwri (95)

DANIELE. H E K N A I Y DBzologiral ~L, Sitencr\ RP\roich C m t u ? , Uiiiivnily (4 North Carolina School of Medicine, Chapel Hill, North Carolina 27514 ( 185) ROBERTL. ISAACSON, Center f o i Neuiobetiazm,ul Sc irizces und Dupurtmenl oJ P\jrholoRy, State Univrruty (4 Neui York ut B l , g h u r m ~ Biirglimton, , N t ~ i York 13901 (339) Phurrriacolokgy,Wushznglon Unnirrsity School of Medzcznr, St. Louih, Micwuri 63110 ( 1 )

EUGENE M. JOHNSON, J R.,Department of

D. KARCHER,Laboruio~yof Nru~ochrrri~\try, Ror u-Hutlge Fourdution, U m w situire Instellzng Antuwrpen, B-2610 Antztwrp, Belgiuui (95)

A. LO\.V~NJTH A L, La borutory of Neil rorli ern I $1I?, Bo, n-B u nge Foil ndution, Unraerutaire lnslrlling AiItu)rr&vi, B-2h10 A n t u w j ~ Bdgiiiiii , (95) 'Present address: Departmeiit ()r Psychiatry ;ind HehavioixI Sciences. Medical Univcrsiry 01' South Carolina, Charleston, Souih Carolina 2!1425.

ix

X

CON I K I R I ’ I O K S

DANIELLU’rTINGER,2 Biological Sciences Research Center und The Neurobiology Program, Universily of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 (185) PAMELA TOY MANNI NC, Department of I’harmarology, Washington university School of Medicine, St. Louis, Missouri 63110 (1)

CHARLES B. NEMEKOFF;’ Biological Sciences Research Center, Department of‘ Psychiatry, and The Neurobiology Program, University of North Carolina School of Medicine, Chapel Hill, Norlh Carolina 27514 (185)

M. N ~ P P E Laboratory , of Neworhemistry, B o n i - B u n g Fountlation, Universitaire Instelling Antwerpen, B-2610 Antwerp, Belgium (95) A L B E R T 0 OLIVERIO, Institute of t’sychobiology avid Psycho~harmacology, National Research Council of Italy, 00198 Romp, Italy (277) ROBERT M. POST. Rtologxal Psychiatry Brunch, National Institute oJ Mentul Health, Bethesda, Maryland 20205 (391) A R T H U R J. PRANGE, JR., Biological Sciences Research Center, Department of Psychiatry, and The Newrobiology Program, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 (185)

STEFANOPUGLISI-ALLEGRA, Institute of P.~ychobiolog?rand Psychophamacology,

Nalional Resetirch Council

of

Italy, 00198 Rome, Italy (277)

MORDECHAISOKOI.OVSKY, Department of Biochemistry, George S. Wise Faculty of L f e Sciences, Tel Aviv Utiiversity, Tel Aviv 69978, Ismel ( 139)

A. TASN IER, Laboratory of Neurochernistry, Rorn-B unge Foundation, Un,iver.ritaire Instelling Antiuerpen, H-2610 Antzoerl,, Belgium (95) W. ZIEGLGANSBERGER, Department of Neuropharmarology, M a x Planck Institute f o r Psychiatry, 0-8000 Munich 4 0 , Federd Republic of‘ Germaiiy (243) MARVIN ZUCKEKMAN, Department Newark, Delaware 19711 (391)

of

Psychdogy, Uriiimsity of Dekiware,

‘Present address: Department o f Pharmacology, Sterling-Wiiithrop Rcsearcli Institute, Kensselaer, New York 12144. “Present address: Departments of Psychiatry and PhariiiacoIogy, h i l i e University Medical Center, l)urham, North Carolina 27710.

INTERNATIONAL REVIEW OF

Neurobiology VOLUME 25

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GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS By Eugene M. Johnson, Jr. and Pamela Toy Manning

Department of Pharmacology Washington University School of Medicine

St. Louis, Missouri

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. General Pharmacology of Guanethidine . . . . .............. 111. Discovery of Guanethidine-Induced Sympath y .................. IV. Degree and Specificity of the Destruction of Neurons . . . . . A. Effects of Guanethidine on Sympathetic Neurons in Viiw . . . . . . . . . . . B. Effects of Guanethidine on Sympathetic Neurons iti V i h V. Structure-Activity Relationships .................................. VI. Mechanism of Destruction of Sympathetic Neurons. . . . . . . . . . . . . . . . . . . A. Inhibition of Oxidative Phosphorylation . . . . . . . . . . B. Inhibition of the Retrograde Transport of Nerve Gr C. Immune-Mediated Mechanism. ................................ VII. Strain Specificity of Guanethidine Sympathectomy .................... VIII. Assessment of the Degree of Sympathectomy Produced by Guanethidine . A. Sympathectomy in Neonatal Rats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Sympathectomy in Adult Rats.. ................................ C. Comparison of Guanethidine-Induced Sympathectomy with that Produced by Anti-NGF or 6-Hydroxydopamine . . . . . . . . . . . . . . . . . . . IX. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. References ....................................................

I 2 4 5 5

8 9 13

16

20 23 24 28 29 33 35

1. Introduction

The objective of this review is to examine several aspects of the phenomenon of guanethidine-induced destruction of sympathetic neurons. T h e general pharmacology of guanethidine will be discussed only briefly. Emphasis will be placed on (1) the discovery of guanethidine as a means of producing sympathectomy, (2) structure-activity relationships of guanethidine and its analogs for neuronal destruction, (3) the mechanism by which guanethidine destroys the sympathetic nervous system in the rat, and (4)the utility of guanethidine as a means of producing animals with permanent peripheral sympathectomy. 1 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOI.. 25

Copyright 0 1984 by Academic Prerr, Inc. All rights of reproduction in any form reserved. ISBN 0-12-366825-5

2

EUGENE M. JOHNSON, JR. AND PAMELA TOY MANNING

II. General Pharmacology of Guanethidine

Guanethidine can be considered the prototypical adrenergic neuron blocking agent (see Fig. 1). As their primary effect, guanidinium adrenergic blocking agents act to dissociate the action potential from subsequent release of norepinephrine in sympathetic neurons. The precise molecular mechanism by which the dissociation is produced is not known. The major classes of adrenergic neuron blocking agents are the quaternary ammonium compounds (e.g., bretylium) and the guanidine derivatives, including guanidinium compounds (e.g., guanethidine), amidoxime analogs, and isoquinoline derivatives (e.g., debrisoquine). The structure-activity relationships of adrenergic blocking agents in producing neuron blockade have been reviewed by Schlitter (197’1).The general pharmacology of these agents has been extensively reviewed by Boura and Greene (1965) and by Maxwell and Wastila (1977). The possible mechanisms of adrenergic neuron blockade have been concisely reviewed by Hausler and Haefely (1979) and by Maxwell (1982). The absorption, distribution, metabolism, and excretion of guanethidine has been characterized in laboratory animals and in humans (see Maxwell and Wastila, 1977). The data involve analysis of guanethidine treatment for short periods (often a single dose) and at low doses of the drug that are related to those needed to produce adrenergic neuron blockade and/or antihypertensive effects in humans. The pharmacodynamics of guanethidine administered at high doses for extended periods, as when it is used to produce sympathectomy, have not been systematically examined. A comparison of effects produced with such treatment in neonates and in adult animals has not been carried out. The distribution of guanethidine reflects its highly ionized nature at physiological pH. After administration of a single high dose of radiolabeled guanethidine, its autoradiographic localization indicates that it is largely excluded from the central nervous system by the blood-brain barrier. In the periphery the highest concentrations are found in those tissues that are heavily innervated by sympathetic neurons (e.g., heart, brown fat) (Furst, 1967). This point reflects the basis for its selective effects on sympathetic neurons: Guanethidine is actively accumulated in

Guonethidine FIG. 1. Chemical structure of guanethidine.

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

3

sympathetic neurons by the catecholamine uptake pump. Because guanethidine is highly charged it is largely excluded from other cells, including neurons that do not possess this active transport mechanism. Following intravenous administration in the rat guanethidine, after an initial rapid fall, is eliminated from the body with a half-life of 7 h r (Schanker and Morrison, 1965). In both man and rats guanethidine is extensively metabolized. Three metabolites have been isolated and identified in urine (McMartin, 1969; McMartin and Simpson, 1971). These include the N-oxide and metabolites in which the heterocyclic ring is broken. None of these metabolites have appreciable pharmacological activity (Maitre et al., 1971), indicating that guanethidine itself, not a metabolite, is the active species. The ability of adrenergic neuron blocking agents to alter neuroeffector function is specific for the sympathetic neuron. An exception to this might appear immediately after high-dose intravenous administration of these agents. Effects on other excitable tissues such as motor neurons can occur, probably because of a local anesthetic action that these drugs possess to varying degrees at high concentrations. This may represent a problem if high doses of guanethidine, such as those used to produce sympathectomy, are injected intravenously. This can result in rapid paralysis and death. Guanethidine and other blocking agents are taken up into sympathetic nerve terminals by the catecholamine uptake pump. Tricyclic anti-depressants and other drugs that block the aniine uptake pump prevent the accumulation of guanethidine within sympathetic neurons and thus its inhibitory effect on norepinephrine release. T h e guanidinium drugs acutely cause the release of endogenous norepinephrine from nerve terminals. Guanethidine becomes incorporated into storage vesicles within nerve terminals and can subsequently be released by nerve stimulation. Because of its ability to block norepinephrine uptake and to cause norepinephrine release, guanethidine treatment leads to a reduction in tissue norepinephrine concentrations. However, blockade of neuroeffector function occurs prior to significant depletion of norepinephrine stores. Thus catecholamine depletion is not the major reason for failure of norepinephrine release in response to the action potential. As a consequence of its ability to compete with norepinephrine for uptake into nerve terminals, treatment with guanethidine and other adrenergic neuron blocking agents causes supersensitivity to exogenously administered norepinephrine. As a practical point in the evaluation of permanent sympathectomy due to neuronal death produced by high doses of guanethidine in the rat, a relatively long period of time (several days to weeks) must pass after cessation of treatment before decreased

4

EUGENE M. JOHNSON, J R . AND PAMELA TOY MANNING

tissue catecholamine levels can be used as a reliable index of neuronal destruction. The precise molecular mechanism by which guanethidine prevents norepinephrine release has not been established. Two reviews on this subject (Hausler and Haefely, 1979; Maxwell, 1982) conclude that the most likely mechanism involves membrane stabilization resulting from the weak local anesthetic activity of the agents coupled with marked accumulation of these agents within sympathetic nerve terminals.

111. Discovery of Guanethidine-Induced Sympathectomy

In 1967 Jensen-Holm first reported that chronic treatment of adult rats with guanethidine in doses well in excess of those required to produce adrenergic neuron blockade produced toxic effects on sympathetic ganglia (Jensen-Holm, 1967). The changes observed, which are similar to those reported following axotomy in the rat (Brown, 1958; JensenHolm and Juul, 1970a,b), included increases in ganglionic protein content and decreases in both specific and nonspecific cholinesterases. The decrease in the activity of both cholinesterases was localized histochemically to the neurons within sympathetic ganglia as well as to the preganglionic nerve fibers. The increase in protein content of the ganglia appeared to be due to a satellite cell infiltration (Jensen-Holm and Juul, 1968, 1970a), as there was a dramatic increase in the ratio of small cells to neurons in rats that received 20-25 mg/kg/day of guanethidine for 10 or more days (Jensen-Holm and Juul, 1970b). In addition chronic guanethidine administration produced chromatolytic changes in sympathetic ganglion cells including peripheral dislocation of the nucleus and a partial loss of Nissl substance. Cells with faintly staining cytoplasm, termed foam or ghost cells, were frequently observed. A marked decrease in formaldehyde-induced fluorescence from both neurons and nerve fibers was also observed following guanethidine administration. These effects were not changed by either pre- or postganglionic nerve division, suggesting a direct effect of guanethidine on neurons within the sympathetic ganglia (Jensen-Holm and Juul, 1970a,b). In 1971 the selective destruction of sympathetic neurons following chronic guanethidine treatment was clearly demonstrated in both neonatal (Eranko and Eranko, 1971) and adult rats (Burnstock et al., 1971a). Guanethidine produced destruction of both pre- and paravertebral sympathetic ganglion cells when administered to neonatal rats (20 mg/kg/ day for 8 days). Three weeks after the period of acute cell death the size of both the superior cervical and the celiac ganglia was reduced to ap-

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

5

proximately 10% of the size of ganglia of age-matched controls. Fewer than 2% of the nerve cell bodies in the superior cervical ganglia remained following chronic guanethidine treatment of adult rats for 6 weeks (25-30 mg/kg/day) (Burnstock et al., 197la). T h e ultrastructural appearance of mitochondria in these remaining cells was abnormal. Few fluorescent adrenergic nerve fibers were found outside the central nervous system in these guanethidine-treated rats 4 months after the cessation of treatment, indicating that the destruction was permanent (Burnstock et al., 1971a).

IV. Degree and Specificity of the Destruction of Neurons

A. EFFECTSOF GUANETHIDINE ON SYMPATHETIC NEURONS in Vivo 1. Morphological Effects on Sympathetic Neurons T h e morphological and biochemical alterations induced by chronic high-dose guanethidine treatment resulting in neuronal destruction occur in both neonatal and adult rats. However, as reported in the early studies of Eranko and Eranko (1971) and subsequently by others (Angeletti et al., 1972; Johnson et al., 1976; Klein, 1979a,b), the destruction produced by guanethidine in neonates, although qualitatively similar, is more rapid and more complete than in adult animals. Treatment of neonatal rats with doses of guanethidine of 20-100 mg/kg either daily or every other day produces destruction of the majority of peripheral sympathetic neurons. Various treatment protocols result in a 85-98% decrease in the number of neurons in the superior cervical ganglia. Destruction is specific for sympathetic neurons (Eranko and Eranko, 1971; Angeletti et al., 1972; Johnson et al., 1976). Within the first few days of guanethidine treatment, at which time the neurons exhibit clear morphological alterations, no changes occur in nonneuronal cells within the ganglia with the exception of a granular deposition in some Schwann cells (Heath et al., 1972). In addition guanethidine has no effect, either structural or ultrastructural, on cholinergic neurons or axons, purinergic neurons, sensory neurons including dorsal root ganglia and nodose ganglia neurons, ciliary neurons, parasympathetic neurons, o r cells of the adrenal medulla (Jensen-Holm and Juul, 1971; Heath et al., 1972; Angeletti et al., 1972; Johnson and Aloe, 1974; Heath and Burnstock, 1977). As mentioned previously the most dramatic alteration of guanethidine-treated sympathetic ganglia at the light microscopic level is a marked infiltration of small, round darkly staining

6

EUGENE M. JOHNSON, JR. AND PAMELA TOY MANNING

cells, particularly at the periphery of the ganglia. Prior to the time of this infiltration occasional neurons develop cytoplasmic pallor and vacuolation. However, no apparent decrease in the number of neurons in the superior cervical ganglia occurs until the time of pronounced cellular infiltration. At this time ghostlike neurons are particularly frequent, surrounded by the darkly staining small cell infiltrate. Within a few days a marked decrease in the number of viable neurons within the ganglia occurs (Angeletti et al., 1972; Manning et al., 1982). At the ultrastructural level, prior to the actual destruction of neurons (i.e., prior to the cellular infiltrate), the observed changes, most dramatically pronounced mitochondria1 swelling (Angeletti et al., 1972), are virtually indistinguishable from those that occur in the adult rat (see following paragraphs). Concurrent treatment of neonatal rats with exogenous nerve growth factor prevents the small cell infiltration and the neuronal destruction caused by guanethidine. Nerve growth factor does not prevent the mitochondrial swelling and apparently the accumulation of guanethidine in the neurons (Johnson and Aloe, 19’74).The mechanism by which this protection is produced is not known but is a particularly interesting question. Treatment of adult rats with doses of guanethidine of 20 mg/kg/day or greater for extended periods of time (2-6 weeks) also produces profound and specific effects on sympathetic neurons. However, the response to given doses is more variable (Juul, 1973). As in neonates chronic guanethidine treatment induces histological changes characterized primarily by chromatolysis of neurons accompanied by a prominent small cell infiltrate followed by cell death (Jensen-Holm and Juul, 1970a,b; Downing and Juul, 1973; Juul, 1977). Prior to the time of neuronal destruction guanethidine has been localized by microautoradiographic techniques primarily to neurons within the ganglia. Treatment with desmethylimipramine, which blocks the catecholamine uptake pump, largely prevents the uptake of guanethidine, the chromatolytic changes, and the increase in ganglionic dry weight that is indicative of small cell infiltration (Juul and Sand, 1973).Thus the histological changes that occur with high-dose treatment (greater than 10 mg/ kg/day) appear to require the accumulation of guanethidine to relatively high concentrations within the neurons, and accumulation via the catecholamine uptake pump is required to reach that high concentration (Juul and Sand, 1973). No apparent morphological changes were observed at the ultrastructural level in most sympathetic neurons in the superior cervical ganglia following prolonged guanethidine treatment at low doses (5 mg/kg/day for 18 weeks). However, at doses that produced destruction of neurons

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

7

changes occurred in sympathetic neurons within a few days. With prolonged treatment (15 days or longer) virtually all primary neurons within the superior cervical ganglia exhibited marked degenerative changes (Heath et al., 1972). Alterations in the neuronal mitochondria were consistently described. Mitochondria were swollen and distorted in outline and exhibited a substantial loss of cristae and a dispersion of the matrix. T h e outer mitochondrial membrane was preserved (Burnstock et al., 1971a; Heath et al., 1972; Juul, 1973, 1977; Heath and Burnstock, 1977; Heath and Sobels, 1977; RCvesz and van der Zypen, 1979). Additional ultrastructural changes in neurons included dilated and disoriented endoplasmic reticulum, decrease or absence of free ribosomes, peripheral dislocation of the nucleus, and development of an irregularly shaped nuclear membrane. An increase in the number of mononuclear cells (lymphocytes, plasma cells, and macrophages) but no increase in capsular cells, Schwann cells, endoneural connective tissue cells, or endothelial cells was observed. Phagocytosis of neurons was also observed (Jensen-Holm and Juul, 1971; Heath et al., 1972). In addition, although the adrenal gland accumulates guanethidine to high concentrations by the amine uptake pump, no ultrastructural changes have been described at doses that caused profound changes within the sympathetic neurons of the same animal. No cellular degeneration occurred in the adrenal gland (Jensen-Holm and Juul, 1971; Rev& and van der Zypen, 1979). In both neonatal and adult rats treated with high doses of guanethidine changes in small intensely fluorescent @IF) cells of the superior cervical ganglia have been observed. In neonates Eranko and Eranko (1971) reported a two- to fivefold increase in the number of SIF cells per ganglion. Differing effects have been reported in adult animals. Heym and Grube (1975) described a decrease in dense-core vesicles and catecholamine fluorescence and mitochondrial swelling in the cells. Eranko and Eranko (1971) reported a decrease in the number of SIF cells in ganglia of guanethidine-treated adult rats. In contrast Burnstock et al. (197 la) reported no change in the number of fluorescence intensity of SIF cells in treated adult animals. 2. Species Specijicity of Guanethidine-Induced Sympathectomy Destruction of the sympathetic nervous system produced by chronic treatment with high doses of guanethidine has been observed only in the rat. Treatment of adult guinea pigs (30 mglkglday, intraperitoneally) for 6 weeks does not result in destruction of sympathetic neurons (ODonnell and Saar, 1974). Chronic treatment of adult cats at doses of 40 mg/ kg/day (Downing and Juul, 1973), adult mice at up to 150 mg/kg/day, or adult toads at doses u p to 250 mg/kg/day failed to produce neuronal

8

EUGENE M. JOHNSON, JR. AND PAMELA TOY MANNING

destruction (Evans et al., 1979). Similarly, we have reported that treatment of newborn cats (37.5 mg/kg/day for 6 weeks), rabbits (100 mg/kg/ day for 6 weeks), and hamsters (50 mg/kg/day for 3 weeks) does not result in neuronal death (Johnson et al., 1977). In unpublished experiments we have treated gerbils and chickens using similar treatment protocols and have failed to observe neuronal destruction. Treatment of gerbils with high doses of guanethidine produces dramatic vacuolation of the neurons, but there is no apparent neuronal destruction. We have treated several inbred strains of neonatal mice with high doses of guanethidine for relatively long periods of time in the hope of finding a strain in which guanethidine destroys neurons. T h e inbred strains tested, several of which are predisposed to various autoimmune disorders, included C57BL/6, C57BL/KsJ, BlO.S, BlO.K, BlO.D, AS/N, Beige, SWWJ, D2.GD, NZB, NZW, NZB X NZW, and SJL. Several outbred strains were also tested including CD-1 (Charles River) and Swiss Webster mice obtained from various suppliers. We have consistently failed to observe neuronal death in response to chronic guanethidine treatment (P. 'r. Manning and E. M. Johnson, unpublished data). The failure of guanethidine to sympathectomize species other than the rat does not appear to be because of a failure of guanethidine to accumulate in neuronal cell bodies. The accumulation of guanethidine in cat superior cervical ganglia (Downing and Juul, 1973) and in the ganglia of hamsters and mice (E. M. Johnson, unpublished data) is greater than that required to cause destruction of sympathetic neurons in the rat. More importantly, the early ultrastructural changes (mitochondrial swelling) produced by guanethidine treatment in the rat also occur in cats and in mice, indicating that guanethidine has accumulated within the neurons of these species. These ultrastructural changes appear to be dependent upon accumulation of guanethidine, as they are prevented by concomitant administration of desmethyliniipramine in the rat (Juul and Sand, 1973). However, neuronal destruction does not occur in the cat or mouse. OF GUANETHIDINE ON SYMPATHETIC B. EFFECTS NEURONSin Vztru

Guanethidine, although it accumulates in sympathetic neurons in vitro (Wakshull et al., 1981), does not cause destruction of these cells under normal tissue culture conditions (Eranko et al., 1972; Johnson and Aloe, 1974). In concentrations ranging from 1-36 mg/liter (3-120 p M ) , it does not have a direct cytotoxic effect on sympathetic nerve cells or

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

9

small intensely fluorescent cells in cultured explants. By light microscopic evaluation no differences from control cultures were observed in any cellular components including the outgrowth of supporting cells, Schwann cells, nerve fibers, or nerve cell bodies nor was there any effect on the overall growth of ganglion explants (Eranko et al., 1972). Based on the guanethidine accumulation data of Juul and Sand (1973) following in vivo administration of guanethidine, Eranko and coworkers calculated that the cultures treated with 36 mg/liter of guanethidine were exposed to a higher concentration of guanethidine than that which occurs during high-dose treatment in vivo (60 mg/kg/day for 14 days). Thus the lack of cytotoxicity in zritro did not appear to be because of inadequate concentrations of guanethidine. This interpretation is supported by the finding that radiolabeled guanethidine is taken up by sympathetic neurons in culture (Wakshull el al., 1981); thus the concentrations within the neurons in culture should be greater than or equal to the concentration in the surrounding medium. In addition low concentrations of guanethidine (1-2 mg/liter) caused a marked increase in the number of SIF cells within the explants (Eranko et al., 1972; Heath et al., 1973) but did not induce ultrastructural alterations within sympathetic neurons (Heath et al., 1973). T h e induction of an immune-mediated destruction by guanethidine treatment in vivo was suggested as a possible explanation for the lack of a direct cytotoxic effect of guanethidine in vitro (Eranko et al., 1972), because immune competent cells were not present in the cultures. In contrast to the above results guanethidine has been shown to cause direct cytotoxic effects in culture, but only at extremely high concentrations (40-80 mg/liter o r 250-400 pM) (Hill et al., 1973; Heath ~t al., 1974) or at extremes of pH (8.0) (Johnson and Aloe, 1974; Wakshull et al., 1981). Neither of these conditions occur in vivo. Thus the toxicity seen under these conditions is probably not relevant to the process by which guanethidine exerts its cytotoxic effects in vivo. This suggestion is further supported by the observation that similar toxic effects are seen in vitro with guanidinium adrenergic neuron blocking agents that do not destroy sympathetic neurons in vivo (Johnson and Aloe, 1974).

V. Structure-Activity Relationships

T h e structural requirements of guanidinium compounds for activity as adrenergic neuron blocking agents and thus antihypertensive agents has been the object of considerable study. Hundreds of analogs have

10

EUGENE M. JOHNSON, JR. AND PAMELA TOY MANNING

been examined and the results of these studies have been reviewed (Schlitter, 1977). Relatively little work has been done in an effort to determine the structural requirements of guanethidine analogs for the ability to destroy rat sympathetic neurons. Only two studies have been published, one (Juul, 1973, 1977) in which effects of various analogs were assessed in adult rats and another in which the effects were assessed in neonatal animals (Johnson and Hunter, 1979). No qualitative differences were found between the responses of adult and neonatal rats. T h e great majority of adrenergic neuron blocking agents did not produce neuronal destruction. In addition to guanethidine four other compounds that produced neuronal destruction were identified in these two studies, which assessed the effects of approximately 50 analogs. All of the four active compounds are very similar in structure to guanethidine. All contain a nonaromatic, nitrogen-containing ring with a twocarbon bridge separating the ring nitrogen from an unsubstituted guanidinium group (see Table I). Of the agents that have been used clinically only guanethidine and guanacline produce neuronal destruction in the rat. Others such as debrisoquin, guanoxan, bethanidine, guanachlor, guancydine, bretyliuni, and guanadrel do not produce neuronal destruction in the rat. A major determinant of cytotoxicity appears to be whether or not the drugs accumulate to high concentrations within neuronal cell bodies. The majority of adrenergic neuron blocking agents do not accumulate to an appreciable extent in sympathetic ganglia after chronic treatment at high doses. Representative cytotoxic and noncytotoxic compounds are shown in Table I. It is interesting to note that of the five active compounds that we have examined (Johnson and Hunter, 1979), guanethidine accumulated to the least extent of the five. By comparing the dose response curve for cytotoxicity with that for accumulation, we estimate that guanethidine must accumulate to a concentration of 0.25-0.3 nmol/ pair SCG (- 1.25 nmol/mg protein) to produce neuronal destruction. The apparent necessity for accumulation in the sympathetic neuron is shown by the prevention of neuronal destruction by simultaneous administration to rats of desmethyliniipramine, an inhibitor of the active catecholamine uptake pump (Juul and Sand, 1973). Thus, the failure of the vast majority of adrenergic neuron blocking agents to produce sympathetic neuron destruction could be due to a failure to accumulate within the neuron. Accumulation of guanidinium agents in the neuronal cell body, although apparently necessary for cytotoxicity, is not alone sufficient. In both neonatal and adult rats the compound Ph 88117 (Compound 5 in Table I) accumulates to concentrations comparable to guanethidine, but

TABLE 1 COMPARISON OF THE ACCUMULATION, CYTOTOXICITY, A N D INHIBITION OF MITOCHONDRIAL RESPIRATION BY GUANIDINIUM ADRENERCIC BLOCKING AGENTS ~

~~

Accumulation in ganglia*

Compound

No.

/,NH

CHz- CH,--NH -C,

1

Cytotoxicity"

nrnollpair SCG (neonate)

nmol/mg dry wt in SCG (adult)

Yes

0.56

0.634

500

Yes

N.D.

0.996

N.D.

Yes

1.05

N.D.

*lo00

Yes

0.94

N.D.

>lo00

No

0.39

0.587

40

Inhibitory state 3 respiration (EDMin PLM)

NH, Guanethidine 2

c H 3 G - -

CH,-cH,

/m -m-c< -NHZ

Guanacline

G

N- CH,-CH,-NH-C,

P M

Z

5 M

Z

Ph 881/7 (roniznutd)

TABLE I (continued)

Accumulation in gangliab

Cytotoxicity"

nmoVpair SCG (neonate)

in SCG (adult)

Inhibitory state 3 respiration (EDjo in p M )

No

0.22

N.D.

*loo0

7

No

0.1 1

0.086

100

8

No

0.03

N.D.

50

Compound

No.

C

N-CH,-CH,

6

/m -cH,-~-c<

Wt

NH,

Debrisoquin a

nmoVmg dry

Cytotoxicity defined as ability to destroy sympathetic neurons upon chronic treatment. Data taken from Johnson and Hunter (1979; neonates) and Juul (1973; adults).

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

13

it is not cytotoxic (Juul, 1973; Johnson and Hunter, 1979), despite the fact that as an antihypertensive agent it is of similar potency to guanethidine (Hermansen, 1963). Perhaps the primary utility of this observation will be the use of Ph 88 1/7 as a negative control to test hypotheses of the molecular mechanisms by which guanethidine and other compounds lead to neuronal death.

VI. Mechanism of Destruction of Sympathetic Neurons

The precise mechanism by which guanethidine exerts its cytotoxic effects on sympathetic neurons is unknown. The observation that the destruction of sympathetic neurons by guanethidine can be completely prevented by concurrent treatment of the neonatal rat with nerve growth factor (Johnson and Aloe, 1974) is likewise unexplained but is an intriguing aspect of the problem. Over the years several mechanisms have been advanced to explain the neuronal destruction induced by guanethidine. However, none of these have been proven to be responsible for its cytotoxic effects.

A. INHIBITIONOF OXIDATIVE PHOSPHORYLATION T h e mechanism most often suggested to explain the neuronal destruction caused by chronic guanethidine treatment involves the inhibition of oxidative phosphorylation (Heath et al., 1972; Juul, 1973; Johnson and Aloe, 1974; Heath and Sobels, 1977). At the ultrastructural level the most pronounced degenerative changes that occur in sympathetic neurons following high-dose guanethidine treatment involve the mitochondria. Mitochondria1 damage is a relatively early event; changes are observable following only 2 days of treatment with 30 mg/kg guanethidine in the adult rat (Heath et al., 1972). The mitochondria, to varying degrees in different neurons within the ganglion, characteristically become swollen and lose cristae. The outer mitochondrial membrane remains intact (Heath et al., 1972; Juul, 1973). I n addition these altered mitochondria exhibit a complete lack of cytochrome oxidase activity in the outer mitochondria1 compartment (localized by cytochemical staining following in vim treatment) even though supporting cells within the ganglion exhibit a normal distribution of activity (Heath and Sobels, 1977). Therefore, based on morphological criteria, it was suggested that

14

EUGENE M. JOHNSON, JR. AND PAMELA TOY MANNING

guanethidine interfered with energy metabolism. However, these ultrastructural changes in the mitochondria may be nonspecific as several conditions, i.e., hypoxia and ascorbic acid deficiency, have been shown to produce similar alterations (Rouiller, 1960; Juul, 1977). In addition the early ultrastructural alterations that occur in the mitochondria of sympathetic neurons and that are suggestive of an inhibition of oxidative phosphorylation have been shown to occur not only in the rat but also in the mouse (Angeletti et al., 1972; RCvCsz and van der Zypen, 1979). However, guanethidine does not cause destruction of sympathetic neurons in the mouse (Evans et al., 1979), even in the presence of marked ultrastructural changes in the mitochondria. Several observations provide a basis for the hypothesis that the inhibition of oxidative phosphorylation is responsible for the cytotoxicity of guanethidine, some of which have been mentioned previously. 1. Guanethidine has been shown to accumulate selectively within sympathetic neurons via the amine uptake pump (Schanker and Morrison, 1965); this uptake and the neuronal destruction can be blocked by desmethylimipramine (Juul and Sand, 1973). 2. Guanethidine accumulates within sympathetic neurons in uivo to concentrations of approximately 0.5- 1.O mM (Juul and Sand, 1973). 3. Guanethidine inhibits oxidative phosphorylation in isolated rat liver mitochondria with a EDs0 of between 0.5-0.9 mM (Malmquist and Oates, 1968; Johnson and Hunter, 1979). Since guanethidine accumulates within sympathetic neurons in uivo to concentrations that have been shown to inhibit oxidative phosphorylation in nitro, it is conceivable that guanethidine could exert its cytotoxic effects by this mechanism. However, the inhibition of oxidative phosphorylation does not appear to be the mechanism responsible for neuronal death. T h e relative potencies of various analogs to accumulate in sympathetic neurons in vivo and to inhibit oxidative phosphorylation in vitro as well as their ability to produce cytotoxicity were examined (Johnson and Hunter, 1979). As shown in Table I accumulation of guanethidine or guanidinium analogs within sympathetic neurons appears to be a necessary but not sufficient requirement for producing cytotoxicity; all compounds that were cytotoxic also accumulated. However, Ph 88 1/7 (compound 5), although it accumulated to concentrations that should have been sufficient to cause cell death, was not cytotoxic. Therefore, if the inhibition of oxidative phosphorylation was the mechanism responsible for cytotoxicity, Ph 881/7 should have been a weak inhibitor as it fails to destroy neurons. This was not the case. Ph 881/7 was a much more

GUANETHIDINE-INDUCED DESTRUCTION

OF SYMPATHETIC NEURONS

15

potent inhibitor of oxidative phosphorylation (12 X ) than was guanethidine. Curiously, it did not produce any swelling of the mitochondria at the ultrastructural level (Juul, 1973), thus bringing into question the relationship between changes in oxidative phosphorylation in vitro and the morphological changes seen in vivo. In addition compounds 3 and 4, although they were cytotoxic, were very weak inhibitors of oxidative phosphorylation. This further dissociates the cytotoxicity of guanethidine from its effects on oxidative phosphorylation. Thus it is highly unlikely that inhibition of oxidative phosphorylation is the mechanism responsible for the cytotoxic effects of guanethidine produced in vivo.

B. INHIBITION OF THE RETROGRADE TRANSPORT OF NERVE GROWTH FACTOR It has been proposed that drugs that destroy the sympathetic nervous system do so by preventing the accumulation of retrogradely transported nerve growth factor (NGF) (Johnson et al., 1979). Vinblastine, 6hydroxydopamine, and guanethidine all produce destruction of sympathetic neurons in neonatal animals, and in each case concomitant treatment with nerve growth factor prevents neuronal death (Johnson and Aloe, 1974; Aloe et al., 1975; Chen et al., 1977; Johnson, 1978). However, the mechanisms leading to neuronal destruction appear to be different. Both 6-hydroxydopamine and vinblastine treatment completely prevent the retrograde transport of [ 1251]NGFfrom the periphery to the sympathetic ganglia in neonatal rats, which is consistent with the idea that the lack of trophic factor at the cell body ultimately results in neuronal death. In contrast administration of guanethidine to neonatal or to adult rats only partially prevents the retrograde transport and subsequent accumulation of [ 1251]NGFwithin the superior cervical ganglion (Johnson et al., 1979). In addition in neonates this decrease occurs only after 1 week of guanethidine treatment (but not at earlier times), by which time marked cellular destruction (80-90%) of neurons has already occurred (Manning et al., 1982). Other guanidinium blocking agents including guanoxan and guanedrel, which are not cytotoxic, also partially inhibit the retrograde transport of NGF. Therefore, the inhibition of the retrograde transport of NGF does not appear to be the mechanism by which guanethidine produces destruction of sympathetic neurons. Similarly, the mechanism by which nerve growth factor prevents neuronal death produced by guanethidine must be different than the mechanism by which it protects against 6-hydroxydopamine and vinblastine.

16

EUGENE

M. JOHNSON,

JR. AND PAMELA TOY MANNING

C. IMMUNE-MEDIATED MECHANISM Several observations in the literature suggest or are consistent with the hypothesis that guanethidine may exert its cytotoxic effects by an immune-mediated mechanism. In fact this mechanism was suggested in the early papers of Jensen-Holm and Juul (1971) and Eranko and Eranko (197 1) largely because of the marked small cell infiltration of the ganglia following guanethidine treatment. Lymphocytic infiltration similar to the cellular infiltrate which occurs following guanethidine administration characteristically occurs in certain disease processes that are known to be immunologically mediated (Paterson, 1971). In addition the small cell infiltrate occurs concomitantly with neuronal destruction (Jensen-Holm and Juul, 1970b, 1971). At the light microscopic level this infiltrate consists largely of monocytes, macrophages, and lymphocytes rather than neutrophils (polymorphonuclear cells; E. M. Johnson and P. T. Manning, unpublished observations). Only a few neutrophils were found to be present within the infiltrate when examined at the ultrastructural level (Manning et al., 1983). The cellular composition of the infiltrate over a short time course is consistent with an immune-mediated response rather than representing a secondary response to neuronal necrosis. Guanethidine does not produce destruction of sympathetic neurons (see Section IV,B) under normal tissue culture conditions (Eranko et al., 1972; Johnson and Aloe, 1974), even though it accumulates within sympathetic neurons in vitro (Wakshull et al., 1981). The hypothesis that the neuronal destruction is immunologically mediated can easily account for this observation because immune competent effector cells are not present in either explants of sympathetic ganglia or in cultures of dissociated sympathetic neurons. Also consistent with an immunologically mediated mechanism is the failure of guanethidine, even when injected chronically directly into the brain, to produce death of noradrenergic neurons. The decrease in catecholamine levels after such treatment is completely reversible (Evans et al., 1975). T h e lack of degenerative effects on central noradrenergic neurons could be explained by the relatively immunologically privileged nature of the brain (Green, 1957). Guanethidine produces neuronal cell death only in the rat but not in the mouse or any other species tested thus far (Downing and Juul, 1973; O’Donnell and Saar, 1974; Johnson et al., 1977; Evans et al., 1979). However, the early ultrastructural alterations including pronounced mitochondria1 swelling (presumably due to the direct effects of guanethidine on the sympathetic neurons) also occur in the mouse

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

17

(Angeletti et al., 1972; RCvesz and van der Zypen, 1979). This suggests that the direct effects of guanethidine that result in mitochondria1 changes are not those responsible for cell death. Indeed in the mouse these ultrastructural changes are completely reversible upon cessation of treatment (Evans et al., 1979).

FIG.2. Effects of various amounts of y-irradiation on guanethidine-induced destruction of sympathetic neurons. One-week-old Sprague-Dawley rats were treated with 600, 750, or 900 rads of y-irradiation and/or 50 mg/kg of guanethidine for 5 days, and were killed 2 days later. A, Untreated control; B, guanethidine alone; C, 600 rads + guanethidine; D, 750 rads + guanethidine; E, 900 rads; F, 900 rads + guanethidine. Bar = 100 pm. [Reproduced with permission of publisher; Manning et al. (1982).]

18

EUGENE M. JOHNSON, JR. AND PAMELA T O Y MANNING

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

19

As a result of the preceding intriguing bits of indirect but suggestive evidence, we designed experiments to test the hypothesis that guanethidine-induced destruction of sympathetic neurons occurs by an immunological mechanism. Initial experiments demonstrated that a variety of immunosuppressants, which act by different mechanisms, completely or partially prevented neuronal destruction induced by guanethidine. Cyclophosphamide and y-irradiation (Fig. 2) completely prevented neuronal cell death. Azathioprine and dexamethasone provided partial protection. Immunosuppressants did not prevent guanethidine accumulation nor did they protect against the sympathectomies produced by 6-hydroxydopamine or antibodies to nerve growth factor (Manning et al., 1982). A portion of the cellular infiltrate was identified immunohistochemically as T-lymphocytes using an antibody prepared against purified rat T-cells. Electron microscopic examination of the cellular infiltrate demonstrated a predominance of lymphocytes and macrophages (Fig. 3), which is characteristic of an immune-mediated response as opposed to a nonspecific response to neuronal destruction. An unequivocal demonstration of the immunological basis of neuronal destruction was provided by immune reconstitution experiments. Immune competent (spleen and bone marrow) cells obtained from control syngeneic rats were transferred to lethally irradiated recipients that could no longer respond to guanethidine treatment. Immune reconstitution resulted in the restoration of the capacity of the irradiated animals to respond to guanethidine, establishing that immune competency is required for guanethidine-induced neuronal destruction to occur (Fig. 4).Guanethidine sympathetectomy thus represents a drug-induced autoimmune disorder (Manning et al., 1983). The specific antigen that induces this immune response as well as the specific immune mechanism responsible for the neuronal destruction remain to be elucidated and are currently under study in this laboratory. The lack of neuronal cell death in species other than the rat (see Section IV,A,2) suggests that either guanethidine accumulation does not result in the expression of the analogous antigen in those species or that this analogous antigen is not seen as foreign in those species and hence does not lead to an immune attack. The ability of NGF to protect against FIG. 3. Ultrastructural characterization of the inflammatory infiltrate in rat superior cervical ganglion following guanethidine treatment. A, Widespread neuronal loss, debrisladen macrophages, and numerous infiltration mononuclear cells were present following guanethidine treatment (3500X); B, a mononuclear cell, probably a monocyte, is located between two endothelial cells. A mitotic figure is seen at the lower margin (4100X). [Reprinted with permission of publisher; Manning et al. (1983).]

20

EUGENE M. JOHNSON, J R . AND PAMELA TOY MANNING

FIG.4. Histologicalchanges in the superior cervical ganglion following immune reconstitution. A, Not irradiated + guanethidine; B, irradiated control (850 rads); C, irradiated + guanethidine; D-F, irradiated + spleen and bone marrow cells + guanethidine. [Reproduced with permission of publisher; Manning et al., (1983).]

guanethidine-induced destruction (Johnson and Aloe, 1974) may be the result of an ability to block either antigen expression or antigen recognition. VII. Strain Specificity of Guanethidine Sympathectomy

Variability among different inbred strains of rats has been observed in response to several autoimmune disorders, both spontaneous and experimentally induced. Because guanethidine-induced destruction occurs by an immunologically mediated mechanism, we used morphological criteria to evaluate several outbred and inbred strains of rats as

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

21

neonates for the severity of the response to chronic guanethidine treatment. Based upon our results, we have classified strains of animals as sensitive (S, no viable neurons remaining, massive lymphocytic infiltration), moderate or intermediate (I, some viable neurons remaining, presence of many ghostlike neurons, marked lymphocytic infiltration), and resistant (R, many viable neurons remaining, minimal lymphocytic infiltration). Resistant strains were not entirely resistant, but the amount of neuronal destruction and the degree of small cell infiltration were much less than in the sensitive strains. Preliminary experiments were also conducted using FI hybrids (first generation) bred by crossing sensitive with resistant strains to determine the dominance of the response to treatment (P. T. Manning and E. M. Johnson, unpublished data). The results are summarized in Table 11; several points in this table are worthy of some discussion. TABLE I1

RESPONSE OF VARIOUS STRAINS OF RATSTO CHRONIC GUANETHIDINE TREATMENT Strain Outbreds Sprague-Dawley"

Supplier

Zivic Miller Pittsburgh, Pennsylvania Sasco Omaha, Nebraska Chappel St. Louis, Missouri

Holtzman

Holtzman Madison, Wisconsin

Spontaneously Hypertensive (SHR) Kyoto-Wistar

National Institutes of Health Bethesda, Maryland National Institutes of Health Bethesda, Maryland Charles River Wilmington, Massachusetts

Wistar Inbreds Lewis

M. A. Bioproducts' Walkersville, Maryland Charles River Wilmington, Massachusetts

Sensitivityb

Reference

Manning et al. (1982) P. T. Manning and E. M. Johnson (unpublished) P. T . Manning and E. M. Johnson (unpublished) P. T . Manning and E. M. Johnson (unpublished) Johnson and Macia (1982) Johnson and Macia (1982) Johnson and Macia (1982) P. T. Manning and E. M. Johnson (unpublished) P. T. Manning and E. M. Johnson (unpublished) (continued)

22

EUGENE M. JOHNSON, JR. AND PAMELA TOY MANNING

TABLE I1 (contznued) Strain

Supplier

Fisher 344

Charles River Wilmington, Massachusetts

New England Deaconess Hospital

New England Deaconess Hospital, bred in this laboratory Cox Laboratory Supply Co. Indianapolis, Indiana

AC!

M. A. Bioproductsc Walkersville, Maryland Brown Norway

National Institutes of Health Bethesda, Maryland

Buffalo

Cox Laboratory Supply Co. Indianapolis, Indiana

Maxx

Cox Laboratory Supply Go. Indianapolis, Indiana

Wistar Fiirth

M. A. Bioproducts' Walkersville, Maryland

Sensitivityb

Reference

P. T. Manning and E. M. Johnson (unpublished) P. T. Manning and E. M. Johnson (unpublished) P. T. Manning and E. M. Johnson (unpublished) P. T. Manning and E. M. Johnson (unpublished) P. T. Manning and E. M. Johnson (unpublished) P. T. Manning and E. M. Johnson (unpublished) P. T. Manning and E. M. Johnson (unpublished) P. T. Manning and E. M. Johnson (unpublished)

~~

One-week-old rats were treated with 50 mg/kg/day guanethidine for 2 weeks. Wistar Fiirth rats were treated with 25 mg/kg/day because of the high mortality rate when treated with higher doses. S: Sensitive, no viable neurons remaining, massive lymphocytic infiltration; I: intermediate, some viable neurons remaining, presence of many ghostlike neurons, marked lymphocytic infiltration; R: resistant, many viable neurons remaining, minimal lyrnphocytic infiltration. ' Now Harlan Spragrie Ihwley, Inc.

1. The same outbred strains of rats from different suppliers responded with different degrees of sensitivity to chronic guanethidine treatment. For example the Sprague-Dawley rats from Zivic Miller Breeders (Pittsburgh, Pennsylvania) were extremely sensitive to guanethidine treatment, whereas those obtained from Sasco Breeders (Omaha, Nebraska) responded with intermediate severity and the variability in the response among animals was high, as shown by varying amounts of neuronal destruction and degrees of lymphocytic infiltration. 2. Different strains of inbred rats or the same strain of outbred rats from different suppliers exhibited different degrees of tolerance for the toxic effects of guanethidine (i.e.,weight loss and mortality).

CUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

23

3. Variability in the response of the same inbred strain of rats (i.e., Lewis rats) obtained from different suppliers was low. 4. A particularly interesting inbred strain was the Wistar Furth rat. Although this strain was resistant to guanethidine the pattern of the response differed from that normally observed. One week following treatment the response was intermediate. However, after 2 weeks of treatment the lymphocytic infiltration had largely resolved, leaving an essentially normal-looking ganglion. This resolution of the small cell infiltration occurred despite continued administration of the drug. We speculate that in this resistant strain, despite the continued presence of antigen, immune suppressor mechanisms are activated, which could then limit the extent of neuronal destruction. Unlike some other experimentally induced autoimmune conditions, i.e., experimental allergic encephalomyelitis, the immune response induced by guanethidine does not appear to be determined solely by the major histocompatibility complex. This conclusion is based on the different responses of the Lewis (S) and Fisher 344 (R) rats to guanethidine. Although these two strains have identical haplotypes (111) at the major histocompatibility locus, they responded with differing degrees of severity following guanethidine treatment. In crosses between F344 (R, resistant) and Lewis (S, sensitive) rats and between ACI (R) and Lewis (S) rats, all F1hybrids were sensitive to guanethidine, indicating that resistance to guanethidine treatment is a recessive trait. Further experiments are necessary to examine the genetics of the immune response in greater detail. VIII. Assessment of the Degree of Sympathectomy Produced by Guanethidine

As has been previously discussed guanethidine administration to rats results in the destruction of only a single cell type, the postganglionic sympathetic neuron. Permanently sympathectomized animals have long been used to study the role of the sympathetic nervous system in physiological and pathophysiological processes. Because all available methods have deficiencies, considerable effort has been expended to characterize the degree of sympathectomy produced by guanethidine in the rat. Guanethidine has the advantage over alternative methods of sympathectomy (immunosympathectomy, 6-hydroxydopamine-induced sympathectomy) of inducing destruction of sympathetic neurons in both neonatal and adult animals. The protocols for producing sympathectomy in newborn and adult rats will be discussed separately. The advantages and disadvantages of guanethidine sympathectomy will then be compared to

24

EUGENE M. JOHNSON, JR. AND PAMELA TOY MANNING

immunosympathectomy and the sympathectomy produced by 6-hydroxydopamine.

A. SYMPATHECTOMY IN NEONATAL RATS Treatment of neonatal rats with guanethidine produces a more complete and more rapid sympathectomy than does treatment of adults. The treatment protocols used, the age at which sympathectomy was assessed, and the changes in some of the parameters measured are summarized in Table 111. As previously discussed doses of guanethidine well above those required for adrenergic neuronal blockade must be administered chronically in order to produce neuronal destruction. Doses of 5 mg/kg/day or less, although they produce adrenergic neuron blockade, do not produce morphological effects on neurons, small cell infiltration, or neuronal loss. Doses of 10 mg/kg/day produce modest effects, whereas doses of 20 mg/kg/day or more produce dramatic effects. Most workers have reported that guanethidine is well tolerated. In our experience adverse effects on survival and vitality are greatest when treatment is initiated during the first few days after birth; mortality rates are increased, growth rates are reduced, and frequent runting is observed. Allowing the animal to reach a week of age before starting treatment produces complete sympathectomy with minimal adverse effects (see following paragraph). The most commonly used treatment regimen (Johnson et al., 1976) involves administering guanethidine, 50 mg/kg/day, to neonatal rats 5 daydweek for 3 weeks starting at 1 week of age. The protocol produces a complete and permanent sympathectomy with little growth retardation or mortality. This regimen represents a modification in a protocol (Johnson et ad., 1975) in which we treated animals with 50 mg/kg or 100 mg/kg of guanethidine each day for the first 20 days of life. The lower dose was as effective as the higher dose in producing sympathectomy. The completeness of sympathectomy (Table 111)was verified by several biochemical and functional criteria. Studies that used the same protocol (Johnson et al., 1976) and that provide sufficient data with which to make a comparison have in some cases (Overbeck, 1979; Friedman et al., 1979; Grzanna and Coyle, 1979) reported results comparable to ours but in other cases reported (Levens et al., 1981; Bennett et al., 1982) lesser degrees of sympathectomy. In the latter two studies Wistar rats were used rather than Sprague-Dawley rats. A particularly striking example of the apparent variability in sympathectomy is that reported by Bell and

TABLE 111 EVALUATION OF THE SYMPATHECTOMY PRODUCED BY GUANETHIDINE TREATMENT OF NEONATAL RATS ~

~~~~

Protocol

Strain

Age of evaluation

50 mglkglday 5 dayslweek (age 7-27 days)

S.D.

>9 weeks

30 mgkglday 1-15 days of age 50 mglkglday 1-15 days of age 20 mglkg every other day until 14 days old 50 mg/kg/day 5 dayslweek (age 5-27 days) 50 mglkglday 5 dayslweek (age 5-27 days) 20 mglkglday 1-8 days of age 50 mgkglday 5 dayslweek (age 7-27 days)

“Mongrel albino” Not stated

4 months

Not stated

Not stated; presumably 15 days Adult

S.D.

10 weeks

Results“

Reference

98% & in TOH in SCG; normal gain in body weight; no change in NE levels of whole brain, cerebellum, or spinal cord; >90% 4 NE of heart, spleen, mesentery; >95% J. in response to stimulation on pithed rat; no permanent changes on adrenal TOH or EPI 88% 4 in number of neurons in SCG; 68% 4 heart NE; 77% & intestine NE; 89% .1 vas deferens NE 85-90% .1 NE in heart, spleen, submaxillary glands; 55% 4 NE in vas deferens 8670 on number of neurons in sections with maximal sectional area

Johnson et al. (1976, 1977)

4 in BP rise in response to tyramine; T response to

Simon (1981)

Rodionov et al. (1981) Blaschke and Uvnas (1979) Klein (1 979a)

NE Wistar

Adult

78% & in heart NE

Levens et al. (1981)

S.D.

4 weeks

S.D.

Adult

90% J in volume of SCG; three-fivefold increase in SIF cells in SCG 95% 4 in DBH activity in heart and spleen; 77% & in DBH in pancreas; 90% .1 in NE in heart and spleen

Eraink6 and Erinko (1971) Grzanna and Coyle ( 1 979) (continued)

TABLE 111 (continued)

Protocol N

Q,

Strain

Age of evaluation

Results"

Reference

Very few axons in atrial or mesenteric arteries; very little response to stimulation of vasomotor outflow in pithed rat preparation J Blood pressure (133 k 4 control; 98 2 treated); 95% in plasma NE

Bell and McLachlan (1979) Overbeck (1979)

80 mglkglday sc 1-14 days of age

Wistar

Adult

50 mgikglday ip 5 daydweek (age 7-27 days) adrenal demedullated 50 mglkgiday ip 5 dayslweek (age 7-27 days) adrenal demedullated 50 mglkglday ip 5 dayslweek (age 7-27 days) adrenal demedullated

S.D.

11-12 weeks

S.D.

13 weeks

98% J in DBH activity in SCG

Schmidt et al. (1981)

Wistar

24-26 weeks

Blood pressure unchanged (control 1671114; treated 156i98); depressed baroreceptor reflex; unchanged chronotropic response in right atria to nerve stimulation but depressed left atria (40% 1); 70-90% J in heart NE levels

Bennett et al. ( 1982)

*

50 mgkglday ip

1-21 days of age 50 mglkglday ip 5 daydweek (age 7-27 days) adrenal demedullated 50 mglkglday for 17 days 100 mg/kg/day 1-20 days of age

50 mgtkglday first 20 days of life ~~~~~~~~~~~

86% 4 in uptake in heart synaptic vesicles; unchanged basal heart rate 4 response to tyramine; t response to NE; 98% 4 in NE in heart and spleen

Bareis et al. (1981) Friedman et al. (1979)

17 days

96% .1 in TOH and DBH in SCG

S.D.

> 10 weeks

S.D.

>10 weeks

20-30% 4 body weight; >90% .1 in NE in heart, spleen, vas; 80-90% J. NE in kidney and intestine; no change in brain or adrenal NE; 90% 4 in rise of blood pressure with nerve stimulation in pithed rat; 30 mm Hg 1in blood pressure; no response to sympathetic stimulation of perfused kidney or intestinal smooth muscle; no impairment of response to nerve stimulation of vas; retention of ejaculatory function >90% .1 in NE in kidney, heart, vas, mesentery, and intestine

Sorimachi (1977) Johnson et al. (1975)

S.D.

40-45 days

Dahlhypertensive

8-10 weeks

Wistar

Johnson et al. (1975)

~~~~~~~~~

TOH, Tyrosine hydroxylase activity; NE, norepinephrine; EPI, epinephrine; SCG, superior cervical ganglion; DBH, dopamine phydroxylase activity; S.D., Sprague-Dawley rat; BP, blood pressure; SIF, small intensely fluorescent.

28

EUGENE M. JOHNSON, JR. AND PAMELA TOY MANNING

McLachlan (1979). These workers reported that in Wistar rats treated with 50 mg/kg/day of guanethidine sulfate for the first 14 days of life, there was little effect on the density of adrenergic axons in atria or mesenteric arteries in the majority of animals. However, treatment with 80 mg/kg/day produced a pronounced sympathectomy in those animals. These results are clearly at variance with the results of the other studies summarized in Table 111. Because different strains of rats clearly respond differently to guanethidine (see 'Table II), it is not surprising that variability is observed. When using guanethidine as a means of producing sympathectomy, it is necessary to verify the degree of sympathectomy in the particular animals used for study. This may be equally true of studies using immunosympathectomy or 6-hydroxydopamine-induced sympathectomy, although this question has not been systematically examined. It is also apparent that different strains of rats and even the same strain of rats from different suppliers tolerate (i.e., show evidence of toxicity, mortality, or growth retardation) the administration of guanethidine with varying degrees of success. We have previously described differences (Johnson and Macia, 1979) in the ability of Okomoto strain SHR rats to tolerate the drug with respect to both survival and weight gain. Similarly, we observe marked differences in the ability of outbred Sprague-Dawley rats from different suppliers to tolerate the drug. In our experience those Sprague-Dawley rats that do not tolerate guanethidine as neonates do well when injected with other agents or when treated with guanethidine after reaching adulthood. In summary the data compiled in Table I11 indicate that guanethidine administration to neonatal rats is capable of producing a complete sympathectomy. The drug is generally well tolerated. In this laboratory when the experimental objective is to produce a sympathectomized rat, we continue to use the treatment protocol we reported in 1976 (Johnson et al., 1976). We feel that guanethidine-induced sympathectomy of neonates still represents the most complete and specific method of producing sympathectomy yet developed and is thus the method of choice in studies involving rats. The lesser degrees of sympathectomy reported by some workers indicate, however, that caution must be exercised, and the extent of destruction of the sympathetic nervous system should be independently verified in any particular animal chosen for study.

B. SYMPATHECTOMY IN ADULTRATS Guanethidine is the only method available to produce a permanent chemical sympathectomy in adult rats. However, administration of

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

29

guanethidine to adult animals has several disadvantages compared to treatment of neonates. Most importantly, the degree of sympathectomy produced is less than that achieved in neonates. It also requires treatment of animals for much longer periods, involves the consumption of large quantities of guanethidine, and is more expensive. Fewer data are available that characterize the sympathectomy produced in adult animals. Comparison of the available studies is also difficult because of differences in dosage regimes and in the parameters measured as means of assessing the degree of sympathectomy (Table IV). Because guanethidine is well tolerated by the adult animal and maximal accumulation in sympathetic ganglia is achieved with doses of 40 mg/kg/day (Juul and Sand, 1973), a treatment regimen of 40 mg/kg/day for several weeks appears to be a rational protocol. We have demonstrated that treatment with 40 mg/kg/day for 5 weeks produced about a 90% destruction of the sympathetic nervous system as assessed by several biochemical and functional criteria (Johnson and O’Brien, 1976). Even greater degrees of destruction would presumably be achieved if treatment were continued for longer periods, as suggested by the data of Nielsen (1977b). All of these protocols are obviously longer and more expensive than that required to produce similar destruction of the sympathetic nervous system in neonatal animals.

C. COMPARISON OF GUANETHIDINE-INDUCED SYMPATHECTOMY WITH THAr PRODUCED BY ANTI-NGFOR 6-HYDROXYDOPAMINE Over the last 20 years two other methods of producing permanent sympathectomy in small experimental animals have been developed. Immunosympathectomy, the first method to be developed, is achieved by the administration of antisera against nerve growth factor (anti-NGF) (Levi-Montalcini and Booker, 1960; Levi-Montalcini and Angeletti, 1966). T h e second method was the development of 6-hydroxydopamine-induced sympathectomy (Angeletti and Levi-Montalcini, 1970). Both of these agents produce destruction of sympathetic neurons only when administered to immature animals. T h e vast majority of the literature deals with neonatal mice and rats. Assuming that the experimental objective is to produce a complete peripheral sympathectomy with no adverse effects on other neuronal types, both anti-NGF and 6-hydroxydopamine have deficiencies that are not shared by guanethidine. These differences relate both to the completeness and to the specificity of the neuronal destruction produced. Unlike guanethidine, neither B-hydroxydopamine nor anti-NGF destroy the short adrenergic neurons inner-

TABLE IV EVALUATION OF THE SYMPATHECTOMY PRODUCED BY GUANETHIDINE TREATMENT OF ADULT RATS

Strain

Time between treatment and examination

25-30 mglkg day ip for 6 weeks 25 mglkglday im for 8 weeks 40 mglkglday ip 5 daystweek for 5 weeks

S.D.

4 months

98% 4 in neurons of the SCG

S.D.

Not stated

S.D.

1, 3, and 6 months

30 mglkglday ip for 6 weeks

S.D.

0-10 weeks

25 mglkglday ip for 5 weeks 40 mglkglday for 28 days 40 mgjkglday for 3 months 28 mglkglday ip for 6 weeks

S.D.

0

Wistar

Up to 60 days

Wistar

3 months

Transient loss of ejaculatory function; supersensitivity to NE 8 weeks after cessation of treatment Slight 1in weight gain; 85-90% in TOH in SCG; no change in adrenals; 90% or > .1 in NE of heart, vas, and spleen; no change in NE in brain areas; 90% -1 in response to vasomotor stimulation of pithed rat preparation; abolition of relaxation in intestine after sympathetic stimulation; 10% in blood pressure Initially NE levels undetectable in treated animals, rising to 20% of control by 8 weeks; no effect on BP rise due to physostigmine or carotid occlusion 90-95% in NE and DBH in SCG; >90% .1 in heart particulate N E and DBH No change in BP; approximately 60% 1in neurons in SCG, expressed in cells/mm2 54 mm Hg .1 in BP; 95% & in neurons in SCG

S.D.

14 weeks

Treatment

Effects"

83% .1 NE in heart; 91% in submaxillary gland

4 NE in spleen; 89% 5- Ne

References Burnstock et al. ( 197la) Hepp and Kreye (1973) Johnson and O'Brien (1976)

Blythe et al. (1976) Grobecker et al. (1977) Nielsen (1977a) Nielsen (1977b) Ostman-Smith ( 1976)

TOH, Tyrosine hydroxvlase activity; NE, norepinephrine; EPI, epinephrine; SCG, superior cervical ganglion; DBH, dopamine P-hvdroxvlase activity: S.D., Sprague-Dawley rat; BP, blood pressure.

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

31

vating the male genitalia. Of greater importance, especially for evaluating the role of the sympathetic nervous system in the regulation of the cardiovascular system, is the relative resistance of the sympathetic innervation of the blood vessels to either 6-hydroxydopamine or anti-NGF (Berkowitz et al., 1972; Finch et al., 1973). Following treatment with either 6-hydroxydopamine or anti-NGF, only modest decreases in the concentration of norepinephrine in mesenteric blood vessels occur in animals that show much larger decreases in both heart and spleen norepinephrine content. In contrast guanethidine-sympathectomized animals have similar decreases in norepinephrine content in mesenteric blood vessels, heart, and spleen (Johnson et al., 1975, 1976). Assessment of the functional deficits of the sympathetic innervation of the vasculature is consistent with these biochemical findings. Stimulation of the entire vasomotor outflow in the pithed rat preparation or stimulation of the sympathetic innervation of the renal vasculature indicates that only partial sympathectomy is produced by maximally tolerated doses of 6hydroxydopamine administered to neonatal animals (Finch et al., 1973). In similar experiments rats sympathectomized neonatally with well-tolerated doses of guanethidine showed an almost complete functional deprivation (Johnson et al., 1975, 1976). Thus the available data indicate that guanethidine is capable of producing, at least in some strains of rats, a greater degree of sympathectomy than either anti-NGF or 6-hydroxydopamine. The sympathectomy produced by 6-hydroxydopamine administration is also less specific; in addition to causing destruction of peripheral sympathetic neurons, it also produces destructive effects on central noradrenergic neurons. T h e extent of this destruction and the changes produced in the CNS vary considerably depending upon the age of the animal when injected (i.e., the younger the neonate, the greater the central effects). Because of these direct effects on CNS neurons, the interpretation of experiments aimed at assessing the role of the peripheral sympathetic nervous system in pathophysiological processes such as hypertension are compromised. Similarly, recent studies have shown that anti-NGF adversely affects the developing sensory nervous system. In very immature animals anti-NGF can produce death of sensory neurons (Gorin and Johnson, 1979; Johnson et al., 1980; Aloe et al., 1981). In older animals cell death apparently does not occur, but decreases in putative transmitter levels are produced (Otten et al., 1980; Schwartz et al., 1982). T h e functional implications of the decreases observed in putative transmitter levels within sensory neurons have not been characterized. The time course of the transition to a state of maturity in which anti-NGF no longer produces death of sensory neurons has not been

32

EUGENE M. JOHNSON, JR. AND PAMELA TOY MANNING

fully clarified in any species. The time of this transition will undoubtably vary from species to species and may vary in different ganglia within the same animal. Neonatal sympathectomy produced by guanethidine treatment thus appears to have advantages over either treatment with 6-hydroxydopamine or with anti-NGF with respect to the extent and specificity of the sympathectomy produced. Guanethidine-induced sympathectomy has, of course, one major limitation not shared by the other methods. It produces sympathetic neuronal destruction only in the rat, whereas the other methods are apparently capable of sympathectomizing many species. Probably all mammalian species born in a relatively immature state are susceptible to treatment with 6-hydroxydopamine and anti-NGF. In the case of anti-NGF the additional variable of cross-reactivity of the antibody with the native NGF of the species to be sympathectomized is an important factor (Harper and Thoenen, 1980). However, direct analysis of this point may not be possible because of the lack of availability of NGF of many species. Finally, relatively little data is available on the permanent sympathectomy produced by these agents in species other than small laboratory rodents. Very little work has been done with the other guanidinium compounds that are capable of destroying rat sympathetic neurons, It is not known whether the other compounds are also capable of sympathectomizing only rats. Guanacline (Compound 2, Table I), as reported in the early literature (i.e., 1967-1974), may have somewhat different properties than guanethidine. Two observations in the literature are of particular interest. First, unlike guanethidine, chronic administration of guanacline to the rat (even at doses that do not cause massive sympathectomy), causes the accumulation of autofluorescent lipopigment in sympathetic neurons (Burnstock et al., 1971b). The accumulation of this pigment appears to be irreversible. The relationship of the accumulation of pigment to the cell death produced by high doses of guanacline is not known. Whether similar accumulation of autofluorescent pigments occurs in other species is not known. The second intriguing observation is that of persistent impairment of sympathetic function in patients taking guanacline. Dawborn et al. (1969) reported that 5 of 3’7 patients who took guanacline for periods of 3 to 4 months developed persistent postural hypotension, which had not reversed 18 months after cessation of therapy (longest time reported). Catecholamine excretion remained low in these patients. Similar results were reported by Bock and Heimsoth (1969). Whether the decreased sympathetic function produced in these patients resulted from actual neuronal destruction is not known. It should be noted that despite the fact that the clinical literature on

GUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

33

guanethidine is much larger, there are no reports of persistent effects of guanethidine following the termination of therapy analogous to those described for guanacline. These observations suggest that guanacline has interesting properties not shared by guanethidine and that guanacline may not be speciesspecific in its ability to destroy sympathetic neurons. Since 1974 experiments designed to examine the properties of guanacline appear to have ceased, probably because the drug is not available from the manufacturer (Bayer). We have recently synthesized guanacline in our laboratory and are currently exploring its activity in species other than the rat.

IX. Conclusion

Since the discovery over a decade ago that chronic guanethidine administration produced destruction of sympathetic neurons, studies primarily focusing on three general objectives have been conducted to (1) characterize the degree and specificity and the sympathectomy, (2) utilize the sympathectomy produced as an experimental tool in elucidating the role of the sympathetic nervous system in various physiological and pathophysiological processes, and (3) determine the mechanism by which guanethidine destroys sympathetic neurons. Studies aimed at the first objective have shown that guanethidine, particularly when administered to neonates, produces a permanent, complete, and highly specific sympathectomy (only postganglionic sympathetic neurons are destroyed). Thus it is a useful tool with which to study the second objective, the role of the sympathetic nervous system in various pathological processes. Unfortunately, guanethidine only produces sympathectomy in the rat, limiting its utility as an experimental tool. This unexpected result is perhaps made less surprising by the demonstration that the mechanism of neuronal death is immune-mediated. This fact in and of itself does not explain the species specificity. However, it is consistent, as many autoimmune conditions show striking species and strain specificities. Expanding the number of species in which guanethidine destroys sympathetic neurons would be most useful because species of experimental animals other than the rat, particularly larger species (i.e., dogs and cats), are used in many studies that examine the role of the sympathetic nervous system in pathophysiological processes. In addition other methods of producing permanent sympathectomy (6-hydroxydopamine and anti-NGF) have not been shown to be (and probably would not be)

34

EUGENE M. JOHNSON, .JR.AND PAMELA TOY MANNING

effective in producing permanent sympathectomy in these larger species. One approach toward achieving this goal would be to examine other guanidinium agents in other species because guanethidine analogs have been studied only in rats. Perhaps a more rational approach toward achieving sympathectomy with these agents in species other than the rat will have to await a more detailed analysis of the mechanism@)underlying the neuronal destruction in rats. With the demonstration that the destruction of sympathetic neurons produced by guanethidine is immune-mediated the phenomenon takes on broader implications. The general importance is increased by the ability of a hormone-like substance (nerve growth factor), which normally acts on the sympathetic neuron, to prevent the immune-mediated destruction. Perhaps the only other known case in which a cell-specific, cell-mediated immune destruction is drug-induced is that of chronic low-dose steptozotocin-induced diabetes. A specific cell type (the pancreatic p-cell) is the target of immune destruction and the phenomenon occurs only in some strains of a single species, the mouse. We have previously discussed in some detail the similarities of these two phenomena (Manning et al., 1983). There are several clinical conditions in which an autoimmune attack on the nervous system plays an important role (Paterson, 1971). There are also many drug-induced autoimmune disorders involving various immune effector mechanisms resulting in damage to a variety of tissues. ‘The pathogenetic mechanisms underlying these conditions are poorly understood. Guanethidine-induced destruction of sympathetic neurons offers many advantages as an experimental model of autoimmune-mediated destruction of the nervous system and/or drug-induced autoimmunity. Guanethidine sympathectomy occurs with a very reproducible time course in the neonatal rat resulting in initiation of the response and destruction of the target cells within a short time (7-10 days). The precipitating agent, guanethidine, is readily available and is a completely defined chemical entity. Some information is available regarding structure-activity relationships (including negative controls). Unlike other autoimmune models of nervous system destruction the treated animal is not debilitated and general health and survival of the animals are not affected to any appreciable degree. Additionally, only a single cell type, the sympathetic neuron, is affected and these cells can be isolated in cell culture free of other cell types, thus facilitating in vitro studies of mechanism. We hope that this phenomenon attracts the interest of other workers interested in autoimmunity and that the eventual elucidation of the antigen being attacked and the mechanism by which nerve growth factor prevents the destruction will provide insight into other autoimmune disorders.

CUANETHIDINE-INDUCED DESTRUCTION OF SYMPATHETIC NEURONS

35

Acknowledgments

We would like to thank Ms. Jacquelyn Udell and Ms. Linda Haniniond for their assistance in the preparation of this manuscript and our laboratory colleagues for their many helpful suggestions. E. M. J. is an Established Investigator of the American Heart Association, and P. T. M. is a Fellow of the Missouri and American Heart Associations. References

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Heath, J. W., Evans, B. K., Cannon, B. J., Burnstock, G., and James, V. B. (1972). Virchows Arch. B 11, 182-197. Heath, J. W., Erankb, O., and Erlnko, L. (1973). Acta Phamnacol. Toxicol. 33, 209-218. Heath, J. W., Hill, C. E., and Burnstock, G. (1974).J. Neurocytol. 3, 263-276. Hepp, R., and Kreye, V. A. W. (1973). Br. J. Pharmacol. 48, 30-35. Hermansen, K. (1963). Acta Pharmacol. Toxicol. 20, 201-212. Heym, Ch., and Grube, D. (1975). Anat. Embryol. 148, 89-97. Hill, C. E., Mark, G. E., Eranko, O., Ekinko, L., and Burnstock, G. (1973). Eur.1. PhurmuC O ~ .23, 162-174. Jensen-Holm, J. (1967). Acta Pharmacol. Toxicol. 25, (Suppl. 4), 74. Jensen-Holm, J., and Juul, P. (1968). Br.J. Phumacol. 34, 211-212. Jensen-Holm, J., and Juul, P. (1970a). Acta Pharmucol. Toxicol. 28, 270-282. Jensen-Holm, J., and Juul, P. (1970b). Actu Pharmacol. Toxicol. 28, 283-298. Jensen-Holm, J., and Juul, P. (1971). Actu Phamacol. Toscicol. 30, 308-320. Johnson, E. M. (1978). Brain Res. 141, 105-118. Johnson, E. M.,and Aloe, L. (1974). Bruin Res. 81, 519-532. Johnson, E. M., and Hunter, E. F. (1979). Biochem. Pharmacol. 28, 1525-1531. Johnson, E. M., and Macia, R. A. (1979). Circ. Res. 45, 243-249. Johnson, E. M., and OBrien, F. (1976).J. Pharmucol. E x f . T h . 196, 53-61. Johnson, E. M., Cantor, E., and Douglas, J. R. (1975).J. Pharmacol. Ex$. Ther. 193, 503512. Johnson, E. M., OBrien, F., and Werbitt, R. (1976). Eur. J. Phamacol. 37,45-54. Johnson, E . M., Macia, R. A., and Yellin, T. 0. (1977). Lye Sci. 20, 107-112. Johnson, E. M., Macia, R. A., Andres, R. Y., and Bradshaw, R. A. (1979). Brain Res. 171, 46 1-472. Johnson, E. M., Gorin, P. D., Brandeis, L. D., and Pearson, J. (1980). Science (Washington, D.C.) 210, 916-918. Juul, P. (1973). Actu Pharmucol. Toxicol. 32, 500-512. Juul, P. (1977). In “Drug Design and Adverse Reactions” (H. Bundgaard, P. Juul, and H. Kofod, eds.), pp. 63-76. Academic Press, New York. Juul, P., and Sand, 0. (1973). Actu Pharmacol. Toxicol. 2, 487-499. Klein, R. M.(1979a). Cell Tissue Kinet. 12, 41 1-423. Klein, R. M. (1979b). CeU T i w e Kind. 12,649-657. Levens, N. R., Peach, M. J., and Carey, R. M. (1981).J. Clin.Invest. 67, 1197-1207. Levi-Montalcini, R., and Angeletti, P. U. (1966). Pharmacol. Rev. 18, 619-628. Levi-Montalcini, R., and Booker, B. (1960). Proc. Natl. Acad. Sci. USA 46, 384-391. Mattre, L., Staehelin, M., and Brunner, H. (1971).J. Pharm. Pharmacol. 23, 327-331. Malmquist, J., and Oates, J. A. (1968). Biochem. Phamacol. 17, 1845-1854. Manning, P. T., Russell, J. H., and Johnson, E. M. (1982). Brain Res. 241, 131-143. Manning, P. T., Powers, C . W., Schmidt, R. E., andJohnson, E. M. (1983).J. Neurmci. 3, 7 14-724. Maxwell, R. A. (1982). Br. J . Clin Phamucol. 13, 35-44. Maxwell, R. A., and Wastila, W. B. (1977). Handb. Exp. Pharmacol. 39, 161-261. McMartin, C. (1969). Biochem. Plwrmacol. 18, 238-243. McMartin, C., and Simpson, P. (1971).Clin. Pharmacol. Ther. 12, 73-77. Nielsen, G. D. (1977a). Acta Pharmacol. Toxicol. 41, 203-208. Nielson, G . D. (1977b). Acta Phurmacol. Toxicol. 41,209-217. ODonnell, S. R., and Saar, N. (1974). Eur. J . Pharmacol. 28, 251-256. Ostman-Smith, I. (1976). Neuroscience 1,497-507. Otten, U., Goedert, M., Mayer, N., and Lembeck, F. (1980). Nature (London) 287, 158- 159.

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Overbeck, H. W. (1979). Hypertension 1, 486-492. Paterson, P. Y. (1971). In “Immunological Diseases” (M. Samter, ed.), pp. 1269-1298. Little, Brown, Boston, Massachusetts. RCvtsz, E., and van der Zypen, E. (1979). Acta Anat. 105, 198-208. Rodionov, J. M., Yarygin, V. N., Mukhammedov, A., Manukhin, B. N., Lebeder, D. B., and Nesterova, L. A. (1981). Ppuegen Arch. European J . Physiol. 392, 206-209. Rouiller, Ch. (1960). Int. Rev.Cytol. 9, 227-292. Schanker, L. S., and Morrison, A. S. (1965). Int. J. Neuropharmacol. 4, 27-39. Schlitter, E. (1977). Handb. Exp. Phnrnugcol. 39, 13-59. Schmidt, R. E., Geller, D. M., and Johnson, E. M. (1981). Diabetes 30,416-423. Schwartz, J. P., Pearson, J., and Johnson, E. M. (1982). Brain Res. 244, 378-381. Simon, G . (1981). A m . J . Physiol. 241, H449-H454. Sorimachi, M. (1977).J.J . Pharmacul. 27,629-634. Wakshull, E., Johnson, M. I., and Burton, H. (1981).J. CellBzol. 79, 121-131.

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DENTAL SENSORY RECEPTORS By Margaret

R. Byerr

Departments of Anesthesiology and Biological Structure Center for Research in Oral Biology University of Washington Seattle, Washington

I. Introduction ................................................... 11. Dental Sensory Axons.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Afferent Axons in Trigeminal Nerves ......................... B. Axonal Size at the Root Foramen . . . C. Intradental Arborization. . . . . ............................... D. Cytochernical Diversity . . . . . . ................. 111. Location of Sensory Nerve Endings. . . . . A. Mature Teeth . . . . . . . . . . . . . . . . . . . . . . . . ......... B. Reinnervated Teeth . . . . . . . . . . . . . . C. Developing Teeth ........................... D. Continuously Erupting Teeth.. ................................. IV. Ultrastructure of Sensory Nerve Endings ...................... V. Neural Relationship to Other Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Schwann Cells and Fibroblasts.. ................................ B. Odontoblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................ C. Axo-Axonic Contacts VI. Sensory Transduction Me sms .............................. A. General Sensory Properties of Teeth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Sensory Transduction in Dentin . . . . . . . . . . . . . C. Sensory Transduction in Pulp .................................. VII. Summary. . . . . . References. . . . . ..........................................

39 40 40 46

49

64 68 74 74 75 79 80 80 81 84

85 87

1. Introduction

The purpose of this review is to summarize what is known about sensory innervation of teeth. Earlier reviews by Windle (1934), Brashear (1937), Bernick (1948a,b), Baume (1980), and especially by Fearnhead (1967) have thoroughly discussed the initial morphological work, so that the present article is primarily concerned with structural studies done after 1960. The general sensory properties of teeth will be discussed briefly, especially in relation to dental sensory mechanisms, but for more 39 1NTERNATION.AL REVIEW OF NEUROBIOLOGY, VOL. 25

Copyright 8 1984 by Academic Press, Inc. All rights of reproduction irr any Corm reserved. ISBN 0-12-366825-5

40

MARGARET R. BYERS

comprehensive reviews of dental neurophysiology, see Anderson et al. (1970), Matthews (1972), Mumford and Bowsher (1976),Anderson and Matthews (1976), Dubner et al. (1978), Sessle (1979), and Narhi et al. (1982d). I n this article, the following questions will be considered 1. What kinds of axons innervate teeth? 2. Where are the sensory nerve endings? 3. What is the ultrastructure of the axoplasm in the nerve endings? 4. What kinds of junctions are formed between sensory nerve endings and other cells such as odontoblasts and do they have functional significance? 5. Where are axo-axonic contacts found in teeth and what function, if any, do they have? 6. What are the possible mechanisms for sensory transduction in teeth?

Finally, what are dental sensory receptors? Is there more than one kind of receptor? Do receptors include everything extending beyond the last perineurial sheath (i.e., almost the entire innervation of each tooth)? Do receptors only include the plexuses of small axons along pulpal blood vessels, the plexuses in peripheral pulp, and the free endings in pulp and dentin? Is receptor activity restricted to the free endings extending beyond the Schwann cells and basal lamina into the odontoblast layer, predentin, and dentin? Does sensory transduction only occur at the beaded regions along the free endings where numerous vesicles and mitochondria are found? To what extent can correlations be made between the structure of sensory nerve endings in teeth and their function?

II. Dental Sensory Axonr

A. AFFERENT AXONSIN TRICEMINAL NERVES Teeth are innervated by bipolar sensory neurons whose cell bodies are in the Gasserian (semilunar) trigeminal ganglion and whose central axons innervate the main sensory nucleus and spinal subnuclei of the trigeminal system (Darian-Smith, 1973; Westrum et al., 1976, 1980, 1981; Johnson and Westrum, 1980; Arvidsson and Gobel, 1981). Their peripheral axons follow maxillary and mandibular branches of the tri-

DENTAL SENSORY RECEPTORS

41

geminal nerve (Robinson, 1979; Fried, 1982). Morphological studies of these nerves and their alveolar branches have found myelinated and unmyelinated axons in a roughly 1 : 1 ratio (Kerr, 1966; Young, 1977). T h e myelinated axons range from 2 to 16 pm in diameter (Windle, 1926; Brashear, 1936; Young, 1977; Fried, 1982), but several studies suggest that the largest axons do not innervate teeth. Pulpectomy was found to cause degeneration or chromatolysis only in the small cell bodies (<30 pm) in the trigeminal ganglion (Windle, 1927; Brashear, 1936), and degeneration of unmyelinated and small myelinated axons in the spinal trigerninal tract (Gobel and Binck, 1977). Many studies found that electrical stimulation of teeth elicited a variety of conduction velocities most of which were in the C-fiber or A-delta range (Wagers and Smith, 1960; Greenwood et al., 1972; Horiuchi, 1974; Horiuchi and Matthews, 1974, 1976; Matthews, 1977) with a few axons conducting at low A-beta velocities of 30-40 m/sec (Wagers and Smith, 1960; Matthews, 1977). This information correlated well with the small diameters of axons at the root foramen and with conduction velocities of nociceptive afferents to suggest that teeth are only innervated by A-delta and C axons. However, it has also been found that 'dental afferent conduction velocity can increase after the action potential leaves the tooth (Horiuchi, 1965), in some cases reaching the 50-60 m/sec (Lisney, 1978; Sessle, 1979; Dong, 1981; Cadden et al., 1982, 1983). This indicates that many axons entering the tooth root are branches from larger parent axons in alveolar nerves (Table I), as might be expected from the relatively poor localization of pulpal sensation in human subjects (Van Hassel and Harrington, 1969). Lisney (1978) demonstrated clearly that the conduction velocity spectrum changes from unimodal in the pulp to bimodal in alveolar and trigeminal nerves. Anderson and Pearl (1974) found maximal velocities of 55-61 m/sec in cats when recording at the trigeminal ganglion, and Cadden et al. (1982, 1983) have recorded velocities of 57.7 m/sec between the root apex and the mandibular nerve. Some of the myelinated dental axons are therefore conducting in the A-beta range according to the classification of Gasser and Erlanger (1927) and may exceed the velocity for nociceptive afferents. T h e unmyelinated dental C fibers are difficult to detect by physiological methods and were not found in many of the early conduction experiments (e.g., Funakoshi and Zottermann, 1963). However, Wagers and Smith (1960), Greenwood et al. (1972), Bessou et al. (1970), Anderson and Pearl (1974), Matthews (1977), Lisney (1978), Cadden et al. (1982, 1983), and Narhi et al. ( 1 98213) all found some dental axons conducting

42

MARGARET R. BYERS

TABLE I

Tyms OF DENTAL AXONS" Intradental axons Function Sympathetic efferents

Neulotransmitter Noradrenalin

Type

Parent axons in trigeminal nerves

Unmyelinated

Unmyelinated

Unmyelinated (C-fiber) Myelinated

C-fiber A-delta

(A-delta)

A-beta

Vasoactive Intestinal Peptide Sensory affcrents (tliermoreceptors, chenioreceptors,

Substance P Acetylcholine

mechanoreceptors) a

References for each entry given in text.

less than 2 m/sec. An interesting characteristic of these axons is that they are especially reactive to heat stimuli (Narhi et al., 1982b). Just as some of the myelinated axons increase their size and conduction velocity after leaving the tooth, some unmyelinated pulp axons increase their size and velocity in alveolar nerves. Jiffry (1981) found that the axons of rat incisor pulp, which are almost all unmyelinated (Bishop, 1981), increased their conduction velocity from 1.4 to 21.8 m/s in the alveolar nerve. Perhaps the innervation of rat incisors is unique, but it is possible that some of the unmyelinated axons in the roots of other teeth also are terminal branches of myelinated parent axons outside the tooth. Finally, it is possible that some of the trigeminal neurons that innervate teeth originate in the mesencephalic nucleus. Mesencephalic trigeminal neurons are primarily involved in dental proprioception o r mastication (Corbin and Harrison, 1940; Jerge, 1963; Darian-Smith, 1973; Cody et al., 1974). It is generally thought that all receptors for dental mesencephalic neurons are in the periodontal tissues (Anderson et al., 1970). It is interesting, however, that when horseradish peroxidase (HRP) is injected into tooth pulp, it travels by retrograde axonal transport to some mesencephalic perikarya as well as to perikarya in the Gasserian ganglion (Chiego et al., 1980).Either the HRP leaked out into

DENTAL SENSORY RECEPTORS

43

the periodontal tissues from the injected pulp or some mesencephalic neurons innervate teeth. Further work is needed to settle this question.

B. AXONAL SIZEAT

THE

ROOT FORAMEN

T h e axonal size at the root foramen (or root apex) has often been examined to find out what kinds of afferent fibers leave the tooth. Small myelinated axons and unmyelinated axons are found; degeneration studies suggest that all of the myelinated axons are sensory (Arwill et al., 1973; Feher et al., 1977), whereas some of the unmyelinated axons are sympathetic (Fried and Hildebrand, 1978), and the rest are sensory (FehCr et al., 1977). In one case myelinated and unmyelinated axons were found at the apices of cat teeth after inferior alveolar nerve resection (Frank et al., 1972), but the postinjury survival time was so long (2 months) that regeneration would already have occurred (Robinson, 1980b; Fried and Erdelyi, 1982). T h e early light microscopists had difficulty distinguishing the unmyelinated axons and concluded that there were only a few relative to the number of myelinated axons (for review see Windle, 1934; Fearnhead, 1967). However, Graf and Bjorlin (1951) were able to make rough estimates of the number of unmyelinated axons at the root foramen using light microscopy as well as to determine the number and size of myelinated axons (Table 11). They found that estimated numbers of axons varied widely from 15 1 to 1296 myelinated axons and from 40 to 650 unmyelinated axons at the root apices of human teeth. The maximal axonal diameter varied from 7 to 13 pm, but the great majority of the axons (83-9996) were equal to o r less than 4 pm in diameter. Similar variation in axonal number and size have been found in quantitative electron microscopic studies of apical root pulp of different teeth (Table 11). In most of these studies axonal diameter was less than 7 pm, with most axons in the 2-4 pm range. However, Jones and Anderson (1982) have reported axons as large as 15 p m in juxta-apical nerves, and some of the light niicroscopic studies have found large axons at the root foramen (Graf and Bjorlin, 1951; Graf and Helniquist, 1955). This suggests that the larger axons of alveolar nerves, whose existence is shown by conduction studies (Lisney, 1978; Cadden et d.,1982), may extend into the root in some cases before arborizing into smaller axons. Some of these differences probably depend on the maturation of the teeth, because axonal size gradually increases in niature cat incisors (Fried, 1982).

