monograph series

3

AMINERGIC

HYPOTHESES

OF BEHAVIOR:

REALITY OR CLICHÉ?
The NIDA Research Monograph series is prepared by the

Research Division of the National Institute on Drug Abuse. Its

primary objective is to provide critical reviews of research problem

areas and techniques the content of state-of-the-art conferences,

integrative research reviews and significant original research. Its

dual publication emphasis is rapid and targeted dissemination to

the scientific and professional community

EDITORIAL ADVISORY BOARD

Avram Goldstein, M.D.

Jerome Jaffee, M.D.

Reese T. Jones, M.D.

William McGlothlin, Ph.D.

Jack Mendelson, M.D.

Helen Nowlis, Ph.D.

Lee Robins, Ph.D.

Addiction Research Foundation

Palo Alto, California

College of Physicians and Surgeons

Columbia University, New York

Langley Porter Neuropsychiatric

Institute

University of California

San Francisco, California

Department of Psychology, UCLA

Los Angeles, California

Alcohol and Drug Abuse Research

Center

Harvard Medical School

McLean Hospital

Belmont, Massachusetts

Office of Drug Education, DHEW

Washington, D.C.

Washington University School of

Medicine

St. Louis, Missouri

NIDA RESEARCH MONOGRAPH series

Robert DuPont, M.D. DIRECTOR, NIDA

William Pollin, M.D. DIRECTOR, NIDA RESEARCH DIVISION

Robert C. Petersen, Ph.D. EDITOR-IN-CHIEF

Eunice L. Corfman, M.A. MANAGING EDITOR

Rockwall Building 11400 Rockville Pike Rockville, Maryland, 20852
aminergic hypotheses

of behavior:

reality or cliché?

edited by

bruce kenneth bernard, ph.d

school of pharmacy

university of connecticut

storrs, connecticut

november 1975

national institute on drug abuse, 11400 rockville pike, rockville, maryland 20852
Reproductions are

of Plates from the Seventh Book

of the De Humani Corporis Fabrica

of Andreas Vesalius of Brussels,

first issued at Basel, 1543.

With permission of the

New York Academy of Medicine.

Library of Congress Cataloging in Publication Data

Main entry under title:

Aminergic hypotheses of behavior, reality or cliche?

(NIDA research monograph series; no.3)

1. Amines--Physiological effect. 2. Psychopharmacology. 3. Pharmacology, Experimental.

I . Bernard, Bruce Kenneth II. Series: National

Institute on Drug Abuse. NIDA research monograph

series; no. 3.

(DNLM: 1. Behavior--Drug Effects. 2. Amines--Metabolism. Wl RE232G no. 3 / QV129 A517)

QP981.A4A44 615'.78 75-33116


FOREWORD

The symposium from which the intriguing title of

this volume is drawn poses a question which is

difficult to assess in the aggregate, and for

meaningful resolution might best be redirected

to the symposium participants for their individual

evaluation. The question ...reality or cliche?...

suggests that one might simply sum a set of

subquestions in an algebraic manner and arrive at

a position with which persons of reason may be

comfortable. Alas, while such a path might calm

the spirit, it would only blur resolution of

the question.

The dilemma presented to us may be illustrated

by a story from the book "From Dream to Discovery

(On Being a Scientist)" by Professor Hans Selye

of the Institute of Experimental Medicine and

Surgery, University of Montreal.

A blind beggar asked his friend, "Tell

me, what do they mean by white? What

is white like?"

"White is a color," he was told. "It

is like the snow on the mountain."

"I see," the blind man said. "It is

a cold and soft color."

"No, not always; paper is white, too."

"It is a thin and fragile color, then?"

"It need not be. Milk is also white."

"Is it fluid and nourishing?" the blind

man asked, somewhat bewildered.

"Not necessarily, " his friend explained.

"All sorts of things are white: clouds,

teeth, an old man's beard- your eyes are

white, too, that is why you cannot see

through them."

"Oh, never mind," the blind man sighed.

"It is a cruel color. Perhaps it's just

as well if I don't try to understand it."

v
Is the blind man plagued by his lack of vision,

or is the inability of his friend to perceive

the question and respond to its separate elements

the root of the man's bewilderment and ultimate

chagrin?

I submit that in attempting to provide explanations

for complex behavioral patterns which are subserved

and modulated by equally complex physiologic and

biochemical processes we are assuming a risk that

our response must necessarily be as faulty as that

of the blind man's friend, The ultimate danger,

I feel, is that we may becomes committed to the

proposition that there must be an answer which is

approachable with present tools and concepts.

In order to provide explanations and to answer

questions we fall prey to the measurement of

amounts of things of one type (e.g., brain amines),

which are accessible. What better wav to utilize

such information than to correlate it with other,

less well definable, observations (e.g., patterns

of behavior)? A large part of science appears to

be so directed.

Those who fall prey to this schema do so out of

a noble motive which, unfortunately, only pushes

the meaningful question further into the future.

Our tendency is to frame global questions--out

of our inability to discern the limits of the

more discrete inquiries which we must make.

Does anyone believe, for example, that we can at

this juncture speak meaningfully to the point

of the action of drugs on the brain? Can we

describe experimentally accessible units of

"brain" to which we direct such questions? The

title of this volume and a reading of the

contributions contained herein leave the

impression that this group of scientists have

not hesitated to contravene the conventional and

the doctrinaire in their respective disciplines.

They have accepted and well responded to the

challenge... "reality or cliche?

William F. Bousquet, Ph.D.

