Progress in Brain Research Volume 28 Anticholinergic Drugs

PROGRESS I N B R A I N RESEARCH V O L U M E 28 ANTICHOLINERGIC DRUGS AND BRAIN FUNCTIONS I N ANIMALS AND MAN

PROGRESS I N BRAIN RESEARCH

ADVISORY BOARD W. Bargmann H. T. Chang

E. De Robertis

J. C. Eccles J. D. French H. Hydtn J. Ariens Kappers S. A. Sarkisov

J. P. SchadC F. 0. Schmitt

Kiel

Shail ghai Buenos Aires Canberra Los Angeles Goteborg Amsterdam Moscow Amsterdam Brookline (Mass.)

T. Tokizane

Tokyo

J. Z. Young

London

PROGRESS I N BRAIN RESEARCH V O L U M E 28

ANTICHOLINERGIC DRUGS AND BRAIN FUNCTIONS I N ANIMALS AND MAN EDITED B Y

P. B. B R A D L E Y Department of Experimental Neuropharmacology, The Medical School, Birmingham (England)

AND

M. F I N K Department of Psychiatry, New York Medical College, New York (U.S.A.)

ELSEVIER P U B L I S H I N G C O M P A N Y A M S T E R D A M / LONDON / N E W Y O R K 1968

EL s E v I E R P u B LIs H I N G c o M P A N Y 335 J A N VAN G A L E N S T R A A T , P.O. B O X 21 I , A M S T E R D A M , THE NETHERLANDS

E L S E V I E R P U B L I S H I N G CO. L T D . BARKING, ESSEX, ENGLAND

A M E R I C A N E L S E V I E R P U B L I S H I N G C O M P A N Y , INC. 5 2 V A N D E R B I L T A V E N U E , N E W Y O R K . N.Y. 10017

This volurrie contains the Proceedings of the Vltli Symposiurn on Anticholinergic Drugs and Brain Firnciions in Aiiinials a d Man, held in Conneziion with the Vtli International Congress Collegiuni Intenlationale Neuro-psychopharmacologicuni, at Washi!igton D.C., March 20-31, 1966 (chairmen: P. B. Bradley and M . Fink)

L I B R A R Y O F C O N G R E S S C A T A L O G C A R D N U M B E R 67-25155

W I T H 102 I L L U S T R A T I O N S A N D 13 T A B L E S

COPYRIGHT 0 1968 BY ELSEVIER PUBLISHING COMPANY, AMSTERDAM

ALL RIGHTS RESERVED TI3IS BOOK O R A N Y P A R T T H E R E O F M U S T N O T BE R E P R O D U C E D I N A N Y F O R M

WITHOUT T H E WRITTEN PERMISSION OF T H E PUBLISHER, ELSEVIER PUBLISHING COMPANY, AMSTERDAM, THE NETHERLANDS

PRINTED IN T H E NETHERLANDS

List of Contributors

0. BENESOVA, Department of Pharmacology, Charles University, Prague (Czecholovakia). Z. BOHDANECK?,Department of Pharmacology, Charles University, Prague (Czechoslovakia). BOST, Thudichum Psychiatric Research Laboratory, Galesburg Research KATHRYN Hospital, Galesburg, Ill. (U.S.A.). P. B. BRADLEY, The Medical School, Birmingham (U.K.). J. BURES,Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia). P. L. CARLTON, Rutgers, The State University, New Brunswick, New Jersey (U.S.A.). Z. CUCULIC, Douglas Hospital, Verdun, Quebec (Canada). E. F. DOMINO,Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (U.S.A.). A. T. DREN,Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (U. S.A.). M. FINK,Department of Psychiatry, New York Medical College, New York (U.S.A.). S. GROF,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). 0. GROFOVA, Department of Pharmacology, Medical Faculty of Hygiene, Charles University, Prague (Czechoslovakia). A. HERZ,Deutsche Forschungsanstalt fur Psychiatrie, Max-Planck Institute, Munich (Germany). H. E. HrMwrc~,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, 111. (U.S.A.). R. Yu. ILYUTCHENOK, Institute of Cytology and Genetics, Pharmacological Laboratory, Siberian Branch, Academy of Sciences of the USSR, Novosibirsk (USSR). T. ITIL,Department of Psychiatry of the Missouri Institute of Psychiatry, University of Missouri School of Medicine, St. Louis, Missouri (U.S.A.). E. JACOBSON, Department of Pharmacology, Royal Danish School of Pharmacy, Copenhagen (Denmark). D. KRUS,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). K. KUNZ,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). V. G. LONCO,Istituto Superiore di Sanita, Rome (Italy). A. S. RUDOLPH,Department of Pharmacology and Brain Research Institute, University of Tennessee Medical Units, Memphis, Tennessee (U.S.A.).

VI

LIST OF CONTRIBUTORS

K. RYSANEK,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). A. SCOTTIDE CAROLIS, Istituto Superiore di Saniti, Rome (Italy). J. SKALA,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). V. V~TEK, Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). M. VOJTECHOVSK?, Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). Z. VOTAVA,Department of Pharmacology, Medical Faculty of Hygiene, Charles University, Prague (Czechoslovakia). R. P. WHITE,Department of Pharmacology and Brain Research Institute, University of Tennessee Medical Units, Memphis, Tennessee (U.S.A.). A. WIKLER,Lexington, Kentucky (U.S.A.). K. YAMAMOTO, Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (U.S.A.). a

Other volumes in this series:

Volume 1: Brain Mechanisms Specific and Unspecific Mechanisms of Sensory Motor Integration Edited by G. Moruzzi, A. Fessard and H. H. Jasper Volume 2: Nerve, Brain and Memory Models Edited by Norbert Wiener? and J. P. Schade Volume 3 : The Rhinencephalon and Related Structures Edited by W. Bargmann and J. P. Schade Volume 4: Growth and Maturation of the Brain Edited by D. P. Purpura and J. P. Schade Volume 5 : Lectures on the Diencephalon Edited by W. Bargmann and J. P. Schade Volume 6: Topics in Basic Neurology Edited by W. Bargmann and J. P. Schade Volume 7: Slow Electrical Processes in the Brain by N. A. Aladjalova Volume 8: Biogenic Ainines Edited by Harold E. Himwich and Williamina A. Himwich Volume 9: The Developing Brain Edited by Williamina A. Himwich and Harold E. Himwich Volume 10: The Structure and Function of rhe Epiphysis Cerebri Edited by J. Ariens Kappers and J. P. Schadd Volume 11: Organization of the Spinal Cord Edited by J . C. Eccles and J. P. Schade Volume 12: Physiology of Spinal Neurons Edited by J . C. Eccles and J. P. Schade Volume 13 : Mechanisms of Neural Regeneration Edited by M. Singer and J. P. Schadt Volume 14: Degeneration Patterns in the Nervous System Edited by M. Singer and J. P, Schade

VIlI

Volume 15 : Biology of Neuroglia Edited by E. D. P. De Robertis and R. Carrea Volume 16: Horizons in Neuropsychopharmacology Edited by Williamina A. Himwich and J. P. Schadk Volunie 17 : Cybernetics of the Nervous System Edited hy Norbert Wiener1 and J. P. Schadt Volume 18 : Sleep Mechanisms Edited by I<. Akert, Ch. Bally and J. P. Schade Volume 19 : Experimental Epilepsy by A. Kreindler Volume 20: Pharmacology and Phvsiology of the Reticular Formation Edited by A. V. Valdman Volume 21 A : Correlative Neurosciences. Part A: Fundamental Mechanisms Edited by T . Tokizane and J. P. Schadt Volume 21B: Correlative Neurosciences. Part B: Clinical Studies Edited by T . Tokizane and J. P. Schadt Volume 22: Brain reflexes Edited bij E. A. Asratyan Volumc 23 : Sensory Mechanisms Edited by Y . Zotterrnan Volume 24: Carbon Monoxide Poisoning Edited by H . Bour and I. McA. Ledingham Volume 25: The Cerebellum Edited by C. A. Fox and R. S. Snider

Volume 26: Deve/opnretitul Ncur01og.v Edited by C. G. Bernhard Volume 21: Structure and Function of the Limbic System Edited by W. Ross Adey and T. Tokizane Volume 28 : Anticholinergic Drugs Edited by P. B. Bradley and M. Fink Volume 29: Brain-Barrier Systeni Edited by A. Lajtha and D. H. Ford Volume 30: Cerebral Circulation Edited by W. Luyendijk

Contents

................................ Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary; Brain, Behavior and Anticholinergic Drugs . . . . . . . . . . . . . . . . . . List of Contributors.

Introduction A. Wikler (Lexington, Kentucky, U.S.A.)

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

The effect of atropine and related drugs on the EEG and behavior P. B. Bradley (Birmingham, U.K.) . . . . . . . . . . . . . .

...........

Neuropharmacological comparison of subcortical actions of anticholinergic compounds P. R. White and A. S . Rudolph (Memphis, Tennessee, U.S.A.) . . . . . . . . . . An examination of a possible cortical cholinergic link in the EEG arousal reaction Z . Cuculic, Kathryn Bost and H. E. Himwich (Galesburg, Illinois, U.S.A.) . . .

V

XI XI1 1

3

. . .

14

......

27

Influence of atropine, scopolamine and benactyzine on the physostigmine arousal reaction in rabbits Z. Votava, 0. BeneSovB, Z . Bohdanecky and 0. Grofova (Prague, Czechoslovakia) . . . . . 40 Brain acetylcholine and habituation P. L. Carlton (New Brunswick, N.J., U.S.A.)

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

48

The effect of physostigmine and atropine on some behavioral and electrophysiological functions in rats J. BureS (Prague, Czechoslovakia) . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Some actions of cholinergic and anticholinergic drugs on reactive behaviour A. Herz (Munich, Germany) . . . . . . . . . . . . . . . . . . . . .

.......

73

Experimental psychoses induced by benactyzine in alcoholics M. Vojtkhovsky, D. Krus, S. Grof, V. Vitek, K. RySanek, K. Kunz and J. Skala (Prague, Czechoslovakia) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Anticholinergic hallucinogenics: Laboratory results vs. clinical trials V. G. Longo and A. Scotti de Carolis (Rome, Italy) . . . . . .

. . . . . . . . . . . 106

Role of cholinergic mechanisms in states of wakefulness and sleep E. F. Domino, K. Yamamoto and A. T. Dren (Ann Arbor, Michigan, U.S.A.) Cholinergic brain mechanisms and behaviour R. Yu. Ilyutchenok (Novosibirsk, USSR)

. . . . . . . 113

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

134

EEG and behavioral aspects of the interaction of anticholinergic hallucinogens with centrally active compounds T. Itil and M. Fink (St. Louis, Missouri, U.S.A.) . . . . . . . . . . . . . . . . . . . 149 Discussion E. Jacobson (Copenhagen, Denmark)

........................ Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Author Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169

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

179

Subject Index.

171

175

This Page Intentionally Left Blank

Preface

The papcrs contained in this volume represent the contributions to a Symposium entitled Anticholinergic drugs and brain functions in animals and man (Anticholinergic Drugs), of which Dr. Fink and I were the Chairmen, and which took place at the Fifth Meeting of the Collegium Internationale Neuro-Psychopharmacologicum (C.1.N.P) held in Washington in March, 1966. When we were first approached by the Programme Committee for this meeting in 1965, and asked if we would jointly organise a symposium in the general field of ‘neuropharmacological effects of drugs in animals and man’, we had no difficulty whatsoever in finding a suitable topic. The problem of the so-called “pharmacological dissociation” between EEG and behaviour was one which had concerned both of us at various periods over the past twelve years, and the time seemed to be ripe for discussion. Nor did we have much difficulty in deciding upon participants, although this was to some extent a reflexion of the small number of active workers in this field. Unfortunately, not all of those we invited were able to participate. However, the Symposium was very well attended and the discussion both vigourous and prolonged, indicating that the topic was of interest to many who were not actively working in the field. It was for this reason, and because the Symposium seemed to form a complete entity in itself, that we decided to publish the papers in a volume separate from the Proceedings of the rest of the meeting. We are grateful to the Editors of the Proceedings for accepting this suggestion and for publishing in their Proceedings only the abstracts of the papers presented here. The contributions appear in the approximate order in which they were given at the meeting, our intention being to first outline the problem of ‘dissociation’ as it was first reported for anticholinergic drugs in animals, and then to analyse the phenomenon in terms of the underlying pharmacological, neurophysiological and biochemical mechanisms, ending with behavioural (psychological) studies and related clinical observations in man. It proved impossible to record the discussions but some aspects of these have been summarised by Dr. Fink in the following pages. We believe this to be the first time this topic has been dealt with in a comprehensive manner and we trust that the reader will find the collection of papers in this volume both informative and interesting, even if they do not provide the complete answer to the problem. P. B. BRADLEY The Medical School Birmingham

Summary

Brain, Behavior and Anticholinergic Drugs MAX FINK Departnient of Psychiatry, New York hfedicat College, 5 East lOZnd Street, New York, New York 10029

In the decades since its introduction, the use of the scalp-recorded electroencephalogram in psychiatry has been marked by recurrent enthusiasm and disillusionment. No single “controversy” has stimulated as much recent discussion as concepts relating EEG changes to behavior, with two views prominent-that the EEG is a sensitive index of specified behaviors, and that the EEG may be related to certain behaviors, but “dissociation” is as likely as “association.” The controversy has been particularly sharp in studies of anticholinergic drugs, and it is this controversy which is illuminated by this symposium. * The electroencephalogram is a physiological expression of consciousness, sensitive to momentary changes in awareness, alertness, and concentration. It is readily modified by active thought such as in problem solving and in anticipation of action; in response to external stimuli such as repetitive flashes and clicks, and eye opening and closure; and to those biochemical changes in the central nervous system after hyperventilation, apnea (hypoxia) or with the introduction of many drugs. The changes in the EEG and in behavior are usually concurrent, with the changes in one measure often definab!e by changes in the other. A prominent association of EEG and behavior is seen in sleep. Sleep is generally accompanied by high voltage slow frequencies, often in regular trains, and wakefulness by low voltage fast frequencies or, in man with eyes closed, by well modulated alpha activity. These observations in sleep are readily confirmed in animals as well as man. But, these associations have not always been observed. At times, with “sleeplike” behavior the EEG exhibits low voltage fast frequencies; or, when the behavior is one of restlessness and movement with open eyes, the EEG exhibits high voltage slow waves. This “dissociation” of EEG and behavior was reported particularly after anticholinergic drugs by Wiklerll, Funderburk and Case4, Bradley and Elkesl and Rinaldi and Himwichg. In part, it is the generalization of these observations which has led to the expectation that little, if any, association between EEG and behavior is to be expected.

* A preliminary review of this problem was undertaken at the 1965 meeting of the Society for Biological Psychiatry and published in Cholinergic Mechanisms in Mental Illness: Anticholinergic Hallucinogens, in Recent Advances in Biological Psychiatry, ed. J. Wortis, Plenum Press, N.Y., VIII: 155-198,1966,

SUMMARY

XI11

The relation of EEG stages and sleep has been clarified, however, by the specific descriptions of associations between rapid eye movements, reports of dreaming and EEG patterns of low voltage fast activity7. Knowledge of this association has stimulated more detailed, quantitative studies which quickly demonstrated that rapid eye movements were also accompanied by increased muscular activity, tachycardia, increased respiratory rate and penile erection. These studies have been repeated in animals, and while dream reporting cannot be confirmed, the other associations were clearly defined. Thus, the definition of specific behaviors and specific EEG patterns has resulted in the definition of meaningful associations. Concurrent repxts suggest another close relationship between EEG and behavior in studies with psychoactive drugs. From results with subjects receiving mescaline, n-allylnormorphine and morphine, Wikler suggests that “regardless of the nature of the drug administered, shifts in the pattern of the electroencephalogram in the direction of desynchronization occur in association with anxiety, hallucinations, fantasies, illusions, or tremors, and in the direction of synchronization with euphoria, relaxation and drowsiness.”lO With the use of quantitative EEG measurements in clinical studies of psychoactive drugs, this association has become more firmly established as each new agent is identified with a specific “signature” of EEG changes2.3. That these patterns are related to the therapeutic range of compounds and even to the individual therapeutic response serves to re-enforce concepts of association. Essays with anticholinergic drugs were included in these studies of psychoactive compounds and these exhibit definable EEG patterns which are consistent with the same principles observed with other psychoactive comp0unds5~~. Are the descriptions of “dissociation” of EEG and behavior characteristic of a specific class of compounds, the anticholinergic hallucinogens, and “association” characteristic of other centrally active compounds? Perhaps, the different descriptions may be related to differences in the subjects, i.e. animal species and man, to the techniques of observation, or to distinctions in terminology and definition? Reviewing the reports of this symposium suggests that each factor may have contributed to the present controversy. Wikler suggests the problem is based on a misunderstanding. He indicates that the central nervous system may be organized with neuronal systems subserving the EEG being distinct from those subserving ideation, mood, awareness, motility and sensations; and further, that the synchronizing-desynchronizing mechanisms serve to maintain “cortical homeostasis.” These concepts are not directly related to learning processes-the identification of learning and the EEG was an “association” that was never claimed-and the controversy of dissociation is based on this error. Other reporters modified conditioning, learning and habituation processes with cholinergic and anticholinergic drugs. Herz reports that cholinergic drugs may facilitate learning to a limited extent while anticholinergics interfere with memory, especially recent memory processes. It is probable that these drugs principally affect active processes, since little change is induced by anticholinergic drugs after

* Nonsubscripted citations are to reports in this volume.

XIV

M. F I N K

consolidation of learning when greater degrees of inhibition may yet result after cholinergic drugs. Similar observations are reported by Burei who concludes that, while cholinergic mechanisms are central to responses in learned behavior, the cholinergic systems may be duplicated by parallel, non-cholinergic ones. Carlton postulates the organism inundated by excessive stimuli and in the “habituation” process necessary for survival, stimuli are selectively filtered-both out and in-and this process is seen as dependent on cholinergic mechanisms. Thus, each observer reports changes in learned behavior with centrally active cholinergic and cholinolytic drugs. But these detailed behavioral observations are accompanied by limited electrophysiological studies which are primarily suggestive. Another technique of study is of neuronal firing ra?es after the topical or iontophoretic application of drugs. Neurons selectively sensitive to cholinergic drugs are described as central to the alerting process by Cuculic, Bost and Himwich and also by Bradley. Votava, using the conventional implanted electrode technique in rabbits, describes the electrophysiological changes evoked by cholinergic drugs. Each of these studies illuminates the site of action of cholinergic drugs but only limited concurrent behavioral data is provided. The question of “dissociation” is most actively joined in the reports by Bradley, Longo, Domino, Itil and Fink. Bradley summarizes his earlier animal studies indicating that the differences in the EEG and the behavioral data may result from measurements of inappropriate behavioral indices. Atropine may have little effect on behavior as defined by states of wakefulness and sleep, but may become clear in other behavioral tasks-as in conditioned avoidance tests. But, after describing the technique, Bradley reports that at the dosages of atropine used, no relation of EEG and behavior change was observed. He concludes that “either the presence of slow waves in the electrocorticogram has no particular relevance for behavior or the cortical mechanisms which are affected by drugs producing dissolution are not those concerned with behavioral responses.” Longo and deCarolis review many clinical and animal experiments utilizing EEG and behavioral observations with anticholinergic drugs, and suggest that the induced syndromes are similar regardless of the anticholinergic drug used. They emphasize that these systems are highly dependent on dose and conclude that the behavioral changes do result from alteration of acetylcholine as a central mediator. (In this conclusion they respond to another question-that of cholinergic mediators in central cellular activity.) Three observers utilize the agonist-antagonist model to study EEG-behavioral interactions. Domino and his co-workers illustrate many direct relations between the EEG arousal or sleep state and the overt motor behavior in various species in whom different cholinergic and anticholinergic drugs were administered in different sequences. This technique is expanded by White and Rudolph who use two electrophysiological methods-spontaneous EEG and the evoked response to shock stimuli repetitively applied to the sciatic nerve or to the pontine reticular formation. The best elaboration of central activity is in response to the use of physostigmine as an agonist to expose

SUMMARY

xv

differences in centrally active drugs. As with the reports of other animal studies, behavior is assessed in terms of sleep-wakefulness or by gross motor movements. Itil and Fink use the agonist-antagonist model in psychiatric patients, and in the quantitative analysis of the resting EEG utilize detailed analog frequency analyzers or digital computer methods. The EEG changes induced by atropine and Ditran are shown to be only superficially similar to that of natural sleep, differing in rate and amount of spindling, persistence of fast activity and in the response to external stimuli. They administer anticholinergics in three dosage ranges and find the changes in EEG and in behavior to be directly related to dose. But the relation between changes in EEG and behavior become most marked when a second drug is administered after an anticholinergic drug. The apparent similarities between alert, active behavior and the motor restlessness of the delirious state are clearly defined, since in one instance the subjects are correctly oriented and in the second, very poorly so. The problem of “dissociation” or “association” of EEG and behavior remains. But these arguments illustrate issues which have contributed to the controversy : 1. Limited observations. Many studies provide either meticulous electrophysiological measures or refined behavioral measures but rarely both in the same experiments. 2. Nonqudntitative assessment. Both in the behavioral and the electrophysiological measures there is limited utilization of available quantitative techniques. For example, more extensive use may be made of the computer methods of EEG analysis, as in period analysis for drug effects and the definition of sleep states3, evoked potentials for studies of anticipation and set9 or spike counts as an index of altered consciousness. The conditioning and learning paradigms, altered interactive behavior in multiorganismic situations and quantitative speech measures provide more quantitative indices of behavior which are available. 3. Definition of relevant behavior. It was in the definition of dream behavior in sleep and of anticipation in evoked potential studies that the relevant behavior was defined. Surely, simple motor activity of an animal can no longer be seen as a relevant definition of “sleep”, nor is the equation of overt behaviors among subjects or species without specific analysis tenable. 4. Dose-drug relations. Cholinergic mechanisms appear to be highly sensitive to agent and to dose, and the equation of agents, as may be done in studies of sedatives and hypnotics, is not successful with the cholinergic and anticholinergic compounds. 5. Species specificity. The equation of central effects of these compounds and the resulting behavior in a wide variety of species ignores the probable differences in metabolism in different animal classes, and in species of a single animal class. While much electrophysiology has been learned from studies in animal species, the behavior critical to man is neither prominent nor readily measurable in other animal classes -such behaviors as learning, speech, memory, alertness, dreaming, fantasy, anxiety, and mood, to name a few-and it is these behaviors which may yet relate to changes in central electrophysiology. It is such behaviors that must be studied, rather than sleep-wakefulness continuum or gross motor activity alone. The “controversy” concerning “dissociation” of EEG and behavior with anticholinergic drugs may have resulted from many factors-misunderstanding of theory,

XVI

M. F I N K

limited techniques of observation and quantification, problems of terminology, and species differences. It is also true that there is a paucity of theories relating brain function to behavior, and this lack has led to undue dependence on simple models described almost two decades ago. The failure to define more tenable hypotheses may result from the difficulties in undertaking such studies in the principal relevant species, man, as well as the associated factors described above. Where studies have been undertaken in man, as in the studies of sleep, of the evoked potential and perception of shape or anticipation of stimulus, and of the scalp EEG and the clinical efficacy of new drugs, most satisfactory EEG-behavioral relations have been defined. It is probable that with additional study in man, association of EEG and behavior will become better defined and the question may change from the conditions of dissociation to questions of the relevant behaviors which may be directly related to specific electrophysiological measures; and, conversely, the specific electrophysiological changes accompanying significant behaviors in man. REFERENCES 1 BRADLEY, P. B. AND ELKES,J. (1957) The effects of some drugs on the electrical activity of the brain. Brain, 80, 77-1 17. 2 FINK,M. (1963) Quantitative electroencephalography in human psychopharmacology 11: Drug patterns. EEG arid Behavior, C. Glasir, Editor, New York, Basic Books, Inc., (pp. 177-197). 3 FINK,M., SHAPIRO, D., HICKMAN, C. AND ITIL, T., (1967) Quantitative analysis of the electroencephalogram by digital computer methods. 111. Applications to psychopharmacology. Corrrpulers in Psychiatry, N. Kline and E. Laska (Editors), New York, Grune and Stratton, (in przss). 4 FUNDERBURK, W. H. AND CASE,T. J. (1951) The effect of atropine on cortical potentials. Electroenceph. cfin. Neurophysiol., 3, 21 3-225. 5 ITIL,T. (1966) Quantitative EEG changes induced by anticholinergic drugs and their behavioral correlates in man. Recent Advances in Biologicnll‘sychiatry, Vol. 8 . J. Wortis, Editcr, New York,

Plenum Press (pp. 151-173). 6 ITIL,T. AND FINK,M. (1966) Anticholinergic drug-induced delirium: Experimental modification, quantitative EEG and behavioral correlations. J . Nerv. Ment. Dis., 143, 492-507. 7 KLEITMAN, N. (1963) Sleep and wakefulness. Chicago, University of Chicago Press. 8 RINALDI,F. AND HIMWICH, H. E. (1955) Cholinergic mechanisms involved in function of rnesodiencephalic activating system. A.M.A. Arch. Nertrol. Psychiat. (Chic.), 73, 396402. 9 WALTER, W. G . (1964) Slow potential waves in the human brain associated with expectancy, attention and decision. Arch. Psjchiat. Nervenkr., 206, 309-322. 10 WIKLER, A. (1954) Clinical and electroencephalographic studies on the effects of mescaline, n-allylnormorphine and morphine in man. J . Nerv. Ment. Dis.,120, 157-175. 11 WIKLER, A. (1952) Pharmacologic dissociation of behavior and EEG “sleep patterns” in dogs: morphine, n-allynorphine, and atropine. Proc. SOC.Exp. Biol. Med., 79, 261-265.

1

Introduction A. W I K L E R Lexington, Kentucky ( U . S . A . )

I am grateful to Prof. Bradley and Dr. Fink for this opportunity to clarify the meaning and some of the implications of the term “EEG - behavioral dissociation’’ which I first used in 1952 (Wikler3). In the studies reported in that paper, dogs that had been given fairly large doses of atropine (7.2 mg/kg) exhibited EEGs characterized by continuous highvoltage slow activity interrupted by spindlebursts, regardless of whether they were quiet and appeared to be dozing or whether they were barking and struggling. When the animals were quiet and dozing, the EEG pattern resembled that commonly seen in such a behavioral state, but the failure of the EEG to change in the direction of desynchronization when the dogs were barking and struggling was a phenomenon for which I was quite unprepared by my “association’ of behavioral arousal with a low voltage fast EEG pattern. The term “dissociation”, therefore, referred to the failure of the EEG pattern to shift in the direction expected from generally observed “associations.” It should be emphasized, however, that under atropine, the dogs’ sleep-waking behavior was not always “dissociated” from the EEG - rather, periods of “association” and “dissociation” alternated at irregular intervals. Independently, Bradley and El kesl.2 reported analogous phenomena in the cat, not only with atropine but also with scopolamine and eserine. In my 1952 paper, 1 concluded that while they were normally interlocked, the mechanisms that subserve synchronization and desynchronization of the EEG were distinct from those that subserve sleeping and waking behavior. In other words, the events that characterize behavioral awakening (opening the eyes, moving about etc.) or behavioral sleep (closing the eyes, muscular relaxation, etc.) are not, respectively, consequences of desynchronization or synchronization of spontaneous cortical electrical activity. In a later paper4, I reported findings on the effects of mescaline, N-allylnormorphine and morphine in man, which prompted me to speculate further on the functions of the EEG-synchronizing and desynchronizing mechanisms. Briefly, it was found that while under these drug conditions, EEG changes in the direction of desynchronization were always accompanied by arousal, anxiety, imagery and/or hallucinations, and EEG changes in the direction of synchronization were always accompanied by relaxation, euphoria and/or drowsiness, such behavioral changes often occurred without any detectable change in the spontaneous EEG. Furthermore, when EEG changes did occur, they were always diffuse and nonspecific with respect to the particular drug condition that obtained - e.g., a desynReferences p . 2

2

A. W I K L E R

chronized EEG under N-allylnormorphine could not be distinguished from a desynchronized EEG under mescaline, nor a synchronized EEG under N-allylnormorphine from a synchronized EEG under morphine, although the behavioral changes observed were quite distinct for each drug. On the basis of these, and the earlier findings with atropine in dogs, I concluded that the mechanisms that desynchronize or synchronize the spontaneous electrical activity of the cortex are distinct, not only from those that subserve waking and sleeping, but also from those that subserve ideation, mood, level of awareness, motility and sensation. Rather, the diffuse, non-specific changes in the EEG that sometimes accompanied behavioral changes under mescaline, N-allylnormorphine or morphine, suggested that the function of the EEG-synchronizing and desynchronizing mechanisms is to preserve “cortical homeostasis” -i.e., to regulate electrical background activity in such a way that limits are set on the excitability of behavior-related cortical neuron systems in both directions (preventing excessive excitation or excessive inactivation). I t stands to reason that if such an hypothesized “cortical homeostatic” mechanism is disrupted, e.g. by atropine, scopolamine or eserine, certain aspects of behavior may be disrupted too. Thus, it is not surprising that a number of investigators have found that in animals, learning is impaired by doses of atropine (or scopolamine) that also produce synchronization of the EEG. To say, however, that in such instances there is no “dissociation” between EEG and behavior is quite irrelevant, as no one has claimed that there is an EEG pattern that is characteristic of the “learned” or “unlearned” state. Certainly the cortical EEG-synchronizing-desychronizingmechanisms have some function but I do not believe they are specifically concerned with learning. Whether or not the ‘‘cortical homeostasis” hypothesis has any validity is something for further research to determine. Be that it as may, I am confident that new insights concerning the functional significance of the EEG will be furnished by the papers on the program at this meeting.

REFERENCES

1 BRADLEY, P. B. AND ELKES,J. (1953) Some effects of diisopropyltluorophosphate on the electrical activity of the brain of the cat. J. Physiol., 121,51 P. 2 ELKES,J., ELKES,C. AND BRADLEY, P. B. (1954) The effect of some drugs on the electrical activity of the brain, and on behaviour. J. Ment. Sci., 100, 125-128. 3 WIKLER, A. (1952) Pharmacologic dissociation of behaviour and E.E.G. “sleep patterns” in dogs: morphine, N-allylnormorphine, and atropine. Proc. SOC.exp. Biol., N . Y., 79, 261-265. 4 WIKLER,A. (1954) Clinical and electroencephalographic studies on the effects of mescaline. N-allylnormorphine and morphine in man. J. Nerv. Ment. Dis.,120, 157-175.

3

The Effect of Atropine and Related Drugs on the EEG and Behaviour P. B. B R A D L E Y The Medical School, Birmingham (England)

There normally exists a good correlation between the pattern of electrical activity recorded in the electroencephalogram in man or the electrocorticogram in animals and the functional state of the organism in terms of wakefulness and sleep. Thus, the electrical patterns associated with different states of wakefulness and of sleep are wellknown, since they have been described for many species by numerous authors (Fig. 1).

A

1‘

I . 500pV

1sec

Fig. I . Electrocorticogram of a normal unanaesthetized cat in different behavioural states. A: Arousal from the drowsy state by a sensory stimulus “S’.B: Intermediate “quiet” state (frorn BRADLEY, P. B. AND HANCE, A. J., 1957). References p. 12-13

4

P. B. B R A D L E Y

Wikler, in 1952, was the first to demonstrate that this correlation between EEG and behaviour is lost when atropine is administered in a sufficiently large dose. He used unanaesthetized dogs with implanted electrodes and he coined the term “pharmacological dissociation” to describe this phenomenon (Wiklerls). Our own studies3 of which we published a preliminary report in 1953 and those of Funderburk and Case12 showed that a similar phenomenon could be observed in the cat. Since then, the dissociation has been demonstrated in other species, including the rat, rabbit and monkey. However, in our studies in unanaesthetized cats carrying chronically implanted recording electrodes4, we were able to show that atropine is not the only substance which produces this dissociation5. Thus, whilst atropine produced a pattern of high-voltage slow waves together with spindle burst activity in the electrocorticogram of animals which were wide-awake or showed behavioural excitation, physostigmine produced the opposite effect. Animals which had been given the latter drug showed a pattern of low voltage fast activity in the electrocorticogram but were not necessarily alerted behaviourally (Fig. 2). It has been suggested that because an animal which has been given atropine can still go to sleep and the electrical activity is then appropriate to its behavioural state, we should not use the term “dissociation”. However, when such an animal is roused by a sensory stimulus, its electrocorticogram shows no change. Similarly, when physostigmine is administered the animal can still show patterns of wakefulness and sleep but when it does sleep, there is no slowing of its electrocortical patterns. In these circumstances, the term “dissociation” is applied appropriately. It is worth considering the dose levels at which these effects appear. In the case of atropine sulphate, the dose required to produce complete dissociation in the cat is between 2.0 and 3.0 mg/kg by intraperitoneal injection. Smaller doses, between 1.O and 1.5 mg/kg cause bursts of slow activity to appear in the electrocorticogram but these are less persistent and can often be blocked by sensory stimuli. In the rat and rabbit, larger doses of the order of 1.5.0-20.0 mg/kg are required. These doses are considerably larger than those which produce peripheral parasympathetic blockade. Physostigmine, on the other hand, produces its dissociation at relatively low dose levels. In the cat, this is between 0.05 and 0.1 mg/kg when given intraperitoneally and the EEG dissociation appears with doses below those which cause parasympathetic stimulation. Higher doses will cause the usual autonomic effects and consequently changes in behaviour. Another interesting feature about the central effects of these drugs is that there seems to be a mutual antagonism between them. Thus, the effects of atropine can be reversed by a subsequent administration of physostigmine and vice versa. There are therefore differences, both qualitative and quantitative, between the peripheral and central effects of these two drugs and it is possible that these may reflect differences in the nature of peripheral and central cholinergic receptors. In our investigations with these drugs we used not only unanaesthetized chronic preparations but also the acute encPphaIe isolP and cerveau isole‘ preparations in which it was possible to show that the effects of atropine and physostigmine on the electrocorticogram were present after transection of the midbrain at the intercollicular levels, thus demonstrating that the integrity of the reticular activating system was not essential

EFFECT OF DRUGS ON

EXF?307/6.

EEG

A N D BEHAVIOUR

5

A

I

500p , . p

C

Fig. 2. Electrocorticogram of an unanaesthetized cat following administration of physostigmine and atropine. A: Control record with arousal response at “S”. B: 10 minutes after 0.08 mg/kg physostigmine sulphate (intraperitoneally). C: 20 minutes after the subsequent injection of 3.0 mg/kg atropine sulphate (intraperitoneally) (from BRADLEY, P. B. AND ELKES,J., 1957).

for their actions. In fact, we suggested that “a cholinergic mechanism is unlikely to be dominant in the mechanisms concerned with wakefulness and a r ~ u s a l ” We ~ . also administered atropine by the intraventricular route and showed that under these References p . 12-13

6

P. B. B R A D L E Y

circumstances no dissociation was produced (Fig. 3). The most striking effects were observed when atropine and physostigmine were used in combination with other drugs, particularly drugs whose central actions are not related to cholinergic mechanisms. For example, an animal which had been given both amphetamine and atropine remained alert and showed behavioural excitation whilst the electrocorticogram was

EXP.307/5

A

1sec

1

B

Fig. 3. Electrocorticogram of an unanaesthetized cat following intraventricular injection of atropine. A: Control record with arousal response. B: 17 minutes after the injection of 300 pg of atropine sulphate into the lateral ventricle (from BRADLEY, P. B. AND ELKES,J., 1957).

dominated by the slow wave activity. The amphetamine produced its normal effect on behaviour and atropine its effect on the electrocorticogram. On the other hand, an animal which had been given a combination of chlorpromazine and physostigminee showed the reduced motor activity and unresponsiveness associated with chlorpromazine, but only low amplitude fast activity was present in the electrocorticogram characteristic of physostigmine administration (Fig. 4). These combinations clearly show superimposition of drug effects and suggest that different receptors must be responsible for electrophysiological and behavioural effects in these cases,

EFFECT OF D R U G S O N

EEG

A N D BEHAVIOUR

7

IS

C

Fig. 4. Electroencephalogram of unanaesthetized cat following administration of chlorpromazine and physostigmine. A: Control record with arousal response. B: 17 minutes after 4.0 mg/kg chlorpromazine hydrochloride (intravenously). C: 25 minutes after the subsequent injection of 0.2 mg/kg P. B. AND HANCE,A. J., 1957). physostigmine sulphate (intraperitoneally) (from BRADLEY,

Using the ence'phale isole' preparation and stereotactically oriented stimulating electrodes in the brain stem reticular formation, Dr. Key and myself7 were able to quantify the dissociation produced by atropine and physostigmine (Fig. 5 ) since the threshold for desynchronisation of the electrocorticogram by electrical stimulation of References p. 12-13

8

P. B. B R A D L E Y

the brain stem was progressively raised by increasing quantities of atropine, whilst the threshold for behavioural arousal remained unchanged. Physostigmine had the opposite effect (Fig. 5 ) . In these experiments there was never complete blocking of EEG desynchronisation even with high doses of atropine, although such a blockade has been observed in the rabbit14. This difference must be due to the different species used. 0

E.EG. arousal

T

Behavioural arousal

400r

0

I

I

I

I

1

2

3

0

,

/

J

1

mg/kg

Fig. 5. The effects of atropine and physostigmine on thresholds for behavioural arousal (solid circles) and electrocortical activation (open circles) produced by electrical stimulation of the reticular formation. The mean percentage change in threshold is plotted against dose. Atropine was administered in increasing doses. The arrow indicates the point at which injections of physostigmine were started. (from BRADLEY, P. B. AND KEY,B. J., 1958).

Other substances were also found to produce dissociation, e.g. hyoscine, benactyzine, imipramine* and various derivatives of atropine13 but in most cases the dissociation was less marked. For example, although hyoscine is some ten times more potent than atropine it also caused an elevation of the threshold for behavioural arousal and this indicates the well-known sedative action of this drug (Fig. 6). Therefore, we should not speak of hyoscine as producing dissociation. Some recent studies have confirmed our findings with atropine, hyoscine and imipramine and have shown that other antidepressant drugs, such as amitryptyline and nortryptyline as well as desmethylimipramine, produce some degree of dissociationlo. These authors point out that all anticholinergic antidepressant drugs have some sedative action and the only other compound apart from atropine which produces complete dissociation is Ditran. Neither Ditran nor atropine are antidepressants but psychotomimetics. Turning now to the behavioural effects of these drugs, it occurred to us that whilst atropine had no apparent effect on behaviour insofar as states of wakefulness and

EFFECT OF DRUGS ON

0

EEG

AND BEHAVIOUR

9

I 2 3 4 5 6 7 8 9 1 0 m g h

400r

0

1

2

3

5

4

0

6

7

8

I

9 10

ms/ks EEGarousal Behavioural arousal

0

1

2

3

4

5

6

7

8

9

1

0

mg/kg

Fig. 6. The effects of drugs on thresholds for behavioural arousal (solid circles) and electrocortical activation (open circles) produced by stimulation of the brain stem reticular formation. The mean percentage change in threshold is plotted against dose. A: benactyzine; B: imipramine; C : hyoscine (from BRADLEY, P. B. AND KEY,B. J., 1959).

sleep were concerned, the effects of the drug might become apparent if sufficiently sensitive behavioural tests were used. We therefore set out to investigate the actions of atropine on the performance of animals using different tests under conditions where dissociation of electrocortical activity and behaviour was apparentl. The rat was chosen for these investigations as it is easier to use this animal in large numbers. In the first experiment a simple conditioned avoidance response was used, the conditioned stimulus being auditory. After the animals had been trained to give 95 % correct responses, half of them were operated upon and cortical recording electrodes implanted. Following recovery from the operation both groups were given a further training session and divided into two subgroups. The subgroups were then paired so that each consisted of equal numbers of operated and unoperated animals. The two new groups so formed were treated as “control” and “experimental”, the former being injected with normal saline and the latter with atropine sulphate. The References p . I2-13

10

P. B. B R A D L E Y

electrocorticogram was monitored in the animals with implanted electrodes and whcn the dissociation had appeared in the animals receiving atropine all were tested again for the conditioned avoidance response. In this experiment, no significant difference was found in the performance of the animals treated with atropine as compared with those injected with saline and there was no difference between the animals with implanted electrodes as compared with unoperated ones. There was some scatter of the reaction times in this experiment but no consistent trend. A conditioned avoidance test was also used in the second experiment but this involved a discrimination between two different auditory stimuli. One, a bell, being the positive conditioned stimulus, and the second, a buzzer the negative conditioned stimulus. The experimental plan was the same as for the previous experiment. Again, there was no significant difference in the responses of atropine-treated animals as compared with the saline-treated controls, nor between operated and unoperated animals. Since the conditioned avoidance response represents a very rigid type of behavioural response with a strong motivational aspect, we thought that a different kind of test involving a different kind of motivation might show up effects of atropine on behaviour. The third experiment therefore consisted of a maze in which animals were required to run from one end to the other for a food reward. Both running times and reaction times were measured and the mean for a number of trials taken for each animal. As before, half the animals were operated upon and electrodes implanted so that the electrocorticogram could be monitored. Again, half of each group was injected with atropine and the other half with saline. No significant changes in either the running times or the reaction times were produced by atropine in doses which induced dissociation in the EEG. We concluded from the results of these experiments that the presence of the dissociated EEG as produced by administration of atropine did not modify the behaviour of the animals within the limits of the methods used. This is somewhat surprising since more moderate doses of anticholinergic drugs have been shown to have effects on behaviour. However, from the results of our experiments it would appear that either the presence of slow waves in the electrocorticogram has no particular relevance for behaviour or the cortical mechanisms which are affected by drugs producing dissociation are not those concerned with behavioural responses. Finally, I should like to review some investigations of the nature of cholinergic mechanisms at the neuronal level. These investigations have been mainly directed towards elucidating the possible role of acetylcholine as a synaptic transmitter in the central nervous system. They have involved the use of the micro-iontophoretic technique in which multibarrelled microelectrodes are used to record the activity of a single cell and various substances which are contained in the other barrels of the micropipette are applied by iontophoresis (see Curtisll). It is believed that the effects observed on the neuronal activity are due to a direct action of the substance ejected on the cell whose activity is being recorded. Our own studies with this method have been carried out on neurones in the brain stem reticular formation in unanaesthetized decerebrate cat@. We have found that acetylcholine can produce both excitatory

EFFECT OF D R U G S O N

EEG

AND BEHAVIOUR

11

and inhibitory effects (Fig. 7). Thus, in a study of over six hundred neurones, 35% were found to be excited by application of acetylcholine, 22 % were inhibited and the remaining 43 % were unaffected by this substance2. These results indicate that there AC h

15

246810

B

20

4osec-G

ACh

I

I

3b

5 10

20

40

I

&

60 sec

Fig. 7. The effects of acetylcholine, applied by micro-iontophoresis to two different neurones in the brain stem of a decerebrate cat with a current of 75 nA. A : a neurone showing excitation; B: a neurone showing inhibition.

may be more than one type of cholinoceptive receptor in the central nervous system and this view is supported by the finding that the excitatory and inhibitory effects of acetylcholine differ pharmacologically. In comparing the effects of muscarine and acetylcholine on the same neurone, it was found that neurones excited by acetylcholine were also excited by muscarine to a similar extent and that muscarine inhibited all neurones inhibited by acetylcholine but with a greater and more prolonged effect. Nicotine was found to have excitatory effects on neurones excited by acetylcholine but did not inhibit neurones whose activity was inhibited by acetylcholine. Thus, it seems that the inhibitory effects of acetylcholine are exclusively muscarinic whereas its excitatory effects are both muscarinic and nicotinic in nature. Atropine was included in a study of antagonists to acetylcholine and blocked both excitatory and inhibitory effects. However, atropine was found to have an inhibitory action of its own and it appeared that this was a non-specific effect since it was present in neurones which were not cholinoceptive, i.e. atropine inhibited the activity of neurones which did not respond to application of acetylcholine. A similar non-specific effect was found in the case of physostigmine which caused excitation of neuronal activity independently of References p . 12-13

12

P. B. B R A D L E Y

the response to acetylcholine (Fig. 8). This drug potentiated the effects of acetylcholine, both excitatory and inhibitory. The excitatory action of physostigmine itself was present even where acetylcholine was ineffective.

Fig. 8. The effects of iontophoretically applied physostigmine on the activity of a single neurone in the brain stem and on the response to acetylcholine. The effect of physostigmine is excitatory but the inhibitory action of acetylcholine is also potentiated.

These experiments show that a number of neurones in the brain stem are cholinoceptive and that cholinergic receptors in the central nervous system are probably of more than one type. The results support the view that acetylcholine has an important role in the brain, probably as a synaptic transmitter. However, the finding that both atropine and physostigmine, the two substances which produce dissociation between behaviour and electrical activity, have effects on the neurones unrelated to the presence of cholinergic receptors suggests that we must also consider the possibility that the dissociation is unrelated to cholinergic mechanisms in the brain. REFERENCES BRADLEY, P. B. (1964) In: AnimalBehaviour andDrug Action, Eds. H. Steinberg, A. V. S . de Reuck and J. Knight, J . & A. Churchill Ltd., London, p. 338-344. BRADLEY, P. B., DHAWAN, B. N. AND WOLSTENCROFT, J. H. (1966) Pharmacological properties of cholinoceptive neurones in the medulla and pons of the cat. J. Physiol., 183,658-674. BRADLEY, P. B. A N D ELKES,J. (1953a) The effect of atropine, hyoscyamine, physostigmine and neostigmine on the electrical activity of the brain of the conscious cat. J. Physiol., 120, 14 P. BRADLEY, P. B. AND ELKES, J. (1953b) A technique for recording the electrical activity of the brain in the conscious animal. Electroenceph. Clin. Neurophysiol., 5,451-456. BRADLEY, P. B. AND ELKES,J. (1957) The effects of some drugs on the electrical activity of the brain. Brain, 80, 77-1 17. BRADLEY, P. B. AND HANCE, A. J. (1957) The effect of chlorpromazine and methopromazine on the electrical activity of the brain in the cat. Electroenceph. Clin. Neurophysiol., 9, 191-215.

EFFECT OF D R U G S O N

EEG

A N D BEHAVIOUR

13

7 BRADLEY, P. B. AND KEY,B. J. (1958) The effect of drugson arousal responses produced by electrical stimulation of the reticular formation of the brain. Electroenceph. Clin. Newophysiol., 10,97-110. 8 BRADLEY, P. B. AND KEY,B. J. (1959) A comparative study of the effects of drugs on the arousal system of the brain. Brit. J. Pharmacol., 14, 340-349. 9 BRADLEY, P. B. AND WOLSTENCROFT, J. H. (1965) Actions of drugs on single neurones in the brainstem. Brit. med. Bull., 21, 15-18. 10 BRIMBLECOMBE, R. W. AND GREEN,D. M. (1967) Central effects of imipramine-like anti-depressants in relation to their peripheral anticholinergic activity. Znt. J. Neuropharmacol., 6 , 133-143. I 1 CURTIS,D. R. (1965) Actions of drugs on single neurones in the spinal cord and thalamus. Brit. med. Bull., 21, 5-9. 12 FUNDERBURK, W. H. AND CASE,T. J. (1 951) The effect of atropine on cortical potentials. Electroenceph. Clin. Neurophysiol., 3, 213-223. 13 HANCE,A . J. (1956) Studies on the effects of drugs on the electrical activity of the brain and behaviour. Ph. D. Thesis, University of Birmingham. 14 RINALDI, F. AND HIMWICH, H. E. (1955) Alerting responses and actions of atropine and cholinergic drugs. Arch. neurol. Psychiat., 73, 387-395. 15 WIKLER,A. (1952) Pharmacologic dissociation of behaviour and E.E.G. “sleep patterrs” in dogs: morphine, N-allylnormorphine, and atropine. Proc. SOC.exp. Biol. N . Y., 79, 261-265.

14

Neuropharmacological Comparison of Subcortical Actions of Anticholinergic Compounds R.P. WHITE A N D A. S. R U D O L P H Department of Pharmacology and Brain Research Institute, University of Tennessee Medical Units,Memphis, Tennessee (U.S.A.)

Numerous reports attest to a direct action of the belladonna alkaloids on the cerebral cortex. They preferentially accumulate in cortical tissue6l and produce a greater percentage loss of acetylcholine in the cerebral cortex than in subcortical areaslg920. The local effects of cholinergic drugs topically applied to the cortex are antagonized by the belladonna alkaloidsQ~37~41 and atropine administered by close arterial injection will unquestionably produce unilaterally cortical EEG synchrony41. Systemically administered, these alkaloids block the EEG activation pattern, but not the behavioral flight reaction derived from hypothalamic stimulation in rabbits3’; an obvious ‘dissociation’ between electrocorticogram and behavior. However, it should be emphasized that these alkaloids abolish conditioned behavior in rabbits32935 and induce abnormal behavior in dogs5~59963.Indeed, many behavioral effects produced by these alkaloids in dogs (e.g., blindness, intermittent sleep, absence of placing reflexes) resemble those produced by decortication5~59.Since the normal electrocorticogram is absent in decorticate dogs, it is not too surprising that some drugs might cause an EEG ‘sleep’ pattern without inducing sleep behaviorally. The experiments of Loeb et ~ 1 . 3 0show the difficulty of demonstrating, at least electrographically, a subcortical site of action for these compounds. Single shock responses recorded from the midbrain reticulum are not reduced by atropine. Moreover, electrostimulation of the reticular formation will still inhibit thalamocortical recruiting indicating that ieticulo-thalamic functions are not impaired by atropine. These investigators confirmed that the EEG activation pattern obtained from highfrequency stimulation of subcortical structures is abolished by atropine, but emphasize this effect might be produced by atropine at the cortical level. On the other hand, there is much data indicating the belladonna alkaloids exert important subcortical effects. Di-isopropylfluorophosphate (DFP) given unilaterally into the nucleus caudatus produces specific contraversive body movements which are antagonized by atropine58. Rhythmic leg movements induced by electrostimulation of the subthalamus are inhibited by the belladonna alkaloidszg. The EEG synchrony produced by atropine given at the cortical level does not prevent the EEG activation induced by electrostimulation of the reticular formation, but atropine administered intravenously abolishes this reticular effect on the cortex, indicating a subcortical

SUBCORTICAL ACTIONS OF ANTlCHOLINERGICS

15

action of atropine is necessary to obtain complete blockade of the EEG activating mechanism41. Physostigmine given intravenously can abolish single shock responses recorded from the midbrain reticulum, an effect which is completely antagonized by the belladonna alkaloids61. However, in all studies with intact animals, including responses from single neurons7, such results may depend on important reverberating or corticofugal connections with various subcortical areas, thus making it difficult to assess which sites are primarily affected by these drugs. Also, injections of drugs directly into the brain substance may excite receptors not normally reached by the vascular route nor normally excited by adjacent neurons. It is significant, therefore, to find reports indicating that the belladonna alkaloids exert actions in animals in which the telencephalon and/or most of the prosencephalon have been extirpated and that cholinergic mechanisms are operant in the brainstem. Teuchmann47 found that scopolamine reduced spinal flexor reflexes in thalamic (decorticate) cats, but not in decerebrate (midcollicular transected) or spinal cats. These results clearly indicate a diencephalic site of action of scopolamine. DeMaarlO confirmed this finding with both scopolamine and atropine, further showing by removing most of the prosencephalon that the ventro-caudal portion of the diencephalon (roughly the area of the subthalamus) was the site for this inhibitory action. Further evidence that there are cholinergic receptors in this general area was afforded by Desmedt and Schlagll who showed that eserine greatly increased the discharge of single midbrain neurons in cats with transections through the posterior aspects of the diencephalon. The administration of acetylcholine directly into the lateral reticular formation causes EEG activation and behavioral alertness2], providing further evidence that EEG arousal may result from excitation of subcortical cholinergic me~hanisms33~42~5~. Also, some unilateral body movements produced by the injection of DFP into one carotid artery persist after complete removal of the diencephalon57, indicating again that cholinergic receptors are present below the thalamus. Most reports suggest that the newer anticholinergic psychotomimetics, the piperidyl benzilates, have similar central actions to the belladonna alkaloids. Both groups of drugs, for example, block the EEG activation caused by physostigmine53,55,61 and reduce brain acetylcholine content20 in laboratory animals to a degree that appears related to their reported psychotogenicity in humans. The piperidyl benzilates appear to differ in effect only quantitatively from the belladonna alkaloids, i.e., in their affinity for different cholinergic receptors53. They have similar behavioral effects in dogs17159 and man26. Both groups of compounds also block the electrographic effects of physostigmine recorded from the midbrain reticulum in intact rabbits61. However, since physostigmine clearly produces electrographic signs of excitation above the midbrain in rabbits45, it is possible that the midbrain electrographic changes reported were secondary to drug effects on structures more anterior in the neuraxis. The work described here was performed, therefore, to ascertain whether anticholinergic agents could antagonize the electrographic effects induced by physostigmine on ‘spontaneous’ brain waves and on single shock responses recorded from the midbrain reticulum of rabbits in which all activity of the prosencephalon was eliminated. References p . 24-26

16

R. P. WHITE A N D A. S. R U D O L P H METHODS

Data were obtained from thirty-one adult albino rabbits. All drugs were injected through a cannulated saphenous vein with dosages computed from the active base. Surgery was performed under local anesthesia (1 procaine) to avoid the effects that residual amounts of general anesthetics may exert on the drugs studied61. Great care was exercised so that no alarm reactions (body movements, blood pressure changes) were observed during the surgical procedures. In one group of rabbits some peripheral signs of action of the anticholinergics were studied; namely, effects on pupil size, light reflex and bradycardia induced by excitation of the peripheral end of the transected right vagus nerve (Table 1). TABLE I COMPARISON OF ANTICHOLINERGIC BLOCKADE AT PERIPHERAL AND CENTRAL SITES IN THE RABBIT. NUMBERS IN PARENTHESES ILLUSTRATE MG/KG DOSES GIVEN YIELDING THE RESULTS INDICATED. PHYSOSTIGMINE GIVEN AFTER THE ANTICHOLINERGIC AGENT

I.V.