TABLE I1

DENTAL AXONSAT

THE

ROOTAPEX

Number of axons b b

rp

Reference Graf and Bjorlin (1951)

Johnsen and Johns (1978)*

Johnsen and Karlsson (1974)

Tooth Human permanent Incisors Cuspids Bicuspids Bicuspids Human permanent Incisors Canines Human deciduous Incisors Canines Cat permanent Incisors Cat deciduous Incisors

Age

M"

Unm

24-44 years 18-42 21 12

257-991 390- 1296 1135 151-457

200-300 40-250 400 95-650

35-55 years 35-55

359 361

7-8

2 4

46 82

7-8

247 2 19 439 f 71

1-2 years

126 f 31

5-8 weeks

22

* 16

Axonal diameter (Pm)

M" 1-10 1-9 1-13 1-7

1591 2 728 2240 2 966

0.5-6.7 0.5-6.7

2083 2 1031 2521 2 722

0.5-6.7 0.5-6.7

432

2

Unm

63

149 2 110

-

-

Fried and Hildebrand (1981a,b)’

Jones and Anderson (1982) Holland and Robinson (1982) Bueltmann et al. (1972)b

Cat permanent Incisors

Cat deciduous Incisors Cat permanent Canines Cat permanent Canines Marmoset permanent Incisor core Incisor periphery Canine core Canine periphery