Department of Pharmacology and Toxicology

Purdue University, Lafayette, Indiana

currently: Director of Biological Research

Searle Laboratories, Chicago Illinois
CONTENTS

FOREWORD V

O. Hornykiewicz 13

INTRODUCTION 1

ABSTRACTS 7

BRAIN MONOAMINES AND PARKINSONISM

SUPERSENSITIVITY TO DOPAMINERGIC

AGONISTS INDUCED BY HALOPERIDOL

J.E. Thornburg and K.E. Moore 23

BLOOD PLATELET MONOAMINE OXIDASE ACTIVITY

IN ANERGIC SCHIZOPHRENICS

E.G. Shaskan and R.E. Becker 29

THE ROLE OF SEROTONIN AND NOREPINEPHRINE

IN SLEEP-WAKING ACTIVITY

P.J. Morgane and W.C. Stern 37

A ROLE FOR DOPAMINE

IN THE PSYCHOPHARMACOLOGY

OF ELECTRICAL SELF-STIMULATION

B.R. Cooper and G.R. Breese 63

AGGRESSION AND THE BRAIN MONOAMINES:

WHAT ARE THE ANSWERS, BUT OF MORE IMPORTANCE,

WHAT ARE THE QUESTIONS?

B.K. Bernard 71

PHARMACOGENETIC STUDIES OF THE SEROTONERGIC SYSTEM IN ASSOCIATION

WITH CONVULSIVE SEIZURES IN MICE

B.E. Ginsburg and P.Y. Sze 85

A MODEL FOR THE NEUROBIOLOGICAL MECHANISMS

OF ACTION INVOLVED IN LITHIUM PROPHYLAXIS

OF BIPOLAR AFFECTIVE DISORDER

A.J. Mandell and S. Knapp 97

EFFECTS OF DISULFIRAM ON THE

AMFHETAMINE-INDUCED BEHAVIORAL SYNDROME

IN THE CAT AS A MODEL OF PSYCHOSIS

A. Sudilovsky 109

THE EFFECTS OF HEROIN ON

CATECHOLAMINE METABOLISI IN MAN

J.J. Schildkraut, R.E. Meyer, P.J. Orsulak, S.M. Mirin,

M. Roffman, P.A. Platz, E. Grab, M.E. Randall, M.McDougle 137

CONCLUSION 147

vii

INTRODUCTION

The Title, "Aminergic Hypotheses of Behavior: Reality

or Cliche?", describes the central question addressed

by this monograph, namely, what is the status of our

current views on the relation between the function of

the brain monoamines and their effect on behavior?

The idea of developing such a publication emerged in

early 1974 following invitations to speak at the First

world Congress of Biological Psychiatry (Buenos Aires,

Argentina; September, 1974) and the Fifth LatinAmerican Congress of Pharmacology and Therapeutics

(Lima, Peru; October 1974) on my research into the

aminergic correlates of aggressive behavior. After

discussions with researchers in related fields, a

workshop entitled "The Functional Significance of

Brain Monoaminergic Systems-Pharmacological and Biochemical Approaches" was organized and co-chaired by

my good friend Dr. Sabit Gabay and myself at the

Thirteenth Annual Meeting of the American College of

Neuropsychopharmacology held in San Juan, Puerto Rico

in December 10-13, 1974. This monograph contains

several selected papers presented at that time and

additional ones that were solicited for their appropriateness to the title topic.

The articles are arranged in an order such that the

monograph progresses along a continuum of behaviors

from those which are well-defined as causally related

to the brain amines to others in which such a relationship is still highly speculative. Interspersed between the "area topic" articles, are papers narrower

in scope and of a more technical nature, which have

been chosen to explore in greater detail some point

raised in the previous article. Articles of the first

type are written by Drs. Hornykiewicz, Morgane and

Stern, Bernard, and Sudilovsky; articles of the second

type include those written by Drs. Thornburg and Moore,

Shaskan and Becker, Cooper and Breeze, Ginsburg and

Sze, Mandell and Knapp and Schildkraut, et al..

The casual relationship between the brain monoamines

and parkisonism is probably one of the most welldefined findings in brain aminergic research to date.

The first chapter, written by Dr. Oleh Hornykiewicz,

deals with the relationship between brain dopomine

and parkinsonism in addition to proposing an anatomical distinction between idiopathic and post1
encephalic parkinsonism. Furthermore, although pathologies of the dopaminergic neuronal system are central to the clinical entity, roles for norepinephrine

in the akinesia and serotonin in the depression associated with the disorder are suggested. This paper

concludes with a discussion of four pathophysiological

and neuropharmacological factors which may impinge on

the efficacy of l-dopa induced amelioration of clinical

symptoms. Although conclusive human evidence for one

of those proposed factors, supersensitivity of the

dopwnine receptor, is difficult to obtain, data from

animal studies such as found in the second paper by

Drs. Thornburg and Moore and supportive of this hypothesized change in receptor responsiveness. Employing

pharmacological manipulations which mimic the proposed

result of parkinsonism, these author conclude that

supersensitivity of dopamine receptors may play a role

in these behavioral phenomena. Continuing the examination begun in the first paper of the relation

between amines and affective disorders is the third

paper written by Drs. E. Shaskan and R. Becker. In

this study the authors attempted to differentiate

schizophrenics from normals on the basis of high and

low monoamine oxidase levels and although each MAO

grouping occurred in normals, alcoholics and schizophrenics, the distribution of these groupings were

similar across subjects.