--

-~

Drug

Vagal blockade

Light reflex

Mydriasis

Evoked pot. of midbrain animal

__

EEG from intact rabbit

Atropine Physostigmine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) None ( 0.1)

Yes 0.5) Yes ( 0.1)

Present ( 5.0) Sleep ( 5.0)* Present [ 0.2) Sleep ( 0.1)

Scopolamine Physostigmine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) None ( 0.1)

Yes ( 0.5) Yes ( 0.1)

Present ( 2.0) Present ( 0.2)

Sleep ( O S ) * Sleep ( 0.3)

JB-329 Physostigmine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) None ( 0.2)

Yes ( 0.5) Yes ( 0.1)

Present ( 2.0) Present ( 0.2)

Sleep ( OS)* Sleep ( 0.4)

JB-318 Physostigrnine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) Yes ( 0.1)

Yes ( 0.5) Less ( 0.1)

Present ( 2.0) Present ( 0.2)

Sleep ( 0.5)* Sleep ( 0.1)

JB-340 Physostigrnine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) None ( 0.1)

Yes (0.5 ) Yes ( 0.1)

Present (20.0) Reduced ( 0.2)

Sleep (50.0) Alert ( 0.1)

JB-305 Physostigmine

No (12.0)

Yes

(15.0)

Mild (15.0) N o ( 0.1)

Present (15.0) Reduced ( 0.2)

Sleep (15.0) Alert ( 0.1)

* Dosages known to consistently

block EEG effects of at least 0.1 mg/kg of physostigmine55.

In the second group, the midbrain was isolated functionally from the prosencephalon. First, the common carotid arteries were tied ;this procedure did not cause unconsciousness. The cerebrum was carefully ablated with a sharp scalpel and the diencephalon was transected from the midbrain by electrocautery and in most cases completely removed (Fig. 1). The animal was then curarized (1 mg/kg d-tubocurarine), put under artificial respiration, and placed in a stereotaxic instrument. Three electrodes were inserted into the midbrain with the help of the stereotaxic atlas of FiflcovS and MarSala14. The exposed surface of the superior colliculus, however, was the starting point for placing most of the electrodes (e.g., 3P and 2L from rostra1 and medial aspects, respectively). The depth of the electrodes was usually 7 mm from the surface

SUBCORTICAL ACTIONS OF ANTICHOLINERGICS

17

of the superior colliculus, although the morphology of the response rather than a predetermined site was selected for the recordings. Electrode position was verified by sectioning the fixed brain (Fig. I). The EEG and evoked potentials were obtained from these electrodes. The evoked potentials were produced by single shocks applied to one sciatic nerve (0.1 msec duration with supramaximal voltage) and/or to the pontine region of the tegmentum. These were recorded on the EEG tracings or by means of a cathode ray oscilloscope, the latter recorded photographically (Fig. 2). In some experiments one additional electrode was placed in the fluid rostra1 to the midbrain

Fig. 1. Gross appearance of the brain and the histological position of the electrodes in the nucleus reticularis tegrnenti of the midbrain from which recordings were obtained.

but no biological activity was recordable. The exposed surfaces of brain were covered with a thin layer of warm mineral oil. Further details on methods used in this study for obtaining single shock responses may be found elsewhereGI. The anticholinergic drugs studied were : scopolamine hydrobromide, atropine sulphate, JB-329 (Ditran), JB-318, JB-340, and JB-305. The J B compounds were References p . 24-26

R. P. W H I T E A N D A. S. R U D O L P H

18

”’!!!t

EVOKED RESPONSE

EEG

CONTROL

CONTROL

-*a C

.

.

CONTROL

CONTROL

H.F.S. SCIATIC N.

0.2 MG

PHYSOSTIGMINE

CRo

0 . 2 MG. PHYSOSTIGMINE

RECOVERY

t

50 MSEC.

2 MG. SCOPOLAMINE

44

0.2 MG. PHYSOSTIGMINE

+‘“J--y’w.’r

9

2 HG.

JE-310

4

0.2 MG.

0 . 2 MG.

PHYSCSTIGMINE

PHVSOSTICMINE

Fig. 2. EEG pattern and the single shock responses obtained from the midbrain of four rabbits in which the diencephalon and cerebrum were removed (see Fig. 1). Tracings at A show control (extreme left) recordings of the EEG, which is low in amplitude, and the single shock responses superimposed (abovedots)upontheEEG,andthesingle shockresponseasit appears on the cathode ray oscilloscope. Subsequently seen (from left to right) is the abolition of the single shock responsa resulting from high frequency (30 c/s) stimulation of one sciatic nerve without a change in the EEG; recovery; and lastly that 0.2 mg/kg of physostigmine also abolishes the evoked potentials. Tracings at B show (left to right) control recording; attenuation of the evoked responses by physostigrnine; restoration of the shock responses by scopolamine; and lastly that after scopolamine, physostigmine no longer affects theevokedresponses. Tracingsat Cshowcontrol responses; that JB-318 didnotchange these responses but blocked the usual effect of physostigrnine. Tracings at D show that, in contrast to JB-318 or scopolamine, the anticholinergic agent JB-340 fails to antagonize the usual effect of physostigmine.

piperidyl benzilates obtained from the Lakeside Laboratories, Milwaukee, Wisconsin ; the first two are psychotomimetics whereas the last two are not2. Their structural formulae are given elsewherez955. All these drugs in adequate dosage produce EEG synchrony in intact rabbit@. The doses used in these experiments were comparatively high to assure a reasonable effect in the ablated animals since a dose-response study was not feasible because of the high mortality of the midbrain preparation. The agonist employed was physostigmine sulfate because it is a cholinergic substance with known CNS stimulant properties53. However, prostigmine bromide was also administered to some animals to ascertain the degree to which cholinergic stimulation peripherally might contribute to the central phenomena studied. Mean blood pressure was recorded on a Grass Polygraph from one femoral artery. The blood pressure rose abruptly 20 to 70 mm Hg immediately following the brain transection by electrocautery and gradually retcrned to control value or to below normal. In the latter case, saline was administered to restore the blood pressure to control value. Normal body temperature was maintained by use of a heating pad.

SUBCORTICAL ACTIONS OF ANTICHOLINERGICS

19

RESULTS

The single shock responses recorded from the reticular formation of the midbrain animal may be easily seen superimposed on the EEG pattern and readily recorded on the cathode ray oscilloscope (Fig. 2). These evoked potentials are remarkably stable in amplitude and shape in most of the ablated animals. However, as the frequency of electrical stimulation is increased to about 6 c/s or more they are reduced in amplitude (attenuated) and at higher frequencies (20-60 c/s) are abolished (Fig. 2). Virtually the same effects were obtained whether the stimuli were applied to the sciatic nerve or in the brainstem, caudal to the recording electrodes. Evoked responses did not always appear in every midbrain lead, but those obtained from different leads resemble each other in response to high frequency stimulation and to the drugs administered. An EEG was recorded from all leads placed in the midbrain. Physostigmine (0.2 mg/kg) produced changes in the single shock responses similar to those obtained with high frequency stimulation ; namely, attenuation, to less than 50 % of control, or less often, abolition of the evoked potential (Fig. 2). Scopolamine, atropine, JB-329 and JB-318 clearly antagonized this action of physostigmine (Fig. 2, Table I) whether given before or after physostigmine. All of these anticholinergic agents restored the evoked response, in the latter case before one-half of the dose was administered, indicating the doses given (Table I) were higher than necessary to reverse the physostigmine effect. Furthermore, these compounds completely antagonized the effects of an additional dose of physostigmine (Fig. 2). In contrast, JB-305 and JB-340 failed to block the attenuation of the evoked response produced by physo,tigmine (Fig. 2, Table 1). Neostigmine failed to mimic the effect of physostigmine, suggesting that the latter’s action is central in origin. This suggestion is strengthened by the finding that JB-340, which possesses strong anticholinergic properties peripherally (Table I), failed to alter this central manifestation of physostigmine (Fig. 2). Other differences between central and peripheral actions among the anticholinergic compounds are shown in Table I. The very low amplitude (4 to 33 p V ) EEG tracing obtained from the midbrain of these animals was not significantly changed by any of the anticholinergic drugs or by electrostimulation (Fig. 2). Even anesthetic doses of pentobarbital (25 mg/kg) when administered slowly did not increase the amplitude. These findings are in contrast to those obtained in intact rabbits where a midbrain EEG pattern is seen which closely resembles the electrocorticogram in magnitude and in response to these drugs and to electrical stimulation53. The low amplitude may reflect a reduction in blood flow in these ablated animals. On the other hand, a continuum of frequencies that shift spontaneously from about 3.5 to 8 c/s was usually evident with the most common frequency range being 4 to 7. Also, a typical high amplitude (180 pVor more) seizure pattern was obtained by injecting Metrazol (5-15 mg/kg) and, in some animals, occurred spontaneously. These last two findings, coupled with the fact that the single shock response was prominent in these ablated animals, suggest that the low amplitude electroreticulogram may be caused by a reduction in the number of midbrain neurons References p . 24-26

20

R. P. W H I T E A N D A. S. R U D O L P H

being discharged “spontaneously” as a result of the destruction of reverberating circuits normally active in the intact brain. DISCUSSION

The four anticholinergic compounds (atropine, scopolamine, JB-329, and JB-3 18) which blocked the attenuating effects of physostigmine on the evoked potentials are well known psychotomimetics. These findings agree with previous studies indicating that drugs which block the EEG activation caused by physostigmine are either psychotomimetics or antiparkinson agents in man53J4*55>62.Moreover, the literature clearly indicates that such compounds exert both actions clinically53.Parsidol (ethopropazine), for example, is a phenothiazine used in the treatment of Parkinson’s disease which, in contrast to the ataractic phenothiazines, will block the EEG activation induced by physostigmine62, will arrest the pseudoparkinsonism produced by chlorpromazine29, and in adequate dosage will induce hallucinations39. Similarly, benactyzine readily blocks the EEG effects of physostigmine54; may be used to treat parkinsonism24; and is capable of producing psychotic episodes51. Lastly, the psychotomimetic JB-3 18 has an antitremor action in patients’. The present study affords new evidence that anticholinergic compounds can antagonize an effect of a cholinergic drug at the midbrain level. Although the changes induced by physostigmine in the midbrain reticulum may be indirect, e.g., secondary to excitation of other brainstem structures, the results clearly show that these changes take place in the absence of the prosencephalon and indicate they are central in origin. Therefore, the antagonism of atropine, scopolamine, JB-329 (Ditran) and JB-3 18 to this effect ofphysostigmine can occur centrally at sites below the diencephalon. This is not true of JB-340, which exerts its strong anticholinergic actions only peripherally. These results, coupled with the finding that JB-318 is less active on the iris than JB-340, but more active centrally in antagonizing physostigmine, emphasize that central and peripheral cholinolytic properties of a drug may be of a different order of magnitude and that inferences concerning the central actions of these drugs should not be based upon results obtained from peripheral tissue53. In high doses, for example, Darstine (mepiperphenidol) mimics many of the peripheral effects of atropine in humans but is far less active centrally60; whereas, the newer anticholinergic psychotomimetics (piperidyl benzilates) are evidently superior tools for neuropsychiatric research because they produce more gradual EEG and psychological changes as the dose is increased, induce richer hallucinogenic episodes, and have less autonomic side effects than the belladonna alkaloidss3. There is a growing body of pharmacological evidence indicating that a family of related cholinergic receptors are involved in synaptic transmission. There is, for example, pharmacological evidence indicating both muscarinic and nicotinic receptors are capable of independently causing EEG activationz5150but that nicotinic receptors may not be present in the cerebral cortex37. Atropine readily blocks the EEG arousal caused by acetylcholine42 but does not block the effects of this substance on the Renshaw ce1112. Conversely, dihydro-/3-erythroidine inhibits the Renshaw neuron

SUBCORTICAL ACTIONS OF ANTICHOLINERGICS

21

but fails to block EEG activation. At least two distinct cholinergic receptors are involved in sympathetic ganglionic transmissionl3. Also, atropine and scopolamine inhibit spinal flexor reflexes only at subthalamic sites; whereas, caramiphen produces a similar inhibition at areas below this level of the neuraxislo. The intracerebral injection of cholinergic substances will produce a wide variety of behavioral effects (rage, sleep, arousal, catatonia, etc.) depending on the area injected 21.It is not surprising, therefore, that differences have been reported among the “anticholinergic psychotomimetics” including the belladonna alkaloids. A survey of the literature, however, indicates these differences are quantitative in nature rather than qualitative53. These compounds apparently also have a dual action in impairing central cholinergic mechanisms: they block the usual EEG effects of cholinergic agents55 and they decrease brain acetylcholine content20. These two actions appear to be related; the most potent blockers of cholinergic drugs seem to be most active in decreasing brain acetylcholine. Moreover, the effects on the EEG53,55*59, behavior55959 and on brain acetylcholine levels20 caused by the anticholinergic psychotomimetics reach a maximum with comparatively low doses. Since scopolamine36.59 and atropine59 d o not produce notable sedation in intact rabbits even in enormous doses59 but do produce a “Iissive” effect in decorticate rabbits36959, it is apparent that the subcortical and cortical actions of the drugs differ in this species. The subcortical effects of these drugs may be related to their antiparkinson actions in humans. At least, our findings lend support to the hypothesis that antiparkinson agents counteract a subcortical hyperactive cholinergic mechanism24,28,49,62. The early work of Veit and Vogt48 may help explain why low doses of these belladonna alkaloids produce sedation in dogs and in higher doses disorientation or deliriumlike behavior59. They found the concentration of scopolamine in the cerebral cortex and midbrain to be comparable after low doses of scopolamine (2 mg/kg), but after high doses (10 mg/kg) the concentration in the cortex was about 2.5 times greater than in the midbrain. Therefore, in low doses normal function may be inhibited throughout the neuraxis producing effects on the EEG and midbrain reticular evoked responses similar to those produced by other sedatives in animals53-61. In low doses both are also sedatives in man32138~60.In higher doses, only the functions of the cerebrum may be further impaired significantly, producing in dogs many of the characteristics of decortication5.59 without producing classical signs of anesthesia, either behaviorally, electroencephalographically, or on midbrain evoked responses34.53. Indeed, the behavioral changes induced in dogs (blindness, slow compulsive gait, etc.) resemble those obtained with LSD256. Moreover, some motor effects produced by amphetamine are enhanced8359 suggesting that adrenergic or non-cholinergic mechanisms may be “released” during atropine or scopolamine toxicity. However, the so-called stimulation produced by these drugs does not mimic that induced by amphetamine in dogs59 and has no reliable analeptic value clinically. Indeed the “stimulation” seen with these alkaloids is interrupted by periods of sleep in dogs, monkeys and man53. Moreover, the EEG activation caused by adrenergic drugs is blocked by anticholinergic agents56 suggesting some central “adrenergic” systems depend ultimately on cholinergic References p . 24-26

22

R. P. W H I T E A N D

A. S. R U D O L P H

processes. Also, the belladonna alkaloids greatly potentiate the depressant actions of ether (personal observations) and pentobarbital69 in dogs. They also potentiate the action of barbiturates in rats19 and monkeys5Q.Such synergism is a common characteristic of CNS depressants. Hence the “excitation” or disorientation induced by toxic doses of the belladonna alkaloids may be considered “pseudostimulation” caused by an inhibition of cholinergic mechanisms and an imperfect “release” of non-cholinergic mechanisms. Most reports dealing with the mechanism by which anticholinergics produce central actions are consonant with the hypothesis that they inhibit cholinergic systems and that their behavioral effects may result from a functional “imbalance”4,53. The tricyclic antidepressants (e.g., desipramine) evidently possess mild anticholinergic actions centrally, reducing the effects of cholinergic agents3.40, while simultaneously may enhancing the actions of adrenergic agents40943, so that a dual a~tion4~~43~46~53 account for their clinical efficacy. In this regard, it would be of interest to administer to endogenously depressed patients small doses of both atropine and amphetamine to ascertain whether this combination is also beneficial to such patients. In humans, of atropine toxicity the EEG synchrony64 and hallucinogenic manife~tationsl~9~~964 revert to normal after the intramuscular administration of 4 mg of physostigmine, presumably by restoring cholinergic functions centrally. Similarly, the belladonna alkaloids and physostigmine are antagonists in their effects on conditioned behavior of laboratory animals22135. Another cholinergic drug, tetrahydroaminoacrin (THA), in 60 mg doses i.v. will antagonize the hallucinations, stupor and other effects of 10 mg of Ditran (JB-329) in humanslB.Moreover, the psychotomimetic effects of Ditran are changed to a coma-like condition with small doses of chlorpr0mazine2~, and perhaps because the sedative properties of chlorpromazine are intensified or “released”, adrenergic phenomena are antagonized. The independent nature of the EEG and the evoked responses seen in this study indicate that different processes or neurons are involved in each phenomenon. The evoked phenomenon is also more specific, being obtained only in certain leads; whereas, an EEG was obtained from all locations of the midbrain. Independent variations between the EEG and evoked responses are also evident in intact a n i m a W . It is possible that the neuroglia contribute significantly to the EEG pattern16, and evoked potentials are specific signals so that, at least under the influence of drugs, they may vary independently. Since the anticholinergic agents failed to alter the EEG pattern from these “midbrain animals”, but do change this pattern in intact rabbits61, it is possible that their main site of action is on cholinergic links above the midbrain23v40.52.On the other hand, such drugs may not be able to affect these abnormal waves because impulses responsible for normal EEG patterns were destroyed, thereby preventing any cholinolytic action at the midbrain level. In this regard, atropine fails to change the electrocorticogram obtained from the acute “isolated hemisphere” preparation42 so that the drug apparently does not affect the electroencephalogram in such abnormal preparations. From more physiological experiments, however, Rinaldi41 concluded that atropine must have actions both at cortical and midbrain sites,

SUBCORTICAL ACTIONS OF ANTJCHOLINERGICS

23

Although it is questionable whether the results described here are specifically related to the many diverse behavioral effects produced by anticholinergic compounds, they do provide evidence that the midbrain reticulum is affected - probably directly by cholinergic and anticholinergic agents. They further show that pharmacological changes may be induced in the midbrain reticular formation that are not dependent on higher centers and demonstrate the importance of testing anticholinergic compounds upon a background of cholinergic stimulation. Alone, none of the anticholinergic drugs given systemically depress midbrain evoked responses, but neither do ataractics or sedatives61. However, the latter two groups of drugs fail to block the effect ofphysostigmine on single shock responses recorded from the midbrain of intact rabbits; whereas, the piperidyl benzilates and belladonna alkaloids are antagonistic to physostigmine53~61.Similarly, atropine alone will not inhibit synaptic activity of the cerebral cortex, but will block the effects of acetylcholine given by close arterial injection44. Lastly, our positive findings question the implication of Giarman and Pepeu20 that scopolamine has no important action on the “rostral” midbrain because in rats it failed to reduce significantly acetylcholine levels in this area. The fact that these investigators found a reduction of 14% in “rostral” midbrain agrees with the report of Veit and Vogt48 that scopolamine does enter the midbrain. Also, with higher doses Veit and Vogt found about 2.5 times more scopolamine in the cortex than in the midbrain. Giarman and Pepeu showed, similarly, that scopolamine reduced acetylcholine levels of the cerebrum 2.5 times greater than the 14% in the midbrain. Our findings indicate that at least some anticholinergic agents enter regions of the brainstem to antagonize the actions of cholinergic agents and support the inferences of 0 t h e r s 7 J 5 ~ ~that 8 ~ ~chclinoceptive ~ neurons are present in the brain below the diencephalon which may be pharmacologically altered by anticholinergic compounds. SUMMARY

Electrographic recordings were obtained from the midbrain reticular formation of rabbits in which the prosencephalon (cerebrum and diencephalon) was extirpated. These recordings consisted of “spontaneous” brain waves (EEG) and evoked potentials produced by applying single shock stimuli to one sciatic nerve or to the pontine region of the reticular formation. Physostigmine (0.2 mg/kg) significantly reduced or abolished the single shock responses. In contrast, atropine, scopolamine, and four piperidyl benzilates (JB-329, JB-3 18, JB-340, JB-305) did not reduce the amplitude of the evoked potentials. However, atropine, scopolamine, JB-3 18 and JB-329 completely blocked the effect of physostigmine on the single shock responses; whereas, JB-340 and JB-305 had no such effect. Possible relationships between the ability of anticholinergic compounds to produce psychotic episodes or ameliorate Parkinson’s disease and their ability to block the electrographic effects of physostigmine in experimental animals were discussed. It was concluded that the reduction of the single shock response caused by physostigmine was central in origin because (1) this same effect was obtained by high freReferences p . 24-26

24

R. P. W H I T E A N D A. S. R U D O L P H

quency stimulation of the pontine reticulum, (2) it was not produced by neostigmine, and (3) JB-340, a compound which exerts strong anticholinergic actions peripherally, was unable to block this action of physostigmine. The “spontaneous” electrical activity recorded from the midbrain reticulum in these experiments was low in amplitude and was not significantly changed by the above drugs. Pentobarbital also failed to change these patterns but a Metrazol seizure pattern could be induced. Important differences between recording antagonistic actions of drugs on single shock responses and on “spontaneous” electrical activity was therefore emphasized. It was also stressed that the use of a suitable agonist (e.g., physostigmine) may be necessary to reveal significant effects of, and differences among, many centrally acting drugs. REFERENCES 1 AeooD, L. G. (1957) Some relations between chemicalstructure andphysiologicalaction of mescaline and related compounds, in Neuropharmacology. Josiah Macy, Jr. Foundation, New York, pp. 229-234. 2 ABOOD,L. G., OSTFELD, A. AND BIEL,J. H. (1959) Structure-activity relationship of 3-piperidyl benzilates with psychotogenic properties. Arch. int. Pharmacodyn., 120, 186-200. 3 BENESOVA, O., BOHDANECK~, Z. AND GROFOVA, I. (1964) Electrophysiological analysis of the neuroleptic and antidepressant actions of psychotropic drugs in rabbits. Znt. J. Neuropharmacol., 3,479-488. 4 BIEL,J. H., NUHFER, P. A., HOYA,W. K., LEISTER, H. A. AND ABOOD,L. G. (1962) Cholinergic blockade as an approach to the development of new psychotropic agents. Ann. N. Y.Acad. Sci., 96, 251-262. 5 BIJLSMA, U. G. AND BROUWER, J. E. (1928) Die Wirkung des Skopolamins in Kombination mit Cyanid, Kohlenoxyd und Luftverdunnung, Arch. exp. Path. Pharmakol., 138, 190-207. 6 BOGDANSKI, D. F., WEISSBACH, H. AND UDENFRIEND, S. (1958) Pharmacological studies with the serotonin precursor, 5-hydroxytryptophan. J. Pharrnacol., 122, 182-194. 7 BRADLEY, P. B. (1957) Microelectrode approach to the neurnpharmacology of the reticularformatiorr. Psychotropic Drugs. Eds. S. Garattini, V. Ghetti. Elsevier, Amsterdam, 207-216. 8 CARLTON, P. L. AND DIDAMO, P. (1961) Augmentation of the behavioral effects of amphetamine by atropine. J. Pharmacol., 132, 91-96. 9 CHATFIELD, P. 0. AND PURPURA, D. P. (1954) Augmentation of evoked cortical potentials by topical application of prostigmine and acetylcholine after atropinization of cortex. EEG Clin. Neurophysiol., 6, 287-298. 10 DEMAAR, E. W. J. (1956) Site and mode of action in the central nervous system of some drugs used in the treatment of Parkinsonism. Arch. int. Pharmacodyn., 105, 349-365. 11 DESMEDT, J. E. AND SCHLAG, J. (1957) Mise en evidence d’elements cholinergiques dans la formation reticulee mesendphalique. J . Physic/. (Paris), 49, 136-1 38. 12 ECCLES, J. C., ECCLES, R. M. AND FATT,P. (1956) Pharmacological investigations on a central synapse operated by acetylcholine. J. Physiol. (Lond.), 131, 154-169. 13 ECCLES, R. M. AND LIBET,B. (1961) Origin and blockade of the synaptic responses of curarized sympathetic ganglia. J. Physiol., 157, 484-503. E. AND MARSALA, J. (1962) Sereotaxic atlases for the cat, rabbit and rat. In J. BureS 14 FIFKOVA, et al., Electrophysiological Methods in Biological Research. Academic Press, New York, Appendix I: 426-467. 15 FORRER, G. R. (1958) Atropine coma therapy: Report of a death. J. Michigan State Med. Soc., 57, 996-998. 16 GALAMBOS, R. (1961) A glia-neural theory of brain function. Proc. Nut. Acad. Sci.,47, 129-136. 17 GERSHON, S. AND BELL,C. (1963) A study of the antagonism of some indole alkaloids to the behavioural effects of “Ditran”. M e d exp., 8, 15-27. 18 GERSHON, S. AND OLARIU,J. (1960) JB-329 - A new psychotomimetic. Its antagonism by tetrahydroaminacrin and its comparison with LSD, mescaline and sernyl. J. Neuropsychiat., 1,283-292.

SUBCORTICAL ACTIONS OF ANTICHOLINERGICS

25

19 GIARMAN, N. J. AND PEPEU,G. (1962) Drug-induced changes in brain acetylcholine. Brit. J. Pharmacol., 19, 226-234. 20 GIARMAN, N. J. AND PEPEU,G. (1964) The influence of centrally acting cholinolytic drugs on brain acetylcholine levels. Brit. J. Pharmacol., 23, 123-1 30. 21 HERNANDEZ-PEON, R., CHAVEZ-IBARRA, G., MORGANE, P. J. AND TIMO-IARIA, C. (1963) Limbic cholinergic pathways involved in sleep and emotional behavior. Exper. Neurul., 8, 93-1 11. 22 HERZ,A. (1967) Some actions of cholinergic and anticholinergic drugs on behaviour. This volume. 23 HIMWICH, H. E. AND CUCULIC, Z. (1967) An examination of a possible cholinergic link in the EEG arousal reaction. This volume. 24 HIMWICH,H. E. AND RINALDI,F. (1957) The antiparkinson activity of benactyzine. Arch. int. Pharmacodyn., 110, 119-127. 25 ILYUTCHENOK, R. J. (1963) Problems of chemical perceptibility of the bruin stem reticular formation. Psychopharmacological Methods, Eds: Z. Votava, M. Horvath, 0. Vinaf. Pergamon Press, Oxford, England, pp. 115-122. 26 ISBELL, H., ROSENBERG, D. E., MINER,E. J. AND LOGAN, C. R. (1964) Tolerance and cross tolerance to scopolamine, n-ethyl-3-piperidyl benzylate (JB-318) and LSD-25. Neuropsychopharmacology, 3,440-446. 27 ITIL,T. M. (1966) Quantitative EEG changes induced by anticholinergic drugs and their behavioral cxrelates in man. Rec. Adv. B i d . Psychiat., 8, 151-173. 28 JENKNER, F. L. AND WARD,JR., A. (1953) Bulbar reticular formation and tremor. Arch. Neirrol. Psychiat. (Chicago), 70,489-502. 29 KRUSE,W. (1960) Treatment of drug-induced extrapyramidal symptoms. Dis. New. Syst., 21, 79-8 1, 30 LOEB,C., MAGNI,F. AND ROW,G. F. (1960) Electrophysiological analysis of the action of atropine on the central nervous system. Arch. ital. Biol., 98, 293-307. 31 LONGO,V. G. (1956) Effects of scopolamine and atropine on electroencephalographic and behavioral reactions due to hypothalamic stimulation. J. Pharmacol., 116, 198-208. 32 LONGO, V. G. (1966) Mechanisms of the behavioral and electroencephalographic effects of atropine and related compounds. Pharmacol. Rev., 18, 965-996. 33 LONGO,V. G. AND SILVESTRIM, B. (1957) Effects of adrenerkic and cholinergic drugs injected by the intra-carotid route on electrical activity of brain. Proc. Soc. exp. Biol., 95, 43-47. 34 LONGO,V. G. AND SILVESTRINI, B. (1958) Contribution a l’etude des rapports entre le potentiel reticulaire Bvoque, l’etat d’anesthtsie et l’activite electrique cerebrale. EEG Clin. Neurophysiol., 10,111-120. 35 MCGAUGH, J. L., DEBARAN, L. AND LONGO,V. G. (1963) Electroencephalographic and behavioral analysis of drug effects on an instrumental reward discrimination in rabbits. Psychophurmacofogia, 4, 126-1 38. 36 MEHES,J. (1929) Studien iiber den Skopolaminschlaf und seine Verstarkung durch Morphium. Arch. exp. Path. Pharmakol., 142, 309-322. 37 NICKANDER, R. C. AND YIM,G. K. W. (1964) Effects of tremorine and cholinergic drugs on the isolated cerebral cortex. Int. J. Neuropharmacol., 3, 571-578. 38 OSTFELD, A. M. AND ARUGUETE, A. (1962) Central nervous system effects of hyoscine in man. J. Pharmacol., 137, 133-139. 39 PFEIFFER, C. C., (1959) Parasymphathetic neurohumors; possible precursors and effect on behavior. Int. Rev. Neurobiol., 1, 195-244. 40 RATHBUN, R. C. AND SLATER, I. H. (1963) Amitriptyline and nortriptyline as antagonists of central and peripheral cholinergic action. Psychopharmacologiu, 4, 114-125. 41 RINALDI,F. (1956) Direct action of atropine on the cerebral cortex of the rabbit. Progr. Brain Res., 16, 229-244. 42 RINALDI,F. AND HIMWICH, H. E. (1955) Cholinergic mechanisms involved in function of mesodiencephalic activating system. Arch. Nrurol. Psychiat., 73, 396-402. 43 SIGG,E. 3.(1962) The pharmacodynamics of imipramine. The first Hahnemann Symposium on Psychosomatic Medicine. Lea and Febiger, Pub., 671-678. 44 SIGG,E. B., DRAKONTIDES, A. B. A N D DAY,C. (1965) Muscarinic inhibition of dendritic postsynaptic potentials in cat cortex. Int. J. Neurupharmucol., 4,281-289. 45 STEINER, W. G. AND HIMWICH, H. E. (1962) Central cholinolytic action of chlorpromazine. Science, 136, 873-874. 46 SULSER, F., BICKEL, M. H. AND BRODIE, B. B. (1964)The action of desmethylimipramine in counteracting sedation and cholinergic effects of reserpine-like drugs. J. Pharmacol., 141, 321-330.

26

R. P. WHITE A N D A . S. RUDOLPH

47 TEUCHMANN, J. (1949) The action of diallilbarbituric acid and of scopolamine on the spinal reflexes of the decapitated, the decerebrated and the decorticated cat. Arch. in/. Pharmacodyn., 79, 257-262. 48 VEIT,F. AND VOGT,M. (1935) Verteilung von Arzneistoffen auf verschiedene Regionen des Zentralnervensystems, zugleich ein Beitrag zu ihrer quantitativen Mikrobestimmung im Gewebe. Arch. f: exper. Path. u. Pharmakol., 178, 534-559. 49 VERNIER, V. G. AND UNNA,K. R. (1956) Theexperimental evaluationofantiparkinsoncompounds. Ann. N . Y.Acad. Sci., 64,690-704. 50 VILLARREAL, J. E. AND DOMINO,E. F. (1964) Evidence for two types of cholinergic receptors involved in EEG desynchronization. The Pharmacologist, 6, 192. 51 VOJTECHOVSKY, M. (1967) Experimental psychosis induced by benactyzine. This volume. 52 WHITE,R. P. (1963) Relationship between cholinergic drugs and EEG activation. Arch. in/. Pharmacodyn., 145, 1-17. 53 WHITE,R. P. (1966) Electrographic and behavioral signs of anticholinergic activity. Rec. Adv. Biol. Psychiat., 8, 127-139. 54 WHITE,R. P. AND BOYAJY, J. D. (1960) Neuropharmacological comparison of atropine, scopolamine, benactyzine, diphenhydramine and hydroxyzine, Arch. in/. Pharmacodyn., 127, 260-273. 55 WHITE,R. P. AND CARLTON, R. A. (1963) Evidence indicating central atropine-like actions of psychotogenic piperidyl benzilates. Psychopharmacologia, 4, 459-47 I . 56 WHITE,R. P. AND DAIGNEAULT, E. A. (1959) The antagonism of atropine to the EEG effects of adrenergic drugs. J. Pharmacol., 125, 339-346. 57 WHITE,R. P. AND HIMWICH,H. E. (1957) Analysis of forced circling induced by DFP and ablation of cerebral structures. Am. J. Physiol., 189, 513-516. 58 WHITE,R. P. AND HIMWICH, H. E. (1957) Circus movements and excitation of striatal and mesodiencephalic centers in rabbits. J . Neurophysiol., 20, 81-90. 59 WHITE,R. P., NASH,c. B., WESTERBEKE, E. J. AND POSSANZA, G . 3. (1961) Phylogenetic comparison of central actions produced by different doses of atropine and hyoscine. Arch. int. Pharmacodyn., 132, 349-363, 60 WHITE,R. P., RINALDI, F. AND HIMWICH, H. E. (1956) Central and peripheral nervous effects of atropine sulfate and mepiperphenidol bromide (Darstine) on human subjects. J. Appl. Physiol., 8,635-642. 61 WHITE,R. P., SEWELL, H. H., JR. AND RUDOLPH, A. S. (1965) Drug-induced dissociation between evoked reticular potentials and the EEG. EEG Clin. Neurophysiol., 19, 16-24. 62 WHITE,R. P. AND WESTERBEKE, E. J. (1961) Differences in central anticholinergic actions of phenothiazine derivatives. Exp. Neurol., 4, 317-329. 63 WIKLER, A. (1952) Pharmacologic dissociation of behavior and EEG “sleep patterns” in dogs: Morphine, N-allylnormorphine and atropine. Proc. Soc. exp. Biol., N . Y., 79, 261-264. 64 WILSCN, W. P. (1961) Observations on the effect of toxic doses of atropine on the electroencephalogram of man. J. Neuropsychiat., 2, 186-190.

27

An Examination of a Possible Cortical Cholinergic Link in the EEG Arousal Reaction Z. CUCULIC*, K A T H R Y N BOST

AND

H. E. H I M W I C H

Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, lllii7ois 61401 (U.S.A.)

Knowledge concerning the function of the reticular formation received a great impetus with the discoveries of Moruzzi and Magoun27 and their schools. The part played by the thalamic diffuse projection systems has also been intensively studied, especially by Jasperl5. Because these two systems may function together in the production of EEG arousal, Rinaldi and H i m ~ i c hsuggested ~ ~ ? ~ ~the term mesodiencephalic activating system (MDAS) to denote their combined action. They also suggested that the EEG arousal reaction is cholinergic i n nature for not only was the response evoked by cholinergic drugs but it was blocked by atropine. Other cholinolytics, like benztropine methane sulfonate (Cogentin) and benzilic acid diethylaminoethylester (benactyzine)l3J4 also prevent EEG arousal. In seeking a rostral link in the MDAS, it was found that EEG arousal could still be obtained after post-collicular post-pontine secti0n3~.With a more rostral transection, at the pre-collicular pre-pontine level, thus excluding the midbrain, Steiner and Himwich38 observed that the administration of acetylcholine was followed by EEG arousal which was blocked by atropine. In this regard it is pertinent that Smirnov and Ilyutchenok36 found that atropine-like drugs applied topically to the cortex blocked EEG arousal, thus indicating the possibility of cholinergic structures at or close beneath the cortical surface, a suggestion of importance in establishing a cortical cholinergic link in EEG arousal. In agreement with these workers are the observations of Rinaldi30, who administered atropine not only topically but also by injection into a cortical artery. The present report is concerned with further studies of the above suggestion. METHOD A N D MATERIAL

Our experiments were performed on 90 adult albino rabbits weighing between 2.5 to 3.0 kg. The rabbits were prepared under ether and local 0.2 % pontocaine anesthesia and were studied under artificial respiration, using small doses of curarz sufficient to establish physical immobilization but not to affect the electrical phenomenon. The electrocardiogram was recorded throughout the experiment and blood pressure

*

Present address: Douglas Hospital, Verdun, Quebec (Canada).

References p. 38-39

28

z.

CUCULIC

et al.

readings were obtained from the femoral artery by means of a mercury manometer. A polyethylene cannula was inserted in the femoral vein for the intravenous administration of the drugs. The cortical electrodes were of the silver ball type with flexible insulated silver wires and placed in Plexiglas holders. In some experiments, deep coaxial electrodes were implanted according to the maps of Sawyer, Everett and Green33a for the rabbit. The recordings were registered from the following areas: anterior cortex (motor), posterior cortex (limbic), caudate nucleus, hippocampus and thalamus (see Fig. 1). All drugs were dissolved in distilled water and for

SAGITTAL SUTURE

SAGITTAL SUTURE

Fig. 1. Schematic representation of the electrode sites used in present experiments. a, b, c, d are cortical leads. In these experiments monopolar coaxial electrodes were used. The deep electrodes include nucleus caudatus (NC), thalamus (TH), reticular formation (RF) and hippocampus (H). Four different arrangements of electrode sites are presented: A, B, C, D.

topical application were placed on the exposed cortex in gel-foam pledgets (6 x 8 mm) saturated with the solution of the drug used. The pH was adjusted to approximately 5. The drugs used and the concentrations employed were : benactyzine 0.3 %, scopolamine 0.3 %, atropine 0.34.5 %, eserine 0.1 %, pilocarpine 0.1 %, Metrazol 0.5 %, pontocaine 2 % and 1 %, carbocaine 1 %, and strychnine 0.05%. The intravenous injections included cholinolytic drugs given in the following ranges : scopolamine 0.9-1 .O mg/kg, benactyzine 1.5-2 mg/kg, atropine 1-3 mg/kg. The dosage ranges of the cholinergic agents included i.v. eserine at 0.1-0.2 mg/kg and i.v. pilocarpine 14 mg/kg. One convulsant was given i.v., strychnine 0.1-0.3 mg/kg. The ranges of the other two drugs used i.v. were d-amphetamine 4.5-6 mg/kg and 5-hydroxytryptophan with a toral dose of 33 mg. In each experiment the dose employed was sufficient to produce the required results, whether blocking or activation. To stimulate the midbrain reticular substance, direct current pulses of a frequency of 300/second and a duration of 2 msec were employed. The voltage varied from 1 to 10 with total durations from

EEG

AROUSAL AND CORTICAL CHOLINERGIC LINK

29

3 to 10 seconds. The topical effects of the local anesthetics, pontocaine and carbocaine, were examined against the EEG alerting produced by 0.1 mg/kg eserine i.v. RESULTS

A . Results o j topicul application of the cholinolytic drugs (benactyzine, scopolamine, atropine) upon cortical alert patterns previously established with i.v. eserine or pilocarpine In 24 animals, soon after the topical application of gel-foam pledgets saturated with the anticholinergic agents, the alert pattern evoked by eserine (16 experiments) and pilocarpine (8 experiments) changed to high slow waves but there were differences between the three cholinolytic drugs. Following the application of atropine, a period of from 5 to 6 minutes elapsed before the synchronization was observed. Scopolamine required about 3 minutes and benactyzine synchronized the brain waves during the first 2 minutes of application. With simultaneous cortical and subcortical recordings (12 experiments) the synchronization was seen only in the area of the cerebral cortex EFFECT OF TOPICAL APPLICATION OF BENACTYZJNE O N THF AROUSAL REACTION EVOKED BY ESERINE I.V. a

b C

EKG 1 0 0 p v ~ I-SCC.

R.M

\

C

N

.

R s=zc-s

Fig. 2. Blocking effect of topical application of benactyzine (0.3 % solution) to the left cortical sites (a, c) on the arousal reaction evoked by i.v. eserine (0.2 mg/kg). For the designation of electrode sites, refer to Fig. 1, C.

where the cholinolytic drug was applied (Fig. 1, B,C,D) Other cortical as well as subcortical areas (nucleus caudatus, hippocampus, thalamus) continued unchanged (Fig. 2). Moreover, of the three drugs, benactyzine proved to be the most effective in the complete elimination of alerting and was especially superior to atropine which intermittently permitted alerting patterns to break through the inhibition. The blocking action of benactyzine was most pronounced in the immediate vicinity of the coronal suture. Single topical applications of benactyzine or scopolamine were followed by periods of EEG synchronization which endured for 30-45 minutes. But after atropine References p. 38-39

30

z.

C U C U L I C et

al.

the synchronization lasted only 15-20 minutes. After these periods, however, alerting procedures evoked the EEG arousal in all leads although the arousal was still somewhat less pronounced in the area of cortical application of the anticholinergic agents. The differences between the inhibitory influences exerted by benactyzine on eserine or pilocarpine alerting were slight.

B. Eflect of the topical application of the cholinolytic drugs upon the cortical EEG resting pattern In 6 animals the areas of application of benactyzine revealed waves of comparatively higher amplitude and lower frequency than the other cortical regions; these differences endured for approximately 60 minutes. In 6 other rabbits, however, the resting patterns did not disclose differences between the sites of application and the contralateral control area until after the usual alerting procedures when the treated portions of the cerebral cortex failed to show alerting either to sound or pain or the intravenous administration of eserine, similar to the patterns observed in Fig. 2. C . Results o j the topical application of cholinolytic drugs on the EEG arousal evoked by the electrical stimulation of the midbrain Approximately 2 minutes after the topical application of benactyzine (8 animals) the strength of the current stimulating the midbrain reticular formation had to be increased from 3-4 volts with a duration of 5 seconds to a current of from 6-8 volts with the same duration before the arousal reaction could be evoked. But 10 minutes after the application of benactyzine even a current of 10 V for 10 seconds could no longer induce the alert pattern though it was seen clearly in non-treated areas (Fig. 3).

EFFECT OF TOPICAL APPLICATION OF BENACTYZINE ON THE AROUSAL REACTION EVOKED BY ELECTRICAL STIMULATION OF THE MIDBRAIN RETICULAR FORMATION

d

EKG

Fig. 3. Contrasting effects of previous application of benactyzine (0.3 % solution) to left cortical areas (a, c) on the arousal reaction subsequently evoked by electrical stimulation of the midbrain reticular formation (direct current pulses with a frequency of 300/second and a duration of 2 msec; 4 volts with a total duration of 5 seconds). For the designation of electrode sites, refer to Fig. 1, B.

EEG

AROUSAL AND CORTICAL CHOLINERGIC LINK

31

D . Effects of topical application of’ eserine followed by the intravenous administration of either benactyzine or scopolamine In 5 animals after the topical application of eserine, spiking appeared in the area of application in approximately 5 minutes. Subsequently, in most instances the intensity increased to seizure-like outbursts. These seizure patterns were triggered by the intravenous administration of benactyzine or of scopolamine as well as by various alerting procedures. E. Eflects of topical application with pentamethylenetetrazol (Metrazol) followed by i.v. eserine on EEG alerting In 5 rabbits the topical application of pentamethylenetetrazol (0.5 solution) was followed by the appearance of rounded waves of high amplitude and slower frequency though sometimes spikes were also observed as well as seizure-like patterns. But the usual EEG arousal patterns evoked by 0.2 mg/kg of eserine continued interspersed between the abnormal waves similar in general design to that shown in Fig. 4 where the abnormal waves are caused by strychnine. F. Effects of topical applications of strychnine against the intravenous administration of benactyzine, eserine and pilocarpine In 7 experiments after the application of strychnine, sporadic spikes, later becoming s-izurz-like outbursts were observed. The outbursts were readily triggered by peripheral stimulation (sound or pain) or by the intravenous administration of eserine. The intravenous administration of benactyzine intensified the local effects of strychnine but in untreated cortical areas, waves of high amplitude and low frequency indicating synchronization were observed. In 6 of these 7 experiments strychnine spiking spread t o all leads but was most pronounced in the areas of topical application where the frequency was greater and seizure-like outbursts were more numerous. In 6 additional experiments the intravenous administration of eserine (0.2 mg/kg) or pilocarpine (14 mg/kg) induced alert patterns in all leads though in the cortical area covered with the gel-foam pledgets saturated with strychnine, the alerting patterns were interspersed between areas of multiple spiking even more pronounced than with strychnine alone (Fig. 4). G. Interactions resulting from the topical application of benactyzine and the intra-

venous administration of strychnine Benactyzine was applied topically prior to the intravenous administration of strychnine in 6 animals. Following strychnine, blood pressure increased from 80 mm Hg to 160 mm Hg, two minutes after the injection. Simultaneously EEG alert patterns were observed in the areas other than those of topical application. This alert pattern lasted for 3 minutes after the first injection (0.1 mg/kg) but after two or three injections the arousal persisted for 10-1 5 minutes. The arousal reaction was followed by spiking first in the areas of the application of the cholinolytics but later spreading to all areas recorded. References p . 38-39

32

2. C U C U L I C et al. INTER-REACTION OF TOPICAL APPLICATION O f STRYCHNINE AND THE AROUSAL REACTION EVOKED BY ESERINE I.V.

a

c d 100 p

v

L

I-SEC.

EKG OCZC-3

Fig. 4. The arousal reaction evoked by the intravenous administration of eserine (0.2 rng/kg) following previous topical application of strychnine (0.05% solution) on the left hemicortex (areas a, c). For the designation of electrode sites, refer to Fig. I A .

H . Efects of the topical application of benactyzine on the alert pattern following the intravenous administration of amphetanzine In 4 experiments benactyzine was applied topically and in 2 others atropine was used after the amphetamine-induced alert pattern had been established. Approximately two minutes after benactyzine the alert pattern was changed to high slow waves in the treated areas (Fig. 5 ) . In one experiment with successive applications of benactyzine EFFECT OF TOPJCAL APPLICATION OF BENACTYZINE ON THE AROUSAL REACTION EVOKED BY 0-AMPHETAMINE I.V. AMPHETAMINE AROUSAL

AFTER BENACTYZINE

I-SEC.

DLZC-6

Fig. 5. l h e blocking effect of topical application of benactyzine (0.3%solution) to the left cortical areas (a, c) on the arousal reaction evoked by the intravenous administration of d-amphetamine (total dose 9 mg). For the designation of electrode sites, refer to Fig. l,A.

EEG

AROUSAL A N D CORTICAL CHOLINERGIC LINK

33

to all cortical areas, amphetamine arousal was gradually extinguished until all exposed cortical sites exhibited blocked patterns as previously described in B. With atropine the synchronization was not so prominent and required about 10 minutes before blocking occurred.

EKG

5-HTP ACTIVATION a-

b C

EKG EFFECT OF TOPICAL APPLICATION OF BENACTYZINE ON THE AROUSAL REACTION EVOKED BY 5-HTP I.V.

a

>

b C-

&c-2 'OQ+z

EKG

Fig. 6 . Interactions of 5-hydroxytryptophan induced alerting and topical application of benactyzine. Top series of tracings, controls with alerting response to sound of hand clapping. Middle series of tracings, alerting induced by 3 intracarotid injections of 5-hydroxytryptophan (5-HT), 11 mg each. Lowest series of tracings portray effects of the topical application of benactyzine (left cortical areis a, c) on the arousal reaction evoked by 5-HTP. For the designation of electrode sites, refer to Fig. 1, A. References p . 38-39

34

z.

CUCULIC

et al.

I. Effects of topical application of benactyzine on the EEG alert pattern obtained by the administration of 5-hydroxytryptop~an(5-HTP) According to the technique of Schweigerdt and Himwich34, EEG arousal was obtained by intracarotid injections of 11 mg of 5-HTP given three times. Usually the alerting evoked by 5-HTP was not as marked as that produced by eserine nor was the inhibition exerted by benactyzine always as clearly demarcated, but in 3 of 4 experiments a change from the resting pattern followed the intravenous administration of the serotonin precursor and in each benactyzine synchronized the pattern of the cortical surfaces to which it had been applied (Fig. 6). J . Effects of pontocaine and carbocaine on EEG arousal patterns In 4 experiments gel-foam pledgets saturated with 2 % pontocaine were applied to the left side of the exposed cortex causing some diminution of amplitude on that side. Nevertheless arousal patterns continued, occurring apparently spontaneously and after auditory and pain stimuli as well as after i.v. eserine, 0.1 mg/kg. The arousal responses evoked by i.v. eserine continued for 20-30 minutes (Fig. 7) despite the topical application of pontocaine. Four other experiments with 1 % pontocaine INTER - REACTION OF TOPICAL APPLICATION OF 2 % PONTOCAINE A N D THE AROUSAL REACTION EVOKED BY ESERlNE Apparently. spontaneous Control

a r o u d 16 min. after 2 I Pontocaine on a & c

Arousal 3 min. after h e r i n e 0.1 m g / k g I.V.

b-

v

d-

K E G -

100 p v

r , I-See.

9c-zc--7

Fig. 7. Interreaction of topical application of 20/, pontocaine and the arousal reaction evoked by eserine.

yielded similar results except that in addition, biphasic spikes of slow frequencies occurred in the medicated side. In these animals the alert patterns were continuous on the right side of the cortex but on the left side were observed only between the slow spikes, a mixture of frequencies similar to the alerting patterns which occurred between strychnine spikes as illustrated in Figure 4. After 0.1 mg/kg eserine i.v. in one of these four observations, the slow spikes gradually disappeared and the alerting patterns were maintained throughout all observed areas. In four additional experiments 1 % carbocaine saturated gel-foam pledgets were applied to the left side of the exposed cortex with a resulting slight increase in amplitude and slowing of the brain waves on that

EEG

AROUSAL A N D CORTICAL CHOLINERGIC LINK

35

side. Arousal reponses again occurred spontaneously, after auditory and pain stimuli and following the i.v. injection of 0.1 mg/kg eserine. The eserine-induced arousal responses ranged in duration from 16-30 minutes. These 12 experiments illustrate EEG arousal after the application of local anesthetics. DISCUSSION

The purpose of the present experiments was to apply various pharmacologically active drugs on the cerebral cortex and to compare their influences on the EEG arousal reaction. The results have revealed that drugs which inhibit structures innervated by post-ganglionic cholinergic nerves in the peripheral system also block the EEG arousal reaction. In this regard it is well to point out that Dale and Sherrington6, in discussing possibilities for mechanisms of central transmission, pointed to the phenomena of peripheral transmission as a heuristic guide. Many diverse methods have produced evidence in favor of a cortical cholinergic mechanism and our results are discussed in relation to these findings. The present experiments have revealed that topical application of all three cholinolytics used have been effective in blocking EEG arousal; but atropine sulfate was less so than either benactyzine or scopolamine. Here we may refer to Giarman and PepeuQ who found that atropine sulfate was also less effective than scopolamine in reducing the acetylcholine levels of cerebral hemispheres. It is also worthy of comment that such reductions in acetylcholine concentrations did not occur in the subcortical areas of the rostra1 mesencephalon nor of the caudal mesencephalon and myelencephalon. long^^^, studying th.: EEG blocking effects of intravenously administered scopolamine and atropine on the arousal reaction evoked by eserine, found similar correlations, scopolamine being 10 to 15 times as active as atropine. It is important to notice that every drug applied topically in our experiments caused spiking in higher concentrations but only cholinolytic drugs blocked the EEG arousal reaction. The topical application of acetylcholine usually produced a localized depression, often succeeded by enhanced electrical activity8 and in some instances this activity was organized into distinct groups of paroxysmal spikes. There is no doubt that eserine, causing the accumulation of acetylcholine, can evoke alerting but the intravenous or intracarotid administration of anticholinesterase can also produce EEG spikingll. The use of anticholinesterases is associated with accumulations of acetylcholine in the brainl. This is not to say that excessive EEG activity must have an exclusive basis only in large amounts of acetylcholine. Our observations with topical applications of strychnine, pentamethylenetetrazol and 1 % pontocaine revealed abnormal electroencephalographic activity evoked by mechanisms other than those involving acetylcholine. In fact, the EEG changes brought on by these three drugs became apparent only when their influence was prepotent over that of acetylcholine and replaced the EEG alerting pattern evoked by eserine (Fig. 4). These EEG results indicate that eserine induces arousal independently of the abnormalities associated with the topical application of strychnine, thus indicating that they are two unrelated phenomena. References p. 38-39

36

Z . C U C U L I C et

al.