3 months 7 months 17 months 3 years 9 years

2-15 14-60 29-48 3-53 12-37

8-55 76-330 121-202 17-385 37-116

2 months

10-20

80-90

Adult

153

192

Adult

193-529

375-1376

Adult

53-230 9-77 55- 137 38-4 1

50-228 45-121 105- 155 382-39 1 ~

a

M,Myelinated axons; Unm, unmyelinated axons. Axonal diameter for these papers estimated from data on axonal circumference. Number of axons estimated from data on total axons and percent unmyelinated axons.

~~~

1-3.5 1-6 1-8 1-5.5 1-9

0.1-1.6 0.1-0.8 0.1-0.8 0.1-0.6 0.1-0.5

1-5

0.1-1.2

0.75-15.0

0.2-3.0

0.5-6.5 0.5-6.5 0.5-6.5 0.5-6.5

0.1-1.5 0.1-1.5 0.1-1.5 0.1-1.5

46

MARGARET R. BYERS

Bueltmann et al. (1972) found that in apical pulp some axons form a central bundle of myelinated and unmyelinated axons, and others are found in the periphery of the pulp. The peripheral axons are mostly unmyelinated; they could either be preterminal branches that end in roots o r there may be a special set of axons that goes directly to the peripheral root pulp. Many axons branch in the first 1-2 mm of the root pulp so that very different numbers are found depending on the exact site at which the sample is taken. An interesting new study has found that although axonal number and size vary widely for particular teeth from one animal to another [most likely due to age differences (Fried, 1982)], there is very little variation between similar teeth within the same animal, in this case mandibular canines of cat (Holland and Robinson, 1982). Similarly, Graf and Bjorlin (1951) found about the same number of myelinated axons (315 and 390 axons) in cuspid (canine) teeth from a human donor, and permanent incisors from the same cat had fairly similar numbers of myelinated axons (126 -+ 31) (Johnsen and Karlsson, 1974). In a comparison of deciduous and permanent teeth Johnsen and Johns (1978) found no major differences in axonal caliber. Johnsen and Karlsson (1977) found a gradual maturation of axonal number and form during maturation of deciduous and permanent cat teeth, and Johnsen and Karlsson (1974) and Fried and Hildebrand (1981a,b) found more axons in permanent incisors than in deciduous incisors of cats. Although this suggested that fully developed permanent teeth have a more sophisticated sensory apparatus, permanent teeth are proportionally larger and there is approximately the same innervation density in relation to the size of the permanent teeth as in deciduous teeth (Johnsen and Karlsson, 1974).

C. INTRADENTALARBORIZATION The principal site of intradental arborization is in the crown, as shown by numerous histological studies (for reviews see Windle, 1934; Brashear, 1936; Fearnhead, 1967; Baume, 1980) and electron microscopic studies (Arwill, 1967; Harris and Griffin, 1968; Frank, 1968; Frank et al., 1972; Corpron and Avery, 1973; Dahl and Mjor, 1973; Byers, 1977; Holland, 197’7). Most of the axons in the pulp core are ensheathed by Schwann cells and look like conducting axons, not terminal axons. However, some axons branch to innervate root pulp or dentin

DENTAL SENSORY RECEPTORS

47

(Fearnhead, 1957; Byers and Matthews, 1981; Byers and Dong, 1983). In a quantitative study of axonal number in cat canines a 1 : 3 branching ratio has been found between root apex and midcrown (Beasley and Holland, 1978; Holland and Robinson, 1982). This ratio might have been even greater, because the extensive branching at the crown tip was not included. T h e final arborization of intradental axons in the plexus of Raschkow, the odontoblast layer, and the dentin is discussed in Section 111.

D. CYTOCHEMICAL DIVERSITY Recent work described below has shown that dental innervation has distinct sets of ( 1 ) sympathetic unmyelinated axons containing noradrenalin or vasoactive intestinal peptide or both, (2) large and small cholinergic sensory axons, and (3) small substance-P-reactive sensory axons (Table I). Both the cholinergic and the substance-P sensory axons occur on blood vessels and in the peripheral pulp, so that in teeth each of these may have more than one function. Histochemical and degeneration studies have shown that dental sympathetic axons are unmyelinated and contain high concentrations of noradrenalin, whereas sensory axons are myelinated and untnyelinated and are cholinergic (Christensen, 1940; Kukletova et al., 1968; Pohto and Antila, 1968a,b, 1972; Larsson and Linde, 1971; Arwill et al., 1973; FehCr et al., 1977; Avery et al., 1980). The noradrenergic axons are primarily associated with blood vessels but can also end in other pulp regions including the odontoblast layer and possibly dentin (Avery, 1975; Avery and Cox, 1977; Avery et al., 1980). The cholinergic axons are more numerous and have fine beaded endings forming plexuses along blood vessels o r are terminal axons of various sizes in peripheral pulpal plexuses (Kukletova et al., 1968). T h e cholinergic fibers on blood vessels may be sensory. Alternatively, the paravascular cholinergic fibers may be parasympathetic axons (Chiego et al., 1980) or they may be sympathetic axons with low levels of cholinergic reactivity (Pohto and Antila, 1972). An initial report that acetylcholinesterase activity was present in dentinal tubules (Rapp et al., 1959) seems to have been based on artifacts caused by excessive incubation times (Rapp et al., 1968) and others could not repeat those results (TenCate and Shelton, 1966). Numerous small axons in cat canines have recently been shown to be immunoreactive to substance P (Olgart et al., 1977), (see Fig. l), but the larger myelinated pulp axons were not reactive. The reactivity of small

48

MARGARET R. BYERS

FIG. 1. Irnmunofluorescence micrographs of cat dental pulps after incubation with antiserum to substance P (SP) (A-E) or control serum (F). SP-positive nerves are seen in bundles (arrowheads in A and B) or as single fibers (B-E), sometimes related to blood vessels (asterisk in A). Note that SP-immunoreactive fibers often are running close to myelinated axons (arrow in B and C) but clearly are separated from them. The myelinated

DENTAL SENSORY RECEPTORS

49

fibers was lost after transection of the inferior alveolar nerve but not after extirpation of the cervical sympathetic ganglion. The substance P axons are therefore sensory but do not include the largest sensory axons. Substance P axons were found along blood vessels and throughout the pulp; most axons appeared to terminate in the plexus of Raschkow, but some could be traced as far as the odontoblast layer. Whether they also innervate dentin could not be determined in the excised pulps used for this study. Antidromic stimulation of the substance P axons causes vasodilatation in the pulp and may be involved in neurogenic inflammatory responses (for review see Gazelius rt al., 1977, 1981; Olgart, 1979; Gazelius, 1981). T h e characteristics of substance P-mediated vasodilatation were different from those of cholinergic-mediated vasodilatation, suggesting that these sets of neurons may be distinctly different (Gazelius, 1981). A recent study of capsaicin toxicity also suggests that cholinergic neurons are different from substance P neurons (Gamse et al., 1982). Other substances such as histamine (Kroeger, 1968; Pohto and Antila, 1972; Olgart, 1974), serotonin (Olgart, 1979), prostaglandin (Ahlberg, 1978), somatostatin (L. Olgart, personal communication), and vasoactive intestinal peptide (VIP) (Olgart et al., 1981) affect dental sensitivity, are present in pulp, and play a role in vasodilatation, vascular permeability changes, and inflammatory responses. It is interesting that VIP activity in pulp is abolished by sympathectomy but not by sensory nerve transection, whereas the reverse is true of substance P (Olgart et al., 1981).

111. location of Sensory Nerve Endings

A. MATURETEETH 1. Early Work The general characteristics of pulpal innervation have been known ever since the early histological studies of Retzius (1892, 1893)and others such as Boll and Raschkow in the nineteenth century (for review see axons contain no specific fluorescence. SP-positive fibers may, however, also run completely independent of myelinated axons (D). Close to the odontoblasts (arrows in E) numerous thin, varicose fluorescent fibers are seen. No specific immunofluorescence is observed in sections incubated with control serum (F), only unspecific fluorescence along tissue border, in this case blood vessels (asterisk).(A-E) 2 5 0 X and (F) I O O X . Bar in F: 0 . I mm. [From Olgart el n / . (1977), reproduced with permission.]

50

MARGARET R. BYERS

Fearnhead, 1967; Baume, 1980). Initially, methylene blue staining of vital tissue gave good information that was supplemented later by osmic acid staining o r by the gold or silver impregnation techniques. From these studies it was found that teeth are heavily innervated, the nerves are relatively unbranched in the roots, they arborize extensively in the crown, and they form a plexus of fine beaded terminal axons in the peripheral pulp of the crown. The difficult problem has been to determine whether and to what extent dentin is also innervated. Uncertainty surrounded this question until the past 20 years because the histological techniques could not reliably show dentinal axons. There were three main kinds of technical problems. First, the silver impregnation methods that worked so beautifully to reveal nerves elsewhere did not work well in dentin. T h e chemistry of the tissue altered the way in which the silver was deposited and the presence of argyrophilic connective tissue fibers confused the results. For most investigators the axons could be followed as far as the odontoblastic layer but vanished as soon as they reached predentin or dentin. Several investigators found evidence of innervation of dentin (e.g., Mummery, 1924; Wassermann, 1939; Fearnhead, 1957, 1961, 1963; Rowles and Brain, 1960; Hattyasy, 1961), which in some cases appeared to be in the dentinal matrix rather than in the tubules (Powers, 1952). Others were not able to repeat their work (e.g., Bernick, 1952, 1956), probably because of subtle differences in the exact procedures of tissue fixation, embedding, and staining. Occasionally, stained nerve fibers appeared to enter dentin and then loop back to the pulp (Bernick, 1968). This pattern, however, may be an artifact that occurs only when the contents of dentinal tubules “fall out” of the section during processing and lie across the dentin. This problem has been observed occasionally in some autoradiographic sections (M. R. Byers, unpublished observations) and might allow a positive silver reaction that ordinarily could not occur within the dentinal tubules. It should be noted that nerve loops have not been found in autoradiographic (Byers and Kish, 1976; Byers, 1980; Byers and Matthews, 1981; Byers and Dong, 1983) studies or horseradish peroxidase-labeled teeth (Marfurt and Turner, 1983). T h e second technical problem involved photomicrography. It was impossible to reproduce clearly what the histologists saw in the microscope because of the thickness of the sections. For example the photographs of Mummery (1924), Wassermann (1939), and Fearnhead (1957, 1961, 1963) only approximated what they saw, and supplementary drawings were used. In some cases photos were retouched to show the position of “nerves” more clearly (Powers, 1952). A common source of

DENTAL SENSORY RECEPTORS

51

argument and controversy concerned the accuracy of the published drawings (e.g., Lewinsky and Stewart, 1936; Tiegs, 1938). T h e third technical problem involved tissue sampling procedures. It is now known that dentinal innervation is much greater immediately adjacent to the tip of the pulp horn than in other coronal regions, and it is much less in roots (see Section III,A,4). The highly publicized controversy between Mummery and Hopewell-Smith in the early twentieth century depended in part on the fact that Mummery (1924) often took his samples from the tip of the crown, whereas Hopewell-Smith (1924) sampled roots; not surprisingly, Mummery was the only one to find nerve endings in dentin. Similarly, the electron microscopic studies by Frank (1966, 1968), Arwill (1967), and Arwill et al. (1973) that found dentinal innervation were of samples near the pulp horn tip, whereas Holland’s initial EM studies, which did not find innervation of dentin, were of samples from midcrown or root regions (Holland, 1975, 1976c, 1980b). 2. Electron Microscopy The question of dentinal innervation began to be answered in the 1960s when electron microscopic studies clearly revealed nervelike cells in dentinal tubules (Frank, 1966, 1968; Arwill, 1967; Harris and Griffin, 1968; Avery, 1971, 1975; Frank et al., 1972; Corpron and Avery, 1973; Dahl and Mjor, 1973). This was further clarified when Corpron et ul. (1972) and Arwill et al. (1973) showed degeneration of the nervelike cells in the dentinal tubules after transection of the inferior alveolar nerve. However, the similarity between the structure of some odontoblastic collaterals and that of the terminal axons caused controversy about cell identification in the electron micrographs (for further discussion see Sections I V and V). I n addition the small size of the samples removed for EM made it difficult to determine the full extent of the dentinal innervation.

3. Axonal Transport Mupping Techniques ‘The development of axonal transport mapping techniques in the 1960s greatly increased neuroanatoniical information in general (for review see Cowan and Cuenod, 1975; Schwartz, 1979; Heimer and Kobards, 1981; Ochs, 1982) and provided ways to learn much more about dental innervation. The anterograde axonal transport method depends upon the fact that neuronal protein synthesis occurs almost exclusivel) in the nerve cell body or in the dendrites. Although a few small peptides can be

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synthesized in axons and a small proportion of axonal proteins are made by the Schwann cells, almost all the proteins needed for cell function in the axons and nerve endings are supplied via anterograde axonal transport. Lasek et al. (1968) were the first to realize that this neuronal phenomenon could be used to map nerve pathways by allowing sufficient time for axonal transport of radioactive protein and then detecting that radioactivity by autoradiography. An important aspect of autoradiographic mapping of rapid axonal transport is that only the neurons with their perikarya near the injected precursor (e.g., [3H]proline) will have sufficient production and transport of radioactive protein to be detected by the relatively insensitive photographic emulsion. Axons passing through the injected zone are not significantly labeled, and neurons in other regions do not pick up radioactive precursor or protein in sufficient amounts from the isotope that leaks into the general circulation during injection to be detected. It has been found that axonal transport occurs at similar rates in peripheral sensory axons as in efferent axons (Droz, 1965; Lasek et al., 1968; Fink et al., 1975; Brimijoin et al., 1980). In trigeminal axons the rapidly transported material has the same biochemistry as in axons that lead to synaptic endings; the protein moves in the cortical axoplasm, accumulates at a ligature or at the sensory endings, and is affected in the same manner by lidocaine as had been found for efferent axons (Droz, 1965; Fink et ad., 1975; Byers et al., 1975). With survival times up to 1 week transported protein remains within the sensory endings and does not transfer to epithelial cells in vibrissae o r gum, to odontoblasts in teeth (Fink et al., 1975; Byers and Holland, 1977; Byers, 1977, 1979), o r to epithelial cells of cornea (Weill et al., 1975). Instead, silver grains are clustered over the sensory endings, and the adjacent local cells have only background levels of labeling. A similar result was found by Fidone et al. (1975, 1977) for sensory endings in chemoreceptors; as measured by autoradiography, the local glomus cells were not labeled at all but the sensory endings were well labeled. It is quite likely that small amounts of radioactive material leak into adjacent cells, if only because of turnover and reutilization of the radioactive proteins. However, from the point of view of the autoradiographic emulsion, the label in the neighboring nonneural cells, if present, is too dilute to be detected. The anterograde transport method is therefore a reliable one for identifying sensory nerve endings in teeth. Retrograde axonal transport also occurs in dental neurons, so that extracellular markers such as horseradish peroxidase (HRP) are taken up by the sensory endings and transported back to the cell body and central synaptic endings. Retrograde transport of HRP from tooth pulp

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or trigeminal nerves has been very useful for studies of the extent of peripheral branching of dental or orofacial afferents, the somatotopic organization of the ganglion, and the location of central terminals (Arvidsson, 1975; Furstman rf al., 1975; Anderson P t at., 1977; Cox P t nl., 1977; Grant et al., 1979; Chiego et al., 1980; Westruni P t ul., 1980, 1981 ; Wilson et al., 1980; Winfrey et al., 1980; Marfurt, 1981). Recently, Marfurt (1982) showed that HRP can also move from central endings or trigeminal perikarya to corneal sensory nerve endings, in a manner similar to the anterograde transport of HRP to cutaneous endings (Robertson and Aldskogius, 1982). Similar methods can therefore be applied to dental neurons for further understanding of their peripheral arborization (Marfurt and Turner, 1983). 4 . Innervation of Dentin

In 1975 Fink et al. showed that labeled trigeminal nerve endings extended as far as 0.2 mm into dentinal tubules in rat molar crowns. T h e axonally transported protein moved at a rapid rate of at least 288 mm/ day and could be demonstrated in dentinal endings as early as 3 hr after injection of the ganglion. Quinton-Cox (1975) and Weill et al. (1975) also used axonal transport at that time to label nerve endings in cat teeth. The results of Weill et al. were somewhat different from those of Fink et al., for in the former study the labeled nerves only went 10-20 p m into the dentinal tubules, and the pulpal plexus was much denser. The source of Weill’s samples is not mentioned, so perhaps the autoradiograms were of cervical or midcrown regions where nerve penetration into dentin would have been short, as shown later by Byers and Matthews (198 1). T h e heavy labeling of the plexus by Weill may simply have been due to the intense radioactivity achieved in their experiments. In later studies of rat innervation some pulpal plexus axons were similarly well labeled (Byers, 1979, 1980; Byers et al., 1982a). Four other autoradiographic studies were published in 1976-77. Byers and Kish (1976) and Byers (1977) reported further results on innervation of rat molars; Pimenidis and Hinds (1977) reported on labeling studies of rat molars; Menke et al. (1977) described similar experiments on rats. The rats used by Menke et al. unfortunately had a very short postinjection survival time, so that radioactivity was very weak and silver grains representing transported radioactivity were indistinguishable from the background labeling of dentin; most of the quantitative data of Menke et al. do not correlate with the findings of others. Pimenidis and Hinds demonstrated good labeling of dentinal tubules and of nearby pulp. They also showed occasional dentinal tubules labeled near the dentino-enamel junction. We also have seen this labeling pattern in

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slides suffering from drying artifacts (for discussion of these artifacts see Rogers, 1979); when this happens the slides of control teeth also show the same patterns and the entire set of slides must be rejected. We have not seen labeling beyond the inner 0.2 mm of dentinal tubules in autoradiograms free from such artifacts (Figs. 2 and 3). Several of the early observations on labeled dental innervation were confirmed in later studies of rat molars (Byers, 1979, 1980), cat teeth (Byers and Matthews, 1981), and monkey teeth (Byers and Dong, 1981, 1983).

1. Sensory nerves innervate dentin and can extend u p to 0.2 mm into dentinal tubules at the tip of the crown but usually penetrate shorter distances in other coronal regions. 2. Certain kinds of dentin are not innervated (interradicular dentin) or are poorly innervated (reparative dentin). 3. The innervation of tubular dentin (orthodentin) is graded, with the greatest innervation adjacent to the tip of the pulp horn. Fewer nerves are present in mid-crown dentin, even fewer in intercuspal and cervical dentin, and fewest in the dentin of roots. 4. Near the tip of the crown more than 50% of the dentinal tubules can be innervated. 5. T h e nerves are oriented parallel to the dentinal tubule and do not form numerous recurrent loops or perpendicular branches as found in some earlier histological experiments (Bernick, 1968; Langeland and Yagi, 1972). Several criteria are important in order to be certain that silver grains over dentinal tubules represent radioactive axons. The following have been used throughout our studies. 1. Teeth from the ipsilateral (injected) side of the animal must be compared with contralateral posterior teeth (which do not receive transmedian innervation) to monitor background silver grains and the occasional occurrence of drying artifacts. 2. Labeled pulp nerves must be identified and checked to see whether labeling is consistent in serial sections. 3. These nerves must be followed in serial section to show continuity with labeled axons in coronal pulp and adjacent dentin. Other indices of specific labeling of dental sensory axons and endings are that the silver grains are most numerous over the coronal plexus of Raschkow, adjacent odontoblastic layer, and inner dentinal tubules; also EM autoradiography shows very specific labeling of nerve cells in pulp and inner dentin (Byers and Dong, 1983).

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FIG. 2. Autoradiograph of a cusp of a right first molar from a 100-day-old rat. T h e right trigerninal ganglion was injected with [3H]proline 24 hr prior to fixation in order to label axonally transported protein. Linear clusters of silver grains (arrows) show position of sensory endings in dentin and are most numerous along the walls of the cusp, near the tip of the pulp horn (P). T h e reparative dentin (RD), cervical dentin (CD), and intercuspal dentin (IC) are poorly innervated. Bar: 0.2 mm. 7 2 x . FIG. 3. Tip of cusp from a first molar of a year-old rat after 22.5 hr trigeminal axonal transport of [SH]proline-labeled protein. Dentinal attrition has greatly decreased the distance between the occlusal surface (wide arrow) and the dentinal sensory endings (thin arrows) as compared with the younger first molar in Fig. 2. Reparative dentin (RD) is poorly innervated. Bar: 50 pm. 190X.

Examples of autoradiographic labeling of sensory axons in rat molars are shown in Figs. 2-4. Note that (a) the silver grains are most concentrated near the tip of the pulp horn, ( b ) they are sparse over regions containing reparative dentin, (c) they are lined up over the dentinal tubules, ( d ) they extend about 100 to 150 p m into the tubule, and ( e ) in the case of a year-old rat (Fig. 3) they come close to the occlusal surface because of attrition of the dentin and the resultant proximity of

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pulp to the surface. The development of dentinal innervation (discussed in Section II1,C) progresses rapidly in rats, so that within 4 to 5 weeks of eruption the roots are almost fully formed and many coronal tubules contain radioactive nerves (Fig. 4).The same kind of labeling pattern has been found in cat teeth (Figs. 5 and 6) and monkey teeth (Fig. 7) Again the innervation is concentrated at the tip of the pulp horn. When very few axons in a tooth were well labeled, the arborization pattern could be identified for individual axons (Fig. 6). In general, then, the autoradiographic method has shown that dentinal tubules of mature mammalian teeth are well innervated near the tip of the crown and that the axons extend from the plexus of Raschkow across the cell-free zone of Weil into the odontoblastic layer and as far a s 0.2 mm into dentinal tubules (Fig. 8). In midcrown regions fewer tubules are innervated and intratubular distances are shorter. Root dentin can be innervated, but innervation is less frequent than in the crown. Finally, reparative dentin is sparsely innervated, and interradicular dentin is not innervated. These autoradiographic results on nerve distribution in teeth are in agreement with recent electron microscopic studies of human teeth (Lilja, 1979; Byers et al., 1982b).

5 . Contralateral Innervation Autoradiographic studies of dental nerves in cats found labeling in occasional axons in the contralateral maxillary central incisor but not in other contralateral teeth (Byers and Matthews, 1981). This result was in disagreement with physiological studies of Anderson et al. (197’1), Anderson and Pearl (1974), and Pearl et al. (1977) that showed contralateral innervation as far as the canines in cats and with retrograde tracing FIG. 4. Autoradiograph of tip of cusp from first mandibular molar of a 47-day-old rat; tissue postfixed in osmium and embedded in Epon. Linear arrays of silver grains over dentinal tubules show that much sensory innervation has occurred by 4 to 5 weeks posteruption. Axonal transport time after [3H]proline labeling was 4-5 hr. Bar: 20 pm, Byers ( I W ) ) , reprotlured with permission.] 4 6 0 ~ [From . FIG. 5. Silver grains after 24 hr axonal transport of [3HH]prolineplus [3H]leucinelabeled protein label a majority of cross-sectioned dentinal tubules next to the tip of the pulp horn (P) of a paraffin-embedded cat maxillary canine. Beyond the inner 40-60 pin of dentin autoradiographic label decreases to background level. Bar: 20 pni. 3 2 0 ~ . FIG. 6. This central incisor of a cat was contralateral to the injected ganglion. The clusters of silver grains in pulp and dentin show the location of terminals of a single welllabeled axon 3-4 pm in diameter, shown in the inset. Bar: 20 pm. Magnification of both pictures, 3 8 0 ~[From . Eyers and Matthews (1981), reproduced with permission.] FIG.7. This tip of a paraffin-embedded monkey canine was fixed 24 hr after injection of the ipsilateral trigeminal ganglion with [YH]proline.A majority of the dentinal tubules are labeled with silver grains, indicating the presence of sensory endings. Bar: 20 pm. 430x.

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FIG. 8. Schematic drawing showing sensory nerve location in pulp and dentin of a mature mammalian tooth. T h e percentages of innervated dentinal tubules at the specified regions, A-D, are as follows: A, More than 40% (Arwill, 1967; Byers, 1977; Lilja, 1979; Holland, 1981a); B, 4.1-X.S’%,;C, 0.2-1.0%; and D, 0.02-0.2% (Fearnhead, 1957). Note the different structure of the plexus of Kaschkow (Px), cell-free zone (cfz), odontoblastic layer (0), predenrin (P),and dentin in the crown as compared with the root. The innervation of blood vessels (bv) is also indicated. (Modified from Fearnheatl, 1957.)

studies of Chiego et al. (1980). However, the autoradiographic results agreed with retrograde transport mapping studies (Arvidsson, 1975; Westrum et ad., 1980, 1981; Wilson et al., 1980) and with physiological studies (Lisney, 1978; Matthews and Lisney, 1978; Robinson, 1979; Nord and Rolince, 1980) that did not find innervation of contralateral canines by ipsilateral trigeminal nerves. Robinson ( 1980a) has found physiological evidence for rare innervation of the contralateral central mandibular incisor in cats by axons of the ipsilateral lingual nerve.

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We have since mapped sensory innervation in monkey teeth and have found occasional weak labeling of axons in contralateral mandibular canines as well as incisors (Byers and Dong, 1983), which was missed in our preliminary study (Byers and Dong, 1981); none of the contralatera1 maxillary teeth were labeled in those experiments. In the autoradiographic study of cat teeth only 10-50% of the dental axons were labeled on the ipsilateral side, whereas in the monkey teeth 40-80% of axons in ipsilateral teeth were labeled, and there was a very high incidence of labeled neuronal perikarya in the ganglion. This suggests that we may have missed contralateral axons to canine teeth in the cat study because of the low incidence of labeled trigeminal axons and perikarya. Alternatively, the different results in cats and monkeys may be because of species differences. T h e anterograde mapping technique does not reveal all peripheral axons and if those that extend to contralateral teeth are from neurons with weak radioactivity, they would be unlabeled. Similarly, there might be a subset of pulpal/dentinal axons that label poorly, only occasionally, or at a different rate by the retrograde transport techniques, as has been found in other nerves that have asynchronous accumulation of HRP in their cell bodies (Warr et al., 1981). Further work is needed here to determine the exact peripheral distribution of dental axons to contralateral teeth.

B. REINNERVATED TEETH Several interesting facts about dental innervation have been learned in studies of reinnervated teeth. Robinson (1980b, 1981) has studied the return of sensitivity to cat canines after denervation; he found that by 6 to 9 weeks they were reinnervated but conduction velocities were lower than normal. T h e reinnervating axons were from ipsilateral mylohyoid and lingual nerves as well as the contralateral lingual and inferior alveolar nerves (IAN) if a nylon tube prevented regeneration of the ipsilateral IAN (Robinson, 1981). This showed that under these conditions there can be collateral sprouting across the midline from some axons that do not ordinarily innervate the teeth. Histological studies by Sorg (1960) and Machida (1977) suggested that sensory nerves return to their previous positions in pulp and dentin. A study has found essentially normal pulpal axon morphology 2 months after denervation (Fried and Erdelyi, 1982). An autoradiographic study of reinnervated rat molars found a correlation between the return of sensitivity and the reinnervation of dentin and peripheral pulp (Berger et al., 1983; Berger and Byers, 1983); it was found that sensory nerves grew back into dentinal tubules

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to the same depth as in normal molars (see Figs. 9-12). Finally, Ohman (1965) studied young replanted human teeth and found a good correlation between the regeneration of pulp and axons to the pulp/dentin border and sensitivity to external, monopolar electrical stimulation of the replanted tooth. It is interesting that sensitivity of replanted human teeth to external electrical stimuli can return to normal even when the number and caliber of reinnervating axons is less than normal (Ohman, 1965)-

C. DEVELOPING TEETH Studies of developing dental innervation have found the same maturation stages in all teeth including deciduous teeth, but there are very different rates of nerve maturation depending on the rate of tooth development (Wassermann, 1939; Bernick, 1959, 1964; Fearnhead, 1961, 1967; Corpron and Avery, 1973; Johnsen and Karlsson, 1977; Byers, 1980; Fried, 1982). These stages are 1. T h e early developmental period in which a few pioneer axons enter the dental papilla during crown formation. The number of axons increases a bit during crown formation, but there is no development of peripheral plexuses and there are only a few terminals in the odontoblastic layer. At this stage most axons are still associated with blood vessels. 2. T h e eruptive period in which roots form, the tooth erupts to the occlusal plane, dentin thickens, the pulp chamber narrows, and the root apices close to make the mature narrow root foramen. During this period there is a rapid development of sensory innervation to form the plexus of Raschkow and the terminals in the odontoblast layer, predentin, and dentin. This formation begins at the tip of the pulp horn and gradually spreads to include most of the crown. 3. The adult period in which there is a gradual increase in axon numbers and in the density of the pulpal plexus and dentinal innervation. This gradual maturation continues until in very old teeth reparative FIGS.9-12, The cusps of the right first mandibular molar are shown here in the normal adult rat (Fig. 9) and at 2 days (Fig. lo), 7 days (Fig. 11). and 21 days (Fig. 12) after transection of the right inferior alveolar nerve. Autoradiographic labeling of dentin and pulp (arrows) after 24 hr axonal transport of [3H]proline-labeled protein is absent at 2 days after injury; it is beginning to reappear by 7 days. By 2 1 days the sensory innervation has regenerated to pulp and dentin. Bar: 2 0 pm. Figs. 9-10, 3 1 5 X ; Figs. 11-12, 2 0 0 X . [Micrographs courtesy of Dr. R. L. Berger.]

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dentin formation, ingrowth of dentin, o r pulp calcification occlude the pulp chamber causing some loss of axons (Fried and Hildebrand, 1981b). T h e eruptive period takes only a few weeks in molars of mice and rats, so that most of the sensory innervation has formed within a month after tooth eruption (Corpron and Avery, 1973; Byers, 1980; Byers et al., 1982a),but in human teeth several years elapse before the root apex finally closes and the maturation of innervation is correspondingly slow (Fearnhead, 1961; Avery, 1971; Johnson et al., 1983). Finally, the deciduous teeth o f cats (Johnsen and Karlsson, 1977; Fried and Hildebrand, 1981a) and humans (Fearnhead, 1961) have a more rapid eruptive period than do the permanent teeth and a similarly more rapid development of sensory innervation. Thus the development of pulpal and dentinal innervation is closely linked to the rate of development of the tooth structure. T h e sensory axons that innervate teeth are selective in their choice of termination sites. They bypass the interradicular dentin and the dentin of the walls of the crown to go initially to the tip of the cusp (Wassermann, 1939; Fearnhead, 1961). This same pattern is found in replanted developing hamster teeth (Sorg, 1960). Because initial innervation of teeth follows the blood vessels, the final innervation pattern may depend on the vasculature pattern in the pulp, which has the densest subodontoblastic plexuses in the coronal cusp tips and does not make a capillary plexus adjacent to interradicular dentin (Kramer, 1968). The distinctive structure of the plexus of Raschkow, the cell-free zone, and the odontoblastic layer near the tip of the crown (Gotjamanos, 1969) may also be a guide to the development of the coronal sensory arborization and dentinal innervation. T h e first pioneer axons enter the dentinal tubules just prior to tooth eruption, when the odontoblasts of that region have only a few more weeks of rapid dentinogenesis (Byers, 1980). Whether the sensory axons initiate the change in odontoblastic function or whether the odontoblastic change initiates the beginning of dentinal innervation is not known, but when the axons enter the dentin the odontoblasts in that area appear to be still making dentin as rapidly as in adjacent noninnervated regions. The sensory axons enter 0.1-0.2 mm into dentinal tubules, both by their own growth and by the elongation of the surrounding tubule. By the time the dentinal axons have reached a depth of 100 to 150 p m the production of dentin for that region has virtually stopped (Byers, 1980). Some of these developmental events can be seen in Fig. 13. This first molar is from a 105-day-old rat that received intraperitoneal injections of [3H]proline at 15, 21, and 42 days to mark the position of the pulp/ dentin border at those times. These growth lines also reflect the rate of

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FIG. IS. Distal cusp and root of a right first maxillary molar from a 105-day-old rat is shown in A. 'I'he position of the pulp-clcntin border was marked by intraperitoneal injection of'[:'H]proIine when the rat was 15,21, and 42 days old, shown at higher niagnification in B. The sensory innervation was labeled by [:'H]proline injected into the trigeminal ganglion at 104 days for 24 hr axonal transport prior t~ fixation. The dentinal growth lines at 15, 21, and 42 days show the life history of.tlie sensory innervation: axons at the cusp tip (arrows) entered dentin at about 15 clays, inidcrown dentin around 2 1 t o 42 days, and intercuspal dentin between 42 and 105 days. Reparative dentin (KD) grew much niore from 21 to 105 days than did the adjacent innervated orthodentin. Bar: 0.2 mni. A, 35x; B, 9ox.

dentinogenesis for each area by the intensity of incorporation of isotope into the dentinal matrix. The sensory innervation was then labeled at 104 to 105 days by axonal transport of [3H]proline. The location of labeled nerves in relation to the growth lines indicates that the tip of the cusp was beginning to be innervated at about 15 days; by 21 days the

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dentin for that zone was almost fully formed; by 42 days, many midcrown regions had been innervated. Only in the intercuspal region was much tubular dentin made after 42 days. The reparative dentin at the tip of the cusp has grown a great deal in relation to neighboring tubular dentin, is only poorly innervated, and appears to have destroyed most of the innervation in that area, perhaps because it occludes many dentinal tubules. A final point of interest concerns the maturation of dental sensitivity. Some clinical studies have found that newly erupted teeth are as sensitive as mature teeth (Nordenram, 1970); however, it can be difficult to control such studies, and more recent reports found a two- to threefold increase in sensitivity (decrease in stimulus threshold) between the time of eruption and root closure (Fulling and Andreasen, 1976; Klein, 1978; Johnsen et al., 1983). Once deciduous and permanent teeth are fully erupted with root apices closed, their sensitivity is similar (Johnsen et al., 1979). We have recently studied the maturation of the digastric reflex response to electrical stimulation of rat molar teeth (Byers et al., 1982a). We found a rapid two- to threefold increase in sensitivity during the 1-2 month eruptive period, followed by a plateau in sensitivity during the next year. This sensory maturation correlated both with the closure of the root apices and with the density of the coronal dentinal innervation. It did not correlate with the distance between sensory nerves and the occlusal surface, which changes during attrition of rat molars from more than 500 p m at eruption to only 10-20 pm in 1-year-old rats (Byers, 1980; Byers et ad., 1982a).

D. CONTINUOUSLY ERUPTING TEETH T h e innervation of continuously erupting teeth, such as rat incisors, differs in many ways from that of mature posteruptive teeth such as rat molars or primate teeth. In general 1. There are fewer sensory axons.

2. Those axons are mostly unmyelinated. 3. They are usually associated with blood vessels deep in the pulp. 4. They mostly terminate in the incisal third of the pulp. 5. They have varicosities, or “beads,” near their endings. 6. They do not form a plexus of Raschkow.

7. They have not usually been found among odontoblasts. 8. They do not enter the dentin.

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There are important differences among the continuously erupting teeth of different species, so the findings will be described in relation to specific species. 1 . Rat Incisors Light microscopic studies using silver or osmium stains (Wassermann, 1939; Bernick, 1956; Hattyasy, 1956, 1959; Bishop, 1981), histochemistry (Pohto and Antila, 1968b), or autoradiography (Byers and Kish, 1976) have found rat incisor axons to be sparse, thin, and associated with blood vessels deep in the pulp. A plexus of Raschkow has not been found (Wassermann, 1939; Hattyasy, 1959); instead, axons terminate individually near pulp vessels (Hattyasy, 1956, 1959). The axons do not innervate dentin and in most cases have not been found near odontoblasts. However, Weatherford (1939) found some endings in the odontoblast layer of very young rat incisors, and Sakai (1974) found some axons just beneath the odontoblast layer. Preliminary results from an autoradiographic study of rat incisors (Fig. 14A, B) show clusters of silver grains near blood vessels or odontoblasts in the incisal end of the pulp; EM autoradiographs show radioactivity in many unmyelinated axons and a few myelinated axons in that region (Fig. 14C, D). Winfrey et al. (1980) found that axons of rat incisors cannot be labeled by application of HRP to deep dentinal cavities in the crown, but in a similar study of innervated cat dentin (Wilson el aL, 1980) many sensory perikarya were labeled by retrograde axonal transport. This suggests that rat incisor innervation is restricted to pulp. The types of axons innervating rat incisors are very interesting. Many early studies assumed that the axons were all sympathetic because they were unmyelinated and near blood vessels (Bernick, 1956; Hattyasy, 1956, 1959). T h e histochemical studies of Larsson and Linde (1971) confirms that many of the perivascular endings contain noradrenalin. However, recent use of histochemistry (t'ohto and Antila, 1968b), degeneration methods (Gregg and Dixon, 1973; Sakai, 1974), retrograde axonal transport (Furstman et al., 1975), and anterograde transport (Byers and Kish, 1976) have shown that many of the axons in rat incisor pulp are sensory. Studies of the base of the tooth suggest that nerve fibers branch into numerous thin axons soon after entering (Weatherford, 1939; Bernick, 1956). T h e diameters of those axons vary (Weatherford, 1939; Hattyasy, 1956; Bishop, 198 1 ) . The thorough electron microscopic study by Bishop (1981) has shown that the numbers of unmyelinated axons are fairly constant from the base to the incisal third of the pulp (range: 233-328 unmyelinated axons) and that myelinated axons are rare (none in two teeth and one or two myelinated axons in the

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FIG. 14. Sensory nerve endings (thin arrows) at the incisal end of rat incisors from year-old rats are shown in light microscopic autoradiograms (A,B) and EM autoradiograms (C,D) after 24 hr axonal transport of [SH]proline-labeledprotein. Note the proxirnity of the nerve endings to the occlusal surface (wide arrow, A) and to the incisal odontoblasts (A,B). Most labeled axons were unmyelinated (C), but a few were small myelinated axons (D). Bars: A, 0.2 mm; D, 1.0 pm. A, 70X; B, 180X; C and D, 6250x.

other two teeth) (see also Fig. 14). Analysis of conduction velocities shows, however, that many of these axons must have originated from myelinated parent axons; for example, conduction is faster from the incisor base to the inferior alveolar nerve (21.8 mlsec) than within the incisor (1.4 m/sec), (Jiffry, 1981) (Table I). I t is interesting that electrical stimulation of rat incisors and cat canines gives essentially the same localization of 2-deoxyglucose in the brain stem (Shetter and Sweet, 1979; Shetter and Kreinick, 1979) in spite of the apparently different structure of their intradental receptors. Electrical stimulation of rat incisors and cat canines gives a comparable short-latency digastric reflex (Wilson and Reid, 1978; Jiffry, 1981), although the rat incisor also produces a long-latency digastric reflex (Jiffry, 1981). T h e ultrastructure of the terminals of the incisor axons is still not settled. Han and Avery (1965) showed only one unmyelinated axon,

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which contained mitochondria and vesicles. Bishop (198 1) found virtually all the axons in his samples to contain microtubules and neurofilaments but very few mitochondria or vesicles. The characteristic inclusion of vesicles and mitochondria in beaded free endings in other teeth (e.g., Frank, 1966; Byers, 1977) or in skin (Andres and von During, 1973) suggests that perhaps Bishop’s micrographs were of preterminal axons rather than endings, but this question needs further study because some cutaneous nerve endings may have few or no beaded regions (Cauna, 1969) and Bishop’s survey was very thorough.

2. Mowe Incisors Ridehalgh and Stewart (1938) using silver or osmium staining techniques were able to find axons in mouse incisors more easily than in rat incisors. They found that the mouse incisor nerve rapidly subdivides into numerous branches after entering the pulp. They reported many myelinated axons in the pulp but could have had difficulty distinguishing between bundles of unmyelinated axons and myelinated axons. Alternatively, mouse incisors may have more myelinated axons than rat incisors.

3. Hamster Incisors Katele arid James (1963) studied hamster maxillary incisors. ‘I’hey described the branching of the anterior superior alveolar nerve into periodontal nerves and a recurrent branch to the incisor base; the recurrent nerve branched into small fascicles soon after entering the incisor pulp. In the basal region they found some growing tips of axons. Myelinated and unmyelinated axons followed the blood vessels and terminated near the incisal end of the pulp without reaching the odontoblast layer. As in all continuously eruptiiig teeth the incisal tip contained degenerating odontoblasts reacting to attrition of the occlusal surface; some degenerating axons are also found there (Katele and James, 1963). I n rat incisors Bishop (1981) found degenerating axons in the region of pulp degeneration as well as apparently normal axons.

4. Rabbit Incisors and Molars Several investigators found it much easier to demonstrate axons in continuously erupting rabbit incisors or molars than in rat incisors (Ridehalgh and Stewart, 1938; I’ohto and Antila, 1968b). ’I‘hese axons were near blood vessels deep in the pulp and did not reach the odontoblastic layer (Ikeda, 1936). In rabbit teeth l’otito and Antila (19681,) found many axons containing noradrenalin or iicetylctiolinesterase, or

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both, whereas the rat incisors contained no noradrenalin axons and only sparse acetylcholinesterase activity. It has been suggested by some (Wassermann, 1939; Hattyasy, 1956, 1959) that the continuously .erupting teeth are really equivalent to tooth germs prior to root formation and that the need for continuous eruption holds these teeth at an immature stage of development with immature innervation. However, the continuously erupting teeth are different from tooth germs because they have a spectrum of developmental stages from immature odontoblasts near the base to the degenerating senescent cells near the tip. Furthermore, developing rat molars have sensory axons innervating their immature odontoblast layer prior to root formation (Byers, 1980). It has been suggested that the unusual innervation of continuously erupting teeth and their lack of dentinal receptors is consistent with the rapid rates of eruption and attrition of these teeth (Katele and James, 1963).

IV. Ultrastructure of Sensory Nerve Endings

Electron microscopic studies of teeth have found somewhat different results depending on fixation quality and the region of the tooth that was sampled, but the following key points were clearly established between 1963 and 1973, especially by the work of Frank (1966,1968,1969), Frank et al. (1972), Arwill(l963, 1967), Arwill et al. (1973), and Corpron and Avery (1973). 1. All nerve endings in teeth are free endings; there is no special capsular material around the endings and in Inost cases they extend far beyond their terminal Schwann cell and basal lamina. 2. T h e endings are unmyelinated axons with successive dilatations (beads) connected by thin axonal regions. 3. The thin regions contain primarily microtubules and neurofilaments. 4. The enlarged beads contain clear and dense-core vesicles, dense bodies, smooth endoplasmic reticulum, and numerous mitochondria as well as microtubules and neurofilaments; the axoplasm in the beads resembles that of other sensory receptors (i.e., recuptoplusm defined by Andres and von During, 1973). 5. Axons and nerve endings can easily be identified in pulp either along blood vessels or arborizing to form the peripheral plexus of Raschkow; however, after axons cross the cell-free zone and enter the odon-

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toblastic layer, they only rarely have an associated Schwann cell and they usually are difficult to distinguish from the odontoblastic collaterals that are numerous in that layer. 6. Many nerve endings enter dentinal tubules, sometimes following a groove in the odontoblastic process, and they can extend as far as 0.2 mm into the dentin. 7. T h e ultrastructure of the nerve endings and the odontoblastic processes are distinctly different in dentinal tubules; each has a characteristic density, the odontoblastic process is much larger, and there are rarely any organelles in the odontoblastic process other than microtubules and microfilaments. 8. T h e nerve endings frequently occur in clusters with a narrow cleft separating their cell membranes. 9. T h e same basic structure for nerve endings has been found in all types of teeth except rat incisors and in all species studied (man, monkey, cat, rodents, fish), but the incidence varies widely depending on sampling; when samples are taken near the tip of the coronal pulp horn nerve endings are the most numerous. Myelinated and unmyelinated axons in tooth pulp were reported by Matthews et al. (1959) and Engstrom and Ohman (1960) but the first to describe well-fixed nerve endings in teeth were Frank (1966, 1968) and Arwill (1967). In 1966 Frank showed nervelike structures in dentinal tubules (Fig. 15). He found that their axoplasmic organelles were identical to those of other sensory receptors (e.g., Cauna, 1966) and were distinctly different from those of the adjacent odontoblastic process. In 1968 Frank published a lengthy description of very well-fixed nerve endings in pulp, predentin, and dentin near the tip of the pulp horns of extracted human teeth. He followed the beaded nerve endings from the pulp as far as 0.2 mm into the dentin and found that they lay in grooves in the odontoblastic process, they became thinner the further they went into dentin, and their path became increasing tortuous and complex. Initially the nerve endings were parallel to the odontoblast, but at their furthest extent they could be coiled around the odontoblastic process. N o specialized organelles were found in the odontoblastic process adjacent to the nerve endings and in most cases the cells maintained a regular separation of at least 20 nm. The axonal contents of mitochondria and vesicles was so similar to those of other types of sensory receptors that Frank felt justified in identifying them as neural. Arwill, on the other hand, felt that these structures should only be called accessory cells until shown to degenerate after sensory nerve transection. In a preliminary report (1963) Arwill found a few small acces-

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FIG. 15. Cross section through an odontoblastic process (0)at the predentin level. An unmyelinated fiber (F), containing numerous mitochondria and some structures analogous to synaptic vesicles, is found making a close contact with the odontoblastic process. Bar: 0.2 pm. 65,000~.[From Frank (1966). reproduced with permission.]

sory cells adjacent to the dentinal odontoblastic process and then in 1967 showed many beautiful electron micrographs of these accessory cells. His findings were similar to those of Frank, demonstrating the distinctive vesicles and mitochondria in those cells. He then studied the effect of sensory nerve transection on the sensitivity and ultrastructure of cat canines (Arwill et al., 1973)and found that the accessory cells of predentin and dentin degenerated after transection of the inferior alveolar nerve but not after transection of the cervical sympathetic ganglion; the sensitivity of those teeth was similarly eliminated by the sensory nerve injury but not the sympathetic nerve injury. Surprisingly, Frank el al. (1972) had not found nerve loss in dentin following sensory nerve injury in monkeys, but the survival time was so long (2 months) that much regeneration could have already occurred (e.g., Fearnhead, 1961; Robinson, 1YSOb, 1981; Fried and Erdelyi, 1982) or the original transection might not have been complete. Both Frank and Arwill noted the difficulty of distinguishing the free nerve endings from odontoblastic collaterals in the odontoblastic layer, especially those regions of the sensory axons that only contained rnicrotubules and neurofilaments, but their work certainly demonstrated that sensory nerve endings could be found in predentin and dentin. Several other electron microscopists also studied dental nerve end-

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ings at this time. Harris and Griffin (1968) thought they had found three kinds of nerve endings in teeth: (1) perivascular sympathetic unmyelinated endings, (2) free sensory unmyelinated endings in the peripheral plexus, and (3) free beaded sensory endings with terminal expansions. However, their electron micrographs did not support their classification because the fixation quality was poor, most of their samples were apparently of preterminal axon bundles rather than endings, and they seem to have mistaken the cut axons created by sectioning for real nerve endings. In the much more thorough studies of Frank and Arwill using samples that were much better fixed, the nervelike cells varied greatly in size but were interpreted as being different parts of a single type of sensory ending. As for identifying the perivascular endings as sympathetic, histochemical studies now show both adrenergic and cholinergic activity along blood vessels (Pohto and Antila, 1972) as well as substance P-like immunoreactivity (Olgart et al., 1977; see Fig. 1). Thus some perivascular endings are sensory. There may also be at least two kinds of sensory endings in peripheral pulp and dentin, because Fearnhead’s light microscopic work showed that thin axons there form thinner endings than d o the larger myelinated axons (Fearnhead, 1961, 1963). This question was not solved by the micrographs of Harris and Griffin (1968) and needs further work. Subsequently, Avery (1971, 1975), Corpron et al. (1972), Corpron and Avery (1973), Dahl and Mjor (1973), Lilja (1979), and Byers et al. (1982b) all found sensory nerve endings in pulp and dentin with essentially the same structure as was found by Frank and Arwill. T h e beaded nature of the intradentinal endings was especially well documented by Corpron and Avery (1973), as was the development of those nerve endings. Further proof that nerve endings in dentin were sensory came from electron microscopic autoradiography studies (Byers and Kish, 1976; Byers, 1977, 1979; Byers and Dong, 1983). The labeled nervelike cells were found in many dentinal tubules near the tip of the pulp horn, they were usually more electron-dense than the adjacent odontoblastic process, several free nerve endings were often clustered together, and they contained characteristic vesicles and mitochondria in the beaded regions (Figs. 16-19). Although the innervation of dentin is almost entirely sensory (e.g., Arwill et al., 1973) it is also possible that a few endings are sympathetic. Using the false transmitter 5-hydroxydopamine, Avery (1975) and Avery and Cox (1977) have found some noradrenergic endings in the odontoblastic layer and predentin. Most sympathetic axons end along blood vessels but approximately 30% get as far as the odontoblastic

FIG. 16. Silver grains label clusters of axon-like cells in five dentinal tubules (small arrows) from a third molar of a rat after 7 days of axonal transport of [gH]proline-labeled protein. The larger odontoblast processes and the dentin matrix are not labeled. Note that some dentinal tubules containing clusters of axon-like cells are not labeled (arrowheads). Bar: 1.0 pm. 6500~. FIG. 17. EM autoradiograph of a small labeled dentinal nerve ending from a rat third molar after 7 days axonal transport. Bar: 1.0 pm. 20,500x. [From Byers and Kish (1976), reproduced with permission.] FIG. 18. Labeled large nerve ending from a rat third molar after 7 days axonal transport. Note varied vesicles including a large densecore vesicle (Idc),and the mitochondria (ni). Bar: 1.0 pm. 19,000X. [From nyers (1979), reproduced with permission.]

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FIG. 19. EM autoradiograph of labeled sensory nerve endings with varied axoplasniic contents. A, The labeled axon contains a mitochondrion, glycogen (g), and a few vesicles; B, the ending contains many mitochondria, vesicles, and glycogen; two large dense-core vesicles (Idc) are indicated; C, many small clear vesicles (scv) and large clear vesicles (Icv) are present as well as mitochondria and what looks like a multivesicular body (mvb). A gap junction between two ribosome-filled odontoblasts is in B (arrowhead). [SH]proline-labeled axonal transport. Bars: 0.5 p m . 27,000-30,500X. [C from Byers (1979), reproduced with pcrmission .]

layer, mostly in association with blood vessels there (Avery et al., 1980), and some may enter the dentin. In autoradiographic studies of sensory nerve endings there are always many that are not labeled (Fig. 16) (Byers, 1977); in most cases this reflects incomplete labeling of the trigeminal neurons but some of the unlabeled endings may be sympathetic. The innervation of cat canines has been of special interest because those teeth are used for so many physiological studies of dental sensation. When Arwill et al. (1973) took samples near the tip of the crown, they found many nerve endings. Conversely, Holland (1975, 1976d, 1977) did not find nerve endings in dentinal tubules of cat canines, but his studies were of midcrown regions that are now known to have much less dentinal innervation than the cusp tip (Byers and Matthews, 1981 ;see also Figs. 5 and 6). Holland has included the tip of the crown in his recent investigations and has found a high incidence o f nervelike cells in denti-

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FIG.20. Two exaniples from predentin near the coronal tip of a cat canine are shown in A and B. In each figure an odontoblast process is accompanied by other cell processes o f different appearance arid organelle content, probably nerve terminals. Bar: 0.5 pni. 40,000x. [Micrographs courtesy of Dr. G. R. Holland; A, from Holland (IYXla), reproduced with permission.]

nal tubules (Holland, 1981a); their structure (Fig. 20) is similar to that described in other species. V. Neural Relationship to Other Cells

A. SCHWANN CELLSAND FIBROBLASTS Most electron microscopic studies of teeth have found the same kind of relationship between nerve endings and Schwann cells as between nerve endings and fibroblasts. In the nerve bundles of the pulp core both the myelinated and unmyelinated axons have Schwann cells ensheathing them that resemble Schwann cells of the main trigeminal nerve (Young, 1977). The only differences within the tooth core are that

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the perineurial sheath is incomplete or absent (Stockinger and Pritz, 1970; Bishop, 1982) and unmyelinated axons have a greater proportion of their surface in contact with other unmyelinated axons (Holland, 1981b). As the arborizing axons approach the peripheral plexus of Raschkow, most n o longer have a myeliri sheath and the Schwann cell ensheathment is increasingly incomplete. In the plexus and cell-free zone many axons are partly apposed to fibroblast-like cells (no basal lamina) as well as to Schwann cells (with basal lamina) (Frank, 1968; Cahen and Frank, 1970; Byers et al., 1982b). In the odontoblastic layer a few axons are still ensheathed by Schwann cells or fibroblasts (Arwill, 1967; Frank, 1968; Avery, 1971; Corpron and Avery, 1973; Dahl and Mjor, 1973), but most are now completely free. Finally, most axons or endings in the predentin and dentin are also free but occasionally an ensheathing cell is present (Fig. 21). B. ODONTOBLASTS 1. Are There Synapses, Tight Junctions, or Gap Junctions between Odontoblasts and Nerve Endings? No. Prior to the electron microscopic studies of teeth, some dental histologists thought that the odontoblasts were primary receptor cells and transferred information to the nerve endings at synaptic junctions (for review see Fearnhead, 1967; Anderson et al., 1970). A few electron microscopists also thought synapses were present between nerves and odontoblasts (Pischinger and Stockinger, 1968; Roane et al., 1973). However, the teeth used in those studies were poorly fixed and no vesicles could be seen in the odontoblastic cytoplasm. In more thorough studies by others, no synapses were found (e.g., Arwill, 1967; Frank, 1968; Arwill et al., 1973; Corpron and Avery, 1973; Dahl and Mjor, 1973; Byers, 1977; Byers et al., 198213). There is also no evidence in freezefracture studies for any tight junctions (long regions of membrane fusion) between any cells in teeth (Koling et al., 1981). The question of possible gap junctions between nerves and odontoblasts has been more difficult to answer. Most EM studies of teeth reported a regular extracellular cleft between the two cell types, but occasionally unusual junctions were found, especially in studies of extracted human teeth. Frank ( 1968) described occasional desmosome-like junctions and tight junctions between odontoblastic processes and other cells in dentinal tubules; in some cases those other cells looked similar to the odontoblasts (Byers et al., 1982b), but they could also contain vesicles and

FIG.21. In this EM autoradiograph of labeled nerve endings in predentin, an apparent Schwann cell (sc) surrounds one axon. The odontoblast process (asterisk) and collagen of predentin are not labeled. [3H]proline labeled axonal transport. Bar: 0.5 pm.32,OOOX. FIG. 22. A cluster of labeled nerve endings maintains their narrow axo-axonic clefts (solid arrow) and the wider cleft (open arrow) with the odontoblast process (asterisk), even though cell shrinkage appears to be occurring here. Bar: 0.5 pm. 32,OOOx. FIG.23. Optimal views of the wide cleft (open arrow) between free nerve endings and odontoblast process (A) and of the narrow axo-axonic cleft (B) (solid arrows) are shown here. T h e EM autoradiogram in A was tilted 47"; in R, it was rotated 90" and tilted 2'2" using a goniometer stage. Note the poor definition of these clefts if they are not properly aligned with the electron beam. Bar: 0.5 pm. 43,OOOX. [From Byers (1977), reproduced with permission.] FIG. 24. This EM autoradiograph was tilted using the goniometer stage for optimal view of the wide cleft (open arrow) between nerve ending and odontoblast and of one of the axo-axonic close appositions (solid arrow). Bar: 0.5 pm. 27,OOOX. [From Byers (1977), reproduced with permission.]

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mitochondria (Frank, 1969). Junctions of this sort were not found in perfusion-fixed teeth of animals (Corpron and Avery, 1973; Arwill et al., 1973; Byers, 1977, 1979; Byers and Dong, 1983) and might have been caused by retraction of odontoblastic collaterals into the dentin during cleavage and immersion fixation of the extracted human teeth. In 1975 Matthews and Holland reported that stimulation of isolated axons in inferior dental nerves of cats caused conduction in other axons from the same canine tooth, perhaps by electronic coupling. Gap junctions would allow such coupling (Bennett and Goodenough, 1978), and Holland found examples of gap junctions between odontoblastic cell bodies and adjacent pale cells (Holland, 1975; Matthews and Holland, 1975). It was strongly suggested that the pale cells were neuronal (Holland, 1976a,b), even though they usually lacked the vesicles typically found in nerve endings, and they often resembled odontoblastic collaterals. T h e difficulty in distinguishing nerve endings o r axons from the odontoblastic collaterals has been described in Section IV. Subsequent papers continued to suggest that the pale cells were axons or nerve endings that could form gap junctions with each other or with odontoblasts (Holland, 1980b), even though the presence of ribosomes and rough endoplasmic reticulum in those cells clearly indicated that they were odontoblastic (Holland, 1978). However, gap junctions between axons and odontoblasts have not been found in recent studies of cat canines (Holland, 1980a, 1981a), and a study by 'Turner (1982) shows that many of the pale processes may be fibroblastic. In the first EM autoradiographic study of rat molars, it was suggested that some gap junctions might join labeled sensory axons and odontoblasts (Byers and Kish, 1976). However, there was much cellular overlap in these preparations so that it was hard to see extracellular clefts. If the autoradiograms were tilted using a goniometer stage to get an optimal view of the intercellular cleft, no gap junctions were found (Byers, 1977, 1979) (see Figs. 22-23). Studies of gap junctions in other tissues (Larsen, 1977) are interesting because junctions are almost always found between cells of the same type, for example, two liver cells, two kidney cells. They are only rarely found between dissimilar cells and in those cases the two cells are both epithelial and of ectodermal origin (Larsen, 1977). Because the trigeminal nerve endings are ectodermal whereas odontoblasts derive from neural crest mesoderm (Johnson et d., 1973), it would be most L I ~ U S L I ; if I~ there were gap junctions between nerve endings and odontoblasts. It would still be possible for axons to join each other at gap junctions, except for the fact that neurons are a type of cell that only rarely forms gap junctions (Larsen, 1977). It now seems most likely that the numer-

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ous gap junctions in the odontoblast layer (Holland, 1977; Kding et al., 1981) only connect odontoblasts to one another or to fibroblasts (Turner, 1982).

2. Are There Special Junctions Between Odontoblasts and Nerve Endings? Maybe. Most electron microscopic studies of teeth have found a very regular apposition of nerve endings and odontoblasts with a 20-40 nm cleft between the cells (Frank, 1966, 1968; Arwill, 1967; Frank et al., 1972; Corpron and Avery, 1973; Byers, 1977, 1979; Byers et al., 1982b). Especially in predentin and dentin these contact regions are prevalent, the cleft width is regular, and the neuron is usually in a depression or groove in the odontoblastic process so that it is partially surrounded (ensheathed?) by the odontoblastic process (e.g., Figs. 15, 17-20). It is possible that these are sites of adhesion between the odontoblasts and nerve endings. Fearnhead (1961) noted that nerves and odontoblasts are easily separated at sites of cell shrinkage, but when cells have shrunk in EM preparations, the nerve endings and odontoblast often seem to be remarkably attached to one another (Fig. 24), and even the process of separating pulp from dentin or cleaving human teeth fails to disrupt the appositions (Arwill, 1967; Frank, 1968; Byers et al., 1982b). Specialized membrane densities are not found on the odontoblastic or axonal membranes as is the case for many other types of receptors with associated cells (e.g., Cauna, 1966; Gottschaldt et al., 1982; Yeh and Byers, 1983) or abutment sites (Gottschaldt and Valle-Hinz, 1981). Efforts to enhance staining of the nerve-odontoblast appositions using tannic acid or uranyl acetate were not successful (Byers et al., 1982b); instead, the same kind of membrane-associated material was found on all sides of the cells, not just at the appositions. Perhaps the most interesting observation on nerve-odontoblast association is Frank’s (1968) description of the groove in the odontoblast process that is occupied by the nerve ending as it follows an increasingly tortuous course into the dentinal tubule. If the free ending needs an anchoring cell to hold it in the correct site for sensory transduction, or if the nerve ending holds onto the odontoblast process in order to react to odontoblast movement, an adhesive junction would be helpful. Desmosomes have characteristic densities for each specific tissue and, conceivably, adhesion of nerve endings to odontoblasts does not require elaborate membrane-associated dense material. Alternatively, the nerve endings and odontoblast processes might of necessity be close to one another because they share the confined space

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of the dentinal tubules. It is perhaps important that free nerve endings in gingiva (Byers and Holland, 1977), in oral mucosa of the palate (Yeh and Byers, 1983), and in skin (Kruger et al., 1981) maintain a 20-40 nm separation from adjacent epithelial cells. Such a cleft width may simply represent the minimal extracellular “envelope” that free nerve endings require around themselves. Further work is needed here to fully understand the nature of the nerve-odontoblast appositions. C. AXO-AXONIC CONTACTS Many of the EM studies of dental innervation have found extensive regions of apposition between adjacent nerve endings or adjacent axons (Frank, 1966,1968; Arwill, 1967; Frank et al., 1972; Corpron and Avery, 1973; Johnsen and Harshbarger, 1977; Byers, 1977, 1979; Holland, 1980a, 1981b, 1982). The use of the goniometer to tilt EM autoradiograms for optimal orientation of intercellular clefts (Byers, 1977, 1979) showed that the separation of adjacent axons was only 10-15 nm compared with the 20-40 nm separation between nerves and odontoblasts (Fig. 23-24). It has since been found that similar close axo-axonic appositions are conimon in predentin and dentin of human teeth (Byers rt nl., 1982b). It has been suggested (Byers, 1977, 1979) that such close apposition between adjacent axons could allow ephaptic interaction. Such interaction could explain the electrical coupling observed between intradental axons (Matthews and Holland, 1975; Matthews, 197’7).Ephaptic interaction between adjacent neurons was first proposed by Arvanitaki (1942) to describe electrical interaction across extracellular space. It has since been proposed for many sites, for example, for dendritic synchronization of neurons in neocortex (Petsche et al., 1975) or in special cases such as Mauthner fiber inhibition (Bennett, 1977). One of the properties of dental pain is that it often seems to involve the entire tooth; synchronization of the sensory axons in dentin, in the odontoblast layer, and/or in the plexus of Raschkow would be one way to achieve this. Alternatively, the very close apposition of nerve endings or axons in teeth may not have any functional significance. Unmyelinated axons in peripheral nerves in general (Ochoa, 1976) and trigeminal branches in particular (Young, 1977; Holland, 1982) normally have some axo-axonic contact, but there is a tenfold greater amount of contact in the plexus of Raschkow than in the alveolar nerves (Holland, 1982). Axoaxonic contacts are either an important aspect of the dental sensory

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receptor apparatus or else the free nerve endings do not need much separation or insulation from each other to function independently.

VI. Sensory Transduction Mechanisms

A. GENERAL SENSORY PROPERTIES OF TEETH Many studies have shown that dental sensory receptors can be activated by electrical, chemical, osmotic, mechanical, or thermal stimuli applied to the intact tooth or to exposed dentin (for reviews see Anderson et al., 1970; Horiuchi and Matthews, 1976; Scott and Maziarz, 1976; Mumford and Bowsher, 1976; Dubner et al., 1978; Sessle, 1979). Application of suprathreshold stimuli to human teeth usually causes pain; for this reason dental receptors have often been considered to be good models for nociception. However, nonpainful or prepain sensations typically occur when stimulus intensities sufficient to elicit muscle reflexes are near threshold (Mumford and Bowsher, 1976; Matthews et al., 1976; Azerad and Woda, 1977; Sessle, 1979; McCrath et al., 1981; CondesLara et al., 1981; Chudler and Dong, 1983), and there is a distinct difference between the digastric reflex threshold and the escape threshold after electrical stimulation of teeth in awake cats (Wilson and Reid, 1978; Chudler and Dong, 1983). Although dental stimuli can cause nonpainful sensations, they might not be involved in the tactile or the proprioceptive sensitivity of teeth, because these sensations were normal in nonvital teeth (Linden, 1975) and in a patient with congenital insensitivity to pain and absence of sensitivity to electrical stimulation of dentin (Chatrian et al., 1975; for further discussion see Sessle, 1979). Dental pain has been described as either a sharp pain or an aching unpleasant pain (for review see Mumford and Bowsher, 1976). The sharp pain occurs at a lower threshold than the dull ache; this was most clearly demonstrated in studies of human teeth using bipolar intrapulpal electrodes (Azerad and Woda, 1977). Some evidence suggests that sharp pain occurs after movement of fluid in the dentinal tubules and that it depends primarily on low threshold, fast-conducting A-delta mechanoreceptors (Brannstrom et al., 1967; Brannstrom and Astrom, 1972; Narhi, 1978; Olgart, 1979; Gazelius, 1981; Narhi et al., 1982a), whereas dull ache may depend primarily on unmyelinated pulpal receptors (Narhi et al., 1982b). The sensory system is more complex, however, than simply containing one kind of pulpal receptor and another kind of dentinal receptor; experiments by Haegerstam et al. (1975) show that

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some dentinal receptors are cholinergic but that they are different from receptors sensitive to air blast stimuli. In addition some receptors may be polymodal nociceptors. Whether there are also any specifically nonnociceptive, low-threshold receptors in teeth is not yet settled. T h e mechanisms by which dental stimuli are transduced into neural activity are only partly understood. There is evidence for more than one kind of mechanism, and there are some differences in the sensitivity of dentin and pulp. (For other discussions of sensory mechanisms see Anderson, 1968; Brown, 1968; Anderson et al., 1970; Matthews, 1972; Horiuchi, 1974; Brannstrom and Johnson, 1978; Dubner et al., 1978; Sessle, 1979).

B. SENSORY TRANSDUCTION IN DENTIN T h e dentino-enamel junction (DEJ) is highly sensitive, yet most studies of outer dentin do not find either nerve endings or odontoblast processes near the DEJ of mature teeth (Frank, 1968, 1969; Tsatsas and Frank, 1970; Frank et al., 1972; Garant, 1972; Brannstrom and Garberoglio, 1972; Holland, 1976a; Byers and Kish, 1976; Thomas, 1979, 1980). A few investigators have reported cellular processes near the DEJ (Avery et al., 1980), but they may have misidentified peritubular dentin; alternatively, occasional tubules or certain types of teeth may have cells near the DEJ. However, the extensive sampling and fixation controls used by Holland (1976b) strongly suggest that the outer dentin of mature cat canines is acellular. T h e sensitivity of the DEJ of cat canines and other teeth would therefore depend upon the transfer of stimuli through the outer dentin to the cells of the inner dentin or pulp. There are at least six possible mechanisms for sensory transduction in dentin. 1. Hydrodynamic Mechanisms On the basis of the experimental work of Anderson, Brannstrom, and others (for reviews see Anderson et al., 1970; Dubner et al., 1978), the hydrodynamic theory was proposed by Brannstrom; according to this theory movement of fluid in dentinal tubules is the key mechanism for activating sensory nerve endings in teeth (Brannstrom, 1963a,b; Brannstrom el al., 1967, 1979; Brannstrom and Astrom, 1972). There is much experimental work to support the hydrodynamic theory, although some experiments indicate that the action of desensitizing agents on dentin does not always correlate with their ability to impede fluid flow (Greenhill and Pashley, 1981).

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There are at least three kinds of cellular response to fluid movement in dentin that might form the basis for hydrodynamic sensory mechanisms in teeth. a. Odontoblastic Transduction. Many investigators have thought that odontoblasts are the primary receptor cell in teeth. This theory grew out of the observed sensitivity of the dentino-enamel junction and the early histological demonstrations that odontoblastic processes extend far into dentin without any (or many) accompanying sensory nerves (for review see Fearnhead, 1967). The more recent work on dental nerve location (see Section 111) makes it clear that in fact many sensory nerves do go as far as 0.2 mm into dentinal tubules. The odontoblast receptor idea has persisted in the suggestions that there are synapses (Roane et aL, 19’73) or gap junctions (Matthews and Holland, 1975) between nerves and odontoblasts. However, further work has shown that neither of these types of junction occurs between these two kinds of cell (see Sections IV and V). There are, however, two other ways in which odontoblasts might initiate sensory transduction in teeth.

1 . They might release particular ions (e.g., potassium) in response to fluid movement that are then detected by the nerve endings; however, odontoblast potentials do not appear to change during sensory transduction (Winter et al., 1963). 2. Alternatively, odontoblasts are known to move when dentinal fluid moves (Brannstrom et al., 1967), and it is possible that sensory nerve endings react to that movement. An argument against the odontoblastic receptor theory is that in rat incisors the sensory neurites normally end near the odontoblasts that are degenerating at the incisor tip (Bishop, 1981); however, rat incisors have unusual innervation, and their sensory mechanisms might also be unusual. b. Independent Neural Transduction. There is some evidence that the sensory nerve endings are the first cells to react to the movement of dentinal fluid. T h e neurons could respond to electrical potentials that arise when fluid moves in the dentinal tubules (for discussion of these streaming potentials see Mumford, 1976; Eriksson, 1976). Alternatively, nerve endings could react to their own deformation during fluid movement. Matthews (1970) was able to activate a radial nerve inserted into a cat canine pulp using electrical stimulation of exposed dentin, suggesting that contact with the odontoblast layer is not necessary for an intrapulpal nerve to be stimulated. No changes in membrane potentials were found by intracellular recording of cells in the odontoblast layer during sensory transduction (Winter et al., 1963), and dentin is apparently still

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sensitive over regions of pulp necrosis with degenerating odontoblasts (Brannstrom and Astrom, 1972). However, even if the nerve ending is the primary receptor, the odontoblasts would at least play a supporting role since they build and maintain dentinal matrix and fluid. c. Combined Neural and Odontoblastic Transduction. It is also possible that both cell types are needed for sensory response to fluid movement in the dentin and pulp. It may be essential to neural reactivity that the odontoblasts provide a framework to hold nerve endings at the correct site for optimal transduction. Odontoblasts might also maintain a specific electrolyte composition of dentinal fluid upon which optimal neural responsiveness might depend; experiments by Haljamae and Rockert (1970) found unusually high concentrations of potassium and low concentrations of sodium in dentinal fluid. The aggregation of pulpal plexuses and dentinal innervation near the tip of the pulp horn and the loss of neurites from regions where reparative dentin has formed (see Section 111) suggest that the sensory endings might require a specific environment that is established by the cuspal odontoblasts and dentinal tubules. 2. Other Transduction Mechanisms

There are at least five alternatives to the hydrodynamic mechanism for transfer of stimuli across outer dentin to the cells of inner dentin and pulp. It is entirely possible that more than one of these mechanisms occurs in teeth, especially because some nerve endings that respond to hot or cold stimuli do not respond to osmotic stimuli (Matthews, 1977). a. Piezoelectric efsects in dentin were proposed by Liboff and Shamos (197 1) because pressure on dentin was shown to generate current, which might be able to cause neuronal activation; such pressure should occur in response to any mechanical dental stimulus. For further discussion of piezoelectric effects see Eriksson (1976) and Athenstaedt et al. (1982). b. Bioelectric potentials are normally present across enamel and dentin. These vary depending upon the contents of the oral cavity, and it has been suggested that rapid changes in oral ionic concentration may be able to cause dental pain by changes in bioelectric potentials rather than by hydrodynamic mechanisms (Atkinson and Parker, 1969). c. Pyroelectric qfects occur in skin (Athenstaedt et al., 1982) and may also be present whenever there are thermal gradients across teeth. d . Thermal gradients may cause fluid movement (Brannstrom et al., 1967) or they may be detected directly by the nerve endings (Matthews, 1977). In addition the sensitivity to cold and to heat may depend on different nerve endings (Wagers and Smith, 1960; Scott and Tempel, 1965; Matthews, 1972).

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e. Chemical stimuli have been shown to act in dentin by nonosmotic and nonhydrodynamic mechanisms (Horiuchi and Matthews, 1976).

C. SENSORY TRANSDUCTION IN PULP

Discussion of pulpal sensory mechanisms depends partly upon where one chooses to designate the pulp-dentin border. Does pulp include the cellular projections of odontoblasts and neurons into dentinal tubules? Is the border located at the separation between the vascular pulp (including the odontoblastic layer) and the avascular predentindentin? Are the odontoblastic cell bodies really part of dentin because they produce dentin matrix and because they usually adhere to dentin when pulp is extirpated? For a discussion of sensory mechanisms, it seems reasonable to distinguish among (1) the inner pulp consisting of central nerve bundles, vascular neural plexuses, and the peripheral neural plexus of Raschkow; (2) the pulp-dentin border zone consisting of nerve endings in the odontoblastic layer, the predentin, and the inner dentin; and (3)the outer noninnmated dentin. All of the mechanisms discussed above for dentin probably also include the pulp-dentin border zone and in many cases the inner pulp as well. Certainly movement of fluid, mechanical stimuli, and chemical stimuli are as important for pulpal sensory transduction as for dentinal sensitivity. There is an interesting difference, however, between the thermal sensitivity of “pulp” and that of “dentin” that is most likely a difference between inner pulp and the pulp-dentin border. Many studies have shown that application of hot and cold stimuli to teeth causes two different kinds of pain. Studies on human teeth show that cold stimuli usually cause sharp pain, whereas hot stimuli cause a dull ache that is slow to develop (Brannstrom, 196313; Bryant, 1971). It has been suggested that different regions along a single type of receptor might respond differentially to heat or to cold (Scott, 1977). However, single-fiber recordings have shown that the neurons that are sensitive to heat differ from those responding to cold (Wagers and Smith, 1960; Scott and Temple, 1965; Matthews, 1970, 1977). The neurons responding to cold stimuli share many characteristics with those responding to mechanical dentinal stimuli (Scott and Tempel, 1965; Brannstrom and h t r o r n , 1972), whereas heat activates pulpal C fibers but not dentinal mechanoreceptors in intact teeth (Ngrhi et al., 1982b). Two further complications of thermal sensitivity are that when dentin is exposed, some dentinal mechanoreceptors become heat-sensitive (Narhi et al.,

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1982c), and that some neurons may react specifically to cold without causing pain (Griisser et al., 1982). It is likely that future work with single-fiber recording techniques will reveal further complexities of pulpal and dentinal sensory mechanisms. One such finding is that most of the A-delta dentinal mechanoreceptors that were tested were also sensitive to mechanical disturbance of external pulp, whereas only 3 of 10 pulpal heat-sensitive C fibers responded to mechanical stimulation of external pulp, suggesting that they occur deeper in the pulp (Narhi et al., 1982a,b). It is important to note that the normal sensory properties of pulp and of dentin change during inflammation because of sympathetic nerve activity (Edwall and Scott, 197 1; Matthews, 1976), intradental cytochemical changes (Olgart, 1979), or perhaps physical distortion of the nerve endings (Narhi et al., 1982a).

VII. Summary

Teeth are innervated by unmyelinated sympathetic axons, and by unmyelinated and small myelinated sensory axons. Some sensory axons in teeth are terminal branches of larger parent axons, so that conduction from teeth to CNS in trigeminal nerves includes C-fiber, A-delta, and Abeta velocities. Sensory dental axons contain acetylcholine or substance P-like immunoreactivity. The sympathetic axons contain noradrenalin. Other neuropeptides may also be present, such as vasoactive intestinal peptide and serotonin. Dental axons of mature teeth of many species (man, monkey, cat, rodents, fish) are essentially the same, but continuously erupting teeth have smaller and fewer axons. Free sensory nerve endings in mature teeth are found in the peripheral plexus of Raschkow, the odontoblastic layer, the predentin, and the dentin. Free nerve endings are most numerous in those regions near the tip of the pulp horn, where more than 40% of the dentinal tubules can be innervated. Many dentinal tubules contain more than one free nerve ending. Intradentinal axons can extend as far as 0.2 mm into dentin but usually end less than 0.1 mm from the pulp. Some sensory endings also occur along pulpal blood vessels. In continuously erupting teeth nerve endings do not enter the dentin but remain within the pulp. Nerve endings in dentin are labeled by axonal transport. They are therefore as viable and active as the nerve endings in pulp. T h e axoplasm of the free nerve endings contains organelles typical

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of other somatosensory receptors. These organelles are most common in the successive beaded regions along the free nerve endings and include mitochondria, clear and dense-core vesicles, multivesicular bodies, profiles of smooth endoplasmic reticulum, and relatively few microtubules and neurofilaments. The beads can vary in size from about 0.2 to 2.0pm and can have varying amounts of receptor organelles. The interbead axonal regions are thin and contain mainly microtubules and neurofilaments. Nerve endings are associated with companion cells after they leave the coronal nerve bundles; these companion cells include Schwann cells, fibroblasts, and odontoblasts. There is no good evidence of gap junctions or synapses between nerve endings and odontoblasts. Instead, the two cell types form appositions that have a 20-40 nm extracellular cleft and parallel apposed plasmalemmas but no unusual membrane-associated material. No special organeles occur in the odontoblastic cytoplasm at these sites. In predentin and dentin the nerve endings follow an increasingly tortuous groove in the odontoblast processes, maintaining the apposition cleft of 20 to 40 nm along the contact region. There is as yet no evidence for or against odontoblastic-neural interaction(s) at these sites. Axo-axonic contacts occur in trigeminal nerves but become 10 times more common in the terminal arborization of dental axons. More than 40% of the terminal axonal surface may be in contact with other axons in the pulp, and this percentage is even higher in predentin and dentin, in which there are often clusters of free nerve endings. Axoaxonic contacts may allow synchronization of dentinal sensory neurons by ephaptic interactions, as has been proposed for some other neuronal systems such as dendritic bundles in the neocortex. Alternatively, terminal axons and endings may not need much separation to function independently. There is good evidence that movement of fluid in dentinal tubules and pulp triggers sensory transduction, as proposed by the hydrodynamic theory. Whether this fluid movement acts directly on nerve endings or whether the odontoblasts play a role is not known. It is unlikely that odontoblasts are primary receptors, because they do not form junctions with the nerve endings and they do not degenerate when teeth are denervated. Hydrodynamic mechanisms could activate neurons either mechanically or by causing streaming potentials. Other possible mechanisms of sensory transduction in teeth include piezoelectric, bioelectric and pyroelectric effects, chemosensitivity, and thermosensitivity. The thermal sensitivityof pulp and dentin differs: Cold causes sharp pain by activation of fast-conduction fibers that are probably dentinal

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mechanoreceptors, and heat causes dull ache apparently by diffusion of heat into the pulp and activation of pulpal C-fiber nerve endings. There is more than one type of sensory receptor in teeth. T h e Adelta dentinal mechanoreceptors and the heat-sensitive pulpal C fibers can differ in their location, transduction mechanisms, and conduction velocity. Some dentinal nerve endings are activated by chemical rather than by hydrodynamic or thermal stimuli. Some sensory endings form plexuses along pulpal blood vessels, and they probably differ from nerve endings in the plexus of Raschkow, the odontoblastic layer, and the dentinal tubules. The exact site of sensory transduction in dental nerve endings is not yet determined, but there are similarities between the axoplasm in the beaded regions of dental free endings and axoplasm in other types of somatosensory receptor. Acknowledgments

I thank Drs. R. L. Berger, R. M. Frank, G. R. Holland, and L. Olgart for generously providing micrographs; Drs. W. K. Dong, G. R. Holland, M. Narhi, and L. Olgart for preprints and infbrmation about their current work; Drs. W. K. Dong, L. E. Westrum, B. R. Fink, and R. L. Berger for helptul comments on the manuscript and A. Ross for editing it; P. O’Neill, M. Donian, and C. Rubenstein for technical assistance; and L. Goodwin, 1’. Kunz, and S. Garber for preparing the manuscript. This work was supported by NIH Grants DE05 159, DE02600, and Research Career Development Award DE00099. References

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Lisney, S. J. W. (1978).J. Physiol. (London) 279, 509-517. Machida, K. (1977). Shika Gakuho (J. Dent.) 77, 1661-1682. (Cited by Robinson, 1981.) Marfurt, C. F. (1981).J. Comp. Neurol. 203, 785-798. Marfurt, C. F. (1982). Anat. Rec. 202, 118A. Marfurt, C. F., and Turner, D. F. (1983). Brain Res. 261, 1-12. Matthews, B. (1970). Arch. Oral Biol. 15, 523-530. Matthews, B. (1972). Proc. R. Soc. Med. 65, 493-495. Matthews, B. (1976). Adv. Pain Res. Therap. 1, 195-203. Matthews, B. (1977). J . Physiol. (London) 264, 641-664. Matthews, B., and Holland, G. R. (1975). Brain Res. 98, 354-363. Matthews, B., and Lisney, S. J. W. (1978). Brain Res. 158, 303-312. Matthews, B., Baxter, J., and Watts, S. (1976).Brain Res. 113, 83-94. Matthews, J. L., Dorman, H. L., and Bishop, J. G. (1959).J . Dent. Res. 38, 940-946. McGrath, P. A., Sharev, Y., Dubner, R., and Gracely, R. H. (1981). Pain 10, 1-17. Menke, R., Weine, F., Ulinski, P., and Smulson, M. (1977).J. Endodontics 3, 128-134. Mumford, J. M. (1976). “Toothache and Orofacial Pain.” Churchill-Livingstone, London. Mumford, J. M., and Bowsher, D. (1976). Pain 2, 223-244. Mummery, J. H. (1924). Proc. R. SOC.Med. 17, 35-47. Narhi, M. V. 0. (1978). Thesis, Univ. of Kuopio, Finland. Narhi, M. V. O., Hirvonen, T.J., and Hakumaki, M. 0.K. (1982a).ActaPhysiol. Scand. 115, 173-1 78. Narhi, M. V. O., Jyvasjarvi, E., Hirvonen, T., and Huopaniemi, T. (1982b). Pain 14,317326. Narhi, M. V. O., Hirvonen, T., and Hakumaki, M. (1982~).In “Anatomical, Physiological, and Pharmacological Aspects of Trigeminal Pain” (B. Matthews and R. G. Hill, eds.), pp. 67-76. Exerpta Medica, Amsterdam. NPrhi, M. V. O., Hirvonen, T. J., and Hakumiki, M. 0. K. (1982d). Arch. Oral Biol. 27, 1053- 1058. Nord, S. G., and Rolince, D. E. (1980). Exp. Neurol. 69, 183-195. Nordenram, A. (1970). Acta Odontol. Scand. 28, 233-242. Ochoa, J. (1976).I n “The Peripheral Nerve”(D. N. Landon, ed.),pp. 106-158. Wiley, New York. Ochs, S. (1982). “Axoplasmic Transport and Its Relation to Nerve Function.” Wiley, New York. Ohman, A. (1965). Odontol. Tidskr. 73, 166-227. Olgart, L. (1974). Acta Physiol. Scand. 92, 48-55. Olgart, L. (1979). In “Mechanisms of Pain and Analgesia Compounds” (R. F. Beers and E. G . Bassett, eds.), pp. 285-294. Raven, New York. Olgart, L., Hokfelt, T., Nilsoon, G., and Pernow, B. (1977). Pain 4, 153-159. Olgart, L., Lundberg, J. M., and Gazelius, B. (1981).J. Dent. Res. 60B, 1260. Pearl, G. S., Anderson, K. V., and Rosing, H. S. (1977). Exp. Neurol. 54,432-443. Petsche, H., Prohaska, O., Rappelsberger, P., and Vollmer, R. (1975). Adv. Neurol. 12, 53-70. Pimendis, M., and Hinds, J. (1977).J. Dent. Res. 56, 827-840. Pischinger, A., and Stockinger, I.. (1968). Z. Zrllfnrsch. 89, 44-6 I . Pohto, P., and Antila, R. (1968a). Acta Odontol. Scand. 26, 137-144. Pohto, P., and Antila, R. (1968b). Acta Odontol. Scand. 26, 641-656. Pohto, P., and Antila, R. (1972). Int. Dent. J . 22, 228-239. Powers, M. (1952).J. Dent. Res. 31, 383-392.

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CEREBROSPINAL FLUID PROTEINS IN NEUROLOGY' By A. Lowenthal, R. Crols, E. De Schutter, J. Gheuens, D. Karcher, M. Noppe, and A. Tasnier Laboratory of Neurochomirtry Born-Bungo Foundation Univerritairo Instelling Antwerpan Antworp, Belgium

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Quantitative Determinations of Proteins. . . , , , . . . . . . , . , . . . . . . . . . . , . , . . A. Determination of CSF Total Protein. . . , , , . . . . . . , , , . . . . . . . , , . . . . . . B. Proteins Considered Individually . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Tumor Proteins . . . . . . ..................................... D. Nervous Tissue-Specific teins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Calculation of Albumin and IgC Ratios. . . .... . ........ 111. Qualitative Studies . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Electrophoresis Results. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . B. Methodological and Physiological Problems. . . . . . . . . . . . . . . . . . . . . . . . IV. Nervous Tissue-Specific Proteins. . . A. S-100 ....................................................... B. Myelin Basic Protein (MBP)-Antibodies to MBP . , . . . . . . . . . . . . . . C . a-Albumin (GFAP, Astroprotein)-Antibodies to a-Albumin. . . . . . . . . D. Enolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............... E. a2-Glycoprotein (14-3-3). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Enzymatic Determinations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

Lumbar puncture, which is required to examine human cerebrospinal fluid (CSF), was introduced about a hundred years ago in clinical

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Abbreviations: A, albumin; BBB, blood-brain barrier; CNS, central nervous system; CSF, cerebrospinal fluid; EAE, experimental allergic encephalomyelitis; EAN, experimental allergic neuritis; ELISA, enzymo-linked immunosorbent assay; GFAP, glial fibrillary acidic protein; IRMA, immunoradiometric assay; MBP, myelin basic protein; MS, multiple sclerosis; RIA, radioimmunoassay; and SSPE, subacute sclerosing panencephalitis. 95 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL.25

Copyright 8 1984 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-366825-5

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neurology. Since then, the biochemical study of the CSF has increased continuously. In the field of clinical medicine special attention has been given to CSF proteins, which have been studied both quantitatively and qualitatively. Determinations of the enzyme activities of these proteins have also been made. The field of fundamental medical research, however, followed a different path. When one examines texts devoted to the physiological study of the CSF, one can immediately observe that protein studies were rarely made and only those concerning the levels of total CSF protein. Nowhere were qualitative examinations carried out, largely because of the lack of sufficient amounts of CSF. Enzymatic studies were more frequently carried out. This divergence between clinical studies and fundamental research is the reason that there is no response to queries from clinicians concerning the mechanism of the changes of the total protein content or of the qualitative content of these proteins. The answers proposed by clinicians, however, are frequently not accepted by fundamental researchers. A review of what clinicians know and think about CSF proteins could be of interest for both the clinicians themselves and for the biochemists. Therefore, we will discuss here only problems referring to human CSF and mainly human pathological CSF. It is generally accepted that CSF proteins can originate from cells in the CSF through ultrafiltration from the blood, from the cells in the nervous system (glial cells, neurons), or from the choroid plexus. The relative contribution of each of these sources is unknown. One important subject for discussion is the blood-brain barrier (BBB) and what crosses it. In fact, where does this barrier, which gives passage to the proteins, lie? But in human pathology the most important item for discussion is the possibility of intrathecal synthesis of the proteins. Most of the discussions in this area concern the synthesis of the immunoglobulins. Therefore, an important part of this chapter will be devoted to the topic of immunity. Studies of proteins in human CSF can be classified as follows:

1. Quantitative determination of different proteins 2. Qualitative studies a. electrophoresis results b. methodological and physiological studies 3. Determination of nervous tissue-specific proteins 4. Determination of enzymatic activities We shall now discuss these items in detail.

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II. Quantitative Determinations of Proteins

A. DETERMINATION OF CSF TOTAL PROTEIN Accurate determination of CSF total protein remains a problem to which there is no final answer. Different methods are utilized, including the biuret method, biorad, nephelometry, and precipitation with trichloroacetic acid or sulfosalicyclic acid. All of these methods are useful. They usually indicate normal values that vary between 20 and 40 mg/ 100 ml. However, none of these methods can be considered as completely reliable. Comparative studies have been made but without any definitive conclusion (Bleijenberg et al., 1982). T h e low values of the CSF total protein content explain why in pathology one talks only about increases. Technical developments have made it possible to carry out total protein measurements by laser determination (Schliep and Felgenhauer, 1978). This method makes it possible not only to work with small quantities of CSF, but also to recover the CSF, as do other physical methods (for instance, UV spectrophotometry). The laser method is likely to be the technique of the future and can be used in conjunction with immunological techniques: IgG, IgA, IgM, a2-macroglobulin,and ceruloplasmin can also be determined by laser nephelometry (Di Costanzo et al., 1980).

B. PROTEINSCONSIDERED INDIVIDUALLY Certain proteins such as a2-macroglobulin,al-microglobulin (Tagaki et al., 1980), fibrinogen, and several immunoglobulins can be determined in the CSF by Mancini, Laurell, electrodiffusion, and Ouchterlony methods. Ferritin was determined by radioimmunoassay (RIA) (Hallgren et al., 1982) and was found to increase after bleeding or infection, usually in young children. Some results should be emphasized, such as the heterogeneity of an-macroglobulin(Rocchelli et al., 1982). Up to now, these quantitative determinations have, in general, given very little clinical information, but they should be mentioned. Wikkels@and Blomstrand (1 982) claim to have identified specific protein fractions by isofocusing and affinity chromatography using antibodies to human serum but do not give more information concerning these fractions.

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Enzyme immunoassay is available for quantitative determinations of the three main classes of immunoglobulin: IgA, IgC, and IgM. The absolute concentration of the three immunoglobulins increases with age; for IgG and IgA even relative concentrations increase. IgA and IgM CSF levels correlate with serum levels (Kobatake et al., 1980). Up to now studies of IgM have not yielded decisive results (Sindic et al., 1982).

C. TUMOR PROTEINS

Another item we must mention here among these measurements is the determination of proteins such as carcinoembryonic antigen that would allow the diagnosis of cerebral tumors (Hill et al., 1980; Dearnaley et al., 1981; Wasserstrom et al., 1981). These authors try either to apply to the CSF measurements that are made in the serum or to detect specific markers that allow the differential diagnosis of cerebral tumors. These are not yet routine procedures but no doubt will open interesting doors for further research.

D. NERVOUS TISSUE-SPECIFIC PROTEINS Nervous tissue-specific proteins can also be used as markers for brain tumors and other neurological diseases and are discussed at length in Section IV. Table I summarizes normal CSF values for the best-known of these proteins. TABLE I CONTROL VALUESFOR SIXNERVOUS TISSUE-SPECIFIC PROTEINS IN

CSF

Protein

s-100 MBP a-albumin (GFA, astroprotein) Enolase 14-3-3

a*-gl ycoprotein

Control value (ng/ml) Absent in controls <4

0-9 6-14 4 -

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E. CALCULATION OF ALBUMIN AND IGG RATIOS T h e quantitative determinations of serum and CSF albumin and IgG have permitted the calculation of ratios or indexes that may give information concerning the permeability of the BBB and the synthesis of intrathecal proteins. This is an empirical approach to demonstrate the local synthesis of immunoglobulin G within the central nervous system (CNS), which is a generally accepted phenomenon. Cerebrospinal fluid IgG can reflect the intrathecal production of immunoglobulins. At first it was thought that the nervous tissue acted as an immunological tissue. Infiltration of the CNS by plasmocytes, lymphocytes, and immunocytes was shown in subacute sclerosing panencephalitis (SSPE) and multiple sclerosis (MS). These cells are a source of antibody production within the CNS, and their presence could be equated by some authors with a breakdown of the BBB. In order to describe these conditions, empirical calculations have been made and will now be discussed. The CSF IgG index is calculated according to the following formula (Christensen et al., 1978): C S F I-~ Gx 103 serum IgG = CSF IgG index CSF albumin x lo3 serum albumin T h e normal index is variable, accordina to the authors (Link and Tibbling, 1977; Christensen et al., 1978; NoFdal et al., 1979): 0.34, 0.58, and 0.85. It is necessary to establish one’s own normal CSF IgG index. IgG and albumin (A) concentrations should be determined simultaneously to reduce analytical error. An index increase is considered to indicate a breakdown of the BBB. T h e oligoclonal pattern, which will be discussed later, does not always coincide with an abnormal CSF IgG index, and the converse is also true: An abnormal index does not necessarily forecast immunoglobulins with restricted heterogeneity. Rice et al. (1982) showed that IgG and albumin concentration varied during serial sampling of CSF. This should be taken into account in evaluation of the calculated index. T h e CSF IgG index should be considered a reference with which to work for future investigations and was discussed by Eah et al. (1982). According to Tourtellotte et al. (1980) the formula: -

IgG,) - (Alb,,f - Alb,) (IgG,) 369 230 Alb,

X

0.43

1

X

5

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represents the amount of IgG synthesized per day in the CSF derived from extravascular sources. The IgG,.[, IgG,, Alb,,f, and Alb, are expressed in mg/ 100 ml. As described by Tourtellotte: -369 is a ratio constant that quantitatively determines the proportion of CSF IgG that normally passes by filtration from the serum into the CSF across an intact BBB. -1gC,/369 is the IgG that is expected to cross from the serum to the CSF based on the patient’s serum IgG concentration and the nornial CSF/serurn ratio. -230 represents a constant that determines the proportion of CSF albumin that normally passes by filtration froni the serum to the CSF across the intact BBB. -Alb,$r - AlbJ230 is a term that represents the excess CSF albumin that has crossed a damaged BBB. -the formula (IgGJAlb, X 0.43 converts the excess CSF albumin to excess CSF IgG that has crossed a damaged BBB.To calculate the daily IgG synthesis this equation is x 5 to convert froni concentration in mg/100 nil to the aniount present in 500 ml C S F that o n the average is formed each day.

This formula was, as far as we know, never applied to other proteins than the IgGs. It must be emphasized that these ratios, although they give interesting information, may, however, not be accepted without reservations as to their physiological significance. Peterslund and Pedersen (1982) have stressed these reservations and demonstrated that the calculation of ratios can reveal intrathecal synthesis in patients suffering from meningitis, meningism, and aseptic meningitis, as well as in patients who have diseases that cause immune reactions in the CNS such as MS. Ukkonen et al. (1981) reach similar conclusions: In patients with mumps meningitis, they identified in certain cases an intrathecal synthesis of IgG and also IgM mumps antibodies. They did not find a correlation between the intrathecal synthesis of these antibodies and the intrathecal synthesis of IgGs. T h e concentration of specific IgGs against mumps was much less than the total concentration of CSF IgGs. In addition similar calculations applied to other proteins such as transferrin (Kerenyi et al., 1980) lead to the conclusion that this protein could also be synthetized intrathecally. One wonders how far the calculation of these ratios could lead. New formulas were proposed and applied to experimental allergic encephalomyelitis (EAE) data (Suckling et al., 1983). 111. Qualitative Studies

A. ELECTROPHORESIS: RESULTS T h e first qualitative determinations of CSF proteins were carried out indirectly by colloidal reactions. These reactions show both changes in

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the content of total protein and qualitative changes caused by changes in the albumin/globulin ratio. These reactions are known under the names of goldsol, Pandy, Weichbrodt, Nonne, Kafka, etc. Most of these reactions are still listed and discussed but in practice are hardly used. T h e above-mentioned colloidal reactions showed for the first time that there could be qualitative differences between the CSF proteins. That is, in two samples of CSF with the same and very often normal total protein content different proteins can be found. This was clearly revealed by electrophoresis, which was applied first to CSF proteins by Hesselvik (1939) and later by Kabat et al. (1942). As one has access to relatively small quantities of CSF and as the total CSF protein content is very low, technical problems soon arose. T h e free electrophoresis method of Tiselius, which was first applied to CSF proteins, could never be used on a large scale, and the first results were thus relatively irrelevant. Several sophisticated methods were tried, among which one by Booij (1949) was an attempt to overcome the difficulties. The electrophoretic study of the CSF proteins became routine with the use of paper electrophoresis following the method o f CI1.assmann and Hannig. Proteins were then stained as well as lipoproteins and glycoproteins. 'Ten ml CSF was concentrated for this type of electrophoresis. However, a real change occurred when we had the opportunity to be the first, in 1958, to study the CSF proteins using agar gel electrophoresis according to the method of Wieme (Van Sande et al., 1958, 1959; Karcher et al., 1959; Lowenthal, 1959, 1962, 1964; Lowenthal el al., 1960, 1966; Lowenthal and Petre-Quadens, 1963).We were able to work with smaller quantities of CSF (3 ml), although it still had to be concentrated and to reveal the proteins by staining with amidoblack. A possible source of artifacts is the necessity to concentrate CSF. The same method permits immunoelectrophoretic and enzymoelectrophoretic study of the CSF proteins. Enzymoelectrophoresis of lactate dehydrogenase, for example, showed that besides the identified proteins those with enzymatic activity that were not stained by amidoblack could be revealed (Lowenthal et al., 1961, 1966). Agar gel electrophoresis made it possible to describe four different types of electrophoretic patterns: the normal type, a type with increase of fast-migrating a-globulins, a type with increased diffused y-globulins or serum-like type, and a type with fractionation of y-globulins, with or without their increase. Dencker (1966) showed that these y-globulins were in fact IgCs (at the time named 7 S globulins) and that one could confirm indirectly the fractionation of these y-globulins after immunoelectrophoresis. We now know that this fractionation has a physiopathological significance: The

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fractions show that there is formation of homogeneous antibodies through hyperimmunization, as in an abnormal immune reaction (Lowenthal, 1979). A comparative study of the values obtained after agar gel electrophoresis, biochemical studies of CSF, and immunoelectrophoresis was recently made by Kolar et al. (1980). One of the problems being studied at that time by using paper electrophoresis as well as for gel electrophoresis was the existence of specific CSF proteins. Prealbumin and 7-globulin were identified in this way. 1. Normal CSF Pherogram (Fig. 1 )

When comparing the CSF to the serum pherogram, one can immediately recognize the CSF pherogram by the presence of prealbumin, the prevalence of p-globulins over y-globulins, the fractionation of the /3globulins into PI- and 7-globulin and the fact that the y-globulins are present at a low level. Prealbumin and 7-globulin, although present, are seldom seen following electrophoresis of serum, and this has led to the erroneous conclusion that prealbumin and 7-globulin are specific to the CSF proteins. After more elaborate studies prealbumin and 7-globulin were also found in serum. Pherograms of the normal types are seen in patients who have no neurological anomalies as well as in patients who suffer from severe neurological conditions such as hereditary degenerative diseases and amyotrophic lateral sclerosis. In slow viral diseases with spongiform encephalopathies normal pherogram has always been found. The same is

FIG. 1. Serum (A) and CSF (B) protein agar gel electrophoresis.Normal patterns. The second /3-globulin migrates in serum faster than CSF T-globulin (arrow). In CSF prealbumin is observed.

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FIG. 2. a-type CSF protein pherogram.

true for spontaneous as well as experimental (Lowenthal, 1964; Olsson, 1980) spongiform encephalopathies. Williams et al. (1980) were the only group to show the fractionation of the y-globulins in presenile dementia.

2. Pherogram with Fast-Migrating a-globulins (Fig. 2 ) This pherogram appears in acute (toxic, vascular) neurological diseases and can often be correlated with the increase of glial fibrillary acidic protein (GFAP) or a-albumin level in the CSF. It appears also in Tay-Sachs disease or GMp gangliosidosis, where megalencephaly is associated with very high GFAP values in the CNS (Karcher ut al., 1972b).

3. Serum-like Pherogram (Fig.3 ) This appears in all cases where serum proteins enter the subarachnoidal space; it is seen, for example, in compressions, meningitis, diabetes and Guillain-BarrC syndrome. Sometimes one can observe a tendency to fractionation at the level of the y-globulins in Guillain-Barre syndrome. 4. The Fractionation of the y-Globulins (Fig. 4 )

We first described the fractionation of the y-globulins that we mentioned in Section III,A in 1958. In 1966 Dencker proved this pattern to be a fractionation of IgG, using immunoelectrophoresis. This fractionation is found in MS and in many other neurological diseases such as SSPE (Lowenthal, 1964), cysticercosis (Machado et al., 1979), herpetic

FIG. 3. Serum-like type CSF protein pherogram.

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FIG. 4. y-globulin restricted heterogeneity (oligoclonal reaction) in CSF.

encephalitis, trypanosomiasis, neurosyphilis, and filariosis (Lowenthal, 1964). The fractionation, which is not typical of a disease but of an immune reaction, is caused by the formation of antibodies with restricted heterogeneity. The study of K and A light chains show that the fractions are not only of one particular class of IgG: It was not possible to demonstrate complete homogeneity for fractions separated by agar gel electrophoresis. A question of terminology should be considered here. First, we used the expression fractionation of y-globulin or IgG. Later, we spoke of formation of M or myeloma proteins as this disease gives similar patterns in serum and CSF. Many authors named it oligoclonal reaction without definitive proof. In fact, it should be called restricted heterogeneity of the IgG, as shown by experimental work (Lowenthal, 1979). The demonstration of antibodies with restricted heterogeneity appears to be, so far, the most important information to be obtained in neurology by the study of CSF proteins. For clinical purposes the only practical method to reveal it is CSF protein electrophoresis in either agar gel or agarose or the more elaborate isoelectric focusing. A search, however, for new technical methods and information is badly needed and will be discussed in the following section.

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B. METHODOLOGICAL AND PHYSIOLOGICAL PROBLEMS Various questions are raised concerning the origin and the meaning of this immune reaction with restricted heterogeneity of the IgG. We now wish to discuss some of these questions:

1. Which is the most adequate method to show the fractionation of immunoglobulins? 2. What is the physiopathological mechanism responsible for this fractionation? What type of immune reaction is more or less correlated with this fractionation? 1. Discussion of Methods It is important to find out which technique is the most suitable for demonstrating this fractionation: agar gel electrophoresis, agarose electrophoresis, isoelectric focusing, or isotachophoresis. In order to obtain a good electrophoretic separation of the CSF proteins showing the homogeneous y-globulins, the best protocol should be used. a. Nonconcentrated CSF. To reveal the restricted heterogeneity of the IgG agar gel and agarose electrophoresis have been widely used. The first has always given satisfactory results. The second has been improved in order to liberate the immunoglobulin area away from the site of application, and available agarose plates have facilitated its use. These methods can be combined with immunological methods. It is evident that it would be preferable to work with nonconcentrated CSF. An available procedure has been suggested over the last 10 years. In 1972 Kerenyi and Gallyas suggested staining the proteins with silver nitrate. This technique (Glasner et al., 1979; Karcher et al., 1979; ‘I‘asnier et al., 1982a,b) enables us to examine C:SF protein without prior concentration, which has the immediate advantage of preventing the formation of artifacts due to the concentration and allows one to work with very little CSF (10 pl). Clearly, this helps in the examination of the CSF in children and animals. Silver staining can only be carried out with known amounts of total proteins. The different a-globulin fractions are not so clearly seen with the silver staining method as with amidoblack. The pattern in the y region remains the same by both methods. The different pherogram types obtained with amidoblack as described in Section III,A are also seen with silver staining (Fig. 5 ) . In 80% of MS samples received pherograms with increased and fractionated y-globulins were found. The agar gel electrophoresis with silver staining revealed a new pathological pherogram

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FIG.5. CSF protein patterns after silver staining. A, Normal type; B, a-type; C, serumlike type; D, vtype; E, y-globulins restricted heterogeneity.

type: the .r type (Kerenyi et al., 1977), that has an increased relative content of .r-globulin. The possible existence of this type was mentioned earlier by Habeck when he described variations in the P-globulin region using paper electrophoresis. This r type is found in 16%of our cases using the silver staining, whereas it was seldom or never detected by amidoblack staining, probably because of the concentration procedure. Comparing both methods, amidoblack (concentrated CSF) and silver

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staining (nonconcentrated CSF) (Tasnier et al., 1982a,b), similar results were found in 87% of all cases. The 7 type was found in 50% of our patients with chronic alcoholism and in 40% of those with cortical atrophy shown on CAT-scan. Wikkelso et al. (1981) found increased .r-globulin in 18% of senile dementia cases and only in 6% of their multi-infarct patients. Techniques replacing amidoblack by silver staining need further improvement before routine application. Nevertheless, this method has already become indispensable in research, particularly in the examination of guinea pig CSF in chronic relapsing EAE (Karcher et al., 1982b). Other methods for using nonconcentrated CSF have also been presented, including agarose isoelectric focusing (Kostulas and Link, 1982), agarose isofocusing with radioimmunofixation of the IgG (Lasne et al., 1981), agarose electrophoresis with silver staining (Hiraoka et al., 1982), polyacrylamide disc electrophoresis (Casey et al., 1981; Confavreux et al., 1982a), thin layer isofocusing using 40 microliters of CSF with silver staining and direct immunofixation of the IgGs (Confavreux et al., 1982b), and silver staining of bidimensional protein pherograms in polyacrylamide (Merril et al., 1981). These new methods have not yet been introduced in practice in neurology. They can bring useful new information but should be applied to further series of cases. b. isoelectric Focusing. To improve the separation of the immunoglobulins and to study the oligoclonal pattern detectable by the former techniques, acrylamide isoelectric focusing has been used extensively at a p H range of 3.5 to 9.5 on agar gel (Olsson and Nilsson, 1979; Mattson et al., 1981; Kostulas and Link, 1982). The separation of the immunoglobulins was greatly improved and numerous bands were visible. As an identification procedure they were classified as anodic, cathodic, and extreme cathodic, the pH ranges being respectively 3.5-6.4;6.5-8.4; and 8.7-9.5 (Laurenzi et al., 1980). Samples could be compared by running several samples simultaneously and having a control CSF as reference to evaluate the patterns. T h e bands can be scanned by densitometry and their surface areas compared. Visual estimation, however, remains the best procedure. After isoelectric focusing the oligoclonal pattern is consistently increased in the alkaline area (extreme cathodal) for CSF of MS patients (Kjellin and Vesterberg, 1974; SidCn and Kjellin, 1977). A prevalence in this same region of IgG of the K type in MS and SSPE is not excluded (Rocchelli et al., 1981). Some of these bands could be monoclonal immunoglobulins (Mehta et al., 1981). Comparing control and MS CSF, the normal polyclonal pattern consists mainly of bands between pH 6.5 and 8.6 in both samples. In MS

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additional bands are seen, especially between pH 8.7 and 9.5 (Karcher et al., 1972a; Laurenzi et al., 1980). Isoelectric focusing special band pattern does not change throughout the disease, although the intensity of the different bands might differ. There is no correlation with the duration, the degree of disability, or the age of onset for MS. Isoelectric focusing gives considerable information pertaining to the identification, the modification and the characterization of immunoglobulins. It is, however, still too expensive and time-consuming to be used routinely. c. Zmmunofixation. In order to obtain more detailed information, immunofixation has been introduced (Laurenzi and Link, 1979; Stibler, 1979; Laurenzi et al., 1980; Kostulas and Link, 1982; Keir et al., 1982). Five to 50 pl of concentrated CSF are applied to the supporting medium, polyacrylamide o r agarose gel. At the end of the run the polyacrylamide or the agarose gel is covered with a sheet of nitrocellulose and blotted by means of a kilogram weight. A transfer o r print occurs from the gel to the nitrocellulose, which can be further analyzed. After a first incubation with animal monospecific antihuman immunoglobulin of different classes o r subclasses, followed later by an incubation with horseradish peroxidase-conjugated antianimal immunoglobulins, the bands can be identified. Another alternative is to dip the nitrocellulose in the monospecific antihuman immunoglobulins, lay it on the gel, and apply weights (Laurenzi and Link, 1979). T h e antigen-antibody reaction will take place on the surface of the nitrocellulose. After excess proteins are washed away, staining will show bands corresponding to the monospecific antibody used. When these monospecific antibodies are radiolabeled, the bands can be visualized by autoradiography (Kostulas and Link, 1982). Immunofixation allows the comparison of different immunoglobulin patterns after isoelectric focusing and characterizes them with regard to age, sex, evolution of the disease, and the effect of therapy. This gives us more data for clinical investigation, but certain conditions have to be met and the technique standardized to make it reproducible. In addition the monospecific immunoglobulin detected by immunofixation must correspond to the original pattern. d. Crossimmunoelectrophoresis. Crossimmunoelectrophoresis can also help identify the different bands after CSF isoelectric focusing (Stibler, 1977; Livrea et al., 1981). A gel is transferred to a 1% agarose plate containing antihuman IgC monospecific antibodies. T h e second-dimension immunoelectrophoresis develops different profiles of immunoglobulin precipitates corresponding to the fractions detected in the first dimension and helps to identify them. The antibodies used in the second

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dimension need to be carbamylated in order to slow down their migration. In our opinion this very useful and far-reaching technique is not very suitable for immunological studies owing to the fact that antigen and antibodies are of the same type, both immunoglobulins. These techniques characterize the immunoglobulin type and identify the light chains, for instance, IgA, IgG, IgM classes, Igl, Ig2, Igs subclasses and the K and A chains. e. Imprint Electroimmunojxation. For the characterization of the oligoclonal reaction imprint electroimmunofixation (Nordal el ul., 1979) has been used. After electrophoresis of the CSF samples the agarose plate is brought in direct contact with a gel containing virus antigen. It is then incubated 30 min. After separation of the two layers the electrophoretic gel is fixed and stained, and the antigen-containing gel is washed, incubated with labeled antihuman IgG serum, and later autoradiographed. The corresponding bands indicate the immunoglobulin’s specific activity for one particular antigen. An intrathecal IgG synthesis is suggested when there is a difference in reactivity between matched serum and CSF samples with similar IgG concentration. T h e presence of oligoclonal antibodies in areas where none were demonstrable by protein staining was also demonstrated. f. Transfer Electrophoresis. Another method for characterization of the immune reaction is transfer electrophoresis after polyacrylamide electrophoresis (Towbin et al., 1979; Keir et al., 1982), of, for instance, measles virus (Karcher et al., 1981). T h e polypeptides of measles virus are separated by polyacrylamide electrophoresis and transferred electrophoretically to a nitrocellulose sheet. T h e nitrocellulose carrying the polypeptides is incubated with the CSF of patients affected with SSPE who are known to have high CSF measles antibody titers. The second incubation is carried out with peroxidase-conjugated antihuman immunoglobulins. T h e measles virus polypeptides then become apparent. This technique demonstrates that antibodies to all measles polypeptides are present in the CSF of SSPE patients. One of the polypeptides (matrix) is absent in the SSPE virus itself, which is a measles-like virus but therefore different from the wild measles strain (Edmonston). Nevertheless, the antibodies to the matrix are present in the serum and CSF of these patients as shown by using the wild measles strain Edmonston instead of SSPE virus (Karcher et al., 1982a). The CSF immune response to any agent to be run electrophoretically can be investigated by transfer electrophoresis. With this procedure the activity of the CSF immunoglobulins can be tested against a whole set of antigens. g. Zsotuchophoresis. Besides isoelectric focusing followed by various techniques to characterize the CSF immunoglobulins as mentioned in

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previous paragraphs, there exists another method of analysis of cerebrospinal fluid: isotachophoresis (Kjellin and Hallander, 1975; Delmotte, 1977; Tourtellotte et al., 1982). Fifteen microliters of CSF are needed in this discontinuous electrolyte system. T h e area of interest can be chosen by selecting the leading and terminating electrolyte solutions. The separation of different proteins and the immunoglobulins, in particular, can be achieved without any prior concentration of CSF. The identification of the different peaks can only be related to their mobility and surface areas calculated by automated analytic procedures. It has not yet reached or surpassed the diversity that isoelectric focusing offers. All these methods have helped to identify the different immunoglobulin classes and subclasses and their light chains and also give some insight into the CSF immune activity after electrophoresis. They are guidelines for further investigation and will no doubt remain very useful. For the clinician the agar gel and agarose electrophoresis is the best method to visualize the immunoglobulin band in spite of its shortcomings and its limited ability to provide information. Johnson et al. (1977) and Johnson (1980) examined the different methods of separating CSF proteins and demonstrating the anomalies detected in MS. They reached the same conclusion: Agar gel o r agarose electrophoresis is the most efficient method for clinicians. For Gerson et al. (198 1) identification of oligoclonal bands is a better confirmation for MS clinical diagnosis than determinations of myelin basic protein (MBP) or IgG. Bloomer and Bray (1981) consider the identification of oligoclonal bands as superior to calculations of indexes or to using Tourtelotte’s formula. Liskiewicz (1982) indirectly confirmed the superiority of agar gel or agarose CSF protein electrophoresis.

2. Immunological P r o b l e m In 1958 the oligoclonal reaction was shown in the CSF of MS patients (Van Sande et al., 1958, 1959; Karcher et al., 1959; Lowenthal, 1959, 1962, 1964; Lowenthal et al., 1960, 1966; Lowenthal and PetreQuadens, 1963). This observation has since been repeatedly confirmed (Laterre et al., 1970; Link and Muller, 1971; Vandvik and Skrede, 1973; Delmotte and Gonsette, 1977; Johnson et al., 1977; Laurenzi and Link, 1978; SidCn, 1979; Paty et al., 1979). Depending on the technique used, the oligoclonal reaction can be demonstrated in the CSF of 80 to 95%of MS patients. The oligoclonal reaction is shown clearly and consistently in CSF and serum of patients with chronic infections in the CNS such as trypanosomiasis, SSPE, and progressive rubella encephalitis. T h e oligoclonal reaction is found less frequently and at times temporarily in encephalitis, myelitis, polyradiculitis (Guillain-BarrCsyndrome),

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and some meningitis. The reaction was also demonstrated in myasthenia gravis (Adornato et al., 1978), progressive myoclonal epilepsy (Iivanainen et al., 1981), and after cerebrovascular accidents (Rostrom et al., 1981a). In many cases parallel anomalies were seen in serum and CSF (Lowenthal et al., 1971). It can also be found in synovial fluid in arthritis victims (Hedberg, 1971; Vandvik et al., 1977). The formation of these relatively homogeneous IgG fractions can be induced in semiexperimental conditions in ataxia telangiectasia: Patients affected by ataxia telangiectasia who have abnormal levels of IgA can be treated by parenteral administration of IgG. When such patients are injected regularly over a long period, relatively homogeneous antibodies are formed, which can be seen in serum as well as in CSF (Lowenthal et d.,1972). In chronic relapsing EAE similar patterns are also seen (Fig. 6). This disease can be considered as an experimental model for the formation of the relatively homogeneous antibodies and perhaps also for MS. T h e disease can be induced by inoculation with an encephalitogeneous emulsion (spinal cord extract or myelin basic protein) in different animals such as monkeys, rabbits, rats, and guinea pigs. In its chronic relapsing form this disease shows a clinical course and histopathological findings comparable to those seen in MS (Stone and Lerner, 1965; Raine and Stone, 1977; Wisniewski and Keith, 1977). Silver staining applied to agar gel electrophoresis according to Wieme’s technique using small quantities of nonconcentrated CSF made it possible to carry out electrophoretic examination of the CSF (Verheecke, 1065; Kerenyi and Gallyas, 1972; Tasnier et al., 1981a). Agar gel electrophoresis then requires

FIG.6. Guinea pig serum (A) and CSF protein (B) electrophoresis in chronic relapsing EAE. Serum and CSF y-globulins restricted heterogeneity.

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only 10 pliters nonconcentrated CSF. In the chronic relapsing form there is a marked increase of the fractionation of the y-globulins in serum and CSF. T h e anomalies in the serum are parallel to those in the CSF, which suggest a general immune reaction in EAE, not just a local intrathecal reaction. The patterns seen in EAE are comparable to those seen in MS and SSPE. All these data were collected from clinical or experimental material and reveal the difficulty of a physiopathological interpretation. Can one associate MS, SSPE, myeloma, ataxia telangiectasia, and chronic relapsing EAE in the same group? Therefore, the physiopathological process responsible for the appearance of the IgGs with restricted heterogeneity will now be discussed. a. Humoral Immunity. Origin of the CSF ZgC. Most authors find that part of the CSF proteins are formed by cells in the CNS and in the choroid plexus, and part originate in the blood. The CSF cells could also play a role in the synthesis of CSF proteins (Thompson et al., 1982). Until now, it has not been clear whether the oligoclonal reaction in the CSF is the result of a local immune process in the CNS or of a general immune process. Although the high amount of normal polyclonal IgG in serum masks the identification of an oligoclonal reaction, this reaction in SSPE and to a lesser extent in MS can be found in serum (Lowenthal, 1979). Because of the draining of CSF into the serum the oligoclonal IgG in serum could originate in the CSF, which means that the description of the same oligoclonal pattern in CSF and serum does not give any information on the origin of the oligoclonal IgG. There are some data that suggest that in MS the oligoclonal IgGs are synthesized at the level of the MS plaques. Immunoglobulins isolated from MS plaques showed the oligoclonal pattern (Mehta et al., 1982), an observation that will not be accepted without consideration of the technical problems. From CSF of MS patients, lymphocytes can be isolated that can synthesize in vitro oligoclonal IgG (Sandberg-Wollheim, 1974). Although the pathogenetic relevance of the oligoclonal reaction in MS and the way in which the reaction takes place are not known at this moment, it is possible that the cause can be found to a certain extent in the CNS. One can accept that in MS an abnormal immune reaction takes place that consists of the formation of IgG in abnormal quantity and perhaps quality. This would not be the case with the other immunoglobulins. T h e hypothesis of an abnormal immunoregulation in MS was also supported in recent publications by G u s t et al. (1982) and Paterson and Whitacre (1981). T h e last authors focused on the point that the antibody activity of the IgG of restricted heterogeneity has still to be demonstrated. A nonspecific activation of the B cells could for them be responsible for the

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production of these IgGs. They write that “it would be sobering if this phenomenon had no immunological significance and it would remove one of the strongest] best known and most tantalising pieces of evidence suggesting that MS has an immunopathogenetic basis.” This discussion o f the origin of the IgG with restricted heterogeneity in the CSF and especially in patients with MS would not be complete if one did riot mention the following facts.

1. Adinolfi and Haddad (1977), discussing the origin of the albumin, stress the fact that the BBB does not always have the same permeability during development. 2. Intrathecal synthesis is also evoked to explain certain electropherograms seen in medulloblastoma and in acute cerebellar ataxia in children (Siemes et al., 1981) and to explain symptoms seen in systemic lupus erythematosus (Williams et al., 1981; Bluestein et d., 1981). 3. Symptoms seen in vascular diseases with an oligoclonal reaction, as mentioned in Section III,B,2] pose once more the essential question concerning the origin of the IgG with restricted heterogeneity (Rostrom and Link, 1981). 4. T h e presence of relatively homogeneous antibodies in the CSF of patients with myeloma without any damage in the CNS should be mentioned. It seems difficult to imagine that these proteins d o not cross the BBB. In conclusion, one could say that there are sufficient data to show that some proteins and immunoglobulins can be synthesized in the CNS, but it must be stressed that nothing now proves that the intrathecal synthesis takes place in neurons or in glial cells, whereas it probably does take place in blood cells that have penetrated the CNS (Cremer ef al., 1980).The role of the lymphocytes in the CSF should not be forgotten. Determinations of immune complexes lead to similar questions without giving clear information (Sindic el al., 1982; Arnadotir et al., 1982), although Dasgupta et al. (1982) found that the immune complexes increase during MS exacerbations or relapses. However, Salmi et al. (1982) stress that: (1) there is no parallel between serum and CSF immune complexes levels, and (2) immune complexes are not correlated with clinical status and are also found in serum in so-called nonactive MS cases. These authors made a complete review of the literature pertaining to this problem. ZgG restricted heterogeneity: molecular basis. T h e oligoclonal reaction reflects a restricted heterogeneity of IgG. The molecular basis of this restricted heterogeneity is not completely understood. It could differ in

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the various pathological conditions in which the oligoclonal reactions occur. It has not been proven that each of the individual bands that together form the oligoclonal pattern originates from one specially proliferating lymphocyte plasma cell clone. An individual band can be formed by IgG of different types, making the whole polyclonal (Perrini et al., 1979). T h e normal heterogeneity is mainly the result of the heterogeneity of the variable (V) region of the IgG. Therefore, restricted heterogeneity could be caused by a restricted expression of the V gene repertoire. Structural as well as serological data on oligoclonal IgG support this theory. The N-terminal amino acid sequence of the H- as well as the Lchains in SSPE serum IgG has been determined and demonstrates the relative structural homogeneity of the V region for this IgG (Strosberg et al., 1975). Moreover, dominant idiotypes that are present in 1 to 25% of the Ig repertoire in the CSF were defined in MS and SSPE (Ebers et al., 1979; Strosberg et al., 1979; Baird et al., 1980; Gerhard et al., 1981; Nagelkerken et al., 1982). These serological observations also suggest a restricted expression of the V gene repertoire. In addition to the V region, differences in the C region also contribute to the heterogeneity of immunoglobulins. Serologically this heterogeneity is defined by the classes and subclasses of the H-chain and the type of L-chain. Several authors calculated KIA ratios in MS and SSPE and found those ratios to be different from controls (Roberts-Thomson et al., 1976; Bollengier et al., 1978a,b; Eickhoff et al., 1978, 1979). For Mattson et al. (1982) the different bands are predominantly K o r h chains, and mainly K in MS. These authors also found free light chains in the CSF of MS. Altered proportions of the various IgG subclasses have also been described. This could indicate a different expression of C gene for the L- as well as for the H-chains, which can contribute to the formation of the oligoclonal pattern in electrophoresis. It was not shown whether the C restriction is the result of a linkage between C gene and V gene clusters. Although the significance of the oligoclonal reaction is still not clear, there are indications that the pathological meaning is not the same for all diseases in which the reaction takes place. Most authors agree that the oligoclonal pattern in CSF of MS patients remains constant during the course of the disease and does not fluctuate during its development. This could suggest that the oligoclonal reaction is an epiphenomenon. Some authors even suggested and later rejected the hypothesis that MS oligoclonal IgG are nonsense antibodies. However, a transient oligo-