Sleep is a second area in which research has sought

to demonstrate a causal role for the brain monoamines.

The fourth paper, written by Drs. P. Morgane and

W. Stern, reviews the current experimental findings in

relation to different sleep and waking states and concludes that serotonin and norepinephrine are involved

in the "triggering" mechanisms required for these

states of consciousness. The role of dopamine, as

suggested in the first two articles, may be limited

to behavioral arousal. However, as the paper clearly

points out, the exact function of these pututive

neurotransmitters and their interaction have yet to

be elucidated. Several experimental behavioral paradigms, such as the self-stimulation experiments reported in the paper by Drs. B. Cooper and G. Breeze,

provide additional evidence for the concept of behavioral activation induced by dopamine. After

numerous pharmacological manipulations, these authors

2
conclude that although a role for norepinephrine in

electrical self-stimulation cannot be definitively

excluded, it is their belief that doparnine plays the

major role in this behavior.

The papers thus far have for the most part emphasized

single monoamineric systems in relation to a particular

behavior. The concept of brain monoaminergic system

balancing and integration for the control of a particular behavior is emphasized in the next paper which

reviews a third topic area of amine research, aggression. In this article, I employed several hormonally,

anatomically and pharmacologically distinct models of

aggressive behavior and related each to divergent

brain aminergic profiles. Thus, although correlations

between aggressive behaviors and brain amines are

evident, the functional significance of these findings

is highly speculative. Furthermore, the possibility

that different putative transmitter profiles may be

important for different types of aggressive behavior

increases the speculative nature of implications

based on this data. However, the data do suggest the

Cross-model importance of serotonin as a possible

inhibitory transmitter. Support for this proposal is

provided in the paper by Drs. B. Ginsburg and P. Sze

in which the authors demonstrate a convariance in the

genetic inheritances of audiogenic seizure susceptibility (and aggressive behavior) and tryptophan

hydroxylase activity, the rate-limiting step in the

serotonin biosynethetic pathy. This indoleamine

may in fact be a critical factor involved in many types

of animal affective behavior including that of humans.

Paper eight, written by Drs. Mandell and Knapp describes the multiplicity of events by which brain

serotonergic alterations can be induced. Furthermore,

their data suggest that these alterations in serotonergic functioning are the mechanisms by which long

term treatment with lithium acts prophylactically

against both mania and depression. The authors seek

to establish a neurobiological model of lithium's

action, which although based upon a single amine, provides an explanation for both the increased and decreased Synthesis rates required for the observed

behavioral effects.

3
The final area of brain amine research, touched upon

in the previous paper, involves the relationship between brain monoaminergic systems and the behavioral

effects of drugs. The first paper, written by

Dr. A. Sudilovsky, describes the use of pharmacological agents for the production of a behavioral syndrome in animals which is comparable to the behavioral alterations seen in human amphetamine psychosis

and certain forms of schizophrenia. These results

can be interpreted as indicating a common underlying

aminergic mechanism for these two syndromes. The last

paper in this monograph, written by Schildkraut et al.,

examines explicitly the relation of the amines to

drug behavior by examining the relation of heroin use

to observed changes in urinary catecholamines and

metabotites.

The above is, of course, simply an overview of the

contents of this book and is provided as a guide to

the reader in understanding the flow of the entire

text. The implications and perhaps my own editorial

biases can be found at the conclusion of the monograph.

It is my hope that this publication will be of value

not only to researchers immediately concerned with

the function of brain amines, but also to students of

CNS pharmacology and others seeking a current overview of the field. The topics herein will provide the

undergraduate with a foundation for understanding the

pharmacological basis for the rational use of the

therapeutic arsenal presently available to the health

professions. For the advanced student, recent research, of which these articles are evidence, reveals

the broad, heterogenous and fluid nature of presently

held aminergic hypotheses of behavior and provides

bracing indications that older, simpler suppositions

are no longer adequate. This is always a salutory

development and perhaps especially so for many researchers in this field of functional brain amine

dynamics, who have felt that until recently their

research had become inordinately parochial, each

narrowly mining his own lode, and lacking sufficient

concern for relation, perspective and generalizabitity. Nor is this concern recent. It was never

more poignantly illustrated for me than when as a

graduate student with several years of research accomplished I was faced with the following preliminary

exam question:

4
"Many investigators over the past 10 or so

years have been carrying out seemingly sophiscated biochemical experiments measuring such

things as levels and the kinetics of synthesis

and degradation of putative neurotransmitters

peripherally and centrally. The supposed.

objective of these studies is to define drug

action, physiological mechanisms or behavioral

events in terms of the suspected transmitter.

I contend: (a) that this type of work has

degenerated into a method-oriented, correlateconscious cult from which little of value has

emerged, (b) that the total approach is naive

and fundamentally unsound and, (c) that the

likelihood of this type of study yielding the

above information is nil. Respond."

Such a sobering question, implicitly calling for

critical evaluation of the meaning of one's work, is

not easily dismissed. This monograph, with its coverage

of widely divergent behaviors, provides some of the

facts and comparisons that are prerequisite for that

difficult and continuing evaluation.

Acknowledgements of debts owed are bound to be inadequate. Those left unspecified are not therefore unremembered or unthanked. But I wish especially to

tender my gratitude to Dr. Peter Morgane for his support during the trying times of this monograph preparation, to Dr. Robert E. Willette of the National

Institute on Drug Abuse, and to all fellow researchers

who from time to time want to ask, "What is the meaning

of what I do?"