In evoking EEG arousal we used sensory modalities (audition or pain) and electrical stimulation of the midbrain reticular formation as well as pharmacologically active substances. It is significant that chemically induced EEG arousal is mediated by different classes of agents : (a) the anticholinesterase, physostigmine, (b) the directly reactive cholinergic drug, pilocarpine, (c) the adrenergic compound, d-amphetamine and (d) the precursor of serotonin, 5-HTP. The latter is known to provoke arousal associated with a large and rapid rise of serotonin in the midbrain and without any marked changes in norepinephrine levels4. In this connection it will be well to remember the conclusion of Rinaldi and Himwich31, who regarded atropine as a nniversal inhibitor of alerting reactions, when referring to the effects of atropine-induced blocking of EEG activation, whether the source of stimulation was peripheral or central, including the administration of cholinergic substancesl3J4. White and D a i g n e a ~ l thave ~ ~ carried this idea further and showed that atropine blocked the alerting produced by adrenergic agents. In the present experiments topical applications of benactyzine blocked EEG arousal induced by cholinergic and adrenergic agents as well as by 5-HTP, a precursor of serotonin. Though this communication is not directly concerned with the midbrain and medullary portion of the MDAS, we may point out that the reticular formation is not homogeneous pharrnacologically3~4onor in regard to possible neurotransmitters5~28. Irrespective of thc mechanisms involved in EEG arousal it can be blocked by the topical applications of cholinolytics. We must therefore conclude that this type of inhibition of arousal must act upon structures, whether axons, synapses or dendrites, with cholinergic function situated near the cortical surface. These cortical structures present sites where drugs with cholinolytic activities, including chlorpromazine administered intravenously38 prevent the EEG arousal reaction. Histochemical studies have revealed fibers containing acetylcholinesterase and travelling subcortically extending through layers V and IV, 0.8 to 1.3 mm below the cortical surface. KrnjeviE and Silver22 suggest that these fibers provide a cholinergic innervation of some deep pyramidal cells and that the cholinergic fibers represent the final corticopetal link in the MDAS. KrnjeviC and Phillis21 found that acetylcholine-sensitive cells are present in most regions of the cortex but occur in greatest concentration in the primary sensory areas, an observation agreeing with our finding that the topical effects of the cholinolytic drugs were most marked in the region of the coronal suture. Direct evidence for the presence of acetylcholine in cholinergic neurons has been demonstrated for the cerebral cortex and nutria of guinea pig42. Of the three cliolinolytic substances we applied topically, atropine and benactyzine are known to possess local anesthetic properties. Atropine has weak anesthetic properties, but benactyzine is more effective with double the activity of cocainelo. The potency of carbocaine is of the same order as cocaine but pontocaine is much stronger, in fact 15 times as effective as cocainel7. The fact that pontocaine failed to block EEG arousal suggests that the much less effective atropine and benactyzine do not block cortical arousal because of a local anesthetic property but because of a specific cholinolytic activity. Other observations can also be explained by the presence of such cholinergic

EEG

AROUSAL A N D CORTICAL CHOLINERGIC LINK

37

structures just beneath the cortical surfaces. MacIntosh and Oboris25 as well as Mitchell26 have found that excitation of the cortex through sensory nerves or by direct electrical stimulation increased the acetylcholine output in the fluid collected on the surface of the cortex, thus indicating that a cholinergic agent had been liberated from nervous structures within the cerebral cortex. Beleslin et a1.2 in cats under amytal anesthesia, perfused the cerebral subarachnoid space with neostigmine and the acetylcholine appeared in the effluent. It is noteworthy that output of the acetylcholine was most pronounced on the cerebral cortex bounding the ventral parts of the fissures of Sylvius. In contrast the acetylcholine content of the perfusion fluid obtained from interpeduncular fossa and cisterna magna was minimal. KrnjeviC and Phillis19920 and Spehlmann37,using microiontophoretic methods, found that neurons concentrated in the deeper layers of the cortex can be excited with acetylcholine while on the other hand anti-muscarinic substances like atropine and scopolamine are more potent antagonists of acetylcholine than the antinicotinic substances. For this reason KrnjeviElB suggested that the cortex receives a cholinergic innervation with muscarinic properties. Riehl et ~ 1 . ~ 9in, order to obtain more information on cholinergic receptors in the central nervous system, injected muscarine and arecoline in the enckphale isole' preparations of cats and found that belladonna alkaloids blocked peripheral and central effects of muscarine almost equally while with arecoline only peripheral inhibition was obtained. Histochemical studies yield additional evidence for fibers containing acetylcholinesterase activity. Shute and Lewis35 found that such fibers project from subcortical centers to the cortex. KrnjeviC and Silver22 reported that fibers originating from the striatum invade the fetal neocortex and presumably give rise to the cortical horizontal connections which in the adult are found in layers V and IV and are under control of striated and septa1 cells. Further evidence in favor of tangential cortical fibers comes from Hebbl2 who reported that cortical acetylcholinesterase activity is decreased after cortical undercutting. These observations have been confirmed by Rosenberg and EchIin33 in the chronic partially isolated cerebral cortex of the monkey with pial circulation intact. SUMMARY

A group of 90 adult rabbits weighing between 2.5 to 3.0 kg were subjects of an EEG analysis on the effects of various drugs topically applied to cerebral cortex or administered systemically. The topical application of the cholinolytic drugs benactyzine, scopolamine and atropine blocked previously established EEG alerting evoked by eserine or pilocarpine administered intravenously. The cholinolytic drugs also evoked synchronization when alerting was induced by peripheral stimulation as well as after the electrical stimulation of the midbrain. In addition, the cholinolytic drugs were effective in blocking alerting induced by i.v. amphetamine and 5-HTP. Carbocaine as well as pontocaine, and the latter is a far more potent anesthetic than either atropine or benactyzine, failed to interfere with arousal patterns. It is therefore suggested that the ability of the cholinolytic drugs we used to interfere with the EEG alerting References p . 38-39

38

z.

CUCULIC

et al.

reaction is a specific effect and not due to local anesthesia. Thus our experiments bring additional evidence for the concept of structures cholinergic in function and situated close to the cortical surface being the final corticopetal link in EEC alerting.

R E F E R E NCES 1 APRISON,M. H. AND NATHAN,P. (1956) Brain acetylcholine and behavior. Fed. Proc., 15, 5. 2 BELESLIN, D., POLAK,R. L. AND SPROULL, D. H. (1965) The release of acetylcholine into the cerebral subarachnoid space of anesthetized cats. J. Physiol., 177,420-428. 3 BRADLEY, P. B. (1957) Microelectrode approach to the neuropharmacology of the reticular formation. Psychotropic Drugs, S. Garattini and V. Ghetti, Eds., Elsevier, Amsterdam, pp. 207-216. 4 COSTA, E., PSCHEIDT, G. R., VANMETER,W. G. AND HIMWICH, H. E. (1960) Brain concentrations of biogenic amines and EEG patterns of rabbits. J. Pharmacol. Exper. Therap., 130,81-88. 5 DAHLSTROM, A. AND FUXE,K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. Acta physiol. s c a d , 62, Suppl. 232, 1-52. 6 DALE,SIR HENRY(1934) Chemical transmission of the effects of nerve impulses. Brit. Med. J., 1, 835-841. 7 DEROBERTIS, E. (1964) Electron microscope and chemical study of binding sites of brain biogenic amines. Biogenic Amines, Vol. 8, Progress in Brain Research, H. E. Himwich and W. A. Himwich, Eds., Elsevier, Amsterdam, pp. 118-136. 8 ESSIG,C. F., ADKINS, F. J. AND BARNARD, G. L. (1953) Observations on electrocorticographic effects of acetylcholine in monkeys and cats. Proc. SOC.Exper. Biol. Med., 82, 551-553. 9 GIARMAN, N. J. AND PEPEU,G. (1964) The influence of centrally acting cholinolytic drugs on brain acetylcholine levels. Brit. J. Pharmacol. Chemotherap., 23, 123-1 30. 10 GOODMAN, L. AND GILMAN,A. (1965) Pharmacological Basis of Therapeutics, Third edition, Macmillan Company, New York. 11 HAMPSON, J. L., ESSIG,c. F., MCAULEY, A. AND HIMWICH, H. E. (1950) Effects of di-isopropyl fluorophosphate (DFP) on electroencephalogram and cholinesterase activity. EEG Clin. Neurophysiol., 2, 41. 12 HEBEI,C. O., KRNJEVIC,K. AND SILVER,A. (1963) Effect of undercutting on the acetylcholinesterase and choline acetyltransferase activity in the cat’s cerebral cortex. Nature, 198, 692. 13 HIMWICH, H. E. AND RINALDI,F. (1957a) Analysis of the action of benztropine methanesulfonate against parkinsonism. Tranquilizing Drugs, H. E. Himwich, Ed., American Association for Advancement of Science, pp. 41-57. 14 HIMWICH, H. E. AND RINALDI, F. (1957b) The antiparkinson activity of benactvzine. Arch. Intern. Pharmacodynamie, 110, 119-127. 15 JASPER,H. H. (1949) Diffuse projection systems: the integrative action of the thalamic reticular system. EEG Clin. Neurophysiol., 1, 405-420. 17 JORDAN, E. P. (1958) Modern Drug Encyclopedia and Therapeutic Index, Drug Publications, Inc., New York. 18 KRNJEVIC, K. (1965) Actions of drugs on single neurones in cerebral cortex. Brit. Men. Bull., 21, 10-14. 19 K R N JEVIK. ~ , AND PHILLIS,J. W. (1963a) Iontophoretic studies of neurones in the mammalian cerebral cortex, J. Physiol., 165, 274. 20 KRNJEVIC, K. A N D PHILLIS, J. W. (1963b) Actions of certain amines on cerebral cortical neurones. Brit. J. Pharmacol., 20, 471-490. 21 KRNJEVIC, K. AND PHILLIS, J. W. (1963~)Acetylcholine-sensitive cells in the cerebral cortex. J. Physiol., 166, 296-327. 22 KRNJEVIC, K. AND SILVER, A. (1963) The distribution of ‘cholinergic’ fibres in the cerebral cortex. J. Physiol., 168, 39P. 23 KRNJEVIC, K. AND SILVER, A. (1965) A histochemical study of cholinergic fibres in the cerebral cortex, J. Anat., 99, 711-759. 24 LONGO,V. G . (1956) Effects of scopolamine and atropine on electroencephalographic and behavioral reqctioos due to hypothalamic stimulation, J, Pharmacol. Exper. Therap., 116, 198.

EEG

AROUSAL A N D CORTICAL CHOLINERGIC LINK

39

25 MACINTOSH, F. C. AND OBORIS, P. E. (1953) In: XIXInternationalPhysiologicalCongress, Montreal, August 31-September 4 ; Abstracts of Communications, p. 580. 26 MITCHELL, J. P. (1963) The spontaneous and evoked release of acetylcholine from the cerebral cortex. J . Physiol., 165, 98. 27 MORUZZI, G. AND MAGOUN,H. W. (1949) Brain stem reticular formation and activation of the EEG. EEG Clin. Neurophysiol., 1, 455473. 28 PAVLIN, R. (1965) Cholinesterases in reticular nerve cells. J. Neurochem., 12, 515-518. 29 RIEHL,J. L., PAUL-DAVID, J. AND UNNA,K. R. (1962) Comparison of the effects of arecoline and muscarine on the central nervous system. Int. J . Neuropharmacol., 1, 393-401. 30 RINALDI, F. (1965) Direct action of atropine on the cerebral cortex of the rabbit. Horizons in Neuropsychopharmacology, Vol. 16, Progress in Brain Research, W. A. Hirnwich and J. P. Schadk, Eds., Elsevier, New York-Amsterdam, pp. 229-244. 31 RINALDI,F. AND HIMWICH, H. E. (1955a) Alerting responses and actions of atropine and cholinergic drugs. Arch. Neurol. Psychiat., 73, 387. 32 RINALDI,F. AND HIMWICH, H. E. (195513) Cholinergic mechanism involved in function of mesodiencephalic activating system. Arch. Neurol. Psychiat., 73, 396. 33 ROSENBERG, P. AND ECHLIN,F. (1965) Cholinesterase activity of chronic partially isolated cortex. Neurology, 15, 700-707. C. H., EVERETT, J. W. AND GREEN, J. D. (1954) The rabbit diencephalon in stereotaxic 33a SAWYER, coordinates. J . Comp. Neurol., 144, 253-259. 34 SCHWEIGERDT, A. K. AND HIMWICH, H. K. (1964) An electroencephalographic study of bufotenin and 5-hydroxytryptophan. J. Pharmacol. Exper. Therap., 114, 253-259. 35 SHUTE,C. C. D. AND LEWIS,P. R. (1963) Cholinesterase-containing systems of the brains of the rat. Nature, 199, 1160-1164. 36 SMIRNOV, G . D. AND ILYUTCHENOK, R. V. (1962) Cholinergic mechanism of cortical activation. Sechenov Physiol. J. USSR, 49, 127-139. 37 SPEHLMANN, R. (1963) Acetylcholine and prostigmine electrophoresis at visual cortex neurons. J. Neurophysiol., 26, 127-139. 38 STEINER, W. G. AND HIMWICH, H. E. (1962) Central cholinolytic action of chlorpromazine. Science, 136, 783-875. 39 WHITE,R. P. AND DAIGNEAULT, E. A. (1959) The antagonism of atropine to the EEG effects of adrenergic drugs. J. Pharmacol. Exper. Therap., 125, 339. 40 WHITE,R. P., SEWELL, H. H., JR. AND RUDOLPH, A. S. (1965) Drug-induced dissociation between evoked reticular potentials and the EEG. EEG Clin. Neurophysiol., 19, 16-24. 41 WHITTAKER, V. P. (1964) Investigations on the storage sites of biogenic atnines in the central nervous system. Biogenic Amines, Vol. 8, Progress in Brain Research, H. E. Himwich and W. A. Himwich, Eds., Elsevier, New York-Amsterdam, pp. 9CL117. 42 WHITTAKER, V. P. AND SHERIDAN, M. N. (1965) The morphology and acetylcholine content of isolated cerebral cortical synaptic vesicles. J. Neurochem., 12, 363-372.

40

Influence of Atropine, Scopolamine and Benactyzine on the Physostigmine Aroussl Reaction in Rabbits Z . V O T A V A , 0. B E N E S O V A , Z . B O H D A N E C K Y *

AND

0. G R O F O V A * *

Department of Pharmacology, Medical Faculty of Hygiene, Charles University, Prague (Czechoslovakia)

Acetylcholine is one of the most important mediators of nervous transmission not only in the peripheral, but also in the central nervous system. Research on the catecholamines and serotonin has progressed more quickly than that on acetylcholine because of technical difficulties in the determination of this unstable substance. Recently, intensive research on the effect and mode of action of drugs influencing central cholinergic structures has been undertaken using electrophysiological, behavioural and biochemical techniques. As many authors have stated, both cholinomimetic and cholinolytic drugs change the EEG traces in cortical and subcortical structures, the former provoking the arousal pattern, the latter the resting pattern. These results have been recently reviewed by Long0899 and Votaval5. Wiklerlg, using dogs was the first to notice the “dissociation” between the EEG record and the behaviour of animals after the administration of atropine. The EEG patterns resembled those of sleep, whereas the animals were awake or even excited. These results have been later confirmed by many authors. In more detailed studies, it was found that memory was impaired when cholinolytic drugs provoked “sleep” patterns in EEG13J4. The EEG changes as well as behavioural impairment could be restored by administration of physostigmine10111. In previous paperslJ6, we showed that the EEG arousal reaction provoked by administration of physostigmine in rabbits with chronically implanted electrodes could be used as a tool for the evaluation of the central effects of anticholinergic drugs. In the present paper the effects of atropine, scopolamine and benactyzine are compared. METHODS

The techniques employed in these experiments follow closely those previously describedlJ6. Twelve male Chinchilla rabbits each weighing approximately three kilograms were employed throughout the study. The animals were implanted with four cortical and two subcortical electrodes under local tetrocaine anesthesia.

* **

Research Institute of Pharmacy and Biochemistry, Prague. Pepartment of Anatomy, Faculty of Medicine, Prague,

INFLUENCE OF ANTICHOLINERGIC DRUGS ON PHYSOSTIGMINE REACTION IN RABBITS

4I

Nickelplated screws of 3 mm diameter were used as cortical electrodes; two were implanted into the skull in the frontal area (one on the right, one on the left hemisphere) and two similarly in the occipital area. The subcortical electrodes were bipolar (tip separation 1.5 mm), made of enamelled constantan wire, 0.17 mm diameter, fixed in a miniature electric plug and implanted into the hippocampus and thalamus. All electrodes were attached to the skull by dental phosphate cement. The positions of the subcortical electrodes were determined according to the stereotactic atlas of rabbit brain by Fifkovh and Margalas. During the experiments, the electrodes were connected to an 8-channel VEB Dresden electroencephalograph by means of miniature plugs and fine cables. The fixation of the non-anesthetized rabbit during the experiment in the special metal cage is shown in Fig. 1. Details of the implanted electrodes and the plugs for the EEG records are given in Fig. 2.

Fig. 1. Fixation of the rabbit in a special metal cage during experiment.

After completing all experiments (in about three months), the rabbits were killed under thiopental anesthesia, the brains were perfused with 10 % formol, removed and fixed in 10% formol. After two months, the brains were embedded in celloidin, sectioned at 40 micromillimeters and stained with cresyl violet according to Nissl. The location of the electrodes of our group of twelve rabbits in two anterior-posterior levels, i.e. in the level AP 0.5 and AP 4.0 is shown in Fig. 3. The correct localization within the structures of hippocampus (pyramidal layers, fascia dentata) was found in peferences p . 46-47

42

z. V O T A V A et 01.

Fig. 2. Detailed view of the head with chronically implanted electrodes and miniature plugs to be connected to EEG device.

ten out of the twelve rabbits. In two cases the electrodes were placed in the white matter upon the hippocampus. The localization of the electrodes in the thalamus was correct in all twelve rabbits; their tips being located in the anterior nuclei (in AP 0.5 level) or in the posterior nuclei of thalamus (in AP 4.0 level). At the beginning of the experiments, the rabbits were placed in the cage for about one hour to habituate them to the procedure. The tests were started about two weeks after implantation of electrodes and performed at weekly intervals. All drugs were administered into the auricular vein over a two minute period. Prior to this normal (isotonic) saline solution was injected to determine the effect of injection procedure. After an interval of one week, the effect of physostigmine at the dose level of 0.1 mg/kg was tested. This dose of physostigmine evoked a cortical desynchronization and subcortical synchronization (regular sinusoidal waves of 4-7 c/sec, theta activity) for a period of 20 to 40 minutes. No autonomic and/or behavioural effects were observed after this dose of physostigmine. During the following weeks, the drugs to be tested were administered intravenously; (atropine 0.5 and 1.0 mg/kg, scopolamine 0.05,O.l and 0.5 mg/kg, benactyzine 0.1 and 0.5 mg/kg) with again physostigmine at the same dose, i.e. 0.1 mg/kg after a 20 minute interval. The previous administration of anticholinergic drugs curtailed the duration of the EEG arousal reaction, as demonstrated in Figure 4. For the quantitative evaluation of the duration of the EEG arousal reaction, the hippocampal and/or thalamic theta activity was used, as its presence is easily deter-

INFLUENCE OF ANTICHOLINERGIC DRUGS ON PHYSOSTIGMINE REACTION IN RABBITS

43

Fig. 3. Scheme of a typical location of the electrodes in two anterior-posterior levels according to the stereotactic atlas of Fifiovk and Margala (1960). The upper part of the figure represents the cut in the level AP 0.5, the lower part in the level AP 4. AD = nc. anterodorsalis thalami; AM = nc. anterornedialis thalami; AV = nc. anteroventralis thalami; GL = corpus geniculatum laterale; GLV = corpus geniculatum laterale pars ventralis; LA = nc. lateralis anterior; LP = nc. lateralis posterior; M D = nc. rnediodorsalis thalami; PT = nc. parataenialis; R = nc. reticularis thalami; RE = nc. reuniens; SN = substantia nigra; TMT = tractus marnmillothalamicus; VA = nc. ventralis anterior; VL = nc. ventralis lateralis; VM = nc. ventralis medialis; VPL = nc. ventralis posterolateralis; VPM = nc. ventralis posteromedialis; ZI = zona incerta.

mined. Changes in the cortical EEG traces (fast frequency, low voltage waves) were used only for judging the general state of the rabbit. At the end of the experiment, the effect of physostigmine alone was established to determine whether the reaction of the rabbit was changed in the course of the experiment. To eliminate the influence of the sequence of drugs, the rate of the administration was fixed individually for each rabbit according to random analysis. The change in the duration of the theta activity after the pretreatment with test drugs was compared with the effect of physostigmine alone at the beginning and end of the experiment and expressed as the percentage of the duration. The results for the group of animals were statistically evaluated and the significance of differences was determined. References p . 46-47

44

Z. V O T A V A

et nt.

RESULTS

After a short latency time of about one to two minutes, the intravenous administration of physostigmine at the dose level of 0.1 mg/kg evoked an irregular fast-frequency low voltage activity in the cortical leads and regular slow frequency (five to six per second) high voltage (200 to 300 pV) activity in the hippocampus and thalamus, so-called theta activity. This reaction lasted between 20 to 40 minutes in individual rabbits, and was relatively stable for each rabbit (see Fig. 4 upper part). No behavioural reactions were observed, in accordance with the findings described by Bradley and Elkes2. Thus, physostigmine produced a “dissociation” between EEG and behav-

Fig. 4. EEG record of a rabbit as an example of the experimental procedure. The upper part: On the left is the control record, other records are taken after physostigmine administration in 2, 5, 15, 18 and 20 minute intervals. The lower part: On the left is the record after the administration of atropine. Physostigmine was given 20 minutes after atropine injection. Note the curtailing of the duration of theta activity, evoked in the hippocampus and thalamus by the injection of physostigmine, after pretreatment with atropine.

INFLUENCE OF ANTICHOLINERGIC DRUGS ON PHYSOSTIGMINE REACTION IN RABBITS

45

ioural arousal reaction, as described by Wiklerlg for atropine. Blood pressure and heart rate were not changed by this dose of physostigmine, as was stated earlier. The effects of atropine, scopolamine and benactyzine on the EEG traces were qualitatively similar and they differed only in the intensity of their actions. Very soon after the administration of anticholinergic drugs, a consistent shift to low voltage slow activity was noted and a diminution of the 8-10 clsec waves, which formed the alpha activity of the EEG. This pattern was very similar to that observed in the state of drowsiness, although the rabbits were awake. The intensity of this EEG change was independent of the dose administered. After a period of 20 minutes following physostigmine administration, the EEG arousal reaction was shortened, and started after a longer latency time. With higher doses, the EEG arousal reaction was completely eliminated. The patterns in the cortical leads were sometimes changed in shape, but the theta activity remained unchanged (Fig. 4, lower part). The quantitative comparison of the effect of atropine, scopolamine and benactyzine is shown in Figure 5. It is clear that all the drugs tested significantly reduced the EEG theta activity in the hippocampus and thalamus, evoked by administration of PHYSOSTIGMINE

0.1 mg kg1i.v. 10000-

SCOPOLAMINE PHYSOSTIGMINE BENACTYZINE

PHYSOSTlGMlNE

PHYSOSTIGMINE

6040-

'"1 0

0.5

1.0

ILJL

--0.1 0.5

0.05 0 3

0.5 rng/kg iv.

Fig. 5. Shortening of the duration of EEG theta activity evokcd in rabbits by i.v. administration of physostigmine (first shaded column, duration is taken as 100%) 20 minutes after pretreatment with atropine, benactyzine and scopolamine in doses indicated on the abscissa. The straight lines in the columns indicate the fiducial limits for probabilityp = 0.15.

physostigmine. Scopolamine produced the greatest effect, while atropine had the least effect. The quantitative differences among the drugs tested were statistically significant. If the effect of atropine is taken as 1 .O, then the effect of benactyzine is 2.0 and that of scopolamine 12.0. It should be stressed that it is valid only for one time interval, i.e. 20 minutes after intravenous administration. DISCUSSION

As has been stated by Bradley and Elkesz, physostigmine, in a suitable dose, evokes EEG arousal without changing the general behaviour of animal or provoking excitaReferences p. 46-47

46

Z.V Q T A V A et al.

tion. We have used this effect of physostigmine for the evaluation of drugs with central cholinomimetic or anticholinergic action. In the present paper we compared the effect of three anticholinergic drugs, atropine, scopolamine and benactyzine. In accordance with other authors2,7J7Ja we established that the effect of scopolamine exceeded that of atropine more than ten times. Also, benactyzine proved to be about twice as effective as atropine. These ratios of the effects of these three drugs is also in good agreement with the results obtained in behavioural experiments4~5~6~16 or with different central pharmacological effectslz. If the ratio for the central and peripheral anticholinergic effects of atropine, scopolamine and benactyzine is evaluated12 the specificity of the central effect of scopolamine and especially that of benactyzine is evident. SUMMARY

EEG recordings were made in a group of twelve uiianesthetized rabbits with electrodes implanted in cortex (frontal and occipital), hippocampus and thalamus. Physostigmine (0.1 mg/kg i.v.) evoked EEG arousal reaction lasting 20 to 40 minutes, without any change in general behaviour, blood pressure or heart rate. The effects of atropine (0.5 and 1.0 mg/kg), scopolamine (0.05, 0.1 and 0.5 mg/kg) and benactyzine (0.1 and 0.5 mg/kg) were compared against the EEG arousal evoked by physostigmine. The drugs were given i.v. 20 minutes before administration of physostigmine and the shortening of the duration of the EEG arousal was evaluated. The degree of central anticholinergic effect in the drugs studied in this investigation was: atropine 1.O, benactyzine 2.0, scopolamine 12.0.

REFERENCES 1 BENESOVA, O., BOHDANECK+, Z. AND GROFOVA, I. (1964) Electrophysiological anslyses of the neuroleptic and antidepressant actions of psychotropic drugs in rabbits. Int. J. Neuropharmacol., 3,479488. P. B. AND ELKES,J. (1953) The effect of atropine, hyoscyamine, physostigmine and 2 BRADLEY, neostigmine on the electrical activity of the brain of the conscious cat. J. Physiol. (Land.), 120, 148-149. 3 FIFKOVA, E. AND MARSALA, J. (1960) Stereotaxic atlases for the cat, rabbit andrat. In the book: BureS, Petraii and Zachar: Electrophysiological methods in biological research. Czechoslovak Academic Press, Praha, pp. 426467. 4 HERZ,A. (1959) Ueber die Wirkung von Scopolamin, Benactyzin und Atropin auf das reaktive Verhalten der Ratte. Arch. exp. Path. Pharmak., 236, 110-1 12. 5 HERZ,A. (1960) Die Bedeutung der Bahnung fur die Wirkung von Scopolamin und ahn1i:he Substanzen auf bedingte Reaktionen. 2. Biolog., 112, 104-1 12. 6 HOLTEN,C. H. AND SONNE, E. (1955) Action of a series of benactyzine-derivatives and other compounds on stress-induced behaviour in the rat. Actapharmacol. toxicol. ( K b h . ) , 11, 148-155. 7 LONGO, V. G. (1956) Effects of scopolamine and atropine on electroencephalographic and behavioural reactions due to hypothalamic stimulation. J. Pharmacol., 116, 198-208. 8 LONGO, V. G. (1965) Analyse de la dissociation entre les effets des medicaments anticholinergiques sur le comportement et sur I'activite electrique ckrebrale. AclualifPsPharmacologiques, 18,289-308. 9 LONGO,V. G. (1966) Mechanisms of the behaviour and electroencephalographic effects of atropine and related compounds. Pharmacological Reviews, 18, (in press).

INFLUENCE OF ANTICHOLINERGIC DRUGS ON PHYSOSTIGMINE REACTION IN RABBITS

47

10 LONGO,V. G. AND SILVESTRINI, B. (1958) Contribution a l’etude des rapports entre le potentiel rtticulaire evoque, l’etat d’anesthesie et l’activitk electrique ctrebrale. Electroencephal. clin. Neurophysiol., 10, 111-120. 11 MIKHEL’SON, M. YA. (Ed.) (1957) The physiological action of acetylcholine and the search of new drugs. Leningrad, 1957. 12 PARKES, M. W. (1965)An examination of central action characteristic of scopolamine: Comparison of central and peripheral activity in scopolamine, atropine and some synthetic basic esters. Psychopharmacologia, 7, 1-19. 13 ROUGEL, A., VERDEAUX, J. AND GOGAN,P. (1965) Limits of the dissociation between EEG and behaviour. Int. J. Neuropharmacol., 4, 265-272. 14 SADOWSKI, B. AND LONGO,V. G. (1962) EEG and behaviour correlates of an instrumental reward conditioned response in rabbits. A physiological and pharmacological study. Electroenceph. clin. Neurophysiol., 14, 465-476. 15 VOTAVA, Z. (1966) Pharrnacologie des structures cholinergiques centrales. Actualit& Pharmacologiques, 1, (in press). Z., BENE~OVA, O., METYSOVA, J., AND SOUSKOVA, M. (1963) Drug-induced changes of 16 VOTAVA, higher nervous activity in experimental animals. In the book: Psychopharmacological Methods, Votava, Horvath and Vinai (Eds.), Pergamon Press, Oxford, pp. 31-40. 17 WHITE,R. P. AND BOYAJY,L. D. (1960) Neuropharmacological comparison of atropine, scopolamine, benactyzine, diphenhydramine and hydroxyzine. Arch. int. Pharmacodyn., 127, 260-273. 18 WHITE,R. P., NASH,C. B., WESTERBEKE, E. J. AND POSSANZA, G. J. (1961)Phylogenetic comparison of central actions produced by different doses of atropine and hyoscyamine. Arch. int. Pharmacodyn., 132, 349-363. 19 WIKLER, A. (1952) Pharrnacologic dissociation of behaviour and EEG sleep patterns in dogs: morphine, n-allylnormorphine and atropine. Proc. SOC.exp. Biol. ( N . Y . ) , 79,261-265

48

arain Acetylcholine and Habituation * P. L. CARLTON Rutgers, The State University, New Brunswick, New Jersey (U.S.A.)

Habituation is evidently a relatively primitive form of learning and refers to the fact that the effects of a stimulus disappear under certain conditionsll. Thisloss of stimuluseffectiveness follows two simple rules. First, relatively protracted exposure to the stimulus is required. Second, the stimulus must not itself be a biologically significant one nor can it be associated with a second stimulus having such significance. What do I mean by “biological significance”? A stimulus can have such significance in two different senses. It can be demonstrably significant in determining, first, the survival of the individual organism or in determining, second, the survival of the species. For that minority species that can learn instrumental behaviors, such biologically significant stimuli act as rewards. I will return to the relation of habituation and reward later in the paper. In thinking about habituation, an obvious question arises: what events in the brain control habituation? 1 have preferred to ask the question in a slightly more restricted form : what chemical events in the brain underlie the process? An alternative version of the same question asks about the areas of the brain involved. The “chemical” question about habituation can be re-phrased to read :The activity of what naturally occurring substance is required for habituation to take place? About three years ago2, I made the guess that the normal muscarinic activity of brain acetylcholine might be critical in the process. The experiments I want to describe now suggest that there may be something to that guess. The first problem in investigating the idea was to develop a scheme that would provide a behavioral index of habituation. To get such a measure, we utilized the fact that the ongoing behavior of a n animal is disrupted in a novel environment, but that this disruption wanes as habituation takes place; as the environment becomes less novel. The second problem was to find a means for altering the normal activity of brain acetylcholine. Well established pharmacological techniques are, of course, available ; the action of anticholinergics like scopolamine and atropine is to attenuate the muscarinic activity of acetylcholine (ACh). If ACh activity is, in fact, required for habituation, then habituation should be drastically attenuated when ACh activity is attenuated by the anticholinergics. The particular experimental set-up we have used involves two parts. These are

* Research supported by USPHS Grant MH 08585.

49

BRAIN ACETYLCHOLINE A N D HABITUATION PART 1 ___

Saline

PART 2

Scop.

DRINKING e

x

~

~

~

2days c i -+~ ANIMALS D E ~ PlR l ~ 1~ day~ +(allTEST animals In chamber) No druqs

--e

Exposed I n j e c t ion

Fig. 1. Summary of habituation procedure.

summarized in Fig. 1 . (This experiment and all others used Sprague-Dawley rats as subjects.) In the first part, some animals (Group D) were given 15 min exposure to the chamber 15 min after an injection of 0.5 mg/kg scopolamine hydrobromide (i.p.). Other animals (Group B) were not exposed to the chamber, but were removed from their home cages and given a drug injection. Two other groups were also involved. Both received a control injection of saline; one (Group C) was exposed to the chamber, the other (Group A) was not. The second part of the experiment began two days after this treatment. All animals were water-deprived. A day later (72 h after exposure), all animals were placed in the chamber to which only some had prior exposure. One change was made in this chamber: a water bottle was introduced. We recorded the time it took the thirsty animals to start drinking. This procedure has been described in detail elsewheres. Let me stress that no animal was injected before this test; injections were given only during the first part of the experiment. The results of this procedure are shown in Fig. 2. The animals that had not been exposed took a considerable amount of time to start drinking when tested in the absence of a drug. The values for these two groups are not reliably different. In contrast, prior habituation to the test chamber led, not surprisingly, to much faster initiation of drinking in animals given saline before prior exposure. But animals given the same opportunity to habituate at the time of reduced ACh activity failed to show the effect of this exposure. Rather, they behaved as if they had not been exposed at Time to drink in test (sec) I

1

Saline

Not exposed Scop.

Saline

Exposed

=

Scop. 1

Fig. 2. Mean times to make initial contact with a water bottle in a 3-min session. The results indicate that prior exposure decreases initial contact time but that scopolamine, only at the time of prior exposure, attenuates this effect. References p. S940

50

P . L. C A R L T O N

Exposed (Soline)

Unexposed

Exposed

(Stop,)

a 1

3 160

Fig. 3. Time to initiate drinking in a 3-min test session. Each bar denotes the result obtained from a single rat. Animals tailing to contact the drinking tube were assigned a criterion score of 3 min.

all; as if habituation had not taken place. Fig. 3 is a plot of the data for individual animals (each bar denotes the value obtained from a single rat). The two unexposed groups did not differ; their data are in the center of the figure. The data from exposed animals are at the left. The vertical, dashed line indicates the maximumtime todrink in this group. There is virtually 110 overlap between unexposed and previously habituated animals. The differential effect of prior habituation is a substantial one. The effect of scopolamine is of an equally large order of magnitude. Animals given a prior opportunity to habituate following scopolamine behaved much as if they had not had that opportunity. There is virtually no overlap of their data with those from exposed (saline) animals. (There is one exception, as there is i n the unexposed group). Furthermore, these data are well within the distribution of values obtained from unexposed animals. This effect suggests an involvement of acetylcholine in habituation; if normal ACh activity is blocked, so is habituation. Before this suggestion can be taken seriously, however, there are several questions that need to be answered. First, are we talking about a result of attenuated ACh activity, or are we talking about some idiosyncratic and unknown effect of one anticholinergic, scopolamine? A direct way to answer the question is to evaluate the effects of atropine. If both drugs produce comparable results, we are almost certainly dealing with a muscarinic action of ACh. In the study described below, we used a dose of atropine (10 mg/kg, i.p.) that, on the basis of a wide variety of studies, would be expected to produce about the same effect as the dose of scopolamine used in the experiment I just described. The potency ratio of the central effects of atropine to scopolamine is about 15 or 20 to 1. This brings up a second question. If attenuated habituation is indeed related to ACh activity, is it related to brain ACh? ACh acts as a transmitter in both the peripheral and the central nervous systems. But scopolamine has profound peripheral as well as central effects. Are the effects I have described central? A direct answer to the question i s provided by comparing the action of scopolamine with that of methyl scopolamine. Methyl scopolamine and methyl atropine are at least equipotent with their parent compounds in terms of peripheral action. But the methyl compounds pass the blood-brain barrier very poorly. Thus, equimolar doses of scopolamine and

BRAIN ACETYLCHOLINE AND HABITUATION

51

methyl scopolamine will produce comparable peripheral effects, but vastly different central effects. Therefore, any effect of scopolamine that is not produced by an equimolar dose of methyl scopolamine can most reasonably be attributed to an action in the central, not the peripheral, nervous system. This approach has been discussed and documented elsewhere2. A third question has to do with the specificity of the habituation deficit produced by scopolamine. If we are truly dealing with an effect involving ACh, the phenomenon should be produced only by drugs that can attenuate the action of ACh. On the other hand, is the effect a non-specific one? Will any centrally active drug produce the same deficit in habituation? As a start toward answering this question, I selected two common drugs that are known to have profound behavioral effects. These are the stimulant, amphetamine sulphate ( I .O mg/kg, i.p.) and the depressant, sodium pentobarbital (8.0 mg/kg, i.p.). These doses were used in the study described below and were selected because of their clearly established effects on the learned behavior of rats. These doses are not sub-threshold; they are extremely active in altering certain classes of behavior2.4. In the experiment I want to describe now, we used the same technique as that in the first experiment. We studied the effects of scopolamine, methyl scopolamine, atropine, amphetamine, pentobarbital and a control saline injection : six different kinds of injections in all. In all cases, the drug was given to one squad before exposure and to a second squad that was not exposed. Thus, there were twelve groups of animals, six given an injection before exposure and six given the same injection but no exposure. Again, injections were given only before exposure; no injections were given before the subsequent test. The results of this test are summarized in Fig. 4. The data from Time to drinkin test (sec)

Saline

scop. ALi-op

M-Scop. P.Barb.

Arnph.

=

I

I I I

I I I

Not exposed I I I

I I

Fig. 4. Mean times to drink in a 3-min test session. Relative to unexposed animals, exposure following saline results in shorter times, scopolamine and atropine reverse the normal effect of prior exposure, whereas methyl scopolamine, pentobarbital and d-amphetamine are relatively inactive.

the various unexposed groups did not reliably differ; these data were therefore pooled. The overall effect of lack of exposure and consequent habituation is indicated by the vertical, dashed line. The various bars indicate the average amounts of time to drink References p . 59-60

P. L . C A R L T O N

52

taken by the animals that were exposed following an injection. The exposed animals given saline were faster than unexposed rats; scopolamine again washed out this habituation effect. Atropine had an effect comparable to that of scopolamine. The lack of habituation seems to be related to an attenuation of ACh activity. A dose of methyl scopolamine equimolar to that of scopolamine had no effect on habituation. The phenomenon can therefore be most reasonably supposed to reflect an action on brain ACh. Neither amphetamine nor pentobarbital produced a shift in the effect of prior habituation despite the fact that the doses used have profound effects in other behavioral situations. Although dose-response data are certainly required, these results at least suggest a specificity of action peculiar to the centrally active anticholinergics. This lack of effect of amphetamine and pentobarbital, at these doses, does not mean that a deficit might not be obtained at substantially higher doses. A high, hypnotic dose of pentobarbital would, of course, produce a total deficit. Also, it seems likely that higher doses of either pentobarbital or amphetamine would produce a deficit because of dissociationg. The phenomenon of dissociation is discussed more fully below. The point to be made on the basis of the data in Fig. 4 is that other centrally active drugs, at dose levels that do have some behavioral effects, are evidently inactive in this situation. This brings up another question: Are we talking about a deficit in memory‘!’ Is a particular process, called habituation, reduced in exposed animals given anticholinergics - or, do such animals simply not “remember” that they have been exposed? If the phenomenon is truly a more general memory deficit, it should be possible to demonstrate that deficit in a situation in which habituation does not play a role; that is, one involving biologically significant stimuli. I n one experiment of this type, we first trained thirsty rats to drink from a water bottle in a response chamber. On the next day, they were returned to the chamber, but the water bottle had been removed. Four groups, two given saline and two given 0.6 mg/kg scopolamine (i.p.), were involved. Two of these groups (one saline and one scopolamine group) were presented four moderate intensity, 10-sec tones. The inter-

5001

Mean time to criterion licks Pre.tone (100 licks) Tone (10 licks) I I

DOSE

1

Fig. 5 . Effects of prior conditioning following saline or scopolamine injections. Times to complete 100 licks are plotted at the left; time to complete 10 licks in the presence of the tone are at the right. Relative to unshocked controls, animals that had had the tone paired with pain-shock show reliable suppression of drinking in both periods; drug at the time of conditioning had no effect.

BRAIN ACETYLCHOLINE A N D HABITUATION

53

tone interval was one minute. The other two groups were given the same treatment except that, coincident with the termination of each tone, they also received a brief, inescapable painful shock applied through the grid floor of the chamber. All injections were given 20 min before conditioning. Two days later, all animals were returned to the chamber; the water bottle had been replaced, N o animal was injected. We recorded the time it took the animals to take 100 licks of water. With the occurrence of the 100th lick, the tone came on. We recorded the amount of time it took each rat to take 10 more licks. The results of this test are summarized in Fig. 5. Animals that had been shocked showed a generalized fear of the chamber, as indicated by the relative suppression of drinking in the pre-tone period. Scopolamine treatment before the prior fear conditioning had no effect. These animals evidently remembered that the chamber was a “dangerous” place just as well as the controls. The same kind of effect was found in the tone period (at the right of the figure). Fear, based on prior pairings of tone and shock, led to substantial suppression; again there was no differential effect due to drug treatment at the time of conditioning. There was no evidence of a memory deficit due to scopolamine. It could be argued that scopolamine had no effect because the event to be remembered was such a “vivid” one. That is, the degree of conditioning was so great that no other variable could reasonably be expected to affect it. This possibility seems unlikely for two reasons. Firstly, reliable evidence of conditioning was obtained in the pre-tone period. But scopolamine, at the time of conditioning, had no effect on this relatively low-level fear. That generalized fear of the chamber was indeed less than that elicited by the tone is indicated by the fact that, in the pre-tone period, the animals took less time to make 100 licks than they took to make only 10 in the presence of the tone. Tf scopolamine had produced a deficit in memory, it seems likely that some indication of it would have appeared in the pre-tone data. There was not, however, even a hint of such an effect. Secondly, we have tried to obtain some evidence of a memory deficit due to scopolamine in several different conditioning experiments. No such evidence was obtained in any of them. We have also evaluated the effects of atropine using the same technique as that used in the experiment summarized in Fig. 5. In the atropine experiment, we also examined the possibility that a memory deficit might appear against a relatively weak base-line of fear conditioning. To do this, we varied the number of conditioning trials. One group received 4-tone presentations, but no shock; a second received a single tone-shock pairing and a third received 4 pairings. These animals were given a saline injection 20 min prior to conditioning. A second set of three groups was given the same treatment except that atropine (8.0 mg/kg, i.p.) was injected. In the pre-tone period, the effect of tone-shock pairings was a re1iable:one. Prior fear conditioning did produce suppression; this suppression increased as a function of the number of pairings, but no effect of the drug was obtained. Comparable effects were obtained in the tone period, as shown in Fig. 6. Again, a reliable increase i n suppression was obtained with increased numbers of prior conditioning trials; atropineat the time ofconditioning had no effect. If anything, atropine produced slightly improved memory. Furthermore, the relatively lower level Refeiences p.;59-60

54

P. L. C A R L T O N

Training trials

Fig. 6 . Effects of prior conditioning following saline or atropine. The number of shock presentations was varied between groups. No effect of drug was obtained.

of fear obtained with a single pairing was unaltered by drug treatment. This extends the results obtained in the pre-tone period. Although anticholinergics may produce amnesia i n mans, they do not appear to do so in the rat; at least not at the dose levels we have used. This uniform lack of effect suggests that the results we have interpreted as reflecting a deficit in habituation cannot reasonably be attributed to a more general deficit i n memory. Although the lack of effect of prior exposure appears to be due to attenuated activity of brain ACh, but not due to a deficit in general memory, there is still another question as to the validity of interpreting the deficit in terms of habituation. The effect could be due to dissociation. The term dissociation refers to the fact that animals can discriminate a drug-state from a no-drug state; the presence or absence of a drug's effect can have a discriminative control of behaviorg. Thus, the effects I have been describing could be due, not to a deficit in habituation, but toa stimulusdi fference between prior exposure (with drug) and subsequent test (without drug). An analogous situation would be to expose animals witha buzzer sounding and to test them without the buzzer. Longer times to initiate drinking would be expected in tests simply because of the difference in stimuli between exposure and test sessions. Interpretation of the data I have described in terms of dissociation, or stimulus change, seems unlikely for several reasons. First, centrally active doses of amphetamine or pentobarbital might be expected to produce effects like those produced by scopolamine if stimulus change were the only factor involved. But they do not. There is, of course, the unlikely possibility that the change due to scopolamine (or atropine) vs. no scopolamine (or no atropine) is large, whereas the change due to amphetamine (or pentobarbital) vs. no amphetamine (or no pentobarbital) is negligible. A second consideration bearing on the stimulus change interpretation is that no evidence for such a factor was obtained in the conditioning experiments summarized in Figs. 5 and 6. To the extent that stimulus change is a potent variable, there should have been

BRAIN ACETYLCHOLINE A N D HABITUATION

55

less evidence of conditioning in a subsequent no-drug test when prior tone-shock pairings were given following drug injection. No evidence of such an effect was obtained. Still a third result weighs against the stimulus change interpretation. Overton (personal communication) has found that dissociation can, in his experimental set-up, be obtained with atropine, but the lowest dose that produced reliable dissociation was about 20 times greater than that used in our experiments. Thus, a direct test of dissociation due to atropine suggests that the phenomenon I have been discussing is a league apart from that leading to dissociation. Another direct test of dissociation has been carried out by Meyerss. Meyers used low doses of scopolamine roughly comparable to those we have studied. No evidence of dissociation, at these doserevels, was found. We have independently replicated the essential features of Meyers’ experiment. All of these findings indicate that the deficit I have been describing cannot leasonably be attributed to dissociation. Although both memory deficit and dissociation do not seem to be likely candidates for explaining the basic phenomenon, still another factor might be involved. Suppose scopolamine blocked the “extinction of fear” during the initial exposure; in the later test, animals that had had scopolamine might be slow to drink because they were more fearful. That fear does suppress drinking is amply demonstrated by the experiments summarized in Figs. 5 and 6. One qualification of this point is in order. Extinction typically refers to a shift from reward to non-reward: e.g., an animal is first trained to get reward by emitting some response; reward is then discontinued. In the experiments I have described, an animal may indeed fear the novel environment, but he does so, not because of his previous training, but because of his presumably innate reaction (in the rat, at least) to “strangeness”. Thus, if we are, in fact, talking about extinction, we are talking about the waning of unconditioned rather than conditioned responses. Viewed in this way, there seems to be a rather thin line between habituation and “extinction of fear”. In both cases, an animal comes to a new situation with a set of responses to stimuli. As a consequence of exposure to these stimuli, the initial responses disappear. A distinction can, however, be made. A rat may do one of two antagonistic things in a novel environment; he may suppress behavior (because of fear), or he may move about the environment and thereby explore it. As a consequence of this exploration, the animal finds that some stimuli are neither biologically significant themselves nor correlated with others having such significance. These stimuli thus lose their initial control of behavior; the animal no longer explores them. It is in this sense, rather than one having to do with suppression due to fear, that I have used the term habituation. An animal that has already habituated to an environment starts drinking promptly because he does not explore the environment to the extent that the unhabituated animal does. But is this restriction of usage justified? My best guess at the moment is that it is. First, observation of the animals during their initial exposure to the chamber reveals little, if any, suppression of behavior. In fact, we have consistently used an unemotional strain of rats and, in addition, typically gave them a preliminary period of handling and “gentling” before beginning the experiments themselves. If our animals are fearful during initial exposure, they give very little References p.k59-60

56

P. L. CARLTON

evidence of it. What they do do is explore the chamber; animals given scopolamine are, if anything, hyperactive, hyperexploratory. In addition, a direct evaluation of the effects of scopolamine on extinction of fear indicates that the rate of extinction under drug is the same as that in normal animals. In this study, animals were first given tone-shock pairings like those used in the experiments summarized in Figs. 5 and 6. They were then given a single test trial in the drinking situation. All animals showed high levels of suppression in the tone. No drugs had been given up to this point. The animals were then divided into two groups. Both groups were given several extinction sessions in which the drinking tube was not available and in which the tone, but not shock, was presented; one group received scopolamine before each extinction session, the other received saline. All animals were then given a test in which a single tone was presented, the animals were drinking, no shock was delivered and no injections were given. The extent of suppression of drinking was, as usual, taken as an index of conditioned fear. Cycles of multiple extinction sessions (following injection) plus single test (without injection) were continued until there was no suppression of drinking in the tests. No difference due to scopolamine was obtained. Three points should be made about this study. First, the experiment is a very preliminary one based on few animals; a dubious basis for accepting a negative result. Second, the test sessions without injection were also extinction sessions. It could, therefore, be that this extinction, common to both groups, attenuated any difference that might have been produced by scopolamine. Detailed extension and replication of the study are required on both counts. There is a final qualification. This experiment involved conditioned fear, whereas the fear, if any, that could operate in our earlier studies is of the unconditioned variety. Thus, if one is to accept the lack of effect of scopolamine on extinction of fear as indicating that fear is not an important factor in the earlier work, it must be assumed that rules about one kind of fear will apply to the other. The best that one can say at this point is that interpretation of the effect of scopolamine in terms of habituation (as I am using the term) is at least not contradicted by the lack of effect of scopolamine on the extinction of conditioned fear. Let me change the subject somewhat. I began this discussion by describing a largeorder effect of scopolamine. One interpretation of this effect is that the normal activity of brain ACh is required for habituation; if ACh activity is blocked, so is habituation. But is this interpretation reasonable? This question was answered by asking a series of other questions: Is the effect actually peripheral rather then central? Can the effect be obtained with any centrally active drug? Can the effect be interpreted as being due, not to habituation, but to memory deficit or dissociation or fear? The negative outcome of attempts to answer these questions all supports the original interpretation. But there is a serious gap in such a conclusion. If attenuated ACh activity in the brain retards habituation, accentuated AChactivity :(with a cholinesterase inhibitor like eserine) should accelerate it. The gap in the story I have been telling is that we have, as yet, no data bearing on the effects of accentuated ACh activity. Until such data are in hand, the presumed relation of ACh to habituation

BRAIN ACETYLCHOLINE AND HABITUATION

57

can be accepted only with considerable caution. There is, however, some evidence that indicates heightened ACh activity may increase habituation. This evidence is, unfortunately, rather circumstantial. It is based on effects seen in learning situations. What role might habituation play in learning? As others7 have pointed out, the role of habituation is not negligible. Various experiments support the idea that one aspect of learning a complex problem, a maze for example, is the minimization of the behavioral control exerted by stimuli that do not lead to reward. That is, minimization of the impact of stimuli that are not associated with another having biological significance. Put crudely, successful performance hinges, in part, on suppressing the tendency to explore novel aspects of the environment. The animals must habituate to certain aspects of the situation. An experiment by Whitehouse12 will illustrate the point. This study involved the learning of a discrimination problem. Some animals were given atropine before each learning session; others were given eserine and still others, saline. The results are shown in Fig. 7. I have plotted the numbers of correct responses made by drugged Total correct Rs ( % normal) 100 110

120

I

I

Atropine

b I

I

Replotted from

1

Whttehouse,1959

Fig. 7. Overall numbers of correct responses /relative to controls - 100%) in a series of learning trials. Some animals received atropine before each trial, others received eserine, controls received saline. Atropine tended to impair performance, eserine tended to enhance it.

animals as a percentage of those made by normal controls. The numbers of correct responses made in the course of the series of trials reflect the rate of learning. As the figure indicates, atropine retarded learning, whereas eserine accelerated it. This result makes sense if two assumptions are made. First, there is the very reasonable one that habituation is involved in the successful performance seen in complex learning situations. Second, one must grant what the previous experiments have suggested; that the normal activity of brain ACh is involved in habituation. Thus, reducing ACh activity with atropine should lead to reduced habituation (and poorer performance), whereas increasing ACh activity with eserine should lead to increased habituation (and better performance). Although these expectations are generally borne out, Whitehouse found that only the deficit due to atropine was a statistically reliable one. In an experiment preliminary to the one described here, he had, however, found a reliable improvement due to eserine. This divergence is not surprising if one considers some aspects of actions of a cholinesterase inhibitor like eserine. Cholinesterase inhibition can result in accentuated References p . 59-60

58

P. L. C A R L T O N

ACh activity5. But higher levels of ACh can also lead to less neural activitys. Thus, the action of a drug like eserine will be a biphasic one; heightened cliolinergic activity followed, at higher levels of esterase inhibition, by lower levels of activity. This relationship of ACh and effective, aggregate neural activity is schematized in the upper left portion of Fig. 8.

Replotted from ADrison

XI201

-100 I

20 40 6060.80 *I00 ACHE. % normal

Fig. 8. Schematic representation of relations between ACh activity, as a percent of normil, and neural activity (upper left), cholinesterase activity and ACh activity (upper right), and the consequent relation between cholinesterase activity and neural activity (bottom). T h e figure at the upper right is based on data reported by Aprison'. The arrows indicate the relative values at which functionally heightened activity would be obtained.