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clonal reaction can appear during meningitis or encephalitis, which in these cases indicates a more direct relation to the etiology. Antibody specijcity. Information about the specificity of the oligoclonal IgC also indicates a different significance of the reaction in different pathological entities. In SSPE caused by a chronic measles infection most of the oligoclonal reaction is probably specific for measles antigens (Karcher et al., 1972a; Nordal et al., 1978a). In this case the oligoclonal reaction can be taken as a sign of antigen-specific hyperimmunization. I n MS the specificity of the major part of the oligoclonal reaction has not been identified, whereas a minor part is directed against measles, mumps, herpes simplex, rubella virus (Nordal et al., 1978b; Rostrom et al., 1981b), or even to staphylococcal lipoteichoic acid (Aasjord and Nyland, 1981). This suggests that in these cases the oligoclonal reaction could be the result of a polyclonal B-cell activation. This activation itself could be caused by a nonspecific mitogen activity o r by interleukins produced in the course of a specific immune response against an unidentified MS antigen. In MS and in many other diseases essential questions remain: How can one further examine the immunological reaction? How can the specificity of the antibodies be investigated? Which type of IgG is found? Can we receive more information from the study of cellular immunity? The identification of the activity of the antibodies or IgG, in particular the relatively homogeneous IgG of the CSF, will be the first problem. Techniques used for this identification such as immunofixation and transfer electrophoresis have been discussed. As mentioned in a previous paragraph in this section, the percentage of identified activity in MS IgG remains limited at present. This is not so in SSPE, in which most relatively homogeneous antibodies are directed against measles virus. Among the publications on this subject is the work of Vartdal and Vandvik (1982), who identified measles, herpes, and rabies antibody activity in MS CSF but not at the level of the oligoclonal IgC bands. Many different antibodies were found in CSF by Forgharii et al. (1978, 1980). Norrby et al. (1974), however, in CSF studies still assert the theory of intrathecal synthesis of different viral antibodies present in the CSF. Other antigens (brain antigens, oligodendrocytic antigens) (Traugott and Raine, 1981) should also be considered. New methods should be tried using nonconcentrated CSF in order to achieve a quantitative identification of the antibody activity of the homogeneous IgC. This identification of antibody activity in MS could be a basis for further research. We wish to reiterate here results obtained

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with transfer electrophoresis in SSPE CSF: With this method it has been shown that although the SSPE myxovirus lacks the M protein in serum and CSF of SSPE patients, antibodies to this protein or polypeptides are present (Karcher et al., 1982a). A similar technique allowed Newcombe et al. (1982) to identify antibodies to MS brain proteins among the oligoclonal bands. Other assays to identify the antibody specificity in MS or other IgG activities are still in the early stages. For instance, research into antibodies against oligodendrocytes or demonstration of the presence of antibodies cytotoxic to cells infected with measles virus present in serum and CSF of MS patients (Cremer et al., 1979, 1980). More information may be obtained from studies of idiotypes in MS. Idiotypically defined IgG can be detected in CSF as well as in serum in MS. T h e higher ratio for the CSF/serum idiotypic positive IgG than the CSF/serum total IgG again suggests intrathecal synthesis. The persistence of an idiotype in serum and CSF for several years has been described. T h e specificity of the idiotype positive IgC might lead to the antigen identification in MS and in other neurological diseases. Ryberg and Kronvall(l981)and Gerhard et al. (1981) described a major individual idiotype in an MS patient that was expressed on antibodies directed to Theiler virus. Another aspect of the humoral immunity must also be mentioned: the formation of antibodies against nervous tissue specific proteins (GFAP, MBP, S-100,...). This will be treated in the discussion on nervous system-specific proteins. Let us also mention that specific antibody activity of IgM (Heinz et al., 1981) can be demonstrated in tick-borne encephalitis. b. Cellular Immunity.This chapter would not be complete if we did not provide information pertaining to cellular immunity in the CSF, or in other words the presence of lymphocytes in the CSF. We wish to recall the hypothesis that the lymphocytes could be at least partially responsible for the intrathecal IgG synthesis. B- and T-lymphocytes. In the last decade several investigators examined the relation between B and T lymphocytes in the CSF. The identification of B lymphocytes was done by staining cell surface immunoglobulins. T lymphocytes were recognized by their ability to form rosettes with sheep erythrocytes. T h e standardization of E-rosette formation was difficult, which is one explanation for differences in the results. Most authors find high percentages of T lymphocytes in the CSF in comparison to blood (Goasguen and Sabouraud, 1974; Sandberg-Wollheim and Turesson, 1975; Allen et al., 1976; Moser et al., 1976; Naess, 1976; Fryden, 1977; Kam-Hansen et al., 1978; Manconi et al., 1978; Traugott, 1978; Coyle et al., 1980; Clonkowska et al., 1980; Naess and Nyland, 1980;

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Nyland et al., 1980). A more distinct increase is seen in viral meningitis and MS (Sandberg-Wollheim and Turesson, 1975; Allen et al., 1976; Naess, 1976; Levinson et al., 1976; Fryden, 1977; Kam-Hansen et ul., 1978; Manconi et al., 1978; Traugott, 1978; Coyle et al., 1980; Clonkowska et ul., 1980; Nyland et al., 1980; Naess and Nyland, 1980). Kam-Hansen et al. (1978), Naess and Nyland (1980), and Nyland et al. (1980) found significant differences within control groups. Nyland and Naess (19'78) and Nyland et al. (1980) found a significant increase of T lymphocytes in acute Guillain-Barre disease and in optic neuritis, but this was not confirmed by other studies (Fryden, 1977; Kam-Hansen et al., 1978; Traugott, 1978). It should be mentioned that the percentage of T lymphocytes in the blood of MS patients varies from study to study: In some it decreases (Lisak et al., 1975; Naess and Nyland, 1980), but in others it increases (Oger et al., 1975; Kam-Hansen et al., 1978; Offner et al., 1978). Allen et al. (1976) and Clonkowska et al. (1980) found an increase of T lymphocytes in the CSF of MS patients during exacerbation, but this is contradicted by Traugott (1978) and Coyle et al. (1980). Morrell (1979) found a decreased T lymphocyte percentage in MS patients who responded to corticosteroid therapy; Naess and Nyland (198 1) found the opposite in MS responders to ACTH therapy. There is no correlation between increased T lymphocyte percentage, pleiocytosis, and the amount of CSF protein and IgG ratio (Traugott, 1978; Naess and Nyland, 1981). The relative B lymphocyte content is lower in CSF than in blood, but the values found show no correlation with MS or other diseases. All investigators except Scheiffarth et al. (1977) found more T lymphocytes than B lymphocytes in CSF and in blood. The ratio of B to T lymphocytes in the CSF can be useful for the differential diagnosis of meningitis in lymphoma patients. In lymphomatic meningitis the B lymphocytes predominate (Goodson and Strauss, 1979); in fungal meningitis the T lymphocytes predominate (Davies et al., 1978). T lymphocyte subpopulations in the CSF. Active T cells are cells that bind three or more sheep erythrocytes during the E-rosette test. The function of this subpopulation in the immune system is not known, but they are probably IgG-Fc negative (West et al., 1977). Kam-Hansen (1979, 1980) found a significant decrease of active T cells in the CSF of MS patients, whereas others found a small increase (Coyle et af., 1980; Naess and Nyland, 1980). The IgG-Fc positive subclass coincides more or less with the class of suppressor T cells, which inhibit the activity of B and T lymphocytes. They are identified by binding to immunoglobulin-covered red blood cells. T h e percentage of suppressor T cells in the blood is lower than

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normal in patients with active MS (Santoli et al., 1978; Ante1 et al., 1979; Huddlestone and Oldstone, 1979). In the CSF the IgG-Fc positive cells also decrease during MS exacerbations even in patients with normal blood percentages (Manconi et al., 1978; Coyle et al., 1980). A complete comprehension of the occurrence of the different T subpopulations in MS would improve our understanding of the functions of CSF lymphocytes. This could be obtained by the use of monoclonal antibodies, which allow a reliable classification of functional subpopulations (Kung et al., 1979; Bach et al., 1980). Kelley et al. (1981) expressed their doubts concerning a suppressor T cell dysfunction. CSF lymphocyte stimulation. CSF lyniphocytes proliferate less after stimulation by mitogens such as pokeweed mitogen, phytohemagglutinin, o r concanavalin A than lymphocytes from the peripheral blood (Fryden and Link, 1978; Kam-Hansen et al., 1979). Divergent results were found in MS patients (Levinson et al., 1976; Kam-Hansen et al., 1979; Reunanen st al., 1980), which might be explained by different experimental procedures. In infectious meningitis the CSF lymphocytes proliferate when brought into contact with specific antigens of the infectious agent (Malashkhia and Geladre, 1976; Fryden et al., 1978). This might be used as a diagnostic test. CSF lymphocytes of MS patients undergo blast transformation after stimulation with measles antigen (Reunanen et al., 1980). Similar results are found after stimulation with MBP in active and progressive MS and in encephalomyelitis (Lisak and Zweiman, 1977). All investigators found higher transformation with CSF lymphocytes than with blood lymphocytes. Cellular immunity studies in CSF confirm that immunological regulation is disturbed in MS and also in other neurological diseases. The mechanisms and the significance of these changes remain difficult to identify. Better correlations between cellular and humoral immunity studies are needed. A common factor related to cellular and humoral reaction could explain many of the phenomena seen, including the persistence of antigen and the hyperimmune reaction (Karcher et al., 1977; Karcher, 1979), but it has still to be identified. In clinical practice the proposed tests are not yet used and at present only the electrophoretic study of the CSF IgG remains useful for the clinicians. To summarize our immunological presentation one can say that restricted heterogeneity of the IgG is shown more easily in CSF than in serum and is seen in many chronic and acute, spontaneous and experimental neurological diseases. T h e significance of this finding is sometimes difficult to explain. It means a persistent antigenic stimulation of the production by the B lymphocytes of specific or nonspecific antibodies. These antibodies cannot neutralize the causative agent. The pres-

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ence of a factor inhibiting the suppressive activity of the T lymphocytes on the antibody production or inhibiting the antigen-antibody reaction cannot be excluded, especially in chronic diseases such as MS. There is a possibility that the same factor (Karcher et al., 1977; Karcher, 1979) influences the activity of the suppressor T lymphocytes and the antigenantibody reaction. Thus, in many neurological diseases including MS, CSF IgC studies show an immunological dysregulation. This could be a fundamental observation for the understanding of MS physiopathology.

IV. Nervous Tissue-Specific Proteins

One of the first questions concerns the specificity of the nervous tissue proteins. In some cases nervous tissue-specific proteins or related proteins were found in other structures (Hullin et al., 1980; Boston et al., 1982). It can be thought that the specificity is nothing other than quantitative differences in the distribution of these proteins. Antibodies against these proteins are found elsehwere than in CSF. T h e methods required for studies of nervous tissue-specific proteins are still difficult to carry out. A. S-100

S- 100 was the first nervous tissue-specific protein to be described (Moore, 1965). T h e name is derived from the solubility of the protein in 100% ammonium sulfate at neutral pH. The function of the protein is still unknown. Studies proved that the protein has multiple forms that are antigenically related. The cellular localization of S- 100 has led to a certain confusion possibly caused by the multiple forms of the protein. Eng and Bigbee (1977) suggested that contradictory results in the distribution of S- 100 could be due to technical aspects of the immunohistological techniques. T h e solubility of S- 100 in tissue could produce leakage or diffusion, which is why perfusion is preferable to immersion for fixation. A glial localization was shown by Hyden and McEwen (1966), Cicero et al. (1970a), Hansson et al. (1975), and Ludwin et al. (1976). A neuronal localization was demonstrated by Hyden and McEwen (1966), Haglid et al. (1974), and Donato et al. (1975). S-100 can be shown in the cytoplasm of glial cells and in neuronal membranes and glial nuclei. Perhaps this is related to multiple forms of S- 100: T h e small part may be bound to the neuronal membranes and the larger to glial cytoplasm.

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After injuries to cells in the CNS one can expect to find these specific intracellular markers in the extracellular space and later in the CSF. This indicates that the determination of S-100 and other CNS cell markers in the CSF could be useful in clinical neurology. Sensitive methods are necessary as low levels are to be expected in the CSF. Immunological methods would certainly be recommended. S-100 is a protein with a low antigenicity. Antibodies are prepared in general after binding S- 100 to methylated bovine serum albumin (Levine and Moore, 1965). Four methods have been used preferentially to measure S-100: 1 . A microcomplement fixation test (Moore and Perez, 1966a) 2. Uozumi and Ryan (1973) used an RIA to determine S-100 in human brain. Because they did not succeed in preparing antihuman S-100, antibovine S-100 was used instead 3. A two-site immunoradiometric assay (IRMA) described by Miles et al. (1977) 4. An enzyme immunoassay described by Kato et al. (1982) The information about determinations of S-100 protein in CSF is rather scanty (Michetti et al., 1979, 1980; Kato et al., 1982). A first study was undertaken on MS patients (Michetti et al., 1979). Thirteen out of eighteen of these had detectable amounts of S-100 in the CSF. T h e levels of S100 are higher in the CSF of patients with acute exacerbation of MS. S100 was absent in the control group, 1 1 cases with slight psychiatric complaints o r neurological diseases without parenchyme damage. T h e same authors completed their study by examining the CSF of 93 patients, most of whom lacked S-100 (Michetti et al., 1980). The method used was a microcomplement fixation test and the sensitivity was 6 ng/ ml. The assay range varied from 6 to 29 ng/ml. The patients were of both sexes and the age range was 7-66 years. Nine patients with psychic complaints were negative as were six patients with epilepsy. Low concentrations of S-100 were found in patients with diseases of the peripheral nervous system. S-100 was not found in the only Parkinson case nor in the two patients with dementias. One out of three cases with cerebellar bloodflow was positive. Four cases with cerebellar degeneration were negative. The antigen was found in three out of five cases with amyotrophic lateral sclerosis and in all patients with acute encephalomyelitis (Ludwin et al., 1976). There was a significant presence of S-100 in the CSF of 14 out of 17 patients in the acute stage of MS. Nine clinically controlled patients had limited levels of S-100. Six patients from a group of 15 with spinal compression were positive for S-100. Out of eight patients with intracranial tumors (two meningiomas, three gliomas,

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three metastases of nonneuronal origin), only the two meningiomas were positive. From this study one can conclude that S-100 is absent in the CSF of psychiatric patients and patients without a disease of the CNS. The absence of S-100 in the CSF of patients with a disease of the peripheral nervous system can be understood on anatomical grounds. S-100 can be a very good index for cell damage because of its low normal concentration and/or its absence in slow-evolving degenerative processes. It appears in measurable amounts, however, in the CSF of patients with fastevolving processes. T h e group of Kato et al. (1982) used an enzyme-linked immunosorbent assay (ELISA). T h e sensitivity of the method is more satisfactory: 60 pg/ml. T h e amount of CSF needed was less than 100 pliters, but the number of patients tested (10) was rather small. Patients with glioblastoma and hydrocephalus had high levels of S-100. Also notable was the rather good correlation between S- 100 and the neuron-specific enolase levels. Miles et al. (1977) used a two-site IRMA with a high sensitivity (0.1 ng/ml), but results of the determinations in CSF were not discussed. T h e impression one gets from a survey of the literature is that methodological problems predominate, and at this moment no conclusions can be drawn. B. MYELINBASICPROTEIN (MBP)-ANTIBODIES TO MBP

MBP is directly related to myelin. It is one of the major proteins in the myelin with a molecular weight of 18,500. It is the causative agent for EAE. The study of MBP in human neurological diseases has been stimulated by the following observations. 1. Chronic relapsing EAE in guinea pigs progresses with periodic remissions and exacerbations of increasing severity and so resembles MS (Eylar, 1979), and thus appears to be an animal model for MS (Biber et al., 1981). 2. Both EAE and MS serum produce demyelinating effects in culture (Bornstein, 1972). The presence of MBP has been investigated in the CSF in neurological disorders, particularly in cases of demyelinating diseases such as MS (Gerson et al., 1981). T h e methods used for the determination of MBP in CSF are either RIA or IRMA (Nagelkerken et al., 1982). The RIA is

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carried out in competition with a labeled antibody using a second antibody or another technique to precipitate antigen-antibody complexes formed in the first antibody reaction. Immunoradiometric assay consists of an unlabeled antibody coupled to a matrix that reacts with the antigen from the biological fluid and subsequently with a second labeled antibody. Radioimmunoassay is a more sensitive method than IRMA for assaying MBP. T h e determination of antibodies to MBP in human CSF has been achieved using in the first incubation either beads coated with MBP (Ruutiainen et al., 1981), coated plastic tubes (Panitch et al., 1980), or immunoabsorbent (Melse et al., 1983) and for the second incubation either 1251-labeledantihuman IgG or labeled protein A (Linthicum et al., 1981). Myelin basic protein has been determined in the CSF of neurological patients, and this is an additional valuable test for MS although it is not specific. Most cases with detectable MBP are suffering from a disease affecting the myelin sheath. In MS during exacerbation the level of MBP measured in the CSF is markedly increased in the first 10 days. T h e MBP is released into the CSF during acute attacks and gradually returns to normal (Cohen et al., 1980; Biber et al., 1981). It is also found to be increased in retrobulbar neuritis, Guillain-Barrd, cerebrovascular diseases, metachromatic leucodystrophy, central pontine myelinolysis, neuro-Behcet’s disease (Ohta et aE., 1980). In neurosyphilis (Prange el al., 1980) MBP can serve as an indication of demyelinating activity even when the immunological tests are negative; demyelination resulting from irradiation, and chemotherapy can be detected using MBP determination. T h e quantitative measurements of MBP levels in CSF could serve as a parameter to follow the rate of healing after neurosurgery (Alling et aL, 1980). A follow-up after surgery shows that the MBP values return progressively to normal. The MBP in CSF is associated with destruction or breakdown of nervous tissue. As reported by Jacque et al. (1982), MBP increases in encephalitis and returns to normal levels after 3 weeks. According to Kohlschutter (1978) the presence of MBP in the CSF of newborns and children is associated with severe brain damage. The level of MBP depends on the location of the demyelinating activity, on the CSF circulation, and on individual conditions. A slowly progressive compression does not increase MBP level in CSF; an increase of MBP is found in malignant gliomas and medulloblastomas. The localization of these tumors is an important use of the detection of MBP in CSF. In MS the level of MBP in CSF parallels the clinical activity and might be a tool to follow the activity of MS. Antibodies to MBP are readily measured in the CSF of patients affected with MS or SSPE (Panitch et al., 1980; Ruutiainen et al., 1981). I n

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SSPE and MS CSF, with identical IgG concentration, the activity of the antibodies to MBP was increased compared to control. In MS the appearance of antiMBP parallels the observations made for the antigen MBP: T h e highest values are found in patients with acute exacerbation and the lowest in patients in remission. T h e determination of MBP and MBP antibodies in the CSF may be a useful technique to establish whether the active demyelination process is taking place in the CNS. In experimental allergic neuritis (EAN) and EAE antibodies against MBP or similar proteins (PI, P2) were found (Zweiman et al., 1982; Melse et al., 1983). With regard to MBP technical development has clearly brought us further than in the case of S- 100.

C. a-ALBUMIN (GFAP, ASTROPROTEIN)-ANTIBODIES TO a-ALBUMIN A water-soluble acidic protein was extracted for the first time in 1962 from human spinal cord (Van Sande, 1962). It was named a-albumin because it migrated between a-globulin and albumin after agar gel electrophoresis. T h e presence of a-albumin was detected in soluble extracts of normal and pathological nervous tissues in man and animal (Adriaenssens et al., 1968; Karcher et al., 1969; Chamoles et al., 1970; Zeman et al., 1970). High levels were found in edematous tissue and Tay-Sachs megalencephaly (Karcher et al., 197213). a-Albumin and GFAP were shown to be closely related and to share common immunity (Gheuens et al., 1978, 1979). After a trauma reactive astrocytes could be stained with anti-a-albumin serum using immunohistochemical methods (Duchesne et al., 1979). Glial fibrillary acidic protein was isolated for the first time in 1971 from human brain tissue rich in fibrillary gliosis, particularly MS plaques. It is the best known nervous tissue-specific protein (Eng et al., 1971). It is an acidic protein, consisting of one to seven peptides with a molecular weight of 40,500 to 54,000 and cannot be found outside the CNS (Uyeda et al., 1972). It was demonstrated, using immunofluorescence (Bignami et al., 1973) and immunoperoxidase techniques (Ludwin et al., 1976; Mgller et al., 1977), that fibrillary astrocytes react more than the protoplasmic ones and that reactive astrocytes are positive for GFAP after trauma (Bignami and Dahl, 1976). It is now generally accepted that GFAP is essentially part of the intermediary 910 nm fibers of fibrillary astrocytes (Yen et al., 1976; Shelanski et al., 1976). Recent studies suggest that GFAP acts in glial structural differentiation and the formation of processes (Duffy, 1982). Many samples of normal and pathological nervous tissue were stud-

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ied to localize GFAP in man and animal (Dahl and Bignami, 1975; Dahl, 1976). It was shown that GFAP increased in tumors of astrocytic origin (Dittman et al., 1977) and is inversely proportioned with the degree of malignity (Jacque et al., 1979). The relation between GFAP and the neurofilament protein of peripheral nerves has been discussed (Dahl and Bignami, 1976), and later the nonidentity of these two antigens has been established (Bignami and Dahl, 1977; Schachner et al., 1978). T h e term astrop-otein was first used in 1970 (Benda et al., 1970), although in 1966 it was seen in the brain of patients affected with TaySachs or glioma (Bogoch and Belval, 1966). It has been shown to be specific for glial cells. (Mori et al., 1968; Benda et al., 1970; Mori, 1970; Mori and Morimoto, 1973). Astroprotein and GFAP were demonstrated to be biochemically related and share immunological characteristics (Mori and Morimoto, 1975).a-Albumin, GFAP, and astroprotein are identical markers for glial cells, except for minor differences. a-Albumin, astroprotein, and GFAP were determined in the CSF by means of immunological methods: immunodiffusion and electrophoresis (Uyeda et al., 1972), rocket immunoelectrophoresis (Dittman et al., 1977; Jacque et al., 1978), RIA (Mori et al., 1978; Palfreyman et al., 1979; Ruutiainen et al., 1981), and two-site IRMA (Eng et al., 1976; Noppe et al., 1979). Radioimmunoassay and IRMA were compared with regard to their specificity, sensitivity, reproducibility, and efficiency (Noppe, 1980). They are both equally specific and sensitive, but the efficiency of the RIA is limited by radiolysis and proteolysis. The two-site IRMA with coated tubes is difficult to reproduce because antibodies raised against the antigen in two different species are needed. The intra-assay variance (15%) is not considered satisfactory. The two-site IRMA method using CNBr cellulose-coupled antibodies is easy to reproduce, the immunoabsorbent-antibody complex can be stored for a long period of time (15 months), and the intra- and interassay variances are 5 and 15%, respectively, and thus satisfactory. The calibration curve is established for values ranging from 0 to 5.000 ng/ml. Using CSF the astroprotein content of 60 cases of brain tumors (Mori et al., 1978) was compared with 21 samples of tumor-free neurological patients. The normal values for astroprotein were lower than 25 ng/ml. In 58.8% of patients affected with gliomas, the values were increased, as was also the case in other brain tumors (23.3%) and in four patients with congenital hydrocephalus, epilepsy, and cryptococcus meningitis. In 47 patients with intracranial bleeding the values increased compared to those with subarachnoid bleeding (Hayakawa et al., 1979).The astroprotein level reflects the degree of the lesion and allows the clinician to foresee their evolution.

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TABLE I1 CORRELATION BETWEEN

CSF

P R o T E I N PATTERNS AFTER

AGARG E L

ELECTROPHORESIS AND CSF GFAP LEVELS CSF protein electrophoretic pattern

Normal Increased al-globulin Fast migrating cY-globulins Serum-like y-globulins with restricted heterogeneity

Number of cases 489 97 210 285 132

Percent of pathological GFAP levels (10 ng/ml) 8

29 18

17 22

a-Albumin measurements were carried out in a first study of 649 samples (Lowenthal et al., 197’8)and in a second study of 1090 samples (Crols et al., 1982). The values found ranged from 0 to 1.479 ng/ml and a-albumin was seen in almost all samples. The normal values calculated for a control group of 130 patients with no pathology of the CNS were below 10 ng/ml. T h e level of a-albumin in the CSF was not related to sex. It had a tendency to increase with age. The values were significantly lower in samples with normal agar gel electropherograms than in those with abnormal pherograms. In contrast to the first study, in the second study no statistically significant differences could be found between the different abnormal electrophoretic patterns individually (Table 11), although the highest proportion of a-albumin increases were found in the group with fast-migrating a-globulin. High values were seen in cases of encephalitis and astrocytic tumors and somewhat lower values in cases of bacterial meningitis and syringomyelia. In encephalitis the return to normal value is reached within 3 weeks. a-Albumin was increased in patients with astrocytoma with low a-albumin content and high malignancy (Klaes et al., 1982). It seems probable, although additional studies are needed, that a-albumin or GFAP determinations can fill a role as specific markers in astrocytic pathology. Autoantibodies against a-albumin were found in serum of neurological patients affected with polyneuritis (Melse el al., 1983).

D. ENOLASE Enolase is not only a nervous tissue specific protein but also an enzyme, also known as 14-3-2 protein. It can be asked whether, in the case

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of enolase even more than for any other CNS-specific protein, how far specificity should be considered as a qualitative or a quantitative criterion. This specific protein was isolated for the first time by Moore and Perez (1966b). T h e designation 14-3-2 was given on the basis of its elution pattern during ion exchange chromatography. The neuron specificity was revealed by Cicero et al. (1970a,b) and Perez et al. (1970). The protein has an enzymatic (enolase) activity as shown by Bock and Dissing (1975). Further studies demonstrated that the nervous system contains three forms of this protein (Bock and Dissing, 1975; Fletcher et al., 1976; Marangos et al., 1976). The less acidic form was found to be identical to liver enolase, which was previously known as a dimer of two a subunits. The most acidic form is the brain specific enolase, called the a-a form (Fletcher et al., 1976).The subunit structure is unique and only found in neurons and neuroendocrine cells. The third form is an isoenzyme: It is a hybrid a-y form (Zamzely-Neurath and Keller, 1977). This form probably originates during the purification procedures. Three kinds of neurons are recognized by their characteristic enolase patterns (Cimino et al., 1977): (1) those that contain 14-3-2 very early during their development, and keep it; (2) those that never contain 14-3-2 (e.g., motor cortical neurons); and (3) those that contain 14-3-2 during development (e.g., Purkinje cells). Different determination methods have been described: a solid-phase RIA (Marangos et al., 1975; Revoltella et al., 1976; Hullin et al., 1980), a microcomplement fixation test (Kolber et al., 1974), and a microquantitative precipitation test (Minden and Farr, 1967). Determinations of this neuron-specific protein in CSF and serum were carried out by different authors. Brown et al. (1980) applied a RIA to CSF and serum. The lower limit of sensitivity for the RIA was 150 pglml. Of the 64 CSF samples taken at random, 76% had values between 6 and 14 ng/ml. These authors could demonstrate that the standard used and the enolase in CSF were immunologically the same. The clinical data of the patients were not discussed. In serum the enolase values are around 5 ng/ml. Kato et al. (1982) used an ELISA similar to that for S-100 determinations. The result of S- 100 determinations are neuron-specific enolase correlated very well ( r = .74). High S-100 concentrations are often correlated with high 14-3-2 concentrations. Royds et al. (1981) published a study on CSF enzymes and their significance in pathology. The enzymes measured were enolase, aldolase, pyruvate kinase, lactate dehydrogenase, and creatine phosphokinase. Enolase was the most sensitive marker for pathological changes and the only enzyme increased in CSF of low-grade astrocytonia. One

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hundred and twenty-one patients were examined. Nonspecific increases in pathological cases such as three leukemias, one case of Hodgkin's disease, and patients with active malignant tumors are mentioned. T h e group with low-grade astrocytomas had increased enolase and other enzyme values were rather low. Two-thirds of the patients with increased enolase seem to have low-grade astrocytomas. T h e highest values of enolase were found in CSF of patients with fast-developing tumors. Enolase measurements are a good marker for active demyelinization. T h e study emphasizes the importance of quantitative determinations of enolase in CSF. A distinction should be made among the three different kinds of enolase. Parma et al. (1981) discuss a RIA which permits measurements in CSF.

E. a2-GLYCOPROTEIN (14-3-3) 1. a2-Glycoprotein a2-Glycoprotein was described in 1967 (Warecka and Bauer, 1967), and its specificity for glial cells was shown (Vogel, 1972). Any relation with GFAP, a-albumin, and astroprotein seems improbable because of its neuraminic acid content. This antigen is found in higher concentrations in astrocytomas (Warecka, 1975). a2-Glycoprotein was determined in 85 samples of CSF from MS patients compared to 65 other neurological cases (Leonhardt et al., 1976). A correlation between total CSF protein and ap-glycoprotein was shown.

2. 14-3-3 14-3-3, which is a bad immunogen, was determined by Boston et al. (1982). In 10 serum samples in which the mean value was 5 ng/ml, a range of 18.5 to 185 was found. A niean value o f 4 0 ng/ml was found in CSF. Eighty-two patients were examined. ?l'he values for 14-3-3 varied between 5 and 130 ng/ml. T h e highest values were found in cases of tubercular meningitis and in inflammatory processes. 14-3-3 is a relatively unknown nervous tissue-specific protein that is not yet histologically localized. V. Enzymatic Determinations

Enzymatic determinations have always been the weak point in the biochemical study of the human CSF, although hundreds of publications have been devoted to this problem. We gathered more than 600 refer-

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ences after the publication of the two reviews devoted to this subject (Lowenthal, 1968, 1972). Two essential methodological problems have so far limited progress in this field. 1. It is difficult to repeat lumbar puncture in order to obtain new

CSF samples for a follow-up of enzymatic activities.

2. The methods used for enzymatic measurements in CSF are in fact methods that were described for serum and adapted to CSF. These methods do not always take into account the specific biochemical characteristics of the CSF, among others the pH, metabolites concentrations, etc. CSF enzymatic activities should be determined with specific methods. T h e results should always be compared with the total protein content and the amount of CSF cells and expressed in accordance with these two criteria. If the total protein content and particularly the number of CSF cells increase, the enzymatic activities also increase, and the relative increase of these two parameters can be considerable. TABLE 111 CLINICAL SIGNIFICANCE OF

INCREASES OF

Enzyme Adenylate kinase Aldolase and isoenzymes Creatine phosphokinase and isoenzymes Dopamine P-hydroxylase Enolase Esterases (mainly cholinesterase) P-glycuronidase and isoenzymes Lactate dehydrogenase and isoenzymes Lysosomial enzymes Muraminidase (lysozyme) Peptidase Proteolytic enzymes Transaminase

ENZYMATICACTIVITIES I N HUMAN CSF Disease CNS organic lesions"-discrete vascular lesions Meningitis, vascular lesions Organic lesions of the CNS and the meninges Psychiatric diseases Astrocytoma, demyelination Diseases of the myelin, tumors, meningitis Tumors, meningitis, migraines Organic lesions of the CNS and the meninges Amyotrophic lateral sclerosis Organic lesions of the CNS and the nieninges Organic lesions of the CNS and the meninges MS Organic lesicins of the CNS and the meninges

Organic lesions include vascular, tumoral, recent epileptic fits.

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For most measurements the literature mentions creatine phosphokinase, lactate dehydrogenase, and dopamine-P-hydroxylase. Other enzyme activities are discussed, such as adenylate kinase, adenosine deaminase, esterases, hydrolases, and lysozyme. In the literature interest in transaminases has decreased. Table 111 lists some of the 33 enzymes determined in human CSF. This table demonstrates the lack of specificity in the changes of enzyme activity. Based on total enzymatic measurements or isoenzymatic determinations, we find in the literature attempts to differentiate the inflammatory and the noninflammatory, the viral and bacterial processes, and the benign and malignant tumors. The results seem not to be very specific and do not contribute much to diagnosis. Some attempts to identify minimal brain damage due to surgery by adenylate kinase determinations were published (Ronquist and Frithz, 1982) but are still controversial. It has been mentioned already that enolase determination is the most sensitive method for diagnosis of pathological events in the CSF. T h e methods used for these determinations are perhaps the only enzyme determinations that are specific to the CSF.

VI. Conclusions

In conclusion, we can state that there should be a better correlation between fundamental and clinical research in the field of CSF proteins. Specific methods for the study of CSF proteins and enzymes should be developed, and one should not be satisfied with the methods which are used to measure serum proteins. One should take into account the fact that the same methods are not always used for human and animal CSF. An attempt should be made to reconcile these techniques. At the moment the total protein determinations and the IgG restricted heterogeneity are of great importance in routine clinical measurements. It is most probable that, in the near future, the study of specific proteins of the central nervous system will find its place in clinical neurology. However, we remain skeptical about the use of enzymatic determinations. Numerous physiopathological questions such as the origin of some proteins or the significance of the humoral or cellular immunological dysregulation seen in certain neurological diseases such as MS remain without a definite answer and, looking at the present literature, one encounters methodological problems that make the interpretation of the results obtained by many authors rather difficult.

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Acknowledgments

We wish to thank Mrs. S. Hansen, L. Van de Noort, and L. Vanhove for secretarial assistance, and Mrs. G. Seeldraeyers and Mr. J. Caers for skillful technical assistance. We are grateful for financial support from the Born-Bunge Foundation, Ministerie van Onderwijs, Nationaal Fonds voor Geneeskundig, Wetenschappeliljk Onderzoek, and the Universitaire Instelling Antwerpen.

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1121. Raine, C. S., and Stone, S. H. (1977).J. Med. (Westbury, N.Y.) 77, 1693-1696. Reunanen, M., Salmi, A., Ilonen, J., and Herva, E. (1980).Acta Neural. Scand. 62,293-299. Revoltella, R., Bertolini, L., Diamond, L., Vigneti, E., and Grasso, A. (1976).J. Neurachem. 26,831-834. Rice, G. P. A., Armstrong, H., and Ebers, G. C. (1982). Neurology 32, 893-894. Roberts-Thompson, P. J., Esiri, M. M., Young, A. C., and Maclennan, I. C. M. (1976).J. Clin. Pathol. 29, 1105-1 115. Rocchelli, B., Poloni, M., Mazzarello, P., and Delodovici, M. (1981).J. Neurol. 226, 169179. Rocchelli, B., Mazzarello, P., Poloni, M., and Pinelli, P. (1982). Neurosci. Lett. (Suppl. lo), S418. Ronquist, G., and Frithz, G. (1982). Eur. Neural. 21, 318-323. Rostrom, B., and Link, H. (1981). Neurology 31, 590-596. Rostrom, B., Link, H., and Norrby, E. (1981a). Actu Neural. Scund. 64, 225-240. Rostrom, B., Link, H., Laurenzi, M. A., Kam-Hansen, S., Norrby, E., and Wahren, B. (1981b). Ann. Neurol. 9, 569-574. Royds, J. A,, Timperley, W. R., and Taylor, C. B. (198l).J. Neural. Neurosurg. Psychiatry 44, 1129-1 135. Ruutiainen, J., Newcombe, J., Salmi, A,, Dahl, D., and Frey, H. (1981).Actu Neural. Scand. 63,297-305. Ryberg, B., and Kronvall, G. (1981). Eur. Neurol. 20, 374-379. Salmi, A., Ziola, B., Reunanen, M., Sulkunen, I., and Wager, 0. (1982).Acta Neurol. Scand. 66, 1-15. Sandberg-Wollheim, M. (1974). Scand. J . Immunol. 3, 717-730. Sandberg-Wollheim, M., and Turesson, I. (1975). Scand. J . Immunol. 4, 831-836. Santoli, D., Moretta, L., and Lisak, R. P. (1978).J. Immunol. 120, 1369. Schachner, M., Smith, C., and Schoonmaker, G. (1978). Dev. Neurosci. 1, 1-14. Scheiffarth, F., Teuber, J., Druschky, F., and Baenhler, H. W. (1977).Klin. Wachenschr.55, 903-906. Schliep, G., and Felgenhauer, K. (1978).J. Clin. Chem. Clin. Biochem. 16, 631-635. Shelanski, M. L., Yen, S. H . . and Lee, V . M. (1976). I n “Cell Mobility” (R. Goldman, ‘I.Pollard. and J . Rosenbduni. eds.), pp. 1007- 1020. Cold Spring Harbor Lab., Cold Spring Harbor, New York. SidCn, A. (1979). I n “Humoral Immunity in Neurologic-al Diseases” (D. Karrher, A. Lowenthal, and A. D. Strosberg, eds.), pp. 201-210. Plenum, New York. SidCn, A,, and Kjellin, K . G. (l977).,/. Neural. 216, 251-264. Siemes, H., Siegert, M., Jaroffke, B., and Hanefeld, F. (1981). Eur.J. Pediatr. 137, 49-58. Sindic, C. J. M., Cambiaso, C. L., DeprC, A,, Laterre, E. C., and Masson, P. L. (1982).J. Neurol. Sci. 55, 339-350. Stibler, H. (1977).J. Neural. Sci. 32, 331-336.

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MUSCARINIC RECEPTORS IN THE CENTRAL NERVOUS SYSTEM By Mordechai Sokolovsky Department of Biochemistry G o r g e 5. Wire Faculty of life Sciences Tel Aviv University TeI Aviv. Israel

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Direct Receptor Characterization: Radioligand-Binding Studies. . . . . . . . . 111. Binding of Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... A. Kinetic and Equilibrium Measurements . . . . . . . . . . . . . . . . . . . . . . . . .

B. Cooperative and/or Site Interactions . . . . . . . . . . . . . .............................. IV. Binding of Agonists. . . . . . . . . . V. Receptor-Receptor Interactions . . . . . . . . . . . . . . . . . . . . VI. Localization of Muscarinic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Regional distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Post- and Presynaptic Receptors. .............................. C. Ontogenesis................................................ VIJ. Solubilization and Isolation of the Muscarinic Receptors .............. VIII. Structure and Function Relationship of Muscarinic Receptors. . . . . . . . . . IX. Concluding Remarks ........................................... References ...................................................

139 143 147 147 152 156 161 167 167 168 169 171 174 177 178

I. Introduction

Drug receptors are those complex molecular entities in or on cells through which a neurotransmitter or a hormone acts. These receptors perform two vital functions. First, they recognize and discriminate, by a process of high-affinity specific binding, particular biologically active structures and thus permit them to initiate the cascade of biological reactions that ultimately leads to a biochemical or physiological response. This specific binding function is accomplished by virtue of some complementarity in the structure of the neurotransmitter, hormone, or drug and the receptor protein. The second function of receptors is the transduction of a signal, presumably generated by virtue of the binding interaction with the drug. This signal must be conveyed to some appropriate effector, such as an ion channel or enzyme, and thereby alter its activity in a way that leads to the requisite physiological response. Thus, for instance, the P-adrenergic receptor is coupled to adenylate cyclase I39 INTERNAI'IONAL REVIEW OF NEUROBIOLOGY, VOL. 25

Copyright a 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-366825-5

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using a guanine nucleotide regulatory protein as the coupling device (Lefkowitz et al., 1978), the nicotinic receptor regulates permeability of a specific ion channel (Heidmann and Changuex, 1938), and the insulin receptor regulates the transport system for glucose and amino acids (Steiner, 1977). In the past decade cholinergic neurotransmission has received considerable scientific attention. Elucidation of the role of cholinergic systems in neurological and psychiatric disease states has provided impetus for understanding acetylcholine transmission in normal brain functioning. Moreover it has recently become increasingly clear that the cholinergic system might be an important central nervous system (CNS) pituitary modulator of hormone release. Cholinergic agonists and antagonists are now routinely used in the clinical treatment of various diseases. These pharmacological agents provide major tools for experimental approaches to the studies of receptor-ligand interaction. Although agents that alter the synthesis, release, reuptake, o r catabolism of acetylcholine are useful both therapeutically and experimentally, drugs that act directly on the cholinergic receptor as agonists and antagonists have proved most useful in delineating the biochemical, electrophysiological, and behavioral functioning of the cholinergic system. Receptors for acetylcholine (ACh) can be divided into two broad categories, muscarinic and nicotinic. The distinction between these receptors was first made on empirical pharmacological grounds: Certain responses to ACh, such as the changes in endplate potential of skeletal muscle, were mimicked by nicotine and blocked by curare, whereas other responses, such as the inhibitory effect of ACh on the heartbeat, were mimicked by muscarine and blocked by atropine.(See Figs. 1 and 2 for the structure of several muscarinic agonists and antagonists, respectively.) It

0

II CHj-C

-0

-CHI

-CH2

-NICH&

Acetylcholine

"'c\fO\

-CH2

-NlCH&

II H2N-C

Muscarha

CH3

-0 -CH2

-CCHZ-NICH~)~

I

Carbsmylcholine

/

-CH2

-

fi

H6c2

FIG. 1. Structure of scveral muscarinic agonists.

Pilocarpine

MUSCARINIC RECEPTORS IN THE CENTRAL NERVOUS SYSTEM

141

has, however, become clear that muscarinic and nicotinic receptors differ in more interesting ways than simply their affinity for agonists and antagonists. The molecular mechanisms coupling these receptors to their physiological responses appear to be fundamentally different. Muscarinic responses differ from nicotinic ones in having, for example, a much longer latency (- 100 msec compared to 1 msec) and a much longer response (-0.5 sec or longer compared to 30 to 100 msec). As reviewed by Hartzell(1982), muscarinic receptors seen1 to be coupled to several different types of ionic channels in the plasma membrane by a process that probably involves a series of three or more steps. I n addition to being coupled to conventional drug-activated ionic channels that are not affected by membrane potential, muscarinic receptors are capable of modulating the opening and closing of channels that are gated by changes in transniembrane potential. Activation of muscarinic receptors also produces a variety of metabolic responses in postsynaptic cells that may be important in mediating long-term signaling between neurones and their targets. The physiological significance of muscarinic receptors in the parasympathetic nervous system has been well documented (Pepeu and Ladinsky, 1981). Pupillary contraction, the stimulation of sweat, gastric, and salivary glands, and complex cardiovascular events are among the most marked effects of muscarinic agonists. With increasingly sensitive techniques of receptor measurement, muscarinic receptors have been detected in other cells and tissues such a the retina (Hruska et al., 1978), anterior pituitary (Vale et al., 1976; Avissar et al., 1981a), epithelial cells (Rimele et al., 1981), red blood cells (Aronstam et al., 1977b), lymphocytes (Gordon et al., 1978; Shapiro and Storm, 1980; Zalcman et al., 1981), and oocytes (Kusano et al., (1977). Although their physiological significance in these cells is not completely understood, these receptors are functional in the sense that they can trigger a response induced by muscarinic agonists.

FIG. 2. Structure of several muscarinic antagonists.

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MORDECHAI SOKOLOVSKY

I n the central nervous system most of the cholinergic receptors are of the muscarinic type (Krnjevic and Phillis, 1963). Typical muscarinic activity has been found in the cerebral cortex, hippocampus, basal ganglia, cerebellum, medulla-pons, and hypothalamus. In the cerebral cortex the muscarinic activity is mainly excitatory, whereas in the hypothalamus and medulla-pons it is inhibitory (Krnjevic, 1974). From the studies cited above a picture has emerged of the distribution of the muscarinic receptors, their possible physiological significance, and some of the basic features of drug-receptor interactions. In principle drug-receptor interactions are dose-dependent phenomena analogous to enzyme-substrate interactions. The magnitude of the tissue response is usually a nonlinear function of the drug concentration and reaches a finite maximal value (Michaelson and Zeimal, 1973). Accordingly, it has been postulated that the drug interacts with a finite number of receptors and that the response is a function of the degree of receptor occupancy, which in turn is a function of the affinity of the drug for its receptor (Michaelson and Zeimal, 1973). It should be noted that when these assumptions were originally made (Clark, 1926) investigators could not yet demonstrate the existence, quantity, or nature of receptor molecules. Nevertheless, dose-response measurements, in spite of their limitations, have proved to be useful tools in muscarinic receptor research. They have, for example, permitted an estimation of the potencies of muscarinic agonists and antagonists (Triggle, 1971). Research on structure-activity relationships has yielded a variety of potent drugs, some of which are of therapeutic value and others, which are strong poisons, and can be used in research (KoeIle, 1975). As early as the 1960s the benzilic ester derivatives (Fig. 2) were designed, synthetized, and characterized as potent muscarinic antagonists (Abood and Biel, 1962). Drugs of this group, such a& benziloylcholine (Gill and Rang, 1966), quinuclidinyl benzilate (Yamamura and Snyder, 1974a), and N-methyl-Cpiperidyl benzilate (Kloog and Sokolovsky, 1977), subsequently became useful tools in muscarinic receptor research. Of special note are the quaternary nitrogen group and the ester bond (or triple bond in analogs), which are important for muscarinic activity (Michaelson and Zeimal, 1973). Also of interest is the molecular flexibility of muscarinic agonists in contrast with the rigidity and hydrophobicity of muscarinic antagonists (Beckett et al., 1971; Meyerhoffer, 1972); it has been suggested that these characteristics are important in determining agonistic versus antagonistic activity (Weinstein et al., 1975). Other important features of drugs interacting with muscarinic receptors are their stereospecificity and their conformation (Waser, 1961 ; Abood and Biel, 1962; Chotia, 1970).

MUSCARINIC RECEPTORS I N THE CENTRAL NERVOUS SYSTEM

143

Dose-response curves have yielded other important data as well and from these have emerged some of our most fundamental notions about receptor structure and function. These include the concepts of competitive and noncompetitive antagonism (Arunlakshana and Schild, 1959), desensitization (Katz and Thesleff, 1957), and the theory of “spare” receptors, which derived from comparison of dose-response w-ith ligand binding (Stephenson, 1956). We now know that affinity constants for muscarinic antagonists determined by competitive antagonism measurements are almost identical with those measured by direct binding assays (see Hulme et al., 1978). There are obvious limitations to using indirect measurements to determine receptor function. In order to demonstrate that the receptor molecule exists, it was essential to determine its nature (protein, lipid, glycoprotein, etc.) and structure by direct measurement. Accordingly, radiolabeled drug-binding assays were developed (Yamamura and Snyder, 1974a). The use of potent “-labeled antagonists, which bind specifically and with high affinity to the muscarinic receptors, as well as the development of sensitive assays for measuring biochemical signals coupled to receptors have greatly facilitated such studies. This article (which covers literature up to May 1982) will focus on data accumulated over the past 3-4 years on the biochemical characterization of muscarinic receptors in the CNS. The reader is referred to other useful reviews and books on muscarinic mechanisms for information on related topics not discussed here, for example, biochemical signals coupled to muscarinic receptors, desensitization, and degradation of the receptors (Triggle and Triggle, 1976; Heilbronn and Bartfai, 1978; Birdsall, 1981; Pepeu and Ladinsky, 1981; Sokolovsky at al., 1983). II. Direct Receptor Characterization: Radioligand-Binding Studies

Early pharmacological studies indicated that muscarinic antagonists would bind to their receptors with high affinity. It is thus not surprising that 3H-labeled atropine was the first ligand chosen for the specific labeling of muscarinic receptors, in a study carried out on guinea pig ileum (Paton and Rang, 1965). Since then 3H-labeled antagonists such as benzetimide (Beld and Ariens, 1974), scopolamine (Kloog and Sokolovsky, 1978b), and certain esters of benzilic acid-benziloylcholine (Gill and Rang, 1966), 3-quinuclidinyl benzilate (QNB) (Yamamura and Snyder, 1974a), and N-methyl-4-piperidyl benzilate (4NMPB) (Kloog and Soko-

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MORDECHAI SOKOLOVSKY

lovsky, 1977)-have been used as well (Fig. 2). These drugs were labeled to high specific activities, permitting the detection of minute quantities of receptors. All of the 3H-labeled muscarinic antagonists have been found to bind to tissues and tissue homogenates. The reversibility of the binding and the apparently simple binding isotherms (Yamamura and Snyder, 1974a; Kloog and Sokolovsky, 1977, 1978a,b) suggest that the "-labeled antagonists bind to a finite number of receptor molecules. Muscarinic agonists and antagonists inhibited the binding of these tritiated antagonists, whereas drugs without muscarinic activity did not (Yamamura and Synder, 1974a; Bartfai et al., 1976; Birdsall et al., 1978; Kloog and Sokolovsky, 1978b). These findings, as well as the high affinity of the ligands, which correlated well with their antimuscarinic potency, strongly suggested that they specifically bind to the muscarinic receptor as originally postulated. Binding studies with labeled muscarinic agonists and acetylcholine have been fraught with difficulties. It is only in recent years that receptor-specific binding of 'H-labeled oxotremorine-M (Birdsall et al., 1978), cis-methyldioxolane (Ehlert et al., 1980b), and pilocarpine (Hedlund and Bartfai, 1981) have been demonstrated. In studies of radioligand binding, it is of utmost importance to demonstrate that the binding being measured involves a physiological or pharmacological receptor. Because receptors are present in extremely small numbers and because radioligands can adhere to many membrane components, uptake sites, other irrelevant neurotransmitter receptors, and even inorganic material, considerable caution must be exercised in the interpretation of data. Radioligand-binding studies should therefore satisfy certain criteria to reduce the probability of a false positive receptor identification. We shall therefore briefly discuss some aspects of binding assay methodology. Ideally the ligand should remain unchanged (Lea,undegraded) during the binding process. This is in fact the case when muscarinic antagonists are used in experiments with tissue homogenates (Kloog and Sokolovsky, 1978b). The assay method required, then, is one in which bound and unbound "-labeled ligand can be separated. Equilibrium dialysis is one possibility, but it is time-consuming and thus suffers from the serious disadvantage that the receptors might degrade during the period required to reach equilibrium. Centrifugation and filtration, which involve washing of the membranes, are more suitable for receptor-binding assays; it is important to remember, however, that the system is actually not in equilibrium, because the free ligand is washed out. Nevertheless, these methods have been proven to be accurate enough because of the high affinities of mus-

MUSCARINIC RECEPTORS I N THE CENTRAL NERVOUS SYSTEM

145

carinic ligands and the rapid separation of free and bound ligands. In other words, off-rate kinetics are slow relative to speed of separation. Thus, in most cases one can assume that the amount of bound ligand remaining after centrifugation o r filtration does indeed reflect the equilibrium state present in the reaction mixture prior to the process. The amount of free ligand is then calculated by subtracting the measured quantity of bound ligand from the total ligand present. Binding sites in crude membrane preparations are usually heterogeneous. This is because the ligand binds to different membrane components that include the receptor (specific binding) as well as other components, usually of an unknown nature (nonspecific binding). T h e latter includes the “background” of unwashed free 3H-labeled ligand (e.g., the “dead volume” of the membrane pellet after centrifugation). Specific binding is defined as the binding that can be displaced by agonists and antagonists; hence nonspecific binding is the binding that cannot be displaced. If the assay procedure is valid, nonspecific binding is a linear function of ligand concentration because it is usually characterized by low affinity and high capacity. Experiments have shown that for a given antagonist the nonspecific binding in different tissues does not vary significantly. The ratio of specific to nonspecific binding would thus be determined primarily by the quantity of receptors present in the tissue (Kloog and Sokolovsky, 1978b). When the binding of different antagonists in the same tissue was compared, it was found that those possessing a quaternary nitrogen (i.e., N-methylscopolamine) showed less nonspecific binding than those with a tertiary nitrogen (i.e., scopolamine) (Hulme et a/., 1978). In general, reliable analysis of binding data requires that at least half of the total binding be specific at all ligand concentrations (see Section IV). Owing to the high affinity of muscarinic antagonists for their receptors and the high specific radioactivity, it is possible in most cases to work with fairly small quantities of tissue such that the nonspecific binding is negligible. For work with tissues in which the receptor content is low, quaternary drugs are preferred. It should be noted that if racemic mixtures of 3H-labeled ligands are used, the less active isomer contributes far more to the nonspecific than to the specific binding, hence lowering the ratio of specific to nonspecific binding. There are thus clear advantages in using the pure active isomer of the ligand (e.g., (-)-[3H]QNB or a symmetric ligand such as [3H]-4NMPB. Such ligands also minimize difficulties in kinetic measurements (Burgisser et a&., 1981). Binding is usually carried out by the use of filtration technique on the assumption that the ligand-receptor complex dissociates relatively slowly, but in the rat adenohypophysis the rapid dissociation rate of the

146

MORDECHAI SOKOLOVSKY

bound 3H-labeled ligands 4NMPB and Q N B precludes the use of the filtration method for studying the binding properties of muscarinic receptors (Avissar et al., 1981a). It also demonstrates that one cannot simply assume a link between slow dissociation and high affinity for binding sites without verifying it in each case and for every labeled ligand. This need for caution is heightened in the light of previous studies showing that these ligands dissociated relatively slowly from muscarinic receptor-ligand complexes prepared from several brain regions and therefore that their binding properties could be evaluated by the filtration method (Yamamura and Snyder, 1974a; Kloog and Sokolovsky, 1978b; Kloog et al., 1979a). Although the labeling of the receptors in uitro enables one to evaluate many characteristics of the receptor binding site, it is possible that important functional characteristics may be obscured. Thus, changes in the number of receptor sites under varying physiological and pharmacological conditions may occur during the time in which one sacrifices the animal and prepares brain homogenates for receptor assay. As evident from the comparison shown in Table I, the regional distribution of [3H]4NMPB after in uiuo administration is essentially the same as the binding capacities of 4NMPB assayed in uitro (Sokolovsky et al., 1980a,b; Avissar et al., 1981b). TABLE I REGIONALDISTRIBUTION (in vitro) AND ACCUMULATION (in vivo) OF SPECIFIC [YH]-4NMPBBINDING IN MOUSEAND RAT BRAIN^ Binding capacity (pmol/mg protein) Region Mouse Cerebellum Medulla-pons Hippocampus Basal ganglia Cortex Thalamus Rat Median hypothalamus Posterior hypothalamus Preoptic area Adenoh y poph ysis

In viva

In vitro

0.1 ? 0.05 0.4 2 0.06 0.9 2 0.1 1.8 -+ 0.1 0.9 2 0.1 -

0.12 t 0.01 0.29 2 0.05 1.05 2 0.5 1.54 2 0.38 0.91 f 0.06 0.61 2 0.01

0.04

0.35 k 0.05 0.52 t 0.03 0.39 2 0.01 0.17 2 0.01

0.4 2 0.5 0.4 0.17 ?

* 0.05

* 0.04 0.05

"Taken from Avissar et al. (1981a,b) and Sokolovsky et al. (1980a,b)

MUSCARINIC RECEPTORS IN THE CENTRAL NERVOUS SYSTEM

147

Finally, it should be emphasized that the labeling of a putative receptor must be demonstrated for a particular ligand under particular conditions in a particular brain region or tissue. If any of the particulars are changed, then the exclusivity of binding to the putative receptor must be rigorously reestablished. Thus, affinity constants determined for muscarinic antagonists, for example, can vary by factors of up to 300, depending on the ionic composition of the incubation medium (Birdsall et al., 1979c; Kloog et al., 1980a). T h e temperature dependence of the binding properties is much less pronounced in several brain regions. However, an unusual degree of temperature dependence was observed for agonist binding in the hypothalamus, especially in the preoptic area (Galron et al., 1981).

111. Binding of Antagonists

A. KINETICAND

EQUILIBRIUM

MEASUREMENTS

A number of ’H-labeled antagonists with high specific activities (590 Cilmmol) have been used to quantitate the muscarinic recognition sites (receptors) in the central and peripheral nervous systems. These include reversible ligands such as [“Hlatropine (Paton and Rang, 1965; Kloog and Sokolovsky, 1978a), [3H]-3-quinuclidinyl benzilate ([’HIQNB) (Yamamura and Snyder, 1974a), [3H]-N-methyl-4-piperidyl benzilate ([“H]-4NMPB) (Kloog and Sokolovsky, 1977), [3H]methylscopolamine (Hulme et al., 1978), [“H]scopolamine (Kloog and Sokolovsky, 1978b), and [ 3H]benzetimide (Beld and Ariens, 1974). Binding studies performed with these ligands indicated that the number of binding sites available in a given preparation is the same for all antagonists. In the early studies it was suggested that binding of antagonists at equilibrium follows a simple law of mass action (Paton and Rang, 1965; Beld and Ariens, 1974; Yamamura and Snyder, 1974a). T h e binding process was then tentatively described as follows: ki

R + L e R L

(1)

k-1

where R is the receptor, L is the ligand, and k l and k - l are the association and dissociation rate constants, respectively. Results soon indicated that this was clearly an oversimplification.

1. T h e equilibrium dissociation constant of the antagonist, as calculated from equilibrium binding data, did not coincide with that calcu-

148

MORDECHAI SOKOLOVSKY

lated from kinetic measurements (Kcp# k l l k l ) (Yamamura and Snyder, 1974b; Kloog and Sokolovsky, 1978a,b; Kloog et al., 1979a; Klein, 1980; Jones and Sumikawa, 1981). 2. Kinetic studies on the binding of [“]QNB and [‘H]-4NMPB indicated that a simple Langmuir isotherm, which describes binding of those antagonists under equilibrium conditions, may conceal a more complex reaction than a simple bimolecular association of the free receptor with the antagonist. Thus, the dissociation of antagonists from the muscarinic receptors proved to be a multiexponential process, which appeared to indicate that the antagonist could be dissociating from a heterogenous population of binding sites. These phenomena were observed for several antagonists (Galper et al., 197’7; Galper and Smith, 1978; Kloog and Sokolovsky, 1978a,b). 3. T h e observed on-rate constant for [3H]-4NMPB and [JH]QNB binding determined under pseudo-first order conditions was not a linear but a hyperbolic function of the antagonist concentration (Jarv et al., 1979). It was therefore suggested that the receptor may undergo a conformational change upon ligand binding, resulting in a higher-affinity receptor-ligand complex (isomerization) (Galper et al., 1977; Kloog and Sokolovsky, 1977; Kloog et al., 1979a,b; Jarv et ol., 1979, 1980), as follows:

Support for the proposed two-step isomerization model came from a number of equilibrium and kinetic studies of binding as discussed in the following paragraphs. Further support came from the finding that muscarinic antagonist binding was associated with large positive changes in entropy with a value for AS02r,y;of 13 to 40 entropy units (Cavey Pt al., 1977; Kloog and Sokolovsky, 1978a), frequently an indication of conformational changes in macromolecules. Note that the two-step model does not leave room for the preexistence of interconvertible binding sites of the kind R R* (cyclic model). The possibility of such interconversion has been proposed (Kloog and Sokolovsky, 1977) and is discussed in detail later. Nevertheless, both the sequential and the cyclic models give rise to Langmuir-type binding isotherms and are consequently indistinguishable from each other and from the simplest model, in which no isomerization is involved (Frost and Pearson, 1961). Such binding isotherms are indeed observed in most cases. However, as discussed in Sections III,2 and V, Langmuir binding isotherms yielding curvilinear Scatchard plots have also been

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observed (Burgermeister et al., 1978; Avissar et al., 1981a; Ben-Baruch et al., 1982); these reflect either heterogeneity of sites (two o r more classes of noninteracting sites) or negatively cooperative interactions. T h e following observations favored the isomerization model rather than the assumption of a heterogeneous population of muscarinic receptors. Dissociation from the receptor of various labeled antagonists did not vary as a function of receptor occupancy; if there are two independent sites, the dissociation should be nonexponential at low ligand concentration, in which case only the high-affinity sites are occupied, but this did not happen. In dissociation experiments measured by isotopic dilution technique (total ligdnd >> apparent Kd) the possibility of negative cooperativity was also excluded, because the actual occupancy of receptor sites was 100% during dissociation of 3H-labeled antagonist. According to a sequential model of isomerization, the dissociation rate would vary as a function of the preincubation time of receptor and ligand, and time-dependent changes in the muscarinic receptor-ligand complex were demonstrated (Galper et al., 1977; Kloog et al., 1980a; Sokolovsky et al., 1980a; Avissar et al., 198Ib). Further kinetic experiments using [ 3H]-4NMPB and [ 3H]QNB were then carried out, in which the kinetic parameters involved in the first (bimolecular binding) and the second (isomerization) steps were evaluated (Jarv et al., 1979, 1980; Kloog ut al., 1979,). T h e predicted pseudofirst order rate constant of association is a hyperbolic function of the concentration of the antagonist(s), indicating that the interaction involves two equilibria. The first binding equilibrium was reached rapidly; for [3H]QNB and [‘H14NMPB the respective dissociation constants were 2.7 0.4 nM and 6.7 f 2.5 nM in phosphate buffer (0.05 M, pH 7.4). The first binding equilibrium was followed by a slower step involving isomerization of the receptor-antagonist complex. For both ligands the equilibrium constant for this step was about 0.15 nM. T h e overall binding constant obtained as the product of the above constants is in good agreement with the results of equilibrium binding studies (Jarv et al., 1979). One may summarize the findings by stating that ligands that can convert the receptor into a slowly dissociating complex are good antagonists. Identification of the factors that influence the isomerization step may be important for the understanding of biochemical and physiological regulation of the receptor complex. For example, scopolamine binding is characterized by the formation of equal amounts of the two ligand-receptor complexes, whereas in the case of atropine the ratio between the two isomers is 80 : 20 (Kloog and Sokolovsky, 1978a,b).It is known that the behavioral potency of scopolamine is greater than that of

*

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MORDECHAI SOKOLOVSKY

atropine. Does this difference stem from the different populations of receptor-ligand complexes? If so, which is the “physiologically active” component? Does the presence of agonist or antagonist at the pre- or postsynaptic sites “freeze” a certain active conformation of the receptor? In recent years it was noticed that the binding properties of a given muscarinic ligand, as measured directly with 3H-labeled muscarinic antagonist, are not identical in all tissues. A number of ionic, hormonal, and cofactor effects on binding have been observed, and variations in binding parameters may be secondary to variations in levels of these agents among different preparations. There may also be differences in the ratios of various states of the receptor among tissues o r conditions. The simplest example is the dissociation rate of [3H]-4NMPB from muscarinic receptors in homogenates of two mouse brain regions, medullapons and cerebral cortex (Kloog et al., 1979a), in which the half-lives at 25°C of the complexes are -5 min and 20 min, respectively. These differences are also manifested at equilibrium: The apparent dissociation constant (KD)of [3H]-4NMPB is higher in the medulla-pons than in the cerebral cortex. The K D values for the various regions also seem to depend somewhat on the Aature of the ligand. Thus the KD values measured in cerebral cortex, basal ganglia, and hippocampus differ less from K D values in medulla-pons and cerebellum if the ligand used is a benzilate derivative (e.g., 4NMPB, QNB) than if the ligand is a tropate (e.g., atropine, scopolamine). At this stage one can still only speculate on whether this finding is directly related to differences in the large variety of pharmacological effects exhibited by these structural classes of drugs and whether the differences observed between them constitute a significant element in their modes of action. A case of special interest is the binding of the muscarinic antagonist pirenzepine, for which heterogeneity of binding affinities has been observed in different preparations (Hammer et al., 1980). This heterogeneity was interpreted in terms of different proportions of high- and low-affinity binding sites for pirenzepine in the various tissues studied. We have already mentioned that for any given tissue or brain region tested so far, the number of binding sites is the same for the various ’Hlabeled muscarinic antagonists, all of which mutually compete for these sites and can completely displace each other. The data would thus appear to support the concept of functional heterogeneity (Kloog et al., 1979a; Sokolovsky et al., 1980a). I n molecular terms the muscarinic recognition sites are identical whether they are in the cerebral cortex, cerebellum, or heart. Functional heterogeneity could then simply be a manifestation of preexisting interconvertible binding sites. In other words at a given moment the particular state of the binding protein(s) or polypep-

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tide(s) is dictated by the nature and constituents of the membrane milieu as well as by the properties of the binding ligand; it follows that under appropriate conditions the binding properties of the receptors may be altered. This has been indeed demonstrated in several studies: A decrease in the apparent affinity of muscarinic antagonists as a function of the ionic strength of the buffer was observed in several synaptosomal preparations including rat cerebral cortex (Birdsall et al., 1979c) and Torpedo electric organ (Kloog et al., 1980a). Alkylating and thiol reducing agents clearly changed the binding properties of muscarinic antagonists (Aronstam et al., 1978; Hedlund and Bartfai, 1979). As discussed in Section IV, agonist binding to the muscarinic receptors shows properties distinct from those observed with antagonists. For example, guanine nucleotides exert a selective modulatory effect upon high-affinity agonist binding but not upon antagonist binding to brain muscarinic receptors (Berrie et al., 1979; Gurwitz and Sokolovsky, 1980a,b). However, recent observations have indicated that in the rat heart and striatal (Berrie et al., 1979; Wei and Sulakhe, 1980; Ehlert et al., 1981a,b) and adenohypophysis (Avissar and Sokolovsky, 1981) muscarinic receptors show an effect of guanine nucleotides on antagonist binding. Studies of antagonist binding in cardiac (Birdsall et al., 1980b; Hulme et al., 1981) and striatal (Ehlert et al., 1981a,b) membrane under conditions of low ionic strength have suggested the presence of multiple affinity classes of receptors that could be changed to a single affinity class by guanine nucleotides. Guanine nucleotides also affected muscarinic antagonist binding in the adenohypophysis of the male but not the female rat (Avissar and Sokolovsky, 1981). In these preparations 3H-labeled antagonist binding was characterized by curvilinear Scatchard plots and varied among male and female rats at different stages of the estrous cycle; it could be manipulated by ovariectomy of female rats and by androgenization of newborn females (Egozi et al., 1982). Incubation of the male rat preparation with [3H]-4NMPBin the presence of 100 pM GTP yielded linear Scatchard plots. Thus, the sexual dimorphism manifested in the muscarinic antagonist binding, which can be reversed by endocrine manipulation, strongly indicates the occurrence of interconversion of muscarinic binding sites. It should be noted that the effects induced by GTP could be reversed by washing the membranes and was specific to guanine nucleotides. It has also been documented (Burgisser et al., 1982) that in the frog heart antagonist and agonist binding appear to be regulated in a reciprocal fashion by the guanine nucleotide 5'guanylyl imidodiphosphate. According to this report two states of receptor, which display high-agonist/low-antagonistand low-agonistlhigh-antagonist affinities, respectively, are present in the frog heart in

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approximately equal proportion. 5'-Guanyl imidodiphosphate appears to convert the former type of state into the latter state. To summarize, data obtained from muscarinic antagonist binding suggest that ( a ) the number of binding sites available in a given preparation is the same for all antagonists; ( 6 ) binding of an antagonist to the muscarinic receptor results in conformational changes that lead to a higher-affinity receptor-ligand complex (isomerization); (c) effectors (e.g., ions, GTP) can affect the binding of muscarinic antagonists; ( d ) there is apparent regional heterogeneity in muscarinic receptor-binding properties; and ( e ) cooperative interactions were recently demonstrated in the binding of muscarinic antagonists (see following section). B. COOPERATIVE AND/OR SITEINTERACTIONS Two lines of experiments have provided data that support the notion of interactions among binding sites in muscarinic systems. These approaches include studies of the effects of various drugs such as clomiphene, gallamine, or certain oximes on the muscarinic system and ligand competition experiments designed to test the presence of site-site interactions. Mono- and bispyridinium oximes that, in combination with atropine, serve as antidotes to certain types of organophosphorous poisoning (Oldiges and Schoene, 1970) show antimuscarinic activity in guinea pig ileum (Dirks et al., 1970; Kuhnen-Clausen, 1972). These oximes were also shown to inhibit the binding of the antagonist 4NMPB to mouse and rat brain homogenates (Amitai et al., 1980) through a mechanism that could be competitive inhibition. Initial experiments on the displacement of 4NMPB by bisquaternary pyridines in mouse brain homogenates did not fit a simple mass-action model. It was noted in our laboratory (M. Sokolovsky, unpublished) that the displacement of [3H]-4NMPB by several bispyridinium oximes (Fig. 3) exhibited phenomena typical of negative cooperativity or site heterogeneity, that is, curvilinear Scatchard plots and Hill coefficients of 0.58 to 0.72 in various rat brain regions. A similar phenomenon was observed during measurements of the binding of 4NMPB in the presence of constant concentrations of certain oximes such as HGG-42. The dependence of the affinity of [ "14NMPB on oxime concentration was nonlinear and decreased more than expected at higher oxime concentrations. All these results could be explained either by negatively cooperative interactions between the oxime and the muscarinic receptors or by the simple assumption that the

MUSCARINIC RECEPTORS IN THE CENTRAL NERVOUS SYSTEM

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0 II

HON = HC

@

qc-

ICH,-0-CH, I

0

HGG-12.

f l = G

HGG-42,

f l = a

HGG-52,

R = -CH,-CH

/CH, ‘CH,

FIG. 3. Structure of bispyridinium oximes.

oximes distinguish between high- and low-affinity muscarinic binding sites, whereas muscarinic antagonists do not. However, a combination of the experiments described above with photoaffinity labeling using the muscarinic antagonist azido-4NMPB (Amitdi et nl., 1982) could distinguish between these two possibilities. Over 60% of the muscarinic receptors in brainstem and cortex homogenates were inactivated irreversibly with azido-4NMPB. T h e displacement of 4NMPB by HGG-42 in the modified preparations yielded Hill coefficients of 0.92 to 1.0. Because the binding of azido-4NMPB is not negatively cooperative by itself, these results suggest that the effect of the oxime cannot be simply due to site heterogeneity and that negatively cooperative interactions must be involved. It should be noted that such interactions are not necessarily between the muscarinic sites themselves, because the binding sites of the oximes could be nonequivalent with those of the muscarinic antagonist. It is interesting that rather similar observations were reported by Stockton et al. (1983). They utilized the neuromuscular blocking agent gallamine, which was shown to interact with muscarinic receptors in the heart (Clark and Mitchelson, 1976; Birdsall et al., 1981), in displacement experiments analogous to those described above. As in the case of the oximes, negatively cooperative interactions were also detected between gallamine and the muscarinic receptors in various rat tissue homogenates, especially in the heart. These results were explained on the basis

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of an allosteric modulation of the binding of muscarinic ligands by gallamine (Stockton et al., 1983). T h e results obtained with gallamine and with oximes indicate that negatively cooperative interactions can exist in the muscarinic system. These experiments leave open the question whether such interactions exist between one muscarinic binding site and another. The latter possibility has been explored by a newly developed method based on ligand competition experiments (Henis and Levitzki, 1979). The method compares the binding of a primary ligand (whose binding is cooperative) in the presence and absence of a competing ligand. In the case of simple site heterogeneity the competing ligand affects the Hill coefficient and the dissociation constants of the primary ligand to an extent that can be calculated from the dissociation constants measured for each ligand separately. However, if cooperative interactions are involved, the effect of competition on these parameters can deviate significantly from the values predicted from the separate binding curves. This technique was applied to study the binding of muscarinic antagonists to rat adenohypophysis (Henis et al., 1982), because, as was pointed out earlier in this section, in this system the binding of muscarinic antagonists was found to yield curvilinear Scatchard plots (Mukherjee et al., 1980; Avissar et al., 198la), as required for the primary ligand in the technique mentioned above. Competition experiments employing [3H]-4NMPB and QNB in this system showed that inhibition by the competing ligand is three- to fourfold higher than the values expected from the assumption of two classes of noninteracting sites, suggesting the existence of interactions among the muscarinic binding sites occupied by antagonists or more than two classes of binding sites. Similar experiments did not detect such interactions in homogenates of rat medulla-pons, and the effects of the competition on the binding pattern of the primary ligand were as expected for pure competition. This finding is expected in view of the fact that the binding of muscarinic antagonists to medulla-pons and other brain regions yields linear Scatchard plots that give no indication of site heterogeneity or negative cooperativity. Thus, there may be evidence for cooperative interactions among the muscarinic receptors in the adenohypophysis, although it is not yet clear whether such interactions occur in other brain regions. It should also be noted that the interactions observed in the adenohypophysis do not exclude site heterogeneity in this system, and the two phenomena may coexist. Evidence for site heterogeneity is supplied by the effect of GTP on the binding of muscarinic antagonists described in Section III,A. It is worth noting that experiments employing the irreversible antagonist pro-

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pylbenzilylcholine mustard to inactivate part of the muscarinic receptors in rat brain cortex followed by competition experiments between agonist and a labeled antagonist were interpreted to indicate site heterogeneity only for the muscarinic agonists (Birdsall et al., 1978). Although it is theoretically possible that agonist binding will not involve cooperativity, the recent indications of cooperative interaction in the muscarinic system suggest that such interactions may still be present even for agonists, although the experimental procedure was not sensitive enough to observe them. The observations described thus far reported phenomena related to negative cooperativity. Positive cooperativity was also observed in the interaction of the antiestrogenic drug clomiphene with muscarinic receptor (Ben-Baruch et al., 1982). The displacement of the muscarinic antagonist 4NMPB by clomiphene showed cooperativity in the homogenates from various brain regions. T h e results were indicative of binding of more than one molecule of clomiphene and were interpreted in terms of a model based on two separate binding sites for clomiphene such that one of these sites differs from the binding site of muscarinic ligands. T h e former site was proposed to have lower affinity for clomiphene, and the binding of a clomiphene molecule to this site enables clomiphene to bind to the muscarinic site (the high-affinity site for clomiphene). The findings reported thus far suggest the possible involvement of interactions between binding sites in the binding of some ligands to the muscarinic receptors. Such interactions may exist among the muscarinic sites themselves and could thus contribute to the apparent cooperativity in the binding of muscarinic antagonists (and perhaps of agonists, too). Additional binding sites could be interacting with the muscarinic sites in the membrane, as suggested for the effects of clomiphene, gallamine, and oximes on binding to muscarinic receptors, and these interactions could lead to apparent negative or positive cooperativity. More evidence needs to be obtained in order to substantiate these hypotheses in various organs and tissues. Finally, it should be noted that the detection of an oligomeric (dimeric o r tetrameric) structure in rat brain muscarinic receptors (Avissar et al., 1982, 1983) raises the possibility of cooperative interactions between muscarinic sites located on the same oligomer. I t is also possible that in their native state in the cell membrane the muscarinic oligomers are further associated with other molecules, forming a larger oligomeric complex. Such associations could be involved in cooperative effects exerted on the muscarinic system, discussed in Section V.

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IV. Binding of Agonirts

Until recently agonist binding to the receptor was studied only by measuring the competition between a 3H-labeled antagonist present at a constant concentration and varying concentrations of unlabeled agonists; the basic experimental assumption was that the number of binding sites for agonists and antagonists is equal. Data from these experiments could be interpreted by assuming the existence of two populations of binding sites with different capacities and affinities (Birdsall et al., 1978, 1979b, 1980a) without assuming any cooperativity in agonist binding. In addition to the presence of high- and low-affinity agonist binding sites, Birdsall et al. (197913 and references therein) reported that their nonlinear least squares analysis revealed the existence of a superhigh-aflnity binding site in several brain regions. Binding isotherms of ”-labeled agonists have been demonstrated in only a few reports, using [3H]-Nmethyloxotremorine (Birdsall et al., 1978) and cis-[SH]methyldioxolane (Ehlert et al., 1980b). The number of binding sites that could be measured under the experimental conditions employed was much lower than those of 3H-labeled antagonists. The measured “-labeled agonist sites correspond to the superhigh and some of the high-affinity site fractions observed in competition experiments (Birdsall et al., 1978). The other two lower-affinity site fractions could not be determined directly with SH-labeled agonist due to “masking” by high nonspecific binding, which stems from the high concentrations of agonist required for interaction with the lower-affinity sites. Moreover, rapid dissociation rates render direct kinetic studies with labeled agonists even more difficult. Studies of ‘H-labeled antagonist binding do not suffer from such limitations because saturation occurs at low ligand concentrations due to their high affinity for the receptors. I n another study it was found that the number of the partial agonist [’Hlpilocarpine binding sites was as much as four times higher than that of the antagonist [’H]-4NMPB and that the binding curves could also be fitted to a two-site model (Hedlund and Bartfai, 1981). In view of the lack of kinetic experiments with 3H-labeled agonists, it therefore seems very important to determine the stoichiometrical relationship between muscarinic agonist and antagonist binding. The limited accuracy of experimental data and parameter fitting procedures make it extremely difficult to establish the existence of three separate classes of agonistbinding sites in competition experiments with ‘H-labeled antagonist. In most studies only two classes of agonist binding sites are assumed.

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Therefore, in subsequent discussion we refer only to high- and lowaffinity agonist-binding sites. In our laboratory, analysis of agonist binding using data derived from competition experiments proceeds on the assumption that the fractional decrease in the amount of labeled antagonist bound is equal to the fraction of receptor sites occupied by unlabeled agonist. Although this assumption is not necessarily true in general, our estimates of relative abundance and equilibrium binding constants for high- and lowaffinity site classes obtained in this fashion have been confirmed by a complete analysis of the multiple equilibria present in competition experiments under the experimental conditions we use (A. P. Minton, unpublished observations). Birdsall et al. (1978) interpreted their results as pointing to preexisting heterogeneity of the receptor binding sites for agonists: T h e possibility of agonist-induced receptor “desensitization” during assay was excluded by an experiment in which the irreversible antagonist propylbenzilylcholine mustard was used to alkylate low-affinity binding sites, whereas high-affinity sites were protected by using a low concentration of agonist. Following washout of the ligands, the subsequent binding assay demonstrated that most of the remaining receptors displayed high affinity toward the agonist. Note that the coexistence of site heterogeneity and negative cooperativity or isomerization of the receptoragonist complex is theoretically possible. However, unlike the situation with respect to measurement of antagonist binding in which kinetic data implicated both isomerization and negative cooperativity (see Sections II1,A and B), the difficulties involved in direct measurements of agonist binding have precluded the acquisition of sufficiently precise data. However, analysis of agonist competition curves in various brain regions according to the two-state model indicated that in the cortex, hippocampus, and basal ganglia the proportion of high-affinity binding sites for oxotremorine is less than 15%, whereas it is 40-50% for carbamylcholine or acetylcholine. However, in the cerebellum and medulla-pons the proportion of high-affinity sites calculated for these three agonists approximated much more closely to each other. Comparing these results to those observed in other mammalian brains, it is again evident that by the same analysis of the inhibition data for agonists according to the two-site model, the proportion of high-affinity binding sites for oxotremorine in rat cortex and frog cortex is lower (-18%) than that for carbamylcholine (40-50%) (Birdsall et al., 1979b). Thus, agonist binding cannot be described solely in terms of binding of agonist to fixed receptor sites of high and low affinities. We therefore suggested that agonist binding may

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be explained by induced interconversion of the muscarinic receptors by agonists, reflected in differences in the ratio of high- to low-affinity binding sites for different agonists (Sokolovsky et al., 1980b; Egozi et al., 1980). Hence, we have investigated the possible role of guanine nucleotide because it was established in other receptor systems that GTP and its analogs convert high-affinity agonist-binding sites to low-affinity sites (Rodbell, 1980 and references therein). Indeed, for the muscarinic receptors in the medulla-pons preparation guanine nucleotides induce interconversion of high- to low-affinity agonist-binding sites. The effect is observed in those brain regions that have a relatively large proportion of high-affinity binding sites (Sokolovsky et al., 1980c; Gurwitz and Sokolovsky, 1980a,b) (e.g., medulla-pons, cerebellum, female adenohypophysis). In regions enriched in low-affinity sites (e.g., cortex, hippocampus), however, there is little, if any, such effect. The guanine nucleotide-induced interconversion of CNS muscarinic receptor is reversible. In all of these brain regions guanine nucleotides had no effect on antagonist binding. T h e GTP effect was also reported for the receptors in rat heart (Berrie et al., 1979; Wei and Sulakhe, 1980; Ehlert et al., 1981b), and in rat and frog heart (Burgisser et al., 1982). These observations, however, indicated that in addition to inducing a transition of the agonist-binding site, guanine nucleotides enhance antagonist binding. As mentioned in Section 111, two forms of muscarinic receptors have been documented in the frog heart that display high-agonist/low-antagaffinities, respectively. The nucleonist and low-agonist/high-antagonist otide 5’-guanylyl imidodiphosphate appears to convert the former type of site into the latter type (Burgisser et al., 1982). Interestingly, guanine nucleotides affected the antagonist-binding site in male adenohypophysis, although those of the female rat were unaltered. The nucleotide effect on agonist binding was seen only in the female rat and not in the male rat (Avissar and Sokolovsky, 1981). A number of modifying agents and several types of treatments were also found to alter the ratio of high- to low-affinity agonist-binding sites in a given preparation. The use of reagents that react specifically with amino and thiol groups, for example, N-ethylmaleimide, p-hydroxymercury benzoate, Cd2+,revealed that agonist binding is more sensitive than antagonist binding to modification of these groups (Aronstam et al., 1977a, 1978; Hedlund and Bartfai, 1979; Hedlund et al., 1979; Ikeda et al., 1980; Wei and Sulakhe, 1980). In fact Abood and his collaborators were the first to suggest that agonist-binding states could be transposed. Experiments show that drugs such as veratridine and tetrodotoxin, which affect the voltage-dependent sodium channel, lead to the disappearance of high-affinity agonist binding via blockade rather than inter-

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conversion (Hedlund and Bartfai, 1982). Treatments such as depolarization with high K+, gramicidin, or valinomycin, or making the membranes leaky by freezinglthawing cycles, lead to similar disappearance of high-affinity agonist-binding sites (Hedlund and Bartfai, 1982). I n view of the fact that interconversion of high- to low-affinity agonist-binding sites can be induced, if this conversion has any physiological significance, then the opposite reaction (i.e.. low- to high-affinity site conversion) might be identifiable. Indeed, we found that divalent transition metal ions Mn2+,Co2+,and Nig+ can convert agonist-binding sites from low to high affinity with no detectable effect on antagonist-binding parameters (Gurwitz and Sokolovsky, 1980b). The effect of the metal ions is detected only in brain regions containing a large population of low-affinity agonist-binding sites, for example, cortex and hippocampus. In the medulla-pons and other regions that are enriched with a population of high-affinity sites the effect of Mn’+, Co2+,and Ni2+ is minimal or nil. This effect can be reversed by any of the following procedures: (i) removal of the ions; (ii) thermal exposure, or (iii)addition of micromolar concentrations of guanine nucleotide. Agonist-specific reverse regulation by transition metal ions and guanine nucleotide is best demonstrated in cortical preparations. It is unlikely that this effect is due to formation of metal complexes with endogenous GTP, because a low ( M) concentration of GTP completely reverses this conversion. Antagonism between Ca2+and Mn2+also seems unlikely because the presence of 1 mM Ca2+did not alter the effects of Mn2+.It would appear either that Mn2+mimics the action of a possible endogenous modulator or that the transition metal ions might influence the phosphorylation mechanism discussed in Section VIII. The occurrence of two different binding sites for agonists was interpreted in terms of a “physiological” and a “nonphysiological” state of the muscarinic receptors. Birdsall et al. (1978) suggested that the low-affinity conformation involves coupling to an effector molecule that reduces the affinity towards agonists by virtue of the constraint it forces upon the receptor molecule, whereas the high-affinity receptors are not thus constrained. Another suggestion was that the high-affinity receptors might be a degradation product of the low-affinity receptors because the appearance of the former lags during brain maturation (Wamsley et al., 1980).An interesting finding reported recently for the membrane preparation of female rat adenohypophysis suggests that the interconversion between the two states of agonist binding may be of physiological significance. In the proestrous and diestrous states, the GTP effect produces the conventional pattern, that is, high- to low-affinity site conversion. However, at the estrous stage GTP causes the opposite effect, that is,

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conversion from low- to high-affinity sites (Avissar and Sokolovsky, 1981). Thus, it seems reasonable to assume that the physiological role of the different agonist affinity states varies with the particular cell in which the muscarinic receptor resides, and the interconversion is a tool for upand down-regulation of various responses. Kinetic studies on competition between agonist and 3H-labeled antagonist (Hedlund et al., 1980) showed that the first equilibrium ( k J k l ) of Eq. (2) is apparently not affected by agonists such as oxotremorine and carbamylcholine in concentrations that yield substantial inhibition of 3H-labeled antagonist binding. If the sequential binding scheme is correct this may mean that agonists ( B ) bind with equal affinity to the free receptor (R) and the receptor-antagonist complex (RA).

If a ternary receptor-agonist-antagonist complex (RAB) exists, one should be able to determine this by using “-labeled agonist and 3Hlabeled antagonist simultaneously, and indeed the existence of such a complex has been suggested by Hedlund et al. (1980). Transient state kinetics were utilized in our laboratory to evaluate binding rate constants of unlabeled agonists to muscarinic receptors in homogenates of rat medulla-pons (C. Schreiber, D. Gurwitz, Y. I. Henis, and M. Sokolovsky, unpublished). T h e method employed displacement of agonists with the muscarinic antagonists [ 3H]-4NMPB, [‘HI-QNB, and [3H]-N-methylscopolamine, assuming full competition between agonists and antagonists, as found in equilibrium binding studies. However, it could be argued that the low-affinity sites may not be fully occupied by the agonists. This difficulty was minimized by using rat medulla-pons homogenates, in which over 70% of the muscarinic receptors display high affinity toward agonists, and short incubation times with antagonist, in which case receptor isornerization is not yet significant. These experiments yielded association and dissociation rate constants for agonist and antagonist as depicted in Table 11. T h e results indicate that the different association rate constants are the main reason for the different affinities exhibited by various agonists toward the muscarinic receptor. This contrasts with muscarinic antagonists, in which the dissociation rate constants primarily dictate ligand

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TABLE I1 COMPARISON OF RATECONSTANTS FOR THE BINDING MUSCARINIC AGONISTS AND AhTAGONISTS TO R A T MEDULLA-PONSHOMOGENATES

OF

Rate constants

Agents Agonists Carbamylcholine Acetylcholine Oxotremorine-M Oxotremorine Antagonists

QNB NMPB Scopolamine Atropine

hut, (M-lsec-’)

korr (sec-’ x lo3)

0.15

33

2.0

52

4.1

72

7.0

85

3.3 3.8 3.6 4.8

0.2 1.3 2.5 7.5

affinity. T h e major difference between antagonists and agonists is the higher dissociation rate constants of the latter. It should be noted that these results are in accord with the rate theory proposed for drugreceptor interaction (Paton, 1961). For example, one of the predictions of the rate theory is that the dissociation rate of antagonists from the receptor will be slower than that of agonists, because the frequency of complex formation depends on the dissociation rate. I n accord with this prediction the dissociation rate constants of muscarinic antagonists are more than an order of magnitude lower than those for agonists. Furthermore, in accord with the rate theory a “better” agonist not only has a higher association rate constant but also a higher dissociation rate constant.

V. Receptor-Receptor Interactions

As mentioned in Sections I11 and IV, physiological and biochemical data suggest that muscarinic receptors are affected by interactions with other receptors or with regulatory proteins. Guanosine triphosphate and its analogs have been shown to convert muscarinic binding sites from high to low affinity (Sokolovsky et al., 1980~).It appears that the hydrolysis of GTP (which takes place during incubation with the mem-

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brane preparation) is not an obligatory step for the GTP effect, because a nonhydrolyzable analog of GTP can also cause conversion of the receptor sites. It is thus the binding of GTP, and not the energy released on its hydrolysis, that causes a conformational change in the receptor. Because GTP alone does not bind to the muscarinic receptors, it has been suggested (Sokolovsky et ad., 1980c) that the muscarinic complex contains a separate membrane unit that is guanine nucleotide-sensitive. By analogy to other neurotransmitter and hormone receptors this component may be denoted N,., following Rodbell’s notation (1980). Effects similar to those produced by GTP can be obtained simply by warming membranes from medulla-pons preparations for 3 min to 50°C (Gurwitz and Sokolovsky, 1980a), suggesting that both heat and GTP produce a conformational change. This raises the possibility that the G protein is heat-labile. T h e lability of this component is also indicated by its tendency to be partially lost on washing the membrane preparation (D. Gurwitz and M. Sokolovsky, unpublished). It should be noted that in the heart and in N 1E 1 15 neuroblastoma cells muscarinic agonists can inhibit basal adenylate cyclase activity as well as the activation of adenylate cyclase by agonist-occupied @-receptors (Rodbell, 1980 and references therein). This inhibition of adenylate cyclase activity by muscarinic agonists requires Na+, GTP, and the hydrolysis of the latter (Lichtshtein et at., 1979). It is likely that in these preparations the muscarinic receptor associates in the plane of the membrane with a nucleotide-binding protein (N;) that may be identical with the G protein of the adenylate cyclase complex, thereby modifying the P-adrenergic responses of the postsynaptic cell. In the central nervous system muscarinic agonists do not inhibit basal adenylate cyclase activity. Attempts to modify muscarinic agonist-binding parameters and the effects of GTP by treating brain membrane preparations with cholera toxin, which ADP-ribosilates the G protein of the adenylate cyclase complex (Cooper et al., 1981), were unsuccessful (D. Gurwitz and M. Sokolovsky, unpublished observations). Thus, it is tempting to suggest that the reciprocal modulation of agonist and antagonist binding by guanine nucleotide observed with heart and ileum preparations cited earlier might be related to an interaction with the adenylate cyclase system. Such interaction is not predominant in the brain regions investigated so far; guanine nucleotide affects mainly the agonist-binding site. Another study has shown that /I-estradiol may interact with the muscarinic receptor. Variations in muscarinic binding properties occur in the female rats as a function of the estrous cycle (Avissar et al., 1981a,b). These variations occur only in female rat adenohypophysis and preoptic area, indicating specificity. Endocrine manipulations such as ovariec-

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tomy, androgenization of female rats, and estradiol implantation into ovariectomized rats clearly indicate that the steroid hormone is involved in this process (Egozi et al., 1982).P-Estradiol and progesterone, but not cholesterol, interfere with muscarinic binding (Sokolovsky et al., 1981). T h e steroid hormones converted high-affinity muscarinic binding sites to their low-affinity state in the rat adenohypophysis. These and other results led to the model outlined in Fig. 4 for the relationship between the muscarinic system and other receptor systems. We make the assumption that the muscarink receptor (M) and a membrane component (Y) are modulated via a common unit (X) that regulates both M and Y. This arrangement (i.e., M ... X ... Y) is typical of certain tissues, whereas other tissues may be characterized by the presence of M coupled to X (M-X), free uncoupled M, or free uncoupled Y. We further assume that the regulatory unit X is essential for the activation of the system by M and, although it is not essential for the specific functioning of the Y system, coupling of Y to X will cause perturbation of this system. For example, if one of the specific actions of Y concerns an internalization process (as indicated by the arrow at the upper left of Fig. 4), this will be inhibited by coupling of Y to X. Thus, the model does not specify whether the membrane components are freely mobile or organized in domains. T h e identity of the components is inferred from previous observations. In the muscarinic system it is assumed that binding of antagonists and of agonists differ according to the degree of M-X coupling. Thus,

.or0

WM-X

-Yf,,M-X-Y.-’M-X-YY-O I

C

11. cI

0 0 0

iC I C

FIG.4. Model depicting the putative relationship between the muscarinic system and other receptor systems. 0 and 0 ,Y agonist and antagonist, respectively; 0 and a, muscarinic agonist and antagonist, respectively; M, muscarinic receptor; Y, membranal component (e.g., another receptor); X, protein molecule that regulates both M and Y; C, ligand that binds to X and simultaneously or thereafter to M or Y (e.g., clomiphene, oximes andlor gallamine).

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antagonists will bind similarly to both M and M-X, whereas agonists will bind with low affinity to M and high affinity to M-X. A candidate for X is, for example, the GTP regulatory unit, which need not be identical to that coupled to adenylate cyclase, whereas candidates for Y include other receptor systems and an ionic channel. Evidence for the proposed model is so far circumstantial, but its usefulness can be illustrated in an attempt to interpret the interaction between the muscarinic and the steroid sex hormone systems. 1. In the presence of muscarinic agonist the coupling is basically between M and X. Under such conditions the Y molecule becomes uncoupled and, in the presence of its endogenous substrate (for example, P-estradiol), the binding might be followed by internalization and release of the estrogen into the cytosol. It follows that, in the presence of muscarinic agonists, P-estradiol activity should increase, and experiments carried out in the presence of oxotremorine do indeed indicate an increase in luteinizing hormone release (Libertun and McCann, 1973). The presence of a muscarinic antagonist should therefore result in an opposite effect; reports in the literature indicate that antimuscarinic drugs such as atropine, acting at the hypothalamic-pituitary level, inhibit ovulation (Libertun and McCann, 1973; Strobl et al., 1980). 2. According to the scheme, progesterone or P-estradiol interaction with Y will cause coupling between Y and X, thereby causing dissociation of M from the three-component complex. In such a case (i.e., uncoupling of an M-X complex) we would expect that M would exhibit the agonist low-affinity state while retaining its inherent antagonistic character. Indeed in tissue or cells in which Y is the estrogen receptor (e.g., in the pituitary) the presence of estradiol results in the conversion of the muscarinic agonist-binding site to its low-affinity state (Sokolovsky et al., 1981). Estradiol does not have this effect in the preoptic area or the medulla-pons, suggesting that Y in these brain regions may be specific for another substrate. Indeed progesterone had the same effect on the muscarinic system in the preoptic area and pituitary, indicating the presence of a Y specific for this steroid in these regions. In contrast medullapons was unresponsive to both estradiol and progesterone, suggesting that the Y component there is not specific for any of the steroids investigated so far. Whether the Y component in the medulla-pons is absent or specific for some other neurotransmitter or hormone remains to be determined. 3. In those cases where X is sensitive to guanine nucleotides o r temperature their effect is on that M-X (or Y-X) arrangement that is characterized by agonist high-affinity state. Uncoupling of the arrangement

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by GTP will result in interconversion to a low-affinity state. Conversely, the effect of transition metal ions is coupling of M and X. 4. Now let us assume the existence of a ligand that can bind to X and that this unit can become coupled to either M or Y or to both. Thus, in a tissue containing these elements, this M-X-Y coupling will modify some of the properties of both M and Y; in a tissue in which only M o r Y exists, the binding properties of ligands to their respective receptor (M or Y ) will be modified. A candidate for an X-coupling ligand might be the antiestrogenic nonsteroidal drug clomiphene, which affects the muscarinic system (Ben-Baruch et al., 1982); this effect was exerted by more than one clomiphene molecule and in a cooperative manner. The most likely explanation is that the first clomiphene molecule binds to X, leading to conformational changes such that, when the second clomiphene molecule enters the 4NMPB zone of binding (Fig. 4), the complex formed has a much higher dissociation constant, k o ~and , dissociation of ["]-4NMPB is facilitated (Ben-Baruch el al., 1982). Furthermore, unlike the steroid sex hormones, which exert their effects via the Y component, clomiphene is unlikely to discriminate between the brain regions and will affect all muscarinic systems containing M-X, as was indeed observed. The model also suggests that the binding of P-estradiol or progesterone to the membrane component Y should be affected by the presence of clomiphene. Indeed an acceleration occurs in dissociation of the receptor-ligand complex (Strobl et al., 1980).Other likely candidates for the X-coupling ligand are gallamine and bispyridinium oximes (see Section 111,B). This model, which offers an explanation for the effects of agonists or antagonists specific for one system on those of other systems, proposes a general modus operandi for physiological dialogs among different systems. Thus, it could account for the interplay between cholinergic and other neurotransmitter systems. It should be noted that Watanabe et al. (1978) reported that in membrane preparations of dog myocardium muscarinic agonists could increase P-adrenergic agonist binding up to threefold. This interaction was blocked by muscarinic antagonists and required the presence of GTP. In fact, GTP and (carbachol + GTP) produced equal and opposite effects on P-agonist binding. Muscarinic agonists have also been shown to have a small effect on cyI agonist binding in the heart .(Yamada et al., 1980). I n other systems dopaminergic agonists (e.g., apomorphine) increased the binding of an antagonist to muscarinic receptors in the CNS (Ehlert et al., 1981b), whereas muscarinic-dopaminergic synergism was observed in the retina (Brown and Rietow, 1981).

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Interaction of muscarinic receptors with other membrane-bound macromolecules such as vasoactive intestinal peptide (VIP) receptor and with ion channels have been reported. In rat cerebral cortex vesicles carbamylcholine acting on muscarinic receptors caused a biphasic depolarization-hyperpolarizion, that correlated with binding to high- and low-affinity muscarinic receptors, respectively. Drugs that induce depolarization, such as veratridine, blocked the high-affinity binding sites, as did tetrodotoxin, which blocks the ion channels. It has been suggested that the high-affinity muscarinic binding sites interact with the voltagesensitive sodium channel (B. Hedlund and T. Bartfai, unpublished results). Striking effects of VIP have been observed on the binding of antagonists and agonists to muscarinic receptors in cat salivary gland. The elegant studies of Lundberg and co-workers (Lundberg et al., 1980, 1982; Lundberg, 1981) have demonstrated that in this gland VIP and acetylcholine may coexist in and be coreleased from the same neuron. Although VIP per se does not induce secretion, it strongly potentiates and prolongs acetylcholine-induced secretion. Binding studies (Lundberg et al., 1982) have demonstrated that VIP at a physiologically relevant concentration (- 10-8 M ) will increase muscarinic agonist binding by up to 100,000-fold. This effect, which is more than four orders of magnitude greater than any previously reported, appears to be demonstrable only under special conditions of concentration (3-30 nM VIP) and incubation time (- 1-5 min). VIP increased the rate of association of 4NMPB with the muscarinic receptor and the affinity of muscarinic agonists, suggesting interaction between the VIP and the muscarinic receptors. To summarize, the data clearly indicate that the muscarinic receptors may interact with other membrane-bound macromolecules. The scheme shown in Fig. 4, although based on a number of independent lines of evidence, is still quite speculative. Many questions remain about how the system functions. For example, if indeed the X molecule is a nucleotide regulatory protein, do inhibitory and stimulatory receptors share a common pool of G proteins or are there separate pools that interact specifically with inhibitory versus stimulatory receptors? Is it the 42,000-dalton subunit of the nucleotide regulatory protein that represents the molecule denoted as X (Fig. 4) or are other subunits involved (Sternweis et al., 1981)? Finally, is this the same subunit that is involved when the muscarinic receptor is coupled to adenylate cyclase (e.g., in atria, ileum) or are different subunits involved depending on the nature of the coupled macromolecule (Y)? Further research should clarify the interactions, different receptors, and coupling systems.

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VI. localization of Muscarinic Receptors

Physiological, biochemical, and direct binding methods have all been used to localize muscarinic receptors. These studies have yielded considerable information on the regional distribution of muscarinic receptors in the brain and periphery, as well as on their post- and presynaptic localization.

A. REGIONAL DISTRIBUTION In the CNS regional distribution experiments indicated that tissues rich in cholinergic activity are also rich in "-labeled antagonist binding sites (Snyder et al., 1975). This seems to afford confirmation that the drugs actually bind to the physiologically active receptor. Several methods have been used in the localization experiments. In many studies tissues were dissected out, homogenized, and subjected to standard binding assays (e.g., Snyder et al., 1975; Kloog et al., 1979a). I n other experiments radiolabeled ligands were injected into animals, and the amount of 3H-labeled antagonist present in the tissues was then measured directly (Snyder et al., 1975; Sokolovsky et al., 1980a,b). Autoradiography of tissue slices incubated with 3H-labeled antagonists was also used to characterize muscarinic receptors at the light microscopic level (Kuhar and Yamamura, 1976, 1978; Rotter et al., 1979a,b,c); Wamsley et al., 1981). The results obtained from these different techniques were almost identical. Cerebral cortex, basal ganglia, and hippocampus were found to be highly enriched in muscarinic receptors, whereas medulla-pons and cerebellum contained fewer muscarinic binding sites (see Table I). Lesioning techniques have been used to map cholinergic pathways in the CNS (Kuhar, 1976), including the septohippocampal and the habenulo-interpeduncular pathways. Muscarinic receptors have also been found in great profusion on interneurons in the basal ganglia (McGeer et al., 1975). According to iontophoretic studies (Krnjevic and Phillis, 1963), most of the Renshaw cells in the cerebral cortex have muscarinic receptors. Electrophysiological and binding studies indicated the presence of muscarinic receptors on Purkinje cells in the cerebellum (Crawford et al., 1966). The punch technique and 3Hlabeled muscarinic antagonists with high specific activities have been used to localize muscarinic receptors in discrete brain nuclei (Rea and Simon, 1981).

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In a recent study positron emission tomography employing antagonist labeled with carbon-1 1 was used to characterize baboon heart muscarinic receptors in vivo (Maziere et al., 1981). This seems a promising technique for future studies in humans. In the mapping of cholinergic pathways in different brain regions, muscarinic receptor density was compared with that of other cholinergic markers such as acetylcholinesterase (AChE) and choline acetyltransferase (CAT) (Snyder et al., 1975; Kloog et al., 197913). The relationship between receptor density and enzyme activity is not always clear because of their different localization and subcellular distribution. T h e enzyme AChE is found both in the cell soma and in nerve terminals and is both membrane-bound and cytosolic. The enzyme CAT is mainly localized in the nerve terminals. Muscarinic receptors are located on the soma and dendrites of cholinoceptive cells. It thus seems that the distribution of CAT and of muscarinic receptors are better markers for mapping of the cholinergic system than AChE distribution (for a review see Heilbronn and Bartfai, 1978).

RECEPTORS B. POST-AND PRESYNAPTIC For many years it was thought that receptors for neurotransmitters are located exclusively on the postsynaptic membrane. In the classic description of neuron-neuron or neuromuscular junctions, the prejunctional cell would release the neurotransmitter, and the postjunctional cell would receive it via a specific receptor located on its membrane, to which the neurotransmitter would become bound. Physiological and biochemical evidence pointed to the dominance of the postsynaptic muscarinic receptor in both the central and the peripheral systems (i.e., heart, smooth muscle, exocrine glands) (for a review see Heilbronn and Bartfai, 1978). It later became evident, however, that a variety of receptors for neurotransmitters are present on the prejunctional membrane as well (presynaptic receptors) (see Starke, 1980). To investigate the existence of presynaptic muscarinic receptors acetylcholine release was assayed because this neurotransmitter is known to be released from the presynaptic nerve terminals. It was found that ACh release could be diminished by muscarinic agonists and enhanced by muscarinic antagonists: it was therefore suggested that presynaptic muscarinic receptors are present on cholinergic nerve terminals (Polak, 1971; Kilbinger and Wagner, 1975; Szerb, 1975),and this was subsequently confirmed by the use of lesion techniques and radiolabeled ligand binding assays. Presyn-

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aptic muscarinic receptors occur on cholinergic neurons in the CNS (Kilbinger and Wagner, 1975; Szerb, 1975) and at the periphery, for example, in Torpedo electric organ (Kloog et al., 1980a; Sokolovsky et al., 1980b) and rat diaphragm (Abbs and Joseph, 1981). They also exist on adrenergic neurons in the heart (Sharma and Banerjee, 1977) and CNS (Giorguieff et al., 1977; Gurwitz et al., 1980). Chemical lesion of catecholaminergic neurons with the neurotoxin 6hydroxydopamine (6-OHDA) has been used by several authors to study the location of presynaptic muscarinic receptors on these neurons. A decrease in muscarinic receptor density was detected in rat heart following destruction of sympathetic innervation with 6-OHDA (Sharma and Banerjee, 1977). Intracisternal administration of 6-OHDA to mice or rats resulted in massive destruction of catecholaminergic neurons in all brain regions; however, a decrease in muscarinic receptors was seen only in the striatum, medulla-pons, cerebellum, and hypothalamus (Gurwitz et al., 1980; Avissar et al., 1981b). The most convincing demonstration of presynaptic receptors on sympathetic nerves has come from studies using the nerve ligation technique. Forty-eight hrs following ligation of the splenic nerves in dogs there was a massive accumulation of receptors (five times higher than control values) on both sides of the ligature (Laduron, 1980a,b). This pointed to a bidirectional transport of receptors along the axon. After 11 days, however, accumulation remained only on the proximal side of the ligation, suggesting that the muscarinic receptors originate from the perikarya of sympathetic neurons. The same technique was used for studies in sciatic and vagus nerves of rats (Wamsley et al., 1981). Following ligation, muscarinic receptors accumulated mostly on the proximal side of the ligature, with a much smaller buildup on the distal side. To summarize, the data strongly indicate that muscarinic receptors exist not only on the postsynaptic membrane, as classically believed, but also on presynaptic elements as well. It is believed that these presynaptic muscarinic receptors participate in the regulation of neurotransmitter release. C. ONTOGENESIS Numerous studies have been carried out to characterize the ontogenetic pattern of muscarinic receptors in various tissues, with special emphasis on the brain. It has been shown that in both mice (Aronstam et al., 1979; Egozi et al., 1980) and rats (Kuhar et al., 1980) the most rapid postnatal development of receptor density occurs in the medulla-pons,

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with considerably slower maturation in the cerebral cortex, basal ganglia, and hippocampus. Although the apparent affinity of 3H-labeled antagonists does not change significantly during development, there is an increase in the apparent affinity of agonists as evaluated by their ability to compete with SH-labeled antagonists. This is seen most strikingly in the mouse (Egozi et al., 1980) and rat (Soreq et al., 1982) cerebellum, in which the apparent affinity of carbamylcholine towards the muscarinic receptors increases about 20-fold during the first 30 days of life. It is interesting that this is accompanied by a change in the cellular localization of the receptors in this region, as revealed by autoradiographic techniques (Rotter et al., 1979~).Although most receptors are localized in the granular layer in newborn rats, labeled receptors are transiently seen in the intermediate Purkinje cell layer at 2 weeks, and in the adult animal they are found in the molecular layer. It was recently reported that destruction of cerebellar granule cells in newborn rats by X-irradiation leads to minor changes in SH-labeled antagonist-binding parameters (density and affinity); in contrast, as early as 12 days postnatally agonist binding already exhibited the highaffinity properties normally seen only in the fourth week (Soreq et al., 1982). This phenomenon did not occur in the mutant Gunn rat, in which the granular neurons die after completion of the normal cerebellar circuitry. It thus appears that synaptic contacts between different cell populations must be correctly established at a critical ontogenetic stage in order for muscarinic receptor affinity for agonists to develop normally. Another interesting phenomenon observed in the autoradiographic study occurred in the basal ganglia: in newborn rats the receptors appeared in discrete “patches” (Rotter et al., 1979c) that became progressively larger and gradually occupied more and more of the striatal neurophil, so that 10 days after birth they were confluent and the basal ganglia showed a homogeneous distribution of muscarinic receptors. This is reminiscent of a report of the “patchy” appearance of AChE-rich “islands” in the basal ganglia of newborn cats, which also disappeared upon maturation (Graybiel et al., 1981). Altered ontogenesis of muscarinic receptors can be induced by drug treatment. For example, treating newborn mice with a-thyroxine or P-methasone resulted in accelerated accumulation of receptors in several brain regions at 16 days postpartum, with a subsequent reduction in the level at 30 days (BenBaruch et al., 1981). Aging has also been shown to affect muscarinic binding parameters. In adenohypophysis homogenates obtained from aged (20 months old) female rats, ”-labeled antagonist-binding site density is about half of

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that in younger adult animals (Avissar et al., 1981~).This decrease is paralleled by a decrease in the proportion of the high-affinity agonistbinding sites from 57 to 21%. By contrast no changes were seen in either antagonist or agonist binding parameters in any of the rat brain regions studied. It was proposed that these changes are associated with the persistent estrous characteristics exhibited by aged female rats. Decreases in 3H-labeled antagonist-binding site density upon aging were also reported for postmortem human hippocampus (Nordberg and Winblad, 1981). However, no changes were detected in agonist-binding parameters.

VII. Solubiliration and Isolation of the Muscarinic Receptors

T h e first step required to purify receptors is the removal of the proteins from their natural environment, the membrane, in such a way as to retain their biological activity. Once the receptor is solubilized in active form, the specific protein can be purified using conventional methods. Because the concentration of receptors that bind neurotransmitters is very low, kilograms of tissue are needed in order to purify micrograms of receptors; the purification procedure has to be very efficient. Progress in purifying muscarinic receptors was impeded by difficulties in solubilizing the receptor in the active form. Nonionic and ionic detergents that were found suitable for solubilizing the nicotinic receptors (Eldefrawi et al., 1972) destroyed the activity of the muscarinic receptors from mammalian brain. 1% Lubrol-WX, 1% Lubrol-PX, 1% Triton X-100, and Brij each failed in solubilizing active muscarinic receptors from membrane (Beld and Ariens, 1974; Bartfai et al., 1974; Alberts and Bartfai, 1976; Hurko, 1978; Aronstam et al., 1978). T h e loss of binding activity caused by detergents indicates the intimate association of the muscarinic receptor with the lipid membrane. The question of whether the loss of binding activity in detergent-treated neuronal membranes is due to the action of the detergent on the membranebound receptor itself or consequent to the removal of the receptor from the membrane was therefore investigated. Reuss and Lieflander (1979) claimed that after Triton X- 100 treatment even membrane-bound receptors lost their ability to bind ligand and that therefore the loss of binding is not due solely to solubilization. Anionic and cationic detergents such as sodium deoxycholate or cetyltrimethylammonium bromide were found to have the same effect (Reuss and Lieflander, 1979).

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T w o groups found that muscarinic receptors can be solubilized by 2 M NaCl from rat brain (Bartfai et al., 1974) and ox brain (Carson et al., 1977). However, according to other reports, the solubilized protein binds atropine in a nonspecific manner without the characteristics of the muscarinic receptor (Aronstam et al., 1978; Hurko, 1978; Gorissen et al., 1980). T h e first report on the solubilization of active muscarinic receptors was that of Beld and Ariens (1974). Using 1% digitonin, a plant glycoside, 80% of the muscarinic receptors were reportedly solubilized from microsomal pellet of tracheal muscle whereas only 20% were solubilized from bovine caudate nucleus. T h e removal of lipids by hexane, according to this report, significantly improved the yield. Solubilization of the muscarinic receptor by digitonin in active form was confirmed by other groups (Aronstam et al., 1978; Hurko, 1978; Gorissen et al., 1978, 1981; Reuss and Lieflander, 1979; Repke and Matthies, 1980). There appear to be essential constituents in the membrane that if separated from receptor by detergent result in the loss of binding activity. The digitonin probably partially replaces some of these constituents, whereas other detergents do not. According to the first report on solubilization of the muscarinic receptor using digitonin, the residual activity after solubilization was retained in the pellet (Beld and Ariens, 1974), but according to Aronstam et al. (1978), 70% of the activity is destroyed by the digitonin. Hurko (1978) claimed that the digitonin destroys 50% of the binding activity. Carbamylcholine and atropine added before digitonin solubilization did not protect against loss of binding sites. It should be noted that digitonin was found to be more effective in solubilizing other receptors such as the p-adrenergic receptors (Caron and Lefkowitz, 1976), dopamine receptors (Gorissen and Laduron, 1979), and benzodiazepine receptors (Gavish et al., 1979). A combination of digitonin and sodium cholate (Cremo et al., 1981; Herron et al., 1982) solubilized 80% of atrial mu'scarinic receptors. T h e data from our laboratory indicated that this detergent mixture is not more effective than digitonin itself for solubilization of rat brain cortex. Carson (1982) showed that 15-30% of muscarinic receptors can be solubilized from bovine brain by a mixture of 0.1% sodium cholate and 1 M NaCl. We found (Gavish and Sokolovsky, 1982) that rat brain receptors can be solubilized by the zwitterionic detergent 3-[(3-chloroamidopropyl)dimethylamine]- l-propanesulfonate (CHAPS). It is important to note that the homogenates, the CHAPS treated pellet, and the soluble receptors showed similar affinity for antagonists and agonists. CHAPS seems to be a more promising detergent than digitonin for muscarinic receptor solubilization and purification because (i) yields for

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solubilization should be relatively high because 40-50% of the activity was retained in the pellet of membranes treated with CHAPS; (ii) CHAPS-solubilized receptors are more stable, that is, 95% of the activity is retained after 24 hr at 4";and (iii) digitonin precipitates within days whereas the CHAPS remains in solution. T h e molecular weight of the muscarinic receptors has been measured after labeling of the receptor in the membrane state and in the soluble state with radioactive muscarinic ligands that bind to the receptor reversibly (Gorissen et al., 1978; Haga, 1980a,b) or irreversibly (Fewtrell and Rang, 1973; Birdsall et al., 1979a; Amitai et al., 1982) (Fig. 5). Results have differed among laboratories using different ligands and techniques. According to Bartfai et al. (1974), there were two peaks of radioactivity obtained by gel filtration after solubilization by Triton X100 of rat cerebral cortex incubated with ['Hlatropine. T h e ['Hlatropine was bound to proteins with apparent molecular weights of 33,000 and 70,000. Gorissen et al. (1978) reported that digitonin-soluble preparations incubated with ['Hldexetimide and submitted to sedimentation through a sucrose gradient showed a peak with molecular weight of 200,000. Gel filtration of membranes incubated with ['HIQNB and solubilized by Lubrol-PX (Haga, 1980a) showed a peak with molecular weight of 86,000. OH 0

Q N3

Azido-N-methyl-4-piperidylbenzilate

Propylbenzilylcholine mustard

FIG.5. Structure of irreversible muscarinic ligands.

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Covalent binding of 3H-labeled propylbenzilylcholine mustard ([3H]PrBCM) to rat brain followed by solubilization by SDS and gel electrophoresis reveals that the labeled protein has a molecular weight of 83,000 (Birdsall et al., 1979~).T h e same technique applied to bovine caudate nucleus revealed a molecular weight of 75,000 10,000 (Reuss and Lieflander, 1979). On the other hand, [SH]PrBCM bound to 2 M NaCl solubilized rat brain mitochondria1 pellet showed in SDS-polyacrylamide gel electrophoresis a molecular weight of 30,000 (Alberts and Bartfai, 1976). Photoaffinity labeling (Amitai et al., 1982) of muscarinic receptor from rat cerebral cortex with azido-4NMPB discussed in detail in the following section, indicated that the ligand was covalently incorporated into a macromolecule with a molecular weight of 86,000 5,000.

*

*

VIII. Structure and Function Relationship of Murcarinic Receptors

Following the initial studies of photoaffinity labeling with [SH]azido4NMPB, this technique was used to label covalently specific muscarinic binding sites in various brain regions (Avissdr et al., 1982, 1983). Polyacrylamide-SDS gel electrophoresis of membrane preparations from various brain regions that were covalently labeled with azido-4NMPB yielded specific labeling profiles that can be divided into two groups. In preparations from rat hippocampus and cortex only one specifically labeled macromolecule of molecular weight 86,000 2 5,000 was observed, whereas in the rat medulla-pons, cerebellum, and atria two major labeled macromolecules were present, migrating at a position on the gel corresponding to M , = 86,000 & 5,000 and 160,000 ? 10,000. It is interesting that the former two regions contain mainly low-affinity agonist-binding sites, whereas the latter regions exhibit both low- and high-affinity states for agonists (see Section IV,A). Hence this correlation suggests that the two bands of 80,000 and 160,000 molecular weight may correspond to the receptor states that exhibit low and high affinity for agonists respectively. As described in Section IV,A, it was previously shown that interconversion between high- and low-affinity states can be manipulated chemically. For example, in the rat medulla-pons homogenates interconversion from a high-affinity to low-affinity state can be induced by guanine nucleotides. In contrast transition metals (e.g., Mn2+,Ni2+,Co2+)induce conversion from a low- to high-affinity state in cortical o r hippocampal membranes. Photolabeling and analysis of the covalently labeled macromolecule of medulla-pons preparations in the

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presence of p[NH]ppG revealed that the nucleotide treatment decreased the 160,000-MW band with concomitant increase in the 80,000MW band. A similar analysis of cortex preparations in the presence of 1 mM Mn2+ indicated the appearance of the 160,000-MW band in a preparation that originally contained only the 80,000-MW band. Thus, the 80,000- and 160,000-MW components may correspond to receptor states that exhibit low and high affinity for agonists, respectively. One likely possibility is that the 160,000-MW polypeptide is a dimer of the 80,000-MW component. The covalent nature of the bonds forming the heavier component is indicated by the fact that it does not dissociate in the presence of SDS and j3-mercaptoethanol. Treatment of membrane preparations from rat medulla-pons with alkali and hydroxylamine at room temperature leads to the appearance of all the specific labeling in a band with M , = 40,000. Treatment with NaOH at 0°C indicated that although the 160,000-MW band remained almost intact the 80,000-MW band disappeared with a concomitant appearance of a 40,000-MW band (Avissar et al., 1983). Thus, the muscarinic receptors can exist in two forms: (i) a dimer of molecular weight 80,000, composed of two 40,000-MW subunits joined via a covalent bond, a form of the receptor that is proposed to bind agonists with low affinity; (ii) two such dimers combined to form a tetramer (with a molecular weight of 160,000) containing one or more additional covalent bond(s), which may be different from those that exist in the original dimer. The tetramer is proposed to bind agonists with high affinity. The linear Scatchard plots obtained for the binding of antagonists in various brain regions could be explained by the assumption that antagonists do not discriminate between the two states of the receptor and bind to both with equal affinity. On the basis of the available data a tentative scheme which describes the biological architecture of the muscarinic receptors and the transition of receptor occupancy into a physiological response (Fig. 6) was recently suggested (Sokolovsky et al., 1983). For simplicity in this model the tetramer is described as a dimer of dimers in which ligand-sensitive intersubunit interactions are limited to a single binding axis (the one that exists in the original dimer). Thus, step a, the binding of the ligand A (agonist or antagonist), is followed by isomerization of the binding subunit and induces step b, a conformational change in a free subunit. The induced conformational change may or may not lead to cooperativity in step c, the binding of the following ligand molecule, depending on the exact structure of the specific ligand molecule and its interaction with the binding site. Thus for example, antagonists will yield noncooperative binding curves as long as they do not induce a conformational change

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AGONIST LOW A F F I N I T Y

STATE

I I I INTERCONV E R S ION

I I I

I 1 J I AGONIST HIGH AFFINITY STATE

\cb,

FIG. 6. Tentative scheme for ligand binding to the muscarinic receptor. A, a muscarinic ligand (agonist or antagonist); and ligand-free receptor subunits in the and , conformations of muscarink receptor diiner and tetramer, respectively; binding sites after isornerization in the dimeric and tetrameric forms, respectively; and , conformations of free subunits following a conformational change induced by ligand binding to neighboring subunits. This effect may lead to cooperativity.

0 0

D

x

0

that affects their binding to the neighboring subunit. However, if such a conformational change does occur antagonist binding may show cooperativity (see Section 111,B). The agonist-occupied receptors may be coupled to various processes that lead to a physiological response. The scheme depicts such coupling (double dashed arrows) to targets X (e.g., nucleotide cyclases, sex hormone regulation or release) and Y (e.g., Ca2+ channel or PI turnover). T h e model is also in accord with the existence of functional heterogeneity (see Section 111,A). Thus functional heterogeneity may be simply a manifestation of preexisting interconvertible binding sites in which the particular state of the binding protein(s) or polypeptide(s) is dictated by the nature and constituents of the local membrane milieu as well as the properties of the binding ligand. T h e experiments described above indicate that two types of a bond, probably an ester bond, that is alkali-labile contribute to the assembly of the muscarinic receptor. These bonds can be differentiated by their sensitivity to alkali at 0 and 25°C. These results will be consistent with the hypothesis involving glycosidic cross-linking, thioester bond formation (recently shown to exist in complement proteins) (Law et al., 1980), and/ or phosphodiester or pyrophosphate linkage, which is part of the phosphorylation/dephosphorylation mechanism. It is interesting to note in

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this context that one can induce rapid (within minutes) and reversible interconversion between the high- and low-affinity agonist-binding states of the muscarinic receptors. I n view of the correlation between the presence of 80,000- and 160,000-MW bands and the receptor’s states of low and high agonist affinity, it is tempting to suggest involvement of an enzymatic reaction in the covalent modification of the receptor dimers into tetramers and vice versa, which could account for the rapidity and reversibility of the process. A possible candidate for such a modification is a phosphorylating/dephosphorylating reaction. Consistent with this hypothesis is the fact that transition metals known to be involved in enzymatic phosphorylation reactions also induce interconversion of the muscarinic binding state and the recent observation that changes in phosphorylation are induced by muscarinic agonists (Michaelson et al., 1979; Burgoyne, 1980, 1981; Burgoyne and Pearce, 1982). Last but not least, one cannot exclude the possibility of an unique labile peptide bond in the muscarinic receptor similar to the Asn-Gly bond in collagen (Bornstein, 1970). Thus for example, conformational changes occuring during the receptor-ligand associations might induce the formation of a cis amide bond that would be more reactive to nucleophilic reaction then its “native” isomer, the trans amide bond.