A final thanks:

We are who we touch

and who touches us

betty & jack

gida & mike

carie lee & Cathy

Bruce K. Bernard

5
6
ABSTRACT

Hornykiewicz, O. Brain Monoamines and Parkinsonism. IN:

Aminergic Hypotheses of Behavior: Reality or Cliche?,

B.K. Bernard, ed., Washington. National Institute on

Drug Abuse Research Monograph 3, 1975.

In Parkinson’s disease there is a derangement of the metabolism of at least 3 major brain monoamines, namely,

dopamine (DA), norepinaphrine (NE) and serotonin (5-HT).

Of these alterations the severe deficiency of DA in the

striatum is most characteristic, being (a) found in

Parkinsonian syndromes of any etiology and (b) significantly correlated with the degree of cell loss in the

substantia nigra, and the severity of the main symptoms.

On the basis of neurochemical-clinical correlations

Parkinson’s disease may be subdivided into (a) an

asymptomatic stage during which the striatal DA deficiency

may reach a marked degree but can be canpensated by the

remaining DA neurons, and (b) the stage of decompensation

(i.e. clinically manifest disease) which ensues when the

depetion of striatal DA reaches 70% or more. L-Dopa’s

main feature as a specific antiparkinson drug may be seen

in its potential to revert the decompensated stage of the

disease to the stage of functional compensation. This is

in many cases possible because (a) the DA tumover in the

remaining DA neurons is increased, providing for a high

rate of formation (from L-dopa) and release of DA; (b) the

“denervated” striatal receptors are supersensitive to DA;

and (c) the newly-formed DA can be expected to reach a wide

area of the striatum due to the high degree of divergence

of the dopaminergic innervation. Compared with the striatal tal deficiency, the degree of NE and 5-HT decrease in

the Parkinsonian brain is moderate. The decrease in NE

may be due to the (moderate) cell loss in the locus

coeruleus; at present no morphological basis for the

lowering of brain 5-HT is known. The functional significance of the changes in brain NE may be an aggravation of

akinesia. The decrease in brain 5-HT may be related to

aspects of Parkinson’s disease in turn related to

affective behavior and mood.

KEY WORDS: Brain amines Dopamine Norepinephrine

Serotonin Parkinson’s disease

Department of Pharmacology and Department of Psychopharmacology, Clark Institute of Psychiatry, University of

ABSTRACT

Shaskan, E.G. and R.E. Becker. Blood platelet monoamine

activity in anergic schizophrenics. IN: Aminergic Hypotheses of Behavior: Reality or Cliche? B.K. Bernard. ed.,

Washington, National Institute on Drug Abuse Research

Monograph 3, 1975.

Blood platelet monoamine oxidase (MAO) activity was

evaluated in twenty-four anergic, schizophrenic outpatients during a double-blind study comparing a

chlorpromazine-imipramine combination to thio-thixeneplacebo. Platelet MAO activity was determined on blood

samples drawn after a two-week drug-free washout and

once weekly over a four-week on-drug period. Schizophrenic patients could be classified according to their

blood platelet MAO activity into either a low-MAO or a

high-MAO group. In neither group of this population of

schizophrenics did blood platelet MAO activity correlate

with any of the primary or secondary symptoms of schizophrenia . Ten alcoholics and seven volunteer non-patients

could similarly be divided into low- and high-MAO groups.

Mean blood platelet MAO activity for these groups was not

significantly different from the mean values of the low

and high-MAO groups of the schizophrenics. These findings

do not support published reports of low blood platelet

activity as a genetic marker for schizophrenia. Discriminate function analysis of symptomatology ratings at baseline was used to characterize the low- and high-MAO

schizophrenic patient groups. Individuals in the low-MAO

group were distinguished by hyperactivity, anergia and

sleep disturbance.

KEY WORDS: Chlorpromazine Human Imipramine

Monoamine oxydase Platelets Schizophrenia

Thiothixene

Department of Psychiatry, University of Connecticut School

of Medicine, Farmington, Connecticut, 06032.

ABSTRACT

Toronto, Toronto Canada M5T lR8.

ABSTRACT

Thornburg, J.E. and Moore, K.E. Supersensitivity to

dommineraic agonists induced by haloperidol. IN:

Aminergic Hypotheses of Behavior: Reality or Cliche?,

B.K. Bernard, ed., Washington. National Institute on

Drug Abuse Research Monograph 3, 1975.

Haloperidol caused a significant reduction in the spontaneous locomotor activity of mice when added to their diet

for 11 days. Upon removal of the drug from their diet

these mice exhibited withdrawal hyperactivity for several

days that was characterized by an increase in activity over

control or pre-halopericol values. Similar results were

obtained when mice were fed a diet containing pimozide.

Withdrawal hyperactivity was not detected after 1 or 3

days of a haloperidol containing diet, but was maximal

after 6 days of this diet. Dose-response curves of apomorphine-stimulated motor activity and rearing behavior

were shifted to the left when determined in mice during

the period of withdrawal hyperactivity. Dopaminergic

agonists (apomorphine, piribedil, L-DOPA and d-amphetamine)

induced gnawing at lower doses in mice removed from a

chronic haloperidol-containing diet for 2 days than in mice

maintained on a control diet. These results support the

hypothesis that prolonged blockade of central dopaminergic

receptors by neuroleptics causes subsequent behavioral

effects that may be due to the development of enhanced

receptor sensitivity.

KEY WORDS: Apomorphine Haloperidol Supersensitiity

Department of Pharmacology, Michigan State University,

East Lansing, Michigan, 48824.