What is the relation of cholinesterase inhibition and ACh activity? Aprison' has found that ACh activity in brain increased only after levels of enzyme inhibition fell to about 40-60"/, of normal. At lower levels, ACh activity in brain increased sharply. This relationship (based on Aprison's data) is shown at the upper right of Fig. 8. These two relationships generate the curve shown at the bottom of Fig. 8. Cholinesterase inhibition should have no effect until levels of inhibition reach 40-60% of normal. At that point, there should be an increase in functional ACh activity. With still greater enzyme inhibition, there should be an abrupt decline. The decline will be an abrupt one because of the very rapid rise in ACh activity at levels of inhibition below the 40-60% level. That is, heightened ACh activity should occur only within a very restricted range of cholinesterase inhibition. This will be the case because inhibition in excess of this range should rapidly lead to high levels of ACh and, consequently, a functionally lower level of aggregate cholinergic activity (see the schematic at the upper left of Fig. 8). Because dose-response curves differ between animals, it should be a simple matter to obtain only marginal effects due to cholinesterase inhibition; the performance of some animals may be unaffected, facilitated in some, and depressed i n others. Thus, the effect on a group of animals might be minimal. Furthermore, slight variations between experiments could have very substantial and different effects.

BRAIN ACETYLCHOLINE A N D HABITUATION

59

These relations suggest that in learning situations increased cholinesterase inhibition should facilitate performance in the 4 0 4 0 % range, and depress it at lower levels. RusselP has summarized the results of his own experiments i n just these terms : “. . . when behavior is affected it appears to piss through four phases as ChE activity is reduced.

From 60 to 100 per cent activity no significant eyects hzve bsm observed. Thzre is a suggestion in our data that between 40 and 60 per cent activity the bshsvior mpy show a phase of heightened efficiencv .... Further reduction is associated with a rapid loss in efficiency, which might for convenience be referred to as a phase of ‘bzhavioral toxicity’.”

The role of brain ACh in the control of habituation that I have suggested, coupled with the role of habituation in maintaining performance, is thus indirectly supported. Direct evaluation of the effects of cholinesterase inhibition are, nonetheless, required. One way of summarizing what 1 have been suggesting is to think of the organism as being on the inside of a large balloon filled with stimuli. One problem facing the organism is to handle this profusion of stimuli. Nervous systems seem to have evolved so that, rather than processing everything at once, only certain hunks of the stimulus population are selected, and therefore control the organism’s behavior. It appears that the balloon gets selectively deflated to manageable proportions. This filtering process is called habituation, and follows the rule that biologically significant stimuli do not get filtered. Such stimuli are called rewards in certain contexts and for certain species. Habituation may thus play a part in controlling the behavior seen in such situations. A number of experiments support the guess that directly measured habituation requires the normal activity of brain ACh. Furthermore, results obtained in learning situations appear to support this possibility. I should add that I d o not feel that these data are as yet overwhelmingly in favor of the suggestions I have made. The data are only suggestive. What they suggest is that one of the most important things a brain must do, functionally cancel those stimuli that are not to have an impact on the animal’s behavior, involves the action of brain ACh. This possibility seems to account for a reasonable amount of available data; how much more experimental mileage can be got out of it, remains to be seen. REFERENCES 1 APRISON, M. H. (1962) On a proposed theory for the mechanism of action of serotonin in brain. Recent Adv. Biol. Psychiatry, 4, 133-146.

2 CARLTON, P. L. (1963) Cholinergic mechanisms in the control of behavior by the brain. Psycho/. Rev., 70, 19-39. 3 CARLTON, P. L. AND VOGEL,J. R . (1965) Studies of the amnesic properties of scopolamine. Psychon. Science, 3, 261- 262. 4 GELLER, 1. AND SEIFTER, J. (1960) The effects of meprobamate, barbiturates, &hetamine and promazine on experimentally induced conflict in the rat. Psychopharmacol., 1, 482-492. 5 GOODMAN, L. S. AND GILMAN, A. (1960) The pharmacological basis of experimental therapeutics. New York: Macmillan. 6 MCLENNAN, H. (1963) Synaptic Transmission. Philadelphia: Saunders. 7 MEEHL, P. E. AND MACCORQUODALE, K. (1954) In: W. K. Estes et a/. (Eds.), Modern Learning Thcory. New York: Appleton-Century-Crofts. 8 MEYERS, B. (1965) Some effects of scopolamine on a passive avoidance response in rats. Psychopharmacol., 8, 111-1 19.

60

P. L. C A R L T O N

9 OVERTON, D. A. (1964) State-dependent or “dissociated” learning produced with pentobarbital. J. Camp. Physiol. Psychol., 51, 3-12. 10 RUSSELL, R. W. (1958) Effects of “biochemical lesions” on behavior. Actu fsychohgica, 11, 28 1-294. I I THORPE, W. H. (1963) Lenrningadinsrinct inanitnals. Cambridge: Harvard Univ. Press. I2 WHITEHOUSE, J. M. (1959) The effects of physostigmine and atropine on discrimination lenrning in the rat. Unpuhlished doctoral dissertarion, University of Colorado.

61

The Effect of Physostigmine and Atropine on some Behavioral and Electrophysiological Functions in Rats J. B U R E S Instilute of Physiology,

Czechoslovak Academy of Sciences, Prague (Czechoslovakia)

The chemical aspects of brain organization become more and more important for the neurophysiological analysis of learning. Accumulating evidence about the diversity of central transmitters present in the same brain areas leads logically to the assumption that synaptic chemistry may link neurons to circuits subserving specific behavioral functions. In this case, blockade of a certain type of synaptic transmission may eliminate the corresponding behavior rather than induce a diffuse impairment. Although little is known so far about the exact nature of the central transmitter s u b ~ t a n c e s l 4cholinergic ~~~, transmission i n several brain regions has been established beyond any doubt. The distribution of acetylcholine as well as related enzymatic systems (choline-acetylase, acetylcholinesterase) has been thoroughly described. Drugs interfering with the activity of cholinergic synapses either by anticholinergic or excessive cholinomimetic action are readily available and the basic mechanisms of their effects are well understood. The dissociation of EEG and behavior4.26 raised an important question about the correlation of electrophysiological and behavioral events. These are obviously the reasons why so much attention is paid to the behavioral role of the cholinergic systems. The purpose of the present paper is to test some of the current hypotheses about their role. METHODS

All the experiments were performed in rats aged three months. Assuming that cholinergic transmission can be impaired by both acetylcholinesterase blocking agents and by anticholinergic substances, attention was concentrated on the use of physostigmine salicylate and atropine sulphate. Drugs were applied in concentrations the effects of which on the EEG of rats were thoroughly described in our earlier papers6p7. After intraperitoneal injections of physostigmine or atropine 10 and 15 min respectively were allowed for the full development of the drug effect. The testing period did not usually exceed 30 min.

Rcfermres p

,

71-72

62

J.

RURES

RESULTS

Recent rneniory

Interference with cholinergic transmission was repeatedly shown to impair the acquisition of new memory traces without adversely affecting retrieval of overtrained conditioned reactions6~7~12~19. Although the validity of this statement is limited to certain types of behaviour, similar findings lead to the assumption that cholinergic transmission may be involved in short-term storage of the incoming information. Two experiments were performed to verify this hypothesis. In the first the technique of Blodgett and McCutchanz was modified. An H-shaped apparatus was used (Fig. I )

I

I

The animal was alternately started from the points S1 or SZ while the entrance into the opposite alley was closed by the sliding wall W1 or WP respectively. Intermittent electric shocks were applied with a 5 sec delay until the animal escaped into one of the two goals. Under control conditions most animals displayed a clear-cut tendency to alternate the goals when the starting points were alternated. Usually (80 %) the rats preferred Gz when started from S1 and GI when started from SZ (Fig. 2). The habit to alternate left and right turns is well known from maze studies and is usually attributed to factors like “forward-going tendency” and “centrifugal swing”. It involves some kind of short-term storage of kinesthetic, somesthetic and visual signals influencing the behavior of the animal at the choice point. In physostigmine-injected animals, the alternation was significantly impaired (Fig. 2). The choice did not become random but the rat systematically preferred one of the goals irrespective of the start from which it was released. This is illustrated i n Fig. 2b comparing the preference for one of the goals in the ten drug influenced trials. There was no difference in the effect of 0.5 and 0.25 mg/kg physostigmine. Different results were obtained with 6 mg/kg and 15 mgikg atropine which did not significantly impair the goal alternation in spite of dosages eliciting clear-cut EEG synchronization. 111the second experiment, a left-right alternation was elaborated in a group of

B E H A V I O R A L S I G N I F I C A N C E O F C H O L I N E R G I C SYSTEMS

%

63

I

v

0.25-05

C

Ph

,10 9 0 , 0 1 2

6-15

C

At

7

6 3

4

5 5

Fig. 2. The elTect of 0.25-0.5 mg/kg physostigmine (Ph) or 6-1 5 mg/kg atropine (At) on spontaneous alternation. C control conditions. Above : percentage of alternations. Below : percentage of animals displaying various degrees of preference (abscissa) for one of the goals.

10 hooded rats. The animal was placed on the start of a simple T-maze and required to avoid or escape electric shocks by reaching one of the two goals GI and G2. Both doors were opened during the first run. The rat was allowed to stay in the goal compartment for 10 sec and then was returned to the start again. The opposite goal was then accessible. The goals were regularly alternated. A correction technique was used throughout. Entering the alley leading to the incorrect door was considered as an error. Only a sequence of two correct choices was classified as an alternation reaction. The rats mastered the alternation task to a criterion of 9 alternations in 12 consecutive trials during 32 trials of the first day. After daily training the number of criterion trials dropped to 5.8 on the fifth day, at which point the pharmacological experiments were started. On each day the criterion was reached first. Then the drug was applied and after an interval of 10 min (with physostigmine) or 15 min (with atropine) the training was continued until the criterion was attained again. The interruption alone did not adversely affect the performance in control experiments in which saline was applied instead of drugs (Fig. 3). Both physostigmine (0.5 mg/kg) and atropine (6 mg/kg) caused a clear impairment of the alternation habit, while response latency was only slightly reduced. The rats could be retrained to criterion before the drug effect subsided, however, after an average of ten trials (Fig. 4). Similar results were obtained when the alternation delay was prolonged to 30-90 sec. The performance dropped to chance level during the first 5 trials with physostigmine. The atropine effect was similar for the 30 sec delay but considerably less definite for References p. 71-72

J.

RURES

At 10 sec

Ph

64

4 -

n Ph

C

At

Ph

30 5ec

At

90 sec

Fig. 3. The effect of 0.5 mg/kg physostigmine (Ph) or 6 mg/kg atropine (At) on delayed alternation. Columns indicate number of criterion trials under control conditions and after administration of drugs. 10, 30 and 90 sec - the delays used.

75 -

50 -

25

0

-

I

8

16

. A I .

24

32 min

Fig. 4. The eRect of 0.5 rng/kg physostigrnine (Ph) or 6 mg/kg atropine (At) on delayed alternation. The percentage of correct alternation responses (blocks of 5 trials) increases with continued training. The horizontal dashed line corresponds to the pre-drug performance, From left to right 10, 30 and 90 sec delays.

the 90 sec delay. The average number of criterion trials is shown in Fig. 3. Although the dynamics of the drug action must be taken into account, especially when using the 90 sec delay, in most experiments the alternation responding was perfect before the decline of the drug effect. Two additional factors must be considered when interpreting the above results. As the same rats were used throughout, the habit gradually became more and more overtrained. Furthermore the alternation was repeatedly learned under physostigmine or atropine. When the same dosage and delay was used again, the alternation was less impaired by the second drug application than by the first one. The less pronounced effect of drugs in the later phase of the experiment can be explained therefore

B E H A V I O R A L S I G N I F I C A N C E OF C H O L I N E R G I C SYSTEMS

65

by better fixation of the alternation habit, by state-dependent learning and by drug tolerance. The relative significance of these factors must be elucidated by further research. While the spontaneous alternation of left and right turns in the H-shaped maze is disrupted by physostigmine for periods corresponding well to the electrophysiological changes, the more difficult alternation (with delays of up to 90 sec) could be mastered even at the height of the drug effect. Even higher doses of physostigmine (1 mg/kg) or atropine (15 mg/kg), causing a decrement of avoidance reactions from 80% to about 20-30%, did not eliminate the alternation in highly overtrained animals. This points to the conclusion that cholinergic systems are not indispensable for delayed reactions of the above type, when the animal is adequately motivated and when the habit is sufficiently fixed. These experiments do not answer the question about the cholinergic nature of recent memory. Learning of new habits is impaired both by atropine and physostigmine, but this does not necessarily imply that the deficit is due to loss of recent memory. Cholinergic systems are perhaps engaged in normal learning as well as in short-term storage of information not subject to long-term retentions. A decrease of spontaneous goal alternation after scopolamine w d S reported by Meyers and Dominolx in rats. Bradley and Roberts5 found delayed responses in monkeys considerably impaired by atropine but also by atropine methyl nitrate, which does not penetrate the blood brain barrier. When the cholinergic storage is eliminated by drugs, a noncholinergic trace can be formed which is more or less resistent to cholinergic blockadel7. Overtraining has a similar effect, probably because the non-cholinergic system becomes more and more important with the continuing fixation of the engram.

Locus ojaction In spite of many attempts to determine the brain structures primarily affected by cholinergic and anticholinergic compounds the evidence is still inconclusive. As shown by Bradley and Elkes4 and by Rinaldi and HimwichZ4,physostigmine induces cortical desynchronization in cerveuu isole' rats but not in the isolated hemisphere preparation. This points towards involvement of thalamocortical and limbic mechanisms. Participation of the lower brainstem is not ruled out by the above experiments. The reticular threshold for EEG arousal is raised by atropinez4, but not decreased by physostigminel5>20.Behavioral arousal is altered neither by cholinergic nor by anticholinergic drug+. However, the dissociation of EEG and behavior limits the value of electrophysiological evidence for analysing the cholinergic mechanisms of learning. Structures primarily responsible for behavioral symptoms due to cholinergic and anticholinergic substances can also be identified by comparing the results of pharmacological experiments in normal animals and in animals with circumscribed brain lesions. As pointed out by Meyersl7 the atropine effect resembles the behavioral deficits caused by hippocampal lesions. No systematic study of the cholinergic and anticholinergic influences on the behavior of lesioned animals is available, however. An additional difficulty in such experiments is the sensitivity of denervated structures References p . 71-72

66

J. B U R E ~

which h a y considerably distort the usual relationships between the remaining brain centers. We attempted, therefore, to provide further electrophysiological and behavioral data characterizing the mechanism of physostigmine and atropine influences from the above aspects. Electrophysiological experiments The excitability of the cerebral cortex of unanesthetized curarized rats (BureS and Herink, unpublished data) was examined using two techniques. In the first experiment, a penicillin focus was evoked in the frontoparietal cortex by local application of a minute quantity of G-penicillin onto the exposed brain surface. The EEG was recorded from the area of penicillin application and from the symmetrical point in the opposite hemisphere. Within a few minutes high voltage spikes appeared in the region of the focus as well as in the symmetric contralateral cortical area. After the frequency was stabilized (about 25-40/sec), 1 mg/kg physostigmine or 6 mg/kg atropine were injected intraperitoneally and the recording was continued for 10 or 25 min respectively. An injection of the antagonistic drug followed. The results of these experiments are summarized in Fig. 5. Whilst under control conditions the maximum frequency of the penicillin spikes attained at the beginning of the experiment

%

200

-

150 .

130 -

50

0

-

0

10

20

30

rnin

Fig. 5. The effect of physostigmine and atropine on the rate of penicillin spikes generated from a cortical focus. The average rate before drug application is taken as 100 %. Abscissa = time in minutes.

exponentially decayed with a half time of 30-40 min, atropine prevented the decline after a few minutes and later caused an increase of spike frequency. In 50% animals trains of spikes appeared, raising the spiking rate to 50-100/min. Physostigmine, injected 25 min after atropine, further enhanced the seizure-like bursts, the average spike frequency increasing’to 250% of the control level. In experiments in which physostigmine was injected first, the spike frequency slowly decreased in most animals with trains of spikes occurring in only one animal. After atropine injection there was

B E H A V I O R A L S I G N I F I C A N C E OF C H O L I N E R G I C S Y S T E M S

67

---

Fig. 6. Cortical after-discharge before (A) and 5 (B) or 10 (C) min after administration of 1 mg/kg physostigmine. The inset brain scheme illustrates the arrangement of the recording (0, I , 2) and stimulatingelectrodes (S). ECG = electrocardiogram.Time after stimulation in minutes is shown over the record samples in C.

a short-lasting decrease in the discharge rate followed by activation. Spike grouping was observed in 60% of animals after atropine. Somewhat different results were obtained in the second series of experiments in which cortical afterdischarge was evoked by electrical stimulation (lO/sec, 2 msec, 10 sec) of the frontoparietal cortex. After the average duration of the afterdischarge had been determined, physostigmine or atropine was injected and electrical stimuli were applied at 5 min intervals. While the afterdischarge duration was not much changed by atropine it was strikingly prolonged in the physostigmine injected animals (Fig. 6). In most, the afterdischarge developed into continuous paroxysmal activity lasting for the rest of the experiment (Fig. 7), i.e. over several hours in some cases. The seizure continued even after injection of 6 mg/kg atropine which seems to be inadequate to antagonize the physostigmine effect. Only in one case (Fig. 8) did the afterdischarge stop 8 min after atropine administration and its duration returned to References p. 71-72

68

J.

BURES 5 30 min

Ph

C

Ph

At

Fig. 7. Average duration of cortical afterdischarge after application of drugs. Physostigmine does not influence the afterdischarge when applied after atropine, whilst atropine does not stop the afterdischarge prolonged by previous application of physostigmine.

normal values; it could be prolonged again, however, by a fresh injection of physostigmine. Physostigmine administered after preliminary treatment with atropine evoked a moderate increase in the afterdischarge duration but no continuous paroxysmal activity developed (Fig. 8). The above results indicate that atropine and physostigmine affect the excitability of a localised cortical area less than the excitability of the corticothalamolimbic circuits mediating the cortical afterdischarge. The atropine effect is rather inconspicuous, indicating that noncholinergic neurons are probably involved in the afterdischarge mechanism. Similar results were recently obtained with atropine by Berryl. On the contrary, the physostigmine effect was striking, as only EEG activation without any symptoms of seizure activity is induced by lmg/kg3*6.The extremely prolonged afterdischarge is evidently due to a tendency to maintain reverberative activity started by an intense stimulus. This indicates that blockade of acetylcholinesterase interferes with the mechanism causing abrupt cessation of the seizure activity, characteristic in the normal animal. Formation of secondary epileptic foci may explain the extremely long duration of the seizure, far outlasting the physostigmine effect. Curarization may be considered as another factor involved since acetylcholine and d-tubocurarine were shown to act cumulatively in facilitating cortical afterdischargeslO. Hyperventilation has the same effect22. Further research is required in order to reveal the relative significance of the above factors and to determine the structures primarily involved.

Behavioral experiments The significance of cortical mechanism in the behavioral effects of cholinergic and antich olinergic drugs was analysed by comparing their effects on active avoidance learning in normal and functionally decorticated animals. The naive rats were allowed at

B E H A V I O R A L S I G N I F I C A N C E OF C H O L I N E R G I C SYSTEMS

At

I

0

10

20

30

40

,-._.-..

Ph - - _.____ ~

I

50

69

60

I

70 rnin

Fig. 8. Examples of the differential effect of atropine and physostigmine on cortical afterdischarge.

0C 0

0 Ph0

0At 0

Fig. 9. The effect of functional decortication and of physostigrnine and atropine on ..arning of a simple avoidance reaction. Above : number of criterion trials (total columns) and of spontaneous return reactions (shaded columns). Below : Time spent on the grid floor during the first (I) and second (11) exploratory test. For details see text.

first to explore for five minutes a rectangular runway with an electrifiable grid floor in one half and a wooden floor in the other half ofthe apparatus. The times spent in the two parts of the runway were measured and crossings were recorded. An avoidance reaction was then elaborated: the animal was placed on start and required to reach within 5 sec the wooden floor, otherwise intermittent electric shocks were applied until a successful escape reaction was made. The rat was left for 40-60 sec in the goal compartment and then placed on start again. Training continued to the criterion of 9 avoidances in References p . 71-72

70

J.

BURES

10 consecutive trials. Twenty-four hours later the exploration test was repeated. Bilateral cortical spreading depression was evoked by local application by 25 % KCl onto the exposed cerebral surface 10 min before the first exploration. Drugs were applied at the same time. The results are illustrated in Fig. 9. The exploratory behavior of normal rats was not changed either by physostigmine or by atropine. The animals preferred to stay on the grid floor where they spent the major part ofthe 300 sec exploratory period. Learning the avoidance reaction was slower after physostigmine but not after atropine. The retention test revealed decreased preference for the grid floor in the control group but not in the animals learning under atropine. Although learning was considerably impaired under bilateral cortical spreading depression, most rats were able to reach the criterion in less than 50 trials. While physostigmine did not affect the learning ability of the decorticated animals, the atropine effect was very peculiar. The animals learned rapidly to run from the start to the goal within 5 sec, the avoidance criterion being attained with nearly the same speed as in normal animals. After reaching the goal, the animals often returned back to the grid floor, however, where they were shocked again until they finally spent at least 40-60 sec in the goal compartment. Spontaneous returning from the goal area to the electrified grid occurred in all spreading depression groups, but was most pronounced in the atropinized animals. The low number of criterion trials after atropine does not indicate that the learning ability was improved but that the procedure raised the overall activity of the animal and thus increased the probability of avoiding shocks as well as of receiving them by reaching or leaving the goal. This conclusion is also supported by the results of the exploratory test performed 24 hours later: the preference for the grid floor remained preserved and did not differ from that in normal naive animals. Atropine acts as a psychomotor stimulant in mice16 and rat$. Conversely, cholinergic stimulants reduce spontaneous activity11 and decrease amphetamine toxicityla. As the general stimulating effect of atropine is considerably enhanced in functionally decorticated rats, its site of action must be sought mainly at the subcortical level. On the contrary the behavioral impairment due to functional decortication is not further increased by physostigmine, which probably affects the regions directly or indirectly eliminated by the spreading depression process. It can be conceived that functional decortication produces an imbalance between the remaining parts of the two antagonistic systems, resulting in cholinergic predominance, which can be counteracted by atropine. DISCUSSION

In spite of the accumulating evidence, that the projxtion of the reticular arousal system to the cerebral cortex is cholinergic23, and that the principal cholinoceptive structures include the hippocampus, caudate and thalamus21, the results of our experiments indicate that in the rat other mediators can maintain even complex behavioral functions after cholinergic transmission has been effectively impaired. It seems justified to assume, therefore, that noncholinergic systems duplicate most behavioral functions of the cholinergic ones. No definite behavior can be claimed to be

B E H A V I O R A L S I G N I F I C A N C E OF C H O L I N E R G I C SYSTEMS

71

specifically dependent on cholinergic transmission, which diffusely participates in activities of different CNS structures and is perhaps most clearly expressed in the corticothalamolimbic system. In general, excessive stimulation of cholinergic synapses tends to abolish the motor output and results in some degree of sedation while administration of central cholinergic blockers has an opposite effect. As participation of cholinergic systems in organized neural activity is impaired in either case, the above differences are evidently due to the effect on the synaptically connected noncholinergic neurons, the bombardment of which is increased by physostigmine and decreased by atropine. The relative significance of the primary interference with the cholinergic transmission and of the secondary effects on the noncholinergic system is different in various CNS regions and can be revealed by electrophysiological experiments as well as by behavioral tests performed in lesioned animals. SUMMARY

The behavioral significance of cholinergic systems was examined in rats injected with physostigmine salicylate (0.25-1 .O mglkg) or atropine sulphate (6-1 5 mg/kg). Physostigmine but not atropine impaired spontaneous alternation of left and right turns in an H-shaped maze. Both drugs disrupted delayed alternation in a T-maze (delays from 10-90 sec). With continued training, however, the correct responding returned, before the drug effect started to decline. With repeated drug applications the effect became less marked. Atropine increased more than physostigmine the discharge rate of high voltage spikes elicited by local application of penicillin on the motor cortex of unanesthetized curarized rats. Physostigmine caused an extreme prolongation (up to several hours) of cortical afterdischarge, which could be prevented by atropine. Both physostigmine and atropine slightly impaired the acquisition of a simple avoidance reaction in normal rats. The same avoidance reaction required more criterion trials in functionally decorticated animals, the learning ability of which was unaffected by physostigmine and seemingly improved by atropine. The latter drug increased the overall activity of the animal and thus raised the probability of correct as well as of incorrect responding. It is concluded that cholinergic systems are paralleled by noncholinergic ones, which can take over many initially impaired behavioral functions. The mechanism of the cholinergic drug action is discussed and the differential influencing of various CNS levels is stressed. REFERENCES 1 BERRY, C. A. (1965) A study of cortical afterdischarge in the rabbit. 4rch. int. Pharmacodyn., 154, 197-209. 2 BLODGETI-, H. C. AND K. MCCUTCHAN (1944) Choice point behavior in the white rat as influenced by spatial opposition and by preceding maze sequence. J. Comp. Psychol., 37, 51-70. 3 BOHDANECK~, Z., T, WEISSAND E. FIFKOVA (1963) Influence of neocortical and hippocampal spreading depression on “theta rhythm” elicited by physostigmine. Arch. int. Pharmacodvn., 143,23-33. 4 BRADLEY, P. B. AND 5. ELKES(1957) The effects of some drugs on the electrical activity of the brain, Brain, 80, 77-1 17,

72

J.

BURES

5 BRADLEY, P. B. AND M. H. T. ROBERTS (1967) Studies on the effects of drugs on recent memory in animals. Physiol. Behav. In press. 6 BURES,J., Z. BOHDANECK? AND T. WEISS(1962) Physostigmine induced hippocampal theta activity and learning in rats. Psychopharmaccl., 3, 254-263. 7 BURESOVA, O., J. BURES,Z. BOHDANECK~ AND T. WEISS(1964) Effect of atropine on learning, extinction, retention and retrieval in rats. Psychophurmacol., 5, 255-263. 8 CARLTON, P. L. (1963) Cholinergic mechsnisms in the control of behavior by the brain. Psychol. Rev., 70, 19-39. 9 CARLTON, P. L. AND P. DIDAMO (1961) Augmentation of the behavioral effects of amphetamine by atropine. J. Pharmacol. exp. Ther., 132, 91-96. AND G . MOLNAR (1965) The effect of neuromuscular blocking agents on 10 FOHBR,O., G. KLITINA the electrical activity of cats cerebral cortex. Arch. irit. Pharrnacodyn., 158, 277-285. 11 HARRIS,L. S. (1961) The effect of various anti-cholinergics on spontaneous activity of mice. Fed. Proc., 20, 395. 12 HERZ,A. (1960) Die Bedeutung der Bahnung fur die Wirkung von Scopolamin und ahnlichen Substanzen auf bedingte Reaktionea. 2. Biol., 112, 104-1 12. 13 KILLAM, E. K. (1962) Drug action on the brain-stem reticular formation. Pharmacological Reviews, 14, 175-210. K. (1965) Transmitters in the cerebral cortex. Zr.t. Congress, Tokyo, Lectures and 14 KRNJEVIC, Symposia, PIoc. int. union physiol. sci., 23, 435443. (1957) Action of eserine and amphetamine on the electrical 15 LONGO,V. G. AND B. SILVESTRINI activity of the rabbit brain. J. Pharmacol., 120, 160-170. 16 MENNEAR, J. H. (1965) Interactions between central cholinergic agents and amphetamine in mice. Psychophurmacol., 7 , 107-1 14. 17 MEYERS, B. (1965) Some effects of scopolamine on a passive avoidance response in rats. Psychopharmacol., 8, 11 1-1 19. 18 MEYERS, B. AND E. F. DOMINO (1964) The effect of cholinergic blocking drugs on spontaneous alternation in rats. Arch. irt. Pharrnacodyn., 150, 525-529. 19 MEYERS, B., K. H. ROBERTS, R. H. RICIPUTI AND E. F. DOMINO (1964) Some effects of muscarinic cholinergic blocking drugs on behavior and the electrocorticogram. Psychopharrnacol., 5,289-300. 20 MONNIER, M. (1960) Actions klectro-physiologiques des stimulants du systltme nerveux central. 1. Systtmes adrknergiques, cholinergiques et neurohumeurs serotoniques. Arch. int. Pharmucodyn., 124, 281-301. 21 MONNIER, M. AND W. ROMANOWSKI (1962) Les systkmes cholinoceptifs cerebraux - actions de I’acetylcholine, de la physostigmine, pilocarpine et de GABA. Electroenceph. clin. Neurophysiol., 14,486-500. 22 OLIVER, K. L. AND W. H. FUNDERBURK (1965) Possible role of hyperventilation in the CNS effects attributed to tubocurarine. Electrmnceph. clin. Neurophysiol., 19, 501-508. 23 PHILLIS, J. W. AND G. C. CHONG(1965) Ace+ylcholinerelese from the cerebral and cerebellar cortices: its role in cortical arousal. Nature, 207, 1253-1255. 24 RINALDI, F. AND H. E. HIMWICH (1955) Alerting responses and actions of atropine and cholinergic drugs. Arch. Neurol. Psychiat., Chicago, 73, 387-395. 25 ROEERTIS, E. DE (1965) Subcellular localization of transmitter substances and related enzymes in the CNS. Znt. Congress, Tokyo, Lectures and Symposia, Proc. int. union physiol. sci., 23,411418. 26 WIKLER, A. (1952) Pharmacologic dissociation of behavior and EEG “sleep patterns” in dogs: morphine, n-ally1 normorphine and atropine. Proc. SOC. exp. Biol., N . Y., 79, 261-265.

73

Some Actions of Cholinergic and Anticholinergic Drugs on Reactive Behaviour A. HERZ Deutsche Forschungsaristaltfur Psychiatrie, Max-Planck-Institut, Munich (Germany)

The obvious lack of grossly visible behaviour changes following application of cholinomimetic and cholinolytic drugs led to the term of a ‘dissociation’ between EEG-pattern and behaviour4.39. Further investigations of the correlates of physostigmine- and atropine-induced EEG-patterns at the behavioural level, showed that stimulation as well as inhibition of central cholinoceptive structures is followed by distinct but more subtle changes of behaviour. The investigation of reactive behaviour proved to be very suitable for the detection of such subtle behaviour changes caused by substances acting on central cholinoceptive structures. In the case of cholinolytic drugs, the investigation of the acquisition of the conditioned responses proved to be very suitable and allowed the assumption that cholinolytic drugs interfere with memory processes, whilst in investigating cholinergic stimulants, outlines of a more general concept of the meaning of cholinergic stimulation for responding behaviour became evident. 1.

THE D I F F E R E N T I A L EFFECT OF C H O L I N O L Y T I C D R U G S O N A C Q U I S I T I O N A N D R E T E N T I O N O F C O N D I T I O N E D RESPONSES

In 1959 we demonstrated that the action of cholinolytic drugs on a conditioned avoidance response is highly dependent upon the establishment of this reaction and contrasting effects can be obtained whether the experiments are performed on rats in the state of acquisition of the response or on overtrained animals. Fig. 1 shows the performance of the conditioned pole jump in the first session of training. Every minute the conditioning signal, consisting of a tone of 5 sec duration, was given it was followed by an electric shock. After about 10 min the first conditioned avoidance responses (CAR) occurred and 30 min later a performance level of about 80% was reached. An injection of saline was without any effect and did not impair any further acquisition of the CAR, but the application of scopolamine (0.2 mg/kg) completely disrupted acquisition of the conditioned response. At the height of the action the unconditioned response was also partly abolished. Following scopolamine the animals showed a distinct pattern of excitement, especially when the acoustic signal was given: they walked about in the box, searching and sniffing. When the pole was in their field of view, they sometimes jumped onto it. One had the impression that the animals still References p . 54-85

74

A. H E R Z

II

NoCl Scopolominc

100 %

HEr 0.2 m g l k g

v--*-* -----*----- -.

I

I I

I

80

o--o conditioned reaction

n--e unconditioned reaction

I I

I

U al

I

C

I I

60

I

0 . L I -

al

a

*-

LO

O Y

C

u a D 0

20

0

__ 90

60

30

120

150

min 180

Fig. 1. Acquisition of a conditioned avoidance reaction (pole-climbing) during the first training session. Control-injection of NaCl after 30 min, and of scopolamine (0.2 mg/kg) after 40 min.

_____untrained a n i m a l s lo

__ overtruined

a n i mals

::: '

1

5

.

..

b

wl

<

2

..

wl

E

1

9

... :::. :t*

.

.*.*

b 0 e . 003

li)

0.5 0,2

..

** xxx x

Y

x

$D*

-

XYXY XYXY

Y

.

xx

inhibition of the conditioned reaction animal which received scopolomine in the first troining session and a s o v e r t r o t n e d onlmul

o

onimal which received scopolomine only a5 overtrained onimol

x

NaCL -control

Fig. 2. Comparison of the effect of scopolamine in animals during the first training-session and is over-trained animals.

ACTION O F CHOLINERGIC D R U G S

75

knew they ought to do something when the buzzer sounded, but they did not know what they were supposed to do. Thirty to 60 min later the conditioned response reappeared. The inhibition of the CAR was not complete in all animals and depended essentially on the moment when the drug was given during the acquisition procedure. This is shown in Fig. 2, in which the effect of scopolamine application in the first training session (when a 70% performance level was reached) is compared with the effect of scopolamine in overtrained animals which have had 8-12 training sessions. In these overtrained animals scopolamine was mostly ineffective, even after application of very high doses; only in a few cases did scopolamine inhibit the CAR. These were mainly older animals which did not react promptly even before application of scopolamine. This is not a tolerance phenomenon in the pharmacological sense. The result also remained unchanged when the first injection of scopolamine was given in overtrained animals. This effect can only be ascribed to an alteration of the susceptibility of the animals to scopolamine when the CAR becomes more completely established by training. In such overtrained animals scopolamine may cause an even prompter performance

T

e

.o

e

4-

-20%

5 -LO %

l

1

J 0 4 0 min

30-60min

60-90 rnin

Scopolamine l m g / k g o NaCl controls

Fig. 3. Alteration of the reaction time in over-trainedanimals after injection of 1 mg/kg scopolamine compared with control-injection of NaCI. (Means and standard deviation in 8 animals)

of the CAR. This is shown in Fig. 3 where the reaction time, that is the interval between the beginning of the buzzer and the jump to the pole, has been measured. In these experiments rats were selected with an almost 100% performance level so that they received almost no shocks. Under the influence of the drug the reaction time decreased and the pole jump was performed quite promptly. There may be some correlation between this facilitatory effect and the marked scopolamine-induced motor excitement of the animals. On the whole, the same effects were obtained with other cholinolytic drugs (Fig. 4), although with atropine rather high doses were neccessary. This may not only be due to the weaker central cholinolytic activity of this substance but also to the slower penetration into the CNS compared with scopolaminelg. The speed of penetration of a References p. 84-85

76

A. H E R Z untrained animals

overtrained animals

100 50.

0

x

x

x

x

X Y X X

x

x

x

xx

50 o/o 100 0 50 inhibition of the conditioned reaction 0

o A

x

x

xx

o/o

100

atropine benactyzine trihexyphenidyL chlorpromozine

Fig. 4. Comparison of the effect of several cholinolytic drugs in animals during the first training session and in over-trained animals.

substance into the CNS is quite important in experiments in which the drugs were injected at the moment when the 70% performance level in the first session was reached. When a substance enters the CNS slowly a further fixation of the CAR may occur. Other cholinolytic drugs like trihexyphenidyl and benactyzine were also less effective in animals during their first training session and showed no, or only small inhibition when the reaction became well established by training. This is extremely different to the effect of chlorpromazine. With chlorpromazine some small differences may exist in the effect between trained and non-trained animals, but only a small increase in the dose inhibits the CAR in overtrained animals too. The central origin of the effects of cholinolytic drugs is demonstrated by a much lower efficiency of the quaternized scopolamine and atropine homologs which have a peripheral cholinolytic activity even higher than that of the corresponding tertiary amines which enter the CNS readily. Further experiments were performed with a discrimination task. Rats with a high CAR performance level were trained to distinguish the tone of the buzzer from the ringing of the clock; a t the first signal they had to jump at the pole; at the latter signal they were not supposed to jump. When they did jump all the same, they received shocks a t the pole. Fig. 5 demonstrates such an experiment.

77

A C T I O N OF C H O L I N E R G I C D R U G S after training buzzer

:’ 60. 0

E

LO.

a

0.

L

; 20 aJ

Scopolomine Img/kg

10

2 o0

1

I 0

60

120 rnin 180 0

60

min 120

Fig. 5. Acquisition of discrimination between two different acoustic signals in a rat previously trained to the pole-climbing response. The animal had to jump at the buzzer’s sound and not to d o so, when the bell rang. Effect of scopolamine during the first and after 6 training-sessions.

Two hours after the beginning of this discrimination task, the animal had learned not to jump when the bell rang and there was 80% performance of the CAR to the buzzer. After injection of scopolamine the rat jumped to the pole after almost any signal in spite of receiving shocks in the case of bell-ringing. Scopolamine was also ineffective in this task after several (4-6) training sessions. The essmtial result of these investigations is the observation that the effect of cholinolytic drugs on a conditioned response is highly dependent on the state of acquisition of the response at the moment the substance is applied. The experiments of Meyers et al.25, in which the cholinolytic drugs were applied before the beginning of training of the conditioned pole-jump response, confirm our results. Apparently, application of the substances before the beginning of the learning procedure, (as in experiments of Meyers et ul.) or during the act of learning before the reaction has consolidated, (as in our experiments) does not make an essential difference. In the experiments of Pazzagli and Pepeu29 employing a learned performance method in rats, it was seen that the effect of scopolamine was highly dependent on the consolidation of the reaction. A further series of investigations showed that learning of several active or passive avoidance reactions or reward responses in several kinds of animals517~26~31.33.35 is impaired by the action of cholinolytic drugs. This is in accordance with results using the maze technique which also shows increased perseveration or decreased acquisition of maze problems under the action of atropinelike substances11~24~26~38. These experimental results may be connected with amnesia, especially a loss of recent memory, which is an effect observed following application of atropine, scopolamine and other cholinolytic substances in man27y28.I do not refer to the interesting References p . 84-85

78

A.

HER2

correlation between experimental data especially that obtained in rats and psychological investigations in man in this context; but the parallel is obvious and shows that relatively simple experiments in rodents are suitable for studying equivalents of rather complex psychic phenomena in man. As to the site of action of cholinolytic drugs in producing this amnesia, some facts point to medial temporal structures, particularly the hippocampus. A series of electrophysiological and neurological results show the importance of these structures for memory processes which may be discussed in more detail in other contributions of this symposium (see also the discussion of Meyers et ~ 1 . ~ ~ ) . In considering these results one might expect that cholinergic stimulants enhance the acquisition of conditioned responses. Indeed the effects of cholinomimetics and cholinesterase inhibitors were investigated under such aspects. In the experiments of Brimblecombe5 with the action of the anti-cholinesterase sarin, some tendency for increased acquisition of a conditioned response was observed, but not with other cholinomimetic drugs. The acceleration of formation of a new conditioned response in mice by nicotine and arecoline has been reviewed by Michelson26. A significant facilitation of learning was observed by Bovet-Nit@ with nicotine. The importance of dosage is obvious from the experiments of Card08 and Bureg et al.6 in which in lcw doses of physostigmine the acquisition of an avoidance reaction seemed to be facilitated. At higher doses however, learning was abolished, and the inhibition of conditioned responses is manifest not only in the state of acquisition of the reaction but also in overtrained animals though here some higher doses may be neccessary6. This inhibition of conditioned responses by cholinomimetic drugs seems to depend on quite another mechanism which will be discussed below. While there is almost complete agreement that cholinolytic drugs impair and disrupt insufficiently consolidated responses, our further observations that conditioned responses in overtrained animals are not influenced or even enhanced by cholinolytic drugs, do not agree with some results of other investigations. Though our results relating to this question are confirmed by Meyers et al.25 and Bignami2, there are several reports that in fairly fully trained animals also, cholinolytic drugs may abolish conditioned reactions. The reason for these discrepancies may be due to the different techniques and different kinds of animals. It can be easily understood that more complex reactions such as the instrumental reward conditioned r e s p o n ~ e ~ 2or9 conditioned-operant ~~ behaviourl are more accessible to disturbances than the simple pole-jump response applied in our experiments. Species differences, obvious in the different effect of cholinolytic drugs on spontaneous behaviour37, may also be important. In addition, the term “overtrained” is but poorly defined and it is possible that some strains of rats never do get overtrained even to the simple pole-jump response. The conditions seem to be quite different when the effect of cholinolytics on a passive avoidance response are investigated as in the experiments of Meyers23. Here scopolamine not only disrupted the acquisition of the response but also its retention, which means that the rats left the safe platform. From these results it is suggested that scopolamine not only impairs recent memory, but also produces some sort of disinhibition which may be connected

79

ACTION O F CHOLINERGIC D R U G S

with drug-induced motor hyperactivity. This effect may be engaged also in the diminution of the reaction time in overtrained animals observed in our experiments (Fig. 3). Such disinhibition of drug-induced motor hyperactivity seems also to be revealed in our discrimination-experiments, in which the animals respond with a jump to both signals after application of scopolamine. 2.

THE INHIBITORY

EFFECT O F

CHOLINOMIMETIC DRUGS

ON

REACTIVE

BEHAVIOUR

Applying the same avoidance conditioning technique (the pole-climbing response) as in the experiments described previously with cholinolytic drugs, Pfeiffer and Jenney30, demonstrated that cholinomimetic drugs, like arecoline and pilocarpine as well as cholinesterase inhibitors such as eserine inhibit the CAR at doses which leave the escape reaction unimpaired. A similar inhibition has been observed also by employing other avoidance conditioning techniques such as shuttle-box or lever-pressing with cholinomimetic drugs, such as tremorine, oxotremorine, RS 86 (Spiro-('N-methyl-piperidyl-4'), N-athyl succinimid"Sandoz") or other centrally actingcholinesteraseinhibitors9J4J5J8. Inmaninhibitionofaconditionedreactionby arecolinecanalso bedemonstratedl3. Since in these experiments atropine reversed the effects and the quaternary atropine homolog (eumydrine) was much less effective, this inhibition can be attributed to a central muscarine like mechanism. Though differences may exist between the doses effective in animals in the state of acquisition of the response and in overtrained animalse, cholinomimetic drugs and anticholinesterases, in contrast to the cholinolytics, also Muscarine

*-

Pilocorpine

Arecoline 0

.

RS 86

0

0x0-Tremorine

0,001 0.1

1.0

Effective dose

10.0

rng/kg

1000

'Analgesia' ( M i c e )

Fig. 6. Correlation between effective doses of several cholinomimetic substances inducing EEGarousal in rabbits and inhibiting the nociceptive reaction in mice. References I. 84-85

80

A. H E R 2

inhibit well-established reactions. After application of higher doses of some cholinomimetic drugs, like tremorine or cholinesterase-inhibitors, a muscular weakness occurs which cannot be prevented by eumydrine and which may be of central origin. But with other substances like arecoline or RS 86, the level between inhibition of the conditioned and unconditioned response is high enough (see Fig. 7) to make the assumption that muscular components have no definite significance in the inhibition of the conditioned response and the question of the mechanism for this action arises. In our experiments related to this problem, we compared the inhibition of the CAR with the “analgesic” properties of these substances,lg; ChenlO and Lenke21 reported that tremorine shows analgesic properties in mice, an effect which can be reversed by atropine, but not by eumydrine and is therefore attributed to a central muscarinelike mechanism. Other cholinomimetic drugs also inhibit the reaction to nociceptive stimulation. This is shown in Fig. 6, where doses, effective in Haffner’s mouse-tail-test (abscissa) are correlated to the doses effective in rabbits to induce EEG-arousal (ordinate). A good correlation exists between both parameters. It is interesting to note that muscarine, which is highly effective at the periphery, induces EEG-arousal only at very high doses and is almost ineffective in the inhibition of a nociceptive reaction, even when the periphery is protected by eumydrine. This can be attributed to the poor penetration of this lipid-insoluble quaternary amine into the CNS20. Cholinomimetic drugs also inhibit the nociceptive reaction in rats. We measured the increase in the pain threshold to electrical stimulation of the tail and compared the “analgesic” activity with the inhibition of the CAR, using the pole-climbing response in the same animals. In Figure 7 the efficacy of RS 86, a cholinomimetic drug (Sandoz) is correlated in both of the reactions. The ordinate represents inhibition of the conditioned and unconditioned avoidance response, the abscissa the corresponding increase in the threshold to nociceptive stimulation. The points refer

R S 86

0125-025-05-10-20

mg/kg

100,

condlt loned react ion

.-

0

c -D

0

300

200

100

nociceptive reaction

?Jo

LOO

- increase

of pain threshold in percent

A B R S 86 2 0 mg/kg

+ Eurnydrme 5 m g / k g

EB

tbtropine

”

’’

”

Fig. 7. Comparison ot the effect of RS 86 on the conditioned and unconditioned avoidance response with the increase of the stimulation threshold of the nociceptive reaction. The points refer to doses of 0.125; 0.25; 0.5; 1 .O and 2.0 mg/kg. A : Premedication with eumydrine 5 mg/kg; B : Premedication with atropine 5 mg/kg, followed by 2 mg/kg RS 86.

ACTION OF CHOLINERGIC DRUGS

81

to increasing doses from 0.125 to 2.0 mg/kg. The increasing inhibition of the nociceptive reaction was accompanied by a progressive inhibition of the CAR; at smaller doses the unconditioned response was unimpaired. Premedication with eumydrine reduced the effect of 2.0 mg/kg RS 86 in both test reactions to a small extent, while atropine completely abolished the effect of RS 86, demonstrating again the central origin of both reactions. In Fig. 8 the correlation between the inhibition of the nociceptive reaction and the

Trcrnorine 5-10-15-20rng/kg 1

C

,u u

a 0 c

.c 0

L u

10%

,

,

,

,~

-

40 80 120 160 200 LOO 6W 800 1000% nociceptive reaction increase of p a m threshold in percent

-

a Tremorine 20rng/kg + Atropine Smg/kg 0 RS86 20mg/kg + '' v Arecoline 20rnglkg + "

Fig. 8. Comparison of the effect of tremorine, arecoline and RS 86 on a conditioned avoidance response with the increase of the stimulation threshold of the nociceptive reaction. The points refer to doses of 5 , 10, 15, and 20 mg/kg for tremorine, of 0.5, 1 and 2 mg/kg for arecoline (premedication 5 mg/kg eumydrine) and 0.125; 0.25; 0.5; 1.0 and 2.0 for RS 86. The single points in the left corner refer to experiments in which premedication with atropine 5.0 mg/kg was applied with 20 mg/kg tremorine, 2.0 mg/kg arecoline or 2.0 mg/kg RS 86.

CAR, observed in experiments with other cholinomimetic drugs with central activity is shown. Besides RS 86, tremorine and arecoline also inhibit both reactionsin a similar manner. The behaviour of other centrally acting substances is interesting in this context and therefore we studied the influence of a series of substances on these two reactions. Here I only want to show some examples. Fig. 9 represents the effect of chlorpromazine and of morphine. Chlorpromazine as a representative of neuroleptic drugs, inhibits at low doses the CAR alone and an increase in the pain threshold occurs only at high doses; morphine as a typical analgesic drug, inhibits at lower doses preferentially the pain reaction and only at higher doses the CAR. The activity of a stimulant like amphetamine is also interesting (Fig. 10). At high doses both reactions are inhibited, but at small doses there occurs only an increase in the threshold to nociceptive stimulation, while the CAR is not impaired. Here, on the contrary, there is an especially prompt performance of the CAR, and the number of secondary responses, that is the jump without a signal, increases. The reaction-time decreases in a similar manner as previously reported for scopolamine in overtrained animals. Administration of other substances like reserpine, bulbocapnine and pentobarbital influenced both reactions in a quite different manner and only in the case of the References p. 84-85

82

A. H E R Z

cholinomimetic drugs did inhibition of the CAR and inhibition of the nociceptive reaction run parallel. Therefore the question arises whether inhibition of both reactions by cholinominietic drugs is based on a common central mechanism. There are some further observations supporting the concept that central muscarinic stimulation reduces or inhibits reactive behaviour in a more general sense. V a i l l a ~ i t ~ ~ , studying operant behaviour in pigeons, observed abolition of the motor output under the action of physostigmine, an effect which could be reversed by atropine; lie assumes, that central cholinergic systems are concerned with the occurrence or nonoccurrence of responding*. Stark and Boyd34observeddepression ofthe self-stimulation C

..e P

.-c 0

Z5-10-12.5-15 rng/kg

.-0

*'

4

NaCl

Of40

__

-. .

____-----.

- - - -/ r - - - _ _ . - - - -

-.

UR

80 120 160 200"300 400 500 600 760 Nociceptive reactlon-Increase of pain threshold in percent

Fig. 9. Comparison of the effect of chlorpromazine and morphine on conditioned and unconditioned avoidance responses with the increase of the stimulation threshold of the nociceptive reaction. The pointsrefertodosesof0.5; 1.0;2.0;and 4.0rng/kgforchlorprornazine and7.5; 10; 12.5; and 15 rng/kg for morphine. CR: conditioned reaction; UR: unconditioned reaction. c .OlOO. .+.

57

._ n .r

.-

d-Amphetamine 0.5-1-2-4 mg/kg

80-

d-Amphetamine 05 m d k g

4-

8 3.

reaction time

2 sec. cond. reactions 1

0 40 80 120 160 Nociceptive reaction increase Of pain threshold in percent

20 40 60 Minutes after injection

Fig. 10. Left side : Comparison of the effect of amphetamine on a conditioned and unconditioned avoidance response with the increase of the stimulation threshold of the nociceptive reaction. The points refer to doses of 0.5; 1.0; 2.0 and 4 mg/kg. CR: conditioned reaction; UR: unconditioned reaction. Right side : Alteration of the reaction time of the CAR and increase of secondary reactions by 0.5 mg/kg amphetamine. ____

* Grossman16observed inhibition of the CAR after direct application of cholinomimetics into several brain structures.

ACTION OF CHOLINERGIC D R U G S

83

response in dogs with electrodes in the hypothalamus following physostigmine. There may also be a connection with the observation of Feldberg and Sherwood12 that intraventricular injection of physostigmine reduces responsiveness to external stimuli. The idea of Carltonsa whereupon cholinergic systems act selectively by preferentially antagonizing the effects of systems which activate behaviour, especially when behaviour is unrewarded, is also in agreement with these observations. In addition to this outline of a concept of the correlation between central cholinergic mechanisms and responding behaviour, there are some facts which should not be omitted. Loew and TaeschlerZ2 found that RS 86, though inhibiting nociceptive reactions in mice and rats, shared no “analgesic” properties in rabbits and men, and the inhibition of pain reaction in mice could not be observed when the surrounding temperature was increased, an effect which is barely understandable. With cholinesterase inhibitors, the “analgesic” properties below toxic doses are obviously less pronounced than after the application of cholinomimetic drugs like arecoline or tremorhela. Also the difference between the inhibition of the conditioned and unconditioned response seems to be smaller after administration of cholinesterase inhibitors than after application of the cholinomimetic drugs. This points to the fact that the effects are not identical whether central cholinergic receptors are stimulated by endogenously accumulated acetylcholine or by cholinomimetic drugs which reach the receptors from the blood vessel - although in both cases the effects are abolished by atropine, which means that they are muscarinic. It is easy to understand that quantitative differences at least between the action of cholinesterase inhibitors and cholinomimetic drugs result from such differences. Structures with a high acetylcholine-synthesizing capacity must not neccessarily be the same as structures which are largely reached by exogenously applied cholinomimetic drugs. Within the group of cholinomimetics remarkable differences seem to exist ; for instance, between RS 86 which produces no or only a very small pronounced tremor and arecoline or tremorine and oxotremorine which produce a very intense tremor in some kinds of animals, though the central actions of all these substances seem to be muscarinic. CONCLUSIONS

Two different mechanisms appear to exist by whch reactive behaviour may be altered by substances acting on cholinergic structures. The acquisition of the conditioned response is either improved or abolished by cholinolytics. There is some indication that these substances interfere with memory processes, especially with recent memory. Cholinergic drugs seem to facilitate the acquisition of such responses only to a very restricted extent. Also, inhibition of the acquisition may occur at higher doses. After consolidation of the avoidance reaction in rats by training, cholinolytics no longer inhibit the reaction and may even accelerate the performance of the response. When employing other kinds of animals and tests this reversal of the effectiveness of cholinolytics is dependant upon the establishment of the reaction and does not seem to be as complete as in the pole-climbing response in rats. References p . 84-85

84

A. H E R Z

Well-established conditioned responses are inhibited by cholinergic stimulants. The inhibition of the reaction to nociceptive stimulation runs parallel to inhibition of conditioned behaviour. This, besides other experimental results, indicates that central cholinergic (muscarinic) stimulation results in decreased responsiveness in a more general sense.