IX. Concluding Remarks

Radioligand binding studies have proved to be very useful in determining the events that underlie the initial steps of the response of nerve cell membranes to agonists and antagonists. There is a great deal yet to be understood regarding biochemical, physiological, and behavioral roles of muscarinic receptors. It should be evident from the preceding discussion that the muscarinic receptor is likely to be a complex protein regulating a series of reactions in cells as a consequence of its activation by the neurotransmitter acetylcholine. T h e cells themselves, responding to this receptor-mediated stimulation, in turn also regulate the receptor. Knowledge of the properties of the various related enzymes and regulatory proteins is still limited, and so far only a fragmentary pattern of the cascade of events has emerged. It is clear that several enzymes such as phospholipase C and guanylate cyclase and several ion channel proteins are involved in the muscarinic response. It is not clear whether one o r more of these proteins is directly coupled to the receptor molecule itself. In brain tissue it might well be that the recognition sites are coupled to various kinds of amplifier systems by different molecular mechanisms.

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This variety might serve as the molecular basis for the diversity in pharmacological and biochemical profiles of muscarinic receptors located in various brain structures and in other tissues, for example, G protein, ion channels, or calmodulin. Further work on the solubilization and purification of the receptor is needed in order to throw more light on its mode of functioning and to pave the way for studying its interaction with other membranal components. Future investigations will exploit the initial observations of dialog and interplay between the muscarinic receptors and other neurotransmitter and hormone systems (see Fig. 4). One could expect that these investigations will contribute to our quest to understand the mechanism of action of receptors and are likely to have significant clinical and therapeutic implications. It is even possible that muscarinic receptors and their molecular regulation underlie critical brain functions such as learning and memory and that a better understanding of them will contribute to our understanding of disturbances in these functions with aging (for a review see Bartus et al., 1982) and contribute to rational therapy. Acknowledgments

During the last months of 1982, M. Sokolovsky wdS a Fogarty Scholar-in-Residence, Fogarty International Center, National Institutes of Health, Bethesda, Maryland 20205. References

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Vale, W., Rivier, D., Brown, M., Chan, L., Ling, N., and Rivier, J. (1976). In “Hypothalamus and Endocrine Function” (F. Labrie, J. Meites, G. Pelletier, eds.), p. 397. Plenum, New York. Warnsley, J. K., Zarbin, M. A. Birdsall, N. J. M., and Kuhar, M. J . (1980).BrainRes. 200, 112. Wamsley, J. K., Zarbin, M. A,, and Kuhar, M. J. (1981). Brazn Res. 217, 155-161. Waser, P. G. (1961). Phunnacol. Rev. 13, 465-515. Watanabe, A. M., McConnaughey, M. M., Strawbridge, R. A., Fleming, J. W., Jones, L. R., and Besch, H. R., (1978). J . B i d . Chem. 253, 4833-4836. Wei, J.-W., and Sulakhe, P. V. (1980). Naunyn-Schmiedebergs Arch. Pharmacol. 314, 51-59. Weinstein, H., Maayani, S., Srebrenik, S., Cohen, S., and Sokolovsky, M. (1975). Mol. Pharmacol. 11, 671-682. Yarnada, S., Yamarnura, H. I., and Roeske, W. R. (1980). Eur. J. Pharmacol. 63,239-241. Yamamura, H. I., and Snyder, S. H. (1974a). Proc. Natl. Acad. Sci. USA 71, 1725-1729. Yarnamura, H. I., and Snyder, S. H. (1974b). Mol. Pharmacol. 10, 861-867. Zalcman, S.J., Neckers, L. M., Kaayalp, O., and Wyatt, R. J. (1981). L$e Sci. 29, 69-73.

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PEPTIDES AND NOCICEPTION By D a n i e l Luttinger'

Biological Sciencer Rorearch Center and Tha Neurobiology Program Univonity of North Carolina School of Modicino Chapel Hill, North Carolina Daniel E. Hernandez

Biological Sciencos Research Center University of North Carolina School of Medicine Chapel Hill, North Carolina Charles

B. Nemeroff

Biological Sciences Reroarch Centor, Deportment of Psychiatry, and The Neurobiology Program University of North Carolina School of Medicine Chapel Hill, North Carolina

and Arthur J. Prange, Jr. Biologicol Sciences Reroarch Centor, Department of Psychiatry, and The Neurobiology Program University of North Carolina School of Medicine Chopel Hill, North Carolina

I. Introduction

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

11. Algesic Peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Bradykinin .................................................. B. Substance P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Opioid Peptides.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Enkephalins ................................................. B. /%Endorphin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Dynorphin .................................................. D. Other Opioid Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Naloxone-Sensitive Nonopioid Peptides ............................. A. Adrenocorticotropin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cholecystokinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Somatostatin ................................................

186 188 188 190 195 195 203 211 212 2 13 2 13 2 14 215

Present address: Department of Pharmacology, Sterling-Winthrop Research Institute, Rensselaer, New York 12144. SPresmtaddress: Departments of Psychiatry and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710.

185 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 25

Copyrjght 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-966825-5

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V. Naloxone-Insensitive Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Neurotensin. . . . . . . . . . . . . . . . . . .

2 16

C. Tuftsin.. . . . . . . . . . . . . . . .

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

E. Bombesin. . .

A. Thyrotropin-Releasing Hormone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Melanocyte-Stimulating Hormone Inhibitory Factor (MIF-I). . . . . . . . . . C. Melanocyte-Stimulating Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ....................................

225 226 226 227 227 232

1. Introduction

In recent years there has been an exponential increase in our knowledge of biological factors that influence the perception of pain. One area in particular that has contributed to this rapid growth was the novel discovery that nociception can be substantially altered by many peptides. Peptides are smqll chains of amino acids consisting of two or more amino acids that are cdhected by a covalent peptide bond between the carboxyl group of one amino acid and the amino group of another; the formation of each such connection is accompanied by the loss of one molecule of water. Proteins can contain any number of amino acids. If such molecules contain fewer than about 50 amino acids, one usually speaks of peptides; if they contain more than 50, one usually refers to them as proteins, although there is no fixed demarcation. Most of the peptides described in this review have been found to exist in the central nervous system (CNS). T h e neuroanatomical localization o f these peptides is not included in this chapter; however, when such localization is particularly germane, it is described. In general, the peptides described in this review have been reported to be heterogenously distributed in the CNS. T h e amino acid sequence of each of the peptides discussed is illustrated in Table I. Several of them have been found to be released from brain tissue and to bind to highly specific, reversible, and saturable receptors in a variety of brain regions. In addition, these substances are degraded by tissue peptidases, and their iontophoretic application results in alterations in the firing rate of certain CNS neurons. Based on these and other criteria, the hypothesis that they function as neurotransmitters or neuromodulators has been promulgated.

PEPTIDESEQUENCES ~~~

Substance

Peptide sequence

Molecular weight

Brady kinin Substance P Met-enkephalin Leu-en kephalin P-Endorphin (P-Lipotropin 61-91, human) Dynorphin 1-13 (porcine) Kyotorphin (Des,Tyr’)-y-endorphin (P-Lipotropin 62-77) Adrenocorticotropic hormone (ACTH, human)

Arg-Pro-Pro-Gly-Phe-Ser-Pro-PheArg Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-N H2 Tyr-GIy -Gly-Phe-Met

Cholecystokinin (CCK) octapeptide Somatostatin Neurotensin

Asp-Tyr(SOs)-Met-Gly-Trp-Met-Asp-Phe-N HZ Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-=hr-Ser-C~s I pClu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu

1638.12 1084.38

(Arg8)-Vasopressin

CJs-T yr-Phe-Gln-Asn-Chs-Pro-Arg-GIy -NH2

1084.38

Calcitonin (human)

C~s-Gly-Asn-Leu-Ser-Thr-C~s-Met-Leu-Gly-Thr-Tyr-Thr-Gln-AspPhe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-GIy-

341 8.4 1

Bombesin Thyrotropin-releasing hormone Melanocyte-stimulating hormone inhibitory factor (MIF-I) a-Melanocyte-stimulating hormone (a-MSH) Tuftsin

pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH~

Tyr-G1y-Gly-Phe-Leu

1060.24 1347.80 573.75 555.72 3465.63

Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-ProLeu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-GluTyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys 1604.19 Tyr-Arg

Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-LysLys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-A~a-Glu-Asp-

337.39 1696.22 4541.74

Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Gln-Phe 1142.31

Ala-Pro-NHZ pGlu-His-Pro-NHp

1620.12 362.42

Pro-Leu-Gly-NHZ

284.36

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Va1-N HP Thr-Lys-Pro-Arg

1665.05 500.46

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One of the most thoroughly investigated classes of neuropeptides is the opioids, which include fl-endorphin, dynorphin, y-endorphin, desTyr’ y-endorphin, a-endorphin, kytorphin, &endorphin, the exorphins (e.g., caseomorphin) and Met- and Leu-enkephalin. These peptides share many actions with morphine, a clinically effective opiate analgesic. The similarity in pharmacological effects of the opiate analgesics and the endogenous opioid peptides led to the suggestion that these substances play a physiological role in modulating an organism’s response to noxious stimuli. In addition, several other neuropeptides have been implicated in nociception because they have been observed to alter the response of laboratory animals and man to painful stimuli. T h e purpose of this review is to describe and evaluate critically the data concerning peptides and nociception. II. Algeric Peptides

A. BRADYKININ Bradykinin is a peptide (Table I) that has been reported to possess algesic (pain-producing) properties. Guzman et al. (1962) reported that intraarterial injection of bradykinin in both cats and dogs elicited a response that mimics the symptoms of pain produced by mechanical, thermal, or electrical nociceptive stimuli. Inoki et al. (1973, 1979) analyzed the superfusate from exposed tooth pulp of dogs following application of noxious stimulation. Mechanical, thermal, and electrical stimuli were utilized to induce pain. All three of these noxious stimuli increased the concentration of a substance in the superfusate that produced contraction of rat uterine smooth muscle. Characterization of this substance in the superfusate by various analytical techniques provided results consistent with identification of the active substance as bradykinin. In addition, the superfusate produced a painful sensation in man when applied to a cantharidin blister base preparation; the effect was similar to that produced by synthetic bradykinin. An algesic response to intraarterial (femoral) injection of bradykinin has been observed in guinea pigs using vocalization as the dependent variable (Adachi and Ishii, 1979). This algesic effect of bradykinin was dose-dependent. The peptide was extremely potent in this test; the threshold dose was in the range of 0.03-0.3 pg. T h e response to bradykinin had a latency of approximately 7 seconds, and no tachyphylaxis to this effect of the peptide was noted. T h e algesic response induced by intraarterial bradykinin in guinea

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pigs was attenuated by morphine and aminopyrine (Adachi and Ishii, 1979). In dogs Guzman et al. (1964) observed similar results with morphine but not with aminopyrine. Inoki et al. (1973) observed inhibition of release of a bradykinin-like substance from the stimulated dental pulp after treatment with morphine or aspirin. Cyclazocine, a benzomorphan derivative, suppressed the hind limb flexor reflex induced by bradykinin in intact rats (Satoh et al., 1979a). I n addition, the EEG arousal associated with tooth pulp stimulation is inhibited by cyclazocine. These findings agree with the observation of Inoki et al. (1979) described earlier. Further study (Adachi and Ishii, 1979) revealed that the increased vocalization induced by intraarterial acetylcholine, but not that induced by bradykinin, was antagonized by hexamethonium. This suggests that the effects of bradykinin are not mediated by cholinergic mechanisms. Prostaglandins (PGE1 and PGEp) do not induce vocalizations when given alone but do potentiate the effect of a low dose of bradykinin (0.1 pg). Juan and Seewann (1980) obtained similar results in an isolated perfused rabbit ear preparation, in which a prostaglandin antagonist, SC- 19220, reduced the algesic effect of bradykinin. Vasodilators, such as papaverine, aminophylline (both phosphodiesterase inhibitors), and verapamil (a calcium antagonist), also attenuated the algesic response to bradykinin. Low calcium concentrations, like verapamil, attenuated the bradykinininduced algesia (Lembeck and Juan, 1977). However, high calcium concentrations also attenuated the algesic effect of bradykinin. Thus, the role of calcium in bradykinin-induced algesia is unclear at present. Electrophysiological data support the hypothesis that bradykinin plays a physiological role in the processing of noxious stimuli. Belcher (1979) examined the effects of intraarterial bradykinin on the firing rate of cat dorsal horn neurons. Low doses of bradykinin (2.5-15 pg) preferentially activated cells that were also activated by noxious stimuli. Bradykinin was more potent in this regard than histamine, 5-hydroxytryptamine, and acetycholine. Larger doses of bradykinin (30-70 pg) appeared to be less specific; such treatment activated both nociceptive and nonnociceptive cells. Prostaglandin E2 enhanced the effects of bradykinin on nociceptive cells, a finding concordant with observations of vocalization (see previous paragraph). Soja and Sinclair (1980) reported discrepant findings in recordings from cat dorsal horn neurons. Most neurons that responded to noxious mechanical stimuli were also excited by noxious radiant heat, but the firing rate of only a small number of these cells was altered by intraarterially administered bradykinin, and more than half of these were inhibited. Sampling of different neurons within the dorsal horn may account for these discordant results.

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Both PGE2 and serotonin have been found to sensitize cat group I V muscle receptors to bradykinin (Mense, 1981). Because both serotonin and prostaglandin are believed to be released after tissue injury and group IV muscle receptors are activated by noxious stimuli, these findings are consistent with an algesic role for endogenous bradykinin. Study of visceral afferent nerve endings in the cat heart provide evidence that bradykinin may have both nociceptive and nonnociceptive functions (Baker et al., 1980). The nerve endings (AS or C fibers) described were characterized as either mechanosensitive (sensitive to light touch and changes in pressure) or chemosensitive (insensitive to light touch). Intracardiac bradykinin stimulated firing of both types of cells, and the authors suggested that chemosensitive endings appear to modulate cardiac nociceptors, because they were more strongly stimulated by bradykinin than were the mechanoreceptors. It appears that bradykinin in the periphery may play a role as one of the substances released from damaged tissue that produce pain. Its role in nociception within the CNS is unclear.

B. SUBSTANCE P Substance P was discovered in extracts of horse brain and intestine by von Euler and Gaddum in 1931. The peptide was identified using its hypotensive (invivo) and gut-contracting (in vitro) properties in rabbits. Almost 40 years later Chang and Leeman (1970) purified substance P from extracts of bovine hypothalamus and subsequently sequenced (Treager et al., 1971) and synthesized (Chang et al., 1971) the peptide. T h e sequence of the undecapeptide is shown in Table I. Both bioassay and radioimmunoassay techniques have revealed that substance P is distributed heterogeneously in the brain and spinal cord of a variety of mammals. The peptide is preferentially concentrated in the synaptosomal fraction of rat brain after density gradient centrifugation. Substance P is released from rat brain synaptosomes in vitro after electrical stimulation or addition of potassium; this release is calciumdependent (Lembeck el al., 1977).Substance P binding to rat brain membranes has been demonstrated (Nakata et al., 1977). The greatest density of binding sites was found in dorsal spinal cord and midbrain. Hokfelt and his colleagues (19’15) described substance P immunoreactivity in unmyelinated fibers of rat sensory nerves. In a later study the same group (Hokfelt et al., 1977) studied the distribution of substance P and enkephalin in the rat CNS, paying particular attention to its relation to pain pathways. Substance P-containing nerve terminals and

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cell bodies were observed in the periaqueductal gray, nucleus raphe magnus, substantia gelatinosa of the spinal trigeminal nucleus, and the dorsal horn of the spinal cord. T h e distribution of substance P was remarkably similar to that of Leu-enkephalin. Del Fiacco and Cuello (1980), using immunohistochemical techniques, reported that the electrolytic destruction of the Gasserian ganglion resulted in a marked reduction in substance P immunoreactivity in the substantia gelatinosa of the ipsilateral spinal trigeminal nucleus, whereas Leu-enkephalin-like immunoreactivity was not affected. This finding is concordant with those of Otsuka et al. (1975) who observed that dorsal root section selectively depleted substance P concentrations in the dorsal horn of the spinal cord without altering either substance P concentrations in other regions of the spinal cord or the concentrations of L-glutamate or yaminobutyric acid (GABA) in the spinal cord (Fig. 1). These findings are consistent with the hypothesis that substance P is present in primary afferent neurons. T h e hypothesis that substance P functions as a neurotransmitter in sensory afferents has been supported by considerable electrophysiological data. Krivoy et a1. (1963) demonstrated that a crude substance P extract of horse intestine potentiated dorsal root discharge in decerebrate cats. Otsuka and Konishi (1976) demonstrated release of immunoreactive substance P from the neonatal rat dorsal root after electrical stimulation. Approximately half (45%) of the cat dorsal horn neurons tested by Henry et al. (1 975) were excited by iontophoretically applied substance P, whereas the activity of Renshaw cells were depressed by the undecapeptide. These dorsal horn cells were also excited by application

Substance P

L-

Glutomate

GABA

FIG. 1. Effects of dorsal root section on substance P ( X lo-"' mole/@, L-glutamate mol/g) in cat spinal cords. Dorsal roots (below L5) were mol/g), and GABA ( X unilaterally sectioned, and after the survival time of 11-12 days, the distributions of these substances in the spinal cords (L5-SI) were examined. In each map, the lesioned side is represented on the left, and the intact side is on the right. Each value represents the mean of three to six determinations performed in five operated cats. T h e asterisk in substance P denotes p < .01 compared with the intact side. From Otsuka et al. (1975) with permission. (X

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of a noxious heat stimulus (Henry, 1976). Randic and Miletic (1977) confirmed and extended the findings of Henry et al. (1975). Neurons in the cat dorsal horn that were excited by noxious (thermal or tactile) stimuli were also responsive to substance P, whereas neurons responding to light tactile stimuli only were not depolarized by the peptide. Others have also obtained similar results (Sastry, 1979; Zieglgansberger and Tulloch, 1979). In a study designed to evaluate the effects of substance P on single neurons in the trigeminal nuclei oralis and caudalis in cats, Henry et al. (1980) found that all the neurons in the nucleus caudalis that responded to substance P also responded to noxious cutaneous stimuli and/or tooth pulp stimulation. Neurons responsive to innocuous orofacia1 stimulation did not respond to the application of substance P. Capsaicin appears to be toxic to substance P neurons and thus may be potentially useful in evaluating the role of endogenous substance P in the nervous system. Jansco et al. (1977) reported that capsaicin administration to newborn rats resulted in substantial degeneration of unmyelinated primary sensory neurons. In adult rats capsaicin produced a depletion of immunoreactive substance P in the dorsal horn of the spinal cord (Jessell et al., 1978). Gamse et al. (1981) and Nagy et al. (1981) demonstrated that the neurotoxic effects of capsaicin are not specific for substance P neurons. Administration of capsaicin to neonatal rats produced a depletion of somatostatin as well as substance P in sensory nerves and the dorsal half of the spinal cord (Gamse et al., 1981). This effect persisted for at least 4 months posttreatment. In adult rats the effects of capsaicin were slightly different. Both somatostatin and substance P were initially depleted; however, somatostatin but not substance P concentrations returned to normal within 4 months after capsaicin treatment. Several studies have investigated the effects of capsaicin on nociception (Yaksh et al., 1979; Hayes and Tyers, 1980; Nagy et al., 1981). Yaksh et al. (1979) noted that intrathecally applied capsaicin in rats produced a caudal muscle contracture followed by biting and scratching, which persisted for approximately 10 min. Hylden and Wilcox (1981) have observed a similar behavioral sequence in mice following intrathecal substance P injection. These data are consistent with the hypothesis that capsaicin induces an immediate release of substance P. Twenty-four hours after capsaicin treatment, Yaksh et al. (1979) noted that rats exhibited antinociceptive responses when compared to controls in four commonly used analgesia tests, that is, in tail flick, phenylquinone-induced writhing, formalin injection into a hind paw, and hot plate (55°C) tests. These antinociceptive effects persisted for prolonged periods (up to 5 months) following a single intrathecal capsaicin injection as assessed with

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the hot plate and tail flick tests. The rats responded normally when noxious pinch and a 70°C thermal probe to the caudal region of the back were applied. T h e lack of responsiveness in the hot plate test was seen only in rats that showed a depletion of substance P in the lumbar spinal cord. Nagy et al. (198 1) obtained concordant results in rats 4 weeks after intrathecal administration of capsaicin, but in these animals with elevated pain thresholds, both somatostatin and the biosynthetic enzyme for y-aminobutyric acid(GA3A)-containing neurons (i.e., glutamic acid decarboxylase, GAD) were depleted as well as substance P. Hayes and Tyers (1980), utilizing subcutaneous (sc) injections of capsaicin, reported that substance P concentrations were reduced in dorsal horn and skin, but not ventral horn. Although nociceptive pressure and chemical thresholds were elevated in these capsaicin-treated rats, thermal thresholds (tail flick) were not altered. Why Yaksh et al. (1979) found capsaicininduced alterations in thermal but not mechanical antinociception, whereas Hayes and Tyers obtained opposite results, is not clear but may be due partly to the difference in the route of capsaicin administration. Finally, in one report intracerebroventricular (icv) capsaicin administration has been found to alter basal nociception only at a single time point, 72 hr postinjection (Bodnar et aL, 1982a). Moreover, the effects of morphine were attenuated by capsaicin treatment. These effects were associated with no decrease in substance P. In fact, an increase in the concentrationof substance P in the amygdala was noted, suggesting that these effects may not involve substance P-containing neurons. Lembeck et al. (198 la) studied the effects in mice of intraspinal injection of two substance P analogs (i.e., [DPro2,~ P h e ’ ,~Trp~]-substance P and [DPro2, ~Trp’~~]-substance P) with purported antagonistic effects. Tail flick withdrawal latency was increased by both analogs for approximately 15 min postinjection. Substance P produced the opposite effect, a hyperalgesic response, in the tail flick withdrawal test. As described above, intrathecally administered substance P (1-200 ng) produces a biting and scratching response, which Hylden and Wilcox (1981) interpreted as a nociceptive effect. This finding is consistent with the view that substance P acts as a sensory afferent transmitter mediating responses to noxious stimuli. However, Doi and Jurna (1981) have found that intrathecally administered substance P (0.1- 100 pg) increased the latency of the tail flick response in rats. Because this effect was blocked by naloxone, a substance P-opioid peptide interaction was hypothesized to exist (see following paragraphs). In rats suffering from chronic pain induced by adjuvant-induced polyarthritis, sciatic nerve substance P concentrations were found to be increased (Lembeck et al., 1981b). No alteration was observed in sub-

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stance P concentrations in hind paw skin, dorsal root ganglia, dorsal root, o r dorsal spinal cord. The authors postulated that the alterations in substance P concentrations may reflect adaptative changes to a chronic noxious stimulation. It appears that the available data support the hypothesis that substance P is indeed a neurotransmitter of primary afferent neurons. This peptide exerts a powerful depolarizing action on neurons in the spinal cord, its concentration in the dorsal horn is reduced after dorsal root section, intrathecal administration of substance P produces hyperalgesia, and substance P antagonists and neurotoxins produce analgesia. As briefly noted in a previous paragraph, interactions of substance P with opiates and opioid peptides have been studied. Jessell and Iversen (1977) reported that opiate analgesics, including morphine, the stable enkephalin analog (DAla2,Met)-enkephalinamide, and P-endorphin, inhibit potassium-induced substance P release from rat trigeminal nucleus, but not from the substantia nigra. Similar inhibitory effects have been reported by Mudge et al. (1979), using cultured sensory neurons, and by Yaksh et al. (1980), studying release in vivo by activation of high-threshold peripheral afferents. Furthermore, Vacca et al. (1980) have reported that chronic morphine treatment increased the concentration of immunoreactive substance P in neuronal processes in the spinal cord. Duggan et al. (1979) have obtained evidence that the demonstrated opioid peptide-substance P interactions in spinal cord may not be identical in all areas of the spinal cord. They described in anesthetized cats sites in the substantia gelatinosa at which Met-enkephalin reduced excitation induced by noxious stimuli applied to the skin, whereas substance P was without affect. It is essential to recognize that substance P has been reported to produce both analgesia and hyperalgesia, depending in part on the route of administration employed. Mohrland and Gebhart (1979) found that low doses of intraperitioneally (ip) administered substance P (<0.25 mg/kg) induced antinociception in rats as assessed in a tail flick, but not in a hot plate test. Similar results were obtained following microinjection of the peptide into the periaqueductal gray matter. Malick and Goldstein (1978) also observed an increased tail flick latency in rats after substance P injection into the periaqueductal gray matter. This antinociceptive effect was found to be reversed by pretreatment with the opiate antagonist naloxone. Concordant findings after substance P injection intracisternally (ic) (2 ng) or ip (5 ng or 1 pg) in mice have been reported (Stewart et al., 1976). This antinociceptive effect of intracerebrally applied substance P was antagonized by naloxone, and morphine-tolerant mice were unaffected by substance P. Szreniawski et al. (1979) have also

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reported that substance P, administered ip, increased the latency to paw lick on a hot plate and increased the threshold for footshock-induced jump, flinch, and vocalization in mice. These effects were not observed until more than 60 min postinjection. These antinociceptive effects of the peptide were blocked by naloxone (Szreniawski et al., 1979) and by serotonin depletion (Meszafos et al., 1981). In contrast to the findings just described, Growcott and Shaw (1979) and Goldstein and Malick (1979) did not observe alterations in nociception following ip administration of substance P. Substance P has recently been shown to inhibit competitively enkephalin-degrading aminopeptidase activity in vitro (Barclay and Phillipps, 1980). If this occurs in vim, substance P may increase enkephalin concentrations. This may account for substance Pinduced, naloxone-reversible antinociception. Our group (Nemeroff et al., 197913) reported that ic injection of substance P (1 pg) produced hyperalgesia in mice in a tail immersion test. Share and Rackham (198 1) observed reciprocal hindlimb scratching induced by intracerebral substance P in mice. Narcotic analgesics prevented this effect (mixed agonist-antagonists were without affect). Share and Rackham (198 1) suggested that this scratching involves stimulation of nociceptive pathways. These two findings taken together suggest that under certain circumstances supraspinal administration of substance P may induce a hyperalgesic response. Two important variables in determining the effects of substance P on the response to noxious stimuli have been described. T h e first is dose (Frederickson et al., 1978; Frederickson and Gesellchen, 1980). Low icv doses in mice produce analgesia that is naloxone reversible. At higher doses antinociceptive effects are not observed but can be seen when combined with baclofen (a putative GABA agonist). Hyperalgesia is observed when high doses of substance P are combined with naloxone. A second important variable is the animal’s baseline response to noxious stimuli (Oehme ct al., 1980a,b). Substance P-induced antinociception is observed in animals that are relatively sensitive to noxious stimuli, whereas substance P-induced hyperalgesia is often observed in animals that are relatively insensitive to noxious stimuli.

111. Opioid Peptides

A. ENKEPHALINS T h e observations that led to the discovery of the endogenous opioid peptides was based on a series of observations utilizing two main ap-

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proaches. Reynolds (1969) discovered that electrical stimulation of the periaqueductal gray (PAG) of rats produced significant antinociception. These studies were subsequently confirmed and extended by Mayer et al. (197 l ) , who observed that the opiate antagonist naloxone at least partly antagonized stimulation-induced analgesia. This has been confirmed by others including Mayer et al. (1971) and Akil et al. (1976). A second approach, utilizing biochemical techniques, resulted in the identification of specific opiate-binding sites in brain tissue (Goldstein el al., 1971). Shortly thereafter three independent research groups reported on their discovery of stereospecific opiate-binding sites within the CNS (Simon et al., 1973; Pert and Snyder, 1973; Terenius and Wahlstrom, 1975a). Taken together, the findings of naloxone-reversible analgesia after stimulation and the presence of specific opiate-binding sites in brain led to the conclusion that an endogenous opiate ligand must exist. I n 1975 several research groups independently demonstrated that brain and pituitary extracts contained compounds with opiate-like activity (Terenius and Wahlstrom, 1975b; Hughes, 1975; Pasternak et al., 1975; Cox et al., 1975). Further characterization resulted in the successful identification of two pentapeptides (Table I), Met- and Leu-enkephalin (Hughes et al., 1975). In addition /?-lipotropin (/?-LPH),a pituitary peptide comprising 91 amino acids, was shown to contain a fragment (/?LPH6L-91)with opiate activity. This was termed the C-fragment or /?endorphin (Bradbury et al., 1976; Cox et al., 1976; Li and Chang, 1976). T h e amino acid sequence of Met-enkephalin is contained within /?-LPH and more specifically within /?-endorphin. Since that time a veritable explosion in the area of opioid peptide research has occurred, and it is evident that as the field has grown in complexity, it has become increasingly difficult to summarize the various aspects of all the current work on the opiate peptides in a single review. For this reason and the sake of brevity, we will in this section discuss rather succinctly the literature on the enkephalins (Met- and Leu-enkephalin) only as it relates to nociception. T h e early studies demonstrated that icv-administered enkephalins produce antinociception of short duration in the tail flick test in rats (Belluzzi et al., 1976; Buscher et al., 1976). These studies were consistent with the view that the enkephalins participate in physiological pain perception and transmission. For this reason it is clearly of importance to identify the neuroanatomical distribution of enkephalins in areas involved in nociception. T h e techniques employed include specific radioimmunoassays (RIA) for the enkephalins (Kuhar et al., 1973; Childers et al., 1977; Miller et al., 1978; Duka et al., 1978) as well as immunohistochemical techniques (Watson and Barchas, 1979; Sar et al.,

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1978; Hunt et al., 1980). T h e results obtained from such studies must be interpreted with caution. A major confounding variable is, of course, the specificity of the antibody employed. For example, it has been shown that an antibody raised against Met-enkephalin will have considerable cross-reactivity against Met-enkephalin-Arg6-Phe7(Miller, 1983). Similarly, antibodies used to localize Leu-enkephalin have been shown to exhibit cross-reactivity to dynorphins (Goldstein et al., 1979). This problem is exacerbated by the fact that several naturally occurring enkephalin-related molecules have been discovered and characterized and still others may yet be found. The RIA procedures can, of course, be made more specific by use of prior separation techniques. This is unfortunately not the case for immunohistochemical methods. Therefore, it is quite possible that not all the enkephalin-positive staining is indeed specific for either Met- or Leu-enkephalin. The presence of enkephalin-like immunoreactivity in the CNS, peripheral nervous system, and endocrine tissue, in addition to the fact that enkephalin staining has also been found in invertebrates, demonstrates that the enkephalins have a widespread anatomical and phylogenetic distribution (Simantov et al., 1976). However, as noted earlier, we have restricted our discussion of the distribution of the enkephalinergic pathways to those structures believed to be involved in the mediation of pain perception. High levels of Met-enkephalin-like immunoreactivity have been found in the superficial layers of the spinal cord (Elde et al., 1976; Sar et al., 1978; Hunt et al., 1980). These layers also receive substantial input from small diameter myelinated and unmyelinated primary afferent fibers (Ramon y Cajal, 1909; Light and Perl, 1977), some of which certainly transmit information about noxious stimuli (Price and Dubner, 1977). Ruda ( 1982) demonstrated synaptic interactions between dorsal horn neurons that project to the thalamus and enkephalin-containing axonal endings by combining the techniques of immunocytochemical localization of enkephalin and retrograde transport of horseradish peroxidase. Thalamic projection neurons (TPN) are believed to represent a distinct population of dorsal horn neurons (Adams, 1977), the majority of which respond to noxious stimuli (Trevino et al., 1972; Willis et al., 1974; Giesler et al., 1976; Price et al., 1978). T h e relationship of TPN to the enkephalinergic system is potentially important because these neurons may be responsible for the transmission of noxious stimuli from the dorsal horn to higher centers of the CNS. It has been proposed that either intrinsic dorsal horn neurons or neurons with more rostrally located cell bodies could be the source of the

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enkephalin-containing axonal terminals that contact lamina V TPNs (Sar et al., 1978; Hokfelt et al., 1979). Evidence has accrued that supports both views: There is a population of enkephalin-containing neurons in the brainstem that projects to the spinal cord and in addition, certain of the nerve endings identified appear to originate from intrinsic dorsal horn neurons. The opioids appear to act in part by modulating transmission of nociceptive information from the periphery to higher centers by a direct action on the response of TPNs in the dorsal horn to primary afferent input (Ruda, 1982). This postulate is consistent with the previous observations of Fields et al. (1980), who showed that cutting the sciatic nerve, which causes partial destruction of small diameter primary afferents, reduced the number of both p and 6 opiate-binding sites on dorsal roots. This suggests that both types of opiate receptors exist on small diameter primary afferents. Furthermore, there is evidence that both exogenously administered opiates and endogenous enkephalins act both on postsynaptic receptors in dorsal horn and presynaptic receptors to block transmitter release from nociceptive primary afferent terminals, (LeBars et al., 1976; Jessell and Iversen, 1977; Mudge et al., 1979; Duggan et al., 1979). Several investigators (Sar et al., 1978; Barchas et al., 1978; Watson and Barchas, 1979) have studied the distribution of enkephalins in the CNS and have reported high concentrations in the globus pallidus, medial hypothalamus, thalamus, basal ganglia, amygdala, central gray, and nucleus accumbens and very low concentrations in the cerebellum. The pattern of distribution of both Met- and Leu-enkephalin appeared quite similar. Sar et al. (1978) have studied the distribution of enkephalin immunoreactivity in rat brain and spinal cord by using immunoperoxidase methods with antisera specific to Leu- or to Met-enkephalin. Immunoreactive staining for both pentapeptides was observed to be similar in nerve fibers, terminals, and cell bodies in many regions of the CNS. The regions of localization of enkephalin fibers and terminals in the forebrain include the lateral septum, central nucleus of the amygdala, area CA2 of the hippocampus, certain regions in the cortex, the corpus striatum, bed nucleus of the stria terminalis, hypothalamus, thalamus, and subthalamus. In the midbrain they include the interpeduncular nucleus, periaqueductal gray, and reticular formation. In the hind brain they include the parabrachial nucleus, locus coerulus, raphC nuclei, cochlear nucleus, nucleus tractus solitarii, trigeminal nerve nucleus, motor nuclei, and certain cranial nerves. In contrast, enkephalin cell bodies appear sparsely distributed in the telencephalon, diencephalon, mesencepha-

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lon, and rhombencephalon. Regions that contain enkephalins have been reported also to possess opiate receptors. It has been shown that the magnocellular reticular nucleus (NRPG) in the rat projects primarily to lamina 1-111 and V-VII of the spinal horn, areas that receive nociceptive input (Basbaum et al., 1978). Moreover, electrical stimulation of the bulbar reticular formation of the rabbit inhibits neuronal responses to noxious stimulation in lamina V cells of the spinal cord dorsal horn (Takagi et al., 1975). These observations indicate that a descending inhibitory system terminating in the dorsal horn of the spinal cord originates in the NRPG. Since Goldstein and co-workers ( 1971) established rigorous criteria for distinguishing between specific and nonspecific opiate binding, extensive work has been done to identify different types of opiate receptors. Based on differences in the potency of opiate alkaloids and opioid peptides observed in different tissues, Lord et al. (1977) have proposed two categories of opiate receptor, p and 6, which can be differentiated by binding affinities of the various opiates. Using this criterion, Fields et al. (1980) found in rats that primary afferent fibers, dorsal root, and the dorsal horn contain both p and 6 opiate-binding sites because binding to the selective radioligands [3H]morphine ( p sites) and [ ' H ] ~ A l a ~ - L e u ~ enkephalin (6 sites) were observed. Furthermore, transection of the sciatic nerve led to significant reduction in the number of both types of opiate receptors, suggesting that these types of receptors may exist on small diameter primary afferents. Cesselin et al. (1980), using ["HILeuenkephalin, confirmed the distribution of specific binding sites in the dorsal root of the spinal cord. In addition, opioid receptors that preferentially bind ethylketocyclazocine ( K receptor) have been described. Receptors for the enkephalins appear to be widely distributed in the brain (Kuhar et al., 1973; Atweh and Kuhar, 1977; Miller et al., 1978). However, it remains to be determined whether all these different types of opiate receptors are distributed in areas associated with pain control. These results are in agreement with previous findings (Madden et al., 1977; Terenius, 1977) suggesting the existence of multiple receptor sites for Leu-enkephalin, some of which were not affected by a narcotic antagonist. Many electrophysiological studies have been conducted with the enkephalins. In general, these substances appear to depress neuronal firing rates in many different regions of the CNS (Hill and Pepper, 1976, 1977; Denavit-Saubie et al., 1978; Pepper and Henderson, 1980; Muehlechaler et al., 1980; Ono et al., 1980), although some excitatory responses have been observed. The effects of opioid peptides are usu-

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ally, but not always, inhibited by naloxone (Bloom and McGinty, 1981). Apart from the stereospecific opiate and opioid peptide-induced excitation of Renshaw cells (Duggan et al., 1976; Davies and Dray 1978); most effects of opiates on spinal cord neurons are inhibitory (Davis and Dray, 1978); in this respect nerves in laminae I1 and I11 are particularly sensitive to inhibition by opiates. This observation may reflect the presence of enkephalinergic interneurons and opiate receptors in the same area. It is now well established that both Met- and Leu-enkephalin produce an antinociceptive response in a variety of different experimental conditions. However, as first demonstrated by Belluzzi et al. (1976), this effect is of very short duration, possibly because of rapid degradation of the enkephalins following intracerebral injection. There have been some conflicting reports concerning the activity of the enkephalins in tests designed to screen substances with potential analgesic activity (Leybin et al., 1976). In a study comparing the analgesic effect of Met-enkephalin with other lipotropin fragments that contain the entire structure of Met-enkephalin at the NH2 terminus, Graf et al. (1976) observed that the magnitude of the antinociceptive effect was a function of the length of the peptide sequence. Met-enkephalin was the least potent and P-LPH6,-91 the most potent. The met-enkephalin effect was brief, lasting approximately 6-8 min. Injection of morphine or enkephalins into certain areas of the brain (e.g., periaqueductal gray, raphC nucleus) is known to produce naloxone-reversible antinociception (Pert and Yaksh, 1974). This finding, together with what is known about the other chemically defined neurotransmitter pathways, suggests that enkephalins as well as morphine may be acting upon descending noradrenergic (Cannon and Liebeskind, 1979; Rudy and Yaksh, 1980) and serotonergic (Tenen, 1968; Yaksh et al., 1976) pathways. In addition, intrathecal administration of narcotics or enkephalins produces antinociception (Yaksh and Rudy, 19’18; Yaksh et al., 1976) that apparently is mediated in the substantia gelatinosa (Duggan et al., 1977). Thus, the possibility that both spinal and supraspinal sites could be responsible for the induction of antinociception following opioid peptide administration must be considered. The effects observed after injection of the peptide are believed to reflect the response of neurons to an increase in the synaptic concentration of endogenous opioid peptides (Miller, 1983). Several investigators have confirmed and extended the work of Belluzzi et al. (1976) by demonstrating analgesia in the tail flick test in rats after microinjection of the enkephalins directly into the periaqueductal gray (Chang et al., 1976; Malick and Goldstein, 1977). Takagi (1980) has demonstrated that microinjection of enkephalins, as well as morphine,

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20 1

into the nucleus reticularis paragigantocellularis produced a naloxonereversible antinociception in rats, whereas microinjection of enkephalins o r morphine into other more caudal brain regions (e.g., nucleus reticularis pointis caudalis, nucleus reticularis lateralis) were ineffective in producing dose-dependent antinociception. The medial bulbar reticular formation including the nucleus reticularis paragigantocellularis contains cells that respond maximally or exclusively to peripheral noxious stimuli (Pearl and Anderson, 1978); these cells also give rise to the reticulo-spinal pathways (Peacock and Wolstencroft, 1974). Vaught and Takemori (1979a,b) have compared the antinociceptive effects of Met- and Leu-enkephalin and have noted differences in the effects of these two peptides. Leu-enkephalin, but not Met-enkephalin, enhanced morphine-induced antinociception in mice, although neither peptide altered the concentration of morphine in the brain. Further studies suggested that the effects of Leu-enkephalin are receptor-mediated and dependent on the intact peptide. This finding emphasizes that the effects of these two peptides are not exactly identical. As described above, one of the most striking effects of the enkephalins is the brevity their antinociception in rodents. It has been suggested that this is due to the rapid degradation of these peptides by brain peptidases. For this reason, the major strategy employed to obtain more prolonged action of the enkephalins has been directed toward the development of structural analogs that are resistant to biological degradation and also to the development of peptidase inhibitors, that is, compounds designed to produce a competitive antagonism of the enzymatic degradation of enkephalins. In the section that follows research conducted on the structural analogs of the enkephalins is summarized. T h e basic approach has been to synthesize enkephalin analogs with single or multiple substitutions at various positions in the molecule and/or to increase the size of the peptides. Hexapeptide esters derived by addition of Lys or Arg to the Nterminus of Leu-enkephalin methyl ester are equipotent to Leuenkephalin, and analog esters composed of N-terminal Phe, D A s ~or , pyro-Glu have significant activity. Addition of Arg, Gly, Phe, or Tyr to the N-terminus of Met-enkephalin results in very little loss of activity (Morley, 1980). If Met-enkephalin is extended by adding more than one amino acid residue, then the analog becomes weakly active, suggesting that similar extensions of stable enkephalins may provide highly active analogs (Dutta etal., 1978). Single amino acid substitutions in either Leuor Met-enkephalin using D-amino acids have proved to be more effective than those produced by replacements with L-amino acids. In this context the DAla*-substituted congeners have been reported by various investi-

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gators to possess enhanced analgesic potency in comparison to Met-enkephalin (Coy et al., 1976; Malick and Goldstein, 1977). Coy et al. (1976) demonstrated that (DAla2, Met)-enkephalin and (DAla', Met)-enkephalinamide were very potent peptides both tested in vitro in the mouse vas deferens and as indicated by their ability to compete with enkephalin for opiate receptors from homogenates of rat brain. In addition, Malick and Goldstein (1977) demonstrated that (DAla2,Met)-enkephalin induces antinociception following ic injection. Structural or conformational changes of the Gly or Phe moieties in positions three and four, respectively, have indicated that these amino acid residues as well as the Tyr' are essential for the expression of biological activity of the enkephalins including antinociception (Morley, 1980). Only modest increases in potency have been reported as a result of structural changes of position 5 ; these analogs are, however, invariably active. Supporting this finding is the work of Morgan et al. (1977), who reported that Tyr-~Ala-Gly-Gly-NH(CH2)2Phis almost as potent as (DAla2,Met)-enkephalin in the guinea pig ileum assay but only weakly active in the mouse vas deferens assay. Another approach employed has been the synthesis of compounds with multiple substitutions. One of the best studied series of such compounds has the general formula Tyr-X-Gly-Phe(or MePhe)- Y, where X is DAla or DMet and Y represents one of a wide variety of amino acid residues and their derivatives or other organic radicals. One such analog, Tyr-DAla-Gly-Phe-LeuOMe, was shown to be approximately 5-6 times more potent than Met-enkephalin; this study confirmed the importance of the Tyr' and Gly3 residues (Dutta et al., 1978). In summary, the synthesis of enkephalin analogs has been the focus of much attention, which has resulted not only in the synthesis of congeners with enhanced analgesic activity after intracerebral injection but also has raised the possibility of clinical use of these compounds. This latter consideration is exemplified by the findings of Roemer and Pless (1979), who found that substitution of Gly in position two of the naturally occuring Met-enkephalin by DAla, methylation of both Tyr and Phe in position one and four, respectively, and conversion of the Met5 to the corresponding alcohol sulfoxide produced an orally active enkephalin analog with marked antinociceptive activity as assessed in the tail flick test in mice. Considerable attention has been directed toward the development of inhibitors of enkephalin degradation to prolong the biological activity of these opioids. In early studies with the enkephalins it became evident that these peptides are rapidly degraded both in vivo and in vitro (Hughes, 1975). This rapid degradation of enkephalins appears to be

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due to the actions of both an aminopeptidase and a unique endopeptidase termed enkephalinase (Lane et al., 1977). Although initial studies (Benuck and Marks, 1979) suggested that enkephalinase and angiotensin converting enzyme (ACE) were identical, subsequent use of selective inhibitors and gel chromatography methods have clearly indicated that these are in fact two separate enzymes (Goldstein and Snyder, 1979; Arregui et al., 1979). Hudgin et al. (1981) have found that captopril and the free acid of MK-421, inhibitors of ACE, were poor inhibitors of enkephalinase. The activity of brain aminopeptidase(s) that results in the rapid degradation of the enkephalins (and endorphins) is inhibited by complex natural products such as bacitracin, puromycin, amastatin, and bestatin (Hayashi, 1978; Barclay and Phillipps, 1980; Hersh and McKelvy, 1981; Hersh, 1981; Wagner et al., 1981); many of these substances also inhibit the breakdown of several other peptides, for example, thyrotropin-releasing hormone (TRH). Thiorphan ([~~-3-mercapto-2-benzyl-propanoyl]-g~ycine), a specific endopeptidase inhibitor, has been reported to inhibit the in vitro degradation of enkephalins from washed particulate fractions of rat striaturn, to potentiate the analgesic effects of (DAla2, Met5)-enkephalin, and to produce naloxone-reversible analgesia when injected icv in mice (Roques et al., 1980). Yaksh and Harty (1982) have reported that intrathecal administration of thiorphan, although it produced no effect alone, potentiated the analgesic effects of intrathecal administration of (DAla', Met)-enkephalin in rats using the hot plate and tail flick tests. In summary, the existence of a specific brain endopeptidase (enkephalinase) that degrades enkephalins has been established but whether the aminopeptidases that degrade these pentapeptides in vitro act in the degradation of enkephalins in vivo remains the subject of current study.

B. P-ENDORPHIN I n this section literature concerning P-endorphin and nociception is described. One caveat, however, is that many of the techniques utilized cannot easily discriminate between an effect of P-endorphin or one of the other opioid peptides. The following discussion is not meant to be comprehensive but rather attempts to highlight the different techniques utilized to assess the potential physiological role of P-endorphin in nociceptive processes. @-Endorphin (see Table I) is found in high concentrations in the

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pituitary (Teschemacher et al., 1975), and within the pituitary the highest concentrations are present in the pars intermedia (Bloom et al., 1978). Various brain regions contain relatively high concentrations of the peptide, although these concentrations are lower than those found in the pituitary. I n the CNS high concentrations are found in the hypothalamus, midbrain, septum, and limbic areas (Bloom et al., 1978; Krieger et al., 1977). As discussed in the enkephalin section (see Section III,A), there appears to be more than one receptor activated by opioid peptides (Cowan et al., 1979; Wuster et al., 1979). The E receptor seems to be the most specific for p-endorphin (Wuster et al., 1979), although P-endorphin apparently also binds to p and 6 receptors (Vaught et al., 1982). The characteristics of [3H]/3-endorphin binding to rat brain homogenates have been described by Akil et al. (1980). The binding was found to be saturable, stereospecific, and of high affinity. P-Endorphin binding was displaced by sodium, naloxone, and adrenocorticotrophic hormone 124 (ACTH1-P4).T h e dissociation rate of P-endorphin was tenfold slower than that of naloxone. Li et al. (1980) studied P-endorphin binding and analgesic activity of several different P-endorphin analogs and observed a dissociation between binding affinity and analgesic activity. Thus, some analogs effectively bound to receptors with high affinity but did not produce antinociception. This discrepancy might be due to differences in species used for each test (rats versus mice), to rapid degradation of some of the analogs in vzvo, or to some of the analogs having mixed agonist-antagonist properties. The administration of P-endorphin into the CNS has repeatedly been found to produce an antinociceptive response (Bradbury et al., 1977; Szekely et al., 1977; Tseng et al., 19’77, 1979; Foley et al., 1979; Nemeroff et al., 1979b; Veda et al., 1979).p-Endorphin-induced antinociception is antagonized by naloxone and shows cross-tolerance with morphine (e.g., Szekely et al., 1977; Tseng, 1981). /3-Endorphin-induced antinociception has been observed in a variety of mammalian species. In humans intrathecally administered P-endorphin produced a long-lasting analgesic effect (Oyama et al., 1980), and the peptide has been reported to be partially effective after intravenous administration (Catlin et al., 1977). In animals antinociception has been demonstrated in a variety of tests including tail flick, hot plate, acetic acid-induced writhing, and ice water wet shaking techniques (Loh et al., 1976). Intracerebra1 injections of P-endorphin into the periaqueductal gray, nucleus accumbens, anterior hypothalamus, and medial preoptic area have been reported to induce antinociception in rats (Tseng et al., 1980). Intravenous administration of fl-endorphin has also been reported

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to produce antinociception in rodents (Tseng et al., 1976; Holaday et al., 1977). The antinociception was observed in both hot plate and tail flick tests (Tseng et al., 1976). This is of particular interest because the highest concentrations of p-endorphin are found in the pituitary and suggests that release of P-endorphin from this organ may be involved in nociception. Consistent with the antinociceptive effects of exogenously administered P-endorphin are the results obtained in other experimental paradigms. Implantation of the peptide-producing MtT-F4 tumor in rats, which results in high blood concentrations of p-LPH, p-endorphin, ACTH, growth hormone, and prolactin, also results in naloxone-reversible analgesia (Galeano et al., 1980). Furthermore, bacitracin, a nonspecific inhibitor of opioid peptide degradation, induces naloxone-reversible antinociception in mice (Simmons and Ritzmann, 1980) and rats (Patthy et al., 1977). Several studies indicate complex processing of p-endorphin in the pituitary. Thus, P-endorphin appears to be a cleavage product of a larger prohormone, proopiocortin. Proopiocortin appears to be a prohormone for several other hormones as well. These include ACTH, pLPH and a- and y-endorphin, and a-melanocyte-stimulating hormone (a-MSH) (Mains et al., 1977; Rubinstein et al., 1978; Nakanishi et al., 1979). Figure 2 illustrates the relationship of these various peptides. The various hormones, however, seem to be synthesized in a different manner in different regions of the pituitary in both rats and pigs (Vermes et al., 1980; Smyth and Zakarian, 1980). In the rat pars intermedia pendorphin was released in far greater quantities than P-LPH; however,

PRE-PROOPIOMELANOCORTIN (Pre-POMC) y-MSH

I

1

26

.

t 1

77

" u

ACTH

0-Lipotropin

(u-MSH CLIP HM

w

Signal

-1

t 1 I,

t

t

t

1

8

1

132

173

0-MSH

w-

11 215

t 1

235

4 0-END

265

y-Lipotropin

FIG. 2. T h e N-terminus of each peptide is on the left. The positions of paired basic amino acid residues, presumed signals for cleavage by processing enzymes, are indicated by Lys t , Arg d . Horizontal bars indicate the positions of identified peptides from the ACTH (adrenocorticotrophic hormone) and P-END (p-endorphin). Signal indicates the putative signal sequence of the precursor peptide. The numbers indicate the number of amino acid residues in each precursor peptide and the positions of the first amino acid residue of identified biologically active peptides, numbered from the N-terminus of each precursor. t refers to a lysine residue and & to an arginine residue. From Cox (1982) with permission.

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in the anterior pituitary the amount released was approximately twofold higher for @-LPHthan @-endorphin(Vermes et al., 1980). It is interesting that drug-induced release in the two regions was different as well. In the pars intermedia dopamine inhibited and a /3-adrenergic agonist (isoproterenol) stimulated release, whereas in the anterior lobe vasopressin stimulated release. In pigs the anterior pituitary appears to be the source of the majority of the released P-endorphin and predominantly inactive fragments are released from the pars intermedia (Smyth and Zakarian, 1980). Of interest is the finding that a - M S H , derived from proopiocortin, microinjected into the periaqueductal gray produced @-endorphin-like effects including antinociception (Walker et al., 1980). The putative physiological role of P-endorphin in nociceptive processes has been evaluated by several techniques. These include measurement of endogenous P-endorphin concentrations in various pain states and study of stimulation-produced analgesia and stress-induced analgesia as well as the utilization of opiate receptor antagonists. In a study in which human cerebrospinal fluid concentrations of endorphin activity were measured, patients exhibiting mainly organic pain syndromes had significantly lower levels of endorphin activity than patients exhibiting psychogenic pain symptoms or healthy volunteers (Almay et al., 1978). Endorphin activity in cerebrospinal fluid was defined using an opiate receptor assay; however, the structure of the “endorphin” material was not clearly identified as P-endorphin. Other studies utilizing patients with neurogenic, organic pain syndromes have yielded comparable results (Terenius and Wahlstrom, 1975b; Sjolund et al., 1977). The levels of endorphin-like material, measured as described above, in cerebrospinal fluid of patients with chronic pain syndromes were found to vary slightly with pain threshold (von Knorring et al., 1978). Patients exhibiting a high pain threshold had higher concentrations of endorphin-like material than patients exhibiting a low pain threshold. With a kinesthetic figural aftereffect test, subjects can be classified as augmenters or reducers of what is perceived. This difference in response also exists for their perception of painful stimuli. It is interesting that in chronic pain patients augmenters had lower levels of endorphin-like material in cerebrospinal fluid than reducers (von Knorring et al., 1979). Finally, an inverse correlation has been found between the preoperative endorphin-like activity in cerebrospinal fluid and the dose of postoperative analgesic required (Tamsen et al., 1980). Prior to the discovery of opioid peptides, as previously mentioned, it was demonstrated that electrical stimulation of certain brainstem areas induced surgical analgesia in rats (Reynolds, 1969). Akil et al. (1976)

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found that this effect was partially attenuated by naloxone, suggesting the involvement of opioid peptides in the phenomenon. Similar findings have been obtained in humans (Hosobuchi et al., 1977). Patients with intractable pain, in whom narcotic analgesics did not suppress their constant pain, obtained pain relief after electrical stimulation of the central gray. Their pain was alleviated 5-10 min after stimulation. Tolerance to the effects of such stimulation developed within 4 to 5 weeks. However, if stimulation was limited to approximately 1 hr every 4 hr, tolerance did not develop. I n five of six patients naloxone totally abolished stimulation-induced pain relief as measured by the patients’ own estimate of pain. Periaqueductal gray stimulation in humans has been reported to increase the concentration of p-endorphin and ACTH in the cerebrospinal fluid (Hosobuchi et al., 1979); zona incerta stimulation did not produce similar effects. These findings have now been confirmed (Amano et al., 1980). Analgesia is reliably induced by transcutaneous electrical stimulation. Stimulation at sites innervated by the second trigeminal division of the facial nerve has been reported to produce dental analgesia (Chapman and Benedetti, 1977). Naloxone administered after the transcutanous stimulation only partially reversed the analgesia. Frequency of stimulation is apparently an important variable in the involvement of endogenous opiates in transcutaneous stimulation-induced analgesia. Sjolund and Eriksson (1979) found that low-frequency (2 Hz trains) stimulation produced an analgesic effect that was partially reversed by naloxone. I n contrast, high-frequency (100 Hz) stimulation-induced analgesia was not altered by naloxone treatment. Furthermore, an increase in endorphin-like activity in cerebrospinal fluid following low-frequency stimulation (2 Hz) was reported. Walker and Katz (1981a,b) reported that in humans analgesia induced by subcutaneous electrical stimulation (20 Hz) was neither antagonized by naloxone nor did it exhibit crosstolerance with opiates. In rats auricular electrical stimulation produced antinociception as assessed on a hot plate (Pert et al., 1981). Naloxone partially reversed this effect. Furthermore, as in humans, cerebrospinal fluid concentrations of P-endorphin-like radioreceptor activity was increased after auricular electrical stimulation. The agent of this activity apparently is not pendorphin, however, but rather a lower molecular weight compound, which might be a degradative product of @endorphin. In fact, a concomitant decrease in P-endorphin-like immunoreactivity in the medial thalamus and hypothalamus, but not the septum or periaqueductal gray matter, was observed. Electrical transcutaneous stimulation (electroacupuncture) de-

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creased the firing rate of spinal neurons (Lamina V) to a noxious stimulus in cats (Pomeranz et al., 1977); this effect was blocked by naloxone (Pomeranz and Cheng, 1979). The time course for the decreased firing rate was very similar to that observed clinically (i.e., slow onset, long duration). Although the authors reported that hypophysectomy blocked electroacupuncture-induced antinociception in mice (squeak test), Fu et al. (1980), using a different test (writhing), did not confirm this finding. Utilizing a cross-circulation experiment in rabbits, Yang and Kok (1979) observed that electroacupuncture produced a blood-borne substance that induced a naloxone-sensitive antinociceptive response to dental pulp stimulation. T h e mechanism of action of the Oriental art of acupuncture remained obscure until recent times. The discovery of opioid peptides served as a stimulus for research in this field. In the mouse acupuncture produced antinociception as assessed in the phenylquinone-induced writhing test, and the effect is completely blocked by naloxone pretreatment (Fu et al., 1980). These results are similar to those obtained after low-frequency transcutaneous electrical stimulation (e.g.. electroacupuncture), described in the previous paragraph. T h e possible involvement of opioid peptides in both placebo- and hypnosis-induced analgesia has been investigated. Levine et al. ( 1978a) reported that naloxone increased the perception of pain in subjects that respond to placebo with a decrease in pain sensitivity but not in subjects that were placebo nonresponders. The studies of hypnosis-induced analgesia do not clearly support the involvement of opioid peptides in this phenomenon. Goldstein and Hilgard (1975) reported that naloxone pretreatment did not alter hypnosis-induced analgesia, and similar findings were described by Nasrallah et al. (1979), using a high iv dose (50 mg) of naloxone. In a single-blind study of one patient, Stephenson (1978) reported that naloxone attenuated hypnosis-induced analgesia. A wealth of data has accrued regarding stress-induced antinociception. T h e role of opioid peptides in this phenomenon has been extensively studied. Many, but not all, stressors induce antinociception in laboratory animals. These include footshock, conditioning to footshock, immobilization, 2-deoxy-~-glucose administration, cold-water swimming, insulin treatment, and food deprivation (for review see Watkins and Mayer, 1982). Opioid peptides have been implicated in the mechanism of action of some, but not all, of these stressors. Food deprivation has been reported to induce antinociception in a tail flick test in rats (McGivern et al., 1979). Naloxone reduced, but did not completely abolish, this antinociceptive effect. Similarly, pregnant rats exhibit increases in pain threshold prior to parturition, and this effect is blocked by the

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opiate antagonist naltrexone (Gintzler, 1980). However, several confounding variables appear to render analysis of stress-induced analgesia difficult. Chance ( 1980) stated that classical conditioning of “fear” to environmental stimuli associated with the various nociceptive tests evokes an antinociceptive response. This effect was not attenuated by either naloxone or naltrexone, and the antinociceptive response did not exhibit cross-tolerance with morphine (Chance and Rosencrans, 1979a,b; Chance, 1980). However, in opiate binding experiments using [3H]etorphine as the radioligand, there was a reduction in binding in brain tissue of rats subjected to conditioning-induced antinociception (Chance, 1980). It has been suggested that these findings can best be explained by an opioid system functioning in parallel with another system, with either capable of inducing antinociception. Antinociception induced by footshock appears to involve opioid systems only in the case of certain stimulation parameters and specific types of shock. Lewis et al. (1980) found that brief (3 min) inescapable footshock produced increases in tail flick latencies that were not reversed by naloxone, whereas the increase in tail flick latencies produced by prolonged (30 min) intermittent shock was attenuated by naloxone. Watkins and Mayer (1982) have further characterized shock-induced antinociception. They reported that if the front paws of the rat are shocked briefly, then resultant antinociception is attenuated by naloxone pretreatment. In contrast, if the hind paws are subjected to brief shock, the ensuing antinociception is not altered by naloxone. Immobilization stress decreases escape from a hot plate without affecting paw lick latency (Amir and Amit, 1978). Naloxone reversed the effect of immobilization on escape responses. The fact that immobilization did not alter paw lick latency, but increased escape latency was consistent with the notion that certain stressors may alter affective, but not sensory, properties of nociception. Certain stressors appear to induce antinociception by a pituitarydependent mechanism. For instance, Lewis et al. (1981) noted that antinociception induced by prolonged footshock, which is naloxone reversible, is attenuated by hypophysectomy; in contrast, antinociception induced by brief footshock, which is naloxone insensitive, is not modified by hypophysectomy. Antinociception induced by cold-water swim is partly attenuated by naloxone and by hypophysectomy (Bodnar et al., 1979). T h e complexity of stress-induced antinociception is demonstrated by the finding that other stressors such as brief forepaw shock and conditioning to footshock induce naloxone-sensitive antinociception that is not altered by hypophysectomy (Watkins and Mayer, 1982). T h e effects of a variety of stressful stimuli on plasma &endorphin concentrations in rats have been investigated (Mueller, 1981). Testing

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rat tail flick latencies did not alter plasma P-endorphin concentrations; however, testing rats on a hot plate increased the plasma concentration of this opioid peptide. Ether and immobilization stress are two stressors found to increase circulating P-endorphin concentrations. Immobilization increased the plasma concentrations almost tenfold, ether approximately fivefold. This finding compares well with the fact that immobilization produces a naloxone- and hypophysectomy-reversible increase in antinociception (Watkins and Mayer, 1982). Exposure to ether did not produce antinociception; this may be due to the fact that the increases in plasma P-endorphin are not as large as those observed after immobilization stress (Watkins and Mayer, 1982). It is of interest that immobilization did not alter cerebrospinal fluid levels of P-endorphin, whereas Met-enkephalin levels decreased slightly (Kiser et al., 1981). Barta and Yashpal (1981) reported that cold-water swim stress reduced /+endorphin concentrations in the pituitary; an increase in the concentration of the peptide was observed in the amygdala, periaqueductal gray, and nucleus raphC magnus. This form of stress, however, produces naloxone-insensitive antinociception. In humans stress induced by strong pain increases the threshold of the RIII reflex, a nociceptive flexion reflex (Willer, 1980); this effect is blocked by naloxone (Willer and Albe-Fessard, 1980). This concatenation of results suggest that stress in both humans and animals may under certain circumstances activate endogenous opioid systems. In many of the experiments described above naloxone has been used as a tool to assess the role of endogenous opioid peptides as mediators of antinociceptive effects. Several important issues must be considered when naloxone findings are used to assess the role of P-endorphin. First among these is the specificity of naloxone as an opiate receptor antagonist (Sawynok et al., 1979; Hill, 1981). In addition to acting as an opiate antagonist, naloxone in high doses appears to act as a GABA antagonist. Furthermore, using naloxone to assess the involvement of endogenous P-endorphin is complicated by the presence of several endogenous opioid peptides, all of which can produce effects that are attenuated by naloxone. As described in previous paragraphs, naloxone has been utilized as a tool for assessing the role of endogenous opioid activity following electrical stimulation and stress. In the nonstressed organism the results of studies of naloxone have been less than perspicuous. For instance, Goldstein et al. (1976) observed that in the rat naloxone (25 mg/kg, ip) blocked the effects of morphine but when given alone had no effect on the threshold for escape for footshock. Concordant findings were obtained when nociception was measured by threshold of reactivity to for-

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21 1

malin-induced chronic pain (North, 1978). In contrast, Kokka and Fairhurst (1977) observed an enhancement of acetic acid-induced writhing in naloxone-treated rats. Other studies also have reported a heightened sensitivity to painful stimuli following naloxone (e.g., Carmody et al., 19’19; Fanselow and Bolles, 1979; Pilcher, 1980). Of interest is Pilcher’s (1980) observation that chronic treatment with the opiate antagonist naltrexone produced a tolerance to naloxone-induced hyperalgesia. Newer opiate antagonists (diprenorphine, phenylpiperidine, or noroxymorphone) have been reported to produce a hyperalgesia in mice in the hot plate test (Ramabadran, 1982). T h e data pertaining to the influence of naloxone on pain perception in humans is also equivocal. Levine et al. (197813) found that naloxone administered after extraction of wisdom teeth increased the pain perceived. Moreover, naloxone decreased pain perception in placebo responders when given in a low dose (<2 mg) but increased pain perception following a high dose (>7.5 mg) (Levine et al., 1979). Buchsbaum et al. (1977) found that naloxone administered to normal volunteers had an analgesic-iike action in pain-sensitive subjects, whereas pain-insensitive subjects reported increases in the sensation of pain following naloxone. Hyperalgesia has also been reported following naloxone administration to a patient with documented congenital indifference to pain (Dehen et al., 1979). However, in normal subjects (Grevert and Goldstein, 1978) and in chronic pain patients (Lindblom and Tegnef, 1979) no effect of naloxone on pain perception was noted. Data obtained with naloxone must be cautiously interpreted. For instance, negative data is fraught with several possible explanations for the lack of effect of the drug, including dose, nociceptive test, and time between injection and testing. Positive findings must also be cautiously interpreted because of several factors including dose and baseline pain perceptions. One may conclude that naloxone appears to alter pain perception (nociception) under some conditions.

C. DYNORPHIN Dynorphin is an opioid peptide composed of 13 amino acids (Table I) that was isolated from porcine pituitary (Goldstein et al., 1979). It is heterogenously distributed throughout the brain and pituitary. In the rat dynorphin concentrations are highest in the pituitary, followed by hypothalamus, midbrain, thalamus, and pons-medulla (Hollt et al., 1980). Watson et al. (1981), using immunocytochemistry, demonstrated that in brain and pituitary dynorphin exists separately from Leu-

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enkephalin, although the sequence of the latter peptide is contained within dynorphin. However, dynorphin and a-neoendorphin are COlocalized in the supraoptic and paraventricular nuclei (Weber et al., 1981). The distribution of dynorphin in the human brain has been recently described (Gramsch et al., 1982). The distribution generally parallels that found in rats. Although the pattern of distribution of dynorphin was clearly different from that of P-endorphin, it exhibited many similarities with that of Met- and Leu-enkephalin. Herman et al. (1980) have observed antinociception in the tail pinch test in rats following icv or cerebral aqueduct injection of dynorphin. Intraaqueductal administration was at least tenfold more potent than lateral ventricular injection. Although this antinociceptive effect of dynorphin was attenuated by naloxone, it appeared to be less sensitive than @endorphin to the effects of naloxone. Vocalization was diminished for only a few minutes after dynorphin injection. Similarly, Wuster et al. (1980) reported that vocalization thresholds were not significantly altered by icv dynorphin. In a jump-flinch paradigm icv dynorphin induced antinociception (Petrie et al., 1982). The antinociceptive effect of dynorphin was partially attenuated by naloxone, and the peptide exhibited cross-tolerance with P-endorphin and morphine. T h e effect of footshock stress on endogenous dynorphin concentrations has been investigated (Millan et al., 1981). This stress produced a decrease in immunoreactive dynorphin concentrations within 60 min post-stress in the anterior pituitary and frontal cortex; an increase was observed in the hypothalamus. N o such changes were observed in the intermediate lobe of the pituitary, thalamus, pons-medulla, or other brain regions. T h e magnitude of these effects are comparable to changes noted in the concentrations of other opioid peptides following acute stress (see previous section). D. OTHER OPIOID PEPTIDES Kyotorphin is a dipeptide (Table I) that has been localized in rnidbrain, pons-medulla, and dorsal spinal cord of the rat (Veda et al., 1980), regions that have been posited to be among the loci at which opioid-induced antinociception might be mediated. When administered ic to mice, this peptide produces a naloxone-reversible antinociceptive response (Takagi et al., 1979; Rackham et al., 1982). Kyotorphin apparently does not activate opiate receptors in vitro, suggesting an indirect mechanism of action for this dipeptide. DesTyrl-y-endorphin does not appear to acutely alter nociception.

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However, chronic treatment with this peptide has been reported to potentiate the antinociceptive actions of morphine in rats (Neil et d., 1981).

IV. Naloxane-Sensitive Nonopioid Peptides

A. ADRENOCORTICOTROPIN Adrenocorticotropin (ACTH) is an anterior pituitary hormone comprised of 39 amino acids (Table I) that is derived from a high molecular weight precursor (proopiocortin); its classical endocrine action is stimulation of the release and synthesis of glucocorticosteroids (e.g., corticosterone, cortisol) in the adrenal cortex. This peptide has been reported to be present in the CNS as well (Krieger et al., 1977). Terenius (1976) reported that ACTH, like somatostatin, bound to rat brain membrane opiate receptors in a manner resembling partial opiate agonist-antagonists. Snell and Snell (1982), using structural analogs of ACTH, determined that the N-terminal region of ACTH binds to opiate receptors, as assessed with labeled dihydromorphine and naloxone. Stengaard-Pedersen and Larsson (1981) reported similar structural requirements for the affinity of ACTH to receptors that bind enkephalinamide as well as naloxone. In support of the hypothesis of Terenius (1976) of partial opiate antagonistic activity of ACTH are the observations of Bertolini et al. (1979), who reported that icv injection of ACTH produced hyperalgesia in rats. This was observed in two different tests, the hot plate and the nociceptive threshold of electrical stimalation of the tail. Morphine antagonized the effects of ACTH in the hot plate test; the opiate antagonist naloxone, in a dose that had no effect alone, potentiated the hyperalgesic response to ACTH, assessed by nociceptive threshold of electrical stimulation of the tail. Gispen et al. (1976), using a hot plate test, found that several ACTH fragments inhibited morphine-induced analgesia in rats. Amir (1981) has also investigated interactions between ACTH and morphine, using a modified hot plate procedure (46OC,5 min exposure) with mice. Both ACTH and morphine produced biphasic effects when administered iv in this test. A low dose of ACTH (12.5 pg/kg) enhanced jumping (an escape response), whereas a higher dose (200 pg/kg) slightly suppressed the response. Morphine produced similar effects; low doses (<0.625 mg/kg) enhanced jumping, whereas larger doses (>0.625 mg/kg) inhibited jumping. A high dose of ACTH modified the hyperalgesic effect of a low dose of morphine and similarly, a low dose

2 14

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of ACTH attenuated the analgesic effect of larger doses of morphine. These results are consistent with the proposed interaction between ACTH and opiates at the receptor level. Studies utilizing an analog of ACTH4-9 (ORG 2766) suggest that opiate-ACTH interactions may be quite complex. Walker et al. (198 1) observed that injection of ORG 2766 into the periaqueductal gray of rats produced a dose-dependent analgesia in a tail flick test. This effect was not altered by either naloxone o r morphine tolerance. In vitro, ORG 2766 did not alter naloxone binding. B. CHOLECYSTOKININ Cholecystokinin-related peptides have been found in brain and gut. It has now been established that there is not a single naturally occurring cholecystokinin peptide but rather a class of structural related compounds. I n a recent report Beinfeld (1981) has discussed this issue in detail. In rat brain the majority of cholecystokinin immunoreactivity was found to comigrate chromatographically with the cholecystokinin octapeptide sulfate. Lesser amounts of the desulfated cholecystokinin octapeptide and cholecystokinin tetrapeptide were also detected. T h e desulfated C-terminal septapeptide of cholecystokinin has been reported to inhibit [3H]naloxone binding to opiate receptors in rat brain (Schiller et al., 1978). This peptide was 200-fold less potent than Metenkephalin in this regard. The sulfated septapeptide of cholecystokinin was inactive. In a guinea pig ileum opiate receptor bioassay, the desulfated cholecystokinin septapeptide inhibited electrically evoked contractions; the effect was naloxone-reversible. The physiological significance of this finding is unclear because no cholecystokinin septapeptide was detected in rat brain (Beinfeld, 1981). In fact, cholecystokinin octapeptide, which is found in rat brain, induces contraction of the guinea pig ileum that is reversed by opiates and opioid peptides (Zetler, 1979). The effect of the opiate, but not of cholecystokinin, is blocked by naloxone. I n mice, sc administration of cholecystokinin octapeptide produced antinociception in hot plate (minimally effective dose = 50 pg/kg) and acetic acid-induced writhing (minimally effective dose = 750 pg/kg) tests, but not in a tail flick test (Zetler, 1980). The effect of cholecystokinin was dose-related in both tests. T h e antinociceptive effect was rapid in onset (<5 min) and persisted for approximately 45 min. Unlike the effect of cholecystokinin octapeptide on the guinea pig ileum, the antinociceptive effect was antagonized by naloxone. However, morphine tolerance did not alter cholecystokinin-induced antinociception. To de-

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termine if the effect of cholecystokinin octapeptide is mediated within the CNS, the peptide was administered centrally to rats (Jurna and Zetler, 1981). Cholecystokinin octapeptide induced significant antinociception in a tail flick test when injected into the periaqueductal grey (36 ng), caudate nucleus (72 ng), ventromedial thalamus (72 ng), cuneiform nucleus (72 ng), or when injected intrathecally (36 ng). The EDjo after periaqueductal injection and intrathecal injection was 11 and 21 ng, respectively. Naloxone abolished the antinociceptive effect of cholecystokinin octapeptide after administration by both routes. In contrast, cholecystokinin was ineffective in a tail flick test in mice. This discrepancy remains unexplained.

C. SOMATOSTATIN Somatostatin (SRIF), a tetradecapeptide (Table I), like other peptides that influence pituitary hormone secretion, is heterogenously distributed throughout the brain (Kobayashi et al., 1977). Several regions of rat brain implicated in nociception contain significant amounts of SRIF. These include midbrain central gray, amygdala, and medulla. Somatostatin appears to interact with opiate receptors. Terenius (1976) studied the effects of SRIF on dihydromorphine (an opiate agonist) and naltrexone (an opiate antagonist) binding to rat brain membranes. The results obtained are best described as SRIF acting like a partial opiate agonist-antagonist. Other authors have reported that SRIF analogs inhibit stereospecific binding to opiate receptors (Pugsley and Lippmann, 1978). Tritiated naloxone binding was inhibited by two different SRIF analogs, (desAla'Gly2, desamino3, DTrp8, descarboxy14, di~arba~~'~)-somatostatin and (desAla', Gly2, desamind, descarboxy14)somatostatin. Addition of sodium to the incubation medium decreased the affinity of SRIF for the opiate receptor, suggesting that the tetradecapeptide would act as an opiate agonist and not a partial antagonist as suggested by Terenius (1976). Different results were obtained when the effects of SRIF on opiate binding were studied using neuroblastomaglioma hybrid cells (Traber et al., 1977). In this system naloxone binding was not altered by SRIF. Rezek et al. (1978) found that icv administration of SRIF increased the latency of the tail flick response in rats. This effect of SRIF was marginally dose-related, 1 pg producing an effect with a duration of approximately 30 min. Naloxone ( 5 mglkg, ip) antagonized the SRIFinduced antinociception. This is consistent with some of the in vitro opiate binding studies. Another possible explanation for the naloxone

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reversibility of the SRIF-induced antinociception is suggested by the findings of Barclay and Phillipps (1980).They observed that brain tissue incubated in the presence of SRIF competitively inhibited the degradation of Leu-enkephalin. The IC50 for SRIF was 0.7 k M . These findings suggest that SRIF-induced antinociception may simply be due to inhibition of endogenous enkephalin degradation.

V. Naloxone-Insensitive Peptides

A. NEUROTENSIN The distribution of neurotensin in the CNS has been elucidated with both RIA and immunohistochemical techniques. As determined by radioimmunoassay, neurotensin is found in many regions in the rat brain that have been implicated in the animal’s response to noxious stimuli. These include the amygdala, periaqueductal gray, and thalamus (Carraway and Leeman, 1976; Kobayashi et al., 1977). Immunohistochemical studies have confirmed and extended these observations. Uhl et al. (19’79) observed neurotensin in both cell bodies and fibers in the substantia gelatinosa, locus coeruleus, dorsal raphe, and periaqueductal gray, all areas that have been reported to modulate responsiveness to noxious stimuli. These investigators further noted that the distribution of neurotensin was similar to that of enkephalin and substance P in many regions. Dorsal rhizotomy did not alter neurotensin receptor density or distribution within the spinal cord, suggesting that neurotensin binding sites are not located on primary afferents (Ninkovic et al., 1981). Haigler and Spring (1981) have reported that of the cells within the mesencephalic reticular formation that responded to a nociceptive stimulus, one-third responded in a similar manner to iontophoretically applied neurotensin. However, approximately 25% responded to neurotensin in an opposite manner to the response induced by the nociceptive stimulus. Neurotensin-induced antinociception was first described by ClineSchmidt and McGuffin (1977). Intracisternal injection of low doses of neurotensin (0.25-2500 ng) induced antinociception in female mice. This was observed in two different nociceptive tests: hot plate (60°C)and writhing induced by acetic acid. An intravenous injection of 2500 ng did not alter the response of mice in either test. The minimally effective dose of neurotensin was quite different in the two tests. In the hot plate test 25 ng was the lowest effective ic dose, whereas in the writhing test 0.25 ng was effective. The duration of action after 250 ng neurotensin was

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approximately 1 hr. A tenfold increase in dose increased the duration of action to approximately 2 hr. The potency of neurotensin-induced antinociception was compared with that of other peptides in male mice utilizing a tail immersion test (water 48°C) (Nemeroff et al., 1979b). Only P-endorphin produced a greater antinociceptive response. A variety of other peptides including bombesin, Leu-enkephalin, Met-enkephalin, thyrotropin-releasing hormone, somatostatin, bradykinin, luteinizing hormone-releasing hormone, substance P, and melanocyte-stimulating hormone release-inhibiting factor did not produce a significant antinociceptive response in doses equimolar to 1 p g neurotensin. Neurotensin produced a doserelated increase in response latency following ic injection and the duration of action was comparable to that described by Clineschmidt and McCuffin (1977). T h e minimally effective dose was 300 ng. Neurotensin injected ic in mice reduces body temperature (Bissette et al., 1976), but this appears to be unrelated to its antinociceptive effect. Bombesin, injected ic significantly reduced colonic temperature without affecting nociceptive responses in the tail immersion test (Nemeroff et al., 1979b). In addition, oxotreniorine, chlorpromazine, and DL-5-hydroxytryptophan reduced the body temperature of mice but did not alter reaction time on a hot plate (Clineschmidt and McCuffin, 1977). Finally, direct intracerebral injection of neurotensin produces antinociception but not hypothermia at certain loci (see Table 11). Neurotensin-induced antinocisponsiveness is apparently not due to motor impairment, because doses of neurotensin that produce significant antinociception do not affect coordinated motor control on a rotating rod. However, spontaneous locomotor activity is reduced (Cfineschmidt et nl., 1979). Further studies have demonstrated that neurotensin produces antinociception in rats as well as in mice (Clineschmidt et al., 1979). On a hot plate adjusted to 55"C, 25 ng of neurotensin injected ic increased the response latency 30 min postinjection. However, in a tail flick paradigm with a focused heat lamp neither 25 nor 2500 ng of ic administered neurotensin altered response latency 15, 30, or 60 min postinjection. Intracerebroventricular administration of neurotensin to male rats was found to diminish responsiveness to electric footshock (van Wimersma Greidanus et al., 1982). Neurotensin (300 ng) administered 1 hr prior to testing significantly increased the number of rats that did not respond and decreased the frequency of jerking, running, and jumping. Flinches and vocalizations were unaffected by this dose of neurotensin. These researchers suggested that because neurotensin did not affect vocalization, the effect may be due to a decrease in motor responsiveness to noxious stimuli, rather than an analgesic action per se.

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Martin et al. (1981a) studied the effects of icv, ic, or intrathecal neurotensin injection on nociception in rats. Intraventricular administration of 20 to 60 pg of neurotensin produced a significant antinociceptive response on a hot plate (55°C) 30 min postinjection. Similar results were observed after ic injection, the minimally effective dose being approximately 2 pg. Thus with regard to antinociception, neurotensin is approximately ten times more potent after ic injection than after icv injection. A clear dose-response relationship was not evident with either route of administration. Intrathecal administration of neurotensin in doses as large as 80 p g did not significantly alter the hot plate response time of rats 30 or 60 rnin postinjection. Based on these findings, Martin et al. (198la) suggested that neurotensin-induced antinociception in the hot plate test is mediated at supraspinal sites. In contrast, Yaksh et al. (1982) found that intrathecal neurotensin produced antinociception in rats on a hot plate (55°C). The differences in the results of Yaksh et al. (1982) and Martin et al. (198la) may partly be due to differences in the time at which the animals were tested. Martin and his colleagues (1981a) tested the rats 30 and 60 rnin postinjection, whereas Yaksh and coworkers (1982) tested the rats 5, 15, 30, 60, and 120 rnin postinjection. The duration of neurotensin-induced antinociception following intrathecal administration was of relatively short duration (30 min) and dose-related. As described previously, neurotensin injected ic in rats does not induce antinocisponsiveness in a tail flick test (Clineschmidt et a/., 1979). Experiments performed with a comparable tail flick paradigm revealed similar findings after intrathecal administration (Yaksh et al., 1982). I n mice neurotensin is approximately 100-fold more potent after ic administration in the acetic acid-induced writhing test than in the hot plate test. It is therefore of interest that in rats intrathecal administration of neurotensin produces equivalent antinociception in the two tests. In addition, intrathecal neurotensin administration results in an atypical dose-response relationship, a U-shaped curve in the acetic acid-induced writhing test. Ten fig neurotensin was found to be the maximally effective dose; lower or higher doses of the peptide were less effective in this test. Neurotensin induces an antinociceptive response when microinjected into a variety of sites in rats (Kalivas et al., 1982b). Rats were implanted with bilateral cannulae and injected with neurotensin (2.5 pg/ side) and tested on a hot plate (55°C) 10, 30, and 60 min postinjection. Colonic temperature was also measured. As alluded to earlier, it was evident that different regions are involved in neurotensin-induced antinociception and neurotensin-induced hypothermia (Table 11). Neurotensin was effective in producing antinociception after microinjection

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TABLE 11 COMPARISON OF NEUROTENSIN-INDUCED ANTlNOCICEPrION AND HYPOTHERMIA IN VARIOUS BRAINREGIONV ~

Brain region Lateral ventricles Telencephalon Area preopticus medialis Diagonal band of Broca Hippocampus Nucleus accumbens Nucleus amygdaloideus centralis Other amydaloid nuclei Striatum Diencephalon Area anterior hypothalami Area lateralis hypothalami Medial hypothalamus Posterior hypothalamus Dorsal thalamus Lateral thalamus Ventromedial thalamus Bed nucleus of striae terminalis Zona incerta Mesencephalon Rostra1 periventricular gray Caudal periventricular gray Formatio reticularis Nucleus ventralis tegmenti Substantia nigra Rhombencephalon Formatio reticularis, pars medialis Formatio reticularis, pars lateralis Floor fourth ventricle Raphe magnus Tractus spinalis nervi trigemini

~~

Antinociception (number of responders)

Hypothermia (number of responders)

9

3

5

5 6 5 12

4 3 0

4 2

1

I 2

7

5 0 2

0 2

N

6 16 8 6 5 6 5 9 3 6

1

2 0 2 0 0 0

5 0 2

3

0 2 0

3

2 0

14 6 15 12 7

10 1 4 2 1

4 4 8 2 4

4 0 0 0 1

1

1 1

I 1

3

7 3 2

0 8 0 3

a Neurotensin was injected bilaterally at a dose of 2.5 pglside. A 75% increase in response latency after neurotensin compared to saline treatments in the same animal was considered positive. Modified from Kalivas et al. (1982b).

into the central amygdaloid nucleus, the caudal diagonal band of Broca, the rostral medial preoptic area, the rostral mesencephalic periaqueductal gray, the medial pontine reticular formation, and the ventral thalamus. All of these regions have been found to possess both neurotensincontaining cell bodies and fibers (Jennes et al., 1982).

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The antinociceptive response following administration of neurotensin into the central amygdaloid nucleus has been more closely scrutinized (Kalivas et al., 1982a). The neurotensin effect is dose-dependent. In addition, lesioning the stria terminalis, a major efferent and afferent pathway of the amygdala, blocked neurotensin-induced antinociception produced by injection into the central amygdaloid nucleus. Unlike Kalivas et al. (1982b), Martin et al. (1981b) did not observe an antinociceptive response following neurotensin injection into the mesencephalic periaqueductal gray. However, the latter group used only half the dose of neurotensin used by the former. It is of interest to compare neurotensin-sensitive sites in the CNS to those sensitive to opiates and opiate-like peptides. There are several brain regions in which local injection of both neurotensin and opiates or opioid peptides produce an antinociceptive response. These include the central amygdaloid nucleus (Rodgers, 1977), medial preoptic area (Tseng et al., 1980), mesencephalic periaqueductal gray (Yaksh and Rudy, 1978; Bennett and Mayer, 1979), and the mesencephalic reticular formation (Haigler and Spring, 1978). However, the neurotensin-sensitive locus in the medial pontine reticular formation is not responsive to microinjected morphine, although this region has not been extensively examined (Yaksh and Rudy, 1978). Moreover, microinjection of morphine into the nucleus raphe magnus induces antinociception (Dickenson et al., 1979), whereas neurotensin does not (Kalivas el al., 1982b). The effects of various pharmacological agents on the antinociceptive response to neurotensin has been studied. Many compounds have been tested utilizing mice and a hot plate procedure for assessing the antinociceptive response to ic neurotensin (Clineschmidt et al., 1979). Drugs that did not alter neurotensin-induced antinociception included muscarinic cholinergic antagonists (atropine and benztropine), histamine antagonists (chlorpheniramine, pyribenzamine, and cimetidine), a P-adrenergic antagonist (propanolol), an a-adrenergic antagonist (phenoxybenzamine), a dopamine antagonist (haloperidol), a serotonin receptor antagonist (cyproheptadine), and a prostaglandin synthetase inhibitor (indomethacin). A few of the compounds studied enhanced neurotensin-induced antinociception. These included a nicotinic cholinergic antagonist (mecamylamine), a serotonin antagonist (methergoline), and the a-adrenergic antagonist, HEAT (2-[P-(4-hydroxyphenyl)ethylaminomethyl]tetralone hydrochloride). Utilizing a hot plate test and rats, Long et al. (1981) reported that the serotonin-depleting agent parachlorophenylalanine (PCPA) enhanced the antinociception observed after icv neurotensin. Of obvious importance is whether neurotensin-induced antinocicep-

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tion is mediated by endogenous opioid systems. This question has not yet been answered satisfactorily. Clineschmidt and McGuffin (1977) reported that naloxone (1 mg/kg, sc), did not alter ic neurotensin-induced antinociception in either a hot plate or acetic acid-induced writhing test in mice. Osbahr et al. (1981) confirmed and extended these results; naloxone (1-5 mg/kg, ip) did not antagonize ic neurotensin-induced antinociception in mice in the hot plate, acetic acid-induced writhing or the tail immersion test. In rats, opiate antagonists have been reported to alter neurotensin-induced antinociception, although the studies are not directly comparable to those conducted in mice (van Wimersma Greidanus et al., 1982; Yaksh et al., 1982). Two different opiate antagonists, naloxone (1 pg) and naltrexone ( 1 pg), when administered iv antagonized the neurotensin (300 ng, icv)-induced decrease in responsiveness to electric footshock in rats (van Wimersma Greidanus et al., 1982). Yaksh et al. (1982) observed that the antinociceptive effect following intrathecal neurotensin was antagonized in rats in both the acetic acidinduced writhing and hot plate tests. The effect of neurotensin in the writhing test was antagonized by naloxone (1 mglkg, sc), and 15 p g of naloxone administered intrathecally blocked the analgesic effect of intrathecal neurotensin in the hot plate test. There are many potential factors that may contribute to the discrepant findings concerning the effects of naloxone on neurotensin-induced antinociception. However, at present the explanation for these differences is obscure. Other approaches for characterizing potential opiate-neurotensin interactions have been used (Morley et al., 1980; Luttinger et al., 1983). We have investigated the effects of neurotensin in mice rendered tolerant to morphine (Luttinger et al., 1983). Morphine tolerance markedly attenuated the antinociceptive effect of ic neurotensin as assessed with the hot plate test. In addition, a subthreshold dose of ic neurotensin administered concomitantly with a subthreshold dose of ip morphine resulted in significant antinociception. Morley et al. (1980) have obtained results consistent with the notion of an interaction of opiates and neurotensin. In rats morphine addiction increased the concentration of immunoreactive neurotensin in the thalamus, and naloxone-precipitated withdrawal reduced thalamic neurotensin concentrations to control levels. Naloxone administered to naive animals also reduced thalamic neurotensin concentrations. In summary, the interaction of neurotensin with opioid systems is complex and poorly understood. The neural mechanisms involved in tolerance to opiates appear to be capable of influencing both endogenous neurotensin concentrations and the antinociceptive response to neurotensin. The species and/or route of injection have been identified as potentially important variables.

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Much information about the opioid peptides has been provided from experiments with opiate receptor antagonists; unfortunately this is not the case with neurotensin. However, compounds that antagonize the antinociceptive effect of neurotensin have been described (Osbahr et al., 1981; Bodnar et al., 1982b). Thyrotropin-releasing hormone (TRH) has been found to block many of the CNS effects induced by neurotensin including hypothermia, muscle relaxation, and potentiation of ethanol-induced sedation (Osbahr et al., 1979; Nemeroff et al., 1980; Luttinger et al., 1980). Neurotensin-induced antinociception has also been found to be reversed by TRH in mice (Osbahr et al., 1981). I n three tests [tail immersion, hot plate (5 l°C) and acetic acid-induced writhing] the action of neurotensin was completely reversed by TRH. In none of the tests did TRH alone produce a hyperalgesic response. In addition RX77368 (pGlu-His-3,3 dimethyl-Pro-NHB), a TRH analog with enhanced biological activity, inhibits neurotensin-induced antinociception in mice in a hot plate test (D. E. Hernandez, unpublished). Both Holaday et al. (19’78) and Osbahr et al. (1981) observed that P-endorphin-induced antinociception is not altered by TRH. It would appear that neurotensin-induced antinociception, at least in part, is produced by mechanisms distinct from those of the opioid peptides. Antisera to neurotensin have also been used as potential antagonists and as a putative tool for investigating the role of endogenous neurotensin (Bodnar et al., 198213). The antisera is injected icv to bind to endogenous neurotensin and prevent the peptide from acting on its receptors. In rats the tail flick latencies induced by a moderate thermal stimulus were decreased following icv administration of neurotensin antisera, suggesting that endogenous neurotensin may inhibit responsiveness to painful stimuli. However, neurotensin antisera actually increased the response latency to both high and low intensity stimuli. Data collected using this test is difficult to interpret because Clineschmidt et aE. (1979) reported that ic neurotensin does not alter tail flick latencies in rats. Bodnar et al. (1982b) also noted that in a jump-flinch procedure neurotensin antisera did not alter the responsiveness of rats to electric footshock. This null effect produced by neurotensin antiserum does not, of course, preclude the possibility that the peptide exerts a role in nociception.

B. VASOPRESSIN Vasopressin (antidiuretic hormone) is a peptide (Table I) that is released from the posterior pituitary under conditions of dehydration or

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hypovolemia and promotes resorption of water through an action on the renal tubules (Hays and Levine, 1974). In addition, vasopressin has also been found in neurons not associated with the classical neurohypophysial pathway. Vasopressin-containing neurons are present in periventricular regions of' the diencephalon and mesencephalon, regions that have been implicated in pain transmission (Swanson, 1977; Bugs et al., 1978). Lysine vasopressin, injected icv, produced a potent, dose-dependent antinociceptive response in rats in the tail flick test (Berntson and Berson, 1980). A dose of 16 pg produced a significant antinociceptive response that persisted for approximately 2 1 min. A larger dose (100 pg) of vasopressin increased both the potency and duration (approximately 57 min) of the antinociceptive effect. The antinociceptive effect of vasopressin administered icv was not altered by naloxone (2 mg/kg, sc). Berntson and Berson (1980) also reported that vasopressin (16 pg, sc) produced an antinociceptive response that was comparable to that observed when the same dose was administered into the lateral ventricle. Vasopressin has been found to produce comparable antinociceptive effects in mice (Berkowitz and Sherman, 1982). T h e effects of vasopressin were assessed using writhing tests and hot plate tests. Intravenous administration of vasopressin (30 pg/kg) reduced writhing by 80%; larger doses did not produce greater inhibition. T h e duration of the response was less than 1 hr. As was found in rats, in mice an opiate receptor antagonist (naltrexone) did not alter vasopressin-induced antinociception. However, [ 1 -( P-mercapto-P,P-cyclopentamethylene propionic acid), 2-O-ethyltyrosyl-4-valine] arginine vasopressin, an antagonist of the antidiuretic effects of vasopressin, blocked vasopressininduced antinociception in the writhing test but produced no effect by itself. In the hot plate test iv vasopressin produced an antinociceptive response at a hot plate temperature of 50°C, but not 60°C. In contrast to the results in rats, icv administration of vasopressin did not produce an antinociceptive response in mice, suggesting that vasopressin may be acting at a peripheral locus to produce antinociception. It has been observed that iv injection of vasopressin increases the plasma concentration of @-endorphin in the rat (Anhut et al., 1981). However, this is probably not the mechanism of action of vasopressininduced antinociception, because unlike P-endorphin-induced antinociception, vasopressin-induced antinociception is not antagonized by opiate receptor antagonists. The effects of vasopressin on nociception in humans has also been investigated. Lysine vasopressin, administered im, reduced deep pain in 23 out of 24 patients and superficial pain in 20 out of 2 1 patients suffering from an acute case of severe herpes zoster (Forssman et al., 1973).

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This analgesic effect was evident upon repeated injections. T h e duration of action after a single injection of vasopressin was between 12 and 24 hr. Vasopressin was more efficacious in alleviating acute pain than chronic pain. In an interesting report plasma vasopressin levels were found to be elevated in patients complaining of pain (mostly fractures) seen in a surgical emergency department compared to hospital staff and patients visiting the outpatient laboratory for routine blood tests (Kendler et al., 1978). No difference in plasma osmolality was noted between the two groups. The authors suggest that the vasopressin response to pain “may be specific and not necessarily related to the associated stress” because many stressors do not elevate plasma vasopressin in rats. It is interesting to speculate that the increase in vasopressin that accompanies pain may be one homeostatic mechanism that contributes to the attenuation of pain.

C. TUFTSIN Tuftsin is a tetrapeptide (Table I) discovered by Najjar and Nishioka (1970). It is believed to be synthesized in the spleen and is apparently also present within the sequence of @-globulin (Nishioka et al., 1972). Tuftsin has recently been reported to produce antinociception in rats (Herman et al., 198 1). Intracerebroventricular administration of tuftsin (200 pg) produced antinociception in a hot plate test that persisted for approximately 20 min. This effect was not blocked by naloxone.

D. CALCITONIN Calcitonin is a 32 amino acid peptide hormone (Table I) that causes hypocalcemia and hypophosphatemia after peripheral administration. Fischer el al. (1981) have demonstrated that membrane receptors for calcitonin exist in rat brain. Calcitonin binds to these receptors with high affinity and is poorly dissociable. The calcitonin binding sites are heterogenously distributed with high concentrations in the midbrain-thalamus and brainstem, two regions implicated in pain transmission. Calcitonin binding was unaffected by the addition of morphine, naloxone, or three opioid peptides: Met-enkaphalin, Leu-enkephalin, and &endorphin. I n rabbits icv injection of calcitonin has been reported to produce antinociception as assessed by measurement of the voltage necessary for an electrode placed on the upper incisor tooth pulp to elicit a licking reaction (Braga et al., 1978). An icv dose of approximately 2 p g produced an antinociceptive response that was gradual in onset, plateauing

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90 min postinjection and persisting longer than 2 hr. The magnitude of the effect was comparable to that observed after icv injection of morphine (10 pg). The calcitonin-induced antinociception was not altered by naloxone (1 mg/kg, sc). Daily treatment with calcitonin for 5 days did not result in any alteration in the antinociceptive response to calcitonin; in contrast, tolerance to morphine was clearly evident following 5 days of morphine treatment. Injection of both morphine and calcitonin together produced a rapid onset, persistent rise in pain threshold that was greater than that elicited by either agent alone. Finally, as expected from the binding studies described above, calcitonin did not alter r3H]dihydromorphine receptor binding to rat brain membranes. Calcitonin administered icv reduced responsiveness to painful stimuli in mice (Yamamoto et al., 1979; Satoh et al., 1979a). This antinociceptive response was assessed by measuring the amount of pressure applied to the base of the tail necessary to elicit struggling, squeaking, or biting (Yamamoto et al., 1979). The analgesic effect persisted for approximately 60 min. This is a considerably shorter duration of action than that observed in rabbits. As in rabbits, the decreased responsiveness induced by calcitonin in mice is not altered by opiate antagonists, (e.g., levalorphan). T h e antinociceptive effect of calcitonin is attenuated by icv injection of Ca2+(Satoh et al., 1979a). This is of interest because of the effects of calcitonin on serum Ca2+and also because Ca2+exerts a similar effect on morphine-induced analgesia. It is interesting to note that calcitonin administered concurrently with increased dietary Ca2+ has been found to reduce back pain in patients with osteoporosis (Cohn et al., 1971). T h e effects of calcitonin in this case may be specific to the pathophysiology of osteoporosis, however, and not due to a general antinociceptive effect.

E. BOMBESIN Bombesin is a tetradecapeptide (Table I) that was first described in frog skin by Anastasi et al. (1971). It is structurally related to porcine gastrin-releasing peptide, a peptide composed of 27 amino acids (McDonald el al., 1979). In the CNS bombesin-like immunoreactivity has been found by RIA to be present in the central amygdaloid nucleus, periaqueductal gray, and substantia gelatinosa as well as other regions (Moody et al., 1979). These brain regions have been extensively implicated in nociceptive processes. Pert et al. (1980) have described receptors for bombesin in several rat brain regions. One of the regions in which binding sites were observed

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was the periaqueductal gray, a brain region implicated in nociception. Injection of bombesin into the periaqueductal gray of rats produced a dose-dependent increase in tail flick latency as well as an antinociceptive response in the hot plate test. The antinociceptive effect in the tail flick test was maximal 10 min postinjection and persisted for 1 hr. Naloxone did not alter the antinociception observed after bombesin injection into the periaqueductal gray in either the tail flick or hot plate tests. Injection of bombesin (approximately 1 pg, ic) in mice did not produce antinociception in a tail immersion test (Nemeroff et al., 1979a). VI. Peptides with Mixed Effects

A. THYROTROPIN-RELEASING HORMONE Thyrotropin-releasing hormone (TRH) is a tripeptide (Table I) that releases thyroid-stimulating hormone and prolactin from the anterior pituitary. A large literature indicates that TRH produces a variety of direct CNS effects that are not mediated by its actions on the pituitary (for a review see Nemeroff et al., 1979a). In mice and rats TRH does not alter nociception in a variety of tests (Martin et al., 1977; Osbahr et al., 1981). However, in a short communication, antinociception in a writhing test following icv injection of TRH to mice has been reported (Reny et al., 1982).In our discussion of neurotensin (see Section V,A), the fact that TRH antagonized the antinociceptive effect of neurotensin, but not B-endorphin, was mentioned. In mice TRH has been found to inhibit tolerance to opiate-induced analgesia but not hypothermia (Bhargava, 1981). This effect of TRH was shown not to be due to an alteration in brain or plasma morphine concentrations. Thyrotropin-releasing hormone, but not naloxone, antagonizes Ag-tetrahydrocannabinol-induced antinociception in a tail flick test (Bhargave and Matwyshyn, 1980). B. MELANOCYTE-STIMULATING HORMONE INHIBITORY FACTOR (MIF-I)

Melanocyte-stimulating hormone release-inhibiting factor is a tripeptide (Table I) identical to the tail of the oxytocin molecule. It has been hypothesized to function as a hypophysiotropic hormone inhibiting the release of melanocyte-stimulatinghormone from the pars intermedia of the pituitary.

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When administered orally MIF-I (100 mg/kg) was reported to inhibit acetic acid-induced writhing by 32% (Plotnikoff and Kastin, 1974). These data should be cautiously interpreted because this is a very weak analgesic effect, and writhing tests appear to have low specificity (Hendershot and Forsaith, 1959; Chernov et al., 1967). The analgesic effect of enkephalins and morphine in mice in a tail flick test is antagonized by ip injection of MIF-I (Kastin et al., 1979). In contrast, the contractile effect of opioid peptides in a vas deferens assay was not altered by MIF-I. Further investigation revealed that the MIF-Iinduced antagonism of morphine-induced antinociception does not require the presence of the pituitary (Kastin et al., 1980). It is of interest that ic MIF-I did not alter neurotensin-induced antinociception in either a tail immersion or hot plate test (Osbahr et al., 1981).This finding may be construed as further evidence of a dissociation of mechanisms that mediate opioid-induced antinociception and neurotensin-induced antinociception. Finally, chronic treatment with MIF-I has been reported to block the analgesic effect of morphine in the tail flick test in rats and also to partially attenuate the effect of morphine in the hot plate test in mice (Dickinson and Slater, 1980). C. MELANOCYTE-STIMULATING HORMONE a-Melanocyte-stimulating hormone (a-MSH) is a tridecapeptide (Table I) that is derived from proopiocortin. Szekely et al. (1979) reported that a a-MSH administered concomitantly with morphine reduced tolerance to the opiate. Consistent with this finding is the report of Sandman and Kastin (1981) that icv administration of a-MSH produces hyperalgesia in rats in a tail flick test. Doses as low as 0.1 ,ug of the peptide were effective and a larger dose (1 pg) produced a long-lasting (>SO min) hyperalgesia. In contrast, Walker et al. (1980) found that aMSH microinjected into the periaqueductal gray produced a behavioral profile that was similar to that of P-endorphin, including antinociception. It would appear that a-MSH is capable of producing opposite effects on nociception depending on the injection site in the CNS.

VII. Discussion

The preceding text amply documents many peptide-induced alterations in nociception. Whether one or more peptides are involved in the

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physiology of nociception is still unclear. Data required to definitively answer this question have still not been generated, although the results of such inquiries are available for certain peptides. T o determine the endogenous role of a particular compound, several experimental approaches should be utilized. These issues are reminiscent of criteria for establishing identification of neurotransmitters. To demonstrate involvement of a endogenous peptide in nociception, it would be important to demonstrate that:

1. The peptide is present in the organism. 2. The peptide is released when the system is physiologically activated by noxious stimuli. 3. T h e peptide has access to potential anatomical sites of action. 4. The peptide administered at the postulated site of action mimics the response produced by physiological activation of the system. 5. A system for inactivation of the peptide exists. 6. Antagonists of the effects of the exogenously administered peptide should also antagonize physiological activation of the system. It is clear that the greatest variety of experimental approaches has been used in the study of the putative physiological role of the endogenous opioid peptides. Use of both RIA and immunohistochemical techniques have demonstrated that several opioid peptides are localized within regions of the CNS that have been implicated in nociception. Peptidases that degrade opioid peptides have been isolated and in some instances partially characterized. Moreover, inhibitors of certain of these enzymes have been synthesized. It has been established that in a variety of tests in different laboratory animals that, when injected into certain brain areas, the opioid peptides induce antinociception. The suggestion has been made that a variety of stressors and acupuncture induce release of endogenous opioid peptides. Finally, opiate antagonists (e.g., naloxone) can antagonize both exogenous opioid-induced antinociception and the effects of some, but not all, environmental manipulations that induce antinociception. Some of the techniques utilized to determine if endogenous peptides act as physiological mediators of nociceptive processes merit further discussion. A veritable plethora of information concerning the opioid peptide @endorphin is available and perhaps serves as a useful example; this peptide fulfills many of the criteria described above. Determination of the putative role of endogenous fl-endorphin has rested largely on the use of the opioid receptor antagonist naloxone. It is evident that the question of specificity of naloxone as a /3-endorphin antagonist must be addressed. Although it is clear that naloxone blocks the binding of fl-

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endorphin to brain membranes, it produces other effects as well. Naloxone blocks effects produced not only by /3-endorphin, but also those of other opioid peptides. T h e finding that not all /?-endorphin binding sites are sensitive to the effects of naloxone reveals the complexity of this field (Hazum et al., 1979). In addition, certain of the behavioral effects induced by /?-endorphin are not naloxone-sensitive (e.g., see DeWied et al., 1978). In fact, under certain conditions naloxone has even been reported to induce analgesia in humans (Buchsbaum et al., 1977; Levine et al., 1979). Moreover, high doses of naloxone have been reported to block y-aminobutyric acid receptors (Hill, 1981). One must conclude that if analgesia is decreased by naloxone treatment, it is not necessarily mediated by endogenous p-endorphin. Measurement of peptide concentrations in tissue or biological fluids may provide valuable information concerning the role of a peptide in nociceptive processes. If various environmental stimuli induce an antinociceptive response, alterations in the concentration of one or another opioid peptide can obviously yield useful information. For example, acupuncture has been reported to increase CSF concentrations of pendorphin-like activity in humans (Sjolund and Eriksson, 1979). Regional brain concentrations of /?-endorphin are altered after auricular electrical stimulation in rats (Pert et al., 1981). This approach does, of course, have its limitations. The data obtained is correlational and cannot therefore unequivocally demonstrate cause and effect. Data are often collected from an accessible site distal to the proposed site of action (e.g., plasma). T h e changes of peptide concentrations in plasma are seldom of the magnitude required to induce antinociception. This may, of course, be due to the possibility that plasma concentrations represent overflow from active sites and not a means of distribution to the active site. Certainly, much available data is consistent with the notion that endogenous /?-endorphin is involved in mediating some types of environmentally induced antinociception. The evidence that the opioid peptides maintain a certain basal level of nociception in the unperturbed organism is certainly much less compelling. Exogenous administration of several different peptides that do not bind to opiate receptors has been reported to induce a naloxone-reversible antinociceptive response. These include substance P, SRIF, and cholecystokinin. Several different explanations have been promulgated to explain these findings. These peptides may induce the release of an opioid peptide in viva Alternatively, a metabolite of the injected peptide may interact with opiate receptors. Barclay and Phillipps (1980) demonstrated that some of these peptides, if administered in high doses, may

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compete with the enzymes involved in opioid peptide degradation, thereby making more opioid available to act at the receptor sites. Whether this could occur under physiological conditions remains unclear. Several other peptides, including neurotensin, vasopressin, and calcitonin, produce antinociception that apparently does not depend on opioid mechanisms. These findings are concordant with the observation that certain stressors are capable of eliciting antinociception that is not reversed by naloxone. Furthermore, the finding that electrical stimulation-induced analgesia is only partially attenuated by naloxone implies the involvement of one or more of the previously mentioned peptides as well as a nonpeptide. Consistent with this are the findings that neurotension is contained in the periaqueductal gray and that microinjection of this peptide into this region induces antinociception. Although several peptides are believed to produce antinociception, others appear to enhance nociception. These include bradykinin, substance P, and ACTH. Bradykinin appears to primarily exert a hyperalgesic effect, whereas substance P appears to be capable of both increasing or decreasing pain perception. The predominant effect of substance P appears to depend on several factors including baseline responsiveness, dose, and site of action. T h e hyperalgesic effect of ACTH is indeed intriguing because it is apparently derived from the same precursor as /3endorphin. Furthermore, certain environmental stressors have been demonstrated to induce release of both peptides from the pituitary. These effects would not at first glance appear to be harmonious because @endorphin induces antinociception and ACTH, as noted, produces an opposite effect. However, pituitary release may not mimic the effects of P-endorphin and/or ACTH observed after microinjection into the CNS and therefore could be unrelated to the CNS effects of these substances. Electrophysiologic techniques have also been utilized, and their usefulness is probably best exemplified in the studies concerning bradykinin. The effects of bradykinin appear to be at least in part mediated by an action on primary afferent fibers. In studies of more complex neural circuitry within the CNS the significance of an observed electrophysiological change after application of a peptide is harder to interpret. For example, Haigler and Spring (19%) identified CNS neurons that responded to peripherally applied noxious stimuli. The effects of various peptides on these identified cells were then studied. Certain of the neurons were affected by iontophoresis of various peptides, whereas others were not. Furthermore, certain neurons that were not excited by noxious stimuli were affected by peptide application. Such data are difficult to assess. For instance, if only a few neurons responsive to noxious

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stimuli are affected by peptides, is this physiologically significant? The fact that nonnociceptive neurons are also affected by a peptide does not disprove that the peptide may play a physiological role in nociception. Many behavioral responses have been used to assess the role of peptides on nociception. The tests vary from spinally mediated reflexes (e.g., tail flick) to more complex responses to noxious stimuli that are believed to be integrated at higher CNS sites (e.g., hot plate). In fact, responses in the hot plate test have been suggested to be quite complex and have been further analyzed by separating paw licks from escape responses (Amir and Amit, 1978). Any one such behavioral test is of course associated with specific problems that may produce false positives or negatives. A peptide that increased grooming behaviors might increase the incidence of paw licks and therefore if tested on a hot plate might result in a shorter latency to paw lick. This might occur without actually affecting nociception, although it might be interpreted as an antinociceptive effect. To minimize such false interpretations it is probably best to use a variety of tests. Strictly speaking, in animal studies analgesia can not be assessed directly; rather what is measured is an antinocisponsive effect (i.e., diminished response to a noxious stimulus). In this review we have focused on the effects of peptides on nociception. It is important to recall that these peptides produce many effects that are apparently unrelated to nociception. This multiplicity of peptide actions is essential to consider if only one test of nociception is used because any observed effect may actually be due to an unnoticed and/or unmeasured effect of the peptide (e.g., peptide-induced grooming interactions with a hot plate test). It appears that several peptides are capable of altering the perception of andlor response to noxious stimuli. From a teleological viewpoint it is perhaps worthwhile considering why a myriad of peptides may be involved in nociception. Peptides that induce antinociception can be divided into those whose actions are naloxone-sensitive and those that are naloxone-insensitive. The naloxone-sensitive class of nonopioid peptides might not actually bind to opiate receptors. These peptides could conceivably act at other CNS loci in the neural circuitry involved with nociception. For the naloxone-insensitive peptides the most interesting findings may well be those obtained with neurotensin, for which an “antagonist” has been described (Osbahr ct al., 1981). Thyrotropin-releasing hormone has been found to block the antinociception induced by neurotensin, but not P-endorphin, suggesting at least two independent systems. This antagonism of neurotensin-induced antinociception by TRH, based on binding data, does not appear to be mediated at the receptor site. Our findings of a reduced antinociceptive response to

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neurotensin in morphine-tolerant mice, however, suggest that these systems may interact at some level (Luttinger et al., 1983). Alternatively, morphine tolerance may activate systems that oppose the antinociceptive effects of these other peptides. Clearly, it would be of great interest to determine if the antinociception induced by other naloxone-insensitive peptides (eg., vasopressin and calcitonin) is antagonized by TRH. In agreement with the notion of the existence of more than one nociceptive system is the observation that certain stressors induce a naloxone-sensitive, whereas others induce a naloxone-insensitive antinociception (Watkins and Mayer, 1982). In summary, many peptides have been found to alter pain perception in laboratory animals and a few (e.g., P-endorphin) have been found to induce analgesia in humans. In the last decade we have witnessed a large increase in our understanding of nociceptive processes. This information will undoubtedly lead to a better understanding of chronic pain states as well as the development of new analgesics. Acknowledgments

We are grateful to Judy Barnett for preparation of this manuscript. T h e authors are supported by NIMH MH-32316, MH-34121, MH-33127, MH-22536, NIA AG-03701, and NICHHD HD-03110. References

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OPlOlD ACTIONS ON MAMMALIAN SPINAL NEURONS By

W. Zieglgonsberger

Department of Neuraphannacalogy Max Planck Institute for Psychiatry Munich, Federal Republic of Germany

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

I. Introduction. . . . . . . .

A. Opiate Alkaloids. . B. Opioid Peptides . . . . . . . . . . . . . . . 11. Actions on Single Neu ............ A. Neurotransmitte .............. B. Single Unit Reco C. Microiontophoresis and Pressure Ejection . . . . . . . . . . . . . . . . . . . . . . . . . D. In Vitro and in Vizw. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Spinal Site of Action of Opiates ............................. F. Effects of Naloxone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Tolerance and Dependence on the Single Unit Level.. . 111. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . .. ...................................

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I. Introduction

Gross effects of systemic or topically applied opiate agonists demonstrating dose-dependent, stereospecific effects reversed by opiate antagonists like naloxone and naltrexone are legion and have been reviewed with various emphases (e.g., see Jaffe and Martin, 1975; Eidelberg, 1976; Fredericksen, 1977; Herz, 1978; Zieglgansberger and Fry, 1978; Terenius, 1978, 1981; Rossier and Bloom, 1979; North, 1979; Beaumont and Hughes, 1979). These changes include alterations of behavior, mood, mental processes, and endocrine and autoregulatory functions. T h e most extensive studies carried out on the actions of opiate agonists have been related to analgesia, both its nociceptive-reflexive and the emotional-affective components. I n reviewing the enormous amount of literature concerning opioid actions on neuronal systems it was clearly necessary to restrict the areas covered. This review represents an evaluation of the literature rather than an exhaustive documentation of the actions of opioid agonists in the mammalian spinal cord with special emphasis on their antinociceptive action. Biochemical, histochemical, and structure-activity data will 243 INTERNAI'IONAL REVIEW OF NEUROBIOLOGY, VOL. 25

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be reviewed only in outline. A major part is devoted to the illustration of the methods preferentially employed in these studies. A. OPIATEALKALOIDS The pharmacological effects of the plant alkaloid or its synthetic or semisynthetic congenors can be seen after injection of doses that appear to cause no changes in axonal conduction or in the sensitivity of sensory nerve endings (Wagers and Smith, 1960; Cairne and Kosterlitz, 1962; Ritchie and Armett, 1963; Kosterlitz and Wallis, 1964; Williams and Zieglgansberger, 1981a; also see below). At rather high concentrations both opiate agonists and antagonists affected voltage-sensitive ion channels involved in spike generation. Although an action on fibers in peripheral nerve seems unlikely, this possibility has received some attention (Jurna and Grossmann, 1977; Ferreira and Nakarnura, 1979; Chal, 1980; Maruyama et al., 1980; Bentley et al., 1981). However, the physiological and therapeutic relevance of such an interaction with the prostaglandin system described in peripheral sensory nerve endings needs to be substantiated by further experiments. B. OPIOIDPEPTIDES T h e discovery of stereospecific binding sites for opiate. alkaloids in the nervous tissue of mammals including man (Pert and Snyder, 1973a,b; Simon et al., 1973; Terenius, 1973; Goldstein, 1974; Snyder and Matthyse, 1975; Snyder and Simantov, 1977) stimulated the search for endogenous ligands operative at these structures under physiological conditions. The first attempts to isolate and characterize these endogenous substances probably failed because a close similarity to the opiate alkaloid molecule was expected. This search was highlighted by the discovery of the two pentapeptides methionine- and leucine-enkephalin (Hughes et al., 1975) and of the opioid peptides (the term endorphins will be used in a generic sense) of higher molecular weight, some of which share common N-terminal sequences with C-terminal fragments of plipotropin, a neurohormone previously characterized in the pituitary (e.g., see Beaumont and Hughes, 1979; Bloom, 1979a,b,c; Bloom et al., 1979, 1980; Rossier and Bloom, 1979; Hokfelt et al., 1980; Rossier, 1982). Two highly potent opioid peptides that possess the amino acid sequence of Leu-enkephalin at its N-terminal were recently charac-

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terized (dynorphin) (Goldstein et al., 1979, 1981; Tachibana et ul., 1982) (a-and P-neoendorphin) (Kangawa et al., 1981; see Hiillt, 1983). Since the discovery of the first opioid peptide some 20 other endogenous compounds have been isolated and characterized that mimic the naloxone-reversible effects of opiates (Sawynok et al., 1979) in bioassays designed for opiate activity (Wuster et al., 1981) or compete at low concentrations with opiate ligands for binding at opiate-binding sites. A compound termed fraction I (consisting of several subfractions) was found in the cerebrospinal fluid (CSF) of man and gained considerable attention (Terenius and Wahlstrom, 1975; Terenius, 1978) as a peptide involved in nociceptive information processing. Its identity is not yet known, but it is clearly separable from hitherto characterized opioid peptides. It is interesting that it was postulated that a dipeptide (kyotorphin), which has a naloxone-reversible opiate-like effect, acts by releasing opioid peptides rather than interacting directly with the opiate receptor (Satoh et al., 1980; Shiomi et al., 1981). The functional characterization of these neuropeptides awaits further investigation.

1. Distribution, Release, and Metabolism Studies on the rather specific distribution of the opioid peptides in the nervous system and their origin from different precursor molecules (Noda et al., 1982; Comb et al., 1982; Gubler et al., 1982) indicate that the various endorphins subserve different physiological functions. T h e high molecular weight opioid peptides like P-endorphin and dynorphin and the enkephalin-containing systems exist in separate neuroanatomical networks, and there is evidence that, for example, methionine-enkephalin and leucine-enkephalin are not breakdown products of @-endorphin or dynorphin (Austen and Smyth, 1977; Rossier et al., 1977; Watson et al., 1977a,b, 1978; Bloom etal., 1978; Rossier, 1982). This view is further supported by the finding that these opioid peptides interact with a different spectrum of opiate receptor (binding site) subclasses distributed also in a rather specific manner in the CNS (see Section I,A,2). Surprisingly, there is presumptive evidence that even methionine- and leucineenkephalin, which are invariably distributed in parallel in the CNS, can be localized in different neuronal populations (Larson eL al., 1979). A survey of present data suggests that there exists no simple anatomical or functional link between the endorphinergic systems and a particular neurotransmitter system (see following paragraphs). In contrast to the mainly short-axoned interneurons staining for the enkephalins that are present at all levels of the neuraxis, P-endorphinreactive ~naterialis almost exclusively found to originate from the basal