Morgame, P.J. and W.C. Stem. The role of serotonin and

norepinephrine in sleep-waking activity. IN: Aminergic

Hypotheses of Behavior: Reality or Cliche?, B.K. Bernard,

ed., Washington, National Institute on Drug Abuse Research

Monograph 3. 1975.

A critical review of the evidences relating the biogenic

amines serotonin and norepinephrine to the states of slowwave and rapid eye movement (REM) sleep is presented.

Various alternative explanations for specific chemical

regulation of the individual sleep states, including the

phasic events of REM sleep, are evaluated within the overall framework of the monoamine theory of sleep. Several

critical neuropsychopharmacological studies relating to

metabolism of the amines in relation to sleep-waking

behavior are presented. Models of the chemical neuronal

circuitry involved in sleep-waking activity are derived

and interactions between several brainstem nuclei, particularly the raphe complex and locus coeruleus, are discussed. Activity in these aminergic systems in relation

to oscillations in the sleep-waking cycles is evaluated.

In particular, the assessment of single cell activity in

specific chemical systems in relations to chemical models

of sleep is reviewed. Overall, it appears that the biogenic amines, especially serotonin and norepinephrine, play

key roles in the generation and maintenance of the sleep

states. These neurotransmitters participate in some

manner in the “triggering” processes necessary for actuating each sleep Phase and in regulating the transitions

from sleep to waking activity. The biogenic amines are,

however, probably not “sleep factors” or direct inducers

of the sleep states. Rather, they appear to be components

of a multiplicity of interacting chemical circuitry in the

brain whose activity maintains various chemical balances in

different brain regions. Shifts in these balances appear

to be involved in the triggering and maintenance of the

various states comprising the vigilance continuum.

KEY WORDS: Biogenic amines EEG-sleep Norepinephrine

Serotonin Sleep Pharmacology Sleep-waking

Laboratory of Neurophysiology and Psychopharmacology,

Worcester Foundation for Experimental Biology, Shrewsbury,

Massachusetts, 01545.

7
8
ABSTRACT

Cooper, Barrett R. and G.R. Breese. A role for dopamine

in the psychopharmacology of electrical self-stimulation.

IN: Aminergic Hypotheses of Behavior: Reality or Cliche?,

B.K. Bernard, ed., Washington, National Institute on

Drug Abuse Research Monograph 3, 1975

The psychopharmacology of electrical self-stimulation of

the lateral hypothalamus was studied using 6-hydroxydopamine, alpha-methyltyrosine, U-14, 624, and d-amphetamine.

Reduction of brain dopamine, but not norepinephrine, with

6-hydroxydopamine produced an acute depression of responding which eventually recovered to pretreatment levels.

A low dose of alpha-methyltyrosine, which did not affect

responding in control rats, significantly depressed

responding in the rats with brain dopamine reduced. This

treatment did not alter responding of rats with norepinephrine reduced by 6-hydroxydopamine. A dopamine-betahydroxylase inhibitor, U-14,624, depleted norepinephrine

an additional 70% yet failed to alter self-stimulation in

any of the groups. In other experiments, the 6-hydroxydopamine treatment which reduced brain dopamine was found

to block the facilitation of self-stimulation produced by

d-amphetamine. This facilitation of lateral hypothalamic

self-stimulation was not influenced by treatments which

reduced brain norepinephrine. An experiment suggesting

that dopamine is of importance to locus coeruleus selfstimulation is also described. Implications of these data

indicating a role for dopamine in self-stimulation responding are discussed in relation to the “catecholamine

hypothesis of self-stimulation.”

KEY WORDS: Alpha methyltyrosine d-amphetamine

Self-stimulation Lateral hypothalamus 6-OH dopamine

Department of Psychiatry and pharmacology, Biological

Sciences Research Center of The Child Development Institute,

University of North Carolina School of Medicine, Chapel

Hill, North Carolina, 27514.

ABSTRACT

Ginsburg, B.E. and P.Y. Sze. Pharmacogenetic studies of

the serotonergic system in association with convulsive

seizures in mice. IN: Aminergic Hypotheses of Behavior:

Reality or Cliche?, B.K. Bernard ed Washington,

National Institute on Drug Abuse Research Monograph 3, 1975.

Trytophan hydroxylase (TPH) activity was determined in

whole brain from male C57BL/10/Bg and DBA/1/Bg mice at

14 different ages between postnatal days 4 and 33. Brain

TPH activity was higher at every age in C57BL/10/Bg than

in DBA/1/Bg mice, the difference being 30-50% after day 20.

The apparent Km of the enzyme for substrate was identical

(1.4 X 10-3 M) in both strains. The reciprocal F1’s

between DBA/1/Bg and C57BL/10/Bg strains were similar in

TPH activity, being slightly lower than the predicted

midparental value. At 30 days of age, C57BL/6/Bg males

also had high TPH activity, indistinguishable from the

C57BL/10/Bg strain. Audiogenic seizure susceptibility

in these strains and their hybrid F1’s was inversely

correlated with their brain TPH activities. Similarly,

aggression Scores in these genotypes were also inversely

correlated with TPH activities. These results indicate

that seizure susceptibility and aggression in mice may be

related to the serotonergic activity in the brain. In the

case of seizures, ethanol-induced susceptibility to

audiogenic seizures in mice was enhanced by reserpine, and

the effect of reserpine could be reversed by 5-HTP but not

by DOPA. Furthermore, p-chlorophenylalanine also enhanced

such susceptibility, whereas alpha-methyltyrosine had no

effect. In the withdrawal audiogenic seizures in mice

during chronic ethanol treatment, adrenalectomy blocked

the ethanol-induced increase of brain TPH activity and

also prevented the withdrawal seizures. Our results are

consistent with the hypothesis that the serotonergic

system is among the components regulating excitability

in the brain.