REFERENCES B E R R Y , C . A. A N D L. G. STARK (1965) Modification of conditional behaviour by prior experience. P.sychopharniacolagia, 7 , 409-41 5. 2 BIGNAMI, G . (1964) Effects of benactyzine and adiphenine on instrumental avoidance conditioning in a shuttlebox. P.sychopharniacologia,5, 264-279. 3 BOVET-NITTI, F. (1965) Action o f nicotine on conditioned behaviour in naive and pretraiiied rats. In: v. EULER,U. S., (Ed.), Tobacco Alkaloids and related Compounds. Pergamon Press, Oxford, p. 145-162. (1957) The effect of some drugs on the electrical activity of the brain. 4 BRADLEY, P. B. AND J. ELKES Brain, 80, 71- 117. R. W. (1965) Effects of centrally active drugs on acquisition of a passive avoid5 BRIMBLKOMBE, ance reaction in the rat. Neuropsychopharmacology, Vol. 4. Eds. D . Bente, P. B. Bradley, p.333336. 6 BUR& J., Z. B O H D A N E CAND K ~ T. WEISS(1962) Physostigmine induced hippocampal Theta activity and learning in rats. Psychopharniacollogia, 3, 254-263. i BURESOVA, O . , J. BURES,Z. BOHUANECKY AND T. WEISS(1964) Effect of atropine on learning, extinction, retention and retrieval in rats. PsychophannacolloRia, 5 , 255-263. 8 CARDO,D. (1959) Action de I’amphetamine dextrogyre et de eserine sur unconditionnement de fiiite et sur les phenomhes de discrimination. J . Physiol. (Paris), 5 , 845-860. P. L. (1963) Cholinergic mechanisms in the control of behaviour by the brain. 8a CARLTON, PS~C~O~ Rev. U R,70, . 19-39. 9 CHALMERS, R. K. AND C. K. ERICKSON (1964) Central cholinergic blockade of the conditioned avoidance response in rats. Psychopharmacollogia, 6, 3 1-41. 10 CHEN,G . (1958) The anti-tremorine effect of some drugs as determined by Haffner’s method of testing analgesia in mice. J . Pharmacol. exp. Ther., 124, 13. 11 DOMER, R. AND F. W. SCHUELER (1960) Investigations of the amnesic properties of scopolamine and related compounds. Arch. itzt. Pharmacodyn., 127, 449-458. (1954) Behaviour of cats aftcr intraventricular injection of 12 FELDBERG, W. A N D S. L. SHERWOOD eserine and DFP. J . Physiol., 125, 488-500. 13 F R A N K S ,M.,D.S. ~. TROUTONANUS. G . LAVERTY (1958)Theinhibition ofaconditioned response following arecoline administration in man. J . clin. exp. Psychopath., 19, 226. 14 FUNDERBURK, W. AND T. CASE11947) Effect of parasympathetic drugs on the conditioned response. J . Neurophysiol, 10, 179-188. 15 GOLDBERG, M. E., H. E. JOHNSON, J. B. KNAAKA N D H. F. SMITH,JR. (1963) Psychopharmacological effects of reversible cholinesterase inhibition induced by tz-methyl 3-isopropyl phenyl carbamate (Compound 10854). J . Pharmacolrxp. Therap., 141, 244252. 16 GROSSMAN, S. P. (1964) Behavioral effects of direct chemical stimulation of central nervous system structures. Int. J . Neuropharniacol., 3, 45-58. 17 HERZ,A. (1960) Die Bedeutung der Bahnung fur die Wirkung von Scopolamin und Phnlichen Substanzen auf bedingte Reaktionen. Z . B i d . , 112, 104 18 HERZ,A. UND F. YACOUH (1964) Hemmung nociceptiver und bedingter Reaktionen irn Vergleich mit der Wirkung anderer zentral angreifender Substanzen. Psychopharmacollogia, 5 , 115-125. 19 HERZ,A., H. TESCHEMACHER, A. HOFSTErrER A N D H. Ku~z(1965)Theimportanceof lipid solubility for the central action of cholinolytic drugs. Ittt. J . Neuropharmacol., 4, 207-218. 20 HERZ,A., H. HOLZHAUSER UND H. TESCHEMACHER (1966) Zentrale und periphere Wirkungen von Cholinomimetica und ihre Abhangigkeit von der Lipoidloslichkeit. Arch. exp. Path. uncl Pharnzak., 253, 280-297. 21 LENKE,D. (1958) Narkosepotenzierende und analgetische Wirkung von 1,4-Dipyrrolidino-2butin. Nautiyti-Schmierieberg~Arch. Path. und Pharmak., 234, 35-45.

I

ACTION O F CHOLINERGIC D R U G S

85

22 LOEW,D. U N D M. TAESCHLER (1964) Uber die analgetische Wirkung von RS 86, einem Cholinomimeticum mit zentraler Wirkungskomponente. Helv. Physiol. Pharmacol. Acta, 22, C 80-82. 23 MEYERS, B. (1965) Some effects of scopolamine on a passive avoidance response in rats. Psychopharmacologia, 8, 1 1 1-1 19. 24 MEYERS, B. AND E. F. DOMINO (1964) The effect of cholinergic blocking drugs on spontaneous alternations in rats. Arch. int. Pharmacodyn., 150, 3-4. 25 MEYERS, B., K. H . ROBERTS, R . H. RICIPUTTI AND E. F. DOMINO (1964) Some effects of muscarinic cholinergic blocking drugs on behaviour and the electrocorticogram. Psychopharmacollogia, 5, 289-300. 26 MICHELSON, J . M . (1961) Pharmacological evidences of the role of acetylcholine in the higher nervous activity of man and animals. Activify nerv. sup. (Praha), 3, 140-147. 27 MIGDAL,W. AND M. J. FRUMIN (1963) Amnesic and analgesic effects in man of centrally acting anticholinergics. Fed. Proc., 22, 188. 28 OSTFELD, A. M., X. MACHNE A N D K. R. UNNA (1960) The effects of atropine on the Electroencephalogram and behaviour in man. J . Pharmacol. exp. Ther., 128,265-272. 29 PAZZAGLI, A. AND G. PEPEU(1964) Amnesic properties of scopolamine and brain acetylcholine in the rat. Int. J . Neuropharmacol., 4, 291-299. 30 PFEIFFER, C. C . A N D E. H. JENNEY (1957) The inhibition of the conditioned response and the counteraction of schizophrenia by muscarinic stimulation of the brain. Ann. N . Y. Acad. Sci., 66, 753-764. 31 RICCI,G. F. A N D L. ZAMPORO (1964) Studi sul ruolo delle strutture cdinergiche cerebrali nel condizionamento: relazioni fra le risposte condizionate e la reazione di arresto EEG. Riv. Neurol., 34, 283-286. 32 ROUGEUL, A,, J. VERDEAUX A N D P. GOCAN(1965) Limits of the dissociation between EEG and behaviour under atropine-like drugs in cats. In/. J . Neuropharmacol., 4, 265-272. 33 SADOWSKI, B. AND V. G. LONGO(1962) Electroencephalic and behavioural correlated of an instrumental reward conditioned response in rabbits. EEG Clin. Neurophysiol., 14, 465-476. 34 STARK,P. AND E. S. BOYD(1963) Effects of cholinergic drugs on hypothalamic self-stimulation response rates of dogs. Am. J . Physiol., 205, 745-748. 35 STONE,G. (1960) EffeLts of some centrally acting drugs upon learning of escape and avoidance habits. J. Comp. Physiol. Psychol., 53, 33-37. 36 VAILLANT, G. E. (1964) Antagonism between physostigmine and atropine on the behaviour of the pigeon. Naunyn-Schmiedeberxs Arch. Path. und Pharmak., 218, 406-41 6. 37 WHITE,R. P., C. B. NASH,E. J. WESTERBEKE AND J . PASSANZA (1961) Phylogenetic comparison of central actions produced by different doses of atropine and hyoscine. Arch. inf. Pharmacodyn., 132, 349-363. 38 WHITEHOUSE, J. M. 11964) EFects of atropine on discrimination learning in the rat. J. Comp. Physiol. Psychol., 57, 13-15. 39 WIKLER, A. (1952) Pharmacologic dissociation of behaviour and EEG “sleep patterns” in dogs: Morphine, N-allylnormorphine, and atropine. Proc. Soc. Exp. Biol. Med. N . Y., 79, 261-265.

Experimental Psychoses Induced by Benactyzine in Alcoholics M.VOJTYECHOVSKY, D. K R U S ,

s. GROF, v. AND

V ~ T E K ,K. R Y S A N E K , K. K U N Z J . SKALA

Laboratory of Experinrental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague, Czechoslovakia

The clinical use of benactyzine in Czechoslovakia can be traced back to the year 1956. Pharmacological and clinical experience with this drug have been summarized by Votava et al.35 and VinaPovh et al.24. They confirmed its moderate effect as far as the symptomatic control of anxiety in some neuroses is concerned. It appears, however, that the relatively high frequency of psychic side-effects after medium and large doses of benactyzine will prevent its more extensive use in out-patient practice. These side effects have been described in great detail in the earlier publications of Coady and Jewesbury5, Davies6 and others. One of us (Vojt&hovski) had the opportunity to observe, in 1957, an accidental intoxication which developed after the ingestion of 1300 mg of pure benactyzine. The intoxication had the character of a short benign hallucinatory psychosis. Stimulated by this observation, we attempted, at the beginning of 1958, to evoke a n experimental psychosis with.[benactyzine in a volunteer, a young psychiatrist, who had previously experienced two experimental psychoses after LSD and one after mescaline. The selected dose, 200 mg was, however, very large and caused a prolonged psychotic condition, which had the character of a severe delirium with catatonic features and corresponded to the exogenous psychosyndrome as described by Bonhoeffers. Some elements simulated delirium tremens and KorsakoffIn subsequent experipsychosis, others reminded us of atropine into~ication'4~~8~29. ments we attempted to find the optimal dose of benactyzine which would produce an experimental psychosis without alteration of consciousness and enable satisfactory cooperation with the experimental subject. We found that in most experimental subjects this could be achieved with a dose of 40-70 mg of benactyzine26.27~28.29,or in sensitive subjects by as little as 15-25 mg32833, A further 17 volunteers, including all the authors of the present paper, were subjected to the experiment31, and all developed an experimental psychosis lasting 1-6 h. The psychotic symptoms were similar to those recorded in the first experimental subject after the administration of 200 mg benactyzine, only less intense and the lucidity of consciousness was less affected. Somatic and psychic manifestations were similar to those after smaller doses596. The assumption that benactyzine interferes with serotonin metabolism was proved by the decreased urinary excretion of 5-HIAA, which coincided with the maximum psychotic changes (refs. 26, 27). It remained hypothetical, however, whether the decarboxylation of

EXPERIMENTAL PSYCHOSES INDUCED B Y BENACTYZINEI N ALCOHOLICS

87

5-hydroxytryptophan was affected or whether the oxidative deamination of serotonin was blocked28?29.The inhibitory effect of benactyzine on M A 0 in liver and brain25 supports the previous idea of interference with oxidative deamination.

STUDIES ON ALCOHOLICS

In previous c o m r n u n i c a t i ~ n we ~ ~ pointed to a neglected area of comparative psychopathology: the relation between experimental psychoses and naturally occurring symptomatic psychosyndromes, especially those of alcoholic origin. In a joint communication30 we suggested a hypothesis concerning the cholinolytic nature of Bonhoeffer’ss pathogenetic link in delirium tremens (D.T.) and Korsakoff’s alcoholic encephalopathy (K.P.). Our hypothesis resulted from a comparative study of various psychodysleptic substances in auto-experiments and in experiments in healthy volunteers. We tried to compare the manifestations observed in these experiments with the phenomenology of the alcoholic psychoses. In our work we aimed at a verification of some presuppositions of the previously suggested hypothesis about the modeling of acute psychotic reactions in chronic alcoholics as a relative homogeneous group of candidates for a delirium tremens or Korsakoff’s syndrome. We anticipated a heightened susceptibility to anticholinergic hallucinogens, with a richer psychopathological picture than we saw in auto-experiments and in healthy volunteers. Furthermore we wanted to verify the presupposition that anticholinergic hallucinogens precipitate a clinical picture similar to the psychotic episodes connected with abuse of alcohol in anamnesis. It was necessary to show whether the data of Arnold and Hoffz and Ditman and Whittlesey7 on the similarities of LSD intoxication and delirium tremens are in contradiction with our ideas. We therefore included in our study experiments with LSD as well as with benactyzine in the same subjects.

SUBJECTS

These were sixteen chronic alcoholics, males, between the ages of 28-44 years (mean 36). All were beer-drinkers in the terminal stage (according to Jellinek), treated on a volunteer basis in the anti-alcoholic ward, and taking part in three series of experiments (LSD 200 pug, benactyzine 40 mg and placebo) at weekly intervals. Five of these patients had undergone an alcoholic psychosis previously. All had abstained for 3-5 weeks. None took antabuse or emetic apomorphine cure. The motivation of the subjects for the experiments was the possibility of a psychedelic experience after LSD, with a curative effect.

References p . 103-105

88

M.

VOJTECHOVSK?

et a/.

TABLE I S Y A T l S T l C A L ANALYSlS O F T H E M E T H O D S APPLIED I N E X P E R I M E N T S

(Wilcoxon) -

hour& 0.5 ( a ) Bciiaclyzine: placebo Pulse rate BP systol. BP diastol.

Mydriasis Tapping speed Tremor A-test - latencies - reproductions Time estimation Clyde Mood Scale F E C A J D Diiker lest (items) (errors) Benton test (errors) Stroop test (time) (errors) Learning of 5 paired associations: reinforcement 1

Pulse rate BP systol. BP diastol. Mydriasis Tapping speed Tremor A-test - latencies reproductions Time estimation Clyde Mood Scale F E C A J D

-

2

2.5

3

4

5

6

1

-

1.f V -

-

v ti

n h h

3 10

f h ) LSD: placeho

1.5

I,

L

Handwriting Length of words Time of copying A.-J. Questionnaire

1

n

A

89

E X P E R I M E N T A LP S Y C H O S E S I N D U C E D B Y B E N A C T Y Z I N E I N A L C O H O L I C S

TABLE I (continued) hours

0.5

I

1.5

2

-

r 4 -

J.

c

10

Handwriting Length of words Time of copying A.-J. Questionnaire ( c ) Benactyzine:L S D

Pulse rate BP systol. BP diastol. Mydriasis Tapping speed Tremor A-test - latencies - reproductions Time estimation Clyde Mood Scale F E C A J D Diiker test (items) (errors) Benton test (errors) Stroop test (time) (errors) Learning of 5 paired associations reinforcement 1

-

t

,L.

3 10

f d

1. J -

P < 0.025

: 2

= =

-

P < 0.005 (one tailed) increased values decreased values non significant

References p . 103-105

3

a

Diiker test (items) (errors) Benton test (errors) Stroop test (time) (errors) Learning of 5 paired associations Reinforcement 1 2 3

Handwriting length of words time of copying A.-J. Questionnaire

2.5

-

4

5

6

90

M.

VOJTiCHOVSK4 eta!. METHODS

The experiments began at 12 o’clock and the patients were observed for a period of 6 hours. Clinical, physiological, psychological and biochemical methods were used : clinical interview, pulse rate, blood pressure, pupillometry, ataximetry according to Seashore, Stroop’s Color Test, Hand Steadiness Test, Benton’s Test (used with our more detailed scoring), Duker’s Arithmetic and Concentration Test, Word Association Test22, time estimationg, handwriting (copying of text)”, finger painting, Clyde Mood Scale and Abramson-Jarvik’s questionnaire. The day following the experiments the patients filled out the questionnaire according to Ditman7. Some tryptophan metabolites were followed by urinary analysis. The sequence of the experiments was changed according to a Latin square. The evaluation of the results obtained from 13 subjects was analyzed according to Wilcoxon’s nonparametric test (see Table la, b, c). RESULTS

The clinical analysis showed that there are more differences than similarities between LSD and benactyzine intoxication in the group of normals, as well as in that of the alcoholics (Table It).

T A B L E 11 ~~

...__ ~

LSD

Benactyzine

Delirious state Arnnestic syndrome without hallucinations Verbal hallucinations Paranoid psychosis Schizoform psychosis Ecstatical state Drunkenness

Alcoholics N-16 40mg

Conrroh N=16 40-50nig

8++ 3 2 I 0 0 2

15+++ 1 0 0 0 0 0

Alcoholics N-16

Controfc

200,ug

100 /Lg

N=l6

0

1

0 0 7+ t-

0 0 2 4 8+++ 1

1 5 3

The clinical picture of LSD intoxication had the character of a phantasy with depersonalization, jitteryness, anxiety and paresthesia. Some other, common symptom clusters were revealed by Ditman’s questionnaire (Tables 111 and IV). In comparison with normals we saw more paranoid reactions after LSD. During benactyzine intoxications, the syndrome of trivial delirum and the amnesia syndrome prevailed. In comparison with the control group, the reactions of the alcoholics were much less intense in spite of the fact that the doses of LSD were twice as high. In five patients with alcoholic hallucinations in amnesia benactyzine evoked a clinical picture

E X P E R I M E N T A L PSYCHOSES I N D U C E D B Y B E N A C T YZ I N E I N A L C O H O L I C S

91

TABLE III

LSD

S Y M P T O M C L U S T E R S C O M M O N FOR

AND

B

(Ditman's Questionnaire)

L K

R A B N P M 0

Hypnagogy (10) Somatic discomfort (31) Anxiety (13) Unusual body sensations (25) Thoughts, recollections (18) Hostility (6) Depression (12) Perceptual distortions (27) Delusional - paranoid (21)

N-7

N=7

LSD 200 1% I %)

Benactyzine 40 mg ( %I

41 .O 41.0 36.3 32.3 30.0 26.6 25.0 26.3 18.2

36.0 29.0 24.0 26.4 20.5 21.7 25.0 17.4 12.3

T A B L E IV LSD (Ditman's Questionnaire)

SYMPTOM CLUSTERS T Y P I C A L FOR

H J F I G C

Understanding-Meaning (14) Euphoria, humour, relaxation (19) Appreciation esthetic (1 1 ) Mystical sense of wonder (18) Unity of religious feeling (12) Imaginary (16)

A N D NOT FOR

B

N=7

N= 7

LSD

Benactyzine

200 fig ( %)

40 mlg ( %)

37.7 33.3 27.2 25.0 19.2 18.8

9.3 9.4 0.73 8.9 0.33 0.25

which was almost identical with their previous natural psychosis. LSD intoxication in these patients had a completely different character. Paranoid reactions were much less frequent after benactyzine than after LSD. Optical illusions and hallucinations form a primary part of the benactyzine intoxication and are more frequent and have a richer content than those after LSD, occurring with the eyes open in nearly all cases in the volunteers, and in 60 % of alcoholics. Pick's pontine visions and peduncular hallucinations were observed more frequently in alcoholics after LSD than after benactyzine. Auditory hallucinations occurred after benactyzine in seven alcoholics and after LSD only in one. The vegetative signs were anticholinergic in nature with a shorter duration than with LSD. The latency from administration to appearance of the first signs was half as long after benactyzine, as after LSD (Fig. 1). The course of the effects was steadier; the duration of the effect varying from 3 to 5 hours. The main difference between the effects of LSD and benactyzine in the group of alcoholics was in alonger duration of References p . 103-105

M . V O J T ~ C I I O V S Ket ' ~ ul.

92

n

n

PI

B

LSD

Fig. 1. Left : Number of symptoms (means) measured according to Abramson-Jarvik Questionnaire after the administration of LSD, benactyzine (B)and placebo (PI) during the 8 h of observation. RiRh/ : Number of positive answered symptoms in Abramson-Jarvik Questionnaire registered in the whole course of the experiments with LSD, benactyzine and placebo. Each spot represents the value of one patient. 12 47. --

the effects of LSD. The symptoms did not disappear even i n the eighth hour. The qualitative analysis of Abramson and Jarvik's questionnaire showed that several symptoms were much more frequent after LSD than after benactyzine (see Table V). In benactyzine intoxication only abnormal acoustic and visual perceptions were more frequent . Emotional changes measured by the Clyde Mood Scale (CMS) in the third hour of intoxication showed no substantial changes after placebo (Fig. 2), LSD depressed significantly the parameters of Energy (E) and Clearthinking (C) and elevated those of Jitteryness (J) and Depression (D). The main difference between LSD and benactyzine was in a more marked decrease in parameter C and the absence of changes in D in the experiments with benactyzine. I n the sixth hour (Fig. 3) of intoxication the parameter D continued to rise after LSD, whereas after benactyzine the depression of E and C was still present. The Clyde Mood Scale revealed reliably the main effect of benactyzine on the parameters E and C, whereas LSD had a different effect on J and D. Cardiovascular reactivity : Tachycardia appeard in the first two hours only after benactyzine. (Fig. 4). A hypertensive reaction was observed in the 150th minute after LSD, and in a milder degree in the first two hours after benactyzine. In the diastolic hypertensive reactions there were no substantial differences between LSD and benactyzi ne, In the mydriatic effect (Fig. 5 ) there was no substantial difference between benactyzine and LSD, although LSD seemed to be a little more efficient. The neurological signs were more marked after benactyzine than after LSD, showing principally a hypo-adynamia, ataxia, apraxia and dysarthria syndrome. With the higher dose there was floccilegium. Ataxia and hypodynamia was best measured by means of a tapping ataximeter, according to Seashore. Maximal alteration was seen after benactyzine administration i n the first hour (Fig. 6, upper

93

E X P E R I M E N T A L P S Y C H O S E S l N D U C E D BY B E N A C T Y Z l N E IN A L C O H O L l C S

TABLE V Alcoholics N

I Jnner trembling 2 Anxiety 3 Inadequate smiling 4 Pressure in ears 5 Light feelings in hands 6 Dreamy states 7 Palpitation 8 Numb lips 9 Abnormal hearing 10 Abnormal optical perception

dn +4r

F

_I_

E

C

A

J

Benactyzine 40 mg

14 12

9

8 5 4 3 5

9

4

8

3

10

3 h

xx

+I.

LSD 200 pug

I1

D C M S

=

6

1

3 5

7 9

F

E

16

Placebo

2 0 0 0 1

0 I 1 0 0

C

A

J

D C M S

6h

PL

LSD

-10 5

X

LSD

.B

8

10

- 20

Fig. 2. Clyde Mood Scale registered in the third hour after the administration of LSD, benactyzine and placebo. BUJCline : values obtained from measurement before the administration of individual drugs. Fig, 3. Clyde Mood Scale registered in the sixth hour after the administration of LSD, benactyzine and placebo. References p . 103-105

M. V O J T ~ C H O V S Kel~ al.

94

Amm LSD

0

’

0

1

2

3

4

5

PI

6 h

LSO B 0 P DIASTOL PI

Fig. 4. Changes of physiological parameters during six hours of observation after the administration of LSD, benactyzine and placebo. Base line: initial values before diug administration. Fig. 5 . Changes of pupillary diameter (mean increase in mm) after the administration of LSD, benactyzine and placebo during six hours of observation. Base line: initial values before drugs’ administration.

part). There was a smaller decrease after LSD in the third hour. Alteration in psychomotor functions after benactyzine could be detected even by the Hand Steadiness Test during the period of four hours of observation (Fig. 6 , lower part). In this test LSD did not appear to be a tremor evoking drug. Thought disturbances after benactyzine were more marked than after LSD. Conversational consistency was lost, with frequent signs of thought block and incoherence. With higher doses, thought processes were completely disintegrated. The effect of LSD and benactyzine could be very subtly differentiated by Stroop’s Color Test, in which the number of speech errors was significantly higher after benactyzine in both variants, i.e. text reading and denotations of colors (Fig. 7). The clinical picture after benactyzine contained a marked deterioration of intellect, with striking disturbances of arithmetic and writing performance. Duker’s test applied after benactyzine again revealed a significant decrease on the computation speed and increased number of errors (Fig. 8). In the copy test (handwriting) a retardation of words occurred only after benactyzine (Fig. 9). An increase in the length of words was found in both conditions.

EXPERIMENTAL PSYCHOSES INDUCED BY BENACTYZINE I N ALCOHOLICS

,

1

2

3

4

5

6 h

,

,

I

,

,

,

95

sec

250‘ TAPPING

TIME

200 -

Pl 150 -

LSD

100 OD

B

50 -

0-

15#1

25

NUMBER

OF ERRORS

20

0

10

0

0 5

Pl

B

LSD

Fig. 6 . Upper part : Changes in tapping spead (decrease during one minute of the duration) after the administration of LSD, benactyzine and placebo during six hours of observation. Lower purr : Changes of contacts in simple tremornetry after the administration of LSD, benactyzine and placebo. Mean values during six hours of observation, Fig. 7. Upper part: Time for naming of one hundred colois in Stroop test during the second hour of the administration of LSD, benactyzine and placebo. Each spot represents values of one patient. Lower part: Number of errors in Stroop test.

Memory loses its ability to retain recent events, With higher doses there was usually an insular amnesia at the height of the intoxication. Three tests discriminated between the effect of benactyzine and the effect of LSD. Benton’s test of visual-motor retention showed a higher number of errors than after placebo and LSD (Fig. 10). The difference between LSD and benactyzine was also statistically significant. In the test of free associations to 30 verbal impulses (Fig. 11) benactyzine and LSD prolonged the latent periods in a statistically significant way. However, the differences between the substances was not significant. On the other hand only benactyzine significantly disturbed memory, especially in the first hour of observation. In the test of learning ability (5 paired unrelated associations) the results were significantly worse after benactyzine and LSD for the whole learning period as compared with the placebo (Fig. 12). The difference between LSD and benactyzine was in the first two reinforcements. This suggests that benactyzine disturbs mostly the component of concentration and recent retention. In further reinforcements, the learning processes were significantly impaired as after LSD. Disturbances of consciousness with benactyzine had a dreamy nature at moderate Referencer p . 103-105

M.

VOJTECHOVSK~

e f a/.

n

100 -

90 80

LENGTH OF WORDS

TIME

A sec

-

-

2oc

A rnrn

-

15

-

70 -

15C

0

60 0

50 -

0 0 0

40 .

+

10 -

Q O

D O 0

5c

B

Q O

0

CQ

5

-

Pa

0

0-

0

'0

0 0

20 -

30-

0

5

1oc

0

0

P

0

-

B

LSD

LSD

€3

LSD

Fig. 8. Number of correctly solved problems in arithmetic, measured by Duker test after the administration of LSD, benactyzine and placebo. Each spot represents values of one patient after the drug administration. Fig. 9. Lefr: Time of copying of 30 words after the administration of LSD and benactyzine. Right : Length of words in the same handwriting test. Each spot represents increase or decrease of values of individual patient in comparison to measurement in placebo experiment. Base line: placebo values.

20

-

0

18.

0 16 -

0

14 -

0

12

0

u 0

10.

.

80

e.

6-

0 0 000 0

4-

2-

0-

3 00 I00

-@-v

00 100

-

PI

0 0 000

B

Fig. 10. Number or errors in Benton test after the administration of placebo, benactyzine and LSD in individual patient.

EXPERIMENTAL PSYCHOSES I N D U C E D B Y BENACTYZINE I N ALCOHOLICS

d 010

97

LATENCIES

40 20

D:

10

o---o-o~o-o-o

0

PL

IMPAIRED REPRODUCTIONS

n

70 60

20 10 -

0 ‘

PI

B

LSD

Fig. 11. Changes in word association experiment. Upper part : Changes in latency periods during six hours of observation after the administration of placebo, benactyzine and LSD. Base line: initial values before the drugs’ administration. Middk part : Reproductions in the association experiment during six hours of observation. Lower part: Total number of impaired neproductions in word association experiment during six hours of observation. 180 word stimuli after the administration of placebo, benactyzine and LSD in six series were given. Each spot represents values of one patient.

LEARNING

/

0 ’ @/

1

. I * __ 2

3

4

5

6

7

I

8

9

10

Reinforcement

Fig. 12. Mean values in paired association test after the administration of placebo, benactyzine and LSD. Vertical axis : Number of presented paired associations. Horizontal axis : Number of reinforcements. ReJerences p. 103-10s

98

M. V O J T ~ C H O V S K $et

al.

doses with marked confusion at higher doses. Time disorientation was the symptom most frequently observed after benactyzine as well as after LSD. In our experiments with alcoholics the time estimation ( I min) (Fig. 13) was much more disturbed by benactyzine than by LSD, especially in the first hour. The observed shortening of estimation after benactyzine is similar to the findings in severe brain diseaseg. d sec

O

,

1

,

2

3 I

4 *

5

6

h I

PL

L SD

B

30

Fig. 13. Time estimation (one minute interval) after the administration of placebo, benactyzine and LSD during six hours of observation. Vertical axis : underestimation in seconds. Horizontal a.\-is : time in hours. Base line : sixty seconds.

Behavioral changes depended on the phase of intoxication: there was an initial psychomotor inhibition and hypodynamia and abnormal behavior in the phase of hallucinations in the psychotic stage. In the volunteers we often observed floccilegium and diffuse undifferentiated psychomotor agitation. I n alcoholics no such behavior was observed. The final stage was again of psychomotor inhibition. Altered behavior usually prevented contact with the subjects in the culmination period of psychoses, so that the majority of the tests in this phase could not be applied. Intoxication with benactyzine usually did not stimulate the creative aspect of personality so that artistic experience did not result, as with LSD. Benactyzine intoxication appeared to resemble the organic Korsakoff psychosis and delirium tremeiis as opposed to LSD and psilocybin which produce a more functional type of psychosis. We compared the nuclear symptoms of delirium tremens as they are described in Krystal’s paperlg, based on experience with 700 patients, with our findings after the application of LSD and benactyzine in a group of healthy volunteers and alcoholics. Table V1 shows that the basic symptoms of delirium tremens are better imitated by benactyzine intoxication, especially in healthy volunteers. Also, with this drug the alcoholics had more similarities with natural delirium tremens than after LSD adniinistration. The only exception was anxiety and panic, which was more often evoked by LSD.

99

E X P E R I M E N T A L P S Y C H O S E S I N D U C E D BY B E N A C T Y Z I N E I N A L C O H O L I C S

T A B L E VI ~~

Delirium tremens N = 700 ( %I Krystal, I959

Symptoms

Benactyzine intoxication 40 mg ( %) Healthy Alcoholics volunt . N=17 N=I6

LSD-25 f %) 100 Pg

200 PE

Healthy volunt. N=17

Alcoholics N=16 ~

1 Confusion

+ + - tt

SP-

++$

88.5

68.5

2 Disorientation

100 i+ - I + 100

++ 60 +++

3 Anxiety-panic 4 Tremor

82.6

50.0

23.0

18.7

c I

29.0

58

5 Visual hallucinations 6 Tachycardia over lOO/min

I-

It

11.8

++++ 94.4

62.3

i i f 61.0

-1 + t 82.6

+

7 Emesis

+++

S i t

76.7

+ + 18.7 +++ 56.0 + 25.0 +

50.0

30.8 0 0

18.7

11.8

12.5

*

*

11.8

~-

18.7

* 11.8

++

i

40

23.6 -I71.8

37.5

++ 40

+ ++

++ +

t C i

I

*5.9

~

It

In order to compare the results of our objective methods used for the analysis of the effects of LSD and benactyzine in chronic alcoholics more readily, we examined five psychiatric patients with a diagnosis of Korsakoff’s syndrome using the same methods, together with a group matched for age, of schizophrenics in the subacute state. These patients were best differentiated by Benton’s test (Fig. 14). The differences between schizophrenic and LSD, as between benactyzine and Korsakoff’s syndrome were not significant, but differences between alcoholics after placebo and both natural types of psychosis were. Analogous changes were also found when analyzing the association experiment with regard to the incidence of impaired reproduction and learning of five paired associations, particularly during the first and second reinforcement (Fig. 15). ERRORS

20L 15 10 -

5-

-

11, 3P 00 l

0-

0 0 -0-

0 0 0

0.

0

LSD

Sc h

0

KP

Fig. 14. Number in errors in Benton test in alcoholics after the administration of placebo, LSD and benactyzine in comparison to five age-paired schizophrenics (Sch) and five patients with Korsakow psychosis (KP). References p. 103-105

100

M.

VOJTECHOVSKQ

et al.

n

Pl

0

KP

0

-

1 2 3 4 5 6 7 8 9 1 0 Reinforcement

Fig. 15. Upper part : learning of five paired associations (vertical axis) in alcoholics after the administration of placebo, and benactyzine in comparison to patients with diagnosis of Korsakow psychosis (KP) without B. Lower purr : results of the same experiments in alcoholics after the administration of LSD and five schizophrenics without LSD.

In time estimation, a similar significant trend was found i n alcoholics intoxicated with benactyzine (mean -34.5 sec) and in Korsnkoff group (mean -25 sec). On the other hand estimations of schizophrenic patients had an opposite trend, i.e. they overestimated time (mean +9 sec). This effect was not observed after placebo (mean -1 0 sec) or LSD (mean -26.5 sec). The most striking effects of benactyzine which remind one of Korsakoff’s syndrome are therefore the visual motor and verbal association defects of memory (see Table VII). D 1 S C U S S ION

We have not been able to prove our original hypothesis that chronic alcoholics are more susceptible to anticholinergic hallucinogens than normal persons. Similar observations in psychiatric patients after higher doses of atropine were published by Forrerlz. Nevertheless, the clinical picture of benactyzine intoxication in alcoholics has more similarities with delirium tremens, alcoholic hallucinatory states or Korsakoff psychosis than is the case with LSD intoxication. The finding of Ditman and Whittlesey7 as well as the earlier studies of Arnold and H O P are not supported by the results of our experiments. The lower frequency of visual disturbances in our group of alcoholics as well as the absence of more marked changes in behavior after benactyzine, in comparison with normals, led us to the following considerations (Fig. 16): The occurrence of delirium tremens in Czechoslovakia in comparison with neighboring Austria, where almost

EXPERIMENTAL PSYCHOSES I N D U C E D BY BENACTYZINE I N A L C O H O L I C S

101

TABLE VII TIME ESTIMATION

_____-_ !n

A

median maximum minimum

SEC)

~

K.P. N = 5

see

(60

Schizo N=5

+ 8.5 + 15.0

-25.0 1.0 -45.0

*

- 5.0

Statistical analysis (Wilcoxon): Placebo-Sch. P - 0.01 ;Placebo-C P

= 0.05;

_ _ _ Alcoholics ( N

_ 13)

-

=

LSD

B

Placebo

-26.5 + 25.0 -45.0

-34.5 + 12.0 -50.0

-10.0 c 11.0 -35.0

Sch.-K.P. P

-

0.05; Sch.-LSD P

= 0.01;

K . P . 4 n.s.

IMPAIRED REPRODUCTIONS I N W O R D ASSOCIATION TEST

in

Jec

K . P. N - 5

Schizo N - 5

LSD

9 25 8

3 10 0

4 10 0

Alcoholics ( N = 13) B. Placebo

.

median maximum minimum

Statistical analysis (Wilcoxon): Placebo-Sch. P - 0.05; Placebo-K.P. P

=

0.01; Sch.-K.P. P

-

1

Stage of exhaustion

5

1

Dehydratton

3

\'\

6

0.05; Sch.-LSD ns.; K.P.-B n.s*

Loss of Water - 1

I

Lowsait Syndrome (Na def delirum)

/

resistance to infect ion

NO

.

(.poor attachment. to reality)

L i

(3)

( b ) Antichollnerglc

, /

(Hoff etiological lmk (Bon- -?-% hoeffer)

haliuclnogens

Fig. 16 References p . 103-105

9. Psychological causes of delirium

..

Resins in spirits

3

10 17

o r endogenous hallucinogens

EXO-

102

M.

V O J T B C H O V SetK Pal.

40 % of newly admitted alcoholics are in predelirious or delirious conditionsll is considerably lower, being only 1 ”/, of newly admitted alcoholics in Prague1’. The majority of our patients are beer-drinkers in whom delirium tremens occurs only exceptionallylfi. In contrast, in Austria abuse of spirits is much more common. This suggests the question of whether beer could contain some protective substances preventing the development of delirium tremens during chronic abuse of alcohol. Analzying the causes of similarities between psychoses and benactyzine intoxication we took into consideration a remark in Hoff’s handbook of psychiatryl6 about the possible importance of resins in liquors for the development of delirium tremens. We investigated the chemical structure of admixtures occurring in concentrated spirits. The most striking similarities between ingredients of alcoholic beverages and anticholinergic hallucinogens is in the basic chemical structure of the esterified fatty acids and aromatic acids. The basic radicals in the side chain of anticholinergic hallucinogens can be found in admixtures of spirits (di-triethylamine) or in resins (piperidine). Also tropic acid (the base of atropine alcaloids) is present in spirits and admixtures. The most plausible explanation would be the abnormal hydroxylation of the acetate, the main metabolite of alcohol, to hydroxyacetate (glycolate), which is the base of many anticholinergic hallucinogens. There js also the possibility of endogenous origin of the hallucinogens from vanilmandelic acid, which is the final metabolic product of the catecholamine hormones. With regard to the finding of enormous excretion of catecholamines in predelirious and delirious states in alcoholics13 we can anticipate more enhanced production of this acid in a situation where the catecholamine metabolites could be the basic substance for the esterification by alkylamines from the admixtures of spirits or endogenous basic substances such as acetylcholinelike substances. The whole problem is only hypothetically formulated, however. Even if the presence of anticholinergic hallucinogens in some spirits or liquors or those of an endogenous origin, ideally fulfilled the existence of a hypothetical etiological link between the somatic noxious agent and the psychotic complication in delirium tremens, as suggested more than 50 years ago by Bonhoeffer3, this would be an oversimplification. A great number of observations exist suggesting the “etiological link” of a multidimensional character. According to Krystallg there is, in the foreground of pathophysiological findings the disturbance of mineral metabolism and alterations in liver and adrenocortical funtions. Also, the failure of adaptational mechanisms according to Selye’s concept (infection and trauma) can be taken into consideration. According to Gursey and Olson15 and our own previous experiments32333 alcohol produces changes even in the metabolism of serotonin and catecholamines in the brain which are similar to those of reserpine. It is probable that the prerequisite for the development of delirium tremens is the presence of a large number of the conditions mentioned above. A chemical compound, i.e. a precursor of anticholinergic hallucinogens, an ingredient of some liquors, or an endogenous metabolite, can in a potentially delirious patient, act in a substantially

EXPERIMENTAL PSYCHOSES I N D U C E D BY BENACTYZINE I N ALCOHOLICS

103

altered substrate so that even i n quite small quantities it causes a psychotic condition. On the other hand, the same dose ingested by a social drinker or beer drinker would evoke only changes which were hardly distinguishable from inebriety and subsequent hangover. Further experiments are required for the verification or rejection of this hypothesis, especially in drinkers of spirits and liquors immediately after a long abuse of alcohol or after a load of neurohormone precursors.

SUMMARY

The effect of 40 mg of benactyzine, 200 pg of LSD and placebo on some psychic and physiological functions i n 16 chronic alcoholics was investigated. Clinical changes evoked by benactyzine had more similarities with Korsakoff psychosis and delirium tremens than those induced by LSD. This effect was especially evident in five alcoholics with a psychotic episode in anamnesis (alcohol hallucinosis). Clinical observations were supplemented by a series of objective physiological and psychological methods. The effect of benactyzine in comparison with that of LSD was characterized by tachycardia in the first hour, marked tremor until the fourth hour of observation and a greater alteration of time perception in the first hour. Mental activity was more disturbed by benactyzine : reduced computation ability with greater number of errors, retarded copying in handwriting, frequent intraverbal ataxia. Benactyzine disturbed visual motor memory in a very marked way (Benton’s test), verbal retention in the test of free associations and retarded the early learning of paired associations. In the emotional area, measured by Clyde Mood Scale, the most typical differences were found in the parameter of clear thinking. In a number of other parameters the changes after benactyzine were similar to the effects of LSD (blood pressure, mydriasis, latencies in association experiment). On the other hand, the experimental results did not support our original hypothesis about the heightened susceptibility of alcoholics to anticholinergic hallucinogeiis. The delirious conditions evoked by benactyzine in alcoholics were of a milder degree than those observed in another study in 17 healthy volunteers. Also, the psychopathological picture after LSD, especially in the visual area was less marked in alcoholics than in a control group of normals. In alcoholics, however, LSD evoked paranoid reactions more often. The concluding remarks concern the possibility of the eventual exogenous origin of the hypothetical anticholinergic hallucinogen which could participate in the complicated pathogenesis of the naturally occurring alcoholic psychoses.

REFERENCES

1 ABRAMSON, H. A,, JARVIK, M. E., KAUFMAN, M. R., KORNETSKY, C . , LEVINE, A. AND WAGNER, M, (1955) Lysergic acid diethylamide (LSD-25) : I. Physiological and perceptual responses. J. Psycho/.. 39, 3-60. 2 ARNOLD.H. AND HOFF,H. (1953) Untersuchungen uber die Wirkungsweise von Lysergsaurediethylamide (Erste Mitteilung). Wien. Ztschr. Nerveheilk., 6 , 129-150. 3 BONHOEFFER, K. (1909) Zur Frage der exogenen Psychosen. Zbl. Nervetiheilk.

104

M.

V O J T B C H O V S K Q et al.

4 CLYDE, D. J. (1963) Manualfor the Clyde Mood Scale. Bioinetric Laboratory, University of Miami, Coral Gables, Florida. 5 COAUY, A. AND JEWESBURY, E. C. 0. (,1956) A clinical trial of benactyzine hydrochloride as a physical relaxant. Brit. wed. J . , i, 485-486. 6 DAVIES BERESFORD, E. (1956) A new drug to relieve anxiety. Brit. wed. J., i, 480-481. 7 DITMAN, K. AND WHITTLESEY, J. R. 8. (1959) Comparison of the LSD-25 experience and delirium tremens. A.M.A. Arch. gen. Psychiat., 1, 45-47. 8 DITMAN,K. Sort list of LSD. Personal comniunication. 9 DOUST LOVETT, W. J. (1955) Studies of the physiology of awareness. Dis. new. Systm, 16, 363-365. 10 DUKER,H. (1949) Ueber ein Verfahren zur Untersuchung der psychischen Leistungsfahigkeit. Psychol. Forsch., 29, 10-24. I 1 EXNER,K. Personal communication. 12 FORRER, G . R. (1951) Atropine toxicity in the treatment of mental disease. Am. J . Psychiat., 108, 107-1 12. 13 GIACOBINI, E., TZIKOWITZ, S. AND WEGMANN, A. (1960) Urinary norepinephrine and epinephrine excretion in delirium tremens. A.M.A. Arch. gen. Psychiat., 3, 289-296. M. (1956) Experimentdlni psychosa po poiiti 200 mg benactyzinu. 14 GROF,S. AND VOJTLCHOVSKI, (Expeiimental psychosis following a dose of 200 mgms of benactyzine). Cdpsychiut., 51, 369-376. 15 GURSEY, D. AND OLSON,R. F. (1956) Depression of serotonin and noiepinephrine levels in brain stem of rabbit by ethanol. Proc. Soc. exp. Biol., 104, 280-281. I6 HOFF,H. (1956) Lehrbuch der fsychiatrie. Beno Schwabe Vcrlag, Basel/Stuttgart. E., DRDKOVA, S., AND VANA,J. (1965) RozSiFeni psychos zachycenych v psychiatricke 17 TVANYS, peEi velkombtsk6ho obyvatelstva. (Distribution of psychoses registered in psychiatric care in a part of inhabitants of the capital) &I. psychiat., 61, 47-57. 18 JACOBSEN, E. (1955) A new drug effective on the central nervous system. Dan. med. Bull., 2, 159-160. 19 KRYSTAL., H. (1959) The physiological basis of the treatment of delirium tremens. Am. J. Psychiat., 116, 137-147. 20 LEGGE,D., STEINBERG, H., AND SUMMERFIELD, A. (1964) Simple measures of handwriting as indices of drug effects. Perceptual and Motor Skills, 19,549-558. 21 SEASHORE, R. H. (1951) Work and motor performance. In: Handbook of Exper. Psychol. Ed. S . S. Stevens, John Wiley and Sons, Inc. New York. 22 SMIRNOV, D. A. (1953) Das Verbalexperiment in der medizinischen Praxis. Pawlow-Ztschr. hiih. Nerventiitigk., 3,408-41 5. 23 STROOP, J. R . (,1935)Studies of interference in serial verbal reactions. J. exp. Psychol., 18,643-661. 24 VINAROVA,M., VJNAK,0. AND VOJTZCHOVSK+, M. (1958) Klinicke zkugenosti s benactyzinem, novfm l6kem proti uzkosti. (Clinical experiences with benactyzine, a new drug against anxiety.) t a s . Kk. Ces., 97, 1059. 25 V~TEK, VL. AND RYSANEK,K. (1960) Inhibition of Monoamine Oxidase by benactyzine in vitro and in vivo. Nature, 186,204. 26 VOJTECHOVSK~, M. ( I 958) A psychosis caused by benartyzine intoxication. Acta psych iat. scand., 33, 514518. V., RYSANEK, K. AND BULTASOVA, H. (1958) Psychotogenic and hallu27 V O J T ~ C H O V M S K. , ~V~TEK, , cinogenic properties of large doses of benactyzine. Experientiu, 14,222. 28 VOJTECHOVSKY, M., GROF,S., V~TEK,V., RYSANEK,K. AND BULTASOVA, H. (1960) Experimentalpsychose als Folge der Verabreichung von 200 mg Benactyzine. Wien. Ztschr. Nervenheilk., 17, 279-308. 29 VOJT~CHOVSK+, M., RYSANEK, K. AND V~TEK, V. (1960) Experimental psychoses due to high doses of Benactyzine. Psychiat. Neurol., 139,406-41 5. 30 VOJTECHOVSK+, M. AND GROF,S. (1961) Anticholinergni halucinogeny jako model deliria tremens. (Experimental psychosis produced by anticholinergic hallucinogens as a model of delirium tremens). Activ. Nerv. super. (Prague), 3, 219. 3 I VOJT~CHOVSK+, M. (1962) Anticholitiergic drugs with central action. Clinical and experinieratul studies. Doctoral thesis. Charles University, Prague. 32 VOJTECHOVSK-?, M., GROF,S., VITEK,V. AND RYSANEK,K. (1964) Klinische und biochemische Studie der zentralen Cholinolytica, insbesondere des Benactyzins. I. Mitteilung: Psychopathologische Charakteristik nach Verabreichung hoheren Dosen von Benactyzin. Acta psych iat. scand., 40,219-233.

EXPERIMENTALPSYCHOSES I N D U C E D B Y B E N A C T Y Z I N EI N ALCOHOLICS

105

33 VOJTECHOVSK+, M., RYSANEK, K . AND V~TEK, V. (1964) Humoralni faktory v etiologii depresi PO IeEbE alkaloidy rauwolfie a u alkoholiku. Humoral factors in the aetiology of depressions following rauwolfia alkaloid treatment and in alcoholics. &. Psychiat., 60, 81-88. 34 VOJTECHOVSK~, M . (1 965) Co pEinesl vgzkum halucinogenii pro teorii etiopatogenese endogennich psychos. Research on hallucinogenic drugs and the theory of pathogenesis of endogenous psychoses. b s . Psychiat., 61, 257-261. 35 VOTAVA, Z., SRAMKOVA, J. AND HORAKOVA, Z . (1958) Farmakologick6 vlastnosti diethylaminoethylesteru kyseliny benzylove, l5tky k leEb6 anxioznich stavii. (Pharmacological properties of benactyzine, a new drug for the treatment of anxiety states) z'as. ldk. Ees., 97, 22-27. 36 WILCOXON (1949) Some rapid approximate statistical procedures, American Cyanarnid Coinpiny, New York.

I06

Anticholinergic Hallucinogenics : Laboratory Results versus Clinical Trials V . G. L O N G 0

AND

A. SCOTTI D E C A R O L I S

Isfilitto Sirperiore cli Sariild, Rortra (Itnly)

This paper is limited to only one of the many problems involved in thestudy oftheanticholinergic hallucinogenics; more precisely to the relationships that one can postulate between the central effects observed using electroenceplialographic techniques in experimental animals, and the results obtained in the clinic. CLINICAL DATA

Table I sets forth most of the compounds possessing parasympatholytic activity, which have the capacity to provoke transient psychopathological states in man. This information has been derived from observations based on cases of intoxication, from therapy-oriented investigations (usually clinical trials to assess the antispasmodic activity), and from more specific human psychopharmacological studies. Concerning atropine, its psychotomimetic effects have been known and extensively discussed for quite a long time. The table lists only three references, one refers to a monograph and the others to the works of Forrerll and Millerl9 because of the inclusion in their papers ofa comprehensive description of the central effects observed in the course of the “atropine toxicity therapy”. It can be seen from the chronology and content of papers dealing with this topic that up till a few years ago these articles dealt mostly with the side effects occurring in clinical trials of drugs which in the laboratory demonstrated atropine-like activities (refs. I , 4, 7, 12, 16,25). Investigations of a more strictly psychopharmacological character are those of Pennes and Hoch23, Pfeiffer24, Fliigel and Benteg, Fliigel and Itill”, Abood and Bie13, Bente et a1.5, and Voitechovsky et a/.ZA. Comparing the results obtained with these various compounds, several symptoms appear to be common to the group, constituting what has been termed1* the central anticholinergic syndrome. Characteristic of this syndrome are profound behavioural and psychic disturbances, such as impairment of memory, slurred speech, drowsiness, impaired motor ability, a state of confusion and disorientation, feelings of unpleasantness and hallucinations, both auditory and visual. A better definition of the syndrome resulted from investigations aimed at a comparison with another type of “exogenous psychosis”, that caused by the LSD and related drugs. In the work of Pennes and Hoch“, one finds for the first time an attempt at

ANTICHOLINERGIC HALLUCINOGENICS

I07

TABLE I PSYCHOTOMIMETIC EFFECTS OF ANTICHOLINERGIC D R U G S IN H U M A N S

Compourini Atropine Scopolamine Benactyzine Trihexyp henydil Caramiphen Adiphenine Ditran" Ba 1433 (WH4849)b A H R 376c

RO 2-320212" Win 2299e 7360 RPf Hexamidg

dose and route

32-200 mg, S.C. 0.8 mg, S.C. 2.0 mg, oral 15-25 mg, oral 5-12 mg, S.C. I0 mg, oral 25 mg, oral 100 mg, S.C. 10-20 mg, oral and 2-5 mg, i.v. 5-15 mg, oral 20 mg, i.m. 2-4 mg, oral 4 mg, S.C. 2-10 mg, oral 2-5 mg, i.v. 25-75 mg, oral 50-200 mg, i.m. 20-100 mg, i.v.

S.C.

Reference3

7, 11, 19 21 22,24 26 12 24 7, 24 4 1, 2 10,20 9 10 5 25 23 8 16 10

a

I-ethyl-2-pyrrolidyl-methylphenyl~yclopenthyl-glycolate (70%) and I-ethyl-3-piperidil-phenyl c yclopenthyl-gl ycolate (30 %) ; alpha-phenyl-alpha-isopropyl-glycolic acid-3-N, N dimethylaminopropyl ester I-methyl-3-pyrrolidyl-alpha-phenylcyclopenthane glycolate

e

2-diethylaminoethyl cyclopenthyl (2 thienyl) glycolate

1-methyl-3-benziloyl-oxy-quinuclidol f

g

diethylaminoethyl-phenothiazinyl-lO-dithiocarboxylate 5.5 phenylethyl-3-(P diethylaminoethyl) 2,4,6- trioxy-hexahydropyrimidine

clinical differentiation of the various drugs with psychotomimetic activity, a field which has been extended subsequently by other authors. Even though the results of some workers24 tended to emphasize the similarities, the investigations demonstrated to a large extent differences that have been further documented by later studies. The confusional delirium picture following the anticholinergic drugs is always accompanied by a diminution of alertness and isolation from the environment, while LSD often elicits an extravertive attitude. In contrast to the euphoria commonly observed in the LSD-treated subjects, anticholinergics provoke an apprehensive and anxious mood. The motor hyperreflexia provoked by LSD is seldom observed with the anticholinergics, which provoke the appearance of the Babinski sign only at high doses. An extensive treatment of these and other differences can be found in the review of Cerletti et d . 6 , in the paper of Tsbell et al.14, who reported on studies of cross-tolerance between scopolamine, N-ethyl-3 piperidyl benzilate (JB 318) and LSD, and in the investigation of ltil and Fink15 who studied in particular the relationships between clinical and EEG findings, after treatment with Ditran and LSD. In a paper of Vojtechovsky et aI.27 (see also the present symposium page 27), the similarity was stressed References p . 111-112

108

V. G. L O N G O A N D A. SCOTTI D E C A R O L I S

b:lween the c!inical pictures observed i n subjects treated with benactyzine ( 1 5-75 mg, oral) and deliriim tremens and Korsakoff’s syndrome. LABORATORY RESULTS

On the experimental side, there is a vast literature concerning the effects of anticholinergics on the cerebral electrical activity and a n attempt will be made here t o draw some conclusions from the results dealing with the effect of these drugs on the EEG of laboratory animals. There seem to be no qualitative differences between the EEG alterations provoked by the various central anticholinergics. Within a short time after administration, in all animal species, the modifications of the tracing follow a common pattern. Bursts of 8-12 c/sec waves, similar in many aspects to “spindles” intermingle with high voltage slow (2-5 c/sec) waves appearing in the anterior leads. Slow activity is seen also in the posterior and subcortical leads. These changes are very similar to those occurring during rest or sleep and are broadly classified as EEG synchronization (Fig. I).