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hypothalamus and adjacent tissue (arcuate nucleus) and project paraventricularly caudal to the level of the locus coeruleus. Dynorphin fibers project to cortical and limbic areas, midbrain, and brainstem (Watson et al., 1982). In contrast to /?-endorphin, which seems to disappear during ontogenesis (Haynes et al., 1982), dynorphin-reactive material is also present in the adult spinal cord (Botticelli et al., 1981; R. Pzewlocki, personal communication). I n addition in the pituitary and the projections to the diencephalic and mesencephalic areas no P-endorphinergic innervation has so far been substantiated by histochemical observations in mammals. However, this would not preclude a physiological action of /?-endorphin on remote neurons carrying opiate receptors following release into the circulation or into the CSF (Millan, 1981) after, for example, psychic and physical stressors have been applied or CNS stimulation or acupuncture have been employed (Akil et al., 1978; Hosobuchi et d., 1979; Terenius, 1981; Watkins et al., 1982; Watkins and Mayer, 1982). This assumption gains support from the fact that ACTH and /?-endorphin are derived from a common precursor and are coreleased. However, the concentrations commonly found in plasma appear too small to produce an analgesic effect. A dual role for an endogenous opioid peptide both as a neurotransmitter and a neurohormone would not be unique to this class of compounds. In the past several endogenous substances (e.g., norepinephrine) have been accepted as both neurotransmitters and neurohormones (Bloom, 1979~). T h e density of opiate receptors in the nervous system is loosely correlated with the number of perikarya, fibers, and terminals containing enkephalin-reactive material. This finding further supports the view that enkephalin-containing neurons (provided that the degree of crossreactivity with other peptides is not significant) are mainly short-axoned cells that usually d o not project to other brain sites. However, there are several exceptions; in addition to short projections like the striato-pallidal, amygdala-stria terminalis, hypothalamo-neurohypophysial,, entorhinal-hippocampal and intrahippocampal connections including the mossy fiber system, ascending pathways from cells in Lamina I (Glazer and Basbaum, 1979) and descending pathways in the spinal cord have been described (Cuello and Paxinos, 1978; Elde and Hokfelt, 1979; Hokfelt et al., 1979, 1980; Gibson et al., 1981; Bowker et al., 1981). At present there is no information available concerning specific structural and physiological properties of peptidergic fibers, terminals, or cell bodies. However, the processing of the neuropeptides seems to differ from that observed with other putative neurotransmitters: After they are synthetized on the ribosomes they are converted from a precur-

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sor molecule to the final product during axoplasmic transport (for reviews see Hokfelt et al., 1980; Iversen et al., 1980). A feature common to most cells staining for neuropeptides is that they contain large granular vesicles (Goldsmith, 1977). Several of these neuropeptides coexist with other neuropeptides and with classical neurotransmitters such as the biogenic amines in the same neuron (Hokfelt et al., 1980). In this respect these peptidergic systems resemble endocrine cells, which process and store both peptides and biogenic amines (APUD system) (defined by Pearse, 1969). Endorphins are concentrated in the synaptosomal fraction (Simantov et al., 1976; Osborne et al., 1978; Fukata et al., 1980) and can be released from neuronal elements subsequent to depolarizing stimuli (Henderson et ad., 1978; Osborne et al., 1978; Iversen et al., 1978, 1980; Richter et al., 1979; Osborne and Herz, 1980; Yaksh and Elde, 1981) or more physiologically after activation of descending pathways, for example, to obtain pain relief in patients (Akil et al., 1978). The role of opioid autoreceptors supposedly located on the terminals of enkephalinergic neurons in their release is still obscure (Kosterlitz and Hughes, 1975; Goldstein, 1976; cf. Osborne and Herz, 1980). T h e physiological action of the released enkephalins is probably terminated by specific enkephalinases (Goretistein and Snyder, 1980; Guyon et ul., 1980). This view is supported by the finding that enkephalinase blockers enhance the antinociceptive effect of these compounds and elicit a naloxone-reversible analgesia (Stine et al., 1980; Roques et al., 1980).

2 . Multaple Opiate Receptors The highly specific pharmacological actions of opiates suggested an action mediated through specific receptors long before direct experimental evidence was provided. The attempt to solubilize the opiate receptor complex was undertaken by several groups (Simon et al., 1975; Zukin and Kream, 1979; Ruegg et al., 1980; Bidlack and Abood, 1980; Simonds et al., 1980). If this macromolecular complex could be purified to homogeneity without alteration of its structure, this would permit the analysis of receptor/agonist interactions at a molecular level-a goal still not reached. Heterogenous classes of binding sites ( p ,6, K , E , (T) present in different densities in various central and peripheral neuronal tissues have been demonstrated both in vivo (Martin et al., 1976) and in vitro (Lord et al., 1977; Herkenham and Pert, 1980), but there is at present little evidence that specific classes are related to a particular action, for example,

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the preceptor to analgesic actions (for reviews see Kosterlitz et al., 1980; Wuster et al., 1981; Simon, 1981; Zukin and Zukin, 1981). Some subtypes of opiate receptors are probably colocalized on the same neuron (Zieglgansberger et al., 1980; Williams and Zieglgansberger, 198lb; for the myenteric neurons see Egan and North, 1981), and tolerance develops selectively to p- and &preferring agonists (Schulz et al., 1980a,b). Such a lack of cross-tolerance suggests that this adaptive process develops at the level of the binding or transducing system and not in the effector system, which might be involved in the development of dependence (see also Section 11,G).The notion that p and 6 binding sites are closely related (Vaught et al., 1980; Rothman and Westfall, 1981) and might even interconvert from a p to a &preferring state by an allosteric shift (Bowen et al., 1981) is supported by cross-protection experiments (Robson and Kosterlitz, 1979; Smith and Simon, 1980).It remains to be established whether the other subtypes of opiate receptors are independent entities. Surprisingly, there is evidence from electrophysiological experiments that enkephalin-preferring and morphine-preferring receptors might have different distributions in the dorsal horn (Duggan et al., 1981). Binding studies do not permit localization to subsynaptic membranes or nerve terminals. The controversial issue of pre- and/or postsynaptic localization of opiate receptors and potential sites of action will be addressed later in this review (LaMotte et al., 1976; Pollard et d., 1977; Pickel et al., 1980). A most puzzling finding is the obvious axonal transport of “opiate receptors” (Atweh and Kuhar, 1977; Young el al., 1980). It remains unclear whether the binding assay shows “receptors” that are internalized or transported in the axoplasmic flow. Furthermore, it has to be kept in mind that a low number of opiate binding sites does not necessarily make a particular site a low-priority target area for opiate actions. A major interest in synthetic congeners of the natural ligands of the opiate receptor(s) centers around the possibility of finding agonists with high preference for one class of binding site so that after tolerance development the desired, for example, analgesic action can be triggered via the remaining unaffected class of binding sites. Multiple non-crosstolerant subclasses of opiate receptors may also be predicted to be associated with spinal nociceptive mechanisms (T. L. Yaksh, personal communication; Zieglgansberger et al., 1982), for example, the recently synthetized rather selective p-, 6-, and probably K-preferring agonists with dynorphin as a prototype (Kosterlitz et al., 1981; Corbett et al., 1982; Chavkin et al., 1982) might have therapeutic value (see Section 11,E).

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II. Actions on Single Neurons

A. NEUROTRANSMITTER/NEUROMODULATOR It is obvious that the mere presence of specific peptides and their receptors in certain parts of the CNS does not warrant any speculation about a role as intercellular messenger of any type involved in pharmacological or behavioral effects. Current interest centers on whether opioid peptides are credible candidates for neurotransmitter status or are involved in unconventional modes of information transfer. The specific distribution, the pattern of release following synaptic and chemical stimulation, and the distinct actions that some of these peptides exert on neuronal systems gives support to the belief that they are involved in synaptic transmission. Current research suggests that communication among neurons in the mammalian nervous system is not limited to the rapid opening and closing of ionic channels characteristic of most amino acid neurotransmitters. In addition to such fast synaptic transmission, slower processes take place at the synaptic sites that transiently alter, for example, the response of the neuron to other transmitters without changing the membrane potential or membrane conductance (Kupferman, 1979; Siggins, 1979; Hill, 1980). It is obvious that such a mode of neuronal communication cannot give detailed temporal information. However, it would be able to tune a larger array of target cells or change the sensitivity of parts of the surface of a neuron to neurotransmitters released from other or even the same terminals. Such assumptions are in keeping with the emerging concept that the borderline between neurosecretion and neurotransmission is less clear than previously thought. Despite the fact that there is no universal agreement on an appropriate definition, the term neuromodulator is widely used to describe the latter group of agents, which may include the endorphins (see Section 11,E).

B. SINGLEUNITRECORDING Extracellular recording of single unit activity is limited to studies involving changes in the number of action potentials, which are determined by neuronal and nonneuronal inputs. Intracellular recordings offer additional information about subthreshold events and the ionic mechanism of action. Using this type of recording in combination with microiontophoretic application (for a review see Bloom, 1974), it is also possible to make some predictions about the topographic sensitivity of

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the neuronal surface to biologically active agents. These technically difficult studies have been performed in neurons of only a few mammalian species so far. Only recently were opiate-binding sites, stereospecific actions of opiate agonists, and enkephalin-containing neurons described in invertebrate species (Alumets et al., 1976; Brecha et al., 1979; Stefan0 et al., 1980, 1981; Mancillas et al., 1981; O’Shea, 1982). Studies on large invertebrate neurons, which are accessible t o elaborate electrophysiological techniques, might have considerable advantages for the elucidation of ionic mechanisms of action of opioid peptides. C. MICROIONTOPHORESIS AND PRESSURE EJECTION

Most of the data about actions of opioid agonists on single units in the central nervous system derives from extracellular recordings in combination with systemic or microiontophoretic application. With the latter technique, minute amounts of a neuroactive substance can be applied into the immediate vicinity of the appropriate synapses. This route circumvents the diffusional barriers set by the blood-brain barrier. Furthermore, it reduces indirect effects arising from influences upon remote neurons projecting onto the neuron under study and from actions secondary to metabolic and circulatory disturbances due to hypotension and hypercapnia, typical consequences of systemic opiate administration. These quasi-quantitative methods are of vital importance for studies on central actions of opioid peptides because most of these compounds are rapidly degraded and do not penetrate into the CNS in sufficient concentrations, which precludes their systemic application. A major advantage in the interpretation of data obtained with systemic and microiontophoretic application of opiate agonists is the availability of a specific antagonist, which can be applied via the same route in comparable amounts. Although the number of molecules transported out of the micropipette (usually filled with 5 to 100 mM solutions) is linearly related to the current flowing (usually 50-200 nA for 30 to 120 sec, but see Section II,G), the actual concentrations reached in the neuropil are virtually unknown. Systemic applications of opiate agonists or antagonists in combination with local applications, however, provide evidence that both systemic and iontophoretic applications yield comparable concentrations (Satoh et al., 1976a; Johnson and Duggan, 1981b; Williams and Zieglgansberger, 198lb). Unfortunately, nonspecific effects of opioid alkaloids on the spike-generating mechanism also occur occasionally at concentrations reached by rather low iontophoretic currents. The spike-blocking effect can seriously interfere with data acquisi-

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tion if no precautions are taken to monitor the spike shape. T h e stereoisomers levorphanol/dextrorphari and naloxone also produce these nonspecific membrane effects. The availability of a specific antagonist and of active and inactive stereoisomers are therefore of vital importance for the interpretation of observed actions on unitary discharge activity (Goldstein et al., 1971; Goldstein, 1974). These side effects are absent or less marked with the opioid peptides, which display no influence on the shape of the extracellularly recorded action potential even after quite high iontophoretic currents are employed. The major disadvantage of the microiontophoretic technique, the unknown concentration achieved by the current flowing, can be partially overcome by pressure application from micropipettes filled with diluted solutions. Through this microperfusion technique, which does not require high pressure or measurement of the volume ejected, the concentration at the orifice of the micropipette (tip size 1-2 pm) can be determined, and it is obvious that the Concentration reached at the receptive sites is lower. In various studies this technique has been applied to central neurons. It was established (Zieglgansberger et aZ., 1982) that the effective concentrations of opiates reached at the receptive sites utilizing commonly used iontophoretic currents compared favorably to those used in various bioassays such as the guinea pig ileum or the mouse vas deferens ( 1 O-’- 10-9 M). In addition to the mostly unknown but effectively constant diffusional barriers and uptake and dissolution processes in the neuropil, there are other intriguing features that may be even harder to clarify; the diffusing compounds could act on post- and/or presynaptic elements and reach neurons in their vicinity that are synaptically linked to the neuron under study. There is experimental evidence that such indirect effects occur (e.g., in the hippocampus; Zieglgansberger et al., 1979).

Designed to overcome the complexity of the CNS, in nitro preparations for the study of drug actions have created much enthusiasm among neuroscientists. It is assumed that most of the in V ~ U Ophysiology remains intact. As in in U ~ Z Jpreparations O the identification of neurons by stimulation of some of the afferent pathways is possible, and pharmacological actions can be analyzed without the interference of anesthesia or paralyzing agents. However, the current research illustrates that of the whole list of technically possible investigations in ZYL zdro preparations only a few are actually realized, so the gap between the possible and the already

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achieved is still great. Only recently has the adult spinal cord of rat become available as an in vitro preparation (Zieglgansberger and Sutor, 1983).In vitro preparations permit the administration of the opiate agonist and antagonist in known concentrations. This method of application resembles systemic drug administration but might lead to a simultaneous activation of receptors, which does not occur under physiological conditions. The use of tissue cultures of mammalian spinal neurons in neuropharmacology has introduced several advantages, but these preparations certainly have their inherent pitfalls and shortcomings. In these cells the soma-dendritic membrane can be investigated with excellent visualization and recordings from cocultured neurons can be performed. However, despite the fact that coculturing is possible and some of the dissociated spinal neurons grown in tissue culture contain enkephalin-reactive material (Neale et al., 1978), the mostly unidentified neurons in culture bear only modest anatomical similarities to in vivo cells. Furthermore, this tissue is usually derived from immature cells, and their pharmacological responses might be different. Acute and chronic drug treatment of this tissue will yield clear data. In respect to effects seen in the intact organism, however, these data at times may not be relevant.

E. SPINAL SITEOF ACTIONOF OPIATES Systemically administered opiate agonists depress polysynaptic spinal reflexes in a variety of species, including man, in a dose-dependent, stereospecific, and naloxone-reversible manner (for reviews see Martin and Sloan, 1977; Yaksh, 1981; Yaksh and Hamniond, 1981). Monosynaptic reflexes are less or not at all affected by the same doses. However, slightly increased doses of opiate agonists also reduce the effect of Group 11 afferent stimulation (Koll et al., 1963). In most of the investigations concerned with opiates and spinal reflexes, mixed muscle and cutaneous afferents were stimulated, and it remains unclear what fiber types were excited. Furthermore, systemic administration does not permit any conclusion about the neuronal elements involved in such an action. In a number of experiments naloxone alone enhanced reflexes, suggesting the existence of a tonically active endorphinergic inhibition in the spinal cord (see following paragraphs). Several lines of evidence suggest that serotoninergic, noradrenergic, and other still unidentified transmitter systems are involved in opiateand stimulus-produced analgesia (for reviews see Basbaum and Fields,

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1978; Fields and Basbaum, 1978; Yaksh and Hammond, 1981; Yaksh, 1981). The serotoninergic fiber system arises from the raphe nuclei, which also relay most of the descending influences from the periaqueductal gray. The noradrenergic components have their origin in the brainstem. Endogenous opioid peptides have not only been implicated in local spinal circuitry but have also been postulated to be involved in the descending control of afferent nociceptive signals. The relative importance of supraspinal versus spinal effects following opiate application for behavioral analgesia is still under debate. It is still totally obscure how the activation of descending pathways is evoked by these drugs, which are primarily inhibitors of neuronal activity in the brainstem (Basbaum, 1981). T h e classification of a spinal site of action of opiates has led to a major advance in pain therapy. Intrathecal or epidural applications of opiate agonists via chronically implanted catheters induce a long-lasting analgesia restricted to the dermatomes projecting to the spinal segments reached by the diffusing agonist. The analgesic effects reported by the patients are usually not associated with motor and autonomic impairment or respiratory depression, a hallmark of systemic applications of opiates (for a review see Yaksh, 1981). Intrathecally or epidurally applied opiate agonists-in contrast to local anesthetics-most probably act via a depression of neuronal activity in dorsal horn cells that give rise to ascending tracts like the spino-thalamic or the spino-reticular system. Most of these neuronal species are activated by multimodal afferents (A6 and C fibers). These cells’ spontaneous and evoked activity recorded from their cell bodies or from their ascending axons in the dorsolateral tracts is inhibited by systemically o r microiontophoretically administered opiates. Present research suggests a neuronal circuitry operative in the modulation of somatosensory information at this first stage in the dorsal horn that involves opiate and nonopiate systems. A major endorphinergic link that is synaptically activated by descending fibers exists in the spinal cord (Levine et al., 1982; Zorman et al., 1982). Environmental stimuli can activate segmental neuronal circuits as well as descending pathways. There is evidence that electrical stimulation of peripheral nerves, primarily with acupuncture-like stimulus parameters (low-frequency/high-intensity) (Pomeranz and Chiu, 1976; Chapman and Benedetti, 1977; Sjolund and Eriksson, 1979; Cheng and Pomeranz, 1980; Pert et al., 1981; Shimizu et al., 1981), noxious stimulation of other parts of the body (LeBars et al., 1981), and stimulation of various sites in the CNS can induce analgesia (Reynolds, 1969; Mayer et al., 1971; Hosobuchi et al., 1977) that is to some degree reversible by naloxone (Akil et al., 1976; Adams, 1976; Lewis and Gebhart, 1977; for

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reviews see Mayer and Price, 1976; Basbaum and Fields, 1978; Yaksh and Rudy, 1978; Goldstein, 1979; Pert, 1980). In humans no effect of naloxone was reported on pain threshold (El-Sobky et al., 1976; Grevert and Goldstein, 1977; cf. Buchsbaum et al., 1977; Levine et al., 1978, 1979) or analgesia produced by hypnosis (Goldstein and Hilgard, 1975), but naloxone obviously blocks the acupuncture-induced analgesia that reduced the excitation of dorsal horn neurons following noxious stimulation (Pomeranz and Chiu, 1976; for reviews see Goldstein, 1979; Terenius, 1981). Furthermore, the subjects reported slight changes in mood and feeling (Schull et al., 1981). The observation of a naloxone-induced hyperalgesia (see Section I1,F) casts some doubts on the validity of these tests as indications for a direct link with an endorphinergic system. Via a reduction of an (inhibitory) endorphinergic tonus naloxone could also influence antinociception induced by nonopioid drugs such as benzodiazepines or functionally antagonize non opioid systems. Analgesia induced by dorsal column (Abdelmoumene, 1979) or vaginal stimulation (Crowley et al., 1977) is not affected by naloxone, suggesting functional independence from the endorphinergic system (cf. Hill and Ayliffe, 1981). First-order neurons in the dorsal horn that respond to a wide variety of somatosensory stimuli, including synaptic inputs triggered by noxious thermal, mechanical, and chemical stimulation, are widespread throughout the dorsal horn and seem to be under the control of small, predominantly inhibitory interneurons located primarily in the substantia gelatinosa (Lamina 2)(for reviews see Cervero and Iggo, 1980; Wall, 1980; Yaksh and Hammond, 1981). Spontaneous, synaptically or chemically induced firing of neurons in the various laminae and their analog sites in the trigeminal complex has been reported to be predominantly inhibited by microiontophoretically or intravenously applied opiate agonists (Sasa, 1969; Besson et al., 1972, 1975; Conseiller et al., 1972; Kitahata et al., 1974; Calvillo et al., 1974, 1979; Henry and Neuman, 1974; Neuman et al., 1974; Lodge et al., 1974; LeBars et al., 1975, 1976a,b; Duggan et al., 1976a, 1977a,b, 1981; Zieglg~nsbergerand Bayerl, 1976; Davies and Dray, 1976, 1978; Andersen et ad., 1977; Toyooka et al., 19’77, 1978; Belcher and Ryall, 1978; RandiC and Miletit, 1978; Satoh et al., 1979a,b; Jurna and Heinz, 1979; Zieglgansberger and Tulloch, 1979b; MiletiC and RandiC, 1980; Einspahr and Piercey, 1980). In several studies these inhibitory actions on dorsal horn neurons were characterized as naloxone-reversible and stereospecific (Calvillo et al., 1974; Zieglgansberger and Bayerl, 1976). A selective effect of morphine on cells in particular laminae was postulated. However, recent data suggest that probably all laminae receive high-threshold afferents, casting some doubts on the

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relevance of these findings. Systemic or iontophoretically applied opiates preferentially depress impulses triggered by small diameter afferents, which are considered to provide the major nociceptive input. However, spontaneous firing and responses of neurons to nonnoxious stimuli are also influenced (see following paragraphs). In iontophoretic experiments naloxone, the most frequently used antagonist, occasionally displays nonspecific actions on the spike-triggering mechanism when applied with iontophoretic currents sufficient to antagonize opiate actions. Tests employing both the (-) and the (+) enantiomer of naloxone demonstrated that the nonspecific actions are produced by both forms of the molecule, whereas only the (-) enantiomer displays antagonistic actions (Fry et al., 1980a,b; Gruol et al., 1980). Microiontophoretically applied morphine has also been reported to antagonize the inhibitory effects of glycine (Curtis and Duggan, 1969; Dostrovsky and Pomeranz, 1973) on spinal interneurons, an effect that is probably nonspecific because it only became apparent after application of high iontophoretic currents and was not naloxone-reversible (Duggan et al., 1967a,b; Zieglgansberger and Bayerl, 1976). This antiglycine effect can be observed in the absence of any effect on GABA-induced inhibition (Dostrovsky and Pomeranz, 1973, 1976) and may therefore reflect a strychnine-like action of morphine at the glycine receptor. Morphine is structurally related to thebaine, a convulsant and glycine antagonist (Curtis rt al., 1968). In contrast to most other cells in the spinal cord, Renshaw cells were found to be excited by microiontophoretically administered morphine and opioid peptides (Duggan and Curtis, 1972; Davies and Duggan, 1974; Lodge et al., 1974; Davies, 1976; Davies and Dray, 1978), which also potentiated the excitatory action of acetylcholine and D,L-homocysteic acid administered via the same route. Both opiate- and acetylcholine-induced excitations were antagonized by naloxone, suggesting a postsynaptic site of action of these compounds. There is evidence that the excitatory actions of both drugs are mediated via nicotinic acetylcholine receptors, because the excitatory actions of the muscarinic agonist acetyl-P-metacholine were not antagonized by naloxone. At present it is not clear how an interaction between the nicotinic acetylcholine receptor and the opiate receptor is brought about. There seems to be an essential involvement of the opiate receptor, because of the suppression of these responses by naloxone. However, the enhancing effect of morphine is not mimicked by levorphanol (Duggan et al., 1976a) or etorphine (Lodge et al., 1974). These two opiate agonists reduce the excitation of Renshaw cells by both acetylcholine and the amino acids. The relevance of excitatory responses, which seem to be a specific property of these cells, to

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understanding the actions of opiates observed in the spinal cord is still obscure because intravenous morphine excites only some Renshaw cells (Felpel et al., 1968; Duggan et al., 1976a). It has been described by Duggan et al. (1976b, 1977a,b) that opiate agonists have a particularly strong (naloxone-reversible) action inhibiting the excitatory effects of noxious cutaneous stimuli on dorsal horn cells if applied from a separate micropipette into the substantia gelatinosa. There are several possibilities as to the site at which these depressant effects can be produced. First, dendrites of Laminae 4 and 5 neurons that ramify in the substantia gelatinosa could be affected; second, opiate agonists could excite gelatinosa neurons that are inhibitory on Lamina 5 neurons, and third, opiates might interact with presynaptic primary afferent terminals in the substantia gelatinosa and reduce transmitter release, as has been postulated also in other structures (e.g., see Monte1 et al., 1974; Taube et al., 1976). No selectivity for the suppression of noxious stimuli was found for an enkephalin derivative applied via the same route (Duggan et ad., 1976b). From the surprising finding that iontophoretically applied excitant and depressant amino acids failed to mimic the actions of morphine, Duggan et al. (1977b) suggested that the opiate receptors were not located on interneurons of the substantia gelatinosa or on dendrites of deeper neurons. This view has to be revised in the light of more recent morphological findings (see following paragraphs). Laminae 2 and 3 of the dorsal horn of the spinal cord and their rostra1 analogs of the trigeminal system have attracted considerable attention as a potential site for an endorphinergic control mechanism involved in nociception. In all species investigated these structures contain a relatively high density of opiate binding sites (Pert and Snyder, 1974; LaMotte et al., 1976; Atweh and Kuhar, 1977) and cell bodies and terminals staining for the enkephalins (Elde et al., 1976; Hong et al., 1977; Watson et al., 1977b; Hokfelt et al., 1977; Simantov et al., 1977; Sar et al., 1978; Uhl et al., 1979; Glazer and Basbaum, 1981; Ruda, 1982; Sumal et al., 1982; also see Elde and Hokfelt, 1979; Emson, 1979; Hokfelt et al., 1980; Hunt et al., 1982) when compared to the more ventral parts of the spinal cord. The enkephalin content of the dorsal horn of the spinal cord and the trigeminal complex (subnucleus caudalis) was not changed by dorsal root rhizotomy, sectioning of the cord, or destruction of the Gasserian ganglion, with the result that enkephalin was thought to be contained mainly in short-axoned interneurons (Hokfelt et al., 1980). A significant portion of the opiate binding sites ( p and 6 subtypes) (Fields et al., 1980) appear to be on the terminals of primary afferent

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fibers, because dorsal root rhizotomy (LaMotte et al., 1976) or neonatal capsaicin treatment (Nagy et al., 1980) decreased the number of binding sites by about 50%. However, in both types of study degeneration of postsynaptic structures secondarily to the lesioned afferents cannot be excluded (see following paragraphs). Pharmacological studies (Jessell and Iversen, 1977;Jessellet al., 1978; Yaksh et al., 1980) demonstrating reduction of substance P release from slices of trigeminal nucleus following administration of opiate agonists seem to support the assumption of an interaction between primary afferent systems using substance P as a transmitter and opiate peptides at the first stage of integration. Preliminary assumptions concerning the site of this interaction need to be revised in the light of more recent histochemical and ultrastructural data, which suggest an almost exclusive axo-dendritic and axo-somatic interaction between enkephalincontaining neurons in the substantia gelatinosa (stalked and islet cells in cat) (Gobel et al., 1980; Sumal et al., 1982; Bennett et al., 1982) and dorsal horn cells (Ruda, 1982). In the commonly formed triad (primary afferent and labeled and unlabeled dendrites) the enkephalin-labeled structure displayed features of a dendrite and a membrane density associated with unlabeled dendrites. A number of the enkephalin-labeled neurons have vesicle-filled dendrites, characterizing them as islet cells. From these features it might be deduced that enkephalinergic neurons form dendro-dendritic synapses with spino-fugal cells or cells in the cat trigeminal complex (Gobel, 1976). At present these ultrastructural data are in total agreement with a postsynaptic location of opiate receptors on dendrites (Aronin et al., 1981) previously suggested by electrophysiological data (Zieglgansberger and Bayerl, 1976; Zieglgansberger and Tulloch, 1979b). Both cell types are target areas for various transmitter systems including aminergic (see above), GABAergic (Barber et al., 1982), and substance P-containing terminals deriving from small diameter fibers (Zieglgansberger and Tulloch, 1979a; Glazer and Basbaum, 1980; Hunt et al., 1980, 1982; Nicoll et al., 1980b). The question whether the interaction of substance P and other neurotransmitters with the endorphins occurs solely postsynaptically awaits further experimental clarification (Duggan et al., 1979). In an attempt to identify potential presynaptic actions of opiates as postulated in numerous biochemical and pharmacological studies and from the electrophysiological tests of excitability of terminal sites and dorsal root potentials (Sastry, 1978, 1979a,b; Carstens et al., 1979; Pomeranz and Gurevich, 1979; Suzue and Jessell, 1980), intracellular recordings from cell bodies of primary afferent fibers were performed. Unfortunately, these neurons did not react to even enormous doses of

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opiate agonists in a naloxone-reversible manner (Williams and Zieglgansberger, 198 la) (like cells o f the nodos ganglion; Shefner et nl., 1981), invalidating them as a model for presynaptic opiate actions in adult animals. In cultured spinal neurons the action of opiates was studied by several groups. Stimulation of cocultured spinal neurons and dorsal root ganglion cells with an extracellular electrode resulted in a complex multiunit potential recorded in the dorsal horn area of fetal mouse spinal cord explants (Grain et al., 1977). This multiunit potential was markedly attenuated by low concentrations of opiates in a naloxonesensitive, stereospecific manner. In these preparations opiate-binding sites were found preferentially in the neuritic outgrowth. After invasion of the neurites in the dorsal horn opiate binding significantly increased also in this structure (Hiller et al., 1978). In another study synaptic potentials were recorded intracellularly in cultured spinal neurons by stimulating single dorsal root ganglion cells with intracellular current injection (MacDonald and Nelson, 1978). Iontophoretic application of an opiate agonist (etorphine) onto the spinal neurons induced a significant decrease in the synaptically evoked potential. Because quantal number and not quantal size was reduced by opiates, this effect was interpreted as being mediated via presynaptically located receptors. An action of opiates and opioid peptides on cultured dorsal root ganglion cells, which they share with other neuroactive substances, was to decrease the duration of the action potential (Dunlap and Fischbach, 1978; Mudge et al., 1979; Werz and MacDonald, 1982). This effect is thought to be due to an interference with a voltage-sensitive calcium conductance, but other ion species have not been ruled out. Numerous biochemical and pharmacological investigations suggest an important role of Ca2+ ions in opiate actions evoked in the nervous system. Electrophysiological evidence that morphine interferes with the release of transmitter via a presynaptic Ca-dependent mechanism, for example, was obtained from studies performed in the mouse vas deferens, a site in which presynaptic effects of opiates are well established (Illes et al., 1979; Milner et al., 1982). These studies support the hypothesis that opiates interact with calcium and may reduce the availability of Ca2+ions necessary for the stimiilus-release coupling in presynaptic terminals. However, most of the extra- and intracellular recordings performed in combination with extracellular iontophoretic application in various parts of the CNS have established clear inhibitory postsynaptic effects of opioids on most neurons investigated (for reviews see Fredericksen, 1977; Zieglgansberger and Fry, 1978; North, 1979). Before the morphological demonstration of axo-dendritic and axo-somatic synapses between enkephalin-containing synapses and dendrites of dorsal horn

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neurons, a postsynaptic site of action was indicated solely by electrophysiological investigations demonstrating that, for example, depolarizations produced by iontophoretic application of L-glutamate (Zieglgansberger and Puil, 1973; for reviews see Nistri and Constanti, 1979; Curtis, 1979) were markedly reduced by opiate agonists (Zieglgansberger and Bayerl, 1976; Zieglgansberger and Tulloch, 1979b). The mechanism underlying the inhibitory action in spinal neurons is thought to involve a modulatory effect of opiates on the chemically excitable Na+ channel physiologically activated by excitatory transmitters. These findings, initially reported for the cat spinal cord, (Zieglgansberger and Bayerl, 1976; Zieglgansberger and Tulloch, 1979b) were corroborated by studies performed in spinal neuron tissue cultures (Barker et al., 1978, 1980) and several hippocampal cells (Siggins et al., 1982). The relevance of these findings to theories concerning the effects of systemic opiates is still under debate (Dostrovsky and Pomeranz, 1976; Duggan et al., 1976a, 1977b). As in hippocampal cells in vitro (Nicoll et al., 1980a; Gahwiler, 1980; Dingledine, 1980; Siggins and Zieglgansberger, 1981) and cultured spinal (Barker et al., 1978) and sensory neurons (Dunlap and Fischbach, 1978; Mudge et al., 1979), there was no change in membrane potential or input resistance of these cells associated with the application of opiates. Systemic administration of opiate agonists tended to hyperpolarize spinal neurons, a finding interpreted as a disfacilitatory action on converging inputs (Zieglgansberger and Bayerl, 1976). It is interesting that neurons of the locus coeruleus (Pepper and Henderson, 1980; Williams et al., 1982), some myenteric plexus neurons (North et al., 1979), and some dorsal horn cells of neonatal rat spinal cord studied in vitro (Murase et al., 1982) are hyperpolarized by opiate agonists added to the perfusion medium o r applied by pressure to a micropipette containing the compound. Further studies are required to exclude indirect effects mediated via, for example, interneurons in the investigation performed in spinal cells. From their most elegant studies in spontaneously active cells of the nucleus locus coeruleus in vitro, Williams et ad. (1982) suggested that opioid peptides increase the potassium conductance of the postsynaptic membrane. In these investigations reversal potential for enkephalin at - 105 mV was found. The opiate-induced potassium activation was shown to be voltage-dependent: The hyperpolarizing action of the opioid peptides declined sharply at levels below resting potential (in these studies -55 to -60 mV) and was at maximum at membrane potentials close to threshold. indirect effects were ruled out by the use of calcium-free solutions, as in previous studies (Pepper and Henderson, 1980). Some cells in dissociated spinal tissue cultures also displayed hyperpolarizing responses to enkephalins that were associated with in-

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creased postsynaptic conductance. However, this effect appeared at higher concentrations of enkephalin than the blocking action on L-glutamate-induced depolarizations (Barker et aZ., 1980). In the same preparation enkephalins blocked L-glutamate actions and increased the threshold for spike initiation without an apparent change in membrane conductance or membrane potential. Despite considerable experimental efforts, our knowledge about transduction and second-order processes is still rather scanty. The role of, for example, cyclic nucleotides in the membrane processes triggered by opiates is still totally obscure (for a review see Rosenfeld et al., 1979). Based on intracellular recordings of cat motoneurons, Jurna and coworkers (Jurna, 1966);Jurna et al., 1973) postulated that systemically administered opiates might suppress the temporal facilitation of incoming excitatory signals. Subsequent experiments demonstrated that microiontophoretically applied opiates tended to decrease the rate of rise of the excitatory postsynaptic potential (EPSP) evoked by afferent fiber stimulation (Zieglgansberger and Bayerl, 1976; MacDonald and Nelson, 1978). A decrease in rise time of EPSPs induced by opioids might be expected to be much more effective in blocking spike initiation triggered in dorsal horn neurones by slowly rising synaptic potentials (mainly C fiber input) than those produced by powerful fast excitatory potentials (LeBars et al., 1976b; Jurna and Heinz, 1979). Observations compatible with this assumption have been made in cortical, striatal, and bulbar respiratory neurons (Satoh et al., 1976a; Denavit-Saubie et a!., 1978; Zieglgansberger and Fry, 1978) and appear to be characteristic of opiate action. It therefore seems possible to explain the effect of opiate agonists, for example, on noxious mechanical, thermal, and chemical stimulation (small fiber activation) (Mendell, 1966; Torebjork and Hallin, 1973, 1979) and the associated shift in stimulus-response relationship (Toyooka et al., 1978) in terms of the characteristics of the postsynaptic change in the rise time of the synaptic potentials (Zieglgansberger and Tulloch, 1979b). It is evident that opiates do not simply suppress their target cells but change the “gain” of the transmission system (Zimmermann, 1976). Under appropriate stimulation, activity evoked from large diameter fibers can also be suppressed by iontophoretically administered opiates (Zieglgansberger and Tulloch, 1979b). These afferents are usually highly synchronously activated (e.g., by electrical stimulation) and opiate actions are therefore masked (Yaksh, 1981; Yaksh and Hammond, 1981). In the following paragraph a mechanism involving an enkephalinergic gating of substantia gelatinosa neurons over dorsal horn relay neurons of the spino-thalamic system (first postulated by Melzack and

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Wall, 1965) based on findings from various laboratories (Zieglgansberger and Tulloch, 1979b; Zieglgansberger, 1980, 1982) will be described. It is suggested that spontaneously active enkephalinergic interneurons (stalked and islet cells) (Bennett et al., 1982) in the substantia gelatinosa are activated by large fiber input and are inhibited by small caliber fiber input (Cervero et al., 1977; Bennett et al., 1979, 1980; Gobel et al., 1980). Such a tonically active enkephalinergic system could act as a gating mechanism over neurons involved in somatosensory perception, including pain. Small-caliber fiber stimulation would reduce the enkephalinergic tonus and facilitate the perception of noxious stimuli. T h e alerting function of such a stimulus may suggest it to be physiologically relevant. Large diameter fiber stimulation would have the opposite effect: a reduction of nociception. As a consequence of a reduction of the tonic release of enkephalins from these neurons, naloxone should exert its hyperalgesic action (see following section). Electrical stimulation of Lissauer’s tract, which contains axons of substantia gelatinosa cells, reduced polysynaptic ventral root reflexes. Naloxone increased the amplitude of polysynaptic reflexes and diminished the depression produced by stimulation of Lissauer’s tract. These findings by Wall and Yaksh (1978) are consistent with the assumption of a release of enkephalin from intrinsic interneurons (Hokfelt et al., 1980). This enkephalinergic gating system may be activated in patients undergoing transneuronal stimulation employing acupuncture-like (high-intensity/ low-frequency) stimulation parameters (Sjolund and Eriksson, 1979). In contrast low-intensity/high-frequencystimulation causes only in mice a naloxone-reversible increase in nociceptive threshold (Goldstein, 1979; Chance, 1980). In man both dorsal column stimulation and high-frequency transneuronal stimulation induce naloxone-resistant antinociception that notably lasts only seconds.

F. EFFECTSOF NALOXONE The effects of opiate agonists are prevented or reversed by naloxone (Sawynok et al., 1979). For a long time naloxone has been considered to have no important actions of its own. The first clear-cut experimental evidence for an action of naloxone was described by Jacob et al. (1974) after Akil et al. (1972, 1976) had reported an antagonistic effect of naloxone against stimulation-induced analgesia. Jacob et al. (1974) showed that low doses of naloxone decreased the latencies to lick and jump in mice and rats in hot plate tests, suggesting a hyperalgesic action. Various methodological differences might explain the failure of other groups to

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repeat these initial findings, as discussed in a recent review by Jacob and Ramabadran (1981). It appears that the antagonist decreases the pain threshold to thermal, electrical, or mechanically induced nociception and that excitatory responses reported in various systems are due to a reduction of the inhibitory tonus exerted by the endorphinergic system on its target neurons (Berntson and Walker, 1977; Grevert and Goldstein, 1977; Carmody et al., 1979; Henry, 1979, 1980; Rivot et al., 1979; Hill, 1981; Doi and Jurna, 1982). Locally or systemically applied naloxone primarily enhances the nociceptive responses of neurons in various sites including spinal cord dorsal horn (LeBars et al., 1976a,b;Jurna and Grossmann, 1976; Bell and Martin, 1977; Rivot et al., 1979; Henry, 1980; Fitzgerald and Woolf, 1980; Johnson and Duggan, 1981a). Polysynaptic reflexes in animals and man are facilitated by naloxone (Goldfarb and Hu, 1976; Bell and Martin, 1977; Boureau et al., 1978; Willer et al., 1982), and other multineuronal events such as cortically evoked potentials are also altered by naloxone (Buchsbaum et al., 1977; Burks and Dafney, 1977). Tonically released opioid peptides may also be active in the peripheral nervous system (Waterfield and Kosterlitz, 1975; van Nueten et al., 1976) in the control of adenohypophyseal hormone release: naloxone has been shown to inhibit, for example, prolactin release in rats (Grandison and Guidotti, 1977) and stimulate release of luteinizing hormone in immature female rats (Schulz et al., 1981a,b). The observation of a diurnal rhythmn €or pain sensitivity in rats and for naloxone-induced hyperalgesia also provides strong evidence for the existence of an endogenous enkephalinergic tonus regulating nociception (Davis et al., 1978; Woolf, 1980; Millan et al., 1981; Frid et al., 1981; Schull et al., 1981; Willer et al., 1981). AND DEPENDENCE ON THE SINGLE G. TOLERANCE UNIT LEVEL

That neurons can develop measurable tolerance to and dependence on opiate agonists can be seen in a variety of preparations (Collier, 1980). These adaptive changes are most probably a direct consequence of the specific interaction of the opioid peptides with their receptor sites. A loss of responsiveness to the inhibitory actions of opiate alkaloids and enkephalins has been observed upon the repeated or prolonged iontophoretic applications of opioid agonists to neurons recorded in various structures, including spinal cord neurons (Satoh et al., 1976a; Zieglgansberger and Fry, 1978; Fry et al., 1980a,b; Williams and Zieglgansberger, 1981b; Zieglgdnsberger el al., 1982;cf. Williams et al., 1982)(locus coeru-

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leus, in nitro). The inhibitory responses to the agonists waned during the continuing iontophoretic application and the spontaneous and/or glutamate-evoked discharge activity returned within 10 to 60 min to the initial level. Most of these neurons showed an increased discharge activity upon termination of the application of the opiate agonists, which in some cells was so pronounced that excessive depolarization (inactivation of voltage-sensitive Na+ channels) appeared upon the subsequent application of previously submaximal amounts of L-glutamate. If these neurons exist in a state of tolerance and dependence, then they might reasonably also be expected to display a precipitated withdrawal hyperactivity (as seen in studies on reflexes; see Section II,F) upon microiontophoretic application of naloxone. A higher incidence of excitatory responses to microiontophoretically applied naloxone has indeed been reported for neurons in the medial thalamus (Fredericksen et al., 1975), frontal cerebral cortex (Satoh et al., 1976a), and locus coeruleus (Aghajanian, 1978) of chronically treated morphine-toleranddependent rats but also in acutely treated animals (LeBars et al., 1976a,b). In recent studies (Fry et al., 1980a,b; Williams and Zieglgansberger, 1981b) the pharmacological specificity of neuronal responses to microiontophoretically applied naloxone in the brains of naive and of morphine-toleranddependent rats, and on neurons made acutely tolerant to opiate agonists by prolonged microiontophoretic application have been investigated. The actions of (-)-naloxone were compared with those of its enantiomer (+ )-naloxone, a compound lacking specific opiate antagonist activity (Iijima et al., 1978). Application of naloxone to dorsal horn cells, which did not affect or only moderately increased the firing rate in anesthetized animals before desensitization, produced a large increase ( 100-300%) when applied after desensitization (see Section 11,F).(+)-Naloxone was found to be ineffective in precipitating this discharge activity. These results suggest that the excitatory effects following termination of microiontophoretic administration of (-)-naloxone can be regarded as genuine opiate withdrawal responses at the single neuron level (Johnson and North, 1980). Neurons in the dorsal horn of the spinal cord, like cells in the frontal cortex and striatum, that desensitized to the opioid peptides also became subsensitive to morphine. It is of interest that although the cells were desensitized to morphine, they remained sensitive to the inhibitory action of the opioid peptides. The action of iontophoretically applied morphine was tested before and after the cells were desensitized to Metenkephalin. Cells remained less sensitive to morphine for periods of more than 30 min after termination of the iontophoretic Met-enkephalin administration. This lack of cross-desensitization may suggest the

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existence of multiple opiate receptors on the same cell (Zieglgansberger et al., 1980; Williams and Zieglgansberger, 198 lb) (see Section I,B,2),

and that tolerance does not develop primarily in a common effector system. I n order to further characterize this desensitization, the action of systemically applied morphine was studied on neurons that were desensitized to iontophoretically applied morphine o r Met-enkephalin. In cells desensitized to iontophoretically applied morphine or Met-enkephalin the systemic application of the previously effective dose of morphine (2 mg/kg) also did not have a depressant effect on the firing rate. Up to 20 mg/kg of morphine was required to induce an inhibition of comparable magnitude, indicating that neurons desensitized to locally applied morphine or Met-enkephalin also became subsensitive to systemically applied morphine. Because glutamate-induced changes in excitability of the postsynaptic membrane are blocked by opiate agonists (see Section II,E), it follows that at least a portion of adaptive processes that occur during desensitization are taking place on the soma-dendritic membrane of the neuron under study. Judging from the reported results, it appears that these neurons exist in a state of latent hyperexcitability that is masked by the continued presence of opiate but revealed after termination of application or during the withdrawal precipitated by local application of (-)naloxone. Alternatively, the actual depolarizing responses to excitatory transmitters themselves may be enhanced without any changes in the electrical excitability of the membrane (Satoh et al., 1976b). This latter mechanism remains an intriguing possibility especially if, as implied earlier, opiate withdrawal responses are to be regarded as a mirror image of the acute specific inhibitory effects of these drugs, which appear to occur without detectable changes in resting membrane potential or conductance. Resolution of the above questions must presumably await intracelM a r recordings from central neurons in morphine-tolerant/dependent animals. I n the myenteric plexus neurons of chronically morphinized guinea pigs naloxone causes a depolarization of neurons (North and Zieglgansberger, 1978; Johnson and North, 1980), the mirror image of the acute hyperpolarizing action (North, 1979).

111. Concluding Remarks

As in most other sites investigated in the mammalian CNS, topical microapplication and systemic administration of opiate alkaloids and

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opioid peptides yield mainly inhibitory effects on spontaneous or evoked discharge activity of spinal neurons. Most of these inhibitory actions are reversed by naloxone and are, as has been shown in the few tests performed, mediated via stereospecific opiate receptors. Although clear evidence of endorphinergic neurotransmission has yet to be shown, there is evidence that under physiological conditions intrinsic enkephalin-containing neurons of the substantia gelatinosa may exert an inhibitory action on cells giving rise to spino-fugal systems involved in somatosensory perception, including pain. This enkephalinergic system in the spinal cord may provide the basis for clinical applications such as transcutaneous nerve stimulation and stimulus-produced analgesia by activation of descending pathways. A recently established procedure involves the application of minute amounts of opiate agonists including synthetic opioid peptides, modified to provide enzymatic protection and increased bioavailability, close to the segments of the spinal cord receiving the nociceptive input. As in animals, changes in pain threshold were observed in human beings that were associated with little or no effect on motor function, other sensory functions, or autonomic activity. The resulting analgesia was confined to body parts with neuronal input to the area of application, indicating that the effect was spinal and not due to redistribution of the drug to supraspinal sites. A substantial body of evidence favors the view that opiates act at presynaptic sites, a conclusion supported by the demonstration of opiate-binding sites on primary afferents. This observation plus others leads to the idea that there is an interaction between primary afferents containing substance P and enkephalin-containing terminals. However, this view has to be revised in the light of histochemical and ultrastructural data that have established synaptic contacts exclusively between enkephalin-containing elements and the soma-dendritic membrane of spinal neurons. These findings provide evidence in favor of a postsynaptic localization of the opiate receptor, an assumption previously supported primarily by electrophysiological investigations. Like other neurons studied in various central neural sites in the rat, spinal neurons show no cross-tolerance between opiate alkaloids and the opiate peptides. These findings indicate that there is more than one kind of opiate receptor and that these different types can be on the same cell. Furthermore, they favor the assumption that tolerance does not develop in a common effector system. T h e later finding, which was corroborated recently by various other studies employing different approaches, might lead to interesting therapeutic consequences. A major issue awaiting clarification is, why are opiates so selective with respect to aversive stimuli when applied in therapeutic quantities?

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PSYCHOBIOLOGY OF OPlOlDS By Albefio Oliverio. C l o u d i o C a s t e l l a n o . a n d Stefan0 P u g l i s i - A l l e g r a Institute of Psychobiology and Psychopharmacology

National Research Council of Italy Rome. Italy

I . Introduction ................................................... I1 . Neurochemical Correlates of Opiates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Acetylcholine ................................................ B Catecholamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C Serotonin ................................................... D y-Aminobutyric acid (GABA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Genetic Characterization of Opiate Mechanisms....................... A . Opiate-Induced Analgesia and Activation . . . . . . . . . . . . . . . . . . . . . . . . . ..................... B . Opiate Agonists-Antagonists . . . . . . 1V. Endogenous Opioid Systems ...................................... A Brain Distribution of Opioid Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . B Opioid Peptide Biosynthesis .................................... C Interactions with Neurotransmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Neuroendocrine Effects ....................................... E. Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F Analgesic Effects ............................................. G. Relative Potency of Opioid Peptides and Opiates on Opiate Receptors . H . Phylogenetic and Genetic Studies................................ I . Ontogeny of Endogenous Opioids and Opiate Receptors . . . . . . . . . . . . V . Opioids and Behavior . . . . . . . . . . . . . A . Seizures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B Sleep. . . . . . . . . . . . . . . . . . . . . . . . . C. Consummatory Behavior ...................................... D . Endogenous Opioids and Tolerance E. Learningand Memory ........................................ F. Learning Mechanisms of Toleranc C . Social Behavior .............................................. VI . Environmental Effects . . . . . . . . . . . . A. Stressors and Opioid Production ................................ B. Diurnal and Circadian Rhythms in Opiate Production . . . . . . . . . . . . . . . VII . Brain Opiates and Mental Illness................................... References . . . . . . . . . . . . . . . ...................................

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1 Introduction

Until the discovery of endogenous opiates and opiate receptors. morphine and synthetic opioids were primarily of interest for their 277 INTERNATIONAL REVIEW OF NEUROBIOLOGY VOL. 25

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analgesic effects or for the problems connected with tolerance and addiction. The mechanism of action of morphine was typically related to its neurochemical correlates at the brain level. In recent years a large body of experiments has been devoted to the study of the metabolism of opioids and to the characterization of opiate receptors; at the same time a growing number of studies have been concentrated on the involvement of opioid peptides, natural or synthetic, in a number of behavioral patterns ranging from seizures to memory processes, as well as social behaviors and mental illness. This review deals mainly with the psychobiology of opiates by analyzing their neurochemical correlates at the brain level and the role of different receptor sites.

II. Neurochrmical Correlates of Opiates

From a historical point of view, the first attempts to clarify the mechanism of action of natural and synthetic opiates were based on the study of neurotransmitter modifications at the brain level. Different mediators or neurochemicals were taken into consideration: This trend also reflects the development of the investigations on the various neurotransmitters from acetylcholine to GABAergic systems. In this section each mediator is considered separately, even if different neurochemical systems are involved in the action of morphine and other opiates. Because most of these studies preceded the discovery of opiate receptors, generally they do not take into account the differential effects of opiates at the receptor level. A. ACETYLCHOLINE

A number of studies have shown that morphine interferes with cholinergic mechanisms both at the peripheral and at the central level. Reduction of acetylcholine (ACh) release has been in fact demonstrated to follow morphine administration both at peripheral cholinergicjunctions (guinea pig ileum) (Schaumann, 195’7; Cox and Weinstock, 1966; Gyang and Kosterlitz, 1966) and in some brain areas such as the lateral ventricle and the subarachnoid space of cats (Beleslin and Polak, 1965; Beleslin et al., 1965)or rabbits (Beani et al., 1968).Because decrement of ACh release by morphine is abolished by lesioning of the raphe nuclei (Garau et al., 1975), the septum (Pepeu et ad., 1975), and the medial

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thalamus (Jhamandas and Sutak, 1976), the opiate may exert its action at a subcortical level (see Jhamandas and Poulson, 1978). Increase of ACh content after morphine, probably due to the reduction of its release, has been observed by some authors (Giarman and Pepeu, 1962; Maynert, 1967; Crossland and Slater, 1968; Large and Milton, 1970). It must be pointed out, however, that enhanced release of cortical ACh has been demonstrated also in unanesthetized cats (Mullin et al., 1973). Increments or decrements in the turnover rate of ACh (TRACh)following morphine administration, have been described, depending on the animal preparation and the dose of the drug (see Vasko and Domino, 1978). Some studies have been carried out in order to investigate the mechanisms of morphine interaction with cholinergic systems. In particular Sharkhawi (1970), utilizing the rat cerebral cortex, has demonstrated that the cell membrane may be the site where morphine inhibition of the ACh release occurs. Naloxone-reversible reduction of the resting release of cerebral cortical ACh has been shown by Jhamandas et al. (1970) in morphine-injected cats. These authors hypothesized that the increase in ACh release after treatment with a narcotic antagonist may be partly responsible for the acute withdrawal syndrome precipitated by these drugs in morphinized animals. In agreement with these results Domino and Wilson (1973) have demonstrated a remarkable decrease of brain ACh during withdrawal from morphine in rat. In addition to this the suppression of some manifestations of morphine withdrawal by ACh antagonists and the elevation of the steady-state levels of brain ACh in morphine-dependent rats following morphine administration have been shown (Large and Milton, 1970; Collier et al., 1972; Jhamandas and Dickinson, 1973; Hynes et al., 1976). Naloxone-reversible increments of brain TRAC;,, have been observed by Cheney et al. (1975) in mice implanted with morphine pellets. These authors have also demonstrated that increased brain TRACh is associated only with drug dependence, whereas during withdrawal it returns to normal. Some researches have been carried out in which TRACh has been measured in brain structures with known densities of opiate receptors. The results of these researches suggest that the TRAChmay be regulated by opiate receptors located on the membranes of cholinergic neurons (Cheney et al., 1974; Zsilla et al., 1976). Moroni et al. (1977a) have shown that TRACh is reduced (although the content of ACh is unchanged) following the administration of analgesic or cataleptic doses of morphine or P-endorphin in the hippocampus, nucleus accumbens, globus pallidus, and cortex of the rat. Both the analgesic and cataleptic effects and the decrease of TRAc~were, in these experiments, prevented by naltrexone,

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suggesting an involvement of opiate receptors in these actions. It must, however, be pointed out that no effect on TRAChhas been shown following opiate administration in other structures (striatum, amygdala, etc) with high densities of opiate receptors (Moroni et al., 197th; Zsilla t l al., 1977, 1978), suggesting that opiate receptors may not be located on all cerebral cholinergic neurons. Evidence of cholinergic mediation of morphine-induced analgesia has been obtained also by other investigators. Castellano et al. (1975), in particular, have demonstrated that septa1 lesions, which cause a reduction in the levels of ACh in the brain areas that receive cholinergic input from the septum, antagonize the analgesic effects of morphine in the mouse. Vasko and Domino (1978) have investigated in rats the relationship between the development of tolerance to the depressant and the stimulant action of morphine on locomotor activity and brain utilization of ACh (indirect turnover). Their experiments showed that 1.O mg/kg of the opiate increased locomotor activity as well as ACh utilization in nontolerant animals. T h e administration of 10 mg/kg of morphine induced a biphasic effect, that is, a decrease followed by an increase in locomotor activity as well as an initial decrease and subsequent increase in the utilization of ACh both in the whole brain and in single brain structures (hippocampus, thalamus, hypothalamus). Only a decrease in ACh utilization was evident in caudate. Rapid tolerance developed both to the depressant actions of morphine on activity and to the decrease of brain ACh utilization, but only after large doses of the opiate did tolerance to its activity-stimulating effects develop. Tolerance development was also observed to both the increase and the decrease of ACh utilization in the hippocampus, thalamus, and caudate. More recently, (Ebel et al., 1980) a significant increase was observed in choline acetyltransferase (CAT) activity in the corpus striatum of the C57BL/6 strain of mice (C57), whose activity is enhanced by morphine treatment, whereas no effect was observed in the same brain area of mice of the DBA/2(DBA) strain, whose activity is depressed by the opiate. T h e involvement of other neuromediators in the cholinergic effects of opiates has been demonstrated. Jhamandas and Poulson (1978) have shown that a-methyl-p-tyrosine (AMPT), which causes a depletion of brain catecholamines, blocks the inhibitory action of morphine on cortical ACh release, suggesting that the effects of the opiate on the ACh output are dependent on the functional integrity of the noradrenergic neurons. The effect of morphine was enhanced in their experiments by the serotonin depletor p-chlorophenylalanine and (to a lesser extent) by methysergide, suggesting that integrity of serotonergic mechanisms is not essential for the effects of the opiate on the cortical release of ACh.

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It has been demonstrated in rats and mice (McCeer et al., 1974; Groves et al., 1975; Iwamoto et al., 1978) that the dopaminergic neurons of the nigroneostriatal pathway may inhibit cholinergic neurons in the neostriatum and that at the level of the neostriatum a diminution of dopamine (DA) activity during precipitated withdrawal may result in an enhancement of cholinergic mechanisms (i.e., increased ACh utilization and decreased total brain ACh content).

B. CATECHOLAMINES Several investigators have demonstrated that brain catecholamines are involved in the effects of narcotic analgesics. A decrement in the endogenous level of brain catecholamines and an increase in their synthesis (due to increments of the activity of the catecholaminergic neurons) has been shown to follow opiate administration (Gunne, 1963; Takagi and Nakama, 1966; Rethy et al., 1971; Sasame et al., 1972). Rethy et al. (1971) have observed a decrement of brain catecholamine content in rats injected with morphine at doses that enhanced locomotor activity. Potentiation of morphine-induced analgesia after inhibition of catecholamine synthesis has been demonstrated by other authors (Buxbaum et al., 1973). In addition to this it has been shown that, in rats, changes in motor activity induced by morphine are dependent upon a balance between a catecholaminergic system acting to increase activity and a serotonergic system inhibiting it (Buxbaum et al., 1973). T h e same mechanism of action was also shown in opiate-injected mice (Carroll and Sharp, 1972; Villarreal et al., 1973). Involvement of norepinephrine (NE) in morphine-induced behavioral stimulation, stereotyped behavior, tolerance and dependence, and a less prominent role in morphineinduced antinociception have been detected (see Way and Shen, 1971; Takemori, 1976). In addition different studies based on association of opiates with other psychotropic agents also suggest involvement of catecholaminergic systems in morphine-induced hyperactivity (Sansone et al., 1977). Decrements of NE levels following morphine treatment have been reported by several investigators in a variety of animal species (Vogt, 1954; Gunne, 1963; Laverty and Sharman, 1965; Takagi and Nakama, 1966; Reis et al., 1969). Antagonism of the activity-enhancing effects of morphine following selective NE depletion as well as NE mediation of morphine-induced stereotypyes have been shown in the rat by Ayhan and Randrup (1972, 1973). Paalzow and Paalzow (1975) have studied the effects of morphine on different pain reactions, the antagonism of these effects by several

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drugs, and the turnover rate of NE and DA in different brain areas. In their experiments morphine increased the threshold for vocalization; this effect was antagonized by naloxone and yohimbine and increased by AMPT, FLA-63, chlorpromazine, and pimozide administration. Naloxone-reversible increments in the turnover rates of DA (in telencephalic cortex and the diencephalon-mesencephalon-striatum regions) and of NE (in the medulla-pons region) were also observed. T h e effects of morphine on the threshold for vocalization were increased as the NE and the DA activities were decreased. A. S. Bloom et al. (1976) have demonstrated that morphine treatment induces dose-dependent, naloxone-reversible increases of both antinociception and accumulation of newly synthesized DA and NE and that antinociceptive doses of a wide variety of other narcotic analgesics also increase the accumulation of the catecholamines in the brains of mice. Also in mice Fuchs and Coper (1980) have shown that NE is involved in the acute effects of morphine on body temperature and toxicity. Increases in brain NE levels as well as decreases in NE turnover have been shown in chronically morphine-treated rats (Maynert and Klingman, 1962; Gunne, 1963; Guaza et al., 1980). In addition blockade of naloxone-induced jumping in chronically morphinized rats has been observed following reduction of central NE levels (Blasig et al., 1975; Huang et al., 1978). Several researchers have demonstrated that dopamine is involved in the effects of morphine and that morphine may act as a dopamine receptor blocker (see Lal, 1975). Both electrophysiological and behavioral studies suggest that morphine might activate nigrostriatal DA neurons in particular (see Pert, 19’18). Some authors have shown diminution of morphine-induced antinociception following DA depletion and its restoration following treatment with L-DOPA (Ayhan, 1972; Elchisak and Rosencrans, 1973; Nakamura et al., 1973; Blasig et al., 1975). Potentiation of morphine-induced antinociception following 6-hydroxydopamine (6-OHDA) treatment has, however, also been reported (Samanin and Bernasconi, 1972). Involvement of DA mechanisms in morphine-induced hyperactivity has been demonstrated (Trabucchi et al., 1976; Iwamoto, 1981). In particular Iwamoto (1981) has observed disruption of opiate-induced hypermotility following the administration of both pre- and postsynaptic inhibitors of DA neurotransmission. Dopaminergic mediation of morphine-induced catatonic effects has also been shown. A defect in the stimulation of dopaminergic postsynaptic receptors in striatum resulting from an impairment of the extraneuronal release of DA has been linked to catatonia induced by morphine or Viminol administration in the rat by Carenzi et al. (1975). On

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this point it must be emphasized that increases in the turnover rate of DA in rat striaturn (and no changes in NE turnover rate in cerebellum and spinal cord) have been reported following the administration of analgesic doses of morphine o r Viminol (Costa et al., 1973; Della Bella et al., 1973). I n addition increased turnover of DA has been shown to accompany the cataleptic effects of narcotic analgesics. It has been also recently suggested (Moleman et al., 1978) that morphine-induced catalepsy may be due to activation of mesolimbic or mesocortical DA systems by the opiate. In one study (Havemann et al., 1981) systemic morphine administration to rats led to a rapid decrease followed by a slow increase in striatal 3,4-dihydroxyphenylacetic acid (DOPAC) concentration. Kainic acid lesions of the head of the caudate nucleus, which prevented morphine-induced muscular rigidity, slightly inhibited the decrease and markedly enhanced the increase of DOPAC. Several investigations have demonstrated the involvement of DA in the chronic effects of opiates (see Lal, 1975; Laschka et al., 1978). Cox et al. (1976) have shown dopaminergic involvement in naloxone-precipitated withdrawal in rats. In their experiments hypothermia during withdrawal was blocked by pimozide, a substance that blocks dopaminergic receptors; chewing, head shakes, and writhing, however, appeared to be under a dopaminergic inhibitory influence and were potentiated by pimozide. A shift from a decreased dopaminergic activity in rats following acute morphine administrations to an increase of dopaminergic neurotransmission after repeated morphine injections has been demonstrated by Kushinsky (1975, 1981). In addition, decreased dopaminergic transmission during withdrawal has been reported (Grarnsch et al., 1977). Inhibition of dopamine receptor activity following acute narcotic administration and increased response of the receptors to dopamine agents following their chronic treatment has been in general shown (see Lal, 1975). Keduction of DA activity within the striaturn by sc pellet implantation has been described in rats dependent on morphine (Laschka et al., 1978). Sensitivity to dopamine and to ACh agonists has been investigated in rats treated chronically with morphine by Christie and Overstreet (1979). Supersensitivity to dopamine was displayed by the morphine-tolerant subjects: Higher sensitivity to the behavioral suppressant effects of apomorphine, a dopaminergic agonist, and pilocarpine (an ACh agonist) was evident in their experiments. On the contrary, in the withdrawal state morphine-treated tats were less sensitive to both agonists and showed lower affinity for dopimine receptors in the striatuni and fewer muscarinic cholinergic receptors. Hypersensitivity of the dopaminergic system due to chronic treatment with opiates was reflected in enhanced stereotypic gnawing and chewing in response to dopaminergic

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agents following termination of treatment. I t has been demonstrated that this behavioral hypersensitivity is not due to proliferation of postsynaptic DA receptors but is more likely to be linked to the alteration of presynaptic dopaminergic functions (Carlson and Seeger, 1982).

C. SEROTONIN The involvement of 5-hydroxytryptamine (5-HT, serotonin) in the effects of opiates has been demonstrated in numerous reports with conflicting results. Increments or no effects on brain 5-HT turnover have been observed in rats and mice following acute administration of morphine or of other opiates (Loh et al., 1969; Shen et al., 1970; Yarbrough et al., 1971, 1972, 1973; Haubrich and Blake, 1973; Goodlet and Sugrue, 1974; Malec, 1980). Yarbrough et al. (1973) have shown in particular the existence of 5-HT turnover increments following acute morphine administration in both Wistar and Sprague-Dawley rats; the effect was smaller in the former strain. Increments of the level of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) have been described in mice and rats acutely injected with morphine (Bowers and Kleber, 1971; Malet, 1980). N o effect or increments in the cerebral content of 5-HIAA depending on (Malec, 1980) experimental procedure or strains of animals have been reported (Tagliamonte et al., 1971; Haubrich and Blake, 1973). A number of studies have demonstrated 5-HT involvement in morphine-induced antinociception (see Samanin et al., 1978). T h e administration of the 5-HT precursor 5-hydroxytryptophan (5-HTP) has been found to potentiate (Sigg et d.,1958; Spdrkes and Spencer, 197 1 ; Major and Pleuvry, 1971) or to have no effect (Reinhold ct al., 1973) on morphine-induced analgesia in the rat. In the same animal species pretreatment with p-chlorophenylalanine (PCPA) antagonized the analgesic effect of morphine (measured by the flinch-jump technique) (Tenen, 1968). 5-Hydroxytryptamine involvement in the analgesic action of opiates has been demonstrated also by lesion studies. Lesions of midbrain raphC (MR) that produced a marked decrease in forebrain 5-HT antagonized the analgesic and both the hyperthermic and hypothermic effects of morphine (Samanin et al., 1970; Samanin and Valzelli, 1972). In addition morphine administration induced analgesia in rats previously stimulated in the nucleus raphC dorsalis (DR), and this effect was maximally evident 30 min after the end of the stimulation, when the 5-HIAA levels in the brain were highest (Samanin and Valzelli, 1972). In a further series of experiments morphine analgesia was restored in MR-lesioned rats, following ip injections of 5-HTP. Antagonism of the analge-

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sic effect of morphine (12 mg/kg) has been demonstrated in the rat following high doses of PCPA (3 X 200 mg/kg). An elevation of the 5H T levels by administration of its precursor 5-HTP, however, potentiated the analgesia produced by morphine and pentazocine, but did not affect that produced by codeine or fentanyl (Malec, 1980). Involvement of 5-HT in the motor effects of opiates has been demonstrated by some investigations. Antagonism of the activity depression evident in the rat (Holtzmann strain) following the acute administration of morphine (20 mg/kg) has been observed after treatment with PCPA three times per day for 6 consecutive days (Eidelberg and Schwartz, 1970). Working with Swiss Webster mice, Cheney and Goldstein (197 1) have shown potentiation of levorphanol-induced running by PCPA (320 mg/kg) and inhibition of this effect when the levels of 5-HT were restored by administering 5-HT. These results led the authors to postulate the existence of an inhibitory serotonergic component that modulates running activity in mice acutely injected with opiates. In this model increased serotonin would inhibit the running system, whereas decreased serotonin would potentiate it. Buxbaum et al. (1973) showed in the rat (Sprague-Dawley strain) that acute administration of morphine produced an initial depression of motor activity that was reversed to hyperactivity after PCPA pretreatment. Reversal of morphine-induced activity depression was also observed following depletion of 5-HT by destruction of the raphC nuclei. They showed that activity depression by morphine and increased turnover of cerebral 5-HT occurred at the same time and were naloxone-reversible phenomena. Serotonin is involved also in the cataleptic effects of opiates. Potentiation of the cataleptogenic effect of analgesics due to an increased activity of the serotonergic system has been demonstrated (Fidecka and Langwinski, 1979; Malec and Langwinski, 1980). In addition potentiation (Groppe and Kushinsky, 1975; Tulunay et al., 1976) or a biphasic effect (a short-term depression followed by a prolongation) (Malec 1980) of morphine- (but not of codeine or phentany1)-induced catalepsy following elevation of 5-HT levels in the brain by administration of 5-HTP have been reported. Serotonergic mechanisms are also involved in the chronic effects of opiates. Increases in the rate of synthesis of brain 5-HT in rats and mice made tolerant to morphine-induced antinociception and tolerance decrements following inhibition of this synthesis with PCPA have been demonstrated (Way et al., 1968; Shen et al., 1970; Maruyama et al., 1971). Inhibition of the development of physical dependence on morphine by PCPA and enhancement following 5-HT administration have been shown in rats and mice (Shen et al., 1970). p-Chlorophenylalanine-reversible acceleration of tolerance development to morphine-induced an-

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algesia has been observed by some authors (Ho et al., 1972). However, no effect upon the development of tolerance to opiate-induced running and analgesia or upon the development of physical dependence following PCPA treatment has been observed by other investigators (Schwartz and Eidelberg, 1970; Algeri and Costa, 1971; Cheney and Goldstein, 197 1). I n addition, no change in rats or increased 5-HT turnover in mice made tolerant to and physically dependent upon morphine or methadone by sc pellet implantations have been found (Algeri and Costa, 1971; Bowers and Kleber, 1971; Way et ul., 1973). Differences in techniques employed, strain or species of animals, dose, length of treatment, etc (Way et al., 1973), might account for the discrepancies among these results. Contrasting results also exist concerning possible modifications of tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis, in the morphine-dependent animal. Increases or no effect on brain tryptophan hydroxylase activity have been demonstrated (Azmitia et ad., 1970; Knapp and Mandell, 1972; Schecter et al., 1972) following longterm opiate treatment. The possible explanation of these discrepancies might, according to Knapp and Mandell (1972), be that two forms of tryptophan hydroxylase, soluble and particulate, exist and that morphine affects only the latter one. T h e effects of morphine on serotonin precursor uptake were studied in the brain of the rat by Larson and Takemori (1977). These authors found that increases in the rate of central serotonin synthesis after acute administration of the opiate may be due to an increased uptake of 5HTP from the blood to the brain, whereas increased uptake of tryptophan may account for increased 5-HT synthesis following chronic treatment. It has also been postulated (Samanin et al., 1978) that in morphine-dependent rats a central serotonergic hypofunction, probably related to a decrease in the number of serotonin receptors in the brainstem, occurs as a consequence of persistent activation of central serotonin functions by long-term treatment with morphine.

D. y-AMINOBUTYRIC ACID(GABA) Increasing numbers of investigations demonstrate the involvement in opiate effects of GABA (y-aminobutyric acid), an inhibitory transmitter of the central nervous system in both vertebrates and invertebrates (Elliott and Jasper, 1959; Anden et al., 1964; Roberts, 1974). Lin et al. (1973) found that higher levels of GABA were present in some brain regions (subcortical and hypothalamic areas) in rats ren-

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dered tolerant by repeated injections of morphine. Antagonism of morphine-induced antinociception and enhancement of tolerance and physical dependence development has also been demonstrated in mice in which tissue levels of GABA were elevated by administering the compound exogeneously or by slowing its destruction with aminooxyacetic acid (AOAA) (Ho et al., 1973a,b). Inhibition of tolerance and of dependence development has been observed following blockade of postsynaptic sites of GABA receptors by bicuculline (Ho et al., 1973a). Furthermore Ho et al. (1976) have shown in mice that both neuronal and glial GABA systems may be associated with the mechanisms of morphine tolerance and dependence. Administration of 2,4-diaminobutyric acid (an inhibitor of GABA uptake in the neurons) in these experiments antagonized morphine analgesia in both tolerant and nontolerant mice without modifying naloxone-precipitated withdrawal jumping. In addition P-alanine, an inhibitor of GABA uptake in glial cells, potentiated naloxone-precipitated withdrawal jumping in morphine-dependent subjects without affecting morphine-induced analgesia either in nontolerant or in tolerant mice. Involvement of GABAergic mechanisms in opiate-induced analgesia and catalepsy has been also demonstrated. It has been shown in fact that intravenous administration of a GABA agonist (muscimol) potentiates both the analgesic (Biggio et al., 1977) and the cataleptic (Della Bella et al., 1973; Knoll and Zsilla, 1974) effect of morphine and of other narcotic analgesics (Viminol and azidomorphine). It must be, however, emphasized at this point that, in contrast with Ho et al. (1973a, 1976), Yoneda et al. (1976) have shown that AOAA administration potentiates the antinociceptive effect of morphine in mice. In addition to this, only a very weak increase in morphine analgesia (hot plate test) or no effect on morphine analgesia (wire grid test) have been demonstrated in rats following muscimol peripheral administration; Mantegazza et al. (1 979) have shown'that in the rat this CABA agonist antagonizes the antinociceptive effect of subcutaneous or intraventricular morphine administration if it is injected intraventricularly. 'These discrepancies have been interpreted in terms of differences in muscimol distribution within the central nervous system that depend on the route of administration. Further support of GABA involvement in opiate effects has been given by other studies. Evidence for naloxone and opiates as GABA antagonists has been obtained by Breuker et al. (1976) and Dingledine et al. (1978). I n their experiments pretreatment of mice with a subconvulsant dose of naloxone reduced the EDSO for bicuculline convulsions. In addition, bicuculline, naloxone, morphine, levorphanol, and dex-

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trorphan all were found to displace [3H]GABA from GABA receptor sites in homogenates of human cerebellum. On the basis of these results, the authors suggest that the behavioral excitation observable when large amounts of some opiates or opiate antagonists are administered may reflect functional blockade of GABA inhibitory systems. Tzeng and Ho (1978) have added further support to the hypothesis that the GABA system is linked to morphine analgesia, tolerance, and physical dependence. They measured the effects of morphine administration (acute and chronic) on GABA and glutamate levels and the activity of L-glutamate- 1-decarboxylase (GAD) and of GABA-a-ketoglutarate aminotransferase (GABA-T). The measurements were made in the whole brain as well as in discrete brain areas. Their experiments showed that acute morphine administration increased the whole brain GABA content and decreased glutamate levels without affecting GAD or GABA-T activities. Brain glutamate level was unaffected by continuous morphine administration (3-day pellet implantation), which decreased both GABA levels and GAD activity. Tolerant mice showed a slower rate of brain GABA accumulation than did nontolerant mice. When discrete brain areas were considered, the results showed that ( a ) in the cerebral cortex changes in GABA and glutamate levels and GAD and GABA-T activities were similar to those in the whole brain, and (b) in cerebellum both acute and chronic morphine administration led to a rapid fall in GABA and glutamate levels accompanied by a decrease in GAD activity. In addition, GABA-T activity was increased by acute and not affected by chronic treatment. ( c ) In the brainstem acute treatment caused an increase in GABA level and a decrease in GABA-T activity without affecting GAD activity. Moreover, GABA and glutamate levels and GAD and GABA-T activities were all significantly lower in animals treated chronically, and (d) in the hypothalamus the only effect of acute administration was an increase in glutamate levels. T h e mice implanted with morphine pellets for 3 days showed a significant increase of GABA levels; GABA-T activity was inhibited. No change was observed in glutamate level or GAD activity in these animals. In all the remaining areas acute injection of morphine increased GABA concentration and GAD activity and decreased glutamate level. No change was detected in these areas following chronic administration of the opiate. The changes in turnover rate of GABA(TRGABA) following subcutaneous injections of morphine and intraventricular injections of p-endorphin in different brain areas have been studied by Moroni et al. ( l978b). Specifically, the GABA and glutamate content and the TR(;ABA were measured in substantia nigra, caudate, and globus pallidus 30 min

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after the iv injections of 0.29 and 2.9 nmol of P-endorphin. Increases of TF&BA in globus pallidus and substantia nigra were observed following both doses, whereas only the high dose of the polypeptide decreased the TRGABAin the caudate. Morphine had the same effects. Neither &endorphin nor morphine changed the glutamate or GABA content in these areas. All changes were inhibited by naloxone. The authors also studied the association of injected muscimol, a specific agonist of GABA receptors, into the globus pallidus or the substantia nigra with catalepsy. Bilateral injections of muscimol into the substantia nigra failed to induce catalepsy, which was, on the contrary, induced by its intrapallidal administration. These results suggest that opioid-induced catalepsy is related to increased GABA utilization in the globus pallidus and that opiate receptors may inhibit striatal GABAergic neurons. Buckett (1979) has studied the effects of drugs modifying neurotransmitter function on the morphine treatment withdrawal syndrome in rats physically dependent on the opiate. The experiments showed that in subjects withdrawn from morphine the stereotypic behaviors due to dopaminergic stimulation by apomorphine (such as sniffing, licking, and gnawing) showed enhancement, whereas those induced by elevating brain levels of serotonin by 5-hydroxy-~~-tryptophan treatment (wetdog shakes) and of GABA by ~~-4-aminohex-5-ynoic acid or by y-vinyl GABA administration (hypothermia) were not affected. The same author (Ruckett, 1981) has demonstrated that increasing levels of brain GABA (by y-vinyl GABA) may play a part in reducing oral morphine intake of dependent rats. The role of GABA in morphine dependence mechanisms has also been demonstrated by some experiments carried out in rats injected with n-dipropyl acetate (DPA), an anti-petit ma1 drug that is known to inhibit GABA metabolism and to increase GABA concentration in the brain of rats and mice (De Baer el al., 1977, 1980; Van der Laan and Bruinvels, 1981). In particular it has been shown that morphine administration suppresses, whereas naloxone administration releases, the abstinence behavior following DPA administration, which resembles morphine abstinence behavior. Furthermore, o n the basis of this similarity, because a dual role of GABA in both initiation and terniination of DPA-induced abstinence syndrome has been demonstrated (Van der Laan and Bruinvels, 1981), a dual role of GABA in naloxoneprecipitated abstinence has also been suggested. Initiation of abstinence behavior might be due to drug-induced increase of GABA in the nerve terminal, whereas the overflow of GAHA into the synaptic cleft may be responsible for the suppression of this behavior via stimulation of presynaptic autoreceptors.

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111. Genetic Characterization of Opiate Mechanisms

A. OPIATE-INDUCED ANALGESIA AND ACTIVATION The inbred strains of mice have provided a useful tool to investigate the behavioral effects of opiates and the mechanisms of their action (see Oliverio and Castellano, 1981; Barchas and Sullivan, 1981). Eriksson and Kiianmaa (1971) observed higher morphine consumption in C57BL/6 (C57) than in CBA/CA (CBA) mice. In addition, C57 females showed in their experiments greater morphine-seeking behavior as compared with CBA females, and (in the first generation) consumed twice as much morphine as did the males belonging to their own strain. Gebhart and Mitchell (1973) have shown that the analgesic power of morphine was 16 times higher in the CF1than in the CFW strain of mice. Using the C57, BALB/cJ(BALB), and the DBA/2(DBA) strains of mice, which differ both in behavior and brain chemistry (Bovet et al., 1969; Mandell et al., 1973), Oliverio and Castellano (1974) have demonstrated that morphine and heroin administration enhance activity only in the former two strains, whereas the latter is more sensitive to its analgesic action, suggesting the existence of two different sites for the effects observed. When the genetic analysis was extended to the F1 generation (Castellano and Oliverio, 1975), the performances of the C57 and DBA and of the C57xBALB morphine-injected mice were similar to those of the C57 strain as far as both the running and the analgesic effects were concerned. This suggests that the inheritance of the effects of morphine is genetically determined and is regulated by incomplete dominance. The existence of a genetic model based on more than two loci, which controls the running response to morphine, and the possibility that a larger number of genes are involved in the analgesic effect, have been demonstrated in further research carried out with BALB and C57 mice (Oliverio et al., 1975; Shuster etal., 1975). Katz and Doyle (1980) have demonstrated that a single gene may enhance normal sensitivity to opiates. In their experiments morphine injection, which produced stereotyped running and reduced core temperature, induced significantly enhanced responses in the coisogenic pallid line C57BL/6Jpa, which differs at a single locus from the parent C57 line. Strain differences also in the electrocorticographic (ECoG) response to morphine administration have been demonstrated by Oliverio ( 1975) in DBA and C57 mice. Following morphine injection, a dissociation between ECoG activity and behavior (i.e., slow waves and spindles associated with behavioral stimulation) appeared only in the latter strain. Strain-dependent effects have also been demonstrated for enkephalin

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analogs and opioid peptides. Following the administration of the enkephalin analog FK 33-824, dose-dependent depression of activity in DBA mice and biphasic effects in C57 mice have been shown (Castellano, 1981b), suggesting the existence in the latter strain of at least two types of receptors responsible for the effects of this compound on running behavior. In C57 mice peripheral administration of' analgesic doses of dermorphin, a representative of a new class of opioid peptides occurring in the skin of amphibia (Broccardo et al., 1981), resulted in behavioral depression, whereas icv administration induced clear-cut activity-enhancing effects (Puglisi-Allegra et al., 1982a). The same effects were observed after icv administration of shorter analogs of dermorphin and of (DAla2,Leu)-enkephalinamide, whereas @-endorphin induced behavioral depression. Strain- and dose-dependent effects on activity are exerted also by opiate antagonists (Castellano and Puglisi-Allegra, 1982a). In the C57 strain low doses of naloxone have been followed by activity depression. This effect disappeared at intermediate doses and again became evident when drug dosages were increased. On the contrary, activity levels were always depressed by naloxone in the DBA strain. Moreover, naltrexone administration gave rise to dose-dependent activity depressant effects in both strains. I n a first attempt to clarify the mechanisms underlying the effects of opiates on running activity and analgesia following septal lesions, which reduce acetylcholine levels in the brain areas that receive a cholinergic input from the septum (Castellano et a!., 1975), septal lesions antagonized morphine-induced analgesia in both strains, whereas the running syndrome was unaffected. In addition pretreatment of mice with amethyl-p-tyrosine did not change the analgesic effect of morphine, whereas it did antagonize the behavioral stimulation. These results show that the same biochemical and neurophysiological model cannot explain the effects of opiates on running and analgesia and that the septum, through an action on cholinergic mechanisms, plays an important role in the latter effect, whereas the levels of catecholamines influence the action of morphine. Involvement of the catecholaminergic system in opiate effects has been demonstrated by other investigators using inbred mice (Kempf et al., 1974; Filibeck et al., 1980; Siegfried et al., 1982). For example, increments in NE turnover following morphine administration in the pons and medulla of C57 and BALB, but not of DBA mice following morphine, have been reported (Kempf et al., 1974). In the same research no effect on NE turnover was observed in the hypothalamus of any of the three strains. I n addition, DA turnover was not changed in any strain

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when the caudate nucleus was considered but was increased in all of them in the rest of the brain. Using DBA and C57 mice, Trabucchi et al. (1976)have studied the eventual involvement of DA receptors in the analgesic and locomotor effects of morphine. They have determined striatal dopamine-stimulated adenylate cyclase activity and in v i m cyclic 3’:5’-adenosine monophosphate (CAMP)formation in these strains following morphine treatment. The experiments showed that the stimulation of striatal adenylate cyclase induced by DA and apomorphine was much higher in C57 mice than in the DBA strain. Moreover, in uivo injection of morphine induced a dose-dependent increase of the striatal cAMP concentration only in the C57 strain. These authors conclude that ( a ) C57 mice have a more responsive striatal dopamine receptor than the DBA mice, (b) morphine brings about a stimulation of DA receptors only in the C57 strain, and (c) motor activity is directly dependent upon stimulation of dopaminergic systems. In a further series of experiments Racagni et al. (1977)have demonstrated the involvement also of cholinergic pathways in morphine-induced effects. For this purpose they measured the turnover rate of acetylcholine (TRA(:,,)in the striatum and the limbic system of DBA and C57 mice. In these experiments morphine decreased the TRAchin the limbic system (not in the striatum) of DBA mice, whereas TRAC:,, decrements were found only in the striatum of the C57 mice. This was a consequence of the activation of the turnover rate of dopamine after morphine administration, as demonstrated by the authors in the same research. I n DBA and C57 mice Racagni et al. (1979)have studied the release of dopamine in the striatum and the cerebellum as shown by measurements of the 3-methoxytyramine (3-MT) and cyclic adenosine 3’:5’monophosphate (CAMP)levels in the striatum and by cAMP and cyclic guanosine 3’:5’-monophosphate (cCMP) levels in the cerebellum. In these experiments striatal DA release was increased by morphine only in C57 mice, in which the levels of 3-MT were doubled, whereas striatal 3M T concentrations were decreased in the DBA strain but unchanged in the cerebellum. This effect was evident only after 10 nig/kg of morphine. In uitro morphine (5 x M ) did not change the basal activity of the adenylate cyclase and did not antagonize the stimulation of the enzyme by DA in the striatal homogenates of the two strains. In the case of CAMP striatal concentrations were increased by morphine only in C57 mice 10 min after administration of the opiate, whereas no change was detected in the cerebellum, which has no DA terminals. The cerebellar content of cGMP was increased by morphine in the C57 strain and decreased in the DBA strain, which displays a basal cerebellar cGMP content almost five times greater than that of the C57 mice. Finally, the cGMP levels in the striatum were increased by morphine only in the DBA mice.

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In a further series of experiments the same authors measured the striatal cAMP levels in haloperidol- (a DA receptor blocker) or naltrexone(a pure antagonist of morphine)-pretreated mice of the two strains. T h e results showed that the two drugs ( a ) were able to antagonize the increase of CAMP elicited by morphine in the C57 mice, (6) reduced the increase of motor activity elicited by morphine in the C57 strain, and (c) did not alter the steady-state concentrations of striatal CAMP. In addition, haloperidol, which decreased the cerebellar cGMP content in both C57 and DBA mice, antagonized the morphine-induced increase of cerebellar cGMP in the C57 mice and potentiated the effects of morphine in the DBA strain. Finally, in the same experiments naltrexone administration antagonized the morphine-induced increase of cerebellar cGMP in the C57 mice and partially reversed the decrease of cGMP in the DBA strain. It must be emphasized that because cerebellum lacks both opiate receptors (Pert and Snyder, 1973) and dopaminergic terminals (Ungerstedt, 197 l), morphine may conceivably act indirectly by modifying the concentration of cerebellar cCMP. On the whole, the results of this research indicate that biochemical variations in the striatum and the cerebellum (i.e., areas that are involved in motor coordination) of DBA and C57 mice are related more to locomotor behavior than to pain control. Muraki et al. (1982) have demonstrated increased plasma cAMP and cGMP levels following morphine injection in C57 mice in which running fits were evident, whereas no effect was observed in DBA mice, which were depressed by morphine. Moreover, activity stimulation and increase in plasma cGMP levels were evident in BALB mice, whereas in C3H mice the cCMP levels were increased without effects on activity. DBA mice showed a smaller cAMP response to epinephrine than the C57 strain, and both epinephrine and carbachol increased the plasma cAMP and cGMP levels in both strains (Muraki et d.,1982). According to the authors these strain differences might be due to differences in the populations of opiate receptor subtypes in the central nervous system, which could cause differences in morphine-induced activation of the autonomic nervous system. B. OPIATEAGONISTS-ANTAGONISTS Strain-dependent effects have also been observed when the effects of opiate agonists-antagonists, drugs that could be employed in a clinical approach to morphine dependence (Mello and Mendelson, 1980) are investigated (Filibeck et al., 198 1). Buprenorphine, an agonist-antagonist of the morphine type, which has the characteristics of low intrinsic activity with higher affinity for the p receptors, acted as an agonist-

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antagonist to morphine in both C57 and DBA mice. Used alone in the C57 strain, it induced running fits, but it antagonized morphine-induced behavioral stimulation; moreover, in the hot plate test buprenorphine displayed no analgesic activity either when given alone or together with morphine. No effect on locomotor activity was evident when buprenorphine was injected into DBA mice alone, whereas it reversed the behavioral stimulation induced by morphine in this strain. In the same strains of mice, however, a dissociation of the effects on activity and analgesia that depended on the strain considered was evident following the administration of butorphanol, an antagonist analgesic of the nalorphine type, which has no activity at the p receptors. Butorphanol alone did not affect the locomotor behavior of C57 mice, whereas it depressed activity in the DBA strain; moreover, when it was administered following morphine, activity was reduced in the C57 mice, and a further depression of activity was observed in the DBA mice compared with the effects of morphine alone. Butorphanol had no analgesic effect in the C57 mice if given alone and antagonized the analgesic effects of morphine; in the DBA strain, however, butorphanol alone exerted an analgesic effect, although it also antagonized morphine-induced analgesia. IV. Endogenous Opioid Systems

Opioid peptides are endogenous or synthetic peptides characterized by a spectrum of pharmacological activity similar to that of morphine and other narcotic agonist drugs (Beaumont and Hughes, 1979). The discovery of the endogenous opioids stemmed from two main contributions. T h e first one was the recognition that there might be opiatebinding sites in the brain (Goldstein et al., 1979). This idea led to the discovery of opiate receptors by three independent groups, those of Terenius (1973), Simon (Simon et al., 1973), and Snyder (Pert and Snyder, 1973). These observations raised the question: ‘:Why should specific receptors exist in mammals for recognizing morphine-a plant alkaloid?” This question pointed out the possibility that these receptors were receptors for some unknown morphine-like substance normally found in the central nervous system. Another important contribution that led to the recognition of endogenous opioids came from the observation that electrical stimulation of certain brain areas such as the periaqueductal gray would produce analgesia in animals and man (Reynolds 1969; Liebeskind et al., 1973; Mayer and Price, 1976). These results could indicate that electric stimulation produced the release of some mor-

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phine-like substance in the brain tissue stimulated, This possibility was strengthened by the observation that analgesic effects produced by electrical stimulation could be reversed by naloxone, an opiate antagonist that at that time was believed to have no action except the reversal of exogenous opiate effects (Akil et al., 1976; Mayer and Price, 1976). These studies concerning stimulation-induced analgesia, together with the demonstration of opioid receptors in the brain, suggested that there must be an endogenous morphine-like substance. In 1975 Hughes and Kosterlitz and their co-workers (Hughes et al., 1975) discovered enkephalins. In the subsequent years several substances with opiate activity were identified in the brain and in other tissues. The most important of these are P-endorphin, Met-enkephalin, and Leu-enkephalin. Although it was originally believed that the precursors of the enkephalins must be &endorphin and its precursor P-lipotropin (P-LPH), more recent studies indicate that Met-enkephalin is not derived from p-endorphin and that the Leu-enkephalin precursor peptide is unrelated to the molecules of the P-LPH-derived endorphin series (Goldstein et al., 1979; Kangawa and Matsuo, 1979; Stern et al., 1979). Some evidence seems to point to several other opioid peptide systems such as those of aneoendorphin (Kangawa and Matsuo, 1979), dynorphin (Goldstein et al., 1979), and dermorphin (Erspamer and Melchiorri, 1980).

OF OPIOIDPEPTIDES A. BRAINDISTRIBUTION

Distributional studies on the content of enkephalins and endorphins in the brain have described widespread peptidergic neural systems and pathways. These studies have been carried out using radioimmunoassay and immunohistochemical techniques (for a review see Miller and Cuatrecasas, 1979). 1. Enkephalin Radioimmunoassay (RIA) studies have shown that the amounts of Met-enkephalin are 5- 10 times greater than those of Leu-enkephalin in rat brain. Moreover, enkephalin concentrations are low in cortical regions and high in diencephalon. The highest concentrations of these peptides have been found in the corpus striatum (Hong et al., 1977; Yang et al., 1977). These results have been confirmed by several studies in which radioimmunoassay, radioreceptor assay, and biological assay were used (for a review see Bloom and McGinty, 1981). It must be pointed out that the estimates of enkephalin content by RIA are in general agreement with the quantitative regional and histological esti-

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mates of the distribution of opiate receptors (Atweh and Kuhar 1977a,b,c),whereas the distribution of enkephalin and P-endorphin systems does not always parallel that of the opiate receptors. Immunohistochemistry has shown immunoreactive perikarya and fibers in the telencephalon, in the diencephalon, and in the brainstem. The lowest enkephalin levels have been found in the cerebellum. In the spinal cord enkephalin fibers have been found in the dorsal Laminae I, 11, and 111, whereas ventral horn and central gray regions are less densely innervated by enkephalin fibers (for a review see Bloom and McGinty, 1981). At the present time five enkephalin-containing circuits have been proposed, as pointed out by Bloom and McGinty (1981). The first one consists of interneurons in the dorsal horn of the spinal cord; the second one, of the innervation of the globus pallidus from cell bodies in the basal ganglia; the third one, of the innervation of the stria terminalis from cell bodies in the central nucleus of the amygdala; the fourth one, of the innervation of the neurohypophysis by cell bodies in the paraventricular and supraoptic nuclei; the fifth one, of a projection to the spinal cord from preolivary cells in the medulla (see also Miller and Cuatrecasas, 1979). Some discrepancies between enkephalin immunoreactivity and autoradiographically detected opiate-binding sites in cortex, striatum, amygdala, and spinal cord have been observed (for a review see Bloom and McGinty, 1981).

2. @-Endorphin Some evidence indicates that P-endorphin and enkephalins are localized in different neural systems in the brain (Rosier et al., 1977a,b). For instance, in some brain structures such as caudate nucleus, globus pallidus, and caudal brainstem areas containing enkephalin there is virtually no @-endorphin. A controversial aspect of the distribution of endorphin in the CNS is related to the relationship between brain endorphin and pituitary endorphins. Several studies have recently shown that endorphins in pituitary are unrelated to endorphins localized in other brain areas (Rosier et al., 1977a,b; Bloom and McGinty, 1981). Immunohistochemical studies have localized cells containing endorphins and related peptides in the tuberal zones of the hypothalamus that innervate the lateral hypothalamus, preoptic area, medial amygdala, and the midline of thalamus, periaqueductal gray, locus coeruleus, and several lower brain nuclei (Watson et al., 1977; Bloom et al., 1978a,b). Evidence exists indicating two separately regulated stores of P-endorphin in rat pituitary gland (Przewlocki et al., 1978). The anterior lobe (pars distalis) contains pendorphin and adrenocorticotrophin (ACTH), which appear to be simul-

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taneously synthesized, stored, and released in response to stressors o r endocrine manipulation (Guillemin et al., 1977; Hollt et al., 1978a,b). T h e intermediate lobe of the pituitary gland (pars intermedia) contains the highest concentration of pituitary p-endorphin and little or no ACTH (Przewlocki et al., 1978, 1979).

B. OPIOID PEPTIDEBIOSYNTHESIS T h e biochemical steps involved in the biosynthesis of opioid peptides are largely unknown. P-Lipotropin is thought to be the precursor of biologically active peptides such as @-endorphin (p-LPH6,-91), a-endorphin (p-LPH61-76), y-endorphin (@-LPHsl-77),Des Tyrosine''-y-endorphin (P-LPH62-77), Met-enkephalin ( P - L P H B ' ~ ~and ), @-melanotropin (P-MSH) (pLPH41-58)(Bradbury et al., 1976; Chretien et al., 1976; Grif et al., 1976; Li and Chung, 1976; Burbach et al., 1980). The P-LPH precursor proopiocortin, also called proopiomelanocortin, is the common precursor for other neurohypophyseal hormones such as ACTH and a-MSH. (Miller and Cuatrecasas, 1979; Snyder, 1980; Rossier, 1981; Boileau et al., 1981). Although Met-enkephalin is contained in the amino acid sequence of @-endorphin,there is little evidence to support the idea that Pendorphin could be the biological precursor of Met-enkephalin (Miller and Cuatrecasas, 1979; Bloom and McGinty, 1981). T h e same can be said for Leu-enkephalin (Miller and Cuatrecasas, 1979). The precursor(s) of the enkephalins is at present unknown. However, some recent findings have shown that enkephalin precursors with structures completely different from @-endorphin are localized in large amounts in bovine adrenal medulla. One of these molecules contains both the Leuenkephalin and Met-enkephalin amino acid sequences, and it is therefore considered a common precursor of the two enkephalins (Rossier, 1981). Anatomical, electrophysiological, and neurochemical studies have pointed out a role of both enkephalins and p-endorphin in neurotransmission. I n particular these studies (for a review see Smith and Loh, 1981) suggest pre- and postsynaptic mechanisms in the action of opioids. Moreover, it seems well established that p-endorphin is characterized by a hormone-like action. In the pituitary @-endorphin is secreted simultaneously with ACTH during stress, and removal of the pituitary results in decreased levels of the peptide in circulation (Guillemin et al., 1977). Removal of the pituitary as well as the adrenal glands has been shown to potentiate opiate-induced analgesia and increase the degree of tolerance development (Holaday et al., 1977a,b).

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Acute administration of opiate agonists or P-endorphin to animals produced an increase of circulating levels of some pituitary hormones such as ACTH, growth hormone (GH), prolactin (PRL), and melanocyte-stimulating hormone (MSH) (de Wied et al., 1974; Holaday and Loh, 1979; for a review see Holaday and Loh, 1981; Smith and Loh, 1981). C. INTERACTIONSWITH NEUROTRANSMITTERS It has long been known that alkaloid opiates produce changes in release and turnover of different neurotransmitters in specific brain areas (for a review see Holaday and Loh, 1981). In recent years several studies have shown that the effects of opioid peptides on neurotransmitter release and turnover are similar to those previously described for opiates. Analgesic doses of intraventricular P-endorphin have been shown to decrease acetylcholine turnover in the cortex, hippocampus, nucleus accumbens, and globus pallidus, but not in the caudate. If injected into the septum, P-endorphin decreases ACh turnover in rat hippocampus, but not in striatum and cortex; moreover, it does not produce analgesia, indicating that the decrease in ACh turnover in these brain areas is not related to the analgesic effects of P-endorphin (Moroni, 1977a,b). Intrasepta1 injections of @-endorphin do not modify ACh content but do change turnover rates in the hippocampus (Moroni, 1977b). Injected icv, P-endorphin increases ACh content in the hippocampus (Botticelli and Wurtman, 1979). Kumakura et al. (1980) have shown that opioid peptides stored in the axon terminals of the splanchnic nerves in the adrenal medulla may serve as inhibitory neuromodulators of the acetylcholine receptors located on chromaffin cells involved in catacholamine release, indicating a functional effect of opiate peptides on cholinergic systems (see Holaday and Loh, 1981). Loh et al. (1976a) found that pendorphin inhibited DA release in striatum, but such an effect was not observed by Arbilla and Langer (1978). It has been shown that morphine and 6-agonists stimulate DA release in caudate nucleus (Chesselet et al., 1981). P-Endorphin, as well as other opiate peptides, was shown to inhibit the release of DA from tubero-infundibular neurons, a finding that may be related to the increase in prolactin commonly produced by opiates (see Holaday and Loh, 1981). Van Loon and Kim (1978) observed that although @-endorphin inhibited the release of striatal DA, it increased DA turnover. Met-enkephalin, morphine, and P-endorphin injected icv reduced DA turnover in the medial and lateral pallisade zone of the median eminence (Fuxe et a/., 1980). However, they

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produced an increase of NE turnover in several hypothalamic nuclei (Fuxe et al., 1980). P-Endorphin and some opiate peptides reduced NE release from rat cerebral cortical tissue and from slices of rat occipital cortex (Taube et al., 1977; Arbilla and Langer, 1978; Gothert et al., 1979). @-Endorphin decreased GABA turnover in the caudate but increased it in the globus pallidus (Moroni et al., 1979). Met-enkephalin inhibited K+-stimulated release of GABA from brain synaptosomes (Brennan et al., 1980). Nicoll et al. (1980) reported that the stable enkephalin analog DAla', Met5)-enkephalinamide (DALA) attenuates a number of GABAergic inhibitory pathways in CNS (hippocampus, olfactory bulb, spinal cord) without, however, affecting the action of GABA. @Endorphin was reported to increase the release and the turnover of serotonin in brainstem and hypothalamus and decrease both processes in hippocampus (Van Loon and De Souza, 1978). In conclusion, it can be said that endogenous opioids (and other opiates) can either increase or decrease the turnover and/or the release of a neurotransmitter depending on specific anatomical sites.

D. NEUROENDOCRINE EFFECTS Several studies have indicated that the administration of exogenous and endogenous opiates increased the release of ACTH, p-endorphin, MSH, PRL, and GH from pituitary and decreased the release of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH). These effects seem to be mediated at opiate receptor sites in hypothalamus that modulate the release of hypophysiotropic hormones or biogenic amines (for a review see Holaday and Loh, 1981). Some evidence exists to indicate that opiates are also involved in the control of vasopressin and oxytocin release (Bicknell and Leng, 1982). Holaday and Loh (1981) have pointed out three separate systems that could allow for endorphins to increase their secretion via positive feedback. According to these authors, these systems may allow for the biological amplification of P-endorphin release in times of severe stress and could be turned off by tolerance mechanisms.

E. RECEPTORS There is substantial pharmacological and biochemical evidence to support the existence of multiple opiate receptors. It has been shown that many opiate agonists (i.e., morphine, levorphanol) and antagonists

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(i.e., naloxone, naltrexone) are more potent in competing against the binding of [3H]dihydromorphine or naloxone at the receptor site than [3H]enkephalin or 1251-labeled(DAla2, ~Leu~)-enkephalin. However, enkephalins are more potent in competing against the binding of [3H]enkephalin or 1251-labeled(DAla2,~Leu~)-enkephalin. These results have been explained by assuming that there is a single class of opiate receptors that can exist in two conformational states, the agonist state, which preferentially binds agonists, and the antagonist state, which preferentially binds antagonists (for a review see Lord et al., 1977; Chang et al., 1980; Chang and Cuatrecasas, 1981; Simon, 1981; Zukin and Zukin, 1981). Another hypothesis developed in order to explain these results, which is currently believed to be correct (but see Laduron, 1982), is that there are multiple opiate receptors. Of these, the so-called p and 6 receptors hzve been the best characterized. The 6 receptor is pharmacologically defined as the receptor with a high affinity for enkephalin, whereas the p receptor has been defined as the receptor site that preferentially binds to morphine. It must be pointed out that no endogenous ligand for the p receptor has been found. Several studies of the relative binding capacity and affinity of various opiate antagonists and opiatelike peptides have shown marked regional differences in opiate binding sites. The highest density of both p and 6 sites were found in frontal cortex and striatum, which contain about equal amounts of both types of receptors. In the limbic system, hippocampus, and brainstem there are twice as many p as 6 receptors, whereas in the thalamus and hypothalamus this factor rises to between four and five (see Chang et al., 1980). Isolated guinea pig ileum and mouse vas deferens preparations are used in order to evaluate and compare the relative potency of many opiates, enkephalins, and endorphin analogs. The results obtained with these two preparations correlate with the different specificities of p and 6 receptors as measured in brain membrane preparations (Lord et al., 1977; Waterfield et al., 1979).Several studies have shown that mouse vas deferens contains enkephalin receptors (a), whereas guinea pig ileum contains morphine receptors ( p ) .Another interesting preparation is the rat vas deferens (RVD). Herz and his colleagues (Schulz et al., 1979; Wuster et al., 1980) found that this preparation, unlike mouse vas deferens, contains a receptor that is very sensitive to P-endorphin but much less sensitive to enkephalins and morphine. This observation led to the postulation of another type of opiate receptor, the E receptor. Martin and co-workers (Gilbert and Martin, 1976; Martin et al., 1976) postulated three distinct types of opiate receptors: p, K , and cr. Morphine would behave as a typical p agonist, and ketocyclazocine and ethylketocyclazocine (EKC) would be typical agonists for K receptors.

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T h e latter two drugs produce different withdrawal symptoms in comparison with morphine (see Chang and Cuatrecasas, 1981). A typical agonist for u receptors is allylnormetazocine (SKF 10,047). This compound produces some effects (such as delirium and precipitation of abstinence) in morphine-dependent animals that differ from those produced by p and K agonists. There is some recent evidence for a specific binding site for phencyclidine (PCP) (for a review see Zukin and Zukin, 1981). It has also been reported that the PCP site may be identical to the u opiate receptor (Zukin and Zukin, 1981). In studies of binding capacity carried out on guinea pig ileum and mouse vas deferens preparations, it has been observed that the recently discovered opioid d y n ~ r p h i n , - ,(Chang ~ et al., 1980) is 12 times more potent in the guinea pig ileum than in mouse vas deferens. Moreover, it is 730 times more potent than enkephalin, 190 times more potent than morphine, and 54 times more potent than P-endorphin in the guinea pig ileum. These results have suggested that d y n ~ r p h i n , is - ~the ~ true endogenous opiatelike substance for morphine receptors. It has also been shown that dynorphin1-13and dynorphinl-9 are specific endogenous ligands of the K opiate receptor (Chavkin et al., 1982; Corbett et nl., 1982).

F. ANALGESIC EFFECTS One of the most important effects of narcotic drugs is their analgesic action. Thus, after the discovery of endogenous opioids and once the structure of enkephalin was elucidated, the effects of synthetic pentapeptides on pain sensitivity were tested by injecting them icv into mouse and rat brain. T h e results showed some analgesic effects that were, however, weak and extremely transient in comparison to those produced, for instance, by morphine (Belluzzi et d.,1976; Buscher et al., 1976). Such results were due, as it has been subsequently observed, to the rapid metabolism of the peptides (Terenius, 1978). Stable compounds such as P-endorphin or nonmetabolized enkephalin analogs produced clear-cut and long-lasting analgesic effects (Miller and Cuatrecasas, 1979). If stabilized pentapeptides are used, enkephalins appear to be at least as potent as P-endorphin (Wei et al., 1977; Miller el al., 1978). Moreover, it has been shown that many enkephalin analogs and endorphins are more potent than morphine in producing analgesia following icv injections (Loh et al., 1976b; Pert, 1976). Intracerebroventricular injections of (DAla', ~Leu~)-enkephalin (DADL), which has a high affinity for 6 receptors, have been shown to have only 1% of the antinociceptive effects of Tyr-DAla2-Gly-MePhe-Met(0)-ol (Sandoz), whose affinity

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for the p receptors is as high as that of DADL for 6 receptors. The affinity of the Sandoz compound for the 6 receptors, however, is as low as that of DADL for the p receptors (Kosterlitz, 1980).These results may suggest that the EL. receptors are more important for antinociceptive effects than the 6 receptors. It has been proposed that metkephamid, an analog of Met-enkephalin that has a high affinity for the 6 receptors and is a systemically active analgesic, produces analgesia by an action on 6 receptors as well as, or rather than, on p receptors (Frederickson el al., 1981). G. RELATIVE POTENCY OF OPIOID PEPTIDES AND OPIATES ON OPIATE RECEPTORS

T h e naturally occurring opioid peptides, Met-enkephalin and Leuenkephalin, prefer the enkephalin 6 receptors to morphine p receptors. P-Endorphin seems to bind to both types of receptors with equal affinity. The amidation of the carboxy terminal of enkephalin, as in (DAla', Met5)-enkephalinamide, increases the compounds' affinity for the morphine ( p ) receptor but decreases their affinity for the enkephalin (6) receptor. The orally active enkephalin analog, Sandoz FK 33-824 [Tyr~Ala-Gly-N(Me)Phe-Met(O)ol] behaves as a typical morphine-like substance in the binding assay and in the pharmacological assay (Chang et al., 1979) utilizing the guinea pig ileum. The relative potency of morphine and the metabolically stable enkephalin analogs is morphine > Sandoz FK 33-824 > (DAla2,Met5)-enkephalinamide > (DMet2, Pro')enkephalin > (DAla2,~Leu~)-enkephalin for the morphine ( p )receptors and (DAla2, ~Leu~)-enkephalin > (DAla', Met5)-enkephalinamide > (DMet2, Pro5)-enkephalin > Sandoz FK 33-824 1 morphine for the enkephalin (6) receptors. Etorphine is equally active on both binding sites. Opiate antagonists, such as nalorphine, naltrexone, and naloxone, have about 10 to 20 times greater affinity for morphine receptors than for enkephalin receptors. Diprenorphine binds with a very high, albeit similar, affinity to both sites: the binding reaction is half-saturated at diprenorphine concentrations of 0.2 nM (for review see Chang et al., 1980).

H. PHYLOGENETIC AND GENETIC STUDIES 1. InterspeciJic Differences After the initial descriptions of opiate receptors a number of studies dealt with the phylogenetic distributions of opiate binding and endogenous opioids. Binding sites for these peptides have been found in inver-

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tebrate nervous systems (Pert and Taylor, 1980; Stefan0 et al., 1980). It has been shown that both the levels and the types of binding sites for opiates and enkephalins differ according to the species. Buatti and Pasternak (1981) found that the most dramatic differences are those between rats, which have both high- and low-affinity sites, and goldfish, which have only low-affinity sites. According to these authors, the lack of demonstrable high-affinity sites in goldfish may suggest that high-affinity sites are a physically and pharmacologically different receptor. Given that in rats and mice the high-affinity site mediates opiate, enkephalin, and P-endorphin analgesia, the question arises whether the goldfish has a true antinociceptive system and, if so, which receptor subtype mediates it (Buatti and Pasternak, 198 1). An opiate-like heptapeptide (dermorphine) has been extracted from the skin of a frog, Philloniedwa sauvagei (Erspamer and Melchiorri, 1980). It is characterized by the unique feature of having a DAla residue incorporated in the peptide molecule (Montecucchi et al., 1981). Dermorphine has also been found in rodent brain (V. Erspamer, personal communication). This heptapeptide displays a potent depressive action on electrically stimulated contractions in guinea pig ileum and mice vas deferens preparations. Intravenous injection of this opioid produces a potent, long-lasting analgesia in mice. A similar effect was also evident in rats following intracerebroventricular administration. Morphine was shown to be 752 and 2 170 times less potent than dermorphine depending on the analgesia test used (Broccardo rt al., 1981). Moreover, derniorphine was shown to induce hypermotility in mice when injected icv but not iv (Puglisi-Allegra et al., 1982a).

2. Intraspecijic Differences Strain differences in number, type, o r distribution of opiate receptors have been demonstrated in a number of investigations. Researches carried out by Baran et al. (1975) with C57 and BALB mice, their F1 hybrids, and seven recombinant inbred strains showed that ( a ) the strains tested could be divided into three groups on the basis of their receptor number (the group formed by the two progenitor strains and their F, hybrids showing intermediate number of receptors), and (h) a positive correlation existed between the number of receptors and the analgesic response. However, this correlation was not statistically significant, suggesting that strain differences in receptor numbers could not entirely explain the strain differences in the analgesic effects of opiates. Reggiani et al. (1980) has tried to clarify the molecular mechanisms underlying the effects of opiate administration on running activity and analgesia in C57 and DBA mice. They were involved in particular in the determination of which group of opiate receptors is functionally related

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to dopamine neurons. The authors initially investigated the effect of morphine on DOPAC levels in the two strains. Opiate administration increased both the striatal and the limbic levels of DOPAC only in C57 mice. Because the brain levels of morphine were the same in both strains, opiate receptor function was measured in the same animals by using [3H](~Ala2, Met)-enkephalin ([”HIDAME)or [3H]Leu-enkephalin. The results showed a lower striatal opiate receptor binding in DBA as compared with the C57 mice. In additional kinetic studies carried out by incubating the tissues with various [$HIDAME or [3H]Leu-enkephalin concentrations, a significant reduction in the number of binding sites was found only in the striatum of the DBA mice, although the receptor affinity was similar for the two strains. In addition, no significant differences between the strains tested were observed either in the striatum or in other brain areas such as brainstem, forebrain, or neocortex if [“HJdihydromorphine or [SH]naloxone were used as radioactive ligands for opiate receptors. Finally, lesion studies performed with intraventricular injections of 6-OHDA (which induced a comparable 80% reduction of striatal DA content in the two strains) demonstrated a significant reduction of the opiate receptors’ function (measured by [3H]DAME binding) in the C5’1 mice, whereas no effect was evident in the DBA strain. From these results it can be concluded that (a) DBA mice present a lower number of opiate receptors located on the DA terminals of the nigrostriatal pathway, (b) enkephalins modulate the function of the striatal DA pathway impinging upon DA neurons (thus, the so-called dopaminergic effect of narcotics might depend largely on an enkephalinergic-dopaminergic neuronal interaction), and (c) in the two strains tested different populations of receptors are present. On the basis of the hypothesis (Pert and Taylor, 1980) concerning the existence of two types of receptors: type 1, which binds [3H]naloxone and [3H]dihydromorphine preferentially, and type 2, which binds [3H]Leu-enkephalin or [3H](~Ala2, Met)-enkephalin preferentially and which may distinguish the phylogenetically older circuits, the authors suggest that the opiate receptors located on striatal dopaminergic terminals and involved in the effects of morphine on dopaminergic metabolism might be, at least in part, type 2 receptors. These receptors, numerous in the C57 strain, might be important for locomotor stimulation, whereas the type I receptors, more common in the DBA strain, might be important for induction of the analgesic effect following opiate administration. Reith et al. (1981) have found that mice of a recombinant inbred system differ in p- and &type binding in the brain. Crabbe and coworkers (1981) have found that the whole pituitary contents of @endorphin and ACTH vary widely among five inbred strains of mice (BALB/CAnN, C3H/HeN, C57BL/6N, DBA/SN, and AKR/J). Also, if

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peripheral receptors are considered, there is some evidence that p, and 6 receptors have a separate genetic regulation. In C57BL mice morphine is less effective in suppressing contraction of the vas deferens as compared to other strains (Waterfield et aE., 1978). However, enkephalins are more effective in C57BL mice than in other strains.

I.

ENDOGENOUS O Pl O I D S OPIATE RECEPTOKS

ONTOCENY OF

AND

Zhang and Pasternak (1981) have carried out an extensive study on the ontogeny of high- and low-affinity opiate receptors in which they observed that the high-affinity sites preferentially mediate opiate-induced analgesia. In particular they found that high-affinity sites and analgesia appear at about the end of the first postnatal week. Wohltman et al. (1982) found differential postnatal development of p and 6 receptors in rat brain. The I.L receptors appear to develop earlier than 6 receptors (which appear at about Day 12 of postnatal life). The delayed development of 6 receptors correlates with the late appearance of functional opiate-catecholamine coupling, as reported in previous studies. Tsang et al. (1982) measured the development of opiate receptors in whole forebrain, brainstem, and cerebellum by specific [3H]naloxone binding. The binding of these two opiates varied with the brain region as well as with age, but the amount of ['H]naloxone bound in the same region obtained from animals of the same age was greater than that of [3H]Met-enkephalin, These results seem to indicate that in rat brain there are two types of opiate receptors whose heterogeneity is already apparent during early postnatal life. Endorphin levels in rat brain were found to be much higher than enkephalin levels on embryonic Day 16, the highest endorphin values having been found in the diencephalon, midline telencephalon, and midbrain. During the prenatal period, enkephalin content increased at a faster rate than endorphin in all brain regions, and between postnatal Days 6 and 25 both endorphin and enkephalin levels increased, approaching their adult distribution pattern. Moreover, regional distribution or rates of increase of endorphin or enkephalin in these developmental stages did not show any correlation, suggesting that the two opioid systems are characterized by independent patterns of development (Bayon et al., 1979). Patey et al. (1980) studied the postnatal changes in enkephalin levels, enkephalin receptor density, and enkephalin-degrading enzyme activities in cerebral cortex and striatum. Metand Leu-enkephalin levels both increased by seven- to elevenfold in an independent manner compatible with their presence in distinct neuro-

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nal systems. [3H]Enkephalin binding sites increased only fourfold in striatum, as reported for receptor sites labeled with [3H]opiate antagonists. The development of striatal enkephulinme, the particulate enzyme activity cleaving the Gly-Phe bond of enkephalins, paralleled more or less in time that of enkephalin levels and receptors, with a sixfold increase from birth. In contrast total enkephalin-hydrolyzing activity showed little change. The developmental pattern of angiotensin-converting enzyme was clearly distinct from that of “enkephalinase,” confirming that the two enzymes are different species. Research carried out by Filibeck et al. (1982) has attempted to study the development of morphine-induced changes of activity in the mouse in order to determine if ( a ) opiate receptor development in the mouse is paralleled by behavioral modifications in terms of hyperactivity or catatonia, and ( 6 ) if there are time differences in the development of neurophysiological mechanisms responsible for catatonia or excitatory effects. For this purpose, C57 mice were subjected at different ages (from 8 to 60 days) to a 10-min-long measure of spontaneous activity (Animex apparatus), and their performances were compared with those of salineinjected mice. T h e results showed a sharp rise of activity between 16 and 22 days in the control mice, whereas a fall in activity appeared following the third week of age. Morphine (10 mg/kg) enhanced activity in the 818-day-old subjects. Activity depression was evident in the 3-week-old mice, whereas in the older ones (28 to 60 days) a clear enhancement of activity was recorded. Because catecholaminergic mechanisms might be involved in the stimulating or depressant effects of opiates on activity, the results can be explained in terms of maturation of inhibitory (serotonergic) structures in the central nervous system. This maturation, in fact, occurs at about the third week of life (Marbry and Campbell, 1974). In addition, because different populations of opiate receptors might be responsible for the stimulating or catatonic effects of opiates (Chang et al., 1980; Reggiani et al., 1980; Castellano, 1981b; Filibeck et al., 1981), another possible explanation for the results obtained could be that receptors responsible for the inhibitory or excitatory effects of opiates present different ontogenetic processes.

V. Opiates and Behavior

Endogenous opiates are involved in a number of behavioral activities ranging from CNS activation to consummatory activities, learning and memory, and social interactions. The study of these various behavioral activities may be approached from two different points of view:

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1. T h e involvement of opiates and opiate receptors in each of these behavioral patterns. 2. The usefulness of studying a given behavioral o r neurobiological trait in order to clarify or support a model for opiate mechanisms. This two-sided approach results in a niushrooniing number of experinients and reports along these lines. T h e field o f learning and meniol-y is particularly complex since opiates and other nonopioid peptides (or their fragments) are involved in the modulation of memory processes (de Wied, 1980).

A. SEIZURES 1. ElectroenrPphaloff~iic Mod$catioiu and Ekctmconvulsive Shock-Induced Seizures

Morphine injections produce the appearance of intermittent EEG high-amplitude slow-wave activity (Longo, 1962; Khazan et al., 196’7; Khazan and Colasanti, 1971). Intracerebroventricular enkephalin injections have been reported to induce epileptoid spikes and subsequent onset of slow-wave EEG hypersynchrony associated with an increased voltage output and related stuporous behavior (Urca et al., 1977; Tortella et al., 1978). &Endorphin is more potent than enkephalin or morphine in producing the epileptoid EEG spikes and subsequent high-voltage, lowfrequency EEG synchrony (Moreton et al., 1978). It has been reported that /3-endorphin produced “nonconvulsive” limbic seizures when injected icv at very low doses; recording electrodes were placed in limbic structures. During EEG seizures, animals did not show behavioral signs (Henricksen et al., 1978). Holaday et al. (1978a) reported that p-endorphin injected intracerebroventricularly produced overt motor seizures without any evidence of brain seizure activity. I n recent years several studies have dealth with the involvement of endogenous opioids in electroconvulsive shock (ECS), which is employed in the treatment of severe depression and schizophrenia (electroconvulsive therapy, ECT). T h e occurrence of a spectrum of opiate-like effects such as catalepsy, analgesia, hyperthermia, and respiratory depression has been observed following transauricular ECS in unanesthetized rats (Holaday et al,, 1978b, 1979; Belenky and Holaday, 1979, 1980; Holaday and Belenky, 1980; Lewis et al., 1981). It is worth noting that the ECSinduced catalepsy is characterized by a loss of the righting reflex, which is also a characteristic feature of opiate catalepsy (Segal et al., 1977). Naloxone pretreatment was shown not to affect the duration and the intensity of the behavioral tonic-clonic seizures, although it decreases