KEY WORDS: Aggressive behavior Convulsive seizures

Pharmacogenetic Tryptophan hydroxylase Serotonin

Behavioral Genetics Laboratory, Department of Biobehavioral

Sciences, University of Connecticut, Storrs, Connecticut,

06268.

ABSTRACT

Mandell, A.J. and S. Knapp. A model of the neurobiological

mechanisms of action involved in lithiem prophylaxis of

bipolar affective disorder. IN: Aminergic Hypotheses of

Behavior: Reality or Cliche?, B.K. Bernard, ed., Washington, National Institute on Drug Abuse Research Monograph 3,

1975.

The effects of chronic administration of lithium chloride

on the serotonin synthesizing apparatus in rat brain

suggest a theoretical model that could explain how chronic

treatment with lithium is prophylactic against both poles

of affect in manic-depressive disorder. After three to

five days of lithium chloride administration the Vmax

of high affinity uptake of (

14C)tryptophan into striate

synaptosomes increased to 140% of control values, and

tryptophan-to-serotonin conversion activity increased to

about the same degree. These events were followed by an

apparently compensatory decrease in the Vmax of midbrain

activity

cell body and striate nerve ending tryptophan hydroxylase

activity. After 21 days of drug administration (

14C)-

tryptophan uptake remained above control levels, and

soluble midbrain and solubilized striate synaptosomal

enzyme activity remained below control levels, but synaptosomal conversion activity had returned to control levels.

In vitro, drug concentrations from 10 to 53 nM did not

affect the enzyme activity, but did enhance the uptake and

conversion measures. Also, increasing tryptophan levels

either by pre-incubation with L-tryptophan in vitro or by

the administration of L-tryptophan (20 to 60 mg/kg) in

vivo enhanced the uptake and conversion measures. The

data suggest the possibility that lithium pushes two

complementary adaptive mechanisms to their capacities, and

the net result is restricted but balanced function of

serotonergic transmission in the brain.

KEY WORDS: Adaptive capacity Conversion

Lithium Manic depressive disorder Midbrain

Serotonin Striatum Tryptophan

Tryptophan hydroxylase Uptake

Department of Psychiatry, School of Medicine, University

of California San Diego La Jolla California 92037

ABSTRACT

Bernard, B.K. Aggression and the brain monoamines: what

are the answers, but of more importance, what are the

questions? IN: Aminergic Hypotheses of Behavior: Reality

or Cliche?, B.K. Bernard, ed., Washington, National Institute on Drug Abuse Research Monograph 3, 1975.

Experiments involving three models of aggression (shock-induced fighting, ranacide and septal lesion-induced hyperirritability) are employed to demonstrate classically

different sub-types of aggressive behavior. These categories are shown to be distinct entities when compared

on the basis of hormonal dependency, central anatomical

and peripheral autonomic involvement and inhibition or

enhancement through pharmacological manipulations. Invest -

igations into brain monoamine functioning (norepinephrine,

dopamine and serotonin) demonstrate the heterogeneity of

correlations which may exist between aggressive behaviors

and brain amines. Data are analyzed on the basis of

individual amine alterations and changes in monoaminergic

neuronal balances. Thus, higher levels of shock-induced

aggressive behavior is associated with higher NE/5-HT and

DA/5-HT ratioes whereas similar alterations in these biochemical indices occur without observable changes in

ranacide behavior. Septal lesion induced hyperirritability

is correlated with precisely opposite aminergic changes,

namely, decreases in NE/S-HT ratioes. These results

demonstrate the necessity of precise aggressive model

evaluation prior to attempts at biochemical mechanism

elucidation.

KEY WORDS: Dopamine Norepinephrine Serotonin

Septal lesion induced hyperirritability

Shock-induced aggression

Division of Pharmacology and Toxicology, University of

Connecticut, Storrs, Connecticut, 06268.

9
10
ABSTRACT

Sudilovsky, A. Effects of disulfiram on the amphetamineinduced behavioral syndrome in the cat as a model of

psychosis. IN: Aminergic Hypotheses of Behavior: Reality

or Cliche?, B.K. Bernard, ed., Washington, National

lnstitute on Drug Abuse Research Monograph 3, 1975.

We have previously reported that, from a phenomenological

standpoint, the behavioral manifestations of cats chronically intoxicated with amphetamine parallel the evolution

of the paranoid psychosis induced by the drug in humans.

However, certain manifestations in the cat, such as frozen

postures, disjunctive behaviors and postures, catalepticlike phenanena, obstinate progression, loss of righting

reflex and pupillary changes, did not appear to be consistent with the phenomenology of the paranoid psychosis.

Since treatment of schizophrenic patients with disulfiram,

an inhibitor of norepinephrine synthesis that acts at the

level of the enzyme dopamine beta-hydroxylase, thereby

leading to increased dopamine concentrations, had been

found to profoundly exaggerate psychotic symptanatology,

the current experiments were undertaken to examine the

amphetamine behavioral syndrome in the cat as it is modified by pretreatment with disulfiram.