Ba 1433 0.1 m g / k g

Fig. 1. Effects of Ba 1433 on the electroencephalogram of the rabbit. Control. Upper tracing: EEG activation evoked by an cxternal stimulus (hand clap, between the arrows); lower tracing: EEG activation evoked by an electrical stimulation of the reticular substance (at the bar), Ba 1433 0.1 mg/kg: ten minutes after treatment no activation is observed after external stimuli or electrical stimulation. Non-curarized unanesthetized rabbit. Leads: CF: left anterior sensorimotor cortex, CO: left optic cortex, H P: right dorsal hippocampus, RF: left mesencephalic reticular formation. Calibration : 50 pV.

109

ANTICHOLINERGIC HALLUCINOGENICS

%;nib

A

100-

A

90-

8070 60-

5040

-

30-

20100

L_, 1

2

3

56789l,O

4

2

3

5 6789l,O

4

Log. dose

2

3

56789iP

4

1

10

m9h9

70

6050-

10-

30 0 20

-

10-

4

1

2

I

3

I

L

I

r

I 1 1 1

5 6 18910 f

I

2

I

3

I

L

Log. dose

I 1 1 1 1 1

5 6 1 8910 I 01

I

2

1 3

I

I

I

I

I I I T

5 678910 1

b mg/kg

Fig. 2. Relationships between log. dose and the percentage of inhibition of the duration of the activation caused by electrical stimulation of the reticular substance. In the upper graph are presented the data on atropine and scopolamine (Longo 17); in the lower graph are presented the results obtained with Ditran and Ba 1433. Rcjercnra p. 111-1 12

110

V . C . L O N G 0 A N D A. SCOTT1 DE C A R O L I S

I n addition to the changes of the spontaneous EEG, other modifications have been described. After administration of doses slightly higher that those required for synchronization, physiological sensory stimuli no longer have any “activating” or “desynchronizing” effect on the cortical electrical rhythms. At the hippocampal level, the 4-6 c/sec activity characteristic of the “activated” tracing is superceded by irregular slow waves with superimposed 20-30 cjsec low voltage activity. The threshold of activation upon electrical stimulation of the reticular substance or of the non-specific tlialamic nuclei is significantly raised. Another characteristic of the anticholinergic drugs, which should not be disregarded in considering their mode of action, is that at a certain dose level no additional important modifications ofthe tracings areencountered. Since all the anticholinergics cause similar alterations, the only possible approach in the study of the EEG effects of these drugs is the evaluation of their synchronizing potency. To overcome the difficulties i n evaluating the results in the literature, obtained under different experimental conditions and in different animals, a study has been performed in our laboratory using a method which proved to be very satisfactory in assessing the synchronizing potency o f a drug: the blocking of the EEG arousal provoked by electrical stimuli applied to the reticular formation17. In Fig. 2, data are presented dealing with the results obtained with Ditran and Ba 1433 compared with the data obtained in previous experiments dealing with atropine and scopolamine, which indicates that Ditran is the most effective compound in blocking the EEG activation. The high effectiveness of Ditran in provoking EEG synchronization was also demonstrated by White and Carlton2g8. Therefore, on the basis of the results obtained in EEG studies, it can be concluded that a characteristic of these drugs is their specific “synchronizing” effect, assessed i n particular by the method of electrical stimulation o f the reticular substance. We would

_h_

~

~ - - ~ - - ~ - ~ _-_^____*-

+ ~

*

~

~

~

,

,

~

~

&

.

~

~

v

~

~

-

~

- -r-.y-.-_x-I_

___-”-_~._C..---

+

&

~ ?

A

‘

.

~

+

~

A

~ ,

-

-

~

.

’

,

A

.

.

~

-

:

’

“

~

,

L

a

~ ’

~

~

“

~

,

~

~

-

~

,

I

Fig. 3. Effect of lysergic acid diethylamide (LSD) on the EEG activation produced by electrical stimulation of the reticular substance. A . Activation pattern obtained with reticular stimulation (0.4 V, 250 cjsec, 0.1 msec, at the bar). B. After 150 &kg of LSD the reticular stimulation no longer modifies the tracing; the hippocampal ‘theta’ waves, characteristic of the activation, are present only during the stimulation. Unanesthetized, non curarized rabbit. Leads - F : L anterior sensorimotor cortex; P : L posterior sensorimotor cortex; 0 : L optic cxtex; H : L dorsal hippocampus. Calibration: 2 sec, 100 pV. (from: Longo, Electroencephalographic Atlas for Pharmacological Resenrch, Elsevier, Amsterdam, 1962).

* ~

~

~

~

~ ,

h

~

ANTICHOLINERGIC HALLUCINOGENICS

111

like, therefore, to propose this method as a test for ass-ssing the “central anticholinergic action”, thus avoiding the dangerous extrapolations and parallelisms between the central and peripheral cholinergic-blocking properties. One might now consider how these modifications compare with those provoked by the other group of psychotomimetics (LSD and parent compounds). The EEG modifications provoked by LSD (Fig. 3) are strikingly different and consist of a “flattening” of the cortical tracings (I must insist on this definition, since I consider it to be different froin the “desynchronization” described by some other authors) and a disruption of the theta waves of the hippocampus. Hoffmeister et ~ 1 . 1 3have also pointed out these differences which are seen not only in the EEG but also in other animal behavioural tests. The present status of our knowledge does not allow identification of the various systems responsible for these modifications, but to close this presentation, I would like to give some considerations to the question so often raised as to whether the syndrome observed after administration of the anticholinergic psychotomimetics could be attributed to the central cholinergic block or whether it depends upon other properties of these drugs. May I point out the relative homogeneity of the pharmacological effects of all the anticholinergic hallucinogens : ( I ) the peripheral anticholinergic action ; (2) the similarity of the behavioural effects and (3) of the EEG modifications; (4)the antagonism of eserine towards both the EEG and behavioural effects of these drugs (for references see Longols). 1 agree that, within the range of these effects, the different symptoms may vary in intensity, forming particular patterns for each individual drug, but they can still be put within the same frame. I n conclusion, there are several hints leading to the acceptance of the theory that profound disturbances of behaviour result from the administration of drugs able to alter i n some way the normal chain of activity regulated by acetylcholine in its role of central mediator.

REFERENCES 1 ABOOD,L. G., OSTFELD, A. M. A N D BIEL,J. H. (1958) A new group of psychotomimetic agents. Proc. Soc. Exp. Biol., 97, 483486.

2 ABOOD,L. G., OSTFELD, A. M. AND BIEL,J. H. (1959) Structure-activity relationships of 3-piperidyl benzilates with psychotogenic properties. Arch. inf. Pharmacodynarn., 120, 186-200. 3 ABOOD,L. G. AND BIEL,J. H. (1962) Anticholinergic psychotomimeticagents. fat. Rev. Neurobiol., 4, 217-273. 4 ANICHKOV, S. V. (1959) Pharmacology of the central cholinergic synapszs. Symposia and Special Leciures, 21st. Ozf. Cong. Physiol. Sci.,Buenos Aires, pp. 23-27. H., HARTUNC,M. L. AND PENNING,J. (1964) Zur Pathophysiologie und 5 BENTE,D., HARTUNG, Psychopathologie des durch zentrale Anticholinergica erzeugten amentiell-deliranten Syndroms; Klinische, elektroencephalographishceund testpsychologische Ergebnisse mit I-Methyl-3-pyrrodyl u-phenylcyclopentanglycolat-HCI. Arnreim.-Forsch., Suppl., 14, 51 3-518. M. (1963) Psychodysleptica. Schweiz. 6 CERLETTI,A., SCHLACER, E., SPITZER,F. AND TAESCHLER, Apoih. Ztg., 101, 210-240. 7 DE BOOR,W. (1 956) Pharrnakopsychologie und Psychopathologie. Springer, Berlin. 8 FIKK,M. (1960) Effect of anticholinergic compounds on post convulsive electroencephalogram and behavior of psychiatric patients. EEC Clin. Neirrophysiol., 12, 359-369. 9 FLUGEL,F. AND BENTE,D. (1961) Clinicil and electroencephalographic experiences with a new centrally active anticholinergic drug (Bayer WH 4849) (In German). Med. exp., 5, 215-223.

112

V. G. L O N G O A N D A . S C O T T 1 D E C A R O L I S

10 FLUGEL,F. A N D ITIL, T. (I 962) Klinische-elektroencephalographische Untersuchungen mit “Verwirrheit”-hervorrufenden Substanzen. Psychopharniacologia, 3, 79-98. 11 FORRER, G. R. (1951) Atropine toxicity in the treatment of mental disease. Amer. J . Psychiat., 108, 107-1 12. 12 HESS,G. AND JACOBSEN, E. (1957) The effect of benactyzine on the electroencephalogram in man. Acta. pharm. Tox., 13, 125-134. 13 HOFFMEISTER, F., KREISKOTP, H. A N D WIRTH,W. (1964) Untersuchungen mit zentrale wirksamen Anticholinergica. Arzneim.-Forsch., 14, Suppl. pp. 482-486. 14 Issau, H., ROSENBERC, D. E., MINER,F. J. AND LOGAN, R. L. (1964) Tolerance and cross-tolerance to scopolamine, N-ethyl-3-piperidyl benzylate (JB-318) and LSD 25. In: Neuropsychopharrnucology, Vol. 3 . Eds. P. B. Bradley, F. Fliigel and P. H. Hoch, Elsevier, Amsterdam, pp. 440-446. 15 ITIL, T. AND M. FINK(1966) Klinische Untersuchungen und quantitative EEG-Daten bei experimentellen Psychosen. Arzneim.-Forsch., 16, 237-239 16 LAMBERT, P. A., DIEDERICKS, A. A N D CHARIOT, G . (1961) A propos de I’action psychodysleptique d’un derive phenothiazinique, le phhothiazinyl-10-diothiocarboxylate de diethylaminoethyle ou 7.360 R. P. In: Neuropsychopharmacology, Vol. 2, Ed. E. Rothlin, Elsevier, Amsterdam, pp. 374-380. 17 LONCO,V. G. (1956) Effects of scopolamine and atropine on electroencephalographic and behavioral reactions due to hypothalamic stimulation. J. Pharmacol., 116, 198-208. 18 LONGO,V. G . (1966) Behavioural and electroencephalographic effects of atropine and related compounds. Pharmacol. Rev., 18. 965-996. 19 MILLER, J. J. (1956) Syniposium on atropine toxicity therapy; pharmacology procedure and techniques in atropine toxicity treatment of mental illness. J . Merv. Ment. Dis., 124, 260-264. 20 OSTFBLD, A. M., Aeoou, L. G . AND MARCUS, D. A. (1958) Studies with ceruloplasmin and a new hallucinogen. Arch. Neurol. Psychiat., 79, 317-322. A. M., JENKINS, R. AND PASNAU, R. (1959) Dose-response data for autonomic and mental 21 OSTFELD, effects of atropine and hyoscine. Fed. Proc., 18, 430. 22 OSTFELD, A. M. AND ARUGUETE, A.(1962) Central nervous system effects of hyoscinein man. J . Pharmacol., 137, 133-139. 23 P ~ N N EH. S ,H. A N D HOCH,P. H. (1956) Psychotomimetics, clinical and theoretical considerations; Harmine Win-2299 and nalline. Amer. J. Psychiat., 113, 887-892. 24 PFEIFFER, C. C. (1959) Parasympathetic neurohumors; possible precursors and effect on behavior. hit. Rev. Neurobiol., 1, 195-244. 25 SCHALLEK, W. A N D SMITH,T. H. F. (1952) Electroencephalographic analysis of side effects of spasmolytic drugs. J . Pharmacol., 104, 291. 26 VOJTECHOVSKY, M., GROF,S., VfTEK, V. AND RY~ANEK, K. (1964) Klinische und biochemische Studie der zentralen Cholinolytica, inbesondere des Benactyzins. 1. Mitteilung Psychopathologische Charakteristik nach Verabreichung hoherer Dosen von Benactyzin. Acta psych iat. Scund., 40, 219-233. 27 VOJTECHOVSKY, M., V. VITEK ANU K. RYSANEK (1966) Experimentelle Psychose nach Verabreichung von Benactyzin. Arzneim.-Forsch., 16, 240-242. 28 WHIIE,R. P. AND CARLTON, R. A. (1963) Evidence indicating central atropine-like actions of psychotogenic piperidyl benzilates. Psychopharmacologia, 4, 459-471.

113

Role of Cholinergic Mechanisms in States of Wakefulness and Sleep* E. F. D O M I N O , K. Y A M A M O T O

AND

A. T. D R E N

Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (U.S.A.)

INTRODUCTION

There is a great deal of data to support the hypothesis that cholinergic mechanisms are involved in states of wakefulness and sleep. There are many clinical observations with cholinergic agonists and antagonists which indicate that cholinergic functions in the brain are indeed very important, especially for higher cerebral activity. Rinaldi and Himwichlg,20 were among the first to offer conclusive evidence of the cholinergic nature of some elements of the mesodiencephalic activating system. Denisenk04~5, Ilyutchenok and C O W O ~ ~ ~ ~Mickelsonls, S ~ ~ J ~ Valdman21722 J ~ , and others have gathered evidence for the presence of muscarinic (m)and nicotinic ganglionic (n) cholinergic receptor mechanisms in neocortical EEG activation. Especially pertinent is the research of Wikler27, Bradley and Elkesl, and Bradley and Nicholson2 in which dissociation between the EEG and behavioral manifestations of atropine and physostigmine were reported. The problem of dissociation has been dealt with extensively by Longol'. Hernhdez-Pe6n and his as~ociates10,11~~3 obtained evidence that cholinergic mechanisms are not only involved with arousal, but also with sleep in a limbic forebrain-midbrain hypnogenic circuit. Our own data with nicotinel6328, nicotine and are~oline~~329 and hemicholinium7,8, have led us to conclude that cholinergic mechanisms are important in both wakefulness and sleep. It is the purpose of this paper to summarize some of this research. One of the major problems of using most cholinergic agonjsts such as acetylcholine for injection into the body, as opposed to direct injection into the brain, is that these molecules are highly charged and therefore do not readily penetrate the blood-brain barrier. We have obtained evidence that cholinergic agents such as physostigmine, pilocarpine, arecoline, and nicotine which exist in both nonionized as well as ionized forms a t pH 7.4, produce marked EEG activation. As pharmacologists, our concern

* Supported in part by Grant MY-02653 USPHS. Some of the data described in this manuscript have been presented in another form7 at the Limbic System Symposium in Hakone, Japan, September 1965. References p . 132-133

114

E. F. D O M I N O . K . Y A M A M O T O A N D A. T. D R E N

has been lo determine the central and/or peripheral muscarinic (rn) and nicotinic ( n ) choliiiergic sites of action of these agents using drug antagonists as tools. An addilional problem was to identify tlir gross behavioral correlates ofthe EEG activation phenomenon.

METHODS

Cats hcith chronic ind\idling brairi elwtrodees Experiments were performed in cats utilizing a Latin Square design for drug administration at two week intervals. The cats were prepared for placement of indwelling brain electrodes using modifications of conventional techniques. Adult cats of both sexes were used. Surgical preparation of the animals was under pentobarbital sodium anesthesia. Stainless steel wires of 0.22 mm in diameter (insulated except for tips of 0.5 mm) were used as the depth electrodes. Bipolar depth electrodes were inserted into the amygdala and hippocampus using physiological recordings of injury dischargcs by insertion of electrodes for location of the hippocampus, and olfactory-induced waves for the amygdala. Bipolar silver ball electrodes of 0.5 mm in diameter were applied to the epidural surface of the somatosensory cortex. Additional depth electrodes were placed occasionally in the posterior hypothalamus and mesencephalic reticular formation. Each electrode was soldered to a Cannon plug and fixed on the scalp by means of dental cement. Silastic tubing of 0.7 mm i n diameter was inserted into the right jugular vein with the other end fixed to a connector on top of the skull. The animals were allowed to recover for a two-week period before being used for drug studies. I n the meantime, they were given antibiotics prophylactically to reduce infection. At the time of the experiment the EMG of the posterior neck muscles, EKG, and respiratory movements were recorded along with the brain waves on a Grass polygraph. The animals werc each placed in a sound-proof box with a one-way viewing window. Behavioral changes were observed and correlated with EEG activity. In order to promote naturally occurring sleep, the animals were made as warm and comfortable as possible. With care and patience i t was possible to observe all stages of natural sleep. The following drugs were given as an intravenous infusion i n physiological saline solution: acetylcholine chloride, atropine methylnitrate, atropine sulfate, arecoline hydrochloride, 1,l-dimethyl-4-phenylpiperidiniumiodide (DMPP), mecamylamine hydrochloride, (-) nicotine base, pilocarpine hydrochloride, physostigmine salicylate and trimethidinium methosulfate. The structural formulae of these agents are illustrated i n Figs. I , 2 and 3. All drugs were given in doses calculated as base. The actions of these drugs and various combinations on behavior were compared. Each drug was infused in a constant volume of I .5 ml over a one minute period. After completion of a series of experiments the position of each electrode was determined histologically by the iron deposition technique (see Domino, 1955, for details).

C H O L l N E R G l C MECHANISMS I N SLEEP A N D W A K E F U L N E S S

115

CH3

B

H,-c

H,C-C-0-c

H-,

I I

N+-CH

C-0-CH3

CH,

ARECOLINE

ACETYLCHOLINE

PI LO C A R PI N E

PHYSOSTIGMINE

Fig. 1 . Chemical formulae of some predominantly m cholinergic agonists.

DMPP

1\1 I CC T I N E

I Fig. 2. Chemical formulae of some predominantly IZ cholinergic agonists.

Acute dog preparations

Adult animals of both sexes were used. Dogs were prepared under 80% nitrous oxide20 % oxygen as well as local lidocaine anesthesia following immobilization with decamethonium, I mg/kg intravenously. Artificial respiration was maintained with positive pressure ventilation of 300 ml of air/kg/min. End tidal C02 was monitored with an infra-red COZ analyzer. Body temperature was maintained at 37-37.5"C by placing a heating pad on the dog. Temperature was controlled automatically with a References p . 132-133

E. F. D O M I N O , K. Y A M A M O T O A N D

1 I6

A. T. D R E N

CH--CH, N-CH, HC ,-

CH-0-C-CH

CH-CH,

ATROPINE

I C6H5

H,C-CH-CH,

I ? CH-0-C-CH I

CH,OH

I

I C,H,

METHYL

ATROPINE

Fig. 3. Chemical formulae of some 117 and n cholinergic antagonists.

rectal thermistor connected to an electronic control unit. The femoral artery was cannulated for monitoring blood pressure using a Statham P23A pressure transducer and recorded on an Offner polygraph. The femoral vein was cannulated for intravenous administration of drugs. The animal was positioned i n a stereotaxic instrument. Concentric stainless steel needle electrodes with tip exposure of 0.5 mm and an interelectrode distance of0.5 m m were placed i n various subcortical areas. Monopolar and/ or bipolar recordings were taken from neocortical areas Prcz, Prcl. and 01, and from the dorsal hippocampus, and basal amygdala. The midline nasal bone was used as a n indifferent site for monopolar recordings. An intraventricular needle was placed in the left lateral ventricle for drug injections. At least 1-2 hours elapsed after nitrous oxideoxygen anesthesia for elimination of the general anesthetic before drug testing. The following drugs were given either intravenously or intraventricularly : arecoline hydrochloride, atropine methylnitrate, choline chloride, d-tubocurarine chloride, d-amphet-

Fig. 4. Chemical formula of heniicholinium (HC-3), a cholinergic antisynthesis agent.

C H O L I N E R G I C M E C H A N I S M S I N SLEEP A N D W A K E F U L N E S S

I17

amine sulfate, epinephrine hydrochloride, gallamine triethiodide, hemicholinium bromide (HC-3), (-) nicotine, pilocarpine nitrate, physostigmine salicylate, sodium chloride and sodium bromide. The structural formula of HC-3 is illustrated in Fig. 4. All drug dosage was calculated as base unless otherwise specified. At the conclusion of each experiment the animal was sacrificed and the position of each electrode determined histologically by the iron deposition technique.

RESULTS

I . Interaction qf various cholinergic ugonists-antagonists on the awake-sleep cycle of the Cat Large doses (50pglkg) of physostigmine given intravenously to cats in slow wave sleep cause EEG activation of neocortical and limbic structures and behavioral arousa129JO. The EEG manifestations of physostigmine are illustrated in Fig. 5. Before physostigmine injection, the cat showed both awake and slow wave sleep EEG patterns. Following physostigmine given during slow wave sleep, the cat awakened with marked low voltage, fast activity in the neocortex, fast frequency bursts in the amygdala and marked &era activity in the hippocampus. About 45 min after injection a drowsy state appeared which rapidly changed to fast wave sleep. Although physostigmine has marked peripheral as well as central nervous system actions, these EEG effects are apparently primarily central. Methyl atropine slightly reduced the duration of EEG activation of neocortical and limbic structures, and behavioral arousal but did not block these effects. On the other hand, in equal doses, atropine blocked these actions of physostigmine as illustrated in Fig. 6. In contrast the n ganglionic cholinergic antagonists, trimethidinium and mecamylamine, did not block the EEG and behavioral manjfestations of physostigmine (see Fig. 7). It would appear that intravenous physostigmine produces primarily central m cholinergic effects which are blocked by m cholinergic antagonists like atropine that can penetrate the blood-brain barrier. It has been reported previously by Bradley and Elkesl that physostigmine produces EEG and behavioral dissociation. A similar phenomenon was observed by us. A partial explanation of this is the fact that animals under the influence of physostigmine may shift from the awake or drowsy state to fast wave sleep. In Fig. 8 the upper panel shows that normally spontaneous fast wave sleep usually occurs from a previous baseline of slow wave sleep. After physostigmine, fast wave sleep was also seen to occur from the resting or drowsy state29. Pilocarpine is an m cholinergic agonist which should penetrate the blood-brain barrier readily. In doses of 0.15 mg/kg intravenously it produced prompt EEG activation in neocortical and limbic structures as well as a behavioral arousal (see Fig. 9). These actions usually lasted about 15 min. Within 23 min the animal showed a resting EEG and behavioral state which suddenly shifted into fast wave sleep. Following methyl atropine, the duration of pilocarpine induced EEG activation was markedly reduced, but not blocked. In contrast, equal doses of atropine completely blocked the References p. 132-133

118

E. F. D O M I N O , K . Y A M A M O T O A N D A. T. D R E N

EEG actions of pilocarpine (see Fig. 10). In contrast to the effectiveness of atropine in blocking the EEG effxts of pilocarpine, the n ganglionic cholinergic antagonists, trimethidinium and mecainylamine, had no significant blocking actions (see Fig. 1 1). Paradoxically mecamylamine significantly enhanced the mean duration of EEG activation and arousal (P < 0.01). As in the case of physostigmine administration, pilocarpine frequently caused a shift from the awake or drowsy state to fast wave sleep. I n Fig. 12 the upper panel shows that normally spontaneous fast wave sleep occurs from a previous baseline of slow wave sleep. After pilocarpine this was also seen to occur from the resting or drowsy state. TABLE I MEAN D U R A T I O N

17 S.E.

I N M I N U T E S O F EEG A C T I V A T I O N F O L L O W I N G V A R I O U S CHOLINERGIC AGONISTS-ANTAGONISTS

Agonist Antagonist

0.9 % Saline 1.5 ml Methyl atropine 0.3 mg/kg Atropine 0.3 mg/kg Trimethidinium 1.0 mg/kg Mecam ylamine 0.7 mg/kg

No. of cats Acetylcholine 0.007 nig/kg

Arecoline

Pilocarpine Physosrigniine

0.04 inglkg

0.15 niglkg

0.05 tnglkg

13.3&11.3 3 1 . 6 k 2 . 2

8

4.150.5

11.910.8

6

0.3 f O . l J

9.6 t 0.9

2.3

7

0.1+ 0 . 1 3

0.2fo.1~

03

5

4.5 i 0.7

15.0 f 2.8

15.9 & 2.4

6

6.9 i 1.2l

13.7 1 1.8

21.8 4= 2.03 30.2 1 3 . 4

+ 0.23

22.9 f 3.2’ 1.3 f 0.5.’ 29.7 1 5 . 1

Nicotine 0.02 rnglkg

4.7*0.5

3.2 1 0 . 3 2.1 f 0.32 2.7 f 0.2 0.1 &O0.2j

Student “t” test group comparison with saline injection .-: 0.05 a :. 0.01 3 .’: 0.001

Similar studies have been carried out using other cholinergic agonists and antagonists. Table I summarizes the interactions of these agents on the duration of EEG activation which is associated with behavioral arousal. Given alone or following saline pretreatment, all of the cholinergic agonists tested produced varying degrees of EEG activation and behavioral arousal. On the basis of the interactions of these compounds with various cholinergic antagonists, it may be concluded that the actions of acetylcholine, arecoline, pilocarpine and physostigmine are primarily of the m cholinergic, and nicotine of the n cholinergic receptor type. The EEG and behavioral actions of acetylcholine are primarily peripheral. Arecoline and physostigmine have prominent central EEG actions. Pilocarpine has both peripheral and central EEG actions. The nganglionic cholinergic antagonists did not block the EEG effects of m cholinergic agonists. The reverse situation was not true. Administration of the centrally acting m cholinergic antagonist atropine reduced the EEG activating effects of nicotine.

0

3 0

RESP

3

n

n 3

>

2

v1

!!!a! ! ! ! ! ! ! ! ! ! ! ! ! ! ! I ! ! ! ! ! ! ! ! ! ! !

H!!!!!!

I ! !

!

1

I

!

!

!

!

!

! !

I

!

'

l ! ! ! " " I

8

"

I

!

I

"

/

!

"

'

!

I

1

1

1

I

I

!

"

'

1

'

I

! ! ! !

'

H E A D UP

7

p

-

-

/

-

-

-

-

<

-

5

p

I

-

-

50$V 2 5 e c

Fig. 5. EEG effects of physostigmine in the cat with chronic indwelling brain electrodes. Physostigmine was given while the cat was in slow wave sleep. Note the EEG activation and behavioral arousal 16 minutes later. Subsequently the cat lapsed into fast wave sleep. All recordings were bipolar. Symbols : L. POST SIG.-left posterior sigmoid gyrus; L. AMYAeft basolateral amygdala; L. HIP.-left dorsal hippocampus; EKG-electrocardiogram, Leid I1 : EMG-electromyograph of neck muscles; RESP.-thoracic respiration. Time base and voltage calibration are as indicsted. These symbols when used also apply to Fig. 6-12.

120 E. F. D O M I N O , K . Y A M A M O T O A N D A. T. D R E N

E

c

a c

LT

W

Fig. 6. Modification of the EEG effects of physostigmine by nz cholinergic antagonists. Note that methyl atropine did not block the EEG and behavioral effects of physostigmine, but atropine in equal dosage did.

P

5

5

b

AFTER TRlMETHlDlNlUM 1 mg/kg iv

W WAVE SLEEP

n

AWAKE

3: 0

LPOST.SIG.

x

n ?I

C

r

z

n v, v,

Fig. 7. Lack of blockade of the EEG effects of physostigmine by

IZ

ganglionic cholinergic antagonists

P

n CI 0

I

z

0

< P

P P

3 0 -I

0

P

z

0

? 5 C % i rn

- - ~ - - ~ - ~ . - - - - - - - ~ - . - ~ . ~ - - - ~ ~ - - _ ---~ i -

~--./---z -

J ~ I . . r .

...,

--

~~

50.w Z s e c

Fig. 8. Physostigmine induced fast wave sleep from a previous baseline of EEG and behavioral resting state. T h e upper trzclngs illustrate that normal spontaneous fast wave sleep usually begins from a previous baseline of slow wave sleep. After physostigmine fast wave sleep followed the awake or resting state as shown in the lower tracings.

2 J

$ R 4

.

'p

P

".6

---

-

REST

P+-w?-hm-

l

?

p

.

N

FAST WAVE SLEEP

-

h , w - P w - - ; '

P

$

W

f

W

W

t

~

~

~

~

W

W

W

M

w

I

~~~~~~~~~~~~' / ~~~~~~~~~~~~~~

W

' C r

-

2

23min

rn 50 4

G

Fig. 9. EEG effects of pilocarpine in the cat with chronic brain electrodes. Pilocarpinewas given intravenously a t the arrow over a I-min period while the cat was in slow wave sleep. Note the EEG activation and behavioral arousal which occurred during the injection period. Within 23 min the cat lapsed into fast wave sleep from the resting EEG state.

in in

t d

w

I24 E. t . D O M I N O , K . Y A M A M O T O A N D A. T. D K E N

E

W

i

L POST SIG

L

SPONTANEOUS FAST WAVE SLEEP

SLOW WAVESLEEP

-.-c

L AMY

FAST WAVE SLEEP

50

YY

2 rec

Fig. 12. Pilocarpine induced fast wave sleep from a previous baseline of EEG and behavioral drowsy state. The upper tracings illustrate that normal spontaneous fast wave sleep usually begins from a previous baseline of slow wave sleep. After pilocarpine fast wave sleep also followed the drowsy state as shown in the lower tracings.

CONTROL

4 0 MIN AFTER HC-3

Fig. 13. EEG effects of intraventricular hemicholinium in the dog. HC-3 was given into the left lateral ventricle of the brain of a locally anesthetized, decamethonium paralyzed dog on artificial respiration. All surgery was previously psrformcd under nitrous oxide-oxygzn anzsthzjiz. Not: that the initial effects of HC-3 were to produce amygdala spiking and abolition of the theta waves in the hippocampus. The time listed represent the hours after this massive dose of HC-3. Note tendency for partial recovery especially 45 h later. Symbols : L. Prcz-left precruciate gyrus; L. Prcl-left postcruciate gyrus; L. 01-left lateral gyrus; L. AMYG-basal amygdala; L. DORSAL HIPP-left dorsal hippocampus, bipolar recording. All other recordings monopolar to nasion. BP femoral arterial blood pressure; END TIDAL COZwas recorded with an infrared COZanalyzer. Vertical calibration bars-100 microvolts. These symbols when used also apply to Fig. 14 and 15.

HC-3 (5 mg INTRAVENTRIC.)

CONTROL

A

LPrc,b*.-,jw%.e>"M"r-w 8P

PI LOC ARPl NE (500yg/ Kg I.V.)

B

C _1 I O O y V 2 sec

ATROPINE METHYL NO, 0.25mg/Kg I.V.

Fig. 14. Modification of neocortical EEG effects of HC-3 by various cholinergic agonists. HC-3 induced EEG slow waves were antagonized by intravenous arecoline and pilocarpine, but not nicotine. Methyl atropine was given prior to these drugs to prevent hypotension.

E

u,

10

v

nI

0

I

-I

Llc

t-

0

0

z

0

C H O L I N E R G I C M E C H A N I S M S I N S L E E P AND W A K E F U L N E S S

a

KefevrncEv p . 132-133

m mi

0

1

f

4v)

Fig. 15. Failure of adrenergic agents to antagonize HC-3 induced neocortical EEG effects. Although physostigmine antagonized the EEG effects of HC-3, epinephrine) and d-amphetamine even in large doses did not.

129

I30

E. F. D O M I N O , K . Y A M A M O T O A N D A . T. D R E N

2. EfJpcts qf hemicholinium on drug-induced EEG activation in the dog As might be expected from the highly cationic character of hemicholinium (see Fig. 4), intravenous injection of this agent did not produce consistent EEG changes in dogs maintained with adequate artificial ventilation. Enormous doses, 5-10 mg/kg, given intravenously in animals occasionally produced marked EEG slowing and blockade of activation to all afferent stimuli. It was assumed that the marked variability was due to individual differences in blood-brain barrier permeability, brain and/or blood levels of choline, etc. Therefore the drug was given intraventricularly. Via this route, remarkably small amounts of HC-3 produced consistent EEG changes in neocortical and limbic areass.9. As illustrated in Fig. 13, HC-3 (5 mg total dose) caused within 40 min spiking in the limbic areas and abolition of the hippocampal theta rhythm. However, neocortical activation still persisted. After 4 hours both neocortical and limbic areas showed diffuse slow waves and spiking. These persisted for about two days with partial recovery 45 h after injection. During this period both neocortical and limbic system activation was blocked. We have previously reported8, that these effects are partially reversible with choline, and are not produced with intraventricular administration of sodium chloride, sodium bromide, d-tubocurarine, and gallamine indicating the specifity of HC-3 action. Furthermore, following HC-3 i n total doses of both 50pg and 5 mg, brain acetylcholine levels in subcortical structures near the ventricle are reduced by about 50% in 4 h9. These changes in brain acetylcholine content qualitatively parallel the distribution of **C-labelledHC-3 (Domino et a]., unpublished observations). Inasmuch as 5 mg of HC-3, given intraventricularly, does not deplete brain acetylcholine completely, it would be expected that some cholinergic agonists would antagonize the functional deficits of lowered brain acetylcholine. This indeed was found to be the case. The H I cholinergic agonists arecoline (40 pg/kg i.v.) and pilocarpine (500 pglkg, i.v.) reversed the HC-3 induced EEG neocortical slow waves but the ii cholinergic agonist nicotine did not (see Fig. 14). Physostigmine (IOOpg/kg, i.v.)also antagonized HC-3 induced slow waves, but epinephrine (5 pglkg, i.v.) and damphetatnine (2 mg/kg, i.v.) did not (see Fig. 15). These findings are reminiscent of those of White and Boyajy25 and White and D a i g n e a u P with atropine. CONCLUSIONS AND SUMMARY

Pharmacological data on the importance of cholinergic mechanisms in EEG activation and behavioral arousal as well as sleep are impressive. The gross behavioral consequence of the initial EEG activation is clearly a wake-up or arousal state. It has previously been reported by Wikler27, Bradley and Elkesl, Bradley and Nicholson2 and others, that cholinergic agonists and antagonists produce EEG dissociation from gross behavior. Similar findings have been made by us using large doses of atropine and physostigmine. However, it should be pointed out that effective doses of physostigmine and other cholinergic agonists produce initial behavioral arousal that is associated with neocortical and limbic activation. The emphasis in the literature on EEG dissoc-

C H O L I N E R G I C MECHANISMS IN S L E E P A N D W A K E F U L N E S S

131

iation from gross behavior may have been overstated, particularly in relationship to the awake-sleep cycle of the chronic cat. The findings of Bradley and Elkes and others with cholinergic agonists were made at a time when the stage of fast wave sleep as described by Dement3 and Jouvet15 was not generally known. It would appear that in some instances investigators may have been observing fast wave sleep and did not recognize it as such. By use of various m and n cholinergic antagonists with differential abilities to penetrate the blood-brain barrier, it has been possible to determine if the actions of various cholinergic agonists given intravenously were primarily central or peripheral in origin. Four predominantly muscarinic (m) cholinergic agonists (acetylcholine, arecoline, pilocarpine, and physostigmine) and two nicotinic ganglionic (n) cholinergic agonists (DMPP and nicotine) were studied on the awake-sleep cycle of cats. The animals had chronic indwelling brain electrodes in various neocortical and limbic areas. The effects of these compounds were compared before and after the following m and n cholinergic antagonists: atropine, methyl atropine, mecamylamine and trimethidinium. Atropine pretreatment blocked EEG activation induced by acetylcholine, arecoline, pilocarpine and physostigmine, but only reduced that produced by DMPP and nicotine. Atropine also blocked nicotine induced hippocampal theta wave activity. Methyl atropine, an m cholinergic antagonist with predominant peripheral effects, markedly antagonized EEG activation by acetylcholine, but did not block EEG activation induced by other m or n cholinergic agonists. The n ganglionic cholinergic antagonists, mecamylamine and trimethidinium, had no significant effects on EEG activation induced by m cholinergic agonists. On the other hand, the actions of n cholinergic agonists such as DMPP and nicotine were completely blocked by mecamylamine. Trimethidinium blocked EEG activation of DMPP but reduced slightly that of nicotine. In general, the gross behavior of the cats paralleled the initial neocortical EEG effects of these drugs when given in low doses. Another pharmacological approach to studying central cholinergic mechanisms was with the drug hemicholinium (HC-3) which decreases acetylcholine synthesis by interfering with choline transport. Acute dog preparations were used to study the effects of HC-3. The actions of the drug were unpredictable on intravenous administration, but highly reproducible when given intraventricularly in total doses up to 5 mg. HC-3 produced initially amygdala spiking and blockade of hippocampal theta wave activity, but did not affect neocortical activation. This demonstrates a dissociation between the neocortical and limbic activating systems. Eventually neocortical slow waves appeared. The EEG effects of HC-3 are related to lowered brain levels of acetylcholine, because subcortical acetylcholine was reduced approximately 50 % 4 h after drug administration. Exogenous choline produced a delayed and transient reversal of the HC-3 effects. Arecoline, pilocarpine, and physostigmine caused EEG activation following HC-3, whereas nicotine, epinephrine and d-amphetamine were either much less effective or their EEG actions were completely blocked.

References p . 132-133

132

E. F. D O M I N O . K . Y A M A M O T O A N D A. T. D R E N

REFERENCES 1 BRADLEY, P. B.

A N D ELKES, J. (1957) The effects of some drugs on the electrical activity of the brain. Brain, 88, 77- 1 17. 2 BRADLEY,P. B. AND NICHOLSON, A. N. (1962) The effect of some drugs on hippocampal arousal. Electroenceph. cliw. Neurophysiol., 14, 824-8 34. 3 DEMENT, W. (1958) The occurrence of low voltage, fast electroencephalogram patterns during behavioral sleep in the cat. Electroerrceph. cliw. Neurophysiol., 10, 291-296. 4 DENISENKO, P. P. (1961) Cholinergic and adrenergic systems in the reticular formation of the midbrain and the reaction of activation in the cortex. Sechenov Fiziol. Zh. USSR (Big.), 47,609-61 6. 5 DENISENKO, P. P. (1962) Influence ot pharmscological agents upon cholinoreactive and adrenorcactive systems of the reticular formation and other regions of the brain. Proc. 1st I t d . Pharmacol. Meetings. Phartnacolngical Analysis of Central Nervous Action, Paton, W. D. M. Ed, Pergamon Press. 6 DOMINO, E. F. (1955) A pharmacological analysis of the functional relationship between the brain stem arousal and diffuse thalamic projection svstem. J . Pharmacol. Exptl. Therap., 115, 449463. K. (1966) Pharmacologic evidence for cholinergic 7 DOMINO, E. F., DREN,A. T. A N D YAMAMOTO, mechanisms in neocortical and limbic activating systems. Hakone Symposium held in Japan 1965. Progr. Brain Res. In press. 8 DREN, A. T. AND DOMINO, E. F. (1965) Someeffects of hemicholinium (HC-3) on EEG desynchronizating mechanisms in the dog. Pharmacologist, 7 , 143. 9 DREN, A. T. AND DOMINO, E. F. (1966) Effects of Hemicholinium (HC-3) on EEG activation and brain acetylcholine in the dog. Pharmacofogist, 8, 183. 10 H E R N ~ N D E Z - PR. E ~AND N , CHAVEZ-IBARRA, G. (1963) Sleep induced by electrical or chemical stimulation of the forebrain. In “The Physiological Basis of’ Mental Activity.” R. Hernandez-Pebn Ed. Electroenceph. Clin. Neurophysiol. Suppl., 24, 188-198. 1 1 HERNANDEZ-PE~N, R., CHAVEZ-IBARRA, G., MORGANE, P. J . AND TIMO-TARIA, C. (1963) Limbic cholinergic pathways involved in sleep and eniotional behavior. Exptl. Neurol., 8, 93-1 I!. 12 ILYUTCHENOK,R. 1. (1962) The role of cholinergic systems of the brainstein reticular formstion in the mechanism of central effects of anticholinesterase and cholinolytic drugs. Proc. 1st lnt. Pharmacol. Meetiws. Paton, W. D . M. Ed. Pharmacological Analysis of Central Nervous System, 8, 21 1-216. 13 ILYUTCHENOK,R. I. A N D MASHKOVSKII, (1961) Electrophysiological data on cholinereactive elements of the reticular formation of the brain stem. Sechenov Fiziol. Zh. USSR.,47,1352-1359. 14 ILYUTCHENOK, R. I. AND OSTROVSKAYA, R. U. (1962) The role of mesencephalic cholinergic systems in the mechanism of nicotine activation of the electroencephalogram. Bull. Exptl. Biol. and Med., 54, 753-757. 15 J O U V ~ TM. , (1961) Telencephalic and rhombencephalic sleep in the cat. Ciha Foundatiorr Symposiun~on the Nature of Sleep. pp. 188-206. 16 KNAPP, D. E. AND DOMINO, E. F. (1962) Action of nicotine on the ascending reticular activating system. Int. J . Neuropharmacol., 1, 333-351. 17 LONGO,V. S. (1966) Behavioural and electroencephalographic effects of atropine and related compounds. Pharmacol. Rev., 18,965-996. 18 MICKELSON, M . J. (1961) Pharmacological evidences of the role of acetylcholine in the higher nervous activity of man and animals. Activ. Nerv. Super., 3, 2. 19 RINALDI, F. AND HIMWICH, H. E. (1955) Alerting responses and actions of atropine and cholinergic drugs. AMA Arch. Neurol. Psychiat., 73, 387-395. 20 RINALDI, F. AND HIMWICH, H. E. (1955) Cholinergic mechanism involved in function of mesodiencephalic activating system. A MA Arch. Neurol. Psychiat., 73, 396-402. A. V. (1961) Thepharmacology of reticular fortnutior! andsynaptic transmission. pp. 432. 21 VALDMAN, Leningrad. 22 VALDMAN, A. V. (1963) Problems of pharmacology ofreficular formalion and synaptic transmission. pp. 416. Leningrad. 23 VELLUTI, R. AND HERNANDEZ-PEON, R. (1963) Atropine blockade within a cholinergic hypnogenic circuit, Exptl. Neurol., 8, 20-29. 24 VILLARREAL, J. E. A N D DOMINO, E. F. (1964) Evidence for two types of cholinergic receptors involved in EEC desynchronization. Phavmacologist, 6, 192. R. P. A N D BOYAJY, L. D . (1959) Comparison of physostigmine and amphetamine in anta25 WHITE, gonizing the EEG of CNS depressants. Proc. SOC.Exptl. Biol. N . Y., 102, 479-483.

CHOLINERGIC MECHANISMS I N SLEEP A N D WAKEFULNESS

I33

26 WHITE,R. P. AND DAIGNEAULT, E. A. (1959) The antagonism of atropine to the EEG effeects of adrenergic agents. J . Pharmacol. Exptl. Therap., 125, 339-346. 27 WIKLER,A. (1952) Pharmacologic dissociation of behavior and EEG “sleep patterns” in dogs: morphine, n-allylnormorphine, and atropine. Proc. Soc. Exptl. Biol. N. Y. 79, 261-265. 28 YAMAVOTO, K. AYD DOMINO, E. F. (1965) Nicotine-induced EEG and behavioral arousal. Znt. J. Neuropharmacol., 4, 359-373. 29 YAMAMOTO, K. AND DOMINO, E. F. (1967) Cholinergic neocorticil and hippocampal EEG activation, Int. J . Neuropharmacol. (In press)

134

Cholinergic Brain Mechanisms and Behaviour R.YU. TLYUTCHENOK Insritute of Cytology arid Gertetics ( U S S R ) *

The problem of cholinergic mechanisms in the brain is very complicated. In the present study we have tentatively ignored all the complexities and have focused our attention on some of the problems related to the participation of cholinergic mechanisms in behaviour. In what behavioural reactions do cholinergic mechanisms play an important role? Is behaviour correlated with changes i n bioelectrical brain activity and stress reactions? What is the possible mechanism of behavioural changes under the effect of cholinergic drugs? Certainly, it is difficult to give an exhaustive answer to all these questions. Evidence obtained during the past few years has enabled us to gain an understanding of the possible role of cholinoreactive structures in behaviour. CHOLINERGIC MECHANISMS A N D EMOTIONAL BEHAVIOUR

In previous studies it has been demonstrated that the most characteristic changes produced by cholinergic drugs in experimental animals are those of observed emotional behavi ou r. Our very first observations on the effect of tropazine, an anticholinergic drug relieving fear neurosis, focused our attention upon the study of the role of cholinergic mechanisms in behaviour27. At this early stage, it was already hypothetically presumed that cholinergic mechanisms play an important role in the emotional fear reaction. Experiments using an anti-acetylcholinesterase (galanthamine) provided additional data on the participation of cholinoreactive mechanisms in defensive reactions46. This work has shown that in animals galanthamine administration enhances passive defensive reactions. A number of workers have also demonstrated the participation of cholinergic mechanisms in behavioural reactions3.16,".37.47,65. During the past few years A. G . Yeliseyeva, in our laboratory, has studied in detail the role of the cholinoreactive structures in the mechanisms of the emotional fear reaction. Food conditioned reflex

* Pharmacological Laboratory, Experimental Biology Dept., Siberian Branch, Academy of Sciences of the USSR, Novosibirsk, 90, USSR,

CHOLINERGIC BRAIN MECHANISMS A N D BEHAVIOUR

135

Fig. 1. Inhibition of the emotional fear reaction by muscarinic anticholinergic agent (amysil). When the conditioned food reaction was elaborated (A) the dog was given electric shock (B). After that it did not leave the box for a long period of time to respond to the presentation of the conditioned stimulus then it attempted to run out of the room (D-I?), resisted when attempts were made to bring it to the food cup (F). After the administration of amysil “fear” reaction disappeared. On the next day at the presentation of the conditioned stimulus the dog came near the food cup and ate (G-H). The arrow on the light background marked the action of the conditioned stimulus.

conditioning was carried out with unrestrained dogs (Fig. 1 A). After establishing conditioned responses, the dogs received an electric shock the moment they came into References p . 146-148

I36

R. Y U . I L Y U T C H E N O K

contact with their food cup. After one or two electric shocks a conditioned fear reaction was produced (Fig. I B). This fear reaction was manifested each time the conditioned stimulus was present, overlapped by the electric shock (Fig. 1 C-F). Fear reaction of this type can be observed lasting for a few months. This is confirmed by a number of other authors'. Another series of experiments was performed on cats. An auditory signal was combined with an electric shock delivered through the grid floor. Fear reaction was well established after 3 or 4 combined stimuli and was expressed by the following symptoms: the animal stood stock still with the head drawn in, eyes closed and ears retracted. Urination and defecation were commonly observed. Sometimes the response was o f another type: the animal hissed, snarled, raised its paw threateningly and jumped. This aggressive reaction, however, was noted only at the very beginning of the conditioned response elaboration. With the increasing number of trials the aggressive reaction was replaced by a passive-defensive one. In our experiments on dogs, the blockade of muscarinic cholinergic brain structures by amysil (benactyzine) or bensazine immediately after electrostimulation or on the next day (0.5-1 .O mg/kg intramuscular 2-3 times daily for 1-3 days), considerably inhibited the conditioned responses. Fear reaction disappeared at the same time. Thus, immediately after anticholinergic drug administration it was possible to note its inhibitory effect on fear reaction, in spite o f general inhibition of conditioned reflexes. It should be emphasized that an anticholinergic drug does not inhibit the unconditioned food response. During the following days the conditioned food response was restored completely, but the fear reaction was not re-established (Fig. I G, H). In a series of experiments performed on cats we have also noted an isolated inhibition of the fear reaction due to the effect of anticholinergic drugs. In cats, intravenous administration of amysil in a dose of 0.5 mg/kg abolished the conditioned fear reaction after a few minutes. At the same time no change in the unconditioned reaction to electrostimulation appeared. Components of the aggressive reaction, i.e. the raising of paws, sniffing and hissing (in cases where they were noted previously) were also maintained. Concerning the blocking of the adrenoreactive brain structures, it has not been possible to reveal its isolated influence on the emotional fear reaction in animals. When aminazine (chlorpromazine) was administered intravenously in a dose of I mg/kg, the fear reaction was not abolished. When the intravenous dose was increased to 3-5 mg/kg, the response was attenuated with progressive deepening of the sedative state and parallel decrease of the unconditioned defensive response, aggressive response, loss of motor co-ordination and the presence of muscular relaxation. When the unconditioned fear reaction and the capacity to react to other stimuli were maintained to some degree, the fear reaction was not abolished. It may be supposed that chlorpromazine does not block the mechanism of fear reaction, but inhibits the mechanism through which the motor-vegetative response is realized as a result of general reactivity reduction, including the attenuation of response to noxious stimuli. These facts are completely consistent with our previous observations31~",46, according to which the stimulation of the central cholinoreactive brain structures by

CHOLINERGIC BRAIN MECHANISMS A N D BEHAVIOUR

I37

cholinesterase inhibitors induces and intensifies the fear reaction. Thus, when the central cholinoreactive structures are stimulated these reactions appear, whereas their blockade abolishes these responses. It is therefore possible that the activity of the central cholinereactive structures is included in the specific mechanisms of the fear reaction19,31133, Complete and stable inhibition of an emotional reaction takes place not only when the muscarinic cholinoreactive structures of the brain are blocked not only immediately but also when the anticholinergic drugs are administered during the following days. This effect is not related to the blockade of conduction of nerve impulses under the effect of anticholinergic drugs since the fear reaction, having disappeared at the moment when the muscarinic cholinoreactive structures were blocked, is not restored after normalization of their activity. Moreover, the administration of the same dose of this agent after an interval of 14-1 5 days, does not inhibit the emotional fear reaction. To obtain this effect considerably higher doses are required. Thus, when the muscarinic cholinoreactive structures are blocked, not only are recent memory traces vulnerable (minutes and hours after the establishment of the fear reaction), but also those that are more prolonged (in the first days following their fixation). The present data indicates the important role of muscarinic cholinoreactive brain structures in the mechanisms of emotional memory34. The hypothesis we suggest is directly opposed to the generally accepted notion of the adrenergic nature of passive-defensive reactions. As early as 1945, Arnold6 reported that fear is mainly due to sympathetic activation, as the state of excitement and elation are related to moderate parasympathetic activity. Later Solomon and Wynne, Boward, Anokhin and others4,9>59demonstrated the participation of adrenergic mechanisms in defensive reactions. What was the reason for such a ready and widespread recognition of the adrenergic nature of defensive reactions? It is possible that this attitude is the result of a deep-rooted concept according to which stress is considered to be a reaction related to the sympathetic adrenal system. At the same time it is generally known that the defensive reaction to a pain stimulus is one type of stress response. EMOTIONAL BEHAVIOUR A N D STRESS

Bowardg considers that a system related to positive emotions has a parasympatheti function and inhibits the neuroendocrinal response to stress. The stimulation of the “negative system” produces fear, rage, aggression and promotes stressful neuroendocrinal responses and releases a sympathetic effect. in our laboratory have However, experiments performed by Ye. V. Naumenk05~9~~ shown that stimulation of the central adrenoreactive structures by piridrole (pipradol) has no marked effect on the function of the adrenal cortex and does not alter the response of the pituitary-adrenal system to stress (Table I). The stimulating effect of phenamine (amphetamine) on the adrenal system is abolished following mesencephalic section, whereas EEG activation is maintained (Table 11). The level of corticosteroids in the peripheral blood did not change under the effect of phenamine both References p. 146-148

I38

R. Y U . I L Y U T C H E N O K

TABLE I T H E C H A N G E S OF ~ 7 - H Y D R O X Y C O R T l C O S T E R O I D S L E V E L P R O D U C t l ) H Y V A R I O U S S T I M U L I I N G U I N F A PIGS WITH I N l A C T H R A I N .

~~

Ti,eatrrrent

No. of atiinia/~

~

~~