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the opiate-like behavior that occurred following ECS (Holaday et al., 197813; Belenky and Holaday, 1979; Holaday and Belenky, 1980). The respiratory depression and the cardiovascular changes following ECS are also modified by naloxone pretreatment, indicating that endogenous opioids play a role in these physiological systems (for a review see Holaday and Loh, 1981). Tortella et al. (1980) reported that ECS in rats produces the EEG pattern of slow-wave hypersynchrony, which resembles the EEG activity that occurs following icv injections of /?-endorphin. These effects are prevented by naloxone pretreatment. It has been shown that repeated daily ECS results in increases in Met-enkephalin in caudate nucleus, hypothalmus, and limbic areas. On the contrary, repeated daily ECS for 10 days does not modify p-endorphin content in the hypothalamus. Belenky and Holaday (198 1) have investigated the effects of repeated ECS on the naloxone-sensitive opiate-like behaviors that follow the convulsive seizures and found a sensitization to the effects of morphine on catalepsy and tail flick latencies. Conversely, chronic morphine treatment by pellet implantation sensitized rats to the opiate-like effects produced by ECS. According to Holaday and Loh (198 l ) , this cross-sensitivity between repeated ECS and morphine tolerance indicates that repeated ECS and the induction of morphine tolerance may share common neurobiological mechanisms.

2. Kindled Seizures T h e term kindling refers to repeated subconvulsive unilateral electrical stimulation of discrete brain areas over periods of days to weeks. This stimulation leads to a progressive increase of epileptiform discharges that generalize to the centrolateral areas and result in tonic-clonic convulsive seizures (Goddard et al., 1969; Racine, 1972). Morphine was reported to enhance the epileptiform seizures induced by stimulation of the amygdala (a brain area rich in opiate receptors) in kindled rats. Inconsistent results after naloxone treatment have been reported (for a review see Holaday and Loh, 1981), possibly because of the wide variation in doses of the opiate antagonists and stimulation parameters employed in each study. However, studies carried out so far seem to indicate that low doses of naloxone may facilitate kindling (Hardy et al., 1980; see also Holaday and Loh, 1981). 3. Audiogenic Seizures Systemic injections of Met-enkephalin were reported to decrease audiogenic seizures in mice (Plotnikoff et al., 1976), whereas naloxone increased the severity of audiogenic seizures in two different genetically susceptible strains. Collectively, the results indicate that naloxone may

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facilitate kindling, intensify audiogenic seizures, and decrease postictal depression, suggesting that anticonvulsant action of endogenous opioids may be of greater functional importance than their proconvulsant effects (for a discussion see also Holaday and Loh, 1981). This hypothesis is supported by some recent work. Engel and Ackermann (1980) reported that interictal EEG spikes correlate with decreased rather than increased epileptogenicity in amygdaloid-kindled rats. Oliverio and coworkers (Oliverio et al., 1983; S. Puglisi-Allegra, C. Castellano, A. Oliverio, A. Doka, and V. Csanyi, unpublished) have shown that icv injection of morphine, /3-endorphin, and DADL protected C57BL/6 mice against ECS tonic seizures, whereas naltrexone ( 15-20 mg/kg) facilitated the convulsant effects of ECS. Moreover, they observed that immobilization stress (2 hr) produced protective effects against ECS seizures and that those effects w e r e prevented by pretreatment with subthreshold doses of naltrexone. B. SLEEP Natural and synthetic opiates have been shown to decrease rapid eye movement (REM) sleep periods and to modify sleep patterns. No effect of naloxone on sleep patterns (REM parameters) has been reported (Davis et al., 1977; Martin et al., 1979). In a study of sleep-wake periods in rats Tortella et al. (1978) found that intracerebroventricular injections of enkephalins decreased sleeping time, thus mimicking morphine. After injections of opioids rats alternated between a stuporous and an aroused state. T h e results concerning naloxone, which indicate that this opiate antagonist does not produce any effect on sleep parameters, could mean that endogenous opioids play little or no functional role in normal sleep o r that too low doses of the drug naloxone were used. Holaday and Loh (1981) also postulate that because naloxone is usually without pharmacological effect in nonstressed animals, it could be that some stressful situation (e.g., sleep deprivation) is required to activate the endorphinergic system.

C. CONSUMMATORY BEHAVIOR A number of studies have recently indicated an involvement of endogenous opioids in the control of consummatory behavior (King et al., 1979; Ostrowski et al., 1981; Lang el al., 1982). Opiate antagonists, primarily naloxone, have been shown to suppress water and food intake in rats and mice tested in different experimental conditions (Brown and

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Holtzman, 1979; Apfelbaum and Mandenoff, 1981; Carey et al., 1981; Rowland and Bartness, 1982). Morphine has been shown to increase fat consumption (Marks-Kaufman, 1982), whereas there is some evidence that water intake is suppressed by this opiate alkaloid (Frenk and Rogers, 1979). Hynes et al. (1981) found that although intraperitoneal injections of naloxone (0.5-5.0 nig/kg) decreased food and water intake in deprived rats, no significant effects on consummatory behavior were produced by intracerebroventricular administration of the opiate antagonist (0.5-5.0 mg). Morley and Levine (1980) have shown that stressinduced eating in rats was abolished by naloxone, indicating endogenous opioid system involvement in the control of this behavior. Margules P t nl. (1978) found that naloxone abolishes overeating in genetically obese mice (ob/ob) and rats (fklfu). Moreover, they found elevated concentrations of P-endorphin in the pituitaries of both obese species and in the blood of obese rats, whereas brain levels of @endorphin and Leuenkephalin were unchanged. Shimomura and colleagues (1982) have studied the effects of acute and chronic administration of naloxone on food intake of lean and genetically obese (ob/ob) mice. In chronic experiments the food intake of both lean and obese mice was depressed during the first hour after injecting naloxone. However, beginning on the second day of treatment, the lean mice began to eat more food than the untreated controls during the 8-hr feeding period. Food consumption by lean mice reached values 140-200% above the control levels between the fourth and sixth day. In the obese mice the rise in food intake was more gradual and did not reach 200% of the control value until the sixth day. Body weight changes reflected the changes in food intake. In contrast to naloxone, chronic treatment with morphine lowered food intake and blocked the stimulatory effect of naloxone. These findings seem to indicate that opioids play a role in signaling satiety and in regulating long-term energy balance. Sanger and McCarthy (1982) tested naloxone and naltrexone effects on fixed-ratio (FR) responding maintained by small quantities of milk and on the consumption of milk when it was freely available. Both drugs reduced milk consumption at all doses (0.3-30 mg/kg) but produced only small decreases in FR response rates at the highest doses. According to the authors, these results do not support the view of an inhibitory action of opiate antagonists.

D. ENDOGENOUS OPIOIDSAND TOLERANCE Some evidence exists to indicate that endogenous opiates are no different from morphine in producing tolerance and physical depen-

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dence. Acme tolerance to Met-enkephalin challenge in the guinea pig ileum and mouse vas deferens from chronically morphinized animals has been observed (Waterfield et al., 1976). Tseng et al. (1976) found that the analgesic response to @endorphin increased in morphine pelletimplanted rats. Moreover, P-endorphin reversed opiate withdrawal signs in mice. Wei and Loh (1976) showed that long-term, continuous infusion of Met-enkephalin and P-endorphin produced a typical opiate withdrawal syndrome upon naloxone challenge. Moreover, repeated injections of P-endorphin were shown to result in tolerance to its analgesic, cataleptic, and hypothermic responses (Tseng et al., 1977). The evidence concerning the effects of chronic morphine treatment on endogenous opioid concentration predominantly indicates a lack of effect of such treatment on brain and pituitary endorphin levels (for a review see Holaday and Loh, 1981). According to Holaday and Loh (1981), it cannot be ruled out that the induction of opiate tolerance is without effect on endorphin systems because turnover rates for endorphins have yet to be measured.

E. LEARNING AND MEMORY In the last decade work from several laboratories has demonstrated that opiate agonists, antagonists, and endogenous opioid peptides are involved in the acquisition of a number of learned behaviors as well as memory storage and retrieval of aversive and appetitive events (for a review see Riley et al., 1980; Martinez et al., 1981a). The original work of Castellano (1975), who observed that morphine and heroin impair discrimination learning and consolidation in mice, was followed by several studies that led to the general conclusion that morphine agonists impair and antagonists enhance memory and learning (Jensen et al., 1978; Gallagher and Kapp, 1978; Izquierdo, 1979; Castellano, 1980, 1981a; Izquierdo et al., 1980; Martinez and Kigter, 1980) but also by a number o f reports in disagreement with such conclusions (Belluzzi and Stein, 1977; Mondadori and Waser, 1979; Staubli and Huston, 1980). Messing et al. (198 1) have investigated the effects of opiate agonists and antagonists on retention of inhibitory and active avoidance learning tasks. Morphine, naloxone, and naltrexone all improved retention performance if given in equally divided doses immediately and 30 min after training. However, a single administration of morphine immediately after training o r of naloxone 30 min after training impaired retention performance. These results have been interpreted by the authors in terms of the shared ability of these drugs to displace endogenous peptides from their receptor sites, of their interactions with endogenous I

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neurohumors that change as a function of time after a stressful experience, and of possible different functional effects following a single neurochemical manipulation that occurs at different times in the process of memory consolidation. Gallagher and Kapp (1981) have demonstrated that endogenous opioid mechanisms in the amygdala play an important role in modulating memory processes for aversive experiences. Intracerebral injections of the opiate agonist levorphanol into amygdala immediately following conditioning impaired retention of a passive avoidance task measured 24 hr later. This effect was blocked by concurrent administration of naloxone. Moreover, posttrial injection of naloxone produced a dose-dependent facilitation of retention. Memory for various tasks in the rat is depressed by posttraining systemic administration of p-endorphin (1-10 pg/kg) Met-, Leu-, and DesTyr-Met-enkephalin (0.32-10 pglkg) (Izquierdo et al., 1981). Martinez et al. (198 lb) found that adrenal medullectomy, which removes an endogenous store of enkephalin, abolished the effect on avoidance conditioning of systemically administered Leu- and Met-enkephalin, indicating that the adrenal medulla may be an important locus of enkephalin effects on fear-motivated behavior. High doses (100- 1000 pg/kg) of Leu- but not Met-enkephalin restored behavioral activity, suggesting that the impairing actions of the two types of enkephalins on acquisition of the avoidance response are produced by different mechanisms. @-Endorphinand Met-enkephalin (5-25 pg/rat) also caused retrograde amnesia for the shuttle avoidance task when given by intracerebroventricular injection after the training. Their effects were reversed by naloxone. @-Endorphin also produced naloxone-reversible amnesic effects if administered before training (Izquierdo at al., 1981). KovPcs and de Wied (198 1) have demonstrated that subcutaneous injections of @-endorphin facilitated retention of a fear-motivated passive avoidance response in a dose- and time-dependent fashion. This effect was not reversed by naltrexone. Furthermore, two possible endogenous products generated by the biotransformation of P-endorphin, a-and yendorphin produce opposite effects on passive avoidance response. aEndorphin caused a time-dependent facilitation, whereas y-endorphin caused a time-dependent attenuation of information processing. a-Endorphin injected subcutaneously (sc) was found to inhibit the extinction of a pole-jump avoidance task, whereas y-endorphin (sc) produced more rapid extinction. However, in a passive avoidance task a-endorphin (sc) increased retention latencies, whereas y-endorphin (sc) decreased them (Koob et al., 1981). Moreover, using an appetitively motivated task (a continuous reinforcement lever press situation for food reward), Koob and his co-workers found that a-endorphin (sc) delayed and y-en-

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dorphin (sc) slightly facilitated extinction. However, in a runway task with water reward both a-and y-endorphin delayed extinction, an effect that was not blocked by naloxone. According to Koob et al. (198l), the studies on the effects of opioid peptides on learning and memory suggest that centrally derived endorphins act as chemical opiate receptor agonists, whereas low concentrations of peripherally derived endorphins may act on classical opioid receptors to produce hormone-like actions that are not reversible by naloxone. In their studies on a one-trial step-down passive avoidance task, Belluzzi and Stein (for a review see Belluzzi and Stein, 1981)found that intracerebroventricular injections of morphine (20 pg) and Metenkephalin (100-200 pg), but not Leu-enkephalin (100-200 pg), facilitated retention of the passive avoidance response in rats. Naloxone (10 pg) blocked the memory-enhancing effects of morphine but not those of Met-enkephalin. These results seem to be in disagreement with those data that indicate that opiates and opioids produce amnesia when administered after training. According to Belluzzi and Stein (198l), the amnesic or memory-enhancing effects of opiates could result from separate dose-related actions at presynaptic and postsynaptic sites. Low doses could reduce postsynaptic opiate receptor activity by presynaptic inhibition of endogenous opioid release, whereas high doses could increase postsynaptic opiate receptor activity by direct agonist action. Castellano (1980) has reported that low doses of heroin (0.5 mg/kg) injected posttrial were followed by performance improvements in a fivechoice pattern discrimination task motivated by shock in mice of the C57BL/6 and DBA/2 strains, whereas performances were impaired by higher doses (5 mg/kg) only in the latter strain. These results suggest that the genetic makeup may play a role in the effects of the endogenous opioid systems on memory processing. In agreement with this idea Castellano and Puglisi-Allegra ( 1983)have demonstrated that stress exerts a modulating effect on memory consolidation in the mouse and that such an effect is strain-dependent. Immediate postraining immobilization stress resulted in time-dependent consolidation impairment in outbred Swiss Webster and inbred DBA/2 mice but improvement in inbred C57BU6 mice tested in a passive avoidance apparatus. These effects were reversed by naloxone, indicating the involvement of endogenous opioid peptides. McGaugh and his co-workers (Jensen et al., 1981) have pointed out that alterations in opioid systems may contribute to changes in learning and memory processes in aged animals. Some evidence exists indicating that naloxone administration may produce different effects in young and aged rats. Moreover, regionally specific differences in opiate recep-

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tor concentration and apparent binding affinities in young and old rats have been found (Jensen et al., 1981). Riley et al. (1980), reviewing the role of endogenous opioids in animal learning and behavior, suggested that ir, learning paradigms involving stress the stressor elicits the release of endorphins. Such an idea, based on a number of studies on shock-induced analgesia (SIA), is supported by recent findings (see Izquierdo et al., 1981) indicating that pendorphin is released from the rat brain at the rate of 30 to 50 pglbrainl 25 min during various forms of training including shuttle avoidance, habituation training, pseudoconditioning, and the repeated presentation of footshocks alone. According to Riley et al. (1980), a number of learning paradigms such as the conditioned emotional response, preference for signaled shock (Fanselow, 1979), conditioned taste aversion, and learned helplessness not only indicate the mediation of learning by endogenous opioids but also suggest that the stress-induced release of pendorphin and enkephalins (for a review see Amir et al., 1980) modulates the aversiveness of the stressor and thus affects the learning based on this stressor. A possible interaction between endorphins and ACTH and of opioids at opiate- and AC'TH-binding sites (Jacquet et al., 1977; Jacquet, 1978; Jacquet and Wolf, 1981)has been offered as a physiological basis for the mediation of learning by endogenous opioids (Riley et al., 1980).

F. LEARNING MECHANISMS OF TOLERANCE ACQUISITION Kensner and Baker (198 1) reviewed findings concerning the development of opiate tolerance with respect to major behavioral and physiological tolerance models and pointed out that the behavioral phenomena of morphine tolerance can be explained by a two-process model in which learning plays an important role. In one process, involving Pavlovian conditioning, morphine is considered to serve as an unconditioned stimulus. Tolerance is viewed as a conditioned response that is discriminated by an environmental cue accompanying drug administration (Siege1 et al., 1978; Tiffany and Baker, 1981). According to Kensner and Baker (1981), the development of Pavlovian tolerance would be mediated by a negative endogenous opiate feedback circuit that reduces levels of endogenous opiates when organisms are exposed to stimuli that have previously been paired with the exogenous opiate. The second process involves drug habituation in which morphine functions as a conditioned stimulus. Drug habituation tolerance would be a function of iterative

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drug exposure in the absence of unambiguous, salient environmental cues, would develop with massed rather that with spaced drug administration, and would be delayed spontaneously after a decrease of opiate levels.

G. SOCIALBEHAVIOR In recent years some studies have pointed out brain opioid system involvement in the modulation of some motivational mechanisms underlying social emotion and social attachments. This line of evidence stemmed from an approach to the study of brain mechanisms that sustain social motives that emphasized the analogy between social bond formation and narcotic addiction and suggested that the comfort of social contact may be partially controlled by endogenous opioid systems (for a review see Panksepp et al., 1980a).

1. Social Attachment and Interactions The evidence concerning the role of endogenous opioid systems in social attachment arose primarily from studies using two experimental approaches, that is, the study of separation distress and the study of approach attachments. Panksepp et al. (1978a,b) reported that morphine reduced distress vocalization (DV), which young animals exhibit when they are separated from their normal social environments. These effects of morphine have been observed in puppies (Panksepp et al., 1978a), guinea pigs (Herman and Panksepp, 1978), and chicks (Panksepp et al., 1978b). The effects of morphine were reversed by naloxone (Panksepp et al., 1980a). Moreover, naloxone increased DVs in guinea pigs (Herman and Panksepp, 1978) and chicks (Vilberg et al., 1977; Panksepp et al., 1980b). T h e major opioid peptides such as P-endorphin, a-endorphin, y-endorphin, and Met-enkephalin administered intracerebroventricularly in chicks were also shown to reduce DVs as effectively as morphine. Among these peptides, @endorphin exhibits a higher potency than morphine (Panksepp et d,1980a). Panksepp et al. (1980a) found that DVs can be produced by electrical stimulation of brain areas surrounding the anterior commissure, the dorsomedial thalamus, the tissue surrounding the mesencephalic periventricular gray area, and some sites in the central amygdala and dorsomedial hypothalamus. All these brain areas contain endogenous opioid systems. Distress vocalizations induced by electrical brain stimulation (EBS) can be reduced by morphine administration (10-20 mglkg) and enhanced by naloxone (1 mg/kg) (Panksepp et al., 1980a). Taken

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together, these studies indicate that distress vocalization is mediated by neural systems that are confluent with identified opioid systems and confirm pharmacological results concerning the role of endogenous opioids in the control of the distress vocalization process (see Panksepp et al., 1980a). In addition to separation distress, some other behaviors such as the tendency of animals to maintain proximity, maternal behavior, and play have been shown to be influenced by endogenous opioid systems. Low doses of morphine (1 mg/kg) were found to decrease the tendency of both socially housed guinea pigs and rats to spend time close to other members of their species (Herman and Panksepp, 1978; Panksepp el al., 1979). In a study carried out on rats (Plonsky and Freeman, 1982), methadone (1-4 mg/kg sc) was reported to decrease total time spent in contact, to increase the latency to initial contact, and to decrease aggressive grooming without affecting locomotor activity. Naltrexone and naloxone have been shown to increase grooming and grooming invitations in pairs of talapoin monkeys (Fabre-Nys et al., 1982). Panksepp et al. (1979) found that although naloxone has inconsistent effects on proximity measures it tended to increase social contacts. Moreover, naloxone has been reported to increase, whereas morphine decreased the capacity of young chickens to obtain comfort from social contact (Vilberg et ul., 1977). Some results indicate that naloxone disrupts pup retrieval in both mice and dogs (Vilberg et al., 1977; Panksepp et al., 1980a).Juvenile play in rats is decreased by naloxone and increased by morphine. Moreover, both morphine and naloxone have been shown to affect dominance relationships that evolve during play: Morphine increased and naloxone decreased dominance (Panksepp, 1979).These results seem to indicate a role of endogenous opioid systems in the organization and the development of social behavior. It is worth noting that some recent findings have demonstrated that endorphins are contained in the maternal milk and in placenta (Nakai et al., 1978; Houck et al., 1980; Hazum et al., 1981). Although the function of such chemical systems has not been clarified, the possible presence of opioids in milk and placenta may suggest that endogenous opioid systems are involved in infant developmental processes. 2. Social Deprivutaon Several studies have shown that endogenous opioids systems are involved in the behavioral and neurochemical effects of social isolation in rodents. Adler et al. (1975) found that in morphine withdrawal syndrome precipitated with naloxone, isolated rats showed a reduction of jumping and diarrhea in comparison to grouped rats, thus indicating

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that social factors (housing conditions) can affect the abstinence syndrome in morphine-dependent rats. Some reports have shown that body analgesic systems interact with social environment. Brief periods of isolation can increase pain responsivitiy in young rats, at the same time decreasing the analgesic effects of morphine (Panksepp, 1980). However, after a prolonged period of isolation mice and rats exhibited an increase of morphine responsitivity (Kostowski et al., 1977; De Feudis et al., 1978). S. Puglisi-Allegra (unpublished observations) has observed that isolated mice were less sensitive to the hyperalgesic effects of naloxone in the hot-plate test (jumping) in comparison to grouped mice. Some of these effects have been interpreted in terms of opiate receptor proliferation or supersensitivity caused by the absence of environmental sources of opioid stimulation (i.e., social stimuli) for a prolonged period of time (De Feudis et al., 1978; Panksepp et al., 1980a; Riley et al., 1980). It has been reported that social isolation decreased opiate receptor binding in the whole brain of rats (Schenk et al., 1982). The finding that individually housed rats consumed more morphine solution than grouped animals if given a choice between morphine solution and water seems to support the idea that social environment sustains a desirable level of endogenous opioid activity, whereas social isolation reduces endogenous opioid functioning in some brain structures (Alexander et al., 1978; Panksepp et al., 1980a). In adult laboratory animals it is well known that social isolation induces strong and compulsive inter- and/or intraspecific agression between males (Valzelli, 1981). A few studies have pointed to the involvement of endogenous opioid system in the control of some motivational mechanisms underlying the expression of aggressive behavior of individually housed rodents. Opiate antagonists naloxone (1- 1.5 mglkg) and naltrexone (2.5-5.0 mg/kg), administered systemically, have been shown to decrease agonistic behavior dramatically in DBA/2 isolated mice, although they increased the duration of some social activities such as sniff-body, sniff-nose, and following. Moreover, neither drug affected motor activity and self-grooming of paired mice (Puglisi-Allegra et al., 1982~). Antiaggression effects of naloxone in isolated outbred mice have also been reported by Rodgers and Hendrie (1983) and by Lynch et al. (1 983). Other results (Puglisi-Allegra, 1983) indicate that intracerebroventricular injection of naloxone, p-endorphin, morphine, and (DAla2,DLeu'))-enkephah (DADL) induced a decrease of aggressive behavior in isolated DBA/2 resident mice when a group-housed intruder was placed in the resident's cage (resident-intruder paradigm). Although naloxone induced an increase of social behaviors without affecting defense, locomotion, and stereotyped behaviors, P-endorphin, mor-

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phine, and DADL decreased social behaviors and increased defensive behaviors. Moreover, morphine and /3-endorphin decreased locomotor activity, whereas DADL increased it. Naloxone pretreatment reversed the effects of opioids. These results seem to indicate that opioid peptides act on different motivational factors that may be related to the expression of isolation-induced aggressive behavior depending on their affinity for different opiate binding sites. Naloxone injected into the amygdala, olfactory bulb, and olfactory tubercle decreased mouse killing in isolated killer rats (P. Mandel, personal communication), but when injected systemically it had no antiaggression effects on agonistic behavior of isolated rats interacting with an intruder opponent which had also received a high dose of naloxone (25 mg/kg ip) (Kodgers and Hendrie, 1982).

3. Shock-Induced Aggressive Behavior A few experiments have been carried out on another kind of aggressive behavior, that is, that induced by electric shock. Fanselow et al. (1980) found that naloxone pretreatment (3 mg/kg ip) enhanced shockinduced aggressive behavior (SIAB) in rats and that aggressive responses increased with shock intensity. Rodgers (1982) has reported that naloxone exerts a biphasic effect on shock-induced fighting in rats: Small doses (0.1 mg/kg) facilitated aggression and large doses (10 mg/kg) inhibited it. Unlike naloxone, diprenorphine (0.1-10 mg/kg) did not affect defensive fighting. Naloxone at low doses (0.025 and 0.05 mg/kg ip) potentiated SIAB in C57BW6 but not DBN2 mice without affecting pain sensitivity (Puglisi-Allegra and Oliverio, 198 1). These effects of naloxone on SIAB in C57BL/6 mice were potentiated in combination with low doses (0.25-0.50 mg/kg) of apomorphine (Puglisi-Allegra et al., 1982d).

4. Aggressive Behavior and Pain Responsiveness Several studies have shown the involvement of endogenous opioid systems in the control of pain sensitivity, in particular the analgesic effects of opiate and opioids (see Terenius, 1978), and that stress is a critical factor in the activation of opioid mechanisms (see Amir et al., 1980). There is recent evidence that social states and social conflict may have different effects on endogenous opioid mechanisms controlling pain sensitivity. Miczek et al. (1982), using the resident-intruder paradigm, found that mice exposed to repeated attacks by other mice showed a decrease in pain sensitivity, an effect reversed by opiate antagonists that was not observed in morphine-tolerant mice; moreover, mice repeatedly sub-

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jected to defeat were less sensitive to the analgesic effects of morphine than mice not subjected to defeat, indicating a cross-tolerance between morphine analgesia and defeat-induced analgesia. Miczek and his coworkers also found that mice of the CxBK strain, which respond weakly to morphine, displayed only a moderate analgesia following defeat. Rodgers and Hendrie ( 1983) found that agonistic experience resulted in a potent naloxone-reversible (10 mg/kg) analgesia in intruder mice, whereas in residents it produced a moderate hyperalgesic reaction that was very sensitive to naloxone antagonism (0.1 mg/kg). The same authors, however, found that in rats the experience of an agonistic encounter failed to produce analgesia in either resident or intruder animals (Rodgers and Hendrie, 1982). Agonistic behavior in rats has been also reported to prevent foot-shock analgesia (Rodgers and Deacon, 1981). Another kind of aggressive behavior, mouse killing by rats, was found to induce a naloxone-reversible decrease in pain sensitivity (Kromer and Dum, 1980).

5 . Sexual Behavior T h e theory that endogenous opioid systems are involved in the control of sexual behavior stems mainly from studies that have shown that naloxone increases sexual responsivity in some species, whereas opiate and opioids decrease it. Most of these studies have been carried out in male animals and dealt with copulatory behavior and some sexual reflexes such as erection, penile cup formation, and flips (Gessa et al., 1979; Sachs et al., 1981). The effects of naloxone on copulatory behavior in rats have been reported to be mediated by central catecholaminergic mechanisms (McIntosh et al., 1980). Naloxone-reversible postcopulatory hyperalgesia in female rats has been reported (Hendrie and Rodgers, 1982). These results raise the problem of the adaptive significance of sex differences in changes in pain sensitivity following copulatory experience.

VI. Environmental Effects

A. STRESSORS AND OPIATE PRODUCTION

A number of stressful and painful events produce an analgesic reaction (Amir et al., 1980; Bodnar et al., 1980). This phenomenon, defined as stress-induced analgesia, has been related to the psychological and physiological factors that activate endogenous pain control and opiate sys-

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tems. Electrical stimulation of the central gray area has been reported to elicit analgesia comparable to that produced by small doses of morphine (Reynolds, 1969; Akil and Mayer, 1972). It has also been suggested (Madden et al., 1977) that endogenous opiates are released in response to stress and inhibit pain by activating this midbrain system. It has been shown that electrical stimulation of the central gray, which activates centrifugal inhibitory systems, influences the perception of pain in animals. In particular Chance (1980) indicated that autoanalgesia may result from lesion-induced hyperemotionality or from other fear-inducing treatments. T h e antinociception resulting from these treatments was named autoanalgesia because it is behaviorally induced and results from neuronal activity of endogenously synthesized molecules. According to Chance (1980), although endorphins are involved as mediators of autoanalgesia, other neuronal mechanisms must be also involved. Grau et al. (1981) showed that exposure of rats to inescapable shocks produced sequentially an early naltrexone-insensitive and a late naltrexone-reversible analgesic reaction. Activation of the opiate system was necessary and sufficient to produce analgesia 24 hr later on exposure to a small amount of shock. Lewis et al. (1982) also stressed that adrenal medullary enkephalin-like peptides may mediate stress-induced analgesia. As indicated, other mechanisms are implicated in this antinociceptive effect. For example, shock-induced analgesia is markedly attenuated if the rats are shocked in pairs, which elicits fighting behavior (Williams and Eichelman, 1971). I n this regard it has been noticed that electric shock results in large increases in plasma levels of both P-endorphin and ACTH, reflecting their concomitant release from the pituitary (Guillemin et al., 1977); rats shocked in pairs exhibited lower plasma ACTH levels than those individually subjected to shock (Conner et al., 1971). I n order to account for these naloxone-insensitive effects, Bodnar et al. (1980) have collected data suggesting that the endorphins, enkephalins, and opiate receptors interact with the descending serotonergic bulbospinal system to mediate the analgesic responses to opiates and electrical stimulation, whereas some stressors act through the endorphin system, other stressors act through the latter nonopiate pain inhibitory mechanisms. Although acute exposure to a number of stressors results in transient analgesia, chronic exposure results in adaptation of the analgesic response (Bodnar et al., 1980; Grau et al., 1981). However, it has been demonstrated that naloxone-reversible stress-induced analgesia responds to classical conditioning (Oliverio and Castellano, 1982); therefore the endorphin system may be activated not only by stress but also by past experiences. Thus, a number of behavioral situations that do not occur in the presence of stress but are related to it may result in the

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overproduction of endorphins, which induce analgesia and may affect emotional behavior (Amir et al., 1980).

B. DIURNAL A N D CIRCADIAN RHYTHMS IN OPIATE PRODUCTION A number of reports indicate that in humans sensitivity to pain and a responsiveness to analgesia depend on the time of day (Davies, 1974). Furthermore, Frederickson et al. (1977) and Rosenfeld and Rice (1979) have noticed that baseline levels in mouse pain sensitivity showed significant day-night variations. Diurnal rhythms in the analgesic effect of morphine and of the opiate antagonist naloxone were also demonstrated (Frederickson et al., 1977). These findings were interpreted by suggesting that variations in nociception and in the power of analgesic drugs were related to different levels of endogenous opioids and that these levels are controlled by the light-dark cycle. This type of explanation is supported by a number of findings indicating the existence of circadian rhythms in the synthesis of various pituitary hormones (Halberg, 1969) and monoamines whose function and release are controlled by endogenous opioids (Henderson and Hughes, 1976; Loh et al., 1976a). T h e results of Frederickson et al. (1977) prove that there are diurnal variations in nociceptive mechanisms, but they do not indicate whether these mechanisms also present circadian fluctuations, that is, whether pain responsivity shows fluctuations in the absence of an external light synchronizer. Endogenous fluctuations of pain thresholds, an important adaptive mechanism, are strongly suggested by the existence of several other circadian rhythms associated with a number of relevant behavioral, neurophysiological, and neurochemical mechanisms in mammals (BorbCly et al., 1975; Oliverio et al., 1979; Rusak and Zucker, 1979). Recent findings provide evidence for a circadian rhythmicity of opiate analgesia and endorphin production assessed through stress-induced analgesia in mice. The effects of a 12-12 light-dark (L-D) cycle and of constant light (L-L) on nociceptive thresholds and morphineinduced analgesia were studied in two strains of mice. Under the L-D conditions, a diurnal rhythm was observed in the responsivity of mice to nociceptive stimuli and in the analgesic effect of morphine. Under the L-L schedule, clear patterns of daily rhythmicity were evident in both strains for both nociceptive thresholds and responsivity to morphine. Finally, under the L-L schedule the overall responsivity to pain and the antinociceptive effects of morphine were clearly increased in comparison to the pattern evident in the L-D condition. The administration of

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naloxone decreased the nociceptive thresholds, indicating an involvement of the endogenous opioid peptides (Oliverio et d.,1982a). Other data also indicate that a biological clock regulates the production of endogenous opioids: Diurnal and circadian variations in naloxone-reversible stress-induced analgesia were evident in mice subjected to the L-D and L-L condition (Puglisi-Allegra al., 1982b). Data indicate a circadian rhythm in the number of brain opiate-binding sites (Naber et al., 1981). These findings suggest that a number of emotional and social patterns modulated by endogenous opioids may present diurnal and circadian fluctuations, a fact that is relevant owing to their possible involvement in a number of mood disturbances such as depression, which fluctuates throughout the day (Scheving et al., 1974).

VII. Brain Opiates and Mental Illness

The discovery of endogenous opiates raised hopes that an understanding of how these chemicals work in the brain would provide solutions to mental illnesses such as schizophrenia and depression. Efforts to determine the significance of these opiates have been complicated by the awareness that there are a number of different endorphin systems (and other peptides) distributed in various brain areas. Thus although evidence suggests that endorphins may be involved in the etiology of schizophrenia or play a role in this disease and its treatment, the exact nature of this involvement remains to be determined. Both a lack of brain opiates or an excess of these chemicals have been postulated to cause mental illness; at the present time there is evidence for both views, although the theory that some endorphins act as “endogenous neuroleptics” (Verhoeven et al., 1979) and that a labile endorphin system is a requisite condition for the development of psychosis (Amir et al., 1981) seems to be more acceptable than the excess theory. Suggestions that opiates might be useful for treating mental illness predate the discovery of endorphins. Opium was sporadically used to treat depression for many decades; in addition a prophylactic and therapeutic effect of opiates on mental illness in addicts or ex-addicts have been proposed. In fact, a number of clinical studies on addicts seem to indicate that these patients had turned to drugs (morphine and heroin) as a self-medication to relieve their mental disturbances (Verebey et al., 1978). Attempts to wean addicts from opiates gave further support to the self-medication theory: A small but consistent proportion of weaned addicts developed

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psychotic reactions when the dosage was reduced beyond a certain point. The idea that opiates are useful in the treatment of mental illness was supported by experiments indicating that when P-endorphin was injected into the brains of rats it produced effects resembling those of neuroleptic agents used in the treatment of schizophrenia (Jacquet and Marks, 1976). de Wied and his associates (1978) have proposed that a deficiency of one of the endorphins, y endorphin, might underlie schizophrenia. Endorphin was tested in the laboratory on attention-dependent processes that have consistently been shown to differ in schizophrenic patients from those in normal people (Davis et al., 1980). It is difficult to replicate in animal models the dysfunction of attentional processes: One possible model consists of avoidance behavior and its extinction in the rat. The studies by de Wied et al. (1978) indicate that although P-endorphin and Met-enkephalin delayed extinction of avoidance behavior in the rat, yendorphin and desTyr-y-endorphin facilitated extinction, a property also shared by some neuroleptics. In agreement with these findings, in fish selected for high emotional behavior intracisternal injections of opiates blocked their freezing behavior, which is evident in a new environment (V. Csanyi, unpublished observations). Thus, endorphins result in modifications of emotional levels as assessed in different animal models. Opposite behavioral modifications have been reported to result from injecting naloxone (Jacquet and Marks 1976) in different animal species; the resulting antagonism of endogenous opiates increases distress vocalization in guinea pigs (Panksepp et al., 1980a), young chicks (Panksepp et al., 1980a,b), and young mice (D. Robinson, B. D’Udine, and A. Oliverio, unpublished). T h e observation suggesting that endorphins (or some of them) act as endogenous tranquilizers or neuropeptides are supported by some clinical data indicating that desTyr-y-endorphin is therapeutically successful in a fair proportion of cases (Verhoeven et al., 1979). However, it must be pointed out that other investigators have not found such encouraging results with desTyr-endorphin and that the opposite hypothesis, that is, that an excess of endorphins may cause mental illness has also been proposed (F. E. Bloom et al., 1976); experiments based on the injection of P-endorphin into the spinal fluid of rats indicate that endogenous opiates may also produce a condition of rigid catatonia similar to that seen in some schizophrenics. Because of these contrasting results, several investigators have attempted to define the causes of mental illness by measuring endorphin concentrations in the spinal fluid. Wahlstrom and Terenius (198 1) have separated endogenous opiates into two (as yet unidentified) fractions designated I and 11. Fraction I was increased in

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patients with depression but not in those who alternate depression with mania, whereas fraction I1 correlated positively with anxiety and suicidal tendencies in depressed patients. Other investigations have shown increased P-endorphin-like material in plasma and cerebrospinal fluid in schizophrenics (Domschke et al., 1979; Emrich et al., 1979). In order to account for this evidence that either a lack or an excess of opiates may be involved in psychotic behaviors, Wahlstrom and Terenius (1981) suggest that different endorphins participate in a variety of etiological processes and that an involvement of endogenous opiates in mental illness must be discussed in terms of either quantitative or regional differences, such as production of a fragment or a toxin with a special receptor profile. These authors hypothesize that endorphins in one patient may be released in high quantities, thus precipitating a catatonic attack, whereas another patient may have an increased production of endorphins in brain regions associated with hallucinations. Neurochemical studies suggest that opioid receptors are localized on dopamine neuron terminals (Hokfelt et al., 1980). Evidence suggests that disturbances in the mesolimbic dopaminergic system play a role in the pathogenesis of schizophrenia. One might speculate that an imbalance between endorphins and dopamine could be an etiologic factor in schizophrenia. Another possible link between endorphins and dopamine is represented by the stimulating effects of endorphins on prolactin secretion: neuroleptics that block dopamine receptors also stimulate prolactin release (Labrie et al., 1980). The possible relationships between endorphins and dopamine in the etiology of psychotic behaviors is in good agreement with the hypothesis that schizophrenia is linked to a labile endorphin system activated by stress. This theory, also supported by Amir et al. (198l), may account for the interaction between a genetically determined biological defect and a number of environmental stressors that results in overt schizophrenic behaviors. As previously indicated, endorphins have a central role in the defense response of the organism to stress; a number of stresses result in the overproduction of endorphins. If the endorphin system responds physiologically in the global defense against stress, behavioral adjustments to environmental stressors are possible; however, inborn or acquired defects in the endorphin system-a “labile” endorphin system-are conducive to psychosis. This theory takes into account the functional relationships between the endorphin and dopamine systems: Repeated overproduction of endorphins in response to recurrent stressors may result in the development of supersensitivity of dopamine receptors resulting in the dopaminergic malfunctioning that has been implicated in psychotic behaviors (Amir et al., 1981; Baxter and Melnechuck, 1982).

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HIPPOCAMPAL DAMAGE: EFFECTS ON DOPAMINERGIC SYSTEMS OF THE BASAL GANGLIA By Robert L. lsaacson

Center for Neurobehavioral Sciences

and Depaement of Psychology State University of

New York a t Binghomton

Binghamton, New York

I. Introduction ...................................................

11. Behavioral and Anatomical Changes: The Issue of Variability . . . . . . . . . .

The Basal Ganglia and the Hippocampal Formation . . . . . . . . . . . . . . . . . . Biochemical Changes in the Basal Ganglia after Hippocampal Lesions . . . Dopaminergic Intervention with Basal Ganglia Systems. . . . . . . . . . . . . . . . Hippocampal Lesions: Effects on Neuropeptide Actions . . . . . . . . . . . . . . . Dopaminergic Influences on Excessive Grooming . . . . . . . . . . . . . . . . . . . . Dopaminergic Stimulation, Neuropeptides, and Hippocampal Destruction: Their Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ....................................................

111. IV. V. VI. VII. VIII.

339 340 343 346 348 35 1 352 355 357 358

1. Introduction

As emphasized by Steward (1982) in the preceding volume of this series, the understanding of the behavioral effects of brain damage depends on the consideration of the entire ensemble of remaining brain circuitry that may contribute to the behaviors affected by a lesion. In his chapter Oswald correctly pointed out the difficulties in establishing the neural basis for any behavioral pattern and argued that the study of recovery from brain damage should begin with known anatomical alterations observed after the damage. His own research questions whether or not a particular structural change has significance for behavior. Although there is much to be said in support of the neural-to-behavioral strategy, the crucial point may not be whether research begins with morphology or behavior but rather the degree to which the structural changes are known and how well the behavioral changes after lesions have been studied. If both are equally well understood, it probably does not matter which aspect takes precedence, but when more is known about one than the other aspect, the researcher would probably be best advised to begin with that for which the best data are available. 339 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 25

Copyright D 1984 by Academic Press, In'.

All rights of reproduction in any form reserved. ISBN 0-12-966825-5

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Much of the recent work in my laboratory has been on the changes that occur after damage to the hippocampus. It has made use of wellestablished behavioral alterations found after such damage (e.g., see Isaacson, 1982, Chapter 5 ) . Our work also capitalizes on the fact that we know a great deal about the anatomy of hippocampal circuitry, including its projections to the basal ganglia. There is mounting evidence that it is at the interface between the limbic system and the basal ganglia areas that functional changes occur after hippocampal damage that have great significance for behavior. Therefore, in studying the behavioral changes that follow hippocampal damage, we are using an animal preparation well grounded at both the neural and behavioral levels. However, the interpretations of the behavioral changes that follow hippocampal destruction are diverse and little general agreement has been achieved among theorists. Elsewhere, I have argued that no understanding of hippocampal contributions to behavior can be achieved until the functional characteristics of all of the brain’s systems are determined (Isaacson, 1980a). The hope that we can attribute any particular mental or behavioral characteristics to the hippocampus is a vain attempt to achieve the impossible, an attempt to establish a new neurophrenology, as faulty as that promoted by Gall and Spurzheim. We must be content with far less: knowledge of how systems interact with each other and how they respond when disturbances occur in their normal operations. The answer to the question “What does the hippocampus (or basal ganglia) really do?” will only be answerable in terms of its interactions with other systems. If a useful, functional answer is ever produced, it will probably be expressed in terms or symbols of a neuroscience that has not yet been invented or even imagined. Regardless of the interpretations of behavioral alterations after hippocampal damage, it is possible to know and measure them, as well as to try to relate them to changes in remaining neural systems.

II. Behavioral and Anatomical Changes: The Issue of Variability

T o date, most of the behaviors studied in my laboratory after hippocampal damage are those that occur spontaneously and are not dependent on deprivation, training, rewards, or punishments. They do not involve learning or memory processes, at least as these terms are usually used. They are simply those behaviors naturally exhibited by rats placed into relatively large open fields under dim illumination. We felt that by

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34 1

using these spontaneous actions we could sidestep possible lesion-induced effects on responses to deprivation, rewards or reward frequency, or training. It is possible that damage to the brain, especially the limbic system, would have unusual effects on these variables, which can influence performance in many types of tasks. The particular open field we have used has 16 equally spaced holes in the floor into which the animals can poke their heads if they so choose. Our measurements include the recording of activity (locomotion), hole pokes, grooming, and rearing. Both the number of times each behavior is undertaken and their total duration are then recorded. All of these behaviors are influenced by hippocampal destruction, but the most obvious effect is on locomotion. Animals with hippocampal damage usually are two to three times more active than control animals with lesions restricted to the neocortical surface or with sham operations. The other behavioral effects are less dramatic, although usually statistically significant. It should be noted, however, that not all of the lesioned animals exhibit every one of the behavioral changes. As pointed out by Thomas (1971), the effects of lesions of the fornix or hippocampal lesions on behavior are not always the same. This and related results suggest that the effects of lesions can best be described on a probabilistic basis. Differences among animals are not explicable on the basis of differences in the magnitude of lesions, at least as far as can be determined. For example, in examining the behavior of 18 animals with extensive bilateral hippocampal lesions tested in a study (Hannigan et al., 1983) utilizing our open-field, 16-hole apparatus, we found that 85% of the rats exhibited hyperactivity, 72% showed reduced rearing, and 78% demonstrated reduced grooming, but only 58% exhibited enhanced hole-poking. Seventy-two percent of the animals with hippocampal lesions exhibited three or four of the exaggerated symptoms. None of the sham-operated controls exhibited three o r four of the symptoms. Therefore, although the behavioral effects of hippocampal damage are well known and have been found in a variety of laboratories, the fact remains that after comparable central nervous system damage, identical results are not always found. This probably can be interpreted best on the basis of differences among animals existing before o r after the induction of brain damage that are genetic and experiential in nature. The effect of such variables on the behavioral consequences of brain damage have been studied extensively after septa1 area destruction by Donovick and his associates (for a review see Donovick et al., 1981). I believe that the views of Donovick and his co-workers are unpopular because they lead to the rejection of the notion that there should be a

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uniform, constant effect of any particular lesion. Rather, they suggest that lesions disturb collections of neural systems that have been “individualized” by genetic and environmental conditions existing before surgery and in the postoperative period. Schwegler and Lipp (1981) have started to relate differences in microstructure to behavior. They found that rodent strains that exhibited poor avoidance scores also had more mossy fiber terminals making synaptic contacts in o r below pyramidal neurons in the hippocampus. This analysis was extended to the avoidance performances of individual mice. T h e greater the number of mossy fiber synapses, the poorer was the avoidance performance (Schwegler et al., 1981). Thus, the Platonic notion that there should be an ideal arrangement of brain microstructure or prototypic effect of any lesion may have to be abandoned in favor of an anticipated variability of lesion effects based on anticipated variability in the structure and function of the nervous system. The plasticity of neural connections demonstrable after direct nervous system intervention and after environmental treatments has been interpreted as extreme instances of normal changes occurring constantly in the nervous system (Cotman and Nieto-Sampedro, 1982). T h e acceptance of the notion of a continually changing nervous system has important implications. For one, it provides a theoretical basis for understanding how differences in genetic instructions or experiences can lead to variable behavioral expression of “similar” damage in different animals. Taken a step further, it leads us to look at what apparently “similar” anatomic damage means. Because behavior is readily quantified and because relatively large numbers of animals are frequently used in the studies of lesion effects, any variability in the postlesion behavior can be more or less readily detected. Variability in anatomical projections is not easily determined, however, because most studies are descriptive and not quantitative. Experimental neuroanatomy is a high-technology, labor-intense field of research that often strives to establish patterns of connections and interconnections among neural systems. Comparisons are made among animals with similar lesions or injections but only to ensure that the same pattern of results can be obtained in several animals, often using somewhat different approaches or techniques. This is usually enough to establish the overall pattern of neural connections, but it is not enough to establish the limits of normal variability or to determine genetic and experiential correlates of the variations that may exist. Therefore, although the large-scale morphologic patterns of neural structures seem relatively consistent from brain to brain within a species, neuropathologists frequently point out large differences in “normal” human brains.

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Even so, we know little about individual differences among brains and much less about the variability of micromorphology of normal brains. T o properly understand the effects of lesions on brain function, the dynamic microstructure on which the lesion is imposed must be taken into consideration. It is likely that both the behavioral consequences of lesions and the macro- and microstructural changes will have to be explained on probabilistic rather than absolute terms. I n the research to be reported in this chapter our strategy has been to search both for localized physiological changes and for behavioral changes after hippocampal damage, giving neither aspect any particular preference. We have also gone beyond these correlational measures to test hypotheses about the specific nature of the changes through direct interventions with remaining brain systems. To foreshadow what will come later, we have been following the possibility that at least some of the behavioral changes that follow hippocampal damage may be due in part to lesion-induced changes in the basal ganglia (Reinstein et al., 1982; Hannigan et al., 1983). This was based in part on evidence of altered membrane protein phosphorylation (Bar et al., 198 l), timedependent alterations in catecholamine levels and utilization (Springer and Isaacson, 1982), and perhaps dopamine receptor differentiation (Reinstein, 1980) in basal ganglia regions after hippocampal destruction. Evidence that these secondary neural changes may contribute to behavioral changes after hippocampectomy is indicated by the time course of the behavioral changes over the postoperative period and by the restoration of more normal behavior in rats by direct pharmacologic intervention in the basal ganglia (Hannigan et al., 1981; Reinstein et al., 1982).

111. The Basal Ganglia and the Hippocampal Formation

There were several lines of evidence that led us to the opinion that the basal ganglia is a place likely to exhibit important changes after hippocampal destruction. One of these reasons is that some of the behavioral effects of hippocampal destruction are similar to those seen after manipulation of the mesolimbic dopamine system. For example, ventral tegmental area (VTA) lesions produce hyperactivity, difficulties in response suppression, and reductions in “freezing” (Le Moal et al., 1975) much like those seen in animals with hippocampal lesions. It should be noted that there are reasons to believe that the behavioral effects of VTA lesions are due to a reduction of dopamine (DA) activity

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in the nucleus accumbens (Tassin et al., 1978). The injection of dopamine into n, accumbens results in the suppression of locomotor activity in normal animals (Wachtel et al., 1979). On the basis of its development, cytoarchitecture, neurochemical characteristics, and neuronal connections, the n. accumbens is clearly a part of the basal ganglia systems. Despite its early association with the olfactory system, [e.g., Herrick (1926)l it has few connections with the olfactory systems. Indeed, strong arguments can be made for including the olfactory tubercle itself as a component of the striatal complex (Heimer and Wilson, 1975; Heimer et al., 1982). The n. accumbens and the tubercle can be considered together as the ventral striatum. Perhaps the most ventral and medial portions of the caudate-putamen (basal ganglia) of the rat should be thought of as ventral striatum as well (Heimer et al., 1982). Efferents from n. accumbens and the tubercle project monosynaptically to the dopamine-containing cells of the brainstem, including cells in substantia nigra that project to the main body of the caudate nucleus (Somogyi et al., 1981). In addition, the ventral pallidus receives afferents from the ventral striatum in manner analogous to the globus pallidus’ receipt of fibers from caudate-putamen. The main portion of the globus pallidus projects to the ventral anterior and ventral lateral nuclei of the thalamus and these nuclei, in turn, project largely to premotor and motor areas of neocortex. The projection pattern of the ventral pallidus, however, is largely to the mediodorsal nucleus of the thalamus. This region projects to prefrontal, prelimbic, and anterior cingulate cortical regions. Although there are a number of anatomical pathways connecting the limbic and basal ganglia systems (e.g., Nauta and Domesick, 1981), the behavioral changes we have been studying are likely to be related to the monosynaptic connections from the ventral subicular regions (Swanson and Cowan, 1977; Swanson, 1981).These connections are unidirectional and largely ipsilateral. Similar connections to the caudate have not been reported. The ventral striatum and portions of the ventral and medial caudate nucleus receive afferents from the ventral tegmental area (Beckstead, 1979). Even though not all of the cells in substantia nigra and the ventral tegmental area contain dopamine, this suggests that earlier categorical assumptions of substantia nigra dopaminergic projections to the caudate and ventral tegmental dopaminergic projections to ventral striatum were too simple. For example, Fallon and Moore (1978) found that within the substantia nigra dorsally located cells project to the ventral striatum in a fashion similar to those of the ventral tegmental area

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(VTA). T h e more medial and ventral cells of substantia nigra (SN) project to the caudate-putamen. Fallon (1981) has also found that the dorsal substantia nigra cells and the VTA cells exhibit much less collateralization to other brain regions than do cells in the medial and ventral substantia nigra. Furthermore, Fass and Butcher (1981) have shown that the cells in both SN and VTA project in a limited degree to structures on the contralateral side of the brain in addition to their predominantly ipsilateral distribution. Understanding the nature of hippocampal involvement with dopaminergic mechanisms in the basal ganglia requires an appreciation of the neuropharmacology of these systems. This appreciation must in turn include knowledge of the actions of dopamine (DA) and dopaminergic agonists on receptors in the basal ganglia. Unfortunately, the types and distributions of DA receptors are far from clear at this time (for a review see Creese et al., 1982). We have found that the proposals made by Cools and Van Rossum are useful in interpreting the interactions among the ascending DA systems. On the basis of electrophysiological, pharmacologic, and behavioral data, Cools and Van Rossum (1976, 1980) have argued for two subsystems of functionally distinct dopamine receptors in the basal ganglia. Receptors mediating excitatory (DAe) responses are located primarily in the caudate nucleus, whereas others mediating inhibitory reactions (DAi) are found in greatest concentration in the nucleus accumbens. In addition, the DAe and DAi subsystems can be distinguished on the basis of their selective responsiveness to pharmacological manipulations. For example, apomorphine and 3,4dihydroxyphenylamino-2-imidazoline(DPI) are agonists for the DAe and DAi subsystems, respectively, whereas haloperidol and ergometrine are the presumed respective antagonists for DAe and DAi receptors. Another line of evidence we thought suggested that the hippocampal lesions could be exerting effects on DA systems of the basal ganglia arose from pharmacologic investigations of the behavior of lesioned animals. Animals with hippocampal lesions have an altered responsivity to drugs with predominantly dopaminergic effects. These include a diminished responsiveness to d-amphetamine and an exaggerated response to haloperidol. In one study rats with hippocampal lesions gave less response suppression in an operant task than controls after d-amphetamine (Woodruff and Isaacson, 1977). In addition L. Spear and R. Isaacson (unpublished observations) were not able to show an effect of hippocampal damage on amphetamine hyperresponsiveness in an open field. Similar results were also found by Woodruff (1981)in a somewhat different testing situation. However, hippocampally lesioned animals showed dramatic reductions in response rates in operant situations after systemic

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haloperidol. The sensitivity of the animals with hippocampal lesions to dopaminergic manipulations is selective to dopaminergic systems; systemic alteration of either norepinephrine or serotonin does not produce such effects (see Isaacson, 1980b). On the basis of all of these considerations, we undertook to test the hypothesis that hippocampal destruction results in alterations of the forebrain dopaminergic systems, probably in the nucleus accumbens. This being the case, we thought that the effect of the hippocampal lesion would likely be on the DAi receptor systems. We believed that the majority of available data supported the view that the hippocampally lesioned animal could be characterized as one with either an overresponsive DAe system or less responsive DAi system. Whether or not these fine-grain speculations are correct, the hippocampal lesion at least ought to induce measurable changes in physiologic activities of the basal ganglia. Some of our efforts to test these ideas involved measuring the biochemical changes occurring in the basal ganglia after hippocampal destruction.

IV. Biochemical Changes in the Basal Ganglia after Hippocampal lesions

In an early attempt to survey the effects of hippocampal destruction in a large number of brain areas, we evaluated changes in the uptake of [SH]2-deoxy-~-glucose in lesioned animals relative to controls (Reinstein et al., 1979). Only a few brain regions showed statistically significant reductions in specific activity, including the olfactory bulb and the hypothalamus. However, these determinations were made only 30 days after the lesions and had all of the drawbacks of the 2-DGmethodology, particularly the fact that uptake is measured only during the injectionsacrifice period. During this time the animals may be responding to the stress of the handling and injection procedures, and it is possible that the lesioned animals may exhibit unusual behavioral and physiological stress reactions. Therefore, we undertook to determine the effect of hippocampal and neocortical destruction on the endogenous phosphorylation of membrane proteins. This process does not depend on the detection of perturbations in neuronal activity occurring at the time biochemical measurements are made. In this study we determined the effects of hippocampal damage on membrane phosphorylation in nucleus accumbens, caudate, hypothalamus, and dorsal frontal cortex in the rat at 7 and 28 days after surgery (Bar et al., 1981). Animals with either neocortical or hippocampal damage had an increase in in vitro phosphorylation in nucleus accumbens 28

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days after surgery in the 43,000, 48,000, 50,000, and 82,000 MW protein bands. However, at 7 days after surgery, no effect was found in nucleus accumbens after either neocortical o r hippocampal damage. As early as 8 days after surgery, animals with hippocampal lesions showed enhanced phosphorylation in the 48,000 and 50,000 MW protein bands in caudate tissue. By 28 days after surgery, the animals with hippocampal damage evidenced an elevated in vitro phosphorylation of the protein I band (molecular weight 82,000). This is thought to be a DA-sensitive, CAMP-related protein kinase, which is a sensitive indicator of altered synaptic activities (Greengard, 1979).At 28 days after surgery, the frontal cortex also had enhanced phosphorylation of proteins in both the 48,000 and 82,000 MW protein bands. This finding may be of special interest to those who have investigated similarities between animals with corticofrontal and hippocampal lesions. A direct projection from area CAI of the hippocampus to medial anterior frontal cortex in the rat has been reported (Swanson, 1981). It should be noted that in the procedures used by Bar et al. it is impossible to distinguish between an enhanced activity of the protein kinase as measured in the post hoc assay or an enhanced activity in vivo of the corresponding phosphoprotein phosphatase. The latter would result in a relatively dephosphorylated state of the substrate protein before sacrifice. This would provide the protein kinase with more phosphorylatable sites on the protein under optimal kinase conditions in vitro. Nevertheless, this study produced evidence that hippocampal damage results in selectively altered transmission in the caudate 8 days after damage. This effect would not be anticipated on the basis of known, direct synaptic connections. By 28 days after surgery, this caudate effect is selective for the protein I band. We have found that hippocampal lesions result in alterations of DA and DA metabolite content in the caudate and accumbens after hippocampal damage. Springer and Isaacson (1982) examined DA, DOPAC, and HVA levels in the caudate and nucleus accumbens. Increases in DA content were found in the nucleus accumbens that correlated with a decrease in apparent utilization (ratio of metabolites to DA) 7 days after surgery. This is shown in Fig. 1. As can be seen, this was due primarily to the lesion effect on DOPAC. Metabolite levels were also changed in the caudate at this time. However, in this area the effect was largely due to changes in HVA. By 28 days after surgery the biochemical parameters had returned to control levels. These results suggest that the hippocampal formation can influence DA activities in nucleus accumbens and are compatible with a presumed decrease in DA release shortly after the lesion. We are testing this hypothesis more directly at the present time. If this assumption is correct,

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f

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c 0

K L

1

\ 0

50

S

C

H

S

C

H

I .O

B

50

0.5

c

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FIG. 1. The amounts of dopamine (a), DOPAC (b), and HVA (c) found in nucleus accumbens after the sham operative procedure (S), neocortical lesions (C), or hippocampal lesions (H). Determinations were made either 7 (open bars) or 28 days (hatched bars) after surgery as indicated in the figure. (d) shows utilization ratio of dopamine : DOPAC + HVA/Dopamine. * indicates difference significant beyond p < .05; **, difference significant beyond p < .01. From Springer and Isaacson (1982).

we would expect that a reduced release could lead to an increase in DA receptors in the basal ganglia as part of an effort to maintain overall dopaminergic activities at a constant level (i.e., a compensatory model of neuronal activities). In accordance with this idea Reinstein (1980) found preliminary evidence for changes in apparent binding of [3H]haloperido1 in nucleus accumbens that increased in magnitude with longer recovery periods. Alterations in apparent binding characteristics were seen in nucleus accumbens at 7 days after the lesion. A similar change in haloperidol binding was found only at 28 days after hippocampal lesions in the caudate. V. Dopaminergic Intervention with Basal Ganglia Systems

Given the changes found in DA activities of the basal ganglia, the important question is, “Do these changes have significance for behav-

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v)

W

I-

dW

n

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CORTl C AL LESION

FIG.2. Relative amounts of locomotor activity in rats with hippocampal or neocortical lesions that received either saline (open bars) or 10 p g or DPI (hatched bars) into nucleus accumbens. Summary of results presented in Reinstein et al. (1982).

ior?” To try to answer this question, we undertook several studies in which direct application of a dopaminergic agonist was made into nucleus accumbens in animals with hippocampal lesions. The choice of the dopaminergic agent was based on the evidence of Cools and Van Rossum (1980) on the two forms of dopaminergic receptors noted above. As mentioned in Section 111, our view was that the lesion either enhanced DAe activity, reduced DAi activity, or both. In principle the direct application of a dopamine agonist (DPI) selective for the inhibitory system found predominantly in nucleus accumbens should compensate for hippocam pal damage. Accordingly, animals were prepared with bilateral cannulae placed into nucleus accumbens at the same time that bilateral hippocampal lesions, bilateral neocortical lesions, or sham operations were made. At one of three postoperative intervals independent groups of animals were tested to determine if the injection of the DPI would compensate for the loss of the hippocampus. In these studies we have concentrated our attention on the behavioral tasks that require no training, deprivation, or elaborate testing procedures, as discussed earlier. Our results indicated that locomotor hyperactivity in rats with hippocampal lesions could be restored to nearly normal levels by the unilateral administration of DPI into nucleus accumbens (Reinstein et al., 1982). A summary of this effect is shown in Fig. 2. Although the lesion-produced enhancement of locomotion was reduced by the administration of DPI, the effect is not due to a general depressive effect, because grooming, which is usually low in the lesioned

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animals, is increased to normal levels by the drug application. This is shown in Fig. 3. Cools and Van Rossum (1980) also proposed that normal behaviors depend on a balance between excitatory and inhibitory dopaminergic activities in the basal ganglia. In a study of ACTH-induced grooming in the rat Cools (1978)was able to show that similar behavioral deficits can be produced by either the enhancement of inhibitory activities or the reduction of excitatory activities in the dopaminergic subsystems. According to this view, a reduction of excitatory activity in the caudate would produce an effect equivalent to the enhancement of inhibitory activities in the nucleus accumbens. T o test this hypothesis animals were prepared with cannulae placed into nucleus accumbens or into the caudate at the same time that hippocampal, cortical, or sham operations were performed (Hannigan et al., 1983). Soon after the lesions, DPI was injected into the nucleus accumhens region or haloperidol was injected into the caudate nucleus, each in an ascending series of doses. The hypothesis was that there ought to be some dose of haloperidol that when placed into the caudate would mimic the effects of DPI in nucleus accumbens. As in the earlier Reinstein et al. (1982) study, the DPI injections into n. accumbens reduced the hyperactivity usually found after hippocampal damage. In this case the drug doses were smaller than those used before (i-e., 1-5 pg). However, haloperidol injected into the caudate did

W

w

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FIG. 3. The relative amounts of novelty-induced excessive grooming in rats found after hippocampal or neocortical lesions in animals receiving saline (open bars) or 10 pg DPI injections (hatched bars) into nucleus accumbens. Summary of results reported in Reinstein et al. (1982).

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not produce a similar effect. Although there could be several reasons for our failure to find behavioral reciprocity between haloperidol administration into the caudate and DPI into n. accumbens, it may be that the balance theory of Cools and Van Rossum will not be applicable to locomotor activity.

VI. Hippocampal lesions: Effects on Neuropeptide Actions

Another approach to the study of the effects of hippocampal lesions as they affect the forebrain DA systems comes from investigations of the induction of excessive grooming. Gispen et al. (1975) were the first to systematically examine the excessive grooming that follows the intercerebroventricular (icv) administration of ACTH or certain of its longer fragments. They developed a procedure in which permanent cannulae are placed into the interventricular foramen several days before testing. On the test day the neuropeptide to be studied, or its control vehicle, is injected into the foramen a few minutes before the animal is placed into a small observation chamber. The animal’s grooming behaviors are scored every 15 sec for the next hour or so. (The observation period has varied between 50 and 65 min in different studies.) T h e maximum grooming score that can be obtained is four times the number of minutes the animal is observed. With administration of an effective peptide fragment (e.g., 1 p g of ACTH,-24), almost incessant grooming is produced that lasts well over the next hour or so. T h e effect of the ACTH fragments is to extend the duration of the individual grooming episodes rather than to increase the number of times grooming is begun. It is of interest that icv P-endorphin, another neuropeptide, will induce excessive grooming in the opposite manner, increasing the number of bouts but not influencing their duration (Gispen and Isaacson, 1981). T h e effectiveness of the peptide is independent of the steroidogenic properties of the fragment, the integrity of the hypophysis or the adrenals, the environmental conditions at testing, or the motivational state of the animal (Colbern et al., 1978; Jolles et al., 1979; Gispen and Isaacson, 1981). Rats with near total hippocampal lesions show greatly reduced excessive grooming after icv ACTH1-24(Colbern et al., 1977). However, this is not due to an impaired ability to make the grooming response. T h e frequency of grooming bouts initiated by uninjected hippocampal rats in an open field is equivalent to control levels, although the duration of each bout is less than it is in controls (Reinstein et al., 1982). Elstein et al.

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(1981) have shown that the hippocampally lesioned rat is less sensitive to ACTH1-24 but that high levels of excessive grooming can be induced with high doses of ACTH. There is other evidence that the effect of hippocampal destruction on ACTH-induced grooming is mediated through an interaction with DA-related systems. Isaacson and Colbern (198 1) studied animals with hippocampal lesions in which haloperidol was given systemically just before icv administration of 2 p g ACTHL-Z4.Haloperidol reduced the grooming in hippocampally lesioned animals at doses lower than those required to diminish the excessive grooming in control animals. This is in line with other data indicating that such animals are more sensitive to haloperidol treatment than normal or control lesioned rats.

VII. Dopaminergic Influences on Excessive Grooming

I n intact animals the excessive grooming induced by neuropeptides or by certain stressors has been linked to activities in the ascending dopaminergic systems of the brain. For example, lesions of the substantia nigra reduce excessive grooming and injections of ACTH into this area can induce it (see Gispen and Isaacson, 1981). Furthermore, in rats systemic treatment with haloperidol or the long-acting dopamine antagonist flufenazine reduces the excessive grooming found after icv ACTH (Wiegant et al., 1977) and excessive grooming found in novel environments (Green et al., 1979). T h e microinjection of haloperidol into the caudate suppresses ACTH-induced grooming (Wiegant et al., 1977), and other dopaminergic drugs injected into n. accumbens can produce the same effect (Cools et al., 1978). Guild and Dunn (1982) found that the systemic administration of a variety of dopaminergic agents can influence ACTH-induced excessive grooming in the mouse, Our knowledge of the effects of dopaminergic systems on excessive grooming has been advanced by a study of the time course of excessive grooming as it is influenced by various pharmacologic treatments. A complete analysis of the time course of excessive grooming after icv ACTH has been made (Isaacson et al., 1983). From the variety of data taken from different studies it is clear that there are at least two phases of neuropeptide-induced response. The grooming exhibited in the first 30 min or so after the neuropeptide administration is not reduced by systemic administration of either naloxone o r neuroleptics. The excessive grooming exhibited later in the observation is reduced by these drugs. An example is shown in Fig. 4.Animals in this experiment were

HIPPOCAMPAL DAMAGE: EFFECTS ON DOPAMINERGIC SYSTEMS

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FIG. 4. The time course of excessive grooming after icv administration of 1.0 pg ACTH1-24in animals pretreated with 0.10 mg/kg haloperidol (0)(10 min before) or 0.5 mg/kg flufenazine (0)(24 hr before). 0 , Saline or oil vehicle. Data from Isaacson et al. (1983).

given the long-acting DA antagonist flufenazine 24 hr before the icv administration of ACTH. As can be seen in this figure, the reduction in grooming is confined to the last portion of the observation period. Similar effects are produced by pretreatment with haloperidol or naloxone. Direct evidence for a dopamine-basal ganglia connection with neuropeptide-induced grooming has recently been produced. Springer et al. (1982) found that animals with 6-hydroxydopamine (6-OHDA)

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lesions in n. accumbens that were pretreated with desmethylimipramine exhibited less excessive grooming after icv ACTH than intact animals. Furthermore, the reduction in grooming occurred only in the last half of the observation period, that phase of grooming that is reduced by pretreatment with dopamine antagonists. Because lesions of the dopaminergic contributions to n. accumbens will reduce ACTH-induced grooming when it is placed into the ventricular system, we thought that we should reexamine the possibility that intraaccumbens injections of ACTH could induce excessive grooming. There had been comments in the literature to the effect that injections into the caudate (Wiegant et al., 1977) or accumbens (Cools et al., 1978) did not induce excessive grooming. However, no details of the actual experiment with n. accumbens were provided. Therefore, Ryan and Isaacson (1983) prepared animals with bilateral cannulae placed into n. accumbens or with a single cannula placed into the interventricular foraover men. The animals were tested with increasing doses of ACTH days. The results indicate that 50 ng or 80 ng doses of this ACTH 140-

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fragment placed into n. accumbens will induce excessive grooming but that similar amounts would not d o so when given intraventricularly (see Fig. 5). On the basis of this evidence and subsequent replications of the basic experiment, we believe n. accumbens represents another location (besides substantia nigra) in which ACTH fragments can induce excessive grooming. However, we also have reason to believe that it may not be the site at which icv ACTH produces its effects.

V111. Dopaminergic Stimulation, Neuropeptides,

and Hippocampal Destruction: Their Interactions

In the research of Reinstein et al. (1982) we found that DPI injected into n. accumbens could restore the excessive grooming usually found when rats are handled, transported, and placed into novel environments. A natural question that follows this result is whether or not the DPI treatment in ventral striatum could restore the sensitivity of hippocampally lesioned animals to icv ACTH 1-24. T o answer this question, we prepared lesioned and control rats with bilateral cannulae in n. accumbens and a single intraventricular cannula. Animals were tested at 7 or 28 days postoperatively. T h e results indicate that at 7 days after surgery the DPI treatment was without effect on the ACTH fragment-induced grooming. However, by 28 days after surgery, the DPI pretreatment increased the induced excessive grooming to levels similar to that exhibited by control animals. At least at this longer postoperative recovery period, the DPI administration into n. accumbens restores excessive grooming that results from neuropeptide administration and as an aftereffect to novelty, transport, and handling. As best as can be determined, 7-8 days after hippocampal damage there is less DA release in the accumbens, and the major changes in membrane responsiveness are common to both animals with neocortical and hippocampal damage. Because our method of destroying hippocampal tissue requires neocortical damage, the membrane effect could be entirely due to the cortical lesions. It is the time, however, at which DA receptor changes may just be beginning, although the evidence is admittedly inadequate as yet. By 28 days after hippocampal lesions the early biochemical changes in n. accumbens are no longer present, although the application of DPI is very effective in reducing the behavioral effects of the lesion. This may mean that the long-term consequences of hippocampal damage are in changes in receptor number or their responsiveness. These changes could, in fact, be responsible for the

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return of the initial biochemical abnormalities to within normal limits, although leaving an altered functional system. With the discovery that intra-accumbens administration of ACTH could elicit excessive grooming (Ryan and Isaacson, 1983)we decided to determine if hippocampal lesions would also reduce sensitivity to this method of inducing the behavior. Animals with hippocampal and control lesions were implanted with cannulae in n. accumbens. Adrenocorticotrophic hormonel-24was then administered to the animals in this fashion and compared with animals receiving the ACTH fragment in the ventricular system (Hannigan et al., 1983). A summary of the results is presented in Fig. 6. Not only was the sensitivity to intra-accumbens ACTH of the animals with hippocampal lesions not reduced, but an enhancement of about 30% was obtained. The lesioned animals with icv injections evidenced the usual reduction of excessive grooming. These results suggest that the excessive grooming produced by intraventricular and intra-accumbens injections of ACTH may influence different neural mechanisms to cause their behavioral effects. Certainly, they are differently affected by hippocampal destruction. Other differences between accumbens-initiated and icv-initiated grooming are now being studied (J. Ryan, unpublished). These include a greater resistance to the antagonism of excessive grooming by animals receiving ACTH in accumbens relative to the ventricular system. It also appears that intra-accumbens naloxone will block excessive grooming induced by icv ACTH. Therefore, the n. accumbens may be an effective site for naloxone in regard to excessive grooming but one that is reached less easily than

FIG.6. The effects of neocortical (open bars) or hippocampal (shaded bars) lesions on excessive grooming induced by ACTHI-?.,placed into either the interventricular foramen (icv) and nucleus accumbens (accumbens). Data from Hannigan et al. (1983).

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some other site(s) normally influenced by ACTH applied within the ventricular system.

IX. Summary

Although the research topics discussed in this chapter cover a substantial range of areas, methods, and behaviors, there is a consistent central issue: the alterations of brain and behavior subsequent to hippocampal lesions. T h e initial hypothesis that important secondary changes occur in the basal ganglia after hippocampal lesions has been sustained, at least in part, but other and unexpected results have been obtained that both clarify and mystify at the same time. The restoration of normal locomotion to animals with hippocampal damage by the application of a DAi agonist, DPI, to n. accumbens certainly supports our general idea, but the fact that membrane and transmitter changes in accumbens are transitory needs to be explained and perhaps related to alterations in DA receptor populations. T h e fact that intra-accumbens DPI can restore ACTH-induced excessive grooming at 28 days after hippocampal damage supports the idea that progressive changes are occurring in DA receptors in n. accumbens, but we need to know their nature more precisely. To do this, however, will probably require advances in our general knowledge of central DA receptors. One of the more interesting aspects of our observations is that the intra-accumbens administration of DPI restores normal behavioral patterns in several types of behaviors, especially locomotion and the excessive grooming response. These two behaviors are not closely linked in nature because large changes can occur in them independently, given appropriate conditions for testing. On the basis of available evidence both of these behaviors seem linked to activities in forebrain DA systems. This would further support the idea that the hippocampus modulates DA activities in basal ganglia systems. The results that suggest the presence of multiple sites for the central induction of grooming were also unexpected. As evidence mounts in support of this idea, it is likely that the one located in n. accumbens is more influenced by hippocampal destruction than the region(s) affected by icv neuropeptide administration. T h e interactions that take place in the basal ganglia can be extensive. Afferents from the entirety of the neocortical surface, the limbic system, and the projections from the brainstem DA cell groups meet in the striaturn and ventral striaturn. T h e efferents from this region not only

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reach motor cortical and subcortical sites but also project back onto diffusely projecting monoaminergic cells of the brainstem. Perhaps it is not too far-fetched to suggest that the efferents to these latter regions are at least as important as those that influence the motor systems. Perhaps the interactions that occur at the basal ganglia level regulate the activation provided to large numbers of systems through these brainstem cell groups. There has been a great deal of speculation and some evidence brought forward to support the idea that the dysfunctions of the DA striatal axis are causally related to many forms of mental and behavioral disorders. Our data indicate that lesions of the hippocampus, among other areas, can disturb this axis but offer the hope that restoration of normal behavior can be achieved through appropriate intervention procedures. References

Bar, P. R., Gispen, W. H., and Isaacson, R. L. (1981).Pharmacol. Biodem. Behav. 14, 305313. Beckstead, R. M. (1979).J. Comp.Neurol. 184, 43-62. Colbern, D. L.,Isaacson, R. L., Bohus, B., and Gispen, W. H. (1977).Lije Sci. 21,393-402. Colbern, D. L.,Isaacson, R. L., Green, E. J., and Gispen, W. H. (1978).Behau. BioZ. 23, 381-387. Cools, A. R. (1978).Life Sci. 23, 2475-2482. Cools, A. R., and Van Rossum, J. M. (1976).Psychopharmacologia 45, 243-254. Cools, A. R.,and Van Rossurn,J. M. (1980).Life Sci. 27, 1237-1253. Cools, A. R., Wiegant, V. M., and Gispen, W. H. (1978).Eur.1. Pharmacol. 50,265-268. Cotrnan, C. W., and Nieto-Sarnpedro, M. (1982).Annu. Rev. Psychol. 33, 371-401. Creese, I., Morrow, A. L., Leff, S. E., Sibley, D. R., and Hamblin, M. W. (1982).Int. Rev. Neurobiol. 23, 255-302. Donovick, P. J., Burright, R. G., Fanelli, R. J., and Engellenner, W. J. (1981).Physiol. Behav. 26,495-507. Elstein, K.,Hannigan, J. H., and Isaacson, R. L. (1981).Behau. Neural Biol. 32, 248-254. Fallon, J. H.(1981).J.Neurosci. 1, 1361-1368. Fallon, J. H.,and Moore, R. Y. (1978).J . Comp.Neurol. 180, 545-580. Fass, B., and Butcher, L. (1981).Neurosci. Lett. 22, 109-113. Gispen, W.H.,and Isaacson, R. L. (1981).Pharmacol. Ther. 12, 209-246. Gispen, W. H.,Wiegant, V. M., Greven, H. M., and de Wied, D. (1975).Lije Sci. 17,645652. Green, E.J., Isaacson, R. L., Dunn, A. J.. and Lanthorn, T. (1979).Behau. Biol. 27, 546551. Greengard, P. (1979).‘Cyclic Nucleotides, Phosphorylated Proteins, and Neuronal Function.” Raven, New York. Guild, A. L., and Dunn, A. J. (1982).Pkmulcol. Biochem. Behau. 17, 31-36. Hannigan, J. H., Springer, J. E., and Isaacson, R. L. (1981).Neurosci. Abstr. 7, 888. Hannigan, J. H.,Springer, J. E., and Isaacson, R. L. (1984).Brain Res. (in press). Heirner, L., and Wilson, R. D. (1975).In “Golgi Centennial Symposium Proceedings” (M. Santini, ed.), pp. 177-193. Raven, New York.

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Heimer, L., Switzer, R. D., and Van Hoesen, G . W. (1982). Trends NeuroSci. 5, 83-87. Herrick, C. J. (1926). “Brains of Rats and Men.” Univ. of Chicago Press, Chicago, Illinois. Isaacson, R. L. (1980a). In “Neural Mechanisms of Goal Directed Behavior and Learning” (R. F. Thompson, L. H. Hicks, and V. B. Shvyrkov, eds.), pp. 409-423. Academic Press, New York. Isaacson, R. L. (1980b). Physiol. Psychol. 8, 183-188. Isaacson, R. L. (1982). “The Limbic System,” 2nd ed. Plenum, New York. Isaacson, R. L., and Colbern, D. (1981). Physiol. Psychol. 9, 260-262. Isaacson, R. L., Hannigan, J. H., Jr., Brakkee, J. H., and Gispen, W. H. (1983).Bruin Res. m i . 11,289-293. Jolles, J., Rompa-Barendregt, J., and Gispen, W. H. (1979). Horn. Behav. 12, 60-72. LeMoal, M., Galey, D., and Cardo, B. (1975). Brain Res. 18, 190-194. Nauta, W. J. H., and Domesick, V. B. (1981). In “Psychiatry and the Biology of the Human Brain: A Symposium Dedicated to Seyniour Kety” (S. Matthysse, ed.), pp. 00-00. ElseviedNorth Holland, Amsterdam. Reinstein, D. K. (1980). Ph.D. Dissertation, State Univ. New York, Binghamton. Reinstein, D. K., Isaacson, R. L., and Dunn, A. J. (1979). Brain Res. 175, 392-397. Reinstein, D. K., Hannigan, J. H., Jr., and Isaacson, R. L. (1982). Phamncol. Biochem. Behav. 17, 193-202. Ryan, J. P., and Isaacson, R. L. (1983). Physiol. Psychol. 11, 54-58. Schwegler, H., and Lipp, H. P. (1981). Neuiosci. Lett 23, 25-30. Schwegler, H., Lipp, H. P., Van der Loos, H., and Buselmair, W. (1981). Science (Washington, D.C.)214,817-819. Somogyi, P., Bolam, J. P., Totterdell, S., and Smith,A. D. (1981). BrainRes. 217,245-263. Springer, J. E., and Isaacson, R. L. (1982). Brain Res. 252, 185-188. Springer, J. E., Isaacson, R. L., Ryan, J. P., and Hannigan, J. H., J r . (1982). Life Scz. 33, 207-2 1 1. Steward, 0. (1982). Int. Rev. Neurobiol. 23, 197-254. Swanson, L. W. (1981). Brain Res. 217, 150-154. Swanson, L. W., and Cowan, W. M. (1977). J. Comp. Neurol. 172, 49-84. Tassin, J. P., Stinus, L.,Simon, H., Blanc, G., Thierry, A. M., LeMoal, M., Cardo, B., and Glowinsky, J. (1978). Brain Res. 141, 267-281. Thomas, G. J. ( 1 97 1). J . Corn#. Physiol. Psychol. 75, 4 1-49, Wachtel, H., Ahlenius, S., and AndCn, N.-E. (1979). ~sychofihar?nnfohJgy( N . Y . ) 63, 203206. Wiegant, V. M., Cools, A. R., and Gispen, W. H. (1977). E u r . j . Phamncol. 41, 343-345. Woodruff, M. L. (1981). Physiol. P.cychol. 9, 223-230. Woodruff, M. L., and Isaacson, R. L. (1977) Behav. B i d . 20,493-499.

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NEUROCHEMICAL GENETICS By V. Csanyi

Dopartment of Behavior Genetics Lorond E h o r University Budopest, Hungary

Introduction ................................................... Potentials and Limitations of Genetic Analysis ........................ Natural and Experimental Populations. ............................. Heterogeneous Polylineal Populations. .............................. Homogeneous Populations ........................................ A. Quadrilineal Strains .......................................... B. Bilineal Strains............................................... C. Monolineal Strains. ............................. .......... VI. Regulated Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. A. Heterozygous Isogenic Populations . . . . . . . . . . . . . . B. Chimeras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusions.. . . . . . . . . . . ....................... .. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. 11. 111. IV. V.

36 1 362 364 367 369 369 374 380 382 382 384 385 385

1. Introduction

T h e methodology of genetics may help in the study of many kinds of phenotypes, and the well-identified and precisely measurable neurochemical phenotype is particularly suitable for such an approach. Nevertheless, it seems that neurochemists have not yet explored the possibilities inherent in genetics, and special problems of neurochemical genetics intermingle with the problems of behavior genetics (Fuller and Thompson, 1978), pharmacogenetics (Broadhurst, I978), and neurogenetics (Wahlstein, 1977; Breakefield, 1979), the latter embracing all structural aspects of the nervous system. Besides a brief representative summary of the results obtained, the present approach also attempts primarily to show further possibilities. Therefore, I have paid particular attention to the genetic techniques used, attaching less importance to the nature of the species used or to the neurochemical problem itself studied by a genetic approach, nor have I attempted to give an exhaustive, up-to-date analysis of every result. 36 I INTERNAI'IONAL REVIEW OF NEUROBIOLOCY, VOL. 25

Copyright 0 1984 by Acddeillic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-366825-5

362

v. CSANYI II. Potentials and Limitations of Genetic Analysis

The different traits of living creatures can be ascribed to the chemical structures and chemical reactions found in the organism, for which the genetic substance DNA is responsible. Traits classifiable into diverse categories, such as the form, structure, composition, physiology, or behavior of the organism, arephenotypes.The units of the genetic substance influencing the development of the phenotype are called genes, and the specific gene or group of genes determining the phenotype in question of a given individual or strain is called the genotype. Genetic studies aim at revealing the causal relationship between phenotype and genotype. The phenotypes related to the nervous system can be very diverse: They include the different behavioral phenotypes, the parameters of the nervous system’sown structure, and also all biochemical reactions occurring in the neurons. That is why the various groups of phenotypes have typically been studied separately. Behavioral genetics, in general, is concerned only with the genetic study of behavioral phenotypes and neglects the neural substrate. Neurological genetics (neurogenetics) attempts to find relationships between the structure of neuronal networks and the genes, omitting the behavioral phenotype. Neurochemical genetics should be concerned with the relationship between the genes and the chemical reactions occurring in the nervous system. This, however, is only a rough demarcation. The structure of neurons and neuronal networks is also the result of chemical reactions, which means that neurochemistry is also relevant to neuroanatomy and to behavior. Demarcation of neurochemical genetics poses problems not only at the vertical level of organization but at other levels as well. The neurochemical reactions represent only a part of the biochemical reaction complex maintaining the whole organism. Other regulating factors of the organism can also actively participate in the development of the neurochemical phenotype. Applying these concepts to the relationship between the phenotype and the genotype, it is obvious that the individual phenotypes can only rarely be found to be influenced by the effect of a single major gene. In the majority of cases the phenotype is produced by complex epigenetic processes in which several minor genes also can be demonstrated; that is, the phenotypes are in general polygenzc (Roderick and Schlager, 1966). However, if a given gene is considered, it can in almost all cases be shown to exert its effect not only on one trait (ie., one phenotype) but also on several traits. It is then pleiotropic. The relationship between phenotype and genotype is rendered still more complicated by the fact that a single

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gene can exert different effects as part of two different genotypes because the genes are capable of influencing each other’s effects through dominance and epistasis. It is known that the gene effects themselves can be very diverse (Paigen et al., 1975). One single gene can prescribe the structure of a protein (structural gene). The gene may have a regulating function in that it can stimulate or inhibit the functioning of the structural gene (regulator gene). The gene effect may result in the modification of a given protein (processing gene). Finally, the gene may influence the temporal appearance of the activity of a given gene (temporal gene). Relatively few such phenotypes are known in the development of which the causal chain ranging from genes to the manifestation of the trait has been revealed. T h e protein structures appearing as a result of gene effects may play various roles in the development of the phenotype. Of these, the most important one for the neurochemist is the enzyme function, which itself also may be the object of complicated regulatory processes that are by nature extremely sensitive to the environment. If the phenotype can be characterized by quantitative parameters, the parameters can vary between wide limits as the genotype responsible for the phenotype manifests itself in different environments. T h e organism is multifariously associated with the environment. Temperature, food, the chemical influence of the environment, the most diverse social interactions (in the case of the higher animals), etc., all influence the course of various gene activities and thus the phenotype as well. T h e phenotypic response caused by changes in the environment may often exceed and mask the phenotypic manifestation of differences in the genotype. The most important aspiration of genetic methodology is therefore the exploration of environmental effects by strictly controlling the genotype and by utilizing such controlled genotypes for experimental purposes. Last, but not least, a very important factor is time. Each organism has its own time dimension, ranging from the development of the zygote to the death of the individual. In this time domain the phenotype is continuously and irreversibly changing, the rate of change being low in certain periods. A chemical process taking place in an early phase may trigger important chemical changes that otherwise can be observed only in a much later stage of the ontogeny of the organism. These comments on the genetic organization of the individual organism should be supplemented by the statement that the supraorganismic levels are also sites of genetic events. The propagating group of the same species living together is the population, which is characterized by a high degree of genetic variability. This can be attributed to the polymorphism of

the genes. Phenotypic variability-which is the starting point of experimental work-is produced by genetic variability and by the interaction of the regulatory systems that gives rise, conjunction with the environmental factors, to the phenotype. The first and simplest questions the geneticist is confronted with refer primarily to the effect of genetic variability on the phenotype. The ultimate goal is the complete exploration-that is, from the participating chemical reactions down to the molecular structure of the given genesof the processes giving rise to the given phenotype. In most cases, however, this is a remote ideal, the practical goal being much more modest, namely, to find a fixed point in the gene-protein-chemical reactionneural network-structure-behavior hierarchy that could be the starting point for research on the actual mechanism involved. In each case this would unambiguously mean the exploration of a relationship between genotype and phenotype. For this task, the geneticist will apply two paradigms, manipulating genotype and environment alternately. The majority of environmental effects can be recognized by studying the phenotypes of animals with identical genotypes exposed to different environmental conditions. Using animals with different genotypes and identical environments, the effect of genetic differences on the phenotype can be established. Applying these two paradigms, the genotypephenotype relationship can be attributed to the effect of some major gene or genes. In a more complicated case, especially if the contribution of minor genes to the given phenotype is relatively large, the genetic factors can be characterized only by statistical parameters and disclose nothing about the physiological mechanism of the phenotype. Several outstanding summarizing reports have been published on some special fields of genetic analysis (Green and Doolittle, 1963; Roderick and Schlager, 1966; Oliverio, 1977; Fuller, 1979; Quinn and Gould, 1979). In the following sections the genetic variables available to the geneticist, the possible genotypes of experimental animals, and their use mainly will be described.

111. Natural and Experimental Populations

Different parameters are used for the genetic characterization of different animal populations and for the characterization of the individuals within each population. A detailed analysis of these may be found in textbooks (Green and Doolittle, 1963; Wright, 1968; Bodmer and Cavalli-Sforza, 1976; Roderick and Sheridan, 1979). The parameters

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that are most relevant in the planning of neurochemical experiments characterize, in the case of individuals, genetic homogeneity. Each gene occurs generally in several varieties, that is, alleles in populations living under natural conditions. Thus, a considerable part of the genes of a diploid genome are normally in a heterozygous state, containing two different alleles for a given gene. In the case of an individual the homozygosity or heterozygosity of a gene under study therefore can be characteristic. On the other hand, the individual is also characterized by the degree of homogeneity of the complete genome. In natural populations, using different methods (most frequently protein electrophoresis), the heterogeneity has been shown to be 2030% (Williams, 1956; Lewontin and Hubby, 1966; Cavalli-Sforza and Bodmer, 1971; Lewontin, 1974; Rice and O’Brien, 1980; Rice et ad., 1980; O’Brien, 1980; O’Brien et al., 1980). Because electrophoresis shows about one-third of the actual heterogeneity, it is possible that in natural populations heterozygosity may even reach 60-90%. If a greater part of the genome is composed of heterozygous genes, this will ensure some kind of balanced reactivity for the individual. The particular environmental effects are reduced by the genetic buffer capacity (Lerner, 1970). Therefore, individuals in natural populations are in general stronger, more resistant, and less susceptible to extreme reactions than individuals with strongly homozygous genomes. In individuals the degree of total genome heterogeneity cannot be established. However, in natural populations, this can be deduced by the adequate study of a large number of individuals and by statistical analysis. Attempts are also being made to produce artificial populations in such a way that the degree of heterogeneity can be accurately calculated. In both cases parameters referring to the individual are replaced by statistical parameters (Fisher, 1949) characterizing the population to which the individual belongs. T h e statistical index of total genome homogeneity is the inbreeding coefficientF, the value of which can range between 0 and 1. It indicates the probability that an individual carries two alleles of a given gene that are identical by descent, inherited from a common ancestor of both its parents, In a population of unknown origin or in a natural population, the F value is considered 0, disregarding the actual but hardly determinable value of the real homogeneity. Throughout the process of breeding controlled generations, the coefficient of inbreeding can be calculated depending on the type of breeding (Fisher, 1949; Green and Doolittle, 1963). Another important parameter for genetically characterizing populations is the degree of homogeneity among genomes. For this, the index of the genotypic zdentity I-between 0 and l-can be used to indicate the

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probability with which identical genotypes occur in any gene of any two individuals of a population (Nagy and Csanyi, 1982a). Two individuals from the natural population can carry alleles different from each other at several thousand loci. Thus, the probability of the simultaneous occurrence of two identical genotypes is practically zero. In populations of genetically homogeneous individuals, or of FI hybrids originating from inbred parents, the individuals are genetically identical; the value of genotypic identity approaches 1. Populations comprising only individuals with identical genotypes are called isogenzc. Naturally, these can carry heterozygous genes, so their F value can be fairly low. Based on genetic parameters, natural and experimental populations can be classified in various ways. The classification used in this chapter groups populations according to their origin, taking the possible polymorphism, homogeneity, and the degree of genotypic identity into consideration. Populations showing polymorphism in which the individual genes occur in more than two varieties are considered to be polylineal in origin. Natural populations and random as well as outbred laboratory populations belong in these groups. During genetic homogenization, homogeneous strains are produced by various methods of inbreeding. Because the breeding process usually starts with two individuals, the strains produced by inbreeding can be considered to be the combination of at least four different haploid genomes, that is, they are quadrilineal. The bilineal strains can be obtained by combining two genomes. The recombinant inbred strains and those of gynogenetic origin belong to this grouping. The strains derived from some inbred strains by other methods, differing usually only in one or a few genes, are regarded as monolineal. This classification is seen in Table I. As the table shows, the genetic parameters of the various populations may differ significantly. It is, therefore, crucial that the population used in planning of neurochemical experiments be the most suitable for the experimental purpose. Because in general groups, not individuals, are used for experiments, special attention should be paid to the quality of genetic homogeneity of the animals used. Within the experimental group homogeneity must be considered in two senses: the homogeneity of the individual genomes and the homogeneity between the genomes, that is, the degree of genotypic identity within the selected group. The homogeneity of an outbred population is low in both parameters. The individual genomes contain many heterozygous loci, and some of the individuals differ markedly from each other: The value of genotypic identity is low. The group taken from an inbred strain shows maximal homogeneity. The individual genomes are homo-

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TABLE I CENEIIC CHARACTERISTICS OF NAIUKAI. A N D EXPERIMENTAL I’OPULA.I.IONS

Homozygous genes Origin Polylineal Quadrilineal Bilineal”

Monolineal Hybrid

Wild, outbred, random-bred Inbred strains Clones Recombinant inbred gynogenetic strains, congenic strains Coisogenic strains F, and F2 hybrids and backcrossed generations

I

(%I

0

0

10-40

1 0- 1 1

I 1

100 1 0 - 100

1

100

1

1

c)

0- 1

1OOb 10-40’

“ T h e term bilzneul origin for recombinant inbred strains was first used by Bailey (1981). These strains may contain some genes also in the heterozygous state, but this will only slightly change the total genome homogeneity. ‘ Crossing distant strains.

zygous and individuals are genetically almost completely identical. In an

F1 hybrid group obtained by crossing two unrelated inbred strains the homogeneity of each individual is low because the proportion of heterozygous loci is large, but the genetic identity is maximal because the genotypes of individuals are nearly identical.

IV. Heterogeneous Polylineal Populations

With a few exceptions natural populations are heterogeneous to a great extent. T h e homogeneity both within genomes and between individual genomes is low. Heterogeneity is further increased by polymorphism. T h e occurrence of certain genes in two to five or more allelic forms is the cause of the polylineal character. The various phenotypesamong them the neurochemical phenotype-show great individual variability, and reproducible values can be obtained only by using a fairly large number of individuals. In several cases, however, this is still not possible because genetic variability may also depend on geographical position. Local populations may differ considerably in the frequency of each allele. Natural populations are, therefore, rarely used in genetic

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experiments. If they are used, it is mainly for determining the domain of variability of the given phenotype and for making comparisons (Bovet, 1977; Rice and O’Brien, 1980). There are cases in which the experimenter takes advantage of the genetic heterogeneity. The human population can be viewed as a natural population; therefore, in measurements of phenotypes belonging to the realm of toxicology, pharmacology, or neurochemistry, it can be modeled only by genetically heterogeneous animal populations (Cholnoky et al., 1969; Rice and O’Brien, 1980). Similarly, genetically heterogeneous populations are required for selection experiments (see Section V,A,2). Random-bred and outbred experimental populations have been created for such purposes. The breeding plan should .be such that the population preserves its genetic heterogeneity and polymorphism, possibly for several generations, and it should enable reproducible measurements to be taken. For random-bred populations, random mating has been used, and more up-to-date schemes are applied in outbreeding. These should ensure that for each generation produced, the least related individuals mate. If the population contains a sufficient number of mating pairs, it will preserve its genetic variability for a long time (Wright, 1968; Li, 1968; Rice and O’Brien, 1980). The breeding plans and their evaluation and also the nomenclature used (Festing et al., 1972) can be found in the literature on genetics (Green and Doolittle, 1963; Wright, 1968; Li, 1968; King, 1975). Care should be taken in using outbred populations: They should derive from genetically controlled reliable stock because the genetic homogeneity of a breeding population originating from only a few individuals can rapidly increase, thus making reproducible experiments practically impossible. For ensuring reproducibility not outbred but “synthetic,” or “mosaic,’’ populations that have been formed from inbred strains or their hybrids are often used (Cholnoky et al., 1969; Festing, 1974, 1975). Such populations contain adequate genetic variability, and they can be produced in a reproducible way in the same quality. Information concerning their use can be found in an outstanding work on the genetic study of the neuroanatomical phenotypes of the brain (Roderick et al., 1976). There are several phenotypes for the study of which animal models are not suitable or the direct study of the human phenotype is indispensable. Valuable genetic techniques have been elaborated also for such cases. A psychiatric genetic examination (Odegard, 1963) and a biochemical genetic work may be suggested for further study (Weinshilboum, 1979).

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V. Homogeneous Populations

A. QUADRILINEAL STRAINS 1. Random Homogenization If a breeding pair is taken from a natural or outbred population and is propagated according to some inbreeding plan (Green and Doolittle, 1963; Li, 1968) throughout many generations, genes will be randomly selected from the four possible alleles of genes in the parental pair and a given combination will become fixed, resulting in a genetically largely homogeneous strain. In general, after an inbreeding of 20 generations, by sib mating, at which time the value of F is 0.986, the obtained strain can be considered to be inbred, at least in the case of mouse and rat. T h e inbred strains are the purest analytical tools for the geneticist. Individuals can be propagated in unlimited numbers, and their identical genetic constitution is ensured, making possible an accurate reproduction of the experimental results and the determination of variability ascribable to environmental and experimental differences (Festing, 1976). For their use, however, some factors should be considered. T h e homogeneity of inbred strains is never perfect, particularly if inbreeding has been going on for less than 100 generations (Bailey, 1978). This can be ascribed to several causes. One essential cause is the positive selection of heterozygotes. T h e F value reflects an ideal state calculated from the number of generations. I n fact, owing to the influence of traits such as viability and fertility, a permanent selection is taking place, and some of the genes affecting these traits remain in the heterozygous state, that is, F does not in practice reach 1. T h e other essential cause is that mutation constantly gives rise to new allelic varieties, producing heterozygosity as a consequence. The development of heterozygosity is only one of the possible effects of mutation. It is much more important that-owing to mutation-the inbred strains are gradually changing, new alleles are becoming fixed, and consequently the whole genotype of the strain is changing as well. For examinations performed in different laboratories, even if the same strains are used but the populations have been separated for 30 or 40 generations, the results are often not reproducible. There is also a great risk of contamination. Therefore, the maintenance of inbred strains requires constant and permanent control (Krog, 1976; Krog and Moutier, 1978; Festing, 1981). Finally, some comments about the evaluation of experiments on inbred strains are necessary. The frequently cited statement that members

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of an inbred strain actually represent multiple copies of a single individual of the wild population is not correct. The animal carrying a highly homogenized genome is a special, artificial creature. Several of its reactions are extreme; it does not much resemble the wild-type heterozygotes. Its advantage is that it provides identical genotypes in unlimited numbers, permitting the reliable reproduction of experiments and the determination of environmental and genetic variance as well. The above considerations should be borne in mind when evaluating data. T h e most frequent neurochemical type of experiment is the assessment of the difference between strains. If statistically significant differences are obtained in a given phenotype between two or among some homogeneous strains, it is generally assumed that the difference in the phenotype can be ascribed to genetic causes. This is no doubt true. However, in evaluation several factors should be considered. If, for example, there is no difference in a given phenotype between two strains, this does not imply that the two strains are genetically identicaieven with regard to the given phenotype. Such identity can be established only on the basis of crossing experiments. I n the case of a reliable difference, furthermore, it is not by any means certain that the genetic background producing the difference is relevant from the point of view of elucidating the chemical mechanism of the phenotype. Two inbred strains can differ in several thousand genes. Interfering even at quite distant points in the enormously complex metabolic mechanism controlling the organism may give rise to changes in a given phenotype. T h e value of demonstrating these kinds of nonspecific differences is low if, in addition to simply writing an additional report, we also have some other questions in mind. The discovery of such a nonspecific gene effect does not necessarily mean that we “have detected the gene of X or Y phenotype.” Those differences between the individual strains that help in revealing the chemical mechanism of the phenotype under study are the important ones. The geneticist may observe the effect of the allelic variations of an individual given gene, but he or she cannot recognize genes that are identical in the two strains because these cannot be measured by genetic methods. Despite all these problems, demonstration of differences between strains is a widely used method in neurochemistry and is certainly the first step toward further genetic investigations. The phenotypes studied are most diverse: brain enzymes (Pryor et al., 1966), specific proteins (Malup et al., 1978),concentration of metabolites (e.g., cerebral gangliosides) (Seyfried et al., l979), cyclic AMP (Crenberg et al., 1975),catecholamine metabolism (Maeguryn-Davies et al., 1973; Ciaranello and Ax-

NEUROCHEMICAL GENETICS

37 1

elrod, 1973; Barchus et al., 1974; Berger et al., 1979; Grimm et al., 1980; Goldstein et al., 1980), different brain receptors (Ticku, 1979; Robertson, 1979, 1980; East and Dutton, 1980; Boehme and Ciaranello, 1981), and pharmacological phenotypes (Jori and Rutczynski, 1978; Allegra et al., 1980; Frigeni et al., 1981). By chance, examination of the differences between strains may reveal important genetic regulatory mechanisms (Fuller, 1974) in the course of testing the hypothesis that simple single-gene differences underlie the observed phenotypic differences. In this case there is some hope of clarifying the mechanism of action. The plan to be followed is demonstrated by the exploratory work of Ciaranello and his co-workers on the possible role of enzyme regulation in the biosynthesis of catecholamines (Ciaranello and Axelrod, 1973; Barchas et al., 1974; Ciaranello et al., 1974; Ciaranello, 1977). T h e difference between mouse strains was first assessed by measuring the level of phenylethanolamine N-methyltransferase in the adrenal cortex (Ciaranello and Axelrod, 1973) as well as the levels of tyrosine hydroxylase and dopamine-P-hydroxylase, utilizing two substrains of the BALB/c strain, that is, BALB/cJ and BALBkN. In BALBkJ the level of all three enzymes was about double that measured in the other strain. After the differences between strains were established, reciprocal crosses were made. In the FI generation the enzyme levels roughly ranged between the two parental values and progeny of the two reciprocal crosses did not differ from one another: Therefore, maternal effects and sex-linked heredity could be excluded. By crossing the individuals of the F1 generation, the authors produced an F2 generation that, measured in a larger population, showed mean phenotypic values identical with those of the FI mice for all three enzymes. An essential difference could, however, be observed in the F2 animals; the standard deviations of the values measured were high as compared to the relatively low and identical standard deviations obtained in the other populations. The data available so far suggest a genetic background characterized by a relatively simple mode of inheritance. Various genetic models can be adapted to the data obtained (Ciaranello et al., 1974). The model corresponding best to the data postulates an effect of two alleles of one gene on the breakdown of the three enzymes. The allele responsible for the slow breakdown is carried by BALB/cJ; thus BALB/cJ mice have high enzyme levels. BALB/cN carries the variety responsible for the increased breakdown; thus, these mice have lower enzyme levels. I n F1 mice the phenotype is intermediate; this indicates that neither of the alleles is dominant. Based on this hypothetical model, experiments could

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be performed that would show whether the 1 : 2 : 1 ratio of the high, intermediate, and low phenotypic values in the F2 generation could be demonstrated to be due to this mechanism based on the assumption of a single locus. A similar examination could also be made in backcross populations obtained by crossing members of the parental strains with members of the F1 population. Here the expected ratio of intermediate to high or low phenotypic values was 1 : 1. The ratios found in the three populations (F2, BCI , BC2) did not differ significantly from those expected on the basis of the model. The analysis was supplemented by a correlation study in which the correlations between all three enzyme levels within individuals was examined in the F2 population obtained from the F1 hybrid parents. The data suggested that a gene influencing the breakdown of all three enzymes could be detected, two alleles of which affect the breakdown in different ways, producing the observed double difference in the parental phenotypes. It should be noted that the use of two strains that differed only slightly from one another was important to the success of this revealing analysis because the genetic background was nearly identical and did not interact with the phenotype studied. The two substrains differed in their phenotypes owing to the mutation of a gene regulating the breakdown of enzymes involved in catecholamine synthesis.

2. Directed Sebction Although many inbred strains have been available, their genotypes reflected random selection. It is rarely possible to find strains that differ just in a single trait. By directed selection for extreme values of the relevant phenotype, such strains can be more easily produced. Furthermore, if the change of phenotype is followed up during selection throughout several generations, useful genetic information will also be available. The essence of the technique of selection is that from a population possessing a gene pool of diverse variations breeding lines are selected by the assessment of the phenotype in individuals of each generations and by appropriate matings. The selected lines will show the extreme values of the given phenotype, and possibly the allelic variations responsible for these extreme values of the phenotype will increase their proportion. This, of course, can only be successful if the parental population shows a sufficient degree of genetic variability and if the scheme of selection is capable of making use of the offered diversity. The parental population is often a natural one or an outbred, or rather “synthetic,” population that has been produced from inbred strains of known qualities (Roderick et al., 1976; Broadhurst, 1978; Doolittle and Wilson, 1981). In any case it is always worth starting the selection program by

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373

examining whether there is adequate individual variability in the natural population or in the strains used for the synthetic one. Often there are such great differences in such a population-and for a majority of these marked differences, simple genetic factors are responsible-that the first few generations of selection may furnish valuable data permitting the application of simple genetic models (Vaido and Sitdikov, 1979; Overstreet et al., 1979; Flow et aZ., 1981). If the background of a given phenotype is complex, that is, polygenic, data may be obtained only by a systematic selection involving several generations. Theoretically, the selection process is aimed at separating the allelic variants producing the extreme qualities of a given phenotype into individual strains. However, even if strains with evident differences are obtained during selection, this is still not adequate proof that the goal has been achieved. Owing to the interaction between different genes, because of a too rapid increase in homogeneity it may happen that the differences in the phenotype of two selected lines can be attributed to other causes. If at least two lines for each extreme quality are selected, if the degree of inbreeding is kept low, if after reaching the selection plateau the lines are crossed, and if after a certain number of generations the direction of selection is reversed, such an error may easily be exposed. Roderick and his co-workers (1976) selected for extreme values of brain weight in mice. An outstanding evaluation of selection techniques is also included in their work. Studies on selection involving pharmacological phenotypes have been excellently summarized by Broadhurst ( 1978).

3. Selection and Its By-Products

A common procedure is that the strains obtained by selection for a definite phenotype are utilized and other phenotypic traits that were not used as criteria for selection are studied, the changes in which are probably associated with the originally selected phenotype. Thus, for example, norepinephrine concentrations and tyrosine hydroxylase activity in strains that have been selected for resistance or susceptibility to hypertension (Feuerstein et al., 1979),the variability of the cerebellar receptors of Roman High Avoidance and Roman Low Avoidance rat strains, which have been selected for emotional behavior (Overstreet et al., 1979, 1981), and the effect of different drugs on the exploratory activity of these strains (Martin et al., 1981) have been studied. Care should be taken that if in these comparisons differences between the strains are found, this is not taken to imply that the given phenotype and the originally selected phenotype are correlated. Such a correlation is not excluded, but proving a closer relationship would only be possible by evaluating the results of adequate genetic experiments.

374

V. CSANYI

B. BILINEAL STRAINS Data from the comparison of inbred strains of quadrilineal origin will infrequently yield suitable genetic analyses, primarily because of the great degree of genetic variability between the strains. Construction of bilineal strains decreases the initial variability, permitting thereby a theoretically new type of genetic analysis, which today can be regarded as the most up-to-date method in the organismal genetics of vertebrates. 1, Recombinant Inbred Strains

Recombinant inbred strains for studying immunological phenotypes were first produced by Bailey (1971). He selected two different, practically homozygous inbred mouse strains that differed from each other in several traits, that is, BALB/cBy and C5’7BL/6By. These served as the two parental genotypes. He produced F, ,then F2 generations. From the latter, he produced a battery of homozygous strains by systematically mating siblings for 40 generations. These strains, seven in number, carry random combinations of the genotypes of the two parental strains that produced the F1 generation. In fact the strains can be considered to be an F2 generation with the difference that each individual is represented by a strain, and there are no heterozygotes among them. Figure 1 shows the general scheme for producing recombinant strains. By replacing individuals with strains, the researcher can increase the accuracy of the genetic analysis; quantitative phenotypes, which can be evaluated only PARENTAL STRAINS

P

5 0 0

0

0

0

0

H R1

R2

R3

RA

0

0

H R5

R6

F3

F20-40 R7

RECOMBINANT STRAINS

FIG. 1, Schedule for producing recombinant inbred strains.

NEUROCHEMICAL GENETICS

375

by determining the population distribution and by statistical methods, can also be studied. This is of particular importance in studying behavioral and neurochemical phenotypes. The strain battery produced by Bailey was used for studying not only immunological phenotypes but also several other traits, among them neurochemical phenotypes (Klein, 1978; Oliverio, 1979; Bailey, 1981). If the recombinant strain population already exists, the examination is fairly simple. T h e values of the given phenotype are determined in the parental strains and in the recombinant strains. (Bailey produced, together with the recombinant strains, a larger number of congenic strains from the two parental strains; these have been used in studies that are dealt with in Section V,C.) If the experimental results show a statistically reliable difference among the strains, the genetic parameters can be obtained by analyzing the frequency of the categories of the individual phenotypes. T h e reasoning is as follows. Let us assume that the initial parental strains for the given phenotype differ only in a single gene, that is, in two alleles. Both parental strains carry one of the alleles, each in a homozygous state. T h e difference in the parental phenotypes can be attributed to the different effects of the alleles. In this case, after the two alleles have been combined in the F, hybrid and then separated in the course of inbreeding for many generations starting from the F2, the recombinant strains will carry one or the other allele in the homozygous state. Accordingly, the two phenotypes corresponding to the different alleles can be found in almost equal frequency among the recombinant strains. If this is the situation, and if the single-gene model can be confirmed by measurements taken from backcross progeny and eventually of the F, and the F2 generations produced by the parents, the hypothesis can be considered fairly reliable (Fig. 2). The norepinephrine level of the hypothalamus as a phenotype was studied: In one of the parental strains, BALBkBy, and in six of the recombinant strains, statistically the same high values were measured. These levels were more than double those in the other parent, C57BL/6By, and in the seventh recombinant strain. T h e F1 populations obtained by biparental reciprocal crossing yielded progeny with intermediate levels. It is, therefore, probable that the differences observed between the parental phenotypes are due to a single autosomal gene (Eleftheriou, 1974a). Using recombinants, the [SH]corticosteronebinding of the hypothalamus was examined (Eleftheriou, 1974b). In the BALBkBy strain this value was low, whereas in strain C57BL/6By it was high. The two reciprocal hybrids showed an intermediate value; therefore, there was no dominance and no maternal effect. The backcross generations were not homogeneous: The low and intermediate as well as the high and inter-

v. CSANYI

376

P

Fl

F2 Ala

a10

A h

Ala

ala

AIA

0

0

0

0

0

0

Fn

m m m m m p q AIA

a/a X

1 1

FIG. 2. Allelic segregation among recombinant strains.

mediate values occurred in about the same proportion. These groups were also supplemented by further crosses. I n one of them a recombinant strain with a low value was crossed with the parent showing a low value. In the other the parent showing a low value was crossed with a high-value recombinant. The offspring generation was in both cases of a homogeneous distribution, the first belonging to the low category, the second to the intermediate one. Based on these results, the phenotypic difference in coritcosterone uptake of the hypottialamus can be ascribed to the effect of a single gene. More complicated cases have also been examined. If the phenotypic difference can be attributed to two genes, the four alleles of the two genes can produce four homozygous combinations, that is, four phenotypic categories. The effects of two genes were assumed to be affecting the phenotype of the quantity of opiate receptors of the brain (Baran et al., 1975). The maximal number of phenotypic variants P can be calculated from the relationship P = 2L, where L is the number of loci. Table I1 shows with what probability different numbers of phenotypic categories, depending on the number of genes involved, can occur in a given number of recombinant strains. Using recombinant inbred strains, several kinds of phenotypes have been studied more or less successfully, for example, the plasma corticosterone level (Eleftheriou and Bailey, 1972), norepinephrine uptake (Moisset, 1977), lipid depletion of the adrenal gland (Taylor and Meier. 1976) glucuronidase induction in the kidneys (Swank and Bailey, 1973). morphine tolerance and sensitivity (Oliverio et nl., 1975); Shuster et al.,

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NEUROCHEMICAL GENETICS

T A B L E I1

ESTIMATION OF G E N E NUMBER IN A GKCIUI' CONTAINING; 15 RECOMBINANT $TRAINS ON THE BASISOF THE OR SERVE^ PHENOTYPIC FREQUENCIES No. of genes

NO. of phenotypes

2 4 8 16

Probability of the occurrence of at least 2"-' 1 phenotypes

+

1 - 2(112)'~= 1.000 1 - 6(1/2)'5 = 0.9'39 1 - 56( 1/2)15 = 0.998 I - 1820( 112p = 0.556

71

1975), sensitivity to cocaine (Shuster and Bates, 1977), and the effect of various drugs such as chlorpromazine (Castellano et al., 1974) and amphetamine (Oliverio et ad., 1973) on behavior. The genetic background of differences in the behavioral phenotypes of learning (Oliverio et al., 1972) and aggression (Eleftheriou et al., 1974) have also been studied. The method permits, in certain cases, the chromosomal localization of the given genes. If the chromosomal position of a given locus is known, the strain distribution pattern of phenotype (SDP) influenced by the locus can be made to match that of the studied phenotype. In the case of identity, the two phenotypes are produced by the same gene or are influenced by genes located adjacent to one another (Eleftheriou, 1974a). T h e first recombinant strain population of Bailey has since been followed by several others (Taylor and Meier, 1976; Taylor et al., 1977; Taylor, 1978; Oliverio, 1979) and the range of phenotypes involved in the study has been broadened (Oliverio, 1979). Some comments are necessary about the limitations of the method. Work with recombinants requires more effort even if already established strains are used as parents; this is, however, abundantly compensated for by the greater degree of accuracy of the analysis. By using simple Mendelian analysis, it is rarely possible to detect mechanisms more complicated than single-gene effects, particularly in the case of phenotypes measurable only with relatively large errors, such as neurochemical phenotypes. With recombinants, two-gene and three-gene differences can be measured. It is probable that this method can be further developed by producing a subrecombinant group from the constructed recombinant strains by further (Fig. 3) crossing and inbreeding; thus, theoretically polygenic systems can also be mapped if measuring of the phenotype has been accurate enough.

378

v.

CSANYI

P

F1

F2

F”

5s

F2s

F”s

FIG. 3. Schedule for producing subrecombinant inbred strains.

Genetic background, as in the case of simple Mendelian crosses, is liable to render the evaluation of measurements difficult. T h e parental strains differ not only in genes influencing the studied phenotype but also in several hundreds or even thousands of other genes. These recombine, and the random recombinations produced may seriously disturb, in the individual strains, the effect of the locus under study or may even mask it. I n the case of such effects if the studied phenotype is interesting enough, construction of subrecombinant strains is the only possibility. This will considerably homogenize the genetic background.

2. Gynogenetic Strains Work with recombinant strains is rendered fairly difficult by the considerable amount of work and the long time required for their construction. In lower vertebrates methods have been elaborated by which bilineal strains can be produced in a much shorter time. T h e methodgynogenesis-is a kind of parthenogenesis, because the eggs are fertilized with inactivated sperm cells. By use of gynogenesis throughout several generations, strains that are genetically quite homogeneous can be produced. The method has been applied in fish (Purdom, 1969; Nagy et al., 1978, 1979; Streisinger et al., 1981; Gervai and Csanyi, 1982), in frogs (Nace, 1968; Volpe, 1970), and in newts (Jaylet and Ferrier, 1978). During artificial gynogenesis, haploid embryos are typically produced