Following such pretreatment, a faster development of

certain end-stage components of the amphetamine syndrome

was obtained. Thus, on the first day, development of a

Reactive attitude and of more prominent behavioral disjunction occurred with the combined drug administration

as compared with amphetamine alone. In contrast with the

facilitation of these behaviors was the absence of dyskinesias and hyperreflexia on that day. Stereotyped

behavior, loss of motor initiative and hyperkinetic activity were markedly enhanced and appeared with a shorter

latency period on subsequent days of the intoxication

cycle. Loss of righting reflex was an early manifestation

in these animals.

During the later days, the particularly high level of

compulsive activity was evident from the occurrence of

an obstinate progression syndrome and the performance o f

stereotyped movements of the head in the presence of a

crucifixion posture. In general, modification of the

amphetamine effects on behavior was in a direction consistent with comparable features in experimental catatonia and the catatonic form of schizophrenia.

The need to integrate such phenomena in any amphetamine

model of psychosis is stressed and analogies are drawn

with similar features reported in animals treated with

bulbocapnine or other psychotogenic compounds and with

symptoms of human amphetamine psychosis and schizophrenia.

KEY WORDS: Amphetamine Disulfiram

Model Psychosis Stereotypy

E.R. Squibb and Sons, P.O. Box 4000, Princeton,

New Jersey 08540.

ABSTRACT

Schildkraut, J.J., R.E. Meyer, P.J. Crsulak, S.M. Mirin,

M. Roffman, P.A. Platz, E. Grab, M.F. Randall, M. McDougle.

The effects of heroin on catecholamine metabolism in man.

IN: Aminergic Hypotheses of Behavior: Reality or Cliche?,

B.K. Bernard, ed., Washington, National Institute on

Drug Abuse Research Monograph 3, 1975.

In a study of the effects of heroin administration in nine

human subjects, urinary catecholamines and metabolites were

examined during an initial drug-free baseline period, a

ten-day period of heroin administration and a subsequent

period of methadone detoxification. All catecholamines

and metabolites tended to be increased over baseline values

on the first day of heroin administration. However,

markedly different patterns of change emerged on subsequent

days of heroin administration. Norepinephrine and

normetanephrine remained increased throughout heroin

administration. Epinephrine was increased during the early

phase of heroin administration but returned to baseline

values during the latter phase of heroin administration.

After the increase on the first day of heroin administration, metanephrine decreased and substantial decrements

below baseline values occurred during the latter phase of

heroin administration. After increasing on the first day

of heroin administration, 3-methoxy-4-hydroxy-mandelic acid

(VMA) returned to approximately baseline values. During

heroin administration, an increase in 3-methoxy-4-hydroxyphenylglycol (MHPG) excretion was observed in a subgroup

of four of the nine subjects studied. This is in contrast

to the increase in normetanephrine excretion and the

decrease in metanephrine excretion that was observed in the

entire group of nine subjects. It is conceivable that

persistance of, or development of, tolerance might account

for the failure to observe an increase in MHPG excretion in

all of the subjects. It appeared as if the increase in

MHPG excretion began on the day prior to the administration

of heroin in the subgroup of patients with increased MHPG

excretionduring heroin administration, suggesting the

possibility of an anticipatory or conditioned response,

with the anticipation of heroin producing an increase in

MHPG excretion.

KEY WORDS: Catecholamines Catecholamine metabolism

Norepinephrine 3-methoxy-4-hydroxyphenylglycol

Heroin Opiates

Neuropsychopharmacology laboratory, Massachusetts Mental

Health Center, Boston, Massachusetts 02115.

11
12
BRAIN MONOAMINES AND PARKINSONlSM

Oloh Hornykiowicz

INTRODUCTION

The advances made in the last 15 years

in the field of the neurochemistry and

neuropharmacology of Parkinson's

disease provided the basis for the

re-definition of this basal ganglia

disorder as a "Striatal Dopamine

Deficiency Syndrome" [23]. This new

definition stresses the most prominent

chemical abnormality so far detected in

the brain of patients with Parkinson's

disease. However, it is hardly

necessary to point out that this term

does not preclude the concomitant

occurrence of other, less prominent,

biochemical alterations which may be

critically involved in the manifold

clinical aspects of this disease, both

of extrapyramidal and non-extrapyramidal origin. The following discussion

deals with the changes in brain

monoamines in Parkinson's disease.

Other highly intriguing neurochemical

alterations, such as the disturbance of

the gamma-aminobutyric acid/glutamic

acid decarboxylase system [cf. 24,28],

is outside the scope of this

presentation.

compacta of the substantia nigra [9].

It is now firmly established that these

neurons give rise to the large

nigro-striatal DA pathway [18].

Therefore, it is not surprising that in

addition to the decrease in striatal DA

and HVA levels, also the activity of

the (intraneuronally localized)

DA-synthesizing enzymes L-tyrosine

hydroxylase (manuscript in preparation)

decreased in the Parkinsonian striatum.

and L-dopa decarboxylase [26,27] is

DOPAMINE

From a neurochemical point of view the

most conspicuous feature of Parkinson's

disease is the severe deficiency of

dopamine (DA) and its metabolic

endproduct homovanillic acid (HVA) in

the basal ganglia, particularly the

caudate nucleus, putamen (these nuclei

constitute the striatum) and globus

pallidus [9,16] as well as substantia

nigra [20] (Table 1). These changes

are typical of Parkinsons's disease

regardless of etiology, being basically

due to the degeneration of the

melanin-containing neurons in the zona

13

In view of the importance of the

morphological state of the substantia

nigra for the above chemical changes in

Parkinson's disease, it is relevant

that experiments in laboratory animals

suggest the possibility of a

somatotopic organization of the

dopaminergic nigro-striatal projection.