-

~~~

Level of I7-hyctroxycort ico rteroih 111

jig 04

M

tvi

+

~

P -

~~~

Distilled water Naphtyzin (1 mg/kg)

15

36.81 i 4.32 114.26 1 6.1 I

0.001

Distilled water Naphtyzin (3 nig/kg)

13

47.12 4 6.88 161.61 & 12.02

0.001

Distilled water Amphetamine (5 mg/kg)

12

41.54 4 4.68 106.43 & 9.43

0.001

Distilled water Amphetamine (I0 mg/kg)

14

42.45 I 138.69

3.52 9.00

0.001

Dktilled water Piridrol (5 mg/kg)

10

37.30 44.97 i

4.90 6.42

0. I

Distilled water Piridrol (10 mg/kg)

31

49.28 55.90

6.19 5.92

0.1

Distilled water Galanthamine ( 5 mg/kg)

27

35.16 f 3.94 65.14 4.24

0.001

Distilled water Galanthamine (10 mg/kg)

19

24.13 & 3.45 97.17 & 6.92

0.001

~~

+ j

+ +

~~

T A B L E 11 I

Hr

CHANGES O F 17-HYDROXYCORTICOSTEROlDS LEVEL PRODUCED B Y VARIOUS STIMULI I N GUINEA PIGS WITH MIDBRAIN S t C T l O N

Level of Treatriient

No. of

T i i w of

cteterminatioti

I@ O h

ariimalt ~-

Control

17-liy~troxicortico~trroid~ P

M f nz ~ - _ _ _ -

10

I h after section 2 h after section

65.75 70.63

Saline solution into ventricle

24

I h after section 2 h after section (1 h after injection saline solution)

61.95 & 3.83

Amphetamine (10 mg/kg)

21

Galanthamine (10 m g / W

1 h after section

2 h after section (1 h after amphetamine injection) 14

I h after section 2 h after section (1 h after galanthamine injection)

7.33

t 6.68

0. I

0.001 119.86 & 7.52 63.22 4 4.72 0. I

74.74 1 8.01 66.57 & 5.14

0.1 67.80 t 6.00

CHOLINERGIC BRAIN MECHANISMS A N D BEHAVIOUR

139

following pretrigeminal section and in the cerveau isole'. It is therefore reasoned that the changes observed in the functional state of the adrenal cortex under the effect of phenamine are connected with the stimulation of the peripheral adrenoreactive structures. This is confirmed by the rise in the level of 17-oxycorticosteroids in the blood following intravenous administration of naphtyzine, an agent mainly stimulating the peripheral adrenoreactive structures (Table I). The available data allows us to assume that adrenomimetics exert an effect on the pituitary-adrenal system by stimulating the peripheral adrenergic structures. Thus, it becomes apparent that an hypothesis involving the participation of the central adrenoreactive structures in stress reactions lacks sound evidence. On the contrary, the data cited above prove that this hypothesis is inconsistent. At the same time this suggestion turns out to be a significant argument in the formation of a hypothesis on the role of adrenergic mechanisms in the realization of defensive reactions. It should be noted that the administration ofgalanthamine, a cholinesterase inhibitor, also raises the level of corticosteroids in the blood (Table I). But with pretrigemina1 section or in the cerveau isole'the stimulation of adrenal cortex is eliminated under the effect of anti-cholinesterases (Table rT). It may be supposed that the activating effect of the anti-choline sterases and acetylcholine on the hypothalamic-pituitaryadrenal system is also connected with its primary effect on peripheral cholinoreactive structures. The stimulation of these structures is transmitted through the brain stem to the hypothalamus. The hypothalamic regions contain serotonino-reactive structures through which it is probable that the whole hypothalamic-pituitary-adrenal system is activated. The acceptance of such a mechanism as regulating stress responses casts some doubt on the obligatory relations between behaviour and the endocrine reactions. Tn ordinary environments, fear and rage i n animals are usually accompanied by stress reactions. But in experimental conditions it is possible to differentiate these two sets of reactions. A rise in thelevel of 17-oxycorticosteroidsintheblood following naphtyzine administration may take place without concomitant behavioural changes. On the contrary, a marked behavioural change without stress response is observed under the effect of piridrole. Thus, it is possible to produce isolated stress reactions and behavioural changes separately. Tt seems that the functional systems governing behavioiir and stress reactions are not components of one and the same mechanism, although they are intimately interrelated in the organism as a whole. BEHAVIOUR A N D ELECTRICAL ACTIVITY O F THE BRAIN

A correlation between behavioural changes and brain electrical activity is established

most commonly when two such functionally opposed states as wakefulness and sleep are compared. The correlation between EEG and behavioural changes is more readily revealed under the effect of drugs acting on the adrenoreactive structures. Our data confirm the well known fact that adrenergic stimulation in animals is accompanied by EEG activation. In contrast, when the adrenergic mechanisms are blocked, the animal is sedated and slow EEG waves appear. However, it is hardly expedient to draw such Refevences p . 146-148

140

R. YU. I L Y U T C H E N O K

sharp distinctions between the presence of behavioural changes in adrenergic wakefulness responses and their absence in cholinergic responses. The behavioural result of adrenergic drug administration is undoubtedly more clearly demonstrated than that of cholinergic drug administration. But in certain experimental conditions after the use of drugs acting on cholinergic structures, peculiar behavioural changes are observed. Ln open space conditions, under the effect of a tertiary anticholinesterase (galanthamine), altertness gradually changing into restlessness was observed in cats. Sometimes, even in the absence of environmental stimuli, the cat arched its back and piloerection took place. Occasionally the cat jumped back as if something had loomed before it, and a tendency to squeeze itself into all kinds of small spaces was noted. In small doses, these drugs accelerate the elaboration ofconditioned reflexes and intensify them, whereas large doses inhibit conditioned reflexes. Central muscarinic-anticholinergic agents block the EEG activating effect of anticholinesterases as well as behavioural responses in animals. This paper has already dealt with the characteristic effect of anticholinergic drugs on the emotional fear reaction. Thus, chemical stimulation and blocking of cholinergic brain structures are characterized not by the complete absence of behavioural reactions but by the peculiar forms in which they manifest themselves29~46.These changes, however, do not satisfy the usual correlation of EEG and behaviour when excitation is accompanied by EEG activation and a sedative or drowsy state is in its turn accompanied by EEG synchronisation. It is known that these EEG patterns tis appear with activity changes in the ascending reticular activating system. Thus, it is a correlation between behaviour and an EEG pattern that characterizes the changes in the function of the brain-stem reticular formation. The following question then arises : why under the effect of cholinergic and adrenergic drugs is there only one type of correlation between EEG and behaviour? Adrenergic and cholinergic drugs produce different types of EEG activation@; moreover, the mechanism of adrenergic and cholinergic EEG activation is not identical. These structures do not have one and the same location in the reticular formation12,'4,17,20,25,44,45,49,54,65,66.

It has been demonstrated in experiments conducted in our laboratory that adrenoreactive structures are limited mainly to the caudal regions of the midbrain and pons. The activating effect of adrenomimetics in the cerveau isolb is attenuated, while in the premesencephalic section it disappears completely. Serotonino-reactive structures are also found in the caudal regions of the ponto-mesencephalic reticular formation30~31. A summary of recent contributions on serotonin is presented by Garattini and Valzelli22. Cholinoreactive structures are widely distributed throughout the ponto-mesencephalic reticular formation28J1J5. It has been established that in the cerveau isolP (when a portion of the midbrain remains above the section level), galanthamine and eserine not only inhibit acetylcholinesterease activity but also produce a marked EEG activation. This effect was more obvious in the asymmetric section (Fig. 2). When the midbrain was isolated completely (premesencephalic section), the acetylcholinesterase activity ofthe brain areas situated above the section was inhibited to the same extent as in intact animals. In spite of this, EEG activation did not take place (Fig. 3). This

CHOLINERGIC BRAIN MFCHANISMS AND BEHAVIOUR

141

Fig. 2. The influence of galanthamine o n the EEG of a cat with non-symmetrical brain section (on the left - cerveau isole, on the right - premesencephalic section). From above downwards: lef frontal and occipital, right frontal and occipital cortex. A - before, B - 1 min after intravenous administration of 3 mg/kg galanthamine.

allows us to conclude that the presence of EEG activation is dependent on the degree of acetylcholinesterase inhibition, in the ponto-mesencephalic region of the brain"?". Thus, the brain stem reticular formation is a biochemically heterogeneous system. I t has varying neurochemical regulatory mechanisms. The physiological implication of its chemical heterogeneity has not been investigated and is not as yet very clear. Undoubtedly, different chemically sensitive components of the brainstem reticular formation are responsible for the manifestation of the different reactions in the central nervous system. They participate in the mechanisms of electrical activity of the brain and to a certain extent in mechanisms of behaviour. But it is unlikely that the behavioural pattern under the effect of pharmacological agents is the result only of changes in the activity of the brain stem reticular formation. Probably, in the complex behavioural situation, the components correlating with a spontaneous EEG pattern must have a reticular mechanism. Consequently, one may suppose that correlation takes place i n the case when the changes of both electrical activity of the brain and of certain behavioural components result from changes in the activities of the same functional systems of the brain. References p . 146-148

1 42

R. Y U. I L Y U T C H E N O K

L -

.> ._

I

It

.@

U

0

U

cortex

thalamus hypothalamus midbroin mtdulta

r Jintuct brain

a prcmcscncephalic

section

Fig. 3. Galanthamine's influence 011 EEG and acetylcholinesterase of the brain. I

-

intact brain

11 - premesencephalic section. From above downwards: left frontal and occipital, right frontal and

occipital cortex. A - before and B - 4 min. after the administration of 9 mg/kg galanthamine. I11 - The change of acetylcholinesterase in cats after the administration of 9 mg/kg galanthamine. Expressed on 0.001 M of acetic acid per 0.2 ml of brain homogenate.

The activating influence of anticholinesterases is maintained against a background of previous antiadrenergic drug e f f e ~ t s ~ ~ , ~ ~ , 3 5 , ~ 1 , 6The 4 1 6blocking 7. by high doses of chlorpromaLine of EEG activation caused by anticholinesterase is accounted for by the anticholinergic effects of chlorpromazine01. According to our experiments31 this effect of chlorpromazine is related to the blocking of the central muscarinic cholinergic structures. At the same time, signs of chlorpromazine depression prevail in the behaviour of the animals with a background of motor anxiety and fear reaction which are characteristic of the effect of the anticholinesterases. Simultaneously, the conditioned reflexes are inhibited. EEG activation resulting from the excitation of adrenoreactive structures of the mesencephalic reticular formation is easily blocked by small doses of central muscarink anticholinergic agents"'131~~5~~"66. These agents do not attenuate motor activity induced by adrenomimetics. In some experiments, anticholinergic agents somewhat intensify this activity, at the same time considerably inhibiting the orientation reaction. Thus, some of the effects are removed, whilst others are maintained. In order to have some understanding of the role of different cholinoreactive brain

CHOLINERGIC BRAIN MECHANISMS AND BEHAVIOUR

143

structures in the mechanisms of EEG and behavioural changes, the presence of a cholinergic mechanism at the level of the cortex in synaptic transmission from the brainstem reticular formation must be taken into account. EEG changes characteristic of reticular cortical activation are related to these mechanisms. The investigations we have performed in collaboration with G. D. Smirnovs7 have shown that the terminal pathway of the ascending activating system forms cholinoreactive synapses at the cortex. This is most markedly expressed in the antagonistic effects of anticholinergic drugs and anticholinesterases on reticular cortical arousal potentials which have been shown in our laboratory by V. S. Zinevich. The cortical neurones of the ascending activating reticular system have muscarinic proper tie^^^,^^. This is confirmed by the data obtained in our laboratory by M. A . Gilinsky who has demonstrated the presence of an antagonistic effect of anticholinesterase and muscarinic anticholinergic drugs in relation to spike activity of cortical neurones. The data on the role of the cortical cholinergic neurones is confirmed in the works of a number of author~3*~39~~0.~6,60. Thus, the activity of the ascending reticular activating system must be considered as proceeding from the chemical heterogeneity of the brain-stem reticular formation to the homogeneity of cortical neurones of the ascending activating system. We consider that there exist mechanisms for transmitting impulses arising from the mesencephalic reticular formation (when different chemoreactive systems are stimulated) to muscarinic cholinergic cortical n e u r o i i e ~ 3 ~ , ~ ~ . Proceeding from the results on the chemical heterogeneity of the ascending reticular activating system at the brain stem level and on the cholinergic properties of the cortical neurones of this system, the reason for the blocking of adrenergic and serotoninergic EEG activation by anticholinergic drugs is clear. Thus, when central chemoreactive systems are stimulated or blocked, complex mechanisms are involved correlating EEG and behavioural responses. It is necessary to note the orientation reaction changes under the effect of anticholinergic drugs. Surely, it should not be considered that reticular mechanisms alone participate in the production of the orientation reaction. Sokoloff58 is possibly right when he suggests that the mechanism of the orientation reflex cannot be limited to any single part of the brain. However, there is adequate evidence indicating that the brain stem reticular formation plays an important role in the mechanisms of the orientation response. There is no doubt that changes in the activity of the brain-stem reticular formation may to some degree alter the manifestation of the orientation reaction. It is a fact that when anticholinergic drugs block the arousal reaction, the inhibition of the ascending reticular activating system attenuates the orientation response. It is hard to say at this moment whether all the components of the orientation response are attenuated, but the motor components are notably inhibited. Thus, in relation to the orientation response and the spontaneous EEG under the effect of anticholinergic drugs, we obtain a correlation showing that the same reticular mechanisms participate in both cases. But behavioural changes are not only determined by the stimulation or blocking of the ascending activating system. Behaviour is largely influenced by activity changes of the limbic, neuroendocrine, vegetative and other systems under the effect of neurotropic substances. Thus, a correlation should be found between the various Rriferenirs p . 146-148

I44

R. Y U . I L Y U T C H E N O K

behavioural components, together with other electrical changes, having non-reticular mechanisms ; for example, the correlation with changes of electrical activity in the limbic system. Further investigations will possibly establish the presence of correlations between various behavioural components and other electrical physiological phenomena (neuronal activity, impulse transmission to different systems etc.). SOME NEUROC’HEMICAL M E C H A N I S M S A N D B E H A V I O U R A L C H A N G E S

Most or the existing hypotheses on neurohumoral behavioural mechanisms are based mainly on changes in food and defence conditioned reflexes under the effect of adrenergic and cholinergic drugs. Data on the changes in conditioned reflexes under the effects of these agents cannot serve as proof that defensive responses are adrenergic or that food responses are cholinergic in nature. All the drugs stimulating the chemoreactive brain structures in low doses intensify conditioned responses. Large doses inhibit conditioned reflex activity and depression of food-, as well as defencen-coditioned responses is observed. In intermediate doses, agents stimulating cholinergic and adrenergic brain structures raise the conditioned reflexes but do not modify the EEG or behaviour. It may be presumed that either the functional state of the brain cortex alone is changed, or that the sensitivity of the methods of registering the activity of subcortical structures is inadequate when compared to the sensitivity of the conditioned reflex method. With a gradual increase of dose, we can observe simultaneously an intensification of conditioned responses and characteristic behavioural changes, and EEG activation. Finally, owing to the effect of high doses of agents stimulating the chemoreactive brain structures, considerable inhibition of conditioned responses, behavioural changes and a marked excitation of the neurones of the brain-stem reticular formation takes place. There is a prolonged EEG activation which is not observed in normal conditions when it is usually short term. Even electrical stimulation of a nerve induces changes for a few seconds, whereas under the effect of large doses of neurotropic agents, EEG activation is maintained for hours. This by itself is enough to cause marked behavioural changes in the animals. Behavioural changes cannot be looked upon as insignificant intensifications or reductions of conditioned responses. They are disruptions of conditioned reflex activity. The brainstem reticular formation i s blocked under the effect of agents inhibiting cholinergic and adrenergic brain structures. A consequence of this partial chemical deafferentation of the cortex is the reduction of the excitability of cortical neurons. At the same time, the association of afferent signals is disturbed, the animals lose contact with their environment and do not react adequately to environmental stimuli. Thus, under the effect of high doses of agents stimulating the cholinoreactive structures, as well as after the use of agents blocking these structures and exerting a strong influence on reticular mechanisms, we observe very similar changes in conditioned responses with sharply distinct EEG patterns. Behavioural changes will also be different because they depend not only on reticular mechanisms but also upon the mechanisms of activity changes in other functional systems of the brain.

CHOLINERGIC BRAIN MECHANISMS A N D BEHAVIOUR

145

One cannot as yet the judge role of one or tis other humoral mechanisms in complex emotional responses on the basis only of the changes of simple conditioned responses and the EEG. It is necessary to study the role of different chemoreactive structures and the influence exerted upon them by pharmacological agents in different emotional states. At the same time, various alterations of behavioural patterns under the effect of cholinergic agents are not in themselves proof of cholinergic mechanisms. These alterations could be the result of activity changes in any other functional brain system which is not included in the intimate mechanism of the given pattern of behaviour. It may be only reflected action, realized through a wide interrelationship, between the different brain functional systems. A more convincing proof is an isolated change of one of the brain functions, although it is quite difficult to obtain such an isolated effect. It was during the study of fear emotional reaction that a precise difference in the change of this reaction under the effect of drugs blocking the adrenoreactive and cholinoreactive brain structures was observed. The above experiments have shown that antiadrenergic agents do not block the fear reaction in isolation, whereas anticholinergic drugs produce an isolated blockade of this reaction. Such a blockade of the fear reaction by anticholinergic drugs and an intensification of it by anticholinesterases led us to suggest a hypothesis concerning the cholinergic mechanism of this reaction. What are the brain structures to which the chemical differentiation of different biological reactions are related? Some investigators attach great importance to the brainstem reticular formation in mechanisms of defensive reactions14.42. Undoubtedly the reticular formation plays an important role in the mechanisms of emotional behaviour. The effect of neurotropic drugs on the reticular formation cannot explain the emotional reaction as a whole, it can only account for some behavioural components. It seems that the primary area of the c.n.s. involved in emotional behaviour is mainly the limbic ~ystern~~~~23J6.53, whereas at the level of the reticular formation and hypothalamus only primitively organized, relatively indifferent, types of emotions (in the sense of Bradyls) can be formed. It would seem that hypothalamic structures are mainly responsible for the somatic-vegetative components of emotional reactions. It can be deduced, therefore, that the effect of pharmacological agents in altering emotional defensive reactions is related to the mechanisms of the limbic system. Unfortunately, the mechanism of cholinergic drug action upon the limbic system is not quite clear as yet, except for the fact that these substances, besides evoking EEG activation in the cortex and mesencephalic reticular formation, also produce marked EEG changes in the archaecortex and paleo~ortex2~*J3~6~. The presence of cholinergic neurones has been demonstrated by direct registration of neuronal activity in the septum and hippocampus under the effect of eserine or nicotine13~49,52.62~63. But it is still unknown whether the limbic system includes muscarinic neurons. As indicated above, an isolated inhibition of the emotional fear reaction is possible only under the effect of muscarinic anticholinergic drugs. That is why it is important to determine the presence of muscarinic cholinoreactivse tructures in the limbic system. References p . 146-148

I46

R. Y U . I L Y U T C H E N O K

I n the experiments performed in our laboratory by Yu. Ph. Pastukhoff a n intensification of hippocampal spike activity has been shown when muscarinic cholinergic structures were stimulated byarecoline. It is important to note that previous administraation of drugs blocking the central nicotinic cholinergic structures (gangleron) had no effect on spike activity of the hippocampal neurones following subsequent arecoline or galanthamine administration. The presence of muscarinic neurones in the limbic system and the changes in their activity under the effect of amysil or bensazine confirm our hypothesis concerning the important role of niuscarinic cholinergic structures of this system in mechanisms of emotional behaviour and memory. It is possible that, not only behaviour in general, but also, various patterns of emotional behaviour have different neurochemical mechanisms. However, further investigation of the mechanisms of action of these drugs and, consequently, of the role of cholinergic structures in mechanisms of emotional reaction, should be centred in the first place not on the study of their effect on brain regions and nuclei, but on definite functional brain systems. Analysis of data on the ascending reticular activating system allowed us to suggest the hypothesis3lP3Jthat each functional brain system has a characteristic set of mediators. Possibly it is not the anatomical structures that possess chemical specificity. We assume that there are functional brain systems which include functionally integrated neurones of different anatomic structures.

REFERENCES ADEY,W. K. (1959) Intern. Rev. of Nrurobiology, 1, 1-44. ALLIKMETS, L. KH. (1964) Zhurn. Nevropatologii i Psikhiutrii, 61, 1241-1248. ALLIKMETS, L. KH. (1964) Uchenyye zapiski Tartusskogo Cos. Uiziv., Tartu, vip. 163, 123-127. ANOKHIN, P. K . ( I 958) Vnutrenneye rormozhenie kak probleiiia fiziologii, Moskva, Medgiz. ANOKHIN, P. K . (I958) Elektroeticepplialograficheskiy analiz uslovnogo refleksa, Moskva, Megdiz. ARNOLD.M. R. (1945) Physiol. Rev., 52, 35-48. BERITOV,I . S. (1961) Nervnyye mekhanismi povetler.i,ya vi.rshikh pozvoizochnykh zhivotnykli. Moskva, izd-vo AN SSSR. 8 BOROUKLN, Yu. S. (1965) Material; 1 pribaltiyskoiy konferentsii 7kNIL’ov Metl. institiitov i fakul’tetov, Kauns. 50-52. 9 BOWARD, E. W. (1962) Perspect. in Biol. M e d ? 6, 116-127. 10 BRADLEY, P. B. A N D J . ELKES,(1957) Brain, 80, 77-1 17. 1 I BRADLEY, P. B. AND A. J. HANCE,EEC Cliri. Neurophysiol., 1957, 9, 191-215. 12 BRADLEY, P. B. A N D B. J. KEY,(1958) EEC Clin. Neurophysiol., 10, 97-110. 13 BRADLEY, P. B. AND A . N. NICHOLSON, (1962) EEC Clin.Neurophysiol., 14, 824. 14 BRADLEY, P. B. AND J. H. WOLSTENCROFT (1965) Brit. Med. Uull., 21, 15-18. 15 BRADY,J. V. (1958) In: Biologicaland Riocheniical Bases of Behaviour. Ed. by H. F. Harlow and C. N. Woolsey. The University of Wisconsin Press, 193~-235. 16 BUR&, J., 0.BURESOVA, Z. BOHDANECKq AND T. WEIS (1964) Ciba Foundation SyiTipObiUni jointly with the Co-ordinating Committee foi Syrnposis on Animal Behaviour and Drug action, 1 2 3 4 5 6 7

134.142. 17 COURVILLE, J., J . WALSH,A N I ) J. P. CCRDEAU, (1962) Science, 138, 973--974. 18 DELGADO, J . M. R. (1965) In: Pharmacology of Cmditioning, Learning and Retention. Oxford-

London-Edinburg-New-York-Paris-Frankfurt, Pergamon Press, Praha, Czechoslovak Medical Press, 133-156. 19 YELSEYEVA, A. C . (1965) V sb.: “Voprosi eksperimental’tzoy psikhiatrii”, Novosibirsk, 69-70.

C H O L I N E R G I C B R A I N MECHANISMS A N D B E H A V I O U R

147

EXLEY, K. A., M. C. FLEMING, A N D A. D ESPELtEN, (1958) Brit. J. Pharmacol. Chem., 13,485-492. FINK,M. (1960) EEG Clin. Neurophysiol., 12, 359-369. GARATTINI, s. AND I*. VALZELLI,(1965) serotonin, Elsevier, Amsterdam. GREEN,J. D. AND A. ARDUINI(1954) J. Neurophysiol., 17, 533. HERNANDEZ-PEON, R., G. CHAVEZ-IBARRA, P. J . MORGANE AND C. TIMO-IARIA (1963) Exp. Neurol., 8, 93-1 1 1. 25 HIEBEL, G., M. BONVALLET A N D P. DELL,(1954) Sernaine HGpit., Paris, 30, 2346. 26 HIMWICH, H. E., A . MORILLO AND W. G. STEINER (1962) J . Neuropsychiat., 3, suppl. I, 15-26. R. Yu. (1957) Zhurnal vysshey nervnoy deyarel’nosti, 7,2, 254-262. 27 ILYUTCHENOK, R. Yu. ( I 962) In : First International pharmacological meeting “Mode ofaction of 28 ILYUTCHENOK, drugs”. Vol. 8 : Pharmacological analysis of central nervous action. Oxford-London- New-YorkParis, Pergamon Press, 21 1-216. 29 ILYUTCHENOK, R. Yu. (1963) In: Biochemical pharmacology. Prague, Pergamon Przss, suppl. to 12, 270. 30 ILYUTCHENOK, R. Yu. (1961) Psychopharmacological Methods, Symp. Efect Psychotr. Drugs, Prague, Pergamon Press, 115-1 22. R. Yu. (1965) Neyro-gumoral’nye mekhanizmi reticulyarnoy formazii stvola mozga, 31 ILYUTCHENOK, Moskva, izd-vo Nauka. 32 ILYUTCHENOK, R. Yu. (1965) Clinical neurophysiology. EEG-EMG. 6th International Congress of electroencephalography and clinical neurophysiology, Vienna, 5 13-5 1 5. 33 ILYUTCHENOK, R. Yu. (1965) V sb. : Voprosi eksperimentalnoy psykhiatrii. Novosibirsk, 66-68. 34 ILYUTCHENOK, R. Yu. A N D A. G. YELISEYEVA (1966) X v I I l Infern. Psychol. Congress, Biological bases of memory traces, Moscow, pp. 62-64. 35 ILYUTCHENOK, R. Yu. AND M. D. MASHKOVESKIY (1961) Fiziologicheskiy zhur. SSSR, 47, 13521359. 36 ILYUTCHENOK, R.Yu. AND L. N. NESTERENKO (1965) Fiziol. zhuriz. SSSR, 51, 1177-1181. 37 JACOBSEN, E. (1964) In: Psychopharinacological agents. New-York, Academic Press Tnc., pp. 287-300. 38 JUNG,R. (1958) Klin. Wochenschri/t., 36, 1153. 39 KANAI,T. A N D J. C. SZERB(1965) Nature, 205, 4966, 80-82. 40 KRNJEVIC, K. ( I 964) Neuro-Psychopharmacol., 3, 260-264. 41 LABORIT, H.,C. BARONA N D B. WEBER (1965) Agressologie, 6, 655-720. 42 LINDSLEY, D. B. (1951) In: Handbook ofexperimentalpsychology.Ed. S. S. Stevens, New-York J. Wiley & Sons, pp. 473-516. 43 LONGO,V. G. (1962) EEG atlas for pharmacological research. Elsevier, Amsterdam. 44 LONGO,V. G. AND B. SILVESTRINI (1957) J. Pharmacol. Exp. Ther., 120, 160-170. H. W. (1958) The waking brain, Springfield, Illinois, Charles C. Thomas Pub],. 45 MAGOUN, 46 MASHKOVSKIY, M. D. AND R. Yu. ILYUTCHENOK (1961) Zhurn. Nevropath. iPsikhiat.,61, vip. 2, I 66-1 73. 47 MIKHELSON, M. YA. ( I 957) Fiziologicheskaya rol’ atsetilkholina i iziskanye novykh 1ekar.rtvennykh veshchestv, Leningrad, izd-vo Len. rned. ins-ta. 48 MITSKENE, V. P. AND A. M. MITSKIS (1965) Fiziol. zhurit. SSSR, 51, 544-546. M. AND W. ROMANOWSKI (1962) EEG Cliiz. Neurupkysiol., 14, 486-500. 49 MONNIER, 50 NAUMENKO, YE. V. (1965) Problemi endokrinologii i gormonoterapii, 4, 99-104. 51 NAUMENKO, YE. V. AND R. Yu. ILYUTCHENOK (1964) Pharmacol. i toksikol., 6, 670-672. 52 PETSCHE, H. AND CH. STUMPF(1962) Physiologie de I’Hippocampe. Colloq. Int. du CNRS, Paris, 107, 121-141. 53 PRIBRAM, K. H., W. A. WILSON,JR. AND J. CONNORS (1962) Exp. Neurol., 6 , 3 6 4 7 . (1 955) Diseases of the Nervous System, 10, 133- I4 1 . 54 RINALDI,F. AND H. HIMWICH 55 ROTHBALLER, A. B. (1956) EEG Clin. Neurophysiol., 8, 603-621. (1964) Science, 144, 3618, 493-499. 56 SALMOIRAGHI, G. C. AND F. E. BLOOM 57 SMIRNOV, G. D. AND R. J. ILYUTCHENOK (1962) Fiziol. Zhurn. SSSR, 48, 1141-1145. 58 SOKOLOV, TE. N. (1958) Vospriyatiye i uslovniy rejeks, Moskva, izd-vo Mosk. Univ., 59 SOLOMON, P. AND WYNNE (1950) Amer. Psychologist., 5, 264. R. (1963) J. Neurophysiol., 26, 127-139. 60 SPEHLMANN, 61 STEINER, W. G. AND H. E. HIMWICH(1962) Science, 136, 3519, 873-875. 62 STUMPF, CH. (1 964) In: Neuropsychopharmacology, Amsterdam, Elsevier, 241-244. 63 STUMPF, CH., H. PETSCHE AND G. GOGOLAK 1962, EEG Clin. Neurophysiol., 14, 212-219. 20 21 22 23 24

148

R. Y U . 1 I . Y U T C H E N O K

64 VOTAVA, Z., 0. BENESOVA, Z. BOHDANESK+, J. METYSA N D J . METYSOVA (1964) Poutery Hygieizyi Medycyny Do3 wiadczalnej, 18, 925-943. 65 VOTAVA,Z. AND M. V A N I ~ E(1956) K Physiol. Boheino>loven., 5, 460-467. R. P. AND L. D. BOYAJY (1959) Proc. Soc. Exptl. Biol. Med., 102, 479-483. 66 WHITE, 67 WHITE,R. P. AND E. A. DAICNEAULT (1959) J . Pharni. Exptl. Ther., 125, 339-346.

149

EEG and Behavioral Aspects of the Interaction of Anticholinergic Hallucinogens with Centrally Active Corn pounds T. I T I L

AND

M. F I N K *

Departmeut of Psyc’liairy at the Missouri lnvtiiute of Ps.vchiatr.v, ffniver.rii.v of Mi~soiiriSchool o f Merliciize, 5400 Arsenal Street, St. Lwis, Missouri 63139 (U.S.A.)

The changes in EEG and behavior after administration of anticholinergic drugs have been an intriguing problem for mor? than two decades. The EEG alterations induced by atropine, scopolamine, and a wide range of experimental compounds have been difficult to describe and the conclusions have been controversial. Although some investigators could detect but little change in the EEG after giving anticholinergic d r ~ g s ~ , ~most ~ J *agreed , that anticholinergics, especially those with psychotomimetic effects, do produce systematic EEG changes in animals and in man. Both increase of fast activity with desynchronization7,10~21and the increase of slow wave activity with synchronization have been describedspl3J4Js. But most interesting have been the observations of sleeplike patterns with high voltage slow waves and spindle activity in animals3~12,27,~*~35~36 and in man11g25. The discrepancy between sleeplike EEG patterns and apparent waking behavior of animals led to the term “dissociation of EEG and behavior” which has bem postulated to be a special feature of cholinergic mechanisms of the central nervous system. Clinical and EEG correlations after anticholinergic drugs as well as the differences between the anticholinergic-induced sleeplike state and natural sleep have been described in man in earlier reports11,16~17~1x,1g. To provide additional information concerning anticholinergic-induced EEG and behavioral changes, the present investigation was designed to study the interaction of Ditrant and atropine with a variety of centrally active agents, using quantitative methods for the measurement of the EEG changes.

-

* Present Address: Department of Psychiatry, New York Medical College. Aided, in part, by MH-I 1380 and the Psychiatric Research Foundation of Missouri. 7 Ditran (N-ethyl-3-piperidyl-cyclopentyl-phenyl-glycolate-hydrochloride) supplied by Lakeside Labs., Milwaukee. References p . 166-168

I so

T. I T I L A N D M. F I N K MATERIAL AND METHODS

A total of 291 investigations were carried out i n 84 male and female subjects between 17 and 57 years of age. Sixty-five were classified as schizophrenic ctates and nineteen as affective, emotional or personality disorders. For at least two months prior to the acute investigation, these subjects had received no psychotropic medication. The dosages, rate of administration and number of investigations for Ditran and atropine are shown i n Table 1. Second compounds were administered intravenously 30 to 40 min after the anticholinergic drugs. Eye movements, electrocardiogram, electromyogram and pulse rate were recorded simultaneously with the EEG and continuously up to one hour after each drug. Blood pressure was measured for periods of up to three hours. TABLE I COMPOUNDS, D O S A G b \ AND NUMBER O F T R I A L S

_

_

~

~

~

t h a g e (nrglkg) --

-~

Fin/ clriig Ditran Atropine

~

.~

-

Tittle of Adminiwatiotr

( m ~r. .v.)

_

_

Number of Trialr

0.0054.30 0.04 -0.50

5

171

5

77

0.02 -0.25 0.01 -0.50 1-2 llgglkg 0.15 -0.30 0.10 -0.30 0.02 -0.05 0.5 -1.5

10 5 5 5

53 10

10

4 5

Secnird or third tirug

Chlorpromazine Yohirnbine LSD-25 Amphetamine sulfate lmipramine Neostigmine methylsulfate Tetrahydroaminoacridin (THA)

10-20 10-20

5 4

41

EEG records (right occipital to ear or right occipital to right frontal leads) were analyzed quantitatively using an electronic resonant-filter frequency analyzer. The electronic frequency analysis (power spectral density analysis) consisted of the measurement of the mean pen deflection in millimeters in each of 24 frequency bands from 3 to 33 cjsec for each epoch of 10 sec. Six artefact-free samples, each of 10-sec duration, were selected from the record before the iiijection of the anticholinergic drug and between 25 to 35 min after each injection. In several investigations, the EEG records were analyzed by digital computer methods using both period analytic and powcr spectral density methods. The programs used were those developed for the TBM 1710 system, using digitizing rates of 320 samples per second and epoch lengths of 10 to 30 sec. Behavior ratings were done before and 30 to 40 min after each injection, using our psychopathological rating scale16 of 95 single items divided into 15 symptom clusters. ln addition to the behavior rating scale, psychological tests such as the BenderGestalt, carpet sign test, drawing and neurological examinations were carried out.

_

References p. 166-168

CHANGES IN

EEG A N D BEHAVIOR

0

W

Fig. 1. Low voltage slow EEG after Ditran administration

152

T. I T J L A N D M. FINK

RESULTS

Efiect of anticholinergic drugs The EEG and behavioral effects of atropine and Ditran varied with the dose given and could be classified into three distinct patterns. Relatively low doses of Ditran (0.005-0.05 mg/kg) or atropine (0.04-0.30 mg/kg) produced changes of consciousness and a drowsy state. At the same time, subjects exhibited restlessness, sometimes with agitation and poor coordination. Affective and emotional changes of anxiety, fear, depression or euphoria were frequent. Perceptual disturbances and visual hallucinations were occasionally reported. These clinical alterations were associated with a decrease in alpha-activity and increases in low voltage 5-7 cjsec theta-activity and superimposed fast beta-waves in the EEG (Fig. 1). In comparison to Ditran, atropine induced more slow waves and less superimposed fast activity (Fig. 2). These EEG patterns showed some similarity to the drowsiness or low voltage natural sleep EEG pattern (stage B, Loomis et or stage 1, Dement and Kleitmang).

RO RE

:

LF -LE

RF - R E

LP3 w

w

w

-

RP-RE LO-LE

RO-RE

E

EOM

e

4 frequency

w

*

md l l l ru rilllllllb K

G

G

inno

I

yser

-

-

1

-

-

I

-

-

!

-

I^--i

p//d(\&

EMG

Before Drug

”,

13 mins after Atropme I v. ( 0 3 r r g / k g )

H T Age 48 Record N o 4176

Behavior

drowsy, restless and ogllated

-schizophren o

Diagnoe

I

Fig. 2. Low voltage slow EEG after atropine administration.

a

C H A N G E S IN

EEG

153

A N D BEHAVIOR

RO - RE N

-

L

E

L

R F -RE

EOM

EMG

/ /

B e f o r e Drug

5

O

~

k

24mins after Atropine i v (0.3rng /kg)

Behavior stupor-like state with restlessness H T Age 48 Record No 4:76 Diagnosis schlzophrenx

Fig. 3. Slow wave spindle pattern after atropine administration. frequency onolyser chonnei

RO - RE ,

$

A

,

$

h

R

+-v

L F -LE

RF RP-RE RO-RE

EKG EOML

EMG

LF - R F

2 # 50 A V T

Before Drug

I l e c

23mins: a f t e r Ditran i.v. (0.3mg./kg.) Behavior : stupor. like s1o t e , psyc homo tor

H

Age 48 Record No 4374 Diagnosis schizophrenia T

Fig. 4. Slow wave spindle pattern after Ditran administration, References p. 166-168

agitation

154

T. I T I L A N D M. F I N K

With the highest doses of Ditran (0.04-0.30 mg/kg) or atropine (0.25-0.50 mg/kg) in some subjects, states of deep slecp and stupor developed and persistcd for sevei-a1 hours. They exhibited marked changes i n consciousness and impaired orientation. Communication was difficult, memory function was impaired and psychomotor activity was decreased. Accompanying these behavioral changes were markcd alterations i n EEG patterns. After atropine, alpha-activity decreased and 12-14 cjsec spindle activity dominated (Fig. 3). I n contrast to atropine, Ditran produced in the same subject less slow wave activity, more superimposed fast beta-activity and less spindle activity (Fig. 4). Although these EEG patterns are similar to the deep sleep pattern of natural sleep (stages C and D, Loomis et ~ 1 . ~ 3or 3 ; stages 2 and 3, Dement and Kleitmang), a study of the two states in the same patient showed significant differences. Sleep-like patterns induced by anticholinergic drugs are characterized by less spindle activity and more superimposed fast activity than natural sleep (Fig. 5). Also, during anticholinergic sleep-like activity, the EEG responses to acoustic stimulation were inhibited or abolished. Patients could open their eyes when told to do so, but no significant changes i n the EEG were seen (Fig. 6). With thc administration of Ditran in doses bctween 0.04 and 0.25 mg/kg or with atropine between 0.20 and 0.40 mg/kg i n most subjects, states of confusion and delirium

iye movement

--

-

27rnins after 136rng Ditran

I V

(behavior. stupor, sleep-like state with restlessness and confusion)

Natural Sleep

50uv

(behavior: deep sleep ) NN

Aqe 27

Dx S c h zophrenia Record Nos 1368, 1418

Fig. 5. Comparative EEG changes between Ditran-induced sleep-like pattern and natuml sleep in the same patient.

CHANGES I N

EEG

155

AND BEHAVIOR

were observed. Consciousness fluctuated and thought, association, memory and communication were disturbed. Subjects often reported visual hallucinatory phenomena. Motor activity, restlessness and athetoid movements were seen. Heart rate was

RO

Pre Drug Resting E E G

LF

RF

50 .uv

q

w

y 30rnins. after 13.6mg. Ditran

d I.V.

(0.21rng /kg.) N.N

Age 27

Dx.: Schizophrenio Record No.: 1368

Fig. 6. Response on eyes opening before and after Ditran administration. References p. 166-168

T. I T l L A N D M. F I N K

156 R

o

w

Re Drug Resting EEG

X

I

p

m

2 5 m i m after 1.4mg Ditran i.v.

i.,. I ,

+-

+4-

WE DRUG

24mIns. after 1.4mg. Ditran i.v,

M M A o . 42 Dr Schizophralno RbCOrd No I432

Fig. 7. The number of “carpet signs” in 60 sec pre- and post-Ditran administration in relation to the EEG alterations.

Eye mavement

EMG

V

-Ivc-_

3

LF frequency onoiyzer

+

RF

R

0

F

-

w

Resting EEG

50,~-

IBmins after 68mg Ditron I v (behovior confusional - delirious state)

NN

Age 27

Diagnosis. Schizaphrenm Record No 1480

Fig. 8. Ditran-induced (0.10 mg/kg) disorganization of the EEG.

CHANGES IN

EEG

157

A N D BEHAVIOR

Before Difron

.- - - - - - - - 1 5 - 2 5

I1210O l

Dilron

100 -

-i:

90

mins after I v , 2 4 m g ovg

Recurd Nos 26?2,1818. 1246,560, 2699, 580. 638,920, 2545, 2637,1286

-

0

;80 -

0

W

L

0) -

c

8

60-

c u a

E 50-

E

40

-

30

-

1 O

310

I

35

40

I

45

5'0

I

55

$0

710

65

I

80

3b

I

100

11'0 I I 4 5 I I $ ? I 2b0 I 2;s I 3 6 0 I 120 150 180 220 270 330

FHEPUENCIES

Fig. 9. Changes in EEG frequency spectra with Ditran administration in patients with delirium. (Average of 1 1 investigations.)

increased. The skin became dry, pupils dilated and the subjects complained of difficulty in speech and in near vision. Performance on the Bender-Gestalt drawing tests was impaired and the number of figures completed i n a symbol reproduction task was severely reduced (Fig. 7). The increase in psychomotor activity with hallucinations was more pronounced after Ditran while the changes in consciousness were dominant after atropine. In association with these behavioral changes after Ditran, the electroencephalogram exhibited a reduction of alpha-activity and the appearance of high voltage delta- and theta-waves with superimposed 20-40 clsec desynchronized fast betaactivity (Fig. 8). Epileptic activity in the form of high voltage spikes and sharp waves was occasionally observed. In a group of subjects with induced delirium, the frequency analyzer data showed decreases in alpha-activity and increases in slow wave activity (Fig. 9). The increase in superimposed fast beta-activity was poorly reflected i n the analog power spectral density analysis but was evident on visual inspection and in the digital computer period analytic methods (Fig. 10).

Refrrmrrs p. 166-168

158

T. I T I L A N D M. F I N K

90

-

80 7060 -

50

W

-

40 -

I

i-

s

30

-

20 -

10-

----d

~

Be.

t Ditran i.v. Inj

+

50"

+

4'40"

+

9'35"

+39'40" f

+9'10"

MINUTES DITRAN = 3.7mq / 5 m i n s . THA = 6 0 m g / 9mins Leads = R FRONTAL- OCCIPITAL

-

Digitized 3 2 0 S P S DlTRAN SERIES MIP - 7 / 6 4 Fleming - 00 I

Fig. 10. Period analysis. First derivative - percent time.

Ititeruction of anticholinergic drugs with oilier compounds The relation of the EEG patterns to changes in behavior are more clearly defined in the changes induced by the subsequent administration of centrally active drugs to patients during experimental deliria. When intravenous chlorpromazine was given in very small doses (0.02-0.25 mg/kg) 30 to 40 min after Ditran or atropine, the psychomotor aspects of the anticholinergicinduced delirium were reduced and the alterations i n consciousness intensified. The subjects exhibited stupor or coma with a lack of response to acoustic and painful stimuli. Pupils were dilated, deep tendon reflexes diminished, respiration became more shallow and heart rate slowed. Associated with these behavioral changes were further alterations of anticholinergic-

CHANGES IN

22mins a f t e r Chlorpromozine i v (05mg/kg/ 159n11nsa f t e r D i t r o n )

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

.

Behavior: __

EEG

AND BEHAVIOR

I59

sub-coma

H I Age 4 2 Record No. 5 2 0 Diagnosis. !chizophrenia

50-

E E

25

-

gL.1

t

I

I

1

'

1

'

I

'

30 3 5 40 4 5 5 0 55 6o 65 70 80 9

1

0

' 1 ' I 1 1 1 . 1 ' ' 1 1 '~ o ~~ ~ ~ ~1 3 5 ~ ~ ~ ' 6 5 ~ ~ ~ 2 0 0 ~ ~ 0 2 4 5 ~ ~ ~ 3 0 0 ~ ~

FAEOUENCIES

Fig. 11. Changes in EEG frequency spectra with Ditran and following the addition of chlor promazine.

induced EEG changes with increases in amplitude, marked enhancement of slow wave activity, decreases of fast beta-waves and the predominance of 8 to 12 cjsec spindle patterns (Fig. 11). The response to acoustic stimulation, which could not be evoked prior to chlorpromazine administration, reappeared i n the EEG. After yohimbine (0.0 1-0.50 mgjkg) the changes in consciousness induced by Ditran were lessened. Patients became alert and their speech more relevant, but motor restlessness and irritability persisted or even increased. Perceptual distortions were not altered. In the EEG, the slow delta-activity decreased and theta-activity increased. In some instances, beta-activity also increased and some alpha-waves recurred (Fig. 12). With LSD 25 (1-2 pgikg), the Ditran-induced changes in consciousness decreased and psychomotor activity and hallucinatory phenomena increased. Patients became more alert and communicated more easily. In the EEG, Ditran-induced slow wave activity was reduced, amplitudes decreased and fast activity (over 22 cjsec) was potentiated (Fig. 13). With dextro-amphetamine (0.15-0.30 mg/kg), the Ditran-induced changes of consciousness and psychomotor activity both decreased slightly. Patients became alert and responsive, and less irritable. There was a flattening in the amplitude of all frequencies in the EEG with a decrease in theta, alpha and beta activity (Fig. 14). References p . 166-168

T. I T I L A N D M. F I N K

I60

* m L

Before Gruq

50LVT

26rnns after Yohmbvle '62rnins after Ditron)

"1 lo

t

iv

IFec

(05mgIhg)

J+-w-+J--.

F C Age 50 R e c Nu 605 Diagnosis: schiz0phren.a

310

I

35

40

45

40

Tz-'

7'0

55

65

I

RO

9'0

110 '

100

'

120

li5

'

150

Id5 180

260

'

2 4 5 I 360 I 220 270 330

FREOUENCIES

Fig. 12. Changes in t E G frequency spectra with Ditran and following the addition of Yohirnbine.

Imipramine (0.10-0.30 mg/kg) given intravenously 30 to 40 min after Ditran reduced the delirium in some subjects. While thought disorder and hallucinatory phenomena were still prominent, the subjects were more alert, better oriented and more responsive. Psychomotor activity persisted. In some subjects, the EEG exhibited a decrease of slow wave activity, a marked increase of alpha-waves and a persistence of beta-activity (Fig. 15). Intravenous neostigmine (0.02-0.05 mg/kg) did not significantly alter the Ditraninduced psychotic state. The changes in consciousness seemed less and psychomotor activity and agitation were occasionally increased. These central changes were masked by the severe peripheral gastro-intestinal and vascular symptoms. In the EEG there were no significant changes (Fig. 16). Tetrahydroaminacrin (THA) (0.5-1.5 mg/kg) blocked almost completely the Ditran-induced confusional-delirious state. Vigilance was restored, motor restlessness and agitation diminished, the disturbances i n thought and association ameliorated and hallucinatory phenomena were inhibited. In the EEG also, the Ditran-induced changes were reduced after TH A. Slow activity decreased markedly, and fast a-activity recurred (Fig. 17).

CHANGES I N

EEG

AND

BEHAVIOR

Resting EEG

50,uv

161

1"'".

____________.

15mins. acter Ditran i.v. (4.8mg.l

15 rnins. after LSD-25 i.v. (0.2mg.)

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

I60 f

L.W. Age 45 Rec.No.lB8I

140 -

-

120

m W

.

a

2 loo-

0 (0

.B

-"

80

-

0

r u

600)

E E

40-

35

45

55

65

80

100

120

150

180

22.0

270

330

FREQUENCIES

Fig. 13. Changes in EEG frequency spectra with Ditran and following the addition of LSD-25.

DISCUSSION

The behavioral and EEG changes induced by Ditran and atropine depend on the dosage and the pre-drug resting EEG17. Quantitative analyses have shown that the administration of both these anticholinergic drugs is associated not only with an increase in slow wave activity, but also with a concurrent increase of superimposed fast beta-activity. The ratio of slow activity to fast activity is related to the type of clinical syndrome observedlg. When alpha-activity was replaced by 2 to 5 c/sec slow waves and 30 to 50 c/sec fast activity, a confusional-delirious state with increased psychomotor activity occurred. The appearance of 1 to 4 c/sec slow waves and 12 to 18 c/sec fast activity was associated with a stuporous state, characterized by severe changes in consciousness, but less psychomotor activity than in the delirious state. Compared to atropine, Ditran induced greater increases in fast activity but fewer slow waves. This difference was clinically related to greater psychomotor activity but lesser degrees of stupor. References p . 166-158

162

T.ITIL AND M.FINK

/ -

Before Drug

50pv 14mins. a f t e r D i t r a n i v ~ 0 0 5 m p / k g l --'p'q p. Behavior. confusional delirious state

14 mins after Dexlrc- Amphelamine iv ( (43mlns after Ditranl

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

0

3

.....

m

g

f

Behovlor: decrease

k

q

)

~

,

h

.

~

+

of psychamclor a c t i v i t y

W L . A g e . 4 4 R e c o r d No: 1867 Diagnoshs. chronic alcoholism

-

25t l

$0

I

35

410

45

;O

I

55

$0

I

65

;O

80

9'0

I00

Ill0

I20

1i5 I SI 0 IQ5180 I ZdO I 2 4 5 3d0 220 270 330

FREOUENCIES

Fig. 14. Changes in EEG frequency spectra with Ditran and following the addition of dextroamphetamine.