339

NEUROCHEMICAL GENETICS

A SINGLE FEMALE P gynogenesis 61

G6-8 GR,

GR2

GR.,

GR4

GR5

GR6

GYNOGENETIC RECOMBINANT STRAINS

FIG. 4. Schedule for producing gynogenetic recombinant inbred strains.

and usually die after hatching. By using different techniques gynogenetic diploids can be produced. This can be achieved either by the inhibition of the first mitotic division (Streisinger et al., 1981), in which case the diploid embryo produced will be 100%homozygous, o r by preventing the expulsion of the second polar body after fertilization (Nace, 1968; Jaylet and Ferrier, 1978; Gervai and Csanyi, 1982), in which case the gynogenetic diploid individual will not be completely homozygous because, owing to normal meiotic recombination, the polar body and the chromosomes of the maternal pronucleus may carry partly different alleles. Despite recombination, the degree of homozygosity considerably increases, the effect of gynogenesis within one generation corresponds to that of eight to ten generations of sib matings (Nagy et al., 1979). The isogeneity of the gynogenetic populations approaches, after three to four generations, a level similar to that of inbred strains (Nagy and Csanyi, 1982a). By using an adequate combination of sexual generation and of gynogenesis, fairly rapid genetic homogeneity can be achieved (Nagy and Csinyi, 1982b). By gynogenesis gynogenetic recombinants carrying recombinations of biparental gene series can be produced from a single female, the carrier of two haploid genomes, within a few generations (Fig. 4). T h e advantage of constructing gynogenetic recombinant strains is that two completely homozygous inbred strains are not needed to produce them because the recombinant populations can be derived from a single female parent (J. Gervai and V. Csanyi, unpublished). Thus, an individual of any origin-it might as well be a wild type-can be the parental individual of a strain population. T h e genetic models are the same as have been shown for the recombinant inbred strains. If the

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v. CSANYI

gynogenetic recombinants are started from a single animal, the parental populations and F, will naturally be missing, but the data not available from these groups will be compensated for by the larger number of individuals of easily producible recombinant strains and by the progeny of possible crosses made between the recombinant lines (J. Gervai and V. Csanyi, unpublished). The gynogenetic technique also facilitates the production of subrecombinant populations. Although neurochemical studies on gynogenetic animals have not yet been published, apparently, it is certain that this elegant new method is also suitable for such studies. It is also possible that attempts will be made to use gynogenesis with mammals, primarily with the mouse, to produce homogeneous strains. Some individuals have already been produced by techniques used in cellular surgery, (Hoppe and Illmensee, 1977) but a similar method for practical purposes could be worked out by using inactivated sperm as in the case of fish. Parthenogenesis can be successfully applied not only in vertebrates but also in invertebrates; isogenic individuals have been successfully produced in the grasshopper. In the isogenic grasshopper clones produced, certain neurons and the ontogeny of their connections have been studied (Goodman, 1979). Species reproducing by gynogenesis under natural conditions, which are not homogeneous genetically, will be discussed in Section VI,A,P. C. MONOLINEAL STRAINS Under natural conditions the polymorphism of genes is strongly limited by selection. Mutants in the development of which disorders reducing viability and fertility appear will not survive under natural conditions. Under artificial laboratory conditions, however, some of these mutants can reproduce, and they can be of substantial help in revealing the physiological mechanism of the most diverse phenotypes. In the mouse alone over 500 mutants caused by the change of a single gene have been described (Searle, 1977; Womack, 1979). Of these, more than 150 are neurological mutants (Sidman et al., 1965). Mutants in general are derived from inbred strains, or if the given mutation has first occurred in an outbred strain, attempts are made to introduce it into a well-known inbred strain by multiple backcrosses. Therefore, these are considered to be unilineal in origin. Usually two kinds of mutant strains are used. One type differs from the background strain at a single locus that has undergone mutation.

NEUROCHEMICAL GENETICS

38 1

These are coisogenic strains. Alternatively, a population of animals with a mutant genotype may be maintained as an individual entity, a substrain (Altman and Katz, 1979). The two types are not identical, but they both are, in general, largely homozygous. I n the case of semilethal mutations or in those producing sterility, it may occur that the mutant gene can only be maintained in the heterozygous state; however, this heterozygosity involves only the mutant locus. The coisogenic strains are identical with the background strain except at the mutant locus itself. T h e different substrains can generally differ from each other at more loci, partly because of undetected mutations, partly because of the residual heterozygous gene states of the parental strains (Bailey, 1978). The methods of detection of mammalian mutants are developing continuously and are objects of intensive research. Despite the high level of interest and 10 years of intensive work, it cannot be stated that the use of mammalian mutants has been free of problems. A mutant is recommended for laboratory use if the mutation of a (usually unknown) gene causes a severe disorder in the organism that is easily recognizable. After clarification of the inheritance pattern, the first step of the research is the general description of the change produced by the mutation, Unfortunately, most genes have several pleiotropic effects in addition to any obvious main effect. Because of cascade effects, a minor defect in the gene at the protein level may lead to extremely severe changes in the nervous system or the organism. As a consequence the ultimate cause of the disorders observed at the level of the organism can often hardly be determined (Black, 1976). The method of gene substitution can provide interesting data on individual phenotypes. According to this method a strain series that includes the mutant alleles of several loci obtained by adeqaiate crosses is constructed from a defined homozygous inbred strain. T h e effect of mutations on the given phenotype against an identical genetic background are studied. This method was employed by Oliverio and Messeri (1973) on behavioral phenotypes. Sometimes in a fortunate case the study of a single mutant can provide valuable data because of the phenotypic peculiarity of the given mutant (Caviness, 1980). If in the case of the studied phenotype, several independent mutants are available, the systematic comparison of the individual mutant phenotypes may offer a complete developmental picture of the given phenotype, revealing the most essential points of control. Such examinations have been performed in the case of mutations affecting myelination (Bauman, 1980) and in that of mutants influencing the development of the cellular architecture of the brain (Caviness and Rakic, 1978). Here belongs also the class of mutations-although they are probably not attributable to the

382

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change of a single gene-that produce symptoms resembling human epilepsy in very different species, for example, in mouse (Seyfried, 1979; Nelbels, 1979), in rat (Consroe et al., 1979), in the Syrian hamster (Yoon et al., 1976), in hen (Johnson et al., 1979), and in dog (Elmonds et al., 1979)-a11 of which could serve as interesting models. Investigations with species in which the methods of isolating and identifying mutants are more advanced than in the vertebrates represent a more effective method of using mutants. Thus by studying Drosophila mutants, quite specific neurobiological problems can be approached, for example, synaptic transmission (Jan P t al., 1977), photoreceptor function (Pak, 1979), sexual behavior (Hall, 1981), and neurotransmitter mechanisms (Kandel and Ferrus, 1979). The genetics of Nematodu is well developed (Brenner, 1974; Lewis, 1980), and the solution of special neurochemical problems has been started, for example, in the field of examining chemosensory perception (Lewis and Nadgin, 1977) and acetylcholine receptors (Russel et al., 1977; Lewis et al., 1980). The work on a giant protozoan, Paramecium, which is an excellent model of the higher neuron and which provides data for clarifying the relationships between the different ionic channels and changes in membrane potentials (Kung, 1979), should also be mentioned here.

VI. Regulated Heterogeneity

A. HETEROZYCOUS ISOCENIC POPULATIONS In the above investigations homozygous strains were used. Experiment planning has used the advantages of genetic homozygosity. Useful experiments can also be made with heterozygous genotypes if the isogeneity of the individuals used is guaranteed, that is, if there is no genetic variance at all within the group. So far, such types of populations have not been used for neurochemical studies; therefore, this survey will be limited to the most essential information.

1. F1 Heterozygotes Isogenic heterozygotes can be obtained most simply by crossing homogenous inbred strains. In general, more than two strains are crossed in all the possible combinations, and the method is called diallelic crossing. Certain conclusions can be drawn from the statistical evaluation of the progeny phenotypes concerning the quantitative effect on the examined phenotype of the genotypes of the strains used. General and special combining ability can be distinguished. The general combining ability

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plays a decisive role in additive genetic variance. The special combining ability, which can be calculated from the interaction of the individual pairs, is responsible for interactive variance. The data obtained by the diallelic method belong to the sphere of quantitative genetics and can be utilized relatively little in neurochemistry (Murphy, 1979). A behavioral experiment is suggested for further study (Messeri et al., 1972). Prior to performing a larger selection experiment or to planning comparative studies on several strains, useful information can be obtained on the expected reactions or the individual strains even if the technique does not allow any insight into physiological mechanisms.

2. Natural Isogenic Populations Some isogenic populations can be produced only by genetic manipulation. In the case of Gther species, such populations are found under natural conditions. Some species, including the freshwater crustacean Daphnia, propagate parthenogenetically as part of their reproductive cycle. Thus, the offspring obtained from a single mother are isogenic and can be useful in solving several neurobiological problems. Using Daphnia, Levinthal and his coworkers examined the nature of variability observed in neural connections (Macaquo et al., 1975, Levinthal et al., 1976). There are parthenogenetic species among the vertebrates as well; for example, Poecilia formosa is the natural hybrid of P. latipina and P. sphmops (Kallman, 1962; Abramoff et al., 1968), two normally reproducing related species, and it propagates by gynogenesis as an independent species containing only females. The offspring originating from one mother are isogenic with no genetic variance, although with a high degree of heterogeneity within the genome. Poecilia formosa has also been used in studying the ontogenetic problems of the nervous system (Levinthal et al., 1976). A triploid form of the wild type of the common goldfish Carassius auratus gibelio is highly suitable for this type of work. It also reproduces by gynogenesis, and the offspring population descending from a single mother is isogenic. It is particularly well adapted for use in the laboratory, being relatively small in size and easily managable, and may contribute new data to the neurobiological study of the goldfish. The low degree of variability attributed to the isogeneity of the phenotypes of the triploid goldfish has been demonstrated in behavioral studies (Kabai and Csanyi, 1978). 3. Vertebrate Clones

Methods have been worked out in several species for cloning individuals, for example, in frog (Curdon, 1974) and in mouse (Hoppe and Illmensee, 1977; Illmensee and Hoppe, 1981). The method consists of

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transplanting nuclei from somatic cells or embryos into fertilized and enucleated ova that have been developed by special methods depending on the species. T h e individuals developing from a single donor are genetically perfectly identical, that is, isogenic. They are suitable for solving every kind of problem concerning isogeneity, primarily in research on the genetic control of the development of neural structures. The advantage of clones as opposed to F1 hybrids or natural isogenic populations is that by using them the genetic replica of a specific individual can be produced. Consequently, special phenotypes can also be examined that cannot be produced by conventional methods. For experimental purposes an additional advantage would be that the methods of producing clones may become suitable for mass production. B. CHIMERAS

Animal chimeras are quite extraordinary creatures: They are mosaic organisms composed of cells with different genotypes. Their use in neurochemistry would probably yield highly valuable results, as they have proved to be fairly useful in other fields of neurobiology. The first method for producing chimeras was worked out by Tarkowski (1961). Mouse embryos composed of two cells were fused and after fusion transplanted into an adequately prepared female, in which the normal embryonic development took place. Mintz (1965) was the first to use genetically different blastulae for producing chimeras. Since this pioneer work, many results have been achieved and reliable methods have been elaborated. For a more detailed study see McLaren’s (1976) book on mammalian chimeras. Hexa- and octaparental chimeras have also been produced (Petters and Market, 1980). Chimeras can be used multifariously for studying neurobiological problems. For example, one mouse mutant is characterized by the almost complete lack of Purkinje cells in the cerebellum. The gene, the pcd, is a mutation at an autosomal locus. Mullen (1979) studied whether the phenotype characteristic of the mutant can be ascribed to the gene activity taking place in the Purkinje cells or whether the damage is manifested in other cells, the degeneration of the Purkinje cells being a result of that. His chimera experiment with normal and mutant cells helped in deciding whether the mutant gene exerts its effect within the Purkinje cells. A similar examination was performed on chimeras of cells from a mutant suffering from retinal degeneration and cells from normal mice (Wagmann et al., 1971). This latter study indicates what valuable data the mutant, otherwise utilized with much more difficulty, can fur-

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nish when supplemented by the chimera technique. Very different varieties of the chimera techniques have been developed extremely rapidly. Thus, chimeras from different species (Illmensee and Croce, 1978; Illmensee et al., 1978) and from intraspecies cell hybrids (Mullen and Herrup, 1979), those constructed by using malignant cells (Mintz and Illmensee, 1975), and avian chimeras have been produced (Rathborne, 1980). It has also been discovered that certain well-known techniques such as transfusion can be a modest way of producing chimeras (Howard et al., 1980).

VII. Conclusions

Genetics is making rapid progress today and so are the genetic methods considered to be traditional. Research workers engaged in the special fields of biology may select the techniques best suited to their purposes from a complete storehouse of research aids. The methods presented here are mainly those of traditional vertebrate genetics. Methods used in invertebrates have been touched upon only in some cases. Several methods that go beyond the bounds of the organism have also undergone remarkable development. Excellent genetic studies can also be made by using somatic cell cultures and cell hybrids, which have not been dealt with in this summary. References Abramoff, P., Darnell, R. M., and Balsano, J. S. (1968). Am. Nat. 102, 55-568. Allegra, S . P., Mack, G . , Oliverio, A., and Mandel, P. (1980). Prog. Neuro-Psychophanacol. 13,491-502. Altman, F. L., and Katz, D. D. (eds.) (1979). “Inbred and Genetically Defined Strains of Laboratory Animals,” 2 parts. Fed. Am. Soc. Exp. Biol., Bethesda, Maryland. Bailey, D. W. (1971). Transplantation 11, 325-327. Bailey, D. W. (1978). In “Origins of Inbred Mice” (H. C. Morse 111, ed.), pp. 197-215. Academic Press, New York. Bailey, D. W. (1981). In “The Mouse in Biomedical Research” (H. L. Foster, J. D. Small, and J. G. Fox,eds.), Vol. 1, pp. 223-255. Academic Press, New York. Baran, A., Shuster, L. E., Eleftheriou, B. B., and Bailey, D. W. (1975). Life Scz. 17, 633640. Barchas, J. D., Ciaranello, R. D., Dominic, J. A., Deguchi, T., Orenberg, E., Renson, J,,and Kessler, S . (1974). In “Neuropsychopharmacology of Monoamines and Their Regulatory Enzymes” (E. Usdin, ed.), pp. 375-389. Raven, New York. Bauman, N. (1980). Trendc NeuroSci. 3, 82-85. Berger, B., Herve, D., Delphin, A., Barthelemy, C., Gay, M., and Tassin, J. P. (1979). Neuroscience 4, 877-888. Black, I. B. (1976). Brain Res. 105, 602-605.

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THE NEUROBIOLOGY OF SOME DIMENSIONS OF PERSONALITY By Marvin Zuckerman Department of Psychology Univeniiy of Delaware Newark, Delowaro

James C. Ballenger’ Department of Behavioral Medicine and Psychiatry University of Virginia Medical Center Charlo~erville,Virginia a n d Robert

M. Post

Biological Psychiatry Branch National Institute of Mental Health Betherda, Maryland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Personality Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Persons and Situations.. , , . . . . . . . . . . . . . . . . . . . . . . . , , . . . . . . . . . . . .

C. Abnormalities of Personality ..,............. D. Extraversion, Neuroticism, P 11. Arousal and Arousability . . . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . A. Tonic Cortical Arousal.. . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Arousability. . .... ... ... ... C. Augmenting-Reducing of the Average Evoked Potential. . . . . . . . . . . . . D. Orienting, Defensive, and Startle Reflexes . . . . . . . . . . . . . . . . . . . . E. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Biochemical Studies. . . ........................................ A. Gonadal Hormones ........................................... B. Monoamine Oxidase (MAO) . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . C. Monoamine Systems: Animal Studies D. Monoamine Systems: Human Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . E. Endorphins.. .. .. . .. . .. .. .. . . . .. F. Biochemistry of the IV. Conclusions. . . . . , . . . References .....................................................

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Present address: Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, South Carolina 29425.

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MARVIN ZUCKERMAN

et ad.

1. Introduction

Personality may be defined as the total organization of characteristic modes of behavior (habits) and the internal processes that govern such behavior. The internal processes include cognition and feelings (affects) at one level of analysis and neurobiological reactions at another. Although we are a long way from reducing cognition, affects, or habits to neurobiological processes, a beginning has been made in relating the individual differences in reaction that we call “personality” to neurobiological processes. The aim of this chapter is to describe this relatively new area, which integrates personality and neurobiology.

A. PERSONALITY DIMENSIONS When nonpsychologiststhink of a “personality,”they usually conceptualize it as a unique kind of beast. But on more careful consideration, the uniqueness may be found to consist of a particular combination of salient traits that seem to make one person different from another. Some psychologists have attempted to measure the individual traits that constitute the whole personality. Others (e.g., Allport, 1961) have refused to do this, claiming that personality must be approached at the molar level, much in the way that an author of fiction or a psychoanalyst would (although the latter might do a little slicing into id, ego, superego, self, object categories). An idiographic science that studies single individuals may have an intuitive appeal in describing surface phenomena more precisely, but it usually turns out to have little predictive power relative to nomothetic approaches (Meehl, 1954). Following the development of standardized psychometric methods for measuring the hypothetical trait of “intelligence,”some psychologists set out to measure equally hypothetical personality traits. Because it would be too time-consuming and intrusive to follow people about all day or bring them into the laboratory to observe their characteristic modes of reaction in more controlled situations, most psychologists rely on the persons’ descriptions of their own behaviors or internal processes. Such trait questionnaires typically yield a range of scores usually but not always approximating a normal frequency distribution. The reliabilities of such tests are determined by the subject’s consistency of response, either within the items on a given scale taken at one time or in scores on the entire scale administered repeatedly over time. Validity of the scale is a function of correlations with criteria such as ratings of the person by

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acquaintances that are independent of the responses to the test itself. Some would argue that self-reports on similar kinds of tests are not validity but reliability data because the similarity of scores on the two tests may be merely due to a similarity of the content of the items. Because self-observations are subjective, their meaning must be established in terms of their relationships with external criteria. There is no such thing as face validity in the science of personality measurement, whether it is done with interviews, projective tests, or objective questionnaires. Although some tests are developed for very specific purposes and use strictly empirical methods for item selection, most tests have some kind of theory guiding their development. The theory may only be some preconceived idea of how the trait is embodied in characteristic behaviors or attitudes, or the test may be evolved to measure a hypothetical entity representing one construct embedded in a more developed theory (construct validity). T h e latter type of measure yields predictions that are less obvious than those derived from the type representing an isolated construct. If personality traits can be measured in the ways described, then persons can be ranged from low to high on the trait (from the midpoint of the distribution, because there is generally no absolute zero in a psychological scale). Given the bell-shaped normal distribution, most persons will score in the middle range and smaller numbers will be found at the extremes. B. PERSONS AND SITUATIONS It has been said that trait tests are not highly predictive of behavior in specific situations (Mischel, 1968). This assertion is, of course, true for the persons in the middle range on a trait dimension; that is why we d o not usually consider traits on which a person is average when describing her personality in an offhand way. An average level of a trait does not give us much information about that person that would distinguish her from others. Let 'us take the dimension of personality that runs from extreme introversion to extreme extraversion. One behavioral aspect of this trait is sociability. Sociability may be measured behaviorally as the time one voluntarily spends in social interaction with other persons. An introvert spends little time, an extravert considerable time in interaction with others. Given a specific situation, such as a party, the behaviors of the extreme introverts or extraverts will be more consistent than those of the

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more numerous ambiverts. The introvert may be found sitting alone in a corner talking to only one other person. The extravert will have talked to nearly everyone at the party, moving continually from one group to another, loud in his expressions of affect and initiating, terminating, and interrupting conversations with great ease. Obviously, the situation in which behavior is studied is crucial. If we observe our introvert and extravert watching television, there may be little correlation between the personality trait and any behavior they may exhibit. The situations relevant to a trait are sometimes implicit in the definition of the trait. Although person-situation interactions are important, it does not follow that we can measure or even speak of which is more important or how much variance they contribute to behavior (Zuckerman and Mellstrom, 1977); this will be in large part a function of the ranges of personalities and situations studied. The greater the range of either, the more variance will seem to be accounted for by that source.

C. ABNORMALITIES OF PERSONALITY The idea that the abnormal personality may simply represent an extreme of a dimension on which all persons are ranged was an insight developed by Freud (primarily in terms of underlying processes such as defenses). Hans Eysenck (1947, 1967, 1981) and Raymond Cattell (1950) were foremost in bringing the concept of dimensions of personality into scientific terms, providing operational measures of the dimensions and experimental methods for testing the validity of their methods of assessment. On the phenomenal level this continuum is easier to see for some abnormalities than others. Neurosis, for instance, can be seen as the extreme that defines a dimension ranging from extreme emotional instability and dysphoria to extreme self-confidence and imperturbability at the other. Most of us are in the middle range on this dimension, but periodically we may temporarily move in one direction or the other, as when an unavoidable stress pushes us up on the neurotic dimension or when too many drinks produce a psychopathic sense of invulnerability and the reckless behavior that may accompany this sense. In other abnormalities such as schizophrenia, the dimensionality is harder to see, and we seem to have a true discontinuity between the normal and abnormal. Studies of the relatives of schizophrenics and the general population, however, have revealed large numbers of persons with lesser degrees of some of the traits that comprise the clinical condition including looseness of thinking, lack of emotional reactivity, and

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lack of significant interpersonal relationships or any desire for them. When extremes on several of these traits are combined, we see the vulnerability to the condition that is finally manifested in blatant and overt symptoms such as delusions and hallucinations. Some traits (such as neuroticism) are necessarily abnormal at the extremes of the dimension, but others such as sensation seeking or introversion-extraversion are only abnormal (in the adaptive sense) when extremes on those traits are combined with extreme degrees of other traits. Most sociopaths are high sensation seekers (Emmons and Webb, 1974; Blackburn, 1978; Zuckerman, 1978), but most high sensation seekers are not sociopaths. One must have other traits such as an abnormally low degree of empathy, guilt, and anxiety for his sensation seeking to assume sociopathic expressions. These are not traits that are correlated with sensation seeking, hence some sensation seekers may be sociopathic whereas others are not.

D. EXTRAVERSION, NEUROTICISM, PSYCHOTICISM, AND

SENSATION SEEKING

Because there is a large, but not infinite, number of traits that can be distinguished in the language of a given society (Allport and Odbert, 1936), the methods of factor analysis have been widely employed to determine what basic dimensions of personality emerge when the correlations between traits are used to group them into clusters. Because traits may be grouped into narrower or broader groups, the answer to the question of how many trait dimensions are necessary to describe personality depends on the level of analysis at which one wishes to work. More narrow, specific traits may be better for prediction of specific behaviors in specific situations (Zuckerman 1979a), but broader traits may be more reliably identifiable across methods of measurement (Becker, 1960). Broader traits may have lower but wider prediction value and may have more relevance to neurobiological factors of interest. T h e most widely replicated system of personality description is that developed over the last 30 years by Hans Eysenck. Unlike other test developers, who have not gone far beyond the factorial experiments used to derive the questionnaires, Hans Eysenck (1967, 1981) has demonstrated the validity of his dimensions in a variety of naturalistic and experimental studies. Furthermore, Eysenck has developed a biosocial model incorporating brain function and learning theory to explain the traits in his system. There are three broad personality dimensions defined by questionnaire and other methods: introversion-extraversion,

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neuroticism, (emotional stability versus instability), and psychoticism (social deviance, psychopathy, aggressiveness). The third of these has been most recently studied (Eysenck and Eysenck, 1976) and incorporated into the basic questionnaire measure (Eysenck and Eysenck, 1975). Another development consists of an interest in measuring narrower traits, such as impulsivity (Eysenck and Eysenck, 1977) and sensation seeking (Zuckerman, 1971; Zuckerman et al., 1978), in order to study their role in personality structure and their biological bases. Figure 1 shows a description of two of the three dimensions in terms of trait name descriptions. It is interesting that a similar system is found in the writing of Galen, a Greek physician who lived in the second century A.D., who descibed the four temperaments: melancholic, choleric, phlegmatic, and sanguine. Galen conceived of these temperaments as pure types rather than extremes on two dimensions of personality. He had a biochemical theory of these temperaments that related them to the balance of body humors: phlegm, blood, black bile, and yellow bile. This early biochemical theory, like the ancient Greek atomic theory, may have been right in the general idea, if wrong in the details. Wundt (1874) translated these temperaments into two dimensions: changeability and emotionality. H. J. Eysenck (1981) calls the changeability dimension introversion-extraversion and the emotionality dimension neuroticism. This circumplex arrangement of personality is a highly economical way of describing the major variants of traits. One could conceptualize any number of dimensions across any of the quadrants, say from sociaEMOTIONAL

EXHIbITIONIST susPIcIous

HOT-HEADED

SERIOUS

HISTRIONIC

THOUGHTFUL

ACTIVE

CHANGEABLE REASONABLE CON TROLLED PERSISTENT CONTENTED

WONEMOTIONAL

FIG. 1. Dimensions of personality. Reproduced with permission from H . J. Eysenck (1981).

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bility (stable extraversion) to dysphoria (unstable introversion). In fact Jeffrey Gray (1973, 1981) has suggested that the major dimensions of personality that are most closely aligned with biological determinants and certain conditioning phenomena are these diagonals in Eysenck’s system. T h e one described above is called anxiety and the other is called impulsivity.Some of the data presented in this chapter support this revision of Eysenck’s model. Sensation seeking (Zuckerman et al., 1964) was developed as a trait outside of Eysenck’s model even though early studies showed some correlation between this trait and extraversion (Farley and Farley, 1967, 1970). Subsequent studies (Eysenck and Zuckerman, 1978; Zuckerman, 1979b) have shown low to moderate correlations between sensation seeking and the extraversion (E) and psychoticism (P) scales in Eysenck and Eysencks (1975) questionnaire. T h e real convergence between E and sensation seeking came about as a function of a common theoretical construct used for both. The construct of individual differences in the optimal level of stimulation was the basis of the development of the first Sensation-Seeking Scale (SSS) (Zuckerman et al., 1964). This test was used initially with a theory attempting to explain the results of sensory deprivation experiments (Zuckerman, 1969). Eysenck (1963)applied the theory to explain differences in reactions of extraverts and introverts to drugs and subsequently (Eysenck, 1967) made it central to his dimension of extraversion. His theory is that extraverts feel and function better under conditions of high stimulation (up to an optimal point), whereas introverts reach a peak in positive feelings and efficient performance under conditions with relatively lower stimulation. Because stimulation usually produces cortical arousal, a second hypothesis states that extraverts and high sensation seekers function and feel best under conditions of higher arousal than introverts and low sensation seekers. Zuckerman’s theory suggested that the differences might be in the optimal level of arousal, whereas Eysenck‘s theory postulated that introverts were characteristically in states of higher cortical arousal than extraverts and therefore closer to an optimal level even in low stimulation conditions. Conversely, extraverts were supposed to be generally underaroused, therefore requiring higher levels of stimulation to bring them to an optimal level of arousal. The relative levels of tonic arousal (Eysenck, 1967) or arousability (Zuckerman et al., 1974) were believed to depend on the sensitivity of the ascending reticular activating system (ARAS) to stimulation input and the strength of the corticoreticular descending inhibition. Eysenck’s (1967) concept of the neuroticism dimension related it to the arousability of the limbic system centers that govern emotional reac-

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tivity. T h e concept suggests an interaction between extraversion and neuroticism because collaterals from the limbic system also stimulate the ARAS, thereby amplifying cortical arousal as well as autonomic system arousal. The biological basis of the third dimension, psychoticism (P), has not been fully explored, although both Eysenck and Gray feel that the gonadal hormone testosterone may be involved in this dimension because of the sex differences in P and in aggressiveness and sociopathic traits. Work described later in this chapter convinced the authors that the optimal level of cortical arousal was not a viable basis for the sensationseeking trait. Rather, increasing evidence pointed toward the neurochemistry of limbic reward and punishment systems as being somehow involved in sensation seeking. Differences in arousability between high and low sensation seekers were considered secondary to more basic differences in the responsivity of limbic systems as sensitized by their neurochemistry. This is why my book (Zuckerman, 1979b) was entitled Sensation Seeking: Bqrond the Optimal Level of Arousal.

11. Arousal and Arousability

A. TONIC CORTICAL AROUSAL Although a vast behavioral literature on conditioning (Levey and Martin, 1981) and more complex learning and performance (M. W. Eysenck, 1981) has been used to test Eysenck’s arousal theory, this chapter will be concerned only with physiological measures. EEG studies under tonic resting conditions have yielded equivocal results (Stelmack, 1981). However, after assessing this literature Stelmack concluded, “. , . one must concede that the general direction of these inquiries is toward higher levels of cortical activity for introverts under conditions intermediate between semisomnolence and stressful (p. 42).”On peripheral autonomic measures there is the usual inconsistency of results, probably because of variations in methodology. Stelmack (1981) reports some convergence of results for skin conductance levels and spontaneous variations, with introverts generally showing higher tonic levels than extraverts. A study by Cox (1977) found no difference between high and low sensation seekers in any bands of EEG activity during a resting condition. Nor have any differences in many peripheral autonomic measures

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such as skin conductance (Neary and Zuckerman, 1976; Cox, 1977), respiration, and pulse amplitude been found. Although Cox did not find a difference in heart rate, Ridgeway and Hare (198 1) found a higher prestimulus heart rate in low sensation seekers than in highs. Carrol et al. (1982) found a higher systolic blood pressure in low than in high sensation seekers during what was supposed to be a nonthreatening procedure (e.g., a physical examination by a physician). Carrol et al. (1982) tested the optimal level of arousal theory of sensation seeking in a more direct way by using drugs that either elevated or depressed cortical arousal and testing their effects on high and low sensation seekers. All subjects were administered the stimulant D-amphetamine, the depressant diazepam, and a placebo, each drug given on a separate occasion. Affective states and cognitive and motor performance were tested before and after the drug treatment on each occasion. T h e hypothesis, derived from an optimal level of arousal theory, was that high sensation seekers would feel best and perform most efficiently after taking a stimulant drug, whereas positive feelings and maximal performance would be obtained for the low sensation seekers after taking the tranquilizer. The results showed positive effects for both high and low sensation seekers after taking the stimulant and no interaction between the personality trait and drugs. High sensation seekers reported greater enjoyment of all drugs than the lows. There is some evidence supporting the hypothesis that extraverts are generally in a lower state of activation than introverts in conditions in which they are awake but unstimulated. Similar conclusions cannot be drawn for sensation seeking, although there may be some cardiovascular hyperarousal in low sensation seekers in certain kinds of stress situations. Ample evidence is available showing that neurotics, particularly anxiety neurotics, are in high states of arousal even when not stimulated (Lader, 1975). They have been shown to have higher EEG activity, skin conductance, heart rate, and blood pressure. This is congruent with the self-reported symptoms of anxiety neurotics describing uncomfortable cardiac, respiratory, and sweating states. Although laboratory evidence of hyperarousal supports these complaints for clinical neurotics, neuroticism or anxiety trait measures do not often correlate with psychophysiological measures of activation in a normal population except under certain conditions of mild stress (Hodges, 1976). There are a number of problems in peripheral psychophysiological measures of autonomic arousal that limit their effectiveness in less than extremely anxious individuals.

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B. AROUSABILITY Psychophysiological responses to stimulation have revealed differences between introverts and extraverts and between high and low sensation seekers. T h e average evoked potential (AEP) measured from the EEG in response to repeated stimulation such as light flashes or tones has been particularly useful in these studies. T h e early components of the AEP wave form assess the initial impact of stimulation on the cortex and therefore are more valuable than less direct peripheral measurements such as heart rate o r palmar skin conductance for testing Eysenck's theory, which relates extraversion-introversion to cortical arousal. In support of this theory both Hendricksen (1973) and Stelmack et al. (1977) found greater amplitude of the AEP in introverts than in extraverts in response to tones of moderate intensity. However, Rust (1975) was unable to find any correlation between AEP amplitudes in response to a broad range of intensities (55 to 95 dB) and extraversion. Stelmack (1981) believes that his positive results, obtained using a low-frequency tone (500 Hz) rather than the 1000 Hz tone used by Rust, are valid since Stelmack could not find introversion-extraversion differences using a higher frequency (8000 Hz). Stelmack has reviewed similar findings with the electrodermal orienting reflex (skin conductance response), with introverts showing greater response than extraverts to lowfrequency and medium-intensity (dB) tones. An interaction between introversion-extraversion and stimulus intensity is predicted from the optimal level of stimulation theory. The interaction of the trait with stimulus frequency may be due to the greater individual variability (variance) found with low-frequency, as compared to high-frequency tones.

C. AUGMENTING-REDUCING OF THE AVERAGE EVOKED POTENTIAL A usefr_llmeasure of individual differences in the AEP was developed by Buchsbaum and Silverman (1968). The method consists of measuring AEP amplitudes to a range of stimulus intensities from low to high and describing the relationship between AEP amplitude and stimulus intensity as an individual characteristic. A high positive slope function is described as augmenting and a low or negative slope is described as reducing. This augmenting or reducing characteristic of individuals is reliable over time, shows strong genetic control (Buchsbaum, 1974), and constitutes a behaviorally relevant biological trait in cats as well as humans (Hall et al., 1970; Lukas and Siegel, 1977). Studies by Buchsbaum

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(1971), Coursey et al. (1975), Lukas (1982), von Knorring (1981), and Zuckerman et al. (1974) have shown a positive correlation between sensation seeking and augmenting of the AEP. The last three of these studies used forms of the Sensation-Seeking Scale (SSS) that contain subscales, and all three found that one particular subscale of the SSS is more highly related to augmenting than the others. The scale is called Disinhibition and represents the seeking of sensation through social activity rather than through risky sports (Thrill and Adventure Seeking). The Disinhibition subscale correlates more highly than the others with measures of extraversion and impulsivity (Zuckerman, 1979b) and seems to be the one most related to the clinical concept of sociopathy (Zuckerman, 1978). Eysenck’s Personality Inventory (EPI) was also used in the Zuckerman et al. (1974) study, but neither extraversion nor neuroticism correlated with augmenting of the AEP. von Knorring et al. (1974) compared AEP augmenters and reducers on the EPI in a group of depressed patients. Augmenters and reducers did not differ on extraversion, but augmenters scored significantly higher on the neuroticism scale. Barratt and Patton (1983) have reported that a scale devoted to measuring impulsivity exclusively correlated with augmenting of the AEP. It appears that both the impulsivity aspect of sensation seeking as measured by the Disinhibition subscale and the impulsivity aspect of extraversion are related to high arousability in response to high levels of stimulus intensity or augmenting. Low impulsives or low disinhibitors show some kind of inhibitory neural response to high intensities. Both the optimal placement of the EEC active lead at the vertex, and recent work with visual EP tomography (M. S. Buchsbaum, personal communication) suggest that the differential cortical response of augmenters and reducers is localized in the sensory-motor cortex, although the controlling mechanisms may extend caudally.

D.

ORIENTING,

DEFENSIVE, AND STARTLE REFLEXES

In contrast to the weaker electrodermal orienting reflex (OR) of extraverts male high sensation seekers tend to show a stronger electrodermal OR than low sensation seekers in response to visual and auditory stimuli (Neary and Zuckerman, 1976; Feij et al., 1984), although this result could not be replicated for auditory stimuli by Cox (1977) or Ridgeway and Hare (1981). The latter two studies did find a relationship between the heart rate O R (deceleration of heart rate in response to a stimulus) and sensation seeking, particularly that of the Disinhibition

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type. Low disinhibitors showed a marked acceleration of heart rate instead of a deceleration on the first trial. These findings of Ridgeway and Hare for heart rate replicate those of Orlebeke and Feij (1979) relating heart rate responses to the Disinhibition scale. However, Orlebeke and Feij believe that the acceleratory response represents a defensive reflex, whereas Ridgeway and Hare think that the responses of low sensation seekers represent startle reactions as well as defensive components. Although anxious neurotics are characterized by high levels of tonic arousal, they actually show weaker ORs to specific stimuli than more stable persons (Lader, 1979). Persons high on test-measured neuroticism or anxiety trait do not seem to differ from those with low scores on the OR, but state anxiety measured at the time of the experiment did correlate negatively with the electrodermal OR amplitude (Neary and Zuckerman, 1976). So far, there has been little psychophysiological study of the P dimension of Eysenck’s system. To the extent that this dimension is related to sociopathy, we might expect to find EEG and electrodermal hypoarousal and hypoarousability because these are the most common findings with this clinical group (Hare, 1970). Taking manic-depressives as a possible extreme of this dimension, we would expect to find persons high on P to be augmenters of the AEP because individuals with bipolar affective disorders tend to be augmenters (Buchsbaum et al., 1973). Schizophrenics, however, tend to be reducers on the AEP (Landau et al., 1975) and tend to have high tonic levels of arousal. The P dimension of personality is not specific as to the type of psychopathology to be found at the extreme and may represent too broad a dimension to relate to biological dimensions. E. SUMMARY

Introverts and anxious or neurotic persons seem to be characterized by high levels of tonic arousal, both cortical and autonomic. Sensation seeking is not related to tonic arousal except possibly in the cardiovascular system. Introverts tend to be arousable in response to moderateintensity, low-frequency auditory stimuli as do high sensation seekers, who are characteristically more extraverted than introverted. Neuroticism is related to weak arousability in response to novel stimuli, possibly because of the limitation of response ceiling by high tonic levels of arousal. Given these psychophysiological characteristics, we must go further and ask what makes for these general differences in arousal or arousabil-

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ity of neural systems. For these answers we turn to biochemical studies of personality. 111. Biochemical Studies

A. GONADAL HORMONES T h e consistent sex differences and the decrease in sensation seeking with age, particularly on the Disinhibition and Thrill and Adventure Seeking subscales, suggested that differences in gonadal hormones such as testosterone might play some role in the trait. Both Eysenck (1967) and Gray (1973) felt that the P dimension might be affected by testosterone levels because of sex differences in aggressivity, which trait they feel is a major part of P. Daitzman et al. (1978) correlated plasma androgen levels with the SSS form IV in two samples of males and estrogen levels with the SSS in one of the male samples and one small sample of female college students. In both male samples androgen correlated significantly with the Disinhibition subscale. Estrogen correlated significantly with both the General and Disinhibition scales in the male sample and with Disinhibition alone in the female sample. Because the Disinhibition subscale seemed to represent the type of sensation seeking most specifically associated with gonadal hormones, another study (Daitzman and Zuckerman, 1980) was undertaken using male subjects who scored in the high or low range of this subscale and comparing them on plasma testosterone, estradiol, estrone, and progesterone levels. A variety of other personality scales including the Minnesota Multiphasic Personality Inventory (MMPI) (Hathaway and McKinley, 195l), the California Psychological Inventory (Gough, 1957), the Buss-Plomin (Buss and Plomin, 1975) Emotionality-Activity-Sociability-Impulsivity (EASI) scale, the Bem (1977) Sex Role Inventory, the Eysenck and Eysenck (1964) Personality Inventory, and the Human Sexuality Questionnaire (Zuckerman et al., 1976) were also administered. High-scoring subjects on the Disinhibition scale had significantly higher levels of testosterone, 1 7 4 estradiol, and estrone than those with low scores. T h e difference in progesterone levels was not significant. These findings confirmed the results of the prior study in finding a positive relationship between the disinhibition type of sensation seeking and both androgen and estrogen levels. Testosterone and estradiol correlated with a number of other scales used in the study. A factor analysis of the psychological and hormonal measures was done to describe the

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pattern of these correlations. A factor-loading plot of the first two factors is shown in Fig. 2. Factor dimensions are named in terms of the variables loading highest on them. The horizontal dimension (Factor I) was identified as Stable Extraversion versus Neurotic Introversion. Sociability scales identified the positive end of this dimension, and social introversion and neuroticism scales (depression, psychasthenia, neuroticism, emotionality) marked the negative end. Testosterone levels loaded toward the positive end of this dimension. Factor I1 was called Social Deviancy versus Social Conformity. The positive loading scales from the MMPI such as F (response deviancy), Schizophrenia, Psychopathic Deviate, and Hypomania seem to identify Eysenck‘s P dimension. Unfortunately, the EPI used in this study contained E and N scales but not the more recently developed P scale. At the negative end of the dimension we find scales measuring achievement need, conformity, and the need to make a good impression on others. Estradiol levels loaded positively on this dimension. In the quadrant lying between the positive poles of the two factors we find various representatives of impulsive extraversion: hypomania, psychopathy, sensation seeking, and heterosexual experience. In the diagonally opposite quadrant are scales measuring Responsibility and Socialization. This dimension might be called stable introversion versus impulsive (P) extraversion. Testosterone in males seems to be related to stable extraversion, extradiol to social deviancy (P), and both gonadal hormones to sensation seeking and impulsivity dimensions. Gonadal hormones regulate general activity (Broverman et al., 1968) and androgens increase the likelihood of aggressive and sexual behavior in the males of many other species. Maccoby and Jacklin (1974) in an extensive review of the literature on sex differences conclude that aggressiveness is one of the few traits in humans in which sex differences are based in part on a specific biological factor, testosterone. However, the role of testosterone in individual differences in aggressivity in human males is not firmly established (Rose, 1975, 1978).There is evidence (Ehrhardt et al., 1968)showing that prenatal androgenization can influence the later developing personality in girls leading to a greater interest in “rough and tumble play” and outdoor sports and less interest in dolls, babies, and more typical feminine activities. These differences have been found in comparisons with female siblings not influenced by prenatal androgens, suggesting that the familial sex-role attitudes are not a crucial determinant of these behaviors in the androgen-influenced girls. There has been little study of the effects of gonadal hormones on dominance and sociability

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in humans prior to the Daitzman and Zuckerman (1980) study. Ehrenkranz et al. (1974) found that a socially dominant group of prisoners had higher testosterone levels than less dominant and aggressive prisoners. A variety of heterosexual experiences correlated significantly with both testosterone and estradiol in the Daitzman and Zuckerman (1980) study, which is consistent with the role of testosterone in sex behavior in other species. The involvement of estradiol in these behaviors is less clear, although typically only testosterone has been assessed in studies of male sexual behavior. Much of the estradiol in males comes from the conversion of testosterone to estradiol; therefore, both could be simply an indicator of gonadal activity in these males (but levels of the two hormones are not correlated in this study). There is evidence showing that very low androgen levels in postpubertal males do affect sexual arousal, activity, and fantasy but do not necessarily abolish sexual drive completely. As with aggressivity the role of testosterone in the normal range of individual differences in sex drive is not clear. There is evidence that testosterone levels can be affected by sexual arousal in monkeys (Michael and Zumpe, 1978) and men (Pirke et al., 1974); causal relationships are not clear. This is an inevitable problem with correlational studies of testosterone and behavior, as well as with other biochemicals that show day-to-day fluctuations in level. The gonadal hormones play an important role in regulating the metabolism of several central neurotransmitters, and this may account for their role in increasing activity or general drive level. One influence is on the monoamine systems through their inhibitory effects on monoamine oxidase (MAO), a vital enzyme in the degradation of the monoamines norepinephrine, dopamine, and serotonin.

B. MONOAMINE OXIDASE (MAO) The enzyme M A 0 is present in all tissues including brain with the highest brain concentrations in the hypothalamus (Barchas et al., 1972). Studies of humans have relied largely on measurement of M A 0 from blood platelets. Although the relationship between brain M A 0 and platelet M A 0 is uncertain, a broad variety of clinical and behavioral studies in humans and other species suggests that platelet M A 0 must have some significant relationship to brain MAO. In the discussion to follow, references to M A 0 will be to platelet MAO, except where otherwise indicated. M A 0 constitutes a reliable biological trait in humans. Variations oc-

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cur within subjects, but they are small compared with between-subject differences (Murphy et al., 1976). A large range of platelet MA0 levels has been found in normal adult populations (Murphy et al., 1976) and a similar range has been found in newborn infants (Sostek et al., 1981). MA0 levels seem to be strongly under genetic control (Murphy, 1973; Nies et al., 1973; Pandy et al., 1978). Females have higher levels of MA0 than males at all ages between 18 and 60 (Murphy et al., 1976), but these sex differences were not found in newborns (Sostek et al., 1981). Although one study (Robinson et al., 1971) found an increase in M A 0 levels with age, a larger sampling by Murphy et al. (1976) did not show age changes in this enzyme. Significant negative correlations were found between the General Sensation-Seeking Scale (SSS) and MA0 in two studies (Murphy et al., 1977; Schooler et al., 1978). These studies included two groups of normal males and two groups of normal females. T h e correlations were significant in both groups of males and one of the two groups of females. Schalling et al. (1983) used a Swedish translation of the SSS and their own sensation-seeking scale called “Monotony Avoidance.” They found significant negative correlations between their Monotony Avoidance and MA0 and nearly significant relationships with the translated SS scale. Perris et al. (1980) also found significant negative correlations between the Monotony Avoidance scale and M A 0 in a sample of depressed patients. Johansson et al. (1979) compared chronic pain patients who had low or high M A 0 levels. The low MA0 level patients were higher on the General and Experience-Seeking SS scales than the high-MA0 subjects. These negative correlations between sensation-seeking traits and MA0 indicate that high sensation seekers tend to be low on MAO. In both American studies significant correlations were also found between plasma amine oxidase (AO) and the SSS. Some findings have indicated a similar negative relationship between Eysenck’s Extraversion scale and M A 0 in schizophrenics and two samples of normals, one consisting of male students (Gattaz and Beckman, 1981) and one of middle-aged men and women in a rural population (Demisch et al., 1982). I n the latter study the relationship to extraversion was found in males but not in females. The less consistent findings relating sensation seeking and extraversion to MA0 in females could be related to the fact that M A 0 levels are affected by estrogen, and levels of this hormone vary markedly in women during different phases of the menstrual cycle. T h e findings relating M A 0 levels negatively to extraversion and sensation seeking are consistent with behavioral observations of highand low-MA0 monkeys (Redmond and Murphy, 1975; Redmond et al.,

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1979), and humans (Coursey et al., 1979). High-MA0 monkeys in a colony tend to be solitary, inactive, and passive; low-MA0 monkeys tend to be active, to make many social contacts, and to engage frequently in play. Other animal studies on the effect of M A 0 inhibitors, which reduce M A 0 activity, show that these MAOIs produce hyperactivity and increase activity in a novel environment in rodents. Coursey et al. (1979) found that low-MA0 college students of both sexes reported spending more time in social activities than high-MA0 subjects. The low-MA0 males were more likely than high-MA0 males to use drugs, smoke cigarettes, and have a record of conviction for a criminal offense. The sociability findings confirm the extraversion-MA0 relationship at a behavioral level, and the risk-taking antisocial activities characteristic of low MA0 males fit the sensation seeking-MA0 relationship. Low-MA0 male monkeys also tend to engage in socially agonistic and sexual activities more than the high-MA0 types (Redmond et al., 1979). The hypothesis that gonadal hormones regulate MA0 levels (Broverman et al., 1968) has received some confirmation in humans in a study of depressed women treated with estrogen therapy (Klaiber et al., 1979). Estrogen significantly lowered the elevated MA0 activity in these women to 63% of the mean pretreatment level. M A 0 inhibitors (MAOIs) were among the first antidepressant drugs and seem to act primarily by an activation of motivation and activity level in more retarded depressives. Low MA0 levels have been found in bipolar but not in unipolar depressives (Murphy and Weiss, 1972; Wyatt and Murphy, 1976). This result is interesting in view of the following facts. 1. Mania represents a behavioral extreme of sensation seeking.

2. The hypomanic scale of the MMPI is the highest and most consistent MMPI correlate of the SSS (Zuckerman, 1979b). 3. Nonhospitalized and currently well subjects with a history of manic-depressive episodes score higher on the SSS than a control group (Zuckerman and Neeb, 1979). 4. Affective disorders are common in families of low-MA0 students (Buchsbaum et al., 1976). Low MA0 seems to be one factor in bipolar affective disorders, but it is not a sole determining factor, for low MA0 levels are found in the well relatives of these patients (Leckman et al., 1977), as well as in normal sensation seekers and extraverts. Low MA0 levels are also found in chronic schizophrenics (Buchs-

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baum and Rieder, 1979), chronic alcoholics (Sullivan et al., 1977; Major and Murphy, 1978), and chronic drug users (Stillman et al., 1978). Low MA0 levels seem to constitute a nonspecific genetically inherited risk factor that predisposes either group toward a normal extraverted-sensation-seeking personality o r to certain kinds of psychopathology. T h e risk factor may be a lack of stable regulation of the monoamines, which have been implicated in affective disorders and schizophrenia. A review of the effect of MAOIs (Murphy, 1977) show that these MAO-lowering drugs may produce marked behavioral changes including hypomanic behavior, euphoria, hostility, aggression, hallucinations, and paranoid episodes in patient or normals who seem to be particularly vulnerable.

C. MONOAMINESYSTEMS: ANIMAL STUDIES The growing literature linking MA0 with human personality traits (particularly extraversion, sensation seeking, and impulsivity) and narrower human traits (such as sociability) implicate the monoamine systems partially regulated by MA0 in the brain in these traits. As with MAO, studies of living humans must be limited to peripheral measures of these neurotransmitters and their metabolites and enzymes obtained from cerebrospinal fluid (CSF), plasma, and urine. Although a vast literature has accumulated on these indirect measures in abnormal humans (psychiatric patients), the study of their relationships to dimensions of personality in normals is just beginning. However, studies of other species have allowed more direct experimental investigation of biochemical-behavioral relationships. One of the primary methods used to study the roles of brain systems in motivation is intracranial self-stimulation (Olds and Milner, 1954). Poschel and Ninteman (1964) found that administration of an MA0 inhibitor (tranylcypromine) markedly increased rates of response in rats reinforced by stimulation of their medial forebrain bundle. This study was followed by a more comprehensive one (Poschel, 1969), which studied the effects of M A 0 blockage on various areas of the rat brain. The major excitatory effects of M A 0 inhibition were found in the A10 cell group, sometimes referred to as the mesolimbic dopamine area. As in the previous study the area of the medial forebrain bundle showed the highest rates of response to the MA0 blockade. These studies suggest that M A 0 in the brain is involved in regulating the reward potential in the catecholamine systems, and thus the effect of blockading M A 0 is to increase sensitivity to stimuli associated with reward.

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1. Dorsal Norepinephrine (NE) System Originating in the Locus Coeruleus (LC)

There is considerable controversy over the behavioral role of this system. Table 1summarizes some of the views and the kinds of evidence on which they are based. Gray (1982) and Redmond (1977) and Redmond and Huang (1979) believe that this NE system controls responses to signals of punishment or nonreward, characterized as “anxiety” or “fear” (the distinctions made between these last two terms are relevant to humans only). Both of these investigators also believe that this system plays a role in responses to novelty, which Gray conceives of as part of the anxiety function. Mason and Fibiger (1979) feel that the response to novelty is the major function of this system but that it has little to do with fear unless the novel stimuli have been associated with fear responses. Redmond says that the system may function as a novelty detector at low levels of activity and this may not be a fear response. What he implies is an optimal level of arousal theory of the NE system with adaptive attention enhancement at the low end and paralyzing fear response at the high end. In contrast to these views of the dorsal NE system as mediating fear, we have the reward hypothesis of Crow (1977) and Stein (1974, 1978). These researchers have suggested that this system mediates positive reinforcement and sensitizes the organism to stimuli associated with reward rather than those associated with punishment. Ellison’s (1977) view is similar in that he regards this system as associated with “positive affect related to goal directed approach arousal.” However, Ellison, extrapolating from chemical lesioning studies, concluded that NE mediates fear response in situations that are novel to the animal. Thus, Ellison’s view encompasses both the reward and fear hypotheses, suggesting that either type of response may be related to NE depending on whether the animal is in a familiar or novel environment. In the familiar environment of a rat colony NE levels are positively related to activity, social dominance, and aggression. File et al. (1979) and Sheard (1977) have also suggested that the NE system controls some components of aggressive behavior. Panksepp’s (1982) hypothesis is that the NE system mediates “general arousal of all emotive systems.” This hypothesis encompasses the positive emotional arousal of reward and the dysphoric arousal associated with fear, as well as arousal of anger or the positive emotions of expectancy associated with predatory aggression and sex. Aston-Jones and Bloom (1981) have also proposed a relatively nonspecific arousal function for the NE system centered in the locus coeru-

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leus (LC). Recording spontaneous and sensory-evoked discharge from LC neurons in unanesthesized rats, they found a direct relationship between behavioral arousal and orienting and LC discharge. However, discharge decreased during grooming and consummatory activity, as it did in sleep. Pronounced LC discharges were noted in response to nonnoxious but novel visual, auditory, and somatosensory stimuli, and these responses were related to behavioral orienting. Responses habituated with repetition of the stimulus and fluctuations in magnitude of response were related to level of arousal as measured by the EEG. In other words this system functions like a second arousal system (the ventral ARAS being the first), biasing attention and behavior toward the external world. The concept of an optimal level of arousal could be applied to this system as it has been to the ventral reticular system: Intermediate levels of LC discharge in a waking organism may be optimal, whereas higher levels may be related to the inhibition and disorganization characteristic of anxiety. Aston-Jones and Bloom believe that the anxiety hypothesis of the NE-LC function was based on the fact that anesthesized rats are unresponsive to stimuli in the normal range, and only responses to intense stimuli related to anxiety responses were studied. “The present results indicate a much broader range of environmental influences on NE-LC discharge, and therefore a more general role for this system in brain and behavioral activity (p. 899)” (Aston-Jones and Bloom, 1981). T h e reason for the widely diverging views of the role of the NE system can be understood if one studies the column in Table I describing the evidence on which the hypotheses are based. Proponents of the fear hypothesis rely heavily on evidence that antianxiety drugs and NE lesions have similar effects in decreasing inhibition of response produced by stimuli associated with punishment. They also point to startle or feartype responses produced by LC stimulation and decreased by NE lesions. Mason and Fibiger (1979), however, find that the evidence from their studies does not support an antianxiety effect of NE depletion: such depletion merely produced a reduced response to novel stimuli and less activity, exploration, and consummatory behavior in general. The latter findings are compatible with Crow’s (1977) and Stein’s (1974, 1978) views of the reward function of the NE system. Their hypothesis is based largely on the intracranial self-stimulation studies. Drugs that deplete catecholamines (CA) or block CA receptors reduce o r eliminate responding for self-stimulation of CA neurons. When NE is injected into the ventricles of NE-depleted rats, self-stimulation is resumed. However, as Gray and others have pointed out, depletion of NE does not affect learning for natural rewards (e.g., food), and the effect may be specific to the self-stimulation technique. Critics of this method point out that

TABLE I VIEWSOF THE FUNCTION OF THE DORSAL NOREPINEPHRINE (NE) SYSTEM Authors ~~

Role of the system

Evidence from studies of animals

~

1. Antianxiety drugs reduce activity of the system. 2. Stimulation of the locus coeruleus (LC) produces typical reactions seen in monkeys in response to threat. 3. LC lesions reduce “freezing” responses to foot shock. 4. LC lesions decrease magnitude of unconditioned startle. A. Anxiety (fear) response; 1. Antianxiety drugs reduce activity of Gray ( 1982) the system and responses to stimuli controls a “Behavioral associated with punishment or nonInhibition System” reward, but not to actual punishB. Controls orienting ment or nonreward. reflex (response to novel 2. NE lesions have effects similar to stimuli) those of antianxiety drugs (as in 1 C. Increases arousal above). D. Sensitive to signals of 3. NE lesions decrease startle response punishment or nonand partial reinforcement effects reward (presumably based on nonreward). A. Mediates response to 1. LC stimulation activates other Mason and novelty, approach to systems as well as NE one. Fibiger (1979) novel stimuli, and habitu- 2. One-way active avoidance response ation of fear in response not affected by NE depletion. to such stimuli $these 3. Acquisition learning of two-way stimuli are associated avoidance actually improved by NE with fear depletion. 4. Conditioned emotional response (Sidman Avoidance Paradigm) is not altered by NE depletion. 5 . Social interaction test of anxiety is not altered by NE depletion. 6. NE depletion produces more inhibition of activity and consummatory behavior in a novel environment. 7. NE depletion produces less consumption of novel flavored substances. 1. Drugs that release NE increase A. Reinforcement of beCrow (1977) intracranial self-stimulation (SS). haviors associated with Stein (1974, 2. Drugs that deplete NE or block NE reward 1978) receptors decrease SS. B. Guides response selec3. When NE is injected into the vention in line with previtricles in NE-depleted rats, SS is ously rewarded responses restored.

Redmond (1977) A. Anxiety (fear) response, but generally an “alarm Redmond and system” Huang (1 979) B. At low levels of activity a “novelty detector, stimulus enhancer, or attention provider”

(continued )

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TABLE I (continued) Authors

Role of the system

Evidence from studies of animals 4. SS is obtained from electrodes implanted in the LC, as well as other

File ef al. (1979) Sheard (1977)

A. Controls some components of aggressive behavior, not general anxiety

Ellison (1977)

A. Activity and socialization B. Dominance and aggression C. Positive affect related to a goal-directed approach D. Fearful only in novel environments

Panksepp (1 982)

A. General arousal of all

Aston-Jones and Bloom (1981)

emotive systems: fear, panic, rage, and “expectancy” (appetitive behavior: foraging, predatory aggression, sex, selfstimulation). Selectively augments cortical and behavioral arousal in response to external stimulation. Suppresses more tonic, vegetative functions.

areas associated with the NE system. 1 . No effects of NE lesions (by 6OHDA) on anxiety in social interaction test or inhibition of drinking in a novel situation. 2. Stimulation of lateral hypothalamus and arnygdala in cats elicits aggression and increased turnover of NE. 3. Drug-induced release of central NE elicits spontaneous fighting between rats. 4. NE lesions increase shock-elicited fighting and defensive aggression in response to a cage intruder. 1. NE-lesioned (by 6-OHDA) rats in a colony are lethargic, submissive, and nonaggressive and remain in burrows. 2. NE-lesioned rats underconsumed sucrose even when food deprived. 3. NE-lesioned rats in open field test locomote more and rear less than controls. Opposite effects for serotonin-depleted rats (assumed to have elevated NE). 1. Lack of support for a specific role of NE in SS. 2. Evidence for a nonspecific arousal role of NE.

1. Spontaneous discharge of LC neurons varies with sleep stage and waking. Discharge decreases during sleep, grooming and consummatory activity. 2. Pronounced LC response to nonnoxious auditory, visual, and somatosensory stimuli related to behavioral orienting.

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many pathways may be stimulated by the intracranial current other than the one identified as the source of reinforcement. Studies attempting to replicate findings of self-stimulation of the NE-containing neurons of the locus coeruleus (Crow et al., 1972; Ritter and Stein, 1973) have yielded conflicting results (Wise, 1978). Amaral and Routtenberg (1975), for instance, used fine wire electrodes and low intensities of current in order to confine the stimulation to the LC site itself. They could not find rewarding effects of such stimulation. Furthermore, Olds and Fobes (198 1) point out that the self-stimulation in the LC is different than that obtained from other sites such as the medial forebrain bundle. LC selfstimulation requires extensive training and produces little behavioral activation. In contrast, self-stimulation produced by stimulation of dopamine neurons is rapidly acquired and is accompanied by motor excitement suggestive of an anticipation of consummatory reward (Crow, 1977). 2. Dopamanergzc System

There seems to be more general agreement on the role of the dopaminergic system (Table II), although different authors stress different behavioral characteristics. Crow (1977) and Stein (1978) describe the role of the system in energizing or activating behavior directed toward primary rewards or general exploration in search of such rewards. Panksepp’s ( 1982) hypothesis is similar in describing dopamine as biasing the animal toward behavior controlled by positive expectancy (of appetitive reinforcement). Goldberg and Silbergeld ( 1977) regard the system as involved in activation of motor activity in general without specifying any selective direction of the activity. Zigmond and Stricker (1977) say that dopamine and N E systems enhance an organism’s ability to adapt to physiological or psychological stress, its tolerance for strong stimuli, and its capacity to respond adaptively to weak stimuli. Of course, dopamine depletion affects motor pathways, and with complete destruction of dopaminergic pathways we see animals that are akinetic and cataleptic, seemingly without the capacity to feed themselves. However, the fact that such depletion also produces a motivational deficit is evident in the findings that dopamine-depleted rats may show normal motor activity when stimulated by tasty foods and the social stimulation of other rats. When their lives are threatened by immersing them in water, they manage to swim. Under less intense threats to survival they do not adapt, for instance, failing to build nests when chilled o r failing to perform a learned response to avoid painful shock. T h e dopamine system is said to control aggressive (Sheard, 1977) and sexual behavior (Gessa and Tagliamonte, 1974, 1975; Meyerson et al., 1979) in males. The increased sexual response in the male rat may be

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TABLE I1 VIEWSOF THE FUNCTION OF THE DOPAMINERCIC SYSTEMS Authors Crow (1977) Stein (1978)

Role of the systems Energizes or activates behavior directed toward primary biological rewards; activates search and exploration

Evidence from studies of animals 1. Dopamine neurons are a source

of SS behavior. 2. Associated with SS behavior is excitation of a consummatory response syndrome: sniffing, licking, gnawing. 3. This syndrome (above) is also elicited by amphetamine, which releases dopamine (as well as NE). 4. The syndrome is abolished by chemical lesions (6-OHDA) of catecholamine neurons. 5 . The increased locomotor activity induced by amphetamine is selectively abolished by 6-OHDA. A. Ability to adapt to Zigmond and 1. Lesioned (6-OHDA) rats do not physiological or psychoincrease food intake when given Stricker (1977) large doses of insulin, build nests logical stress B. Tolerance for intense when chilled, increase water intake stimuli and responsivity when made hypotensive, or perform a learned response to avoid to weak stimuli C. Activity and incentive painful shock. motivation 2. Severe lesions (90% or more neuron destruction) produce rats that are akinetic, aphagic, and cataleptic. 3. Feeding can be induced by increasing taste incentives and normal activity by placing in a social group or in water. Motor activity, hyperac1. Comparison of active and inactive Goldberg and tivity strains or active and inactive memSilbergeld (1977) bers of a strain show increased (pertains to both monoaminergic receptor activity in catecholaminergic active animals. systems) 2. Isolation induced hyperactivity is accompanied by increased levels of urinary MHPG. 3. Intracerebral and intraventricular injections of NE increase activity. 4. Injection of dopamine or amphetamine into the nucleus accumbens or amphetamine or apomorphine into the olfactory tubercule (mesolimbic dopamine system) increases activity. (continued)

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TABLE I1 (continued) Authors Sheard (1977)

Cessa and Tagliarnonte (1974, 1975) Meyerson (et al. (1979)

Panksepp (1982)

Role of the systems

Evidence from studies of animals

1. Rage behavior in the cat is elicited by L-dopa or amphetamine injection. Sexual arousal and behav- 1. L-Dopa increases mounting behavior stimulated in the ior in the male given an adequate level of testosterone. male but inhibited in the female. 2. Increased dopamine levels increase mounting, intromission, and ejaculation and decrease latency of these reactions. 3. Increased dopamine in female rate decreases lordosis (presenting) 1 . Foraging behavior evoked by Regulates "expectancy" (appetitive reinforcestimulation of lateral hypothalamus is reduced following destruction of ment) system dopamine systems. 2. Amphetamine increases exploratory activities.

A. Aggression B. Sexuality

related to the inhibition of prolactin by dopamine. Increased dopamine has opposite effects in the female rat, inhibiting normal female heterosexual behavior (lordosis) and stimulating a male pattern (mounting). The evidence presented by nearly all of these theorists links the dopaminergic system with reward motivation as expressed in exploratory, consummatory, sexual, and aggressive behavior as well as general activity. The adaptive function of the system in the maintenance of vital life activities is clear.

3. Serotonergic System There is a fair amount of agreement on the role of the serotonergic system (Table 111). Most conceive of it as an inhibitory system that acts in opposition to either of the two catecholamine systems. T h e system is involved in sleep (Jouvet, 1973) and inhibition of general activity (Crow, 1977) and emotional response (Panksepp, 1982) as well as specific consummatory activities and sexual and aggressive activities (Crow, 1977). However, Crow (1977), Stein (1978), and Gray (1982) also feel that the system is involved in fear or sensitivity to signals of punishment. Crow and Stein believe that this is the only transmitter mediating this function,

TABLE 111

VIEWSOF THE FUNCTION OF THE SEROTONERGIC SYSTEMS Authors

Role of the systems

Evidence from studies of animals

Jouvet (1969)

Sleep

Crow (1977)

General inhibition of behavior

Crow (1977) Stein (1978) Gray (1982)

Sensitivity to punishment or signals of punishment Gray sees an interaction between NE and 5-HT systems: NE alerts to important stimuli, whereas 5-HT adds “aversive” identification. Serotonin activity reduces all waking General inhibition of all activities, whereas reduction of seroemotional systems: fear, tonin increases many motivated berage, panic, and “expectancy” haviors. 1. Animals depleted of both NE and 5NA and 5-HT systems are reciprocally related; H T are underresponsive to all posidepletion of one causes tive reinforcers, overresponsive to all an increase in the negative reinforcers. Double depleted other. Both together animals are helpless in the open-field test. Animals depleted only of 5-HT counteract depression are uninhibited and dominant in the and a depletion of both colony environment but fearful in the causes depression. Serotonjn itself is an open field, freezing, staying near antianxiety and antidewalls, and hypervigilant. presssion agent. Inhibition of sexual 1. Increasing 5-HT decreases mounting in the male and lordosis (presenting) arousal and activity in in the female. Increasing 5-HT deboth male and female; a reciprocal effect to creases intromission and increases its that of dopamine in the latency in the male. male.

Panksepp (1982)

Ellison (1977)

Gessa and Tagliamonte (1974, 1975) Meyerson et al. (1979)

Drugs that deplete 5-HT produce insomnia. The effect can be reversed by administration of the serotonin precursor 5-HTP. 1. Drugs that deplete 5-HT produce increased irritability and aggressiveness, increases in sexual activity and social interaction, and hypersensitivity to environmental cues. 2. Lesions of the median raphe nucleus, which reduce 5-HT, result in increases in activity. 3. Lesions of dorsal and median raphe nuclei cause transient increases in food and water intake. 1. Drugs that deplete 5-HT disinhibit response to a stimulus associated with shock but d o not affect responding for reward. 2. This disinhibition (1 above) is reversed by 5-HTP injection. 3. The results described in 1 and 2 are found for shock-conditioned inhibition of drinking.

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whereas Gray sees the system as interacting with the NE system in this function: the NE system acts as an alerting or alarm system, whereas the serotonin system adds the specific identification of the aversive nature of the signal. The adaptive value of the system would seem to be the inhibition of ongoing activity in the face of threat, because the studies of the effects of serotonin depletion show disinhibition of response to stimuli associated with shock. However, Ellison (1977) found that animals depleted of serotonin, although uninhibited and dominant in the social environment of the colony, were quite fearful, hypervigilant, and immobile in an open field situation. T h e bulk of evidence, however, suggests that the adaptive value of serotonin during stress of any duration may be to reduce activity and to conserve energy in apathetic immobility (e.g., catatonia).

D. MONOAMINE SYSTEMS: HUMANSTUDIES It is not the purpose of this review to discuss the vast literature relating the monoamines and their enzymes and metabolites to psychopathology in humans. The dopamine hypothesis of schizophrenia and the implication of norepinephrine and serotonin in depressive disorders have been extensively reviewed by others. Very little work has been done to relate indices of central monoamine activity to normal or abnormal dimensions of personality or behavior in normal subjects. In fact this section will be largely devoted to one recent experiment, which has attempted to study a broad range of biochemical measures to personality in normals (Ballenger et al., 1983; Zuckerman et al., 1983; Ballenger et al., 1984). 1. The NIMH Study

Ballenger et al. (1983) studied the relationships between a number of biochemical measures and personality traits in a normal sample consisting of 43 male and female volunteers from the community. The normality of the subjects was assured by a psychiatric screening interview analyzed by objective research diagnostic criteria. Anyone who met the criteria for any clinical psychiatric diagnosis was excluded from the study. The sample was heterogeneous in age, but this factor was controlled by the use of partial correlations. Dietary factors were controlled by putting all subjects on a low monoamine diet at least 14 days prior to the blood, cerebrospinal fluid (CSF), and urine sampling. Biochemicals were assayed from CSF, blood, and urine. CSF was obtained from a

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lumbar puncture performed after 9 h r of bed rest and fasting in the experimental ward. a. Biochemical Measures. The most direct measure of the activity of the central NE system in this study was norepinephrine (NE) obtained from the CSF. T h e locus coeruleus, that is, the origin of the dorsal ascending NE system, also has descending tracts leading into the spinal cord. 3-Methoxy-4-hydroxyphenethyleneglycol (MHPG) is a major metabolite of NE. In this study MHPG was measured in CSF, plasma, and urine. Dopamine-p-hydroxylase (DBH), an enzyme synthesizing NE and sometimes released with it during the discharge of neural vesicles, was measured in CSF and plasma. Homovanillic acid (HVA), a metabolite of dopamine, and 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of serotonin, were measured from the CSF. Monoamine oxidase (MAO), an enzyme that metabolizes the monoamines, was measured from blood platelets, and amine oxidase (AO) was measured from plasma. Other chemicals including cortisol (measured in CSF, blood, and urine) and calcium (from CSF and blood) were related to personality variables and will be discussed later. 6. Psychologzcal Measures. The psychological variables in this study were drawn from standardized tests that have been validated as measures of broad traits derived from theory and empirical research over the last 30 years. Eysenck and Eysenck's (1975) Personality Questionnaire (EPQ) provided measures of three basic dimensions labeled Extraversion (E), Neuroticism (N), and Psychoticism (P). Although the status of the later-developed P scale is still controversial (Bishop, 1977; Block, 1977), Eysenck and Eysenck (1976) have marshaled a great deal of evidence suggesting its value in assessment. The Minnesota Multiphasic Personality Inventory (MMPI) (Hathaway and McKinley, 1951) was developed more inductively, using clinical criteria, but the scale has proven useful in personality research with normal populations, and some of the subscales seem to be related to fundamental dimensions of personality. The Social Introversion (Si) provides an excellent measure of the dimension running from stable extraversion to neurotic introversion that many investigators (Gray, 1973; Zuckerman, 1979b; Schalling, 1983; Zuckerman et al., 1983) feel is more closely aligned with biological substrates of personality than the stable extraversion-stable introversion dimension measured by the EPQ. The Ego Strength (ES) scale (Barron, 1953), initially designed to predict responses to psychotherapy, seems to be a general measure of emotional stability versus instability that pre-

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dicts adjustment in the normal population as well as in patients (Spiegel, 1969). The Hypomania (Ma) scale, designed to distinguish the group of bipolar affective disorders from others, is elevated in bipolars in the manic state and psychopaths and seems to measure an unstable behavioral pattern closely akin to Eysenck’s P dimension. The General Sensation-Seeking Scale (SSS-Gen, Zuckerman et al., 1964; Zuckerman, 1971, 1979b) and the Disinhibition (Dis) scale from the same test have shown a broad pattern of correlation with psychophysiological measures (Zuckerman et al., 1980), suggesting that they assess a fundamental dimension of personality similar to that assessed by P and Ma but different in some respects. Given the impreciseness of personality trait measurement, it is desirable to have some redundancy in an exploratory study of this kind. c. Results. T h e ideal way to analyze the relationships between two classes of variables in order to assess the correspondence between the basic dimensions of each set is canonical correlation. However, this method requires large numbers of subjects for stable results and is probably not appropriate for this kind of exploratory study. Furthermore, it is necessary to use partial correlations to control for the variables of age and body size (height and weight) that could affect these relationships. For these reasons our initial approach was to examine patterns of bivariate correlations between the biochemical and psychological variables. This was followed by factor analyses of all variables to place the bivariate results in a dimensional framework. Ideally, factor analysis should use large numbers of subjects, so these findings must be regarded as tentative. T h e basic findings of the bivariate partial correlations are described first. CSF calcium correlated positively with extraversion (E scale) and negatively with neurotic introversion (Si scale) and general neuroticism (N scale). The same tendencies were seen in correlations of trait tests with serum calcium, but they were not as strong or consistent. Calcium ions are found throughout the nervous system and seem to facilitate the release of all types of neurotransmitters. However, calcium found in the CSF seems to be inversely related to behavioral and brain excitability (Post, 1983). When calcium is artificially lowered in experimental animals, the subjects became hyperexcitable. Increases in calcium are anticonvulsant and sedative. Although the relation between CSF calcium and levels in brain are unknown, the data suggest that CSF and possibly blood levels in calcium may be involved in the generalized arousal or arousability that is central to Eysenck’s theory of personality. In theory both introverts and neurotics are thought to be hyperaroused, the former because of low thresholds for arousal in the CNS, and the latter because of instability of the autonomic nervous system. The Si scale,

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which measures both introversion and neuroticism, was the one most highly related to CSF calcium in a direction consistent with the theory. Correlations among CSF NE, CSF and plasma MHPG (J'imerson el aL, 1981),and CSF and plasma DBH suggested that all of these variables might be assessing aspects of a common system, the NE one described previously. However, the relationships with pyschological variables were more discrete. Cerebrospinal fluid levels of NE, plasma DBH, and plasma amine oxidase correlated negatively with the SSS General scale. Plasma MHPG and CSF and plasma DBH correlated negatively with various measures of trait anxiety, but the DBH measures (and CSF MHPG) also correlated positively with state anxiety assessedjust prior to the stressful lumbar puncture procedure. These last findings suggested that activity of the NE system might be normally low in anxious persons during nonstimulated states but might respond strongly to stressful situations. The CSF metabolites of dopamine (EIVA) and serotonin (5-HI.4A) showed little relationship with any of the psychological variables. Cortisol assayed from CSF correlated negatively with MMYI measures of Ego Strength and Hypomania, the Psychoticism scale from the EPQ, and the Disinhibition scale from the SSS. Low levels of CSF cortisol are found in individuals scoring high on these measures of nonneurotic impulsivity. The Ego Strength scale is a measure of stability inversely related to neuroticism in this study. It is interesting that serum cortisol showed an opposite pattern of correlation wit.h neuroticism variables, correlating positively with neuroticism arid negatively with ego strength. Factor analysis was used to enable us to visualize the relationships among biochemical and personality traits niore clearly. The first three major factors that emerged fr~omthese analyses were rotated. Figure 3 shows the factor plot compariiig factors I and 11. Factor I is defined by serisatiori seeking, hypomailia, a d psyc-hoticism scales and a urinary riieasure of hIHYG on the positive pole, ant1 I)y (:SF norepinephrine and cortisol at the negative p"le with lower louclings from sex uni cortisol, plasma DBH, and plasma amiiie oxklase at t.liis pole. 'l'his factor migli t be labeled senstrtion sucking--impzrbi-cd~ (P diinension in E: ysenc.k 's sys t e i n ) . 'Hie loadings of the bioc.helriica1 variables at the negative pole of this factor suggest th;it cortisol niay play soiiie regulatory role in the central iiervous system, perliaps iiiliilitiiig tlie impulsive tendeucies involved in seiisat.ior1seeking and Iippmauic teidencies. Cerebrospinal H i d levels of NE loaded negatively, hut a major NE iiietal)olite, X.II1PG iiieasui ecl from urine, loaded positively on this factor. It sliould be iiot.ed i l i a 1 ;~ltliougha high positive coi,relatioii ( r = .7 1, p < . M I ) was founcl hetweeii CSF and i h s i i i a hIIII'C i i i this st.udy a i d ;I illoderate coi relariwi

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was found between CSF NE and plasma MHPG ( r = .53;p < .001), urinary MHPG correlated only with total plasma MHPG. In a previous study (Buchsbaum et al., 1981) positive correlations were found between urinary MHPG and sensation seeking during both resting and stressful periods of time. No correlation was found between urinary MHPG and personality trait measures in this study, and it seems to have ended up at the positive pole of this factor largely because of its negative correlation with CSF cortisol. T h e finding of a highly significant negative relationship between CSF norepinephrine and sensation seeking in the total group, as well as in males and females considered separately, confirms that there is a relationship between activity of the central NE system and sensation seeking. However, the direction of the relationship was a surprise in that the theory (Zuckerman, 1979b), based in part on Stein's (1978) concept of the NE system as mediating reward, suggested that high levels of NE activity would be characteristic of high sensation seekers. T h e additional findings of a significant negative relationship between plasma DBH and sensation seeking, a finding that replicates one by Umberkoman-Wiita et

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al. (1981), is consistent with the SSS-NE finding if one assumes that plasma DBH is, to some degree, an index of DBH activity in the brain. DBH in the brain is involved in the production of NE and is presumably released with NE from vesicles in the neuron when the neuron discharges. If the negative relationship between sensation seeking and NE activity is more than an indication of a low stress response of high sensation seekers to the anticipation of the lumbar puncture, then the theory of the relationship between the NE system and this personality trait must be revised. One possible interpretation is that high sensation seekers engage in sensation-seeking activities in order to stimulate a hyporesponsive system. In essence, this interpretation is similar to the arousal concept of extraversion that postulates that extraverts seek higher levels of stimulation in order to bring an essentially low level of arousal up to more optimal levels. I n this study it was plasma A 0 rather than platelet M A 0 that loaded on the negative pole of the sensation seeking-impulsivity factor. Actually two of the studies relating platelet M A 0 to sensation seeking (Murphy et al., 1977: Schooler et al., 1978) also found negative correlations between plasma A 0 and sensation seeking, although the latter correlations were not as strong and consistent in the four groups. However, if we interpret plasma A 0 as an index of central M A 0 activity, then the results are consistent with the literature described previously in this chapter relating low levels of MA0 to impulsive, extraverted sensation seeking and sociable behavior in humans and monkeys. Factor I1 is defined at the negative pole by the social introversion (Si) scale from the MMPI and at the positive end by the EPQ extraversion scale, CSF and serum calcium, and CSF DBH. Psychologically, the factor seems to be identifiable as the stable extraversion versus neurotic introversion one described by Gray (1973), because the Si scale contains a mixture of anxiety and social introversion items, whereas the E scale from the EPQ is composed largely of sociability items. Calcium in CSF has been inversely related to behavioral and brain excitability (Post, 1983). Lowered levels of calcium in brain have produced hyperexcitability. Eysenck’s concept that the introvert in general and the neurotic introvert in particular is more generally aroused than the stable extravert is supported by the relationships with calcium. The role played by calcium in producing arousal o r dearousal is not clear, but its wide distribution in the neurons of the CNS could affect general behavioral tendencies such as those seen in introversion-extraversion. The negative relationship of CSF DBH to anxious introversion is probably a function of its negative relationship with trait anxiety. Figure 4 shows the factor plot between factor I (sensation seeking-

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et al.

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impulsivity) and factor 111. Factor 111 is clearly identified on the negative pole by the Neuroticism (N) scale of the EPQ and at the positive end by plasma MHPG. Urinary MHPG loads positively on both factors. I t would appear from both the bivariate correlations and these results of the factor analysis that unxious neuroticism is inversely related to MHPG found in plasma. T h e results seem to be the opposite of what one might predict from the hypotheses directly linking activity of the NE system to the trait of anxiety (Redmond, 1977; Gray, 1982). However, the results are compatible with other theories suggesting that at lower levels of turnover the N E system responds to novelty (Redmond, 1977; Mason and Fibiger, 1979). If we assume that neurotics generally avoid novel situations and activities in order to minimize arousal, then we would not expect to find much activity of the NE system except in situations of unavoidable stress, in which the NE system may actually overrespond. d. Summary of NIMH Results. The results of the NIMH study must be regarded as tentative and exploratory until further confirmation is forthcoming. The difficulties of human correlational research utilizing indirect measures of activity of monoamine systems in the brain cannot be overstressed. Despite the surprisingly high relationships between CSF

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NE, MHPG, and DBH and plasma MHPG and DBH, there is no guarantee that these are reliable measures of brain bioaniine and enzyme activity. Furthermore, it is difficult to ascertain whether the biochemical measures represent stable biological traits, temporary reactions to stressful situations, or some combination of trait and state. Experimental studies using plasma and urinary MHPG might clarify some of these questions. For instance, in the Buchsbaum et al. (1981) study urinary MHPG was found to be positively related to the sensation-seeking trait both in stress and nonstressful conditions. Unfortunately, this measure was not related to sensation seeking in the NIMH study. In terms of Eysenck’s dimensions the results of the NIMH study are clear, although their interpretation is still somewhat uncertain. The stable extraversion versus neurotic introversion dimension is negatively related to levels of CSF calcium; the neuroticism dimension is negatively related to plasma MHPG (as are neuroticism-anxiety measures from other tests); and the psychoticism (or impulsivity) dimension (aligned with sensation seeking and hypomania) is negatively related to CSF cortisol and norepinephrine. Although the data provide a rough and tentative map of possible biochemical bases of these personality dimensions, most of the map must still be labeled terra incognita.

E. ENDORPHINS The discovery of endogenous morphine-like peptides (endorphins) in the CNS (Hughes et al., 1975; Terenius and Wahlstrdni, 1975) has opened up an exciting new area for personality research. Because the endorphins have effects quite similar to exogenous morphine compounds, including analgesia and suppression of general arousal, levels of these peptides in the brain could have profound effects on human emotions and behavior. Stein (1978) has theorized that enkephalin is involved in the drive reduction effects of reinforcement, bringing a behavioral episode involving response for reinforcement to a “satisfying conclusion.” It is also possible that excessively high levels of endorphins might suppress arousability and produce an organism that is chronically phlegmatic and inactive and therefore unresponsive to potentially rewarding and novel stimuli: a low sensation seeker. Johansson et al. (1979) studied the relationships between endorphins in CSF and various personality traits including extraversion, neuroticism, and sensation seeking in a sample of 40 patients with chronic pain syndromes. Twenty-three of these patients were diagnosed as organic, and seventeen were diagnosed as having psychogenic pain syndromes.

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MARVIN ZUCKERMAN et

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The patients with pain syndromes of psychogenic origin had significantly higher levels of endorphins than patients with organic pain syndromes. Within the total group endorphin levels correlated positively with neuroticism and negatively with disinhibition and boredom susceptibility subscales of the SSS and a dominance scale from another inventory. If subjects were classified as high or low on endorphins, the lowendorphin subjects were significantly higher on the General SS scale and all five subscales of the SSS. They were also lower on the neuroticism scale. Neither analysis revealed an association between endorphin levels and extraversion. Endorphin levels in the study were not related to age o r sex of subjects, and there were no differences in the personality traits of psychogenic and organic pain patients, so these variables could not account for the findings relating endorphin levels to personality. As is so often the case in trying to extend findings from one population to another, these results could not be replicated in a normal population. I n the NIMH study, discussed in the previous section, Ballenger et al. (1983) assessed total opioid activity and /3-endorphin levels in these healthy normal subjects. Neither variable correlated significantly with any of the personality variables of interest, including extraversion, neuroticism, and sensation seeking.

F. BIOCHEMISTRY OF THE AUGMENTING-REDUCING OF THE EVOKED POTENTIAL T h e relationship of the various biochemical variables to sensation seeking, extraversion, and impulsivity suggests that some of these chemicals might play a role in the augmenting o r reducing of the cortical evoked potential (EP) in response to stimuli of high intensity. It has been suggested that the monoamine systems play a role in arousability with norepinephrine and dopamine augmenting arousal and serotonin dampening or reducing arousal. Endorphins would, of course, be expected to reduce arousal. M A 0 might also reduce arousal by reduction of levels of available catecholamines.

1. M A 0 Negative correlations between platelet M A 0 levels and augmenting of EPs have been found in patient groups (Buchsbaum et al., 1973) and in students showing signs of affective disorder (Haier et al., 1980), but such correlations have not been found in normal subjects in the last mentioned study or even in one study of depressed patients (von Knorring et al., 1977).

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2. Serotonin

von Knorring and Perris (1981) measured a metabolite of serotonin (5-HIAA) and augmenting-reducing of visual EPs in a mixed psychiatric group and found that low 5-HIAA patients had augmenter EP responses, whereas high 5-HIAA subjects tended to be reducers. The same tendencies were seen in a sample of chronic pain patients, but the differences between low and high 5-HIAA subjects did not reach significance. T h e precursor of serotonin, tryptophan, was also related to the EP in a smaller sample of the psychiatric group. Although the difference was in the expected direction, it did not reach significance.

3. Dopamine Patients with low levels of the dopamine metabolite HVA in the CSF tended to be augmenters, and those with high levels tended to be EP reducers (von Knorring and Perris, 1981). This difference is contrary to what one would predict on the hypothesis that dopamine facilitates arousal. 4. Norepinephrine

MOPEG, a norepinephrine metabolite assayed in CSF, was not related to augmenting-reducing of the visual EP in patients with chronic pain syndromes (von Knorring and Perris, 1981). 5. Dopamine-P-hydroxylase (DBH) Serum DBH was assayed in a large group of psychiatric patients and was found to be significantly related to augmenting-reducing; low serum DBH subjects tended to be augmenters and high DBH patients tended to be reducers (von Knorring and Perris, 1981). These findings are consistent with those showing a negative correlation between serum DBH and sensation seeking (Ballenger et al., 1983; Umberkoman-Wiita et al., 1981), because high sensation seekers tend to be augmenters and lows tend to be reducers.

6 . Endorphins A significant relationship was found between endorphin levels and augmenting-reducing in chronic pain patients (von Knorring and Perris, 1981). High levels of endorphins in CSF were related to reducing of the visual EP and low levels of endorphins were related to augmenting. T h e results are consistent with the findings in the same population relating high levels of endorphins to low sensation seeking and low levels of endorphins to high sensation seeking (Johanssen et al., 1979).

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7 . Cortisol According to Shagass and Straumanis (1978), cortisol given to normals diminishes the augmentation of the visual EP (VEP) resulting from attention to a stimulus, but no changes were found in inattentive subjects. T h e negative relationship between augmenting and cortisol is somewhat consistent with the negative relationship between cortisol and the impulsive dimension of personality.

8. Serum Calcium T h e same authors state that low serum calcium is associated with short-latency, high-amplitude VEPs. Although CSF calcium was the measure most highly related to the extraversion dimension, these results confirm the general negative relationship between calcium levels in CSF or serum and neuronal excitability. 9. Summary

There is some evidence relating augmenting of the visually evoked potential to low levels of MAO, serotonin, dopamine, DBH, endorphins, cortisol, and serum calcium. T h e results for MAO, DBH, and endorphins are consistent in direction with the findings relating them to sensation seeking and relating sensation seeking to augmenting of the VEP. Although largely correlational, the results suggest that the capacity of high sensation seekers of the disinhibition variety to respond to high levels of stimulation is related to low levels of neuroregulators that might modulate such response. T h e tendency for high sensation seekers to use drugs of all varieties, including opiates, might represent an attempt to modulate their cortical arousal by exogenous drugs that substitute for endogenous biochemicals such as endorphins. Relevant to this hypothesis is the finding by Murtaugh (1979) that former drug abusers who used primarily depressant drugs (usually opiates like heroin) tended to be augmenters, whereas those who used primarily stimulant drugs (d-amphetamine and cocaine) tended to be reducers. Perhaps the opiate users were sensation seekers with low levels of endorphins, whereas the stimulant users were characterized by low levels of central catecholamines (amphetamine releases catecholaminergic neurotransmitters).

IV. Conclusions

As is the case for most areas of science, the scientific study of personality began with the development of a method of measurement, in this

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case the personality trait questionnaire. This nomothetic approach to the study of personality has been challenged repeatedly since its beginning about 65 years ago when Woodworth developed an inventory to measure psychoneurotic tendencies in recruits during World War I. Earlier criticisms from the projective test advocates (Frank, 1939) have diminished, probably due to the proven inadequacies of their own methods. More recent critics have attacked the notion of the hypothetical trait structure (Mischel, 1968) and the usefulness of broad traits for behavioral assessment. Although assessment of narrower behavioral “traits” (if that term may still be applied) may be useful for the practical assessment needed by therapists (Zuckerman, 1979a), the broader trait assessment remains the cornerstone of attempts to test personality theories in a scientific context. In attempting to elucidate the biological basis of personality, we must begin with quantitative assessment of broad personality dimensions, for which an adequately validated trait questionnaire reniains the method of choice. Although behavior is certainly determined in no small part by specific adaptations to specific situations, modern trait measurement has set itself the goal of prediction of individual consistencies in behavior across broad ranges of situations. Biological factors in temperament are assumed to affect such broad tendencies, biasing behavior in one direction or another with due regard for perceptions of situations based on individual experience. One consequence of the dimensional approach to personality has been the tendency to regard abnormalities of behavior as extreme manifestations of selected groups of traits. While neurotics, individuals with anxiety disorders (DSM 111), and other diagnostic groups continue to be treated within the medical model as qualitatively distinct groups of individuals, there is growing recognition of the view that these disorders are mostly exacerbations of inore consisteiit, long-standing personality tendencies h a t are measurable along diirierisiotis such as neuroticism or anxiety. S e v c d of these broad dimensions of personality, such as Eysencks in troversion-extra\.‘ersioii and neuroticism (emotional instability), Zuckernian’s sensation seeking, and Gray’s iiiiplsivity have explicitly been based on biological-learnirrg theories of personality. Although our conceprs o f the basic dirnensions of persoiiality iiiay have to be (hanged as briii--helr,ivioral interat tioris are Ijetrer uridcrsii)od, these dimensions colistitiite a good stai tirig 1)oint for studies of the hiological basis of pt~r~oiulit y. I lie o i iginal model of pel son;ility folmiilatcti hy Eysenck grew out of. t o i i \ t i L I I I S tieiived f‘rom die beliavioi ist tlieoiies o f lvari Pavlov (1960) .iirtl ( l a r k I l u l l (19-43). -1 he ktercst i l l the tliscoveiies of‘the regulation

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of cortical arousal by the ascending reticular activating system led Eysenck to modify his theory and was the basis for Gray’s and Zuckerman’s initial theories of personality. This new theory centering around the idea of individual differences in an optimal level of arousal resulted in a great deal of psychophysiological research studying the reactions of the cortical and autonomic systems to varying intensities of stimulation. The results provided some evidence that introverts are generally more aroused and arousable (at low-to-moderate stimulus intensities) than extraverts and that these differences in arousability may affect their conditionability. Arousal levels of the autonomic system proved to be a distinguishing characteristic of those forms of neurosis characterized as anxiety disorders and a characteristic of the neuroticism dimension of personality. Differential effects of stimulant and depressant drugs on extremes on these dimensions, introverts and extraverts and neurotics and stable persons, were also deduced from the theory (Eysenck, 1963). However, the cortical differences between low and high sensation seekers were demonstrated at higher intensities of stimulation (Zuckerman et al., 19’74), and differentia1 drug effects could not be demonstrated (Carrol et al., 1982). Similarly, differences in orienting and defensive reflexes of high and low sensation seekers and introverts and extraverts are limited to specific ranges of stimulation. Theories of cortical reactivity must account for the interaction of stimulus characteristics and the characteristics of the reacting nervous system. The intensive study of the limbic systems and their psychopharmacology led to the next advance in biological personality theory. Generalized arousal, although an unquestionable factor in personality, carried a theoretical weight that was clearly an overload. The discovery of separate limbic systems mediating sensitivity to stimuli associated with reward and punishment led to new formulations (Gray, 1973; Zuckerman, 1979b). These theories proposed that inherited individual differences in the reactivity of these systems might furnish the basis for the differential sensitivities to learning based on the anticipations of reward or punishment. This had led to an increased interest by some students of personality in the neuropharmacology of the systems that govern their sensitivity. In particular the recent work showing inverse relationships between the enzyme monoamine oxidase (MAO) and the traits of sensation seeking, extraversion, impulsivity, and sociability in humans and other species pointed to the importance of the monoamine systems in regulating activity, mood, and appetitive and avoidant motivation. Experimental studies of nonhuman species have led to some preliminary speculations on the adaptive functions of these systems. There are

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some widely diverging theories on the role of the dorsal norepinephrine system. Although most investigators agree that this is an activating system sensitizing the organism to novel stimuli, there are major disagreements about its role in motivation. Some suggest that it is the basis of the general trait of anxiety-proneness, or readiness to learn on the basis of avoidance of punishment, whereas others emphasize its role in positive reinforcement or reward. The disagreements rest on the different kinds of methodologies as well as differences in findings. A new emerging viewpoint is that the system mediates general arousal and is responsive to a broad range of environmental stimuli whether or not such stimuli have been associated with reward or punishment or are merely novel. There is more agreement about the role of the dopaminergic system with most investigators agreeing that this is an energizing system, activating behavior toward exploration and search for reward and raising the probability of aggressive and sexual behavior. Zuckerman (1979b) has speculated that activity of this system, together with the noradrenergic system, furnishes the biological basis for the trait of sensation seeking. The serotonergic system is thought by most to play an important role in the inhibition of behavior and general activity; both Stein and Gray, who disagree about the role of the norepinephrine system, agree that the serotonergic system is involved in anxiety o r sensitivity to signals of punishment. In contrast to a growing experimental literature on the role of these systems in animal behavior, study of their role in the consistencies of human behavior that we call personality are just beginning. It is true that a great deal of research has explored the role of the bioamines and their regulating enzymes in various psychiatric disorders, but little has been done to relate the biochemistry of the monoamine systems to personality dimensions in normals. A ground-breaking study done at NIMH is reported in detail in this chapter. The results are encouraging in showing significant relationships between measured personality traits in normals and neurotransmitters and their metabolites and enzymes obtained from CSF, blood, and urine. However, some of the results have shown findings in directions that are not predicted from some of the current theories. In this kind of exploratory study we may question the reliability of the results, the validity of the methods as measures of brain biochemistry, and the theories or models that cannot accommodate the findings. Replication, study of basic methodological assumptions (particularly those presuming a relation between peripheral and central levels of bioamines and enzymes), and some tentative reorganizing of the models are all answers to the questions raised by this research. The continued

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interaction of basic experimental study done by necessity on other species and human correlational study may yet yield an understanding of the neurobiology of personality. References

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A Acct ylcholine, as opiate neurocliemical

correlate, 278-281 Adenylate kinase, in C significaiice, 128 Atlrenocorticotropic hormone nociception and, 213-214 peptide sequence of, 187 Aggressive behavior, opiates antl, 318-319 Albumin, in cerebrospinal fluid, 99-100 a-Albiimin, in CSF, 123-125 Aldolases. in CSF, clinical significance, 128 Algcsir peptides, nociception and, 188- 195 Alkaloids, opiate, effect on spinal iirtiro~is,244 y-Aiiiiiiob~ityricacid (GABA), as opiate neurocheniical correlate. 28(i-289 Aidgesia ol'i;ite-indirced, genetic aspects of.. 290-293 stress-intlucecl, J19-320 Antibody specificity, cerebrospiiial fluid proteiiis and, 115 (ArgH)-vasopi-essiri,peptitle sequence of, I87 Arotisal antl arousability. neurobiology of., 3!)8-403 Axons (dental), 40-49 cytocheniical diversity of', 47-39 intl-aclental arborizaiion of. 4 - 4 7 size of' ill root apex, 44-45 a t root f'orainen, 43, 46 types of. 40-4!) A/i(lo-A-inct h~l-.l-pipcritl\.1t,cnLo;ite, strt1cttiI.c of.. 173

B Basal ganglia, hippocampal formation antl, 342-346

437

Behavior hippocampal lesion effects on, 340-343 opiates ancl, 306-319 Bispyridiniuni oximes, structure of, 153 Bombesin nociception and, 225 peptide sequence of, 187 Brad ykinin nociception and, 188-190 peptide sequence of, 187 Brain opioid peptide distribution in, 295-297 C

Calcitonin nociceptioii ancl, 224-225 peptide sequence of, 187 Calciiiiii, i n serum. evoked potential (EP) ;1ntl, 428 Catecholamines. as opiate neurocheniical correlates, 281 -284 Cellular immunity, cerebrospinal fluid proteins ancl, 116-1 17 Central nervotis system, inuscarinic acceptors in, 139- 183 Cerebrospinal fluid (CSF) antibody specificity antl, 115- I16 cellular immunity and, 116-1 19 ci-ossiiiinitinorlcctrophoresis of', I 08- 10') CSI; Iyiiiphocyte stimulation antl. 1 18 electrophoresis of, 100- 104 abnormal proteins, 103- 104 normal, 102- 103 enzyine detrrmination in, 127-129 a-globulins, 109 y-globulins, 103- 104 i n hiiiiioral immunity, 112-115 ininiunofixation of, 108 i n iininunological problems, 1 10- 119 impitit electroinimunofixation of, 109 isoelcctric focusing of, 107- 108 isotachophoresis of, 109- 110

438

lndex

methods for study of, 96 nervous-tissue specific, 119- 127 a-albumin, 123-125 enolase, 125-127 ap-glycoprotein, 127 myelin basic protein (MBP), 121-123 S-100 protein, 119-121 in noriconcentrated CSF, 105- 107 proteins of, 95-138 qualitative studies on, 100-1 19 quantitative determination of, 97- 100 individual proteins, 97-98 nervous tissue specific, 98 total protein, 97 tumor proteins, 98 T lymphocytes and, 117-118 transfer electrophoresis of, 109 Chimeras, neurochemicdl genetics of, 384-385 Cholecystokinin nociception and, 214-215 peptide sequence of, 187 Cortisol, evoked potential (EP) and, 428 Creatinine phosphokinases, in CSF, clinical significance, 128

D Debrisoquin, structure-activity effects of, 12 Dental sensory receptors, 39-94 axonal transport mapping techniques for, 51-53 axons, 40-49 in contiiiuously erupiing teeth, 65-68 contralateral innervation, 57-59 in dentin, 53-57 in developing teeth, 61 -64 electron microscopy of‘, 51 location of, 49-68 in mature teeth, 49-51 in reinnervated teeth, 59-61 relationship t o other cells, 74-80 axo-axonic contacts, 79-80 odontoblasts, 75-79 Schwann cells and fibroblasts, 74-75 ultrastructure of, 68-74 Dentin innervation of, 53-57 sensory transduction in, 81 -84 hydrodynamic mechanisms, 81-84

(Des; Tyr l ) -y-endorphin nociception and, 212-213 peptide sequence of, 187 Dopamine, evoked potential (EP) and, 427 Dopamine p-hydroxylase (DBH) in CSF,clinical siknificance, 128 evoked potential (EP) and, 427 Dopaniinergic system, effect on behavior, 434-4113 Dynorphin nociception and, 21 1-212 peptide sequence of, 187

E Endorphins evoked potential (EP) and, 427 role in neurobiology of personality, 425-426 P-Endorphin in brain, 296-297 nociception and, 203-21 1 peptide sequence of, 187 Enkephalin(s) in brain, 295-296 nociception and, 195-203 Enolase in CSF, 125-127 clinical significance, 128 Enzymes in cerebrospinal h i d , 127-129 clinical significance. 128 Esterases, in CSF, clinical significance, 128 Evoked potential (EP), biochemistry of the augmenting-reducirig o f , 426-428 Extraversion, as personality trait, 395-398

F 14-3-3 protein, in CSF, 127 G

Genetic analysis, potentials and limitations of, 362-364 Genetics neurochernical, 361 -389 of opiate mechanisms of action, 290-294 P-Clycuronidases. in CSF, clinical significance, 128

439

Index Gonadal hormones in neurobiology of personality, 403-406 Ct-ootiiing, dopaminergic effects on excessive, 352-355 Cuanacline. structure-activity effects of, 11 Guanethidine chemical structure of, 2 oxidative phosphorylatinn inhibition by. 13-15

pharmacology of, 2-4 structure-activity effects of, 9-13 sympathetic neuron destruction by, 1-37 degree and specificity,.5-7, 23-33 discovery, 4-5 immune-mediated mechanism, 16-20 iri V h O , 8-9 mechanism, 13-20 niorphologic effects, 5-7 species-specificity, 7 -8 strain specificity, 20-23 Cuanoxan, structure-activity effects of, 12

H Hamster incisors, sensory receptors in, 67 Hippocampal damage effects on dopaminergic systems o f basal ganglia, 339-359 interaction efftcts, 355-357 neuropeptide action and, 351 -352 Hormones, effect on personality, 403-406

I Immunoglobuliii C , in cerebrospinal fluid, 99-100

K Kyotorphin nociception and, 212 pepritle sequence of, 187

L Lactate dehydrogenases, i n CSF, clinical significance, 128

Learning, opiate effects on, 31 1-314 Leu-etikephalin, peptide sequence of, 187 Locus coeruleus, dorsal epinephrine system in. 410-414 Lysosomial enzymes, in CSF,clinical significance, 128

M cu-h/Ielanocyte-stiinulating hornione nociception and, 227 peptide sequence of, 187 Melanocyte-stimulating hormone inhibitory factor nociception and, 226-227 peptide sequence of, 187 Memory, opiate effects on, 31 1-314 Mental illness, brain opiates and. 322-324 Met-enkephalin. peptide sequence of, I87 Microiontophoresis of opioids into spinal neurons, 250-251 Monoanline oxidase (MAO) evoked potential (EP) and. 426 personality dimensions and, 406-409 human studies on, 418-425 Mouse incisors, sensory receptors in, 67 Muraminidase, in CSF, clinical significance, 128 Muscarinic agonists binding of, 156-161 structure of, 140 Muscarinic antagonists binding of, 147-155 structure of, 141 Muscarinic ligands, irreversible, structure of, 173 Muscarinic receptors binding of, 147-155 cooperative and site interactions, 152-155 kinetic and equilibrium nieasuretncnts, 147-152 in central nervous system, 139- 183 ligand binding in, proposed scheme for. 176 localization of, 167-171 ontogenesis, 169- 171 post- and presynaptic typcs, 168-169 regional distribution, 167- 168 I-atlioligatitl-biriding studies on, 143-147

440

Index

receptor-receptor interaction5, 161 -166 structure-function relationships of, 174-177 Myelin basic proteiii (MBP), in CSF, 121-123

N Naloxone, effect on opioid effects on spinal neurons, 261 -26‘2 Nerve growth factor (NGF), guanethidine inhibition of transport of, 15 Nervous tissue, CSF proteins specitic for, 119

Nervous-tissue specific proteins, in cerebrospinal fluid, 98 Neurobiology of personality, 391-436 arousal and arousability, 398 biochemical studies, 403-428 dopaininergic system, 414-416 endorphin role, 425-426 evoked potential (EP) studies on, 426-428 nionoamine oxidase arid, 406-409, 4 18-425 norepinephrine system, 412-41 3 serotinergic system, 416-418 Neurochcmical genetics, 361 -389 of chimeras, 384-385 o f heterogeneous polylineal populations, 367-368 o f homogeneous populations, 369-382 bilineal strains, 374-380 quadrilineal strains, 369-374 uiiilineal strains, 380-38‘2 of natural arid experimental populations, 364-367 regulated heterogeneity and, 382-385 in heterozygous isogenic populations, 382-384 Neuropeptides, hippocampal lesion effect on activity of, 351 -352 Neurotensin nociception and, 216-222 peptidc sequence of, 187 Neuroticism, as personality trait, 395-398 Neurotransinit t e n opioid interaction with, 298-299 opioids as possible. 249 Nociception, peptides and. 185-24 I

Norepinephrine, evoked poieritial (EP) and, 427 Norepinephrine system in locus coeruleus, 410-414 function, 412-413

0 Odoiitoblasts, dental sensory receptors’ relationship to, 75-79 Opiates agonists-antagonists of, 293-294 analgesic effects of, 301 -302 behavior and, 306-319 social behavior, 315-319 diurnal and circadian rhythm of production of, 32 1 etivironinental effects of, 319-322 genetic characterization of mechanisms of, 290-294 learning and nieinory effects of, 31 1-314 mental illness and, 322-324 iieurochemical correlates of, 278-289 neuroendocrine effects of, 299 phylogerietic and genetic studies on, 302-305 receptors lor, 299-30 I olltogeny of, 305-306 opiate potency and, 302 seizures from, 307-309 sleep effects of, 309 stress-induced production of, 319-321 tolerance to, 310-31 1 learning mechanisms, 314-315 Opioids biosynthesis of, 297-298 in brain, 295-297 definition of, 294 effect on spinal new-om, 243-275 microiontol’t’oresis and pressure in-jection of, 250-251 naloxoiie effects, 261 -262 opiate alkaloids, 244 opioid peptides, 244-248 single unit recording, 240 site of action, ‘252-261 tolerance to, 262-264 interaction with ncurOiraiisiiiiiters, 298-291)

441

Index neuroendocrine effects of, 299 as possible tieurotratisinitters, 249 nociception and, 1!15-213 psychohiology of, 277-335 Oxidative phosphorylation, inhibition by guanethitlinc, 13- 15

P Pain opiates and sensitivity to, 318-319 peptides and perception of, 185-241 Peptidase, in CSF, clinical significance, 128 Peptides nociceptiori and, 185-241 algesic pcptitles, 188-395 naloxone-inseiisitive type, 216-226 naloxone-sensitive nonopioid type, 2 13-2 I 6 opioid peptides, 195-213 opioid, effect on spinal neurons, 244-248 Personality abnormalities of, 394-395 dimensions of, 392-393 schematic of, 396 neurobiology of, 391 -436 PR 881/7 compound, structure-activity effects of, 1 1 Pressure injection of opioids into spinal neurons, 250-251 Proteolytical enzymes, in CSF, clinical significance, 128 Psychobiology of opioids, 277-337 Psychoticism, as personality trait, 395-398 Pulp of teeth, sensory transduction in, 84-85

R Rabbit teeth, sensory receptors in, 67-68 Rat incisors, sensory receptors in, 65-67

Schwmn cells. dental sensory receptors’ relationship to. 74-75 Seizures, from opiates, 307-309 Sensation seeking, as personality trait, 395-398 Sensory receptors, in teeth, 39-94 Serotinergic system, effect on behavior, 416-418 Serotonin evoked potential (KP) and, 427 as opiate neurochemical correlate, 284-286 Sexual behavior, opiate effects on, 319 Sleep, opiate effects on, 309 Somatostatin nociception antl, 215-216 peptide sequence of, 187 Spinal neurons, opioid effects on, 243-275 Substance 1’ nociception and, 190-195 peptide sequence of, 187 Sympathetic neurons guanethidine destruction of, 1-37

T -1. lymphocytes, in cerebrospinal fluids, 117-1 18 Teeth axons of, types, 42 reinnervated, 59-61 sensory properties of, 80-81 sensory receptors in, 39-94 ~l’liyrotropin-releasinghormone nociception and, 226 peptide sequence of, 187 Transamiitase, in CSF, clinical significance, I28 Trigeminal nerves, afferent axons in, 40-43 3-11 ftsin nociception antl, 224 peptide sequence of, 187 Tunior proteins, in cerebrospinal fluid, determination. 98

S S-100 protein, as nervous-tissue specific

protein in CSF, 119-121

V Vasopressin, nociception and, 222-224

CONTENTS OF RECENT VOLUMES The Fine Structural Localization of Biogenic Monoarnines in Nervous Tissue Floyd E. Bloom

Volume 12

Drugs and Body Temperature P c f n Lomar

Brain Lesions and Amine Metabolism Robcrf Y. Moore

Pathobiology of Acute Triethyltin Intox. ication R. Torack, J . Gordon, andJ. Prokop Ascending Control of Thalamic and Cortical Responsiveness M . Stcriade

Morphological and Functional Aspects of Central Monoamine Neurons K j l l Fuxr. 7'nma.c Hijkflt. and llrhan (Inggtrsttdt

Theories of Biological'Etiology of Affective Disorders John M . Davis

Uptake and Subcellular Localization of Neurotransmitters in the Brain Solomon H . Syndrr. Mirhacl J . Kuhar. Alan I. Green, ,]o.igh 7'. Coylr, and Edicvzrd G.Sha.rkan

Cerebral Protein Synthesis Inhibitors Block Long-Term Memory Samuel H . Barondes

Chemical Mechanisms of TransmitterReceptor Interaction John T. Garland and Jack DureN

The Mechanism of Action of Hallucinogenic Drugs on a Possible Serotonin Receptor in the Brain J . R. Smythics, F. Bmington, and R. D . Morin

The Chemical Nature of the Receptor Site-A Study in the Stereochemistry of Synaptic Mechanisms J , R . Smyfhies

Simple Peptides in Brain Isamu Sano

Molecular Mechanisms in Information Processing Geor.ps Ungar

The Activating Effect of Histamine on the Central Nervous System M. Monnicr, R . Saucr, and A . M . Hall

Mode of Action of Psychomotor Stimulant Drugs Jacques M . van Rossum

The Effect of Increased Functional Activity on the Protein Metabolism of the Nervous System B. Jakoubck and B. Smiginovsky' Protein Transport in Neurons Raymond J , Lasck

AUTHOR INDEX-SUBJECT INDEX

Neurochemical Correlates of Behavior M . H.Aprison and J . N.Hingtgm

Volume 13

Some Guidelines from System Science for Studying Neural Information Processing Donald 0. Waiter and Mariin F. Gardinn

Of Pattern and Place in Dendrites Ma d st E. Schcibel and Arnold B . Schcibel

AUTHOR INDEX-SUBJECT tNDEX

442

Contents of Recent Volumes Volume 14

The Pharmacology of Thalamic and Geniculate Neurons J . W. Phillis The Axon Reaction: A Review of the Principal Features of Perikaryal Responses to Axon Inquiry A . R . Liebmnan CO, Fixation in the Nervous Tissue SZt-Chuh Chen.c Reflections on the Role of Receptor Systems for Taste and Smell John G . Sinclair Central Cholinergic Mechanism and Behavior S. N . Pradhan and S. N . Dutta The Chemical Anatomy of Synaptic Mechanisms: Receptors ./. R . Smythies A U T H O R INDEX-SUBJECT I N D E X

Volume 15

Projection of Forelimb Group I Muscle Afferents to the Cat Cerebral Cortex In,Crnar Rosin Physiological Pathways throilgh the Vestibular Nuclei VictorJ . Wilson Tetrodotoxin, Saxitoxin, and Related Substances: Their Applications in Neurobiology Martin H . Evans The Inhibitory Action of y-Aminobutyric Acid, A Probable Synaptic Transmitter Kunihiko Obata Some AspectsofProtein Metabolismofthe Neuron Mei Satake

443

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 Rafael Elul Mathematical Identification of Brain States Applied to Classification of Drugs E. R.,John, P. Walker, D. Cawood, M. Rush, and,J. Gehrmann A U T H O R INDEX-SUBJECT INDEX

Volume 16

Model of Molecular Mechanism Able to Generate a Depolariziation-Hyperpolarization Cycle Clara Torda Antiacetylcholine Drugs: Chemistry, Stereochemistry , and Pharmacology T, D InchandR. W . Brimbltcombe Kryptopyrrole and Other Monopyrroles and Molecular Neurobiology Donald G.Iruine RNA Metabolism in the Brain Victor E. Shashoua A Comparison of Cortical Functions in Man and the Other Primates R . E. Passin,cham and G . Ettlinpr Porphyria: Theories of Etiology and Treatment H. A . Peters, D . J . Cripps, and H . H . Reese SUBJECT INDEX

Volume 17

Epilepsy and y-Aminobutyric Acid-Mediated Inhibition B. S. Meldrum

444

Contents o f Recent Volumes

O n Axoplasmic Flow Liliana Lubin'ska

Synaptosomal Transport Processes Giulio Levi and Maurizio Raiteri

Schizophrenia: Perchance a Dream? J . Christian Gillin and Richard~j.Wyatt

Glutathione Metabolism and Some Possible Functions of Glutathione in the Nervous System Marian Orlowski and Abraham Karkowsky

SUBJECT INDEX

Volume 18

Neurochemical Consequences o f Ethanol on the Nervous System

Arun K . Rawat

Integrative Properties a n d Design Principles of Axons Stephen G. Waxman

Octopamine a n d Some Related Noncatecholic Amines in Invertebrate Nervous Systems H. A . Robertson and A . V. Juorio

Biological Transmethylation Involving S-Adenosylmethionine: Development of Assay Methods and Implications for Neuropsychiatry Ross J . Baldessan'ni

Apormorphine: Chemistry, Pharmacology, Biochemistry F. C. Colparrt, W . F. M. Van Rwrr, and J . E. h f . F I.ryrrn

Synaptochemistry of Acetylcholine Metabolism in a Cholinergic Neuron Bcrtalan Csillik Ion a n d Energy Metabolisrri ofthe Brain ai the Cellular Level LeijHcrtz and Arne Schousboe Aggression and Central Neurotransmitters S. N Pradhan A Neural Model of Attention, Reinforcement a n d Discrimination Learning Stephen Grossbqq

Thymoleptic a n d Neuroleptic D r u g Plasma Levels in Psychiatry: Current Status Thomar R Cooprr. Grorge M Simpson. and J . Hillary L r r SUBJECT I N D E X

Volume 20 Functional Metabolism of Brain Phospholipids C.Bnan Ansell and Sheila Spanner

Isolation and Purification of the Nicotine Acetylcholine Receptor and Its Functiorial Reconstitution into a Membrane EnvironNeurochemical a n d Ne1iro~)harinaroloRi- ment Michael S Briley and,Jean- Pierre Chantcur cal Aspects o f Ikpression B. E Leonard Bioc hemicai Aspects o1Neurotrltrismissi~)ri Marihuana, L.earning, and hleniory Erncsf L . Abel

XJBJECT I N D E X

Volume 19

Do I lippocampal iiesia in .\ninialq? .%'usan 11 Ioersm

in the Developing Brain Josrph T Coyle 3 he Formation, Degradation, and F u n - tion t)f Cyrlir Nurleotilles in the Nervous System John W Ilaly

F h c tuazion Znalysis Louir ,J I )c FflU f

iri

Ntxirihiology

Peptides and Behavior Georges Unxar Biochemical Transfer of Acquired Information S.R. Mitchell, ,J M Beaton. and R. J. RradlPy ,

Aminotransferase Activity in Brain M. Benuck and A . Lajtha T h e Molecular Structure of Acetylcholine and Adrenergic Receptors: An All-Protein Model J . R. Smjthies Structural Integration of Neuroprotease Activity Elena Gabrielescu

Presynaptic Inhibition: Transmitter a n d Ionic Mechanisms R. A . Nicolland B. E. Alger Microquantitation of Neurotransmitters in Specific Areas of the Central Nervous System Juan M. Saauedra Physiology a n d Glia: Glial-Neuronal Interactions R. Malcolm Stewart and Rogn N . Rosenberg Molecular Perspectives of Monovalent Cation Selective Transmembrane C h a n nels Dan W . Ury Neuroleptics and Brain Self-stimulation Behavior Albert Wauquier

Lipotropin a n d the Central Nervous System W . H . Cispm.,J. M . uan Rer, and D dc Wird

Volume 22

Tissue Fractionation in Neurobiochemistry: An AnalyticalTooloraSourceofArtifacts Pierre Laduron

Transport a n d Metabolism of Glutamate and GABA in Neurons and Glial Cells Arnc Schousboc

Choline Acetyltransferase: A Review with Special Reference to Its Cellular and Subcellular Localization Jean Rossier

Brain Intermediary Metabolism in Viuo: Changes with Carbon Dioxide, Development, a n d Seizures Alexander L . Miller

SUBJECT I N D E X

Volume 21

Relationship of the Actions of Neuroleptic Drugs to the Pathophysiology of Tardive Dyskinesia Ross J . Baldessarini and Daniel Tarsy

N,N-Dimethyltryptamine: An Endogenous Hallucinogen Strum A . Barker, ,John A Monti, and Sam url T. Chrir ria n Neurotransmitter Receptors: Neuroanatomical Localization through Autoradiography L . Charlrs M u n i n Neurotoxins as Tools in Neurobiology E . C. McCear and P. L . McCeer

Soviet Literature on the Nervous System a n d Psychobiology of Cetacea Thedore H. Bullock and VladimirS Cureuich

Mechanisms of Synaptic Modulation William Shain and David 0. Carpenter

Binding and lontophoretic Studies o n Centrally Active Amino Acids-A Srarch for Physiological Receptors F. V. DeFeudrs

Anatomical, Physiological, a n d Behavioral Aspects of Olfactory Bulbectomy in the Rat B. E. Leonard and M. Tuiie

446

Contents of Recent Volunies

The Deoxyglucose Method for the Measurement of LocalGlucose Utilization and the Mapping of Local Functional Activity in the Central Nervous System Louis Sokoloff

Sleep Mechanisms: Biology and Control of REM Sleep Dennu 1% McCinty and R e d R. Drucher-Colin INDEX

INDEX

Volume 24 Volume 23 Chemically Induced Ion Channels in Nerve Cell Membranes David A. Mathers and Jeffv L. Barker Fluctuation of Na and K Currents in Excitable Membranes Berthold Neumke Biochemical Studies of the Excitable Membrane Sodium Channel Robert L. Barchi Benzodiazepine Receptors in the Central Nervous System Phil Sholnich and Steven M . Paul Rapid Changes in Phospholipid Metabolism during Secretion and Receptor Activation F. T Crews Glucocorticoid Effects on Central Nervous Excitability and Synaptic Transmission Edward D. Hall Assessing the Functional Significance of Lesion-Induced Neuronal Plasticity onwld Sltward Dopamine Receptors in the Central Nervous System Ian Creese, A. Leslie Morrow, Stuart E . L e f , D a d R . Siblq, and M a d W.Hamblin Functional Studies of the Central Catecholamines T. W.Robbins and B. J . Everitt Studies of Human Growth Hormone Secretion in Sleep and Waking Wallace B . Menddson

Antiacetylcholine Receptor Antibodies and Myasthenia Gravis Bernard W . F u l p i ~ Pharmacology of Barbiturates: Electrophysiological and Neurochemical Studies Max Willow and Graham A. R . Johmton Immunodetection of Endorphins and Enkephalins: A Search for Reliability Algandro Bayon, William J . Shoemakn; Jacqueline F. McGinty, and Floyd Bloom On the Sacred Disease: The Neurochemistry of Epilepsy 0. Carter Snead Ill Biochemical and Electrophysiological Characteristics of Mammalian GABA Receptors Salvatore J . Ennu and Joel P . Gallagher Synaptic Mechanisms and Circuitry Involved in Motoneuron Control during Sleep Muhad H . Chase Recent Developments in the Structure and Function of the Acetylcholine Receptor F. J . Barrantes Characterization of aI-and a2-Adrenergic Receptors David B. Bylund and David C . U'Prichard Ontogenesis of the Axolemma and Axoglial Relationships in Myelinated Fibers: Electrophysiological and Freeze-Fracture Correlates of Membrane Plasticity Stephen C . Waxman, Joel A. Black, and Robert E . Foster INDEX