The relationship between changes in behavior and their EEG correlates became even more distinct when the syndrome induced by anticholinergic drugs was modified by the subsequent administration of various adrenergic or cholinergic blocking or sensitizing agents. Chlorpromazine, given after anticholinergic drugs, even in very low doses (1 to 2 mg), altered the anticholinergic-induced syndrome with extreme inhibition of psychomotor activity and a further decrease in the level of consciousness. Delirium was converted to a state of coma. The EEG counterpart of this state was a marked potentiation of slow wave activity. A similar potentiation of anticholinergic (atropine) induced slow waves by chlorpromazine was observed in animals by Bradley and Hance4, but they reported no behavioral association. Various degrees of alerting occurred with the administration of several compounds subsequent to anticholinergic drugs. Alerting was associated with decreased psychomotor activity after amphetamine, yohimbine and imipramine, increased hallucinatory phenomena and motor activity after LSD, and almost complete blocking of hallucinations as well as motor activity after THA. These behavioral alterations were accompanied mainly by decreases in slow and increases in fast wave activities. After neostigmine, neither behavioral nor EEG changes were observed.

~

~

CHANGES IN

EEG

163

A N D BEHAVIOR

PRE E E G 50pv

38mins after lmipromine i v 2 0 m g ( 7 9 m i n s a f t e r Ditran)

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

y1

-?

"

Behavior. clearing of consciousness, subdelirious

slate

C.B. Age:39 Rec No 1636 Dx.: Personolily Disorder

1201

100 -

u)

0

'D

80-

c

P

60m a c 40-

i 20

-

, 3b

I

35

4b

'

45

;O

'

55

CO-+O

65

'

80

40

'

I:O

100

I

120

If5

'

150

I85

'

180

'

260 I 245 360 I 220 270 33C

FREQUENCIES

Fig. 15. Changes in EEG frequency spectra with Ditran and following the addition of imipramine.

These findings agree only in part with the earlier investigations in animals2Jp5. They do not support the suggestion that the correlation of EEG and behavior changes after cholinergic potentiating and blocking agents is poor in contrast to the relation of EEG and behavior changes after adrenergic sensitizing and blocking agents29. Our results do confirm, however, the reports which emphasize the differences between cholinergic and adrenergic activating me~hanisrns~5.~4,~~.~8.29. These investigations suggest that anticholinergic drugs, particularly those with psychotomimetic properties, exhibit a dual activity. They appear to stimulate two nonspecific subcortical mechanisms with opposite functions. It is possible that the concurrent activation of the medial ascending reticular activating system which exercises mainly cortical inhibition (and is associated with slow activity in the EEG), and the medial thalamic diffuse projection system, which has predominantly a facilitating influence on the cortex (and is associated with fast activity in the EEG), causes confusional delirious behavior. After low doses of anticholinergic drugs, central excitatory mechanisms appear to predominate, while after high doses, inhibitory functions seem to take precedence. In the modification of the anticholinergic syndrome by other compounds, the kind and speed of the functional alterations of these mechaD o & ~ o n , ~ on c

I66-IAR

T. I T I L A N D M. F I N K

164

l3mins afler Neos!igmine i v l 0 0 1 2 m g / i g ) (63rnins after Difran)

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

_-Schov'or.

...........

"

o r a k e and responr:ve E.F. Age 56 Record tlo 864-

A -

175

-

150

-

Diagnosis .~

chronic alcoholism

0 VI

5

125-

v) u

0

- loo._ : - 750 c

0

U u

u c

a

50

-

25

-

E

$0

I

35

4'0

'

45


55

6'0

I

65

7'0

'

80

9'0

I

Ilb

100

'

133

120

'

150

Id5

iao

'

'

260 I 2h5 360 220 270 330

FREQUENCIES

Fig. 16. Changes in EEG frequency spectra with Ditran and following the addition of neostigmine.

nisms are related to the nature of the behavior changes, and appear to be dependent on the EEG characteristics of the compound used. Drugs which induce slow activity and synchronization (chlorpromazine) alier the delirium induced by anticholinergic drugs to a comatose state. Compounds that have EEG acceleration and desynchronization properties (LSD, yohimbine and amphetamine) reduce changes in consciousness but not psychomotor activity. Drugs which inhibit slow and fast activity as well as reproducing alpha rhythm (THA and, to a lesser degree, imipramine) block both the anticholinergic-induced consciousness changes and psychomotor phenomena. In these interactions the dose of the second drug and the time between each injection are important factors. For example, in milligram doses chlorpromazine modifies the Ditran-induced delirium to coma, while in microgram doses it reduces the consciousness changes and psychomotor activityl6.20. Pharmacologic "dissociation of EEG and behavior" reported in animal experim e n t ~ ~ , ~ was , ~ not 7 , substantiated ~ ~ * ~ ~ in our studies. By comparative investigations in the same subjects, it was possible to determine quantitative and qualitative differences between anticholinergic-induced slow wave spindle patterns and spontaneous sleep activity's. Behavior characteristics were also different in these two states. Therefore,

CHANGES I N

EEG

165

A N D BEHAVIOR

the syndrome induced by anticholinergic drugs must be considered a distinct entity rather than a reproduction of natural sleep. Recent studies using avoidance and discrimination tasks also suggest there is no “dissociation” of EEG and behavior after anticholinergic drugs in animals 22~26.3oJ1932.

---

[ o m.-_----------i n s . after Ditran iv. (O.lmg./kg.I

Behavior: delirium -

3 6 m i n s . a f t e r T H A i.v. (1.3rng./kgl ( 7 3 r n i n s . a f t e r Dilran)

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

120

7

Behavior: reduction -

of delirium, increased alertness

’

HI. Age: 42 Record No: 474 Diagnosis: Schizophrenia

c

100 -

V c 0 0

2

-

80-

W

c

”0 -

60-

L

V 0

c

40-

a E E 20-

I $0

’

35

40

’

40

5b

’

55

6’0

’

6 5

710 I 40 I 11’0 1s; 80 100 I20

1$5 I50

180

2bO I 2b5 3bO I 220 270 330

F REOUENCIES

Fig. 17. Changes in EEG frequency spectra with Ditran and following the addition of THA.

Rather than speak of a dissociation of EEG and behavior after anticholinergic drugs the observations, particularly in human studies, suggest a close relationship between changes in the EEG with changes in those aspects of behavior represented by the concepts of “differentiated consciousness” as well as complex, discriminative psychomotor performance6. Taking into account the conditions such as the speed, quality and quantity of EEG changes, these observations are in agreement with the concept advanced by White34 that any drug which imposes a marked EEG alteration in a short period of time, whether adrenergic, cholinergic or otherwise, may be expected to significantly alter important facets of behavior.

References p . 166-168

I66

T. I T I L A N D M. F I N K SUMMARY

After the anticholinergic drugs Ditran and atropine, distinct clinical alterations have been found which vary with the drug and the dosage. Quantitative EEG analysis demonstrated that the clinical changes are accompanied by characteristic EEG alterations. In relatively low doses, Ditran or atropine produce changes of consciousness with a drowsy state. These behavioral changes are associated with EEG alterations of a decrease of alpha-activity, increase of low voltage 5-7 c/sec theta-activity and superimposed fast beta waves. After a moderate to high dose of Ditran and atropine, a confusional-delirious state was observed. With this clinical syndrome, the EEG presented a marked disorganization of alpha activity and an increase of slow and superimposed fast beta waves. After high doses of Ditran or atropine, deep sleep or stuporous behavior was seen in some subjects, with the simultaneous increase of slow activity and the appearance of spindle patterns in the EEG. Comparative investigations in the same subjects have shown that anticholinergicinduced drowsiness or sleeplike states are clinically and electroencephalographically different from physiological drowsiness and sleep. Atropine, in comparison to Ditran, induced more changes in consciousness but less psychomotor activity. The close correlation between EEG changes and behavior alterations, which were obvious by quantitative analysis, were even more prominent when anticholinergic drug induced EEG changes were further altered experimentally. Drugs whichinduce slow activity and synchronization in the EEG, such as chlorpromazine in very small doses, modify the anticholinergic-induced delirium to coma. The EEG showed further slowing, decrease of beta waves, and increase of amplitude and spindle activity (8-12 clsec). In contrast, drugs which produce fast activity in the EEG (yohimbine, amphetamine and LSD) reduce the anticholinergic-induced consciousness changes: slow activity decreased and fast waves were potentiated. THA and, to a lesser degree, imipramine blocked the anticholinergic-induced delirium. Reduction of slow and fast waves and reoccurrence of alpha activity were seen. These investigations have shown that the EEG changes in man have surprisingly accurate correlations with “differentiated consciousness” and complex psychomotor alterations. The important factor is the speed of the induced changes. The mord sudden the EEG changes, the closer the correlation with behavior or vice versa.

REFERENCES AND BABSKII, E. B. (1950) Electrophysiological analysis of the action of acetylcholine on nerve centers. 11. Action of eserine, prostigmine and atropine on the electrical activity of the visual lobes of a frog. Fiziol. Zh., 36, 151-160. 2 BRADLEY, P. B. AND ELKES, J. (1953) The effect of atropine, hyoscyamine, physostigmine and neostigmine on the electrical activity of the brain of the conscious cat. J. Physiol. (Lond.), 120, 14-15. 3 BRADLEY, P.B. AND ELKES, J. (I 957) The effects of some drugs on the electrical activity of the brain. Brain, 80, 77-1 17.

1 ARTEMEV, V. V.

CHANGES I N

EEG

A N D BEHAVIOR

167

4 BRADLEY, P. B. AND HANCE, A. J. (1957) The effect of chlorpromazine and methopromazine on the electrical activity of the brain in the cat. Electroenceph. clin. Neurophysiol., 9, 191-215. 5 BRADLEY, P. B. AND KEY,B. J. (1958) The effect of drugs on arousal responses produced by electrical stimulation of the reticular formation of the brain. Electroenceph. clin. Neurophysiol., 10,

97-1 10. 6 CAZZULLO, C. L. AND MANCIA,M. (1964) Psychopathological aspects of the relation between vigilance and consciousness, Acta Neurochir., 12, 366-378, D., GIURGEA, C. AND DROCON, G. (1955) Electrographic study of the non-specific 7 DANIELOPOLU, pharmacodynamics of the stimulatory effect of atropine on the cerebral cortex. Fiziol. Zh., 41, 60 1-6 1 1. 8 DARROW, C . W., PATHMAN, J. H. AND KRONENBERG, G. (1945) Autonomic function and EEG. Fed. Proc., 41, 16. 9 DEMENT, W. AND KLEITMAN, N. (1957) Cyclic variations of EEG during sleep and their relation to eye movements, body motility, and dreaming. Electroenceph. d i n . Neurophysiol., 9, 673-690. 10 FINK,M. (1960) Effect of anticholinergic compounds on postconvulsive electroencephalogram and behavior of psychiatric patients. Electroenceph. d i n . Neurophysiol., 12,359-369. I1 FLUGEL,F. AND ITIL, T. (1962) Klinisch-elektroencephalographische Untersuchungen mit “Verwirrtheit‘’ hervorrufenden Substanzen. Psychopharmacologia, 3, 79-98. 12 FUNDERBURK, W. H. AND CASE,T. J. (1951) The effect of atropine on cortical potentials. Electroenceph. elin. Neurophysiol., 3, 21 3-225. 13 GIBBS,F. A., GIBBS,E. L. AND LENNOX, W. G. (1937) Effect on the electroencephalogram of certain drugs which influence nervous activity. Arch. Jnt. Med. (Chic.), 60, 154-166. 14 GROB,D., HARVEY, A. M., LANGWORTHY, 0. R. AND LILENTHAL, J. L., JR.(1947) Administration of di-isopropyl fluorophosphate (DFP) to man. 111. Effect on the central nervous system with special reference to the electrical activity of thz brain. B d l . Johns Hopkins Hosp., 81, 257-266. 15 HIEBEL, G., BONVALLET, M., HWE, P. AND DELL,P. (1954) Analyse neurophysiologique de l’action centrale de la d-amphetamine (maxiton). Sem. HGp. Paris, 30, 1880-1887. 16 ITIL,T., NEUBAUER, H. AND KESKINER, A. (1967) Potentiztion of anticholinergic inducad EEG slow wave activity by phenothiazines and the behavioral correlations in man. Presented at the International Congress of EEG and Clinical Neurophysiology, Vienna. (in press) 17 ITIL,T. (1966) Quantitative EEG changes induced by anticholinergic drugs and their behavioral correlates in man. Recent Advances in Biological Psychiatry, Vol. 8 J. Wortis, Editor, New York, Plenum Press (pp. 151-173). 18 I ~ LT., (1966) Anticholinergic drug induced “sleep” and its EEG correlation in man. Presented at the Meetkg of the Associationfor Psychophysiological Study ofSleep, Gainesdle, Florida. (in press) 19 ITIL,T. AND FINK,M. (1965) The questios of dissociation of EEG and behavior. Presented at the American EEG Society, New York. 20 ITIL,T. AND FINK, M. (1967) Anticholinergic drug-induced delirium (experimental modification, quantitative EEG and behavioral correlations). J.N.M.D. (in press). 21 LECHNER, H. (1956) On the influence of anticholinergic drugs on the EEG of recent closed craniocerebral injuries. Electroenceph. clin. Neurophysiol., 8, 714-715. 22 LINDSLEY, D. F., CARPENTER, R. S. AND KILLAM, E. K. (1965) EEG and discrimination performance in cats under atropine. I. Light-dark and pattern discrimination. Fed. Proc., 21, 516. 23 LOOMIS, A., HARVEY, N. AND HOBART, G. A. (1935) Further observations on the potentisl rhythms of the cerebral cortex during sleep. Science, 2, 198-200. 24 MARAZZI, A. S. AND KING,E. E. (1950) Effects of humoral agents on corticil evoked potentials in monosynaptic preparations. Fed. Proc., 163, 732. 25 OSTFELD, A. M., MACHRE, X. AND UNNA,K. R. (1960) The effects of atropine on the electroencephalogram and behavior in man. J . Pharmacol, Exp. Ther., 28, 265-272. 26 Rrccr, G. F. (1963) Electrocortical correlates of avoidance conditioning in the monkey and their modifications with atropine. Biochem. Pharmacol., 12, (Suppl.) 268-269. 27 R~NALDI, F. AND HIMWICH, H. E. (1955) Alerting responses and actions of atropine and cholinergic drugs. A.M.A. Arch. Neurol. Psychiat. (Chic.), 73, 387-395. 28 RINALDI, F. AND HIMWICH, H. E. (1955) Cholinergic mechanisms involved in function of mesodiencephalic activating system. A.M.A. Arch. Neurol Psychiat. (Chic)I 73, 396-402. 29 ROTHBALLER, A. B. (1956) Studies on the adrenaline-sensitivecomponent of the reticular activating system. Ekctroenceph. clin. Neurophysiol., 8, 603-621. 30 ROUCEUL, A., VERDEAUX, J. AND GOGAN,P. (1965) Limits of the dissociation between EEG and behavior under atropine like drugs in cats. Int. J. Neuropharmaeol., 4, 265-272.

168

T. I T I L A N D M. FINK

31 SADOWSKI, B. AND LONCO,V. G. (1962) Electroencephalographic and behavioral correlates of an instrumental reward conditioned response in rabbits. A physiological and pharmacolopical study. Electroenceph. elin. Neurophysiol., 14, 465-476. 32 SEIDEN, L. S., KOENIG, J. AND KILLAM, K. F. (1965) EEG and discrimination performance in cats under atropine. IT. Total luminous flux (TLF) discrimination. Fed. Proc., 24, 516. W. C., GREEN, R. E., MCNAMARA, B. P. AND KROP,S. (1948) Influence of atropine and 33 WESCOE, scopolamine on the central effects of DFP. J. Pharmacol., 92,63-72. 34 WHITE,R. P. (1965) Some motor and electrical signs of drug action. Horizons ir! neuro-psychopharmacology. Progress in B*ain Research. Vol. 16, New York, Elsevier, pp. 169-183. 35 WHITE,R. P., NASH, C. B., WESTERBEKE, E. J. AND POSSANZA, G. J. (1961) Phylogenetic comparison of central actions produced by different doses of atropine and hyoscine. Arch. int. Pharmacodyn., 132, 349-363. 36 WIKLER,A. (1952) Pharmzcologic dissociation of behavior and EEG “sleep patterns” in dogs: morphine, n-allynorphine, and atropine. Proc. SOC.Exp. Biol. Med., 79,261-265. 37 WILSON,W. P. (1961) Observations on the effect of toxic doses of atropine on the electroe ncephalogram of man. 1.Neuro,pychiat., 2, 186-190. 38 WILSON,W. P. AND HUGHES,J. L. (1964) Observations on the effect of JB-329 (Ditran) on the electroencephalogram of man. J . Neuropsychiat., 5, 310-315.

I69

Discussion E. JACOBSEN Department of Pharmacology, Royal Danish School of Pharmacy, Copenhagen

I am a little surprised at the sharp distinction made in some of the papers between the “muscarinic” and “nicotinic” action of acetylcholine. It was presumably H. H. Dale who first distinguished between these two apparently different effe:ts of acetylcholine. When he analysed the action of choline esters, including acetylcholine, in 1914 ( J . Pharmacol., 6, 145), nothing was known about the r6le of acetylcholine as a transmitter of nerve impulses. His description at that time of the twosided effezt was absolutely justified, and has been faithfully passed on from textbook to textbook. Now, it must be realized that the “mus:arinic effe:t” and most, if not all, of the “nicotinic effect” is caused by the same action, presumably a depolarization of a membrane, only in anatomically different places, such as smooth muscles and salivary glands (like muscarine) or peripheral ganglia, endplates of striated muscles and stimulation of the adrenal medulla with ensuing rise of blood pressure (like nicotine). The inhibitory action of acetylcholine in large concentrations which is mentioned in many textbooks as one of the typical nicotinic actions, is in many instances due to a prolongcd membrane depolarization, so that further depolarization cannot take place and stimulation of released or added acetylcholine will remain without effect. It must therefore be realized that both the muscarinic and the nicotinic actions, a t least peripherally arc in principle caused by the same mechanism. Whether or not this is also the case within the central nervous system seems to be ignored. Acetylcholine is acting differently on different neurons, but until the mechanism of this action is known, it will be highly misleading to use terms based on an old descriptive analysis of the peripheral action of acetylcholine.

This Page Intentionally Left Blank

171

Glossary adiphenine 2-Diethylaminoethyl diphenyiacetate HCI AHR-376 I-methylpyrrolidin-3-ylalpha-phenylcyclopentylglycolate HCl Amiti iptyline 5- (3-dimethylaminopropylidene)-l0,1l-dihydro-5H-dibenzo (a, d) cycloheptatriene HCI

amobarbital

arecoline Methyl-n-methyltetrahydronicotinate HBr atropine dl-tropic tropanol amyzyl, a m i d /?-Diethylaminoethylbenzilate HCI

BA 1433 (WH 4849) a-phenyl-a isopropyl glycolic acid-3-N, N dimethylaminopropyl ester benztropine methane sulfonate 3-Diphenylmethoxy-tropanemethanesulfonate

bulbocapnine caramiphen I-Phenylcyclopentanecarboxylicacid-2-diethylaminoethyl ester-1,2-ethanedisulfonate

carbocaine centrophenoxine 2-dimethylaminoeth yl-p-chlorphenoxy acetate

Transentin(e) Spasmolytin

(Ciba) (Robins)

Elavil RO4-1575 Saroten Tryptizol MK 230 (Merck) Amytal am ylobarbitone Dorminal Barbamil (Lilly) (Penick)

Suavitil Tranquilline Lucidyl Nerva t il

Cogentin Cobrentin MK-02 Cogetin Cogentinol

(Merck)

(Merck) (Penick)

G 2747 Merpanit Panparnit parpani t mepivacaine ANP 235 WIN 91212 Lucidril

chlorpromazine 2-Chloro-1O-[3(dimethylamino)propyl]-pheno- Thorazine Megaphen thiazine HCI Aminazine Largactil

(SKF) (Rh6ne-Poulenc) (Bayer)

172

GLOSSARY

Darstine mepiperphenidol desmethylimipramine (desipramine) 10,ll -Dihydro-5-(3-methylaminopropyl)-5 Hdibenz[b,f]azepine HCI

di-isopropylfluorophosphate (DFP) DMPP I1l-dimethyl-4-phenylpiperidinium iodide eserine physostigmine ethopropazine 10[2-(diethylamino)propyl]-phenothiazine

Euniydrine gallamine triethiodide Hexamid 5,5,phenyl ethyl-3-(beta diethylamino ethyl)2,4,6-trioxohexahydro-pyrimidinhydrochloride h yoscine imiprarnine 5-[3-(Dimethylamino)propyl]-I0,l I -dihydro5H-di benz[b,f]azepine

Pertofran(e) Norpramin (3-35020 desmeth ylimipramine JB 8181 Pertofrina (Geigy) norimipramine (Lakeside) Isoflurophate Floropryl dimeth y lphen y lpiperazinium (Burroughs Wellcome) (Merck) (Parke-Davis) ethopromazine Dibutil Parsidol (Bayer) RP 3356 (Warner) SC 2538 (Rbhe-Poulenc) methylatropine Metropine Flaxedil

(Nordmark) scopolamine Tofranil G-22355 Imizin Deprinol

JB 305 N-ethyl-3-piperidyl diphenylacetate JB 318 n-ethyl-3-piperidyl benzilate JB 329 n-et hyl-2-pyrrolidylmethylphenylcyclopentylglycolate HCI and n-ethyl-3-piperidyl phenylcyclopentyl glycolate HCI Ditran JB 340 n-dimethyl-3-piperidyl diphenylacetate LSD-25 mecamylamine mescaline sulfate

3,4,5-Trimethoxy-phenethylarninesulfate methylscopolamine Br

(Geigy) (Lakeside) (Lakeside)

(Lakeside) (Lakeside)

lysergic acid diethylamide Delysid 1ysergide (Sandoz) inversine HCI (Merck) Mezcaline (Penick) scopolamine methylbromide Parnine bromide methylscopolamine nitrate Skopolate Nitrate

173

GLOSSARY

n-allylnormorphine

neostigmine methylsulphate nortriptyline HCI 5-(3-methylaminopropylidene)-10,11dihydro5H dibenzo (a,d) cyclohepten HCI pentamethylenetetrazol 6,7,8,9-tetrahydro-5H-tetrazoloazepine pentobarbital sodium piridrole a,a-Diphenyl-2-piperidinemethanol hydrochloride pontocaine procaine p-Aminobenzoyldiethylaminoethanol psilocybin 3(2-Dimethylamino)-ethylindol-4-ol dihydrogen phosphate reserpine Methyl-18-0-(3,4,5-trimethoxybenzoyl) reserpate

RO 2-3202/2 3-diphenyl acetoxy-quinuclidine sulfate dihydrate RP 7360 Diethylaminoethylphenothiazineyl-1O-dithiocarboxylate RS 86 (spiro[n-methyI-piperidyl-4]n-ethylsuccinimide HBr Sarin isopropyl methylphosphonofluoridate tetrah ydroaminacrin (THA)

thiopental sodium tremorine 1,4-Dipyrrolidino-2-butyne tri hexy phenidyl alpha-Cyclohexyl-gamma-phenyl-gamma (1piperidine) propanol HCI

Nalorphine allorphine an torphine Nalline Anarcon Lethidrone Prostigmin

(Merck) (Roche)

Aventyl desmethylamitriptyline Arentyl (Lilly) pentylenetetrazol Metrazole (Knoll) Nembutal (Abbott) pipradrol hydrochloride Meratran (Merrill) tetracaine (Winthrop) (Abbott) (Sterling-Winthrop) CY-39 psilotsibin lndocybin

(Sandoz)

Serpasil Raused R eserpoid Sandril

(Ciba) (Hoffman-La Roche)

(Rhone-Poulenc) (Sandoz)

tetrahydroaminoacridine hydrochloride hydrate THA aminacrin tacrine (Lakeside) pentothal (Abbott)

Artane benzhexol HCI Pacitane Pargitan Cyclodol (Lederle)

174

GLOSSARY

trimethidinium methosulfate N-(y-trimethylamrnoniumpropy1)- N-methylcamphidinium-dimethvl sulfate tropazine WIN-2299 2-diethylaniinoethylcyclopentyl(2-thienyl) glycolate HCI yohimbine

trimethidinium Tropacin tropine diphenylacetate hydrochloride (Sterling-Winthrop) Quebrachnine corynine aphrodine

175

Author Index Abood, L. G., 18, 19, 20,22, 106 Adey, W. R., 145 Adkins, F. J., 35 Allikmets, L. Kh., 134, 145 Anokhin, P. K., 137, 142 Aprison, M. H., 35, 58 Araguete, A., 21 Arduini, A., 145 Arnold H., 87, 100 Arnold, M. B., 137 Barnard, G. L., 35 Beleslin, D., 37 Bell, C., 15 BeneSova, O., 22,40-47, 142 Bente, D., 106 Beritov, I. S., 136, 145 Berry, C. A., 68 Bickel, M. H., 22 Biel, J. H., 18, 19, 22, 106 Bignami, G., 78 Bijlsma, U. G., 14, 21 Bloom, F. E., 143 Bogdanski, D. F.. 21 Bohdanecky, Z., 22, 40-47, 61, 62, 68, 77, 78, 79, 134, 142 Bonhoeffer, K., 86, 87, 102 Bonvallet, M., 140, 163 Borodkin, Yu. S.. 145 Bost, K., 27-39 Boward, E. W., 137 Boyajy, J. D., 14, 20, 46, 140, 142 Boyd, E. S., 82 Bradley, P. B., 1, 3-13, 15, 36,45, 46, 61, 65, 73, 113, 117; 130, 140, 142, 145, 149, 162, 163, 164 Brimblecombe, R. W., 8, 77 Brodie, B. B., 22 Brouwer, J. E., 14, 21 Bultasova, H., 86, 87 BureS, J., 61-72, 77, 78, 79, 134, 142 BureSovB, O., 61, 62, 77, 134, 142 Cardo, D., 78 Carlton, P. L., 21, 48-60, 65, 70, 83 Carlton, R. A., 18, 20, 21, 110 Carpenter, R. S., 165 Case, T., 79 Case, T. J., 4, 149 Cazzullo, C. L., 165 Cerletti, A., 107

Chalmers, R. K., 79 Chatfield, P. O., 14 Chhvez-Ibarra, G., 15, 21, 113 Chen, G., 80 Chong, G. C., 70 Coady, A., 86 Connors, J., 145 Cordeau, J. P., 140 Costa, E., 36 Curtis, D. R., 10 Courville, J., 140 Cuculic, Z., 22, 27-39 Daigneault, E. A., 21, 36, 142 Dahlstrom, A., 36 Dale, Sir Henry, 35 Danielopolu, D., 149 Darrow, C. W., 149 Davies Beresford, E., 86 DeBaran, L., 14, 22 Dell, P., 140, 163 De Maar, E. W. J., 15, 21 Dement, W., 131, 152, 154 De Robertis, E., 61 Desmedt, J. E., 15 Dhawan, B. N., 11 Didamo, P., 21, 70 Ditman, K., 87, 90, 100 Domer, R., 77 Domino, E. F., 20 62, 65, 77, 78, 113-133 Doust Lovett, W. J., 90, 98 Drdkova, S., 102 Dren, A. T., 113-133 Eccles, J. C., 20, 21 Eccles, R. M., 20, 21 Echlin, F., 37 Elkes C., 1 Elkes, J., 1. 4, 5, 6, 45, 46, 61, 65, 113, 117, 130, 140, 149, 163, 164 Erickson, C. K., 79 Espelien, A. D., 140 Essig. C. F.. 35 Everett, J. W., 28 Exley, K. A., 140 Exner, K., 102 Fatt, P., 20, 21 Feldberg, W., 83 Fi!kovB, E.. 16, 41, 68 Fink, M., 107, 134, 149-168

176

A U T H O R INDEX

Fleming, M. C., 140 Fliigel, F., 106, 149 FohCr, O., 68 Forrer, G. R., 22, 100, 106 Franks, C. M., 79 Frumin, M. J., 77 Funderburk, W.. 79 Funderburk, W. H., 4, 68, 149 Fuxe, K., 36 Galambos, R., 22 Garattini, S., 140 Geller, I., 5 1 Gershon, S., 15, 22 Giacobini, E., 102 Giarman, N. J., 14, 15, 21, 22, 23. 35 Gibbs, E. L., 149 Gibbs, F. A,, 149 Gilrnan, A., 36, 54, 58 Gogan, P., 40, 78, 165 Gogolak, G., 145 Goldberg, M. E., 79 Goodman, L., 36 Goodman, L. S., 54, 58 Green, D. M., 8 Green, J. D., 28, 145 Green, R. E., 149 Grob, D., 149 Grof, S., 86-105, 106 Grofovl, I., 22 Grofovl, O., 40-47 Grossman, S. P., 82 Gursey, D., 102 Harnpson, J. L., 35 Hance, A. J., 3, 6, 7, 8, 142, 162 Harris, L. S., 70 Hartung, M. L., 106 Harvey, A. M., 149 Harvey, N., 152, 154 Hebb, C. O., 37 Hernlndez-Peh, R., 15, 21, 113 Herz, A,, 22, 46, 62, 73-85 Hiebel, G., 140, 163 Hirnwich, H. E., 8, 15, 20, 21, 22, 27-39, 65, 113, 140, 142, 145, 149, 163, 164 Hobart, G. A., 152, 154 Hoch, P. H., 106 Hoff, H., 87, 100, 102 Hoffmeister, F., I 1 1 Hogstetter, A., 75 Holten, C. H., 46 Holzhauser, H., 80 Horakovl, Z., 86 Hoya, W. K., 22 Huve, P., 163 Ilyutchenok, R. I., 20, 27, 113, 134-148 Isbell, H., 15, 107

ltil, T., 22, 106, 107, 149-168 Ivanys, E., 102 Izikowitz, S., 102 Jacobsen, E., 134, 169 Jasper, H. H., 27 Jenker, F. L., 14, 21 Jewesbury, E. C. O., 86 Johnson, H. E., 79 Jordan, E. P., 36 Jouvet, M., 131 Jung R., 143 Kanai, T., 143 Keskiner, A., 149, 150, 164 Key, B. J., 7, 8, 9, 163 Killam, E. K., 165 Killam, K. F., 165 King E. E., 163 Kleitman, N., 152, 154 Klitina, G., 68 Knaak, J. B., 79 Knapp, D. E., 113 Koenig, J., 165 Kreiskott, H., I 1 1 Krop, S., 149 KrjeviE, K., 36, 37. 61, 143 Kronenberg, G., 149 Krus, D., 86-105 Kruse, W., 20 Krystal, H., 98, 102 Kunz, K., 86-105 Kurz, H., 75 Langworthy, 0. R., 149 Laverty, S. G., 79 Lechner, H., 149 Legge, D., 90 Leister, H. A,, 22 Lenke, D., 80 Lennox, W. G., 149 Lewis, P. R., 37 Lilenthal, J. L., Jr., 149 Lindsley, D. B., 145 Lindsley, D. F., 165 Loeb, C., 14 Loew, D., 83 Logan, C. R., 15 Logan, R. L., 107 Longo, V. G., 14, 15, 21, 22, 35, 40, 46, 65, 77, 78, 106-112, 140, 142, 165 Loomis, A., 152, 154 McAuley, A., 35 MacCorquodale, K., 57 McGaugh, J. L., 14, 22 MacIntosh, F. C., 37 McLennan, H., 58 McNamara, B. P., 149

A U T H O R INDEX

Machne, X., 77, 149 Magni. F., 14 Magoun, H. W., 27, 140 Mancia, M., 165 Marazzi, A. S., 163 MarSala, J., 16, 41 Mashkovskiy, M. D., 113, 134, 140, 142 Meehl, P. E., 57 Mehes, J., 21 Mennear, J. H., 70 MetyS, J., 142 MetySova, J., 40, 46, 142 Meyers, B., 55, 62, 65, 77, 78 Michelson, J. M., 77, 78 Migdal, W., 77 Mikhel’son, M. Ya., 40, 113, 134 Miller, J. J., 106 Miner, E. J., 15, 107 Mitchell, J. P., 37 Mitskene, V. P., 140 Mitskis, A. M., 140 Molnar, G., 68 Monnier, M., 65, 70, 140, 145 Morgane, P. J., 15, 21, 113 Morillo, A., 145 Moruzzi, G., 27 Nash, C. B., 14, 15, 21, 22, 46, 78, 149 Nathan, P., 35 Naurnenko, Ye. V., 137 Neubauer, H., 149, 150, 164 Nicholson, A. N., 113, 130, 145 Nickander, R. C., 14, 20 Nuhfer, P. A., 22 Oboris, P. E., 37 Olariu, J., 22 Oliver, K. L., 68 Olson, R. F., 102 Ostfeld, A., 18, 19, 77 Ostfeld, A. M., 21, 106, 149 Ostrovskaya, R. U., 113 Overton, D. A., 52, 54 Parkes, M. W., 46 Pathman, J. H., 149 Paul-David, J., 37 Pavlin, R., 36 Pazzagli, A., 77 Pennes, H. H., 106 Penning, J., 106 Pepeu, G., 14, 15, 21, 22, 23, 35, 77 Petsche, H., 145 Pfeiffer, C. C., 20, 79, 106, 107 Phillis, J. W., 36, 37, 70 Polak, R. L., 37 Possanza, G. J., 14, 15, 21, 22, 46, 78, 149 Pribram, K. H., 145

177

Pscheidt, G. R., 36 Purpura, D. P., 14 Rathbun, R. C., 22 Ricci, G. F., 77, 165 Riciputi, R. H., 62, 77, 78 Riehl, J. L., 37 Rhaldi, F., 8, 14, 15, 20, 21, 22, 27, 36, 65, 113, 140, 149, 163, 164 Roberts, K. H., 62, 77, 78 Roberts, M. H. T., 65 Rornanowski, W., 70, 140, 145 Rosenberg, D. E., 15, 107 Rosenberg, P., 37 Rossi, G. F.; 14 Rothballer, A. B., 140, 163 Rougel, A., 40, 78, 165 Rudolph, A. S., 14-26, 36 Rusell, R. W., 59 RyShek, K., 86-105, 106, 107 Sadowski, B., 40, 77: 78, 165 Salrnoiraghi, G. C., 143 Sawyer, C. H., 28 Schlag, J., 15 Schlager, E., 107 Schueler, F. W., 77 Schweigerdt, A. K., 34 Scotti de Carolis, A., 106-If2 Seiden, L. S., 165 Seifter, J., 51 Sewell, H. H., Jr., 14, 15, 16, 17, 21, 23, 36 Sheridan, M. N., 36 Sherwood, S. L., 83 Shute, C. C. D., 37 Sigg, E. B., 22 Silver, A., 36, 37 Silvestrini, B., 15, 21, 40, 65, 140 Skala K., 86-105 Slater, I. H., 22 Smirnov, D. A., 90 Smirnov, G. D., 27, 143 Smith, H. F., Jr., 79 Sokolov, Te. N., 143 Solomon, P., 137 Sonne, E., 46 SouSkovA, M., 40,46 Spehlmann, R., 37, 143 Spitzer, F., 107 Sproull, D. H., 37 Srhrnkovii, J., 86 Stark, P., 82 Steinberg, H., 90 Steiner, W. G., 15, 36, 142, 145 Stone, G., 77 Sturnpf, Ch., 145 Sulser, F., 22 Summerfield, A., 90 Szerb, J. C., 143

178

AUTHOR INDEX

Taeschler, M., 83, 107 Teschemacher, H., 75, 80 Teuchmann, J., 15 Thorpe, W. H., 48 Timo-Iaria, C., 15, 21, 113 Trouton, D. S., 79 Udenfriend, S., 21 Unna, K. R., 21: 37, 77, 149 Vaillant, G . E., 82 Valdman, A. V., 113 Valzelli, L., 140 Vbna, J., 102 VaniEek, M., 134 Van Meter, W. G., 36 Veit, F., 20, 21 Velluti, R., 113 Verdeaux, J., 40, 78, 165 Vernier, V. G., 21 Villarreal, J. E., 20, 113 Vinai, O., 86 Vinai-ovb, M., 86 Vitek, V., 86-105, 106, 107 Vogel, J. R., 49 Vogt, M., 20, 21 VojtEchovskf, M., 20, 21, 86-105, 106, 107

Votava, Z., 40-47, 86, 134, 142 Walsh, J. 140 Ward, Jr., A., 14, 21 Wegmann, A., 102 Weiss, T., 68, 77, 78, 79, 134, 142 Weissbach, H., 21 Wescoe, W. C., 149 Westerbeke, E. J., 14, 15, 21, 22, 46, 78, 149 White, R. P., 14-26, 36, 46, 78, 110, 140, 142, 149, 165 Whitehouse, J. M., 57, 77 Whittaker, V. P., 36 Whittlesey, J. R. B., 87, 90, 100 Wikler, A., 1-2, 14, 40, 45, 61, 73, 113, 130, 149, 164 Wilson, W. P., 22, 145 Wirth, W., 111 Woktencroft, J. H., 10, I I , 140, 145 Wynne, 137 Yacoub, F., 79, 80 Yamamoto, K., 113-133 Yeliseyeva, A. G., 137 Yim, G. K. W., 14,20 Zamporo, L., 77

Subject Index Acetylcholine, action of m-cholinergic receptor, I19 -, activity, and cholinesterase inhibition, 58 -, brain, alteration in normal activity, 48 -, -, role in habituation, 52,56 - as central mediator, 11 1 -, prolonged membrane depolarisation, 169 -, as transmitter, in CNS, 169 - ,_ ,synaptic, 12 Acetylcholinesterase, activity, blockade of, 68 - ,_ , fibres containing, 37 ACH, see Acetylcholine Activating mechanisms, adrenergic and cholinergic, 163 Activating system ascending reticular, transmitter mechanisms, 143 Adrenergic mechanisms, activating, 163 _ ,- , role in defensive reactions, 139 After-discharge, cortical, evoked by electrical stimulation, 67 Alcoholic, psychoses, compared with effect of psychodysleptics in normals, 87 -, -, naturally occurring, pathogenesis, 103 -, psychosis, compared with Korsakoff’s psychosis, 100 Alcoholics, benactyzine in, 87 -, LSD 25 in, 87 Alert cortical pattern, cholinolytic drugs on, 29 Amnesia, after anticholinergics, in man, 54 -, after cholinolytics, in man, 77 Amphetamine, drinking test and habituation in rats, 51 -, intravenous, 32 a-Amphetamine, effect of Ditran-induced changes, 159 Amygdala, basal, dog, electrodes in, I16 -, cat, bipolar depth electrodes in, I14 Antagonism, central, sites of, 20 -, mutual, between atropine and physostigmine, 4 -, physostigmine-induced, 19 Anticholinergic, central syndrome, characteristics, 106 -, drugs, behavioural effects and cortical mechanisms, 68 -, -, compared with LSD 25,107 -, -, contrast of effect animals and man, 54 -, -, dual activity, 163 -, -, EEG alterations after, 108 -, -, interaction with other compounds, 149 -, -, physostigmine to determine central effects, 40

-, -, sites of action, 65 -, hallucinogenic precursor, in alcoholism, 102 -, hallucinogens, homogeneity of, 11 1 -, -, laboratory results and clinical trials, 106 -, -induced amnesia in man, 54 -, -induced, behavioural and EEG changes, 149 -, subcortical antagonism, 15 Anxiety, LSD-induced, 98 Arecoline, action of m-cholinergic receptor, 118 -, on conditioned avoidance response, 81 -, reversal of HC-3 induced EEG changes, 130 Arousal, EEG, cortical cholinergic link, 27 -, -, cortical cholinergic mechanisms, 35 -, -, physostigmine-induced in rabbits, 40 Atropine, antagonism of physostigmine on single shock responses, 19,20 -, behavioural effects, 1,9 -, blocking of physostigmine-induced EEG activation, 117 - - - pilocarpine-induced EEG activation, 117 -, changes in consciousness after low dose, 152 -, conditioned avoidance response after, 10 -, deep sleep and stupor after high doses of, 154 -, drinking test and habituation in rats, 51 -, drowsy state after low dose, 152 -, EEG changes after low dose, 152 -, effect on evoked cortical after-discharge, 67 - and learning in decorticated rats, 70 - -induced changes, modified by chlorpromazine, 158 -, intraperitoneal, on penicillin-evoked seizure bursts, 66 - maze test after, 10,63 - as rn-cholinergic antagonist, 117 -, memory deficit, 53 -, mutual antagonism with physostigmine, 4 -, non-specific inhibition of, 11 -, subcortical and cortical effects in rabbits, 21 - sulphate, pharmacological dissociation, 4 - on theta activity, 45 Avoidance, learning in normal and decorticated rats, 68 -, response, conditioned, in rats, 73 Awake state, role of cholinergic mechanisms, 1 13 BA 1433, effectiveness in blocking EEG arousal, 110 Beer, compared with spirits in delirium tremens, 102 - drinkers, benactyzine effects, 87

180

SUBJECT INDEX

Beer drinkers, LSD 25 effects, 87 Behaviour and brain electrical activity, 139 - changes, anticholinergic-induced, 149 - -,after Ditran atropine, I52 - EEG dissociation, I , 61, 65, 73, 113, 117, 149, 164 -, emotional, in animals, 134 -mechanisms, and stress reaction, 139 - -, reactive, 73,79, 81 Behavioural changes, neurochemical mechanisms, 144 - effects, atropine, physostigmine, 8-9 - -, conditioned avoidance response, 9 - functions, cholinergic transmission, 70 - -, duplicated, 70 -, index of habituation, 48 Belladonna alkaloids, 14 Benactyzine, in alcoholics, 87 -, behavioural changes after, 98 -, delirium tremens after, 100 -, intravenous, 31 -, Korsakoff’s syndrome after, 100 - on physostigmine-induced theta activity, 45 -, specificity of central effect, 46 Bilateral cortical spreading depression induced in rats. 70 CAR, see conditioned avoidance response Carbocaine on EEG arousal, 34 Cardiovascular effects of LSD 25 and benactyzine in chronic alcoholics, 92 Cationic character of Hemimecholinium, 30 Cat, bipolar depth electrodes in, 114 -, effects of physostigmine i.v., 117 -, - - pilocarpine i.v., I17 -, sleep, fast and slow wave, in, 117 Central actions of piperidyl benzilates, I5 -antagonism of physostigmine-inducedchange, 20 - anticholinergic syndrome, characteristics, 106 - anticholinergics, effects, 40 - cholinoceptive structures, 73 _ - - , compared to peripheral cholinergic receptors, 4 - mediator, role of acetylcholine as, 1 1 1 - muscarinic stimulation, effect on reactive behaviour, 82 - nervous system, action of ACH, 169 - _ - , importance of speed of penetration into, 76 - - -, levels of cholinergic transmission, 71 Chemical aspects of brain organisation and learning, 61 - processes in habituation, 48 Cholinergic activating mechanisms, compared with adrenergic, 163 - drugs, behavioural effects, 68 - -, emotional behaviour in animals, 134 --, sites of action, 65

- functions, in brain, I13 - -, in structures near cortical surface, 36 - link, cortical, possibility in EEG arousal, 27 - mechanism, cortical and EEG arousal, 35 _ - , nature of at neuronal level, 10 _ - , in wakefulness and sleep, 1 13 - nature of recent memory, 65 - predominance after functional decortication in rats, 70 - receptors, involved in synaptic transmission, 20 - -, peripheral and central, 4 _ _ , type of, 12 - transmission, brain, 61 Cholinesterase inhibition, and ACH activity, 58 _ _ , muscarinic action, 83 Cholinoceptive structures, central, 73 Cholinolytic drugs, amnesia following, 77 - -, in animals and man, 78 _ _ , and conditioned avoidance response, 73 _ _ , topical application, 29-31 Cholinomimetic drugs, inhibition of conditioned avoidance response, 78 _ - - - nociceptive reaction in rats, 80 _ - , - in reactive behaviour, 79 - -, muscarinic action, 83 Cholinoreactive mechanisms, in defensive reactions, 134 - structures, distribution and drug effect, 140 _ _ , muscarinic brain, in emotional memory, 137 Chlorpromazine, after atropine, 158 -, after Ditran, 158 - on conditioned avoidance response and nociceptive reaction in rats, 81 Clinical trials with anticholinergic drugs, 186 Clyde, mood scale, in alcoholics, 92 CNS, see Central nervous system Conditioned avoidance response, after atropine, 10

- - _ , behavioural effects, 9 - - -, establishment of, 75

- - -, inhibition of nociceptive reaction, 81

- - -, after physostigmine, 10

- pole-jump in rats, 73 Confusion after Ditran administration, 154 - - atropine administration, 154 Consciousness, changes in, after Ditran and atropine, 152 Cortical after-discharge, 67 alert patterns, cholinolytic drugs on, 29 - cholinergic link, 27 - _ mechanism in EEG arousal, 35 - desynchronisation, physostigmine evoked, 42 - EEG resting pattern, cholinolytic drugs on, 30 - mechanism, in behavioural effects of drugs, 68 - spontaneous electrical activity, 2 - surface, cholinergic function in structures near, 36

-

SUBJECT I N D E X

Decorticated rats, atropine on learning, 70 _ _ , avoidance learning, 78 Decortication, functional, 70 Defensive reactions, cats, 136 Delayed reactions, role of cholinergic systems, 64 Delirium after atropine and Ditran, 154 Delirium tremens, comparison with effects of benactyzine, 98-100 - _ , in alcoholics, 102 Depolarization, prolonged, of membrane, action of ACH, 169 Depression, bilateral spreading, in rats, 70 Desynchronisation, cortical, physostigmine evoked in rabbits, 42 Discrimination test with pole jump in rats, 76, Disinhibition, effect with scopolamine in rats, 78 Dissociation, EEG and behaviour, 1, 44, 61, 65 73,113,117,149,164 -, pharmacological, 4 Ditran, antagonism of physostigmine effect on single shock responses, 19 -, clinical and EEG findings after, 107 -, differential effect of dose level, 152 -, -induced changes, effect of a-amphetamine, LSD-25, Yohombine, 159 - - _ _ _ imipramine, neostigmine, tetrahydroaminocrin, 160 - _ _ , modified by chlorpromazine, 158 -, -induced psychomotor activity, hallucinations after, 157 Dogs, electrographic experiments in, 1 I 5 Dose, level, in pharmacological dissociation, 4 Drinking test, in rats, 49 Drowsy state, induced by low doses ofDitranand atropine, 152 Dual activity of anticholinergic drugs, 160 Duplication of behavioural functions, 70 EEG, see Electroencephalogram Electrical activity of brain, behavioural correlation, 139 - _ _ cortex, spontaneous, 2 - stimulation, after anticholinergics, 110 - _ , threshold of, I10 Electrodes, bipolar depth in cat, 114 -, depth, in dog, 115 Electroencephalogram arousal, blocking of, 1 10 _ - , cortical cholinergic, 35 - _ , by midbrain stimulation, 30 - _ , by physostigmine, 40 - -, possible cortical cholinergic link, 27 -, changes in, limbic, 130 - _ _ , neocortical, 130 -, cortical resting pattern, cholinolytic drugs on, 30 -, functional significance, 2 -, low amplitude, rabbit mid-brain, 19 -, modifications by LSD 25,111

181

-, synchronisation, after anticholinergic drugs, 108

-, variation independent of evoked response, 22 Electroencephalographic activation, after pilocarpine, 11 7 _ _, speed of, 11 5 Electroencephalographic-behavioural‘dissociation’, 1, 44, 61, 65, 73, 113, 117, 149, 152, 157, 164 - effects, physostigmine in ablated rabbits, 15 - techniques, central effects of anticholinergics, 106.108 Emotional behaviour, animal, cholinergic drugs on, 134 _ _, changes, in alcoholics, 92 - memory, mechanisms, 137 Enckphale isolC, preparation , pharmacological dissociation, 7 Eserine, dissociation after, 1 -, distribution of cholinoreactive structures, effects, 140 - -induced arousal, cholinolytic drugs on, 29 -, intravenous and topical application, 31 Eumydrine, premedication with, 81 Evoked response, EEG-independent variation, 22 Excitatory effects of acetylcholine, 11 Experimental psychoses, induced by benactyzine, 86 Extinction of fear, in rat, 55 Extirpation of prosencephalon, in rabbit, 15

Fast wave sleep, 117 Fear, extinction of, in rat, 55 - reaction, blocking, 145 _ - , in dogs, 136 Fibres containing acetylcholinesterase activity, 37 Frontoparietal cortex of rat, penicillin focus in, 66 Functional significanceof EEG, 2 Galanthamine, cholinoreactive mechanisms, 134 -, effects, distribution of cholinoreactive structures, 140 Habituation, behavioural index of, 48 -, chemical basis of, 48 -, learning, role of ACh, 50,56 Hallucinations, after benactyzine, in alcoholics, 91

-, after Ditran, 157 Hallucinogen, anticholinergic, homogeneity of, 111

-, -, precursor, in alcoholism, 102 Hallucinogenic, anticholinergic drugs, laboratory results and clinical trials, 106

182

S U B J E C T IN D E X

HC-3, see Hemimecholinium Hemimecholinium, cationic character, 130 -, intraventricular, 130 Hippocampal, theta rhythm, abolition of, 130 Hippocampus, cat, bipolar depth electrodes in, 1 I4 -, dorsal, dog, electrodes in, 116 Histochemical, evidence of acetylcholinesterase activity, 37 H-Maze, alternation test, in rats, 62 Homogeneity of anticholinergic hallucinogens, 111 5-HTP, see 5-Hydroxytryptophan 5-Hydroxytryptophan-induced EEG alerting, 34

Imiprarnine, effect on Ditran-induced changes, 160 Index, behavioural, of habituation, 48 Inhibition, acetylcholine, effects of, 11 -, atropine, non-specific, 1 1-12 - of central cholinoceptive structures, 73 - - - cholinesterase, 58 - of conditioned response, by cholinomimetic drugs, 78 - of fear reaction, 136 - of nociceptive reaction, in rats, 80 -, physostigmine, non-specific, 11-12 Intravenous physostigmine, 117 Intraventricular hemirnecholinium, 130 JB-318, antagonism of physostigmine, 19

Mecylamine, effect on physostigmine, 117 - - pilocarpine-induced EEG activation, 117 Mechanisms, activating, cholinergic and adrenergic, 163 -, cholinergic, in wakefulness and sleep, 113 -, cortical, spontaneous, electrical activity, 2 Mediator, central, acetylcholine as, 111 Membrane, depolarisation, of ACh, 169 Memory, deficit, atropine and scopolamine, 93 -, effects of LSD 25 and benactyzine, 95 -, emotional, mechanisms, 137 -, recent, possible cholinergic nature, 65 Mesodiencephalic activating system (MDAS), 27 Metrazol, see Pentamethylenetetrazol Methyl atropine, effect on EEG activation, I I7 Methyl scopolamine, on drinking test and habituation in rats, 51 Mid-brain, reticular formation, single shock responses, 19 Muscarinic, action of ACh, 169 -, cholinoreactive brain structures in emotional memory, 137 -, stimulation, central on reactive behaviour, 82

-

N-Cholinergic receptors, action of drugs as, 118 Neocortex, dog, electrodes in, 116 Neostigmine, effect on Ditran-induced changes, 160 Neurochemical mechanism of behavioural change, 144 Neuronal investigation, cholinergic mechanisms, 10 inhibition, atropine and physostigmine, 1 1 Nicotine, action of n-cholinergic receptor, 118 Nicotinic action of ACh, 169 Nociceptive reaction, in rats, 80 Normal volunteers, benactyzine-induced experimental psychoses, 86

Korsakoff's psychosis, comparison with benactyzine intoxication, 98 Laboratory trials, of anticholinergics, 108 Learning avoidance, in rats, 68 -, brain activity, analysis, 61 -, possible role of habituation in, 57 - processes, impairment after LSD in alcoholics, 95 Lesions, brain, behavioural tests, 65 Limbic, spiking, 130 LSD-25, behavioural changes after, 98 -, cardiovascular effects, in alcoholics, 92 -, in chronic alcoholics, 87,98 -, effects compared with anticholinergics, 107 -, - on Ditran-induced changes, I59 -, modification of EEG by, 1 1 1

Maze test, atropine on behaviour, 10 M-Cholinergic, antagonists, 117 -, physostigmine effects, 117 -, receptors, actions of drugs on, 118

Overtraining, effect on conditioned avoidance response, I5 Paranoid reactions after LSD 25,90 Pathogenesis, alcoholic psychoses, 103 Penicillin focus, evoked in frontal parietal cortex of rat, 66 Pentamethylenetetrazol (Metrazol), topical application, 31 Pentobarbital, on drinking test and habituation in rats, 51 Peripheral, compared to central cholinergic receptors, 4 -, nervous system, action of ACh, 169 Pharmacological, dissociation, 1 , 4 -, evidence of cholinergic receptors in synaptic transmission. 20

SUBJECT I N D E X

Physostigmine, antagonized by belladonna alkaloids, 15 -, antagonism of HC-3 induced EEG changes, 139 -, behavioural effects, 8 -, cholinergic transmission after, 61 -, conditioned avoidance response after, 10 -, cortical desynchronisation in rabbits, 42 -, cortical after-discharge, 67 -, -induced changes in single shock response, 19 -, - EEG arousal in rabbits, 40 -, -theta activity, 44 -, intraperitoneal, on penicillin-induced seizure bursts, 66 -, intravenous, central rn-cholinergic effects, in cats, 177 -, in localised cortical area, 68 maze test after, 10,62 --, rn-cholinergic receptor, 1 1 8 -, mutual antagonism with atropine, 4 -, pharmacological dissociation, 4 -, quantitative comparison with scopalamine,

183

-, nociceptive reaction inhibited by cholinomimetic drugs, 80

-, penicillin focus, frontoparietal cortex, 66 -, scopolamine on habituation, 50 Reactive behaviour, effect of central muscarinic stimulation, 82 - -, test of, 73 Recent memory, possible cholinergic nature, 65 Receptors, cholinergic, 12,20, 1 1 8 Reticular activating system, ascending, 143 - formation of midbrain, 19 RS-86, nociceptive reaction in rats, 86 Rostra1 link in mesodiencephalic activating system, 27 Response, conditioned, 77 -, evoked, 22 -, single-shock, 19

-$

45

-, subcortical synchronisation, in rabbits, 42 Pilocarpine arousal, cholinergic drugs on, 29 -, fast wave sleep after, 117 -, intravenous, 31 -, - a-induced EEG activation, 117 -, m-cholinergicagonist, 117 -, - receptor, 118 -, reversal of HC-3 induced EEG changes, 130 Piperidyl benzilates, central actions compared with belladonna alkaloids, 15 Pole-climbing, test of reactive behaviour, 79 Pole-jump, discrimination test, in rats, 76 Pontocaine, on EEG arousal, 34 Prosencephalon, extirpation of, in rabbits, 15 Psychodyslepticdrugs in normals, 87 Psychomotor activity, after Ditran, 157 - functions, alterations in alcoholics after benactyzine, 94 Psychoses, alcoholic, 87 _ , _ , pathogenesis, 103 -, experimental, in normals, 86 Rabbit, cortical and subcortical actions of drugs, 21 -, midbrain, EEG, 19 -, physostigmine-inducedEEG arousal, 40 -, prosencephalon extirpation, 15 -, topical application of drugs, 28 Rats, avoidance learning in, 68 -, chlorpromazine in, 81 -, cholinergic transmission, 61 -, conditioned avoidance pole-jump test, 73 -, drinking test in, 49 -, fear reaction in, 59 -, maze test in, 62

Scopolamine and CAR acquisition in rats, 73

-, behavioural and cortical dissociation after, 1 -, disinhibition effect inrats, 78 -, on drinking test, in rats, 50 -, and memory deficit, 53 -, in overtrained rats, 75 -, physostigmine, antagonism of, 19 -, -, quantitative comparison, 45 Seizure, activity, effect of ACh blockade, 65 Single shock responses, midbrain reticular formation, 19 Sleep, deep, after high doses of Ditran and atropine, 154 -, fast wave, in cats, 117 -, slow wave, in cats, 117 -, role of cholinergic mechanisms, 1 1 3 Speed of drug penetration in CNS, importance of, 76 -, of EEG alteration, importance of, 165 Spirits, compared with beer as possible cause of delirium tremens, 102 Spreading depression, bilateral, cortical, induced in rats, 70 Stimulation of central cholinoceptive structures, 73 -, central muscarinic, 82 -, electrical, threshold of, after anticholinergics, 110 Stress, reaction and behaviour mechanisms, 139 Stupor after high doses of Ditran and atropine, 154 Strychnine, intravenous, 31 -, topical, 31 Subcortical, compared to cortical effects in rabbits, 21 - effects of anticholinergics, 14 _ _ - belladonna alkaloids, 14 -, synchronisation, physostigmine evoked in rabbits, 42 Synaptic transmission, role of ACh, 12

184

SUBJECT INDEX

_ _ , role of cholinergic receptors, 20 Synchronisation, assessment of drug potency for, 110

-, EEG, 108 -, subcortical, physostigrnine evoked in rabbits, 42 Syndrome, central anticholinergic characteristics, 106 Tetrahydroaminocrin, effect on Ditran-induced changes, 160 THA, see Tetrahydroaminocrin Theta activity, after Ditran and atropine, 152 - -, hippocampal, 130 - -, physostigrnine induced, 44 Thought disturbances, after LSD 25 and benactyzine, 94

Threshold of electrical stimulation after anticholinergics, 110 T-Maze, rats in, 63 Topical application of drugs, 28-30 Transmission, cholinergic, in brain, 61 _ ,- , and behaviour, 70 -, synaptic, 12,20 Transmitter, mechanisms in ascending reticular activating system, 143 -, role of ACh as, 169 Tremorine, in rats, 81 Trimethidinium, after physostigmine, 117 -, - pilocarpine, 118 Yohimbine, effect on Ditran-induced changes, 159