Based on these experiments it has been

proposed that the rostra1 portion of

the nucleus projects preferentially to

the caudate nucleus, and the caudal

portion to the putamen [6,12,30,31].

Results obtained in brains of patients

with Parkinson's disease are in

agreement with this conclusion. Thus

in general, in idiopathic Parkinson's

disease the decrease of DA and HVA is

less severe in the caudate nucleus than

in the putamen [9] (Figure 1). This

most probably reflects the fact that,

in general, in idiopathic Parkinson's

disease the rostra1 portions of the

substantia nigra are less severely

degenerated than the caudal parts [19].

In contrast, in postencephalic

Parkinsonism there is a diffuse and

severe cell loss throughout the whole

of the compact zone of the substantia

nigra [l9]; this correlates well with

the finding that in this variety of
Parkinson's disease the DA deficiency pathophysiological significance of this

is severe in degree and of similar interregional difference in the two

magnitude in both the caudate nucleus varieties of Parkinson's disease has

and putamen [9] (Figure 1). The not yet been fully explored.

FIGURE 1

Postencephalitic and idiopathic Parkineonirm (P): Relationship between

the topography and degree of cell loss in the (rostra1 and caudal)

compact zone of the substantia nigra (SN) and the dopamine (DA) levels

in the caudate nucleus (CN) and putamen (P). Numbers related to CN

and P are concentrations of DA in ug/g fresh tissue. The degree of SN

lesion was assessed according to the following scale: = 0; = 1;

= 2-3 = 4; = 5; for further details, cf. ref. (9).

14
In direct support of the concept that con tralateral to the affected side of

Parkinson's disease is a striatal DA the body [5] (Table 1); (b) the degree

deficiency syndrome observations can be of' striatal DA (HVA) deficiency has

cited showing that there. been shown to be positively correlated

cause-effect relationship between the with the severity of symptoms (notably

DA deficiency and the Parkinsonian akinesia and tremor) [9]; and (c)

symptomatology. In this respect three L-dopa, DA's immediate precursor

observations are particularly relevant: substance, is highly efficacious in

(a) in Hemiparkinsonism the improving the main clinical features of

neurochemical changes are more this disease, especially akinesia

pronounced in the striatum [4,11,14].

TABLE 1

BRAIN DOPAMINE(DA) AND HOMOVANIILIC ACID (HVA) IN PARKINSON's DISEASE

Brain

Region

Controls Idiopathic Postencephalitic

Arterioscler- Manganese Hemiparkinson

otic-senile Parkinson- right left

iem

Caudate - D A 2.64 ± 0.30 (28)

nucleus - HVA 3.23 ± 0.27 (8)

Putamen - D A 3.44 ± 0.29 (28)

- HVA 4.29 ± 0.68 (8)

Subetantia - D A 0.49 ± 0.09 (5)

nigra - HVA 1.79 ± 0.18 (5)

Globus - D A 0.42 ± 0.08 (4)

pallidus - HVA 2.12 ± 0.27 (8)

0.43 ± 0.09 (l3) 0.04 ± 0.02 (6) 0.85 ± 0.26 (5)

0.89 ± 0.12 (13) 0.29 ± 0.17 (6) 1.93 ± 0.49 (5)

0.04 ± 0.01 (13) 0.02 ± 0.01 (6) 0.51 ± 0.28 (5)

0.89 ± 0.12 (13) 0.38 ± 0.18 (6) 1.88 ± 0.40 (5)

0.07 ± 0.01* (10) ne n e

0.51 ± 0.15= (5) ne ne

0.10 (3)* n e ne

0.77 ± 0.12 (12) 0.24 ± 0.05 (6) 1.48 ± 0.42 (4)

0.35

ne

0.04

ne

0.01

ne

ne

ne

1.25 0.59

ne ne

0.93 0.13

ne ne

0.01 0.01

n e n e

ne n e

n e ne

* Single case. ** In these cases no etiological differentiation was made. Values am expressed in ug/g wet tissue

(where applicable as means ± S.E.M.); number of examined cases in parentheses. ne = not examined. Data taken

from ref. (5) (9) (16) and (20).

It appears significant that the symptom

of akinesia was positively correlated

with the degree of loss of DA and HVA

specifically in the caudate nucleus,

but not in the putamen or globus

pallidus [9] (Table 2). This is

noteworthy in view of the fact that the

caudate nucleus may receive its

dopaminergic innervation predominantly

from the rostral substantia nigra.

Thus, it may be concluded that the

dopaminergic pathway critically

involved with striatal initiation of

locomotor activity, to a large extent,

has its cell bodies in the rostral

portion of the compact zone of the

substantia nigra and its terminals in

the caudate nucleus.

15
TABLE 2

CLINICO-BIOCHEMICAL CORRELATION IN PARKINSON'S DISEASE: DEGREE

OF AKINESIA VERSUS CONCENTRATIONS OF DOPAMINE (DA) AND HOMOVANILLIC ACID

(HVA) IN CAUDATE NUCLEUS, PUTAMEN AND GLOBUS PALLIDUS

Caudate nucleus

DA

HVA

Putamen

DA

HVA

Globus pallidus

HVA

Degree of akinesia

mild marked

(group A) (group B)

0.58 ± 0.12 (13) 0.22 ± 0.08 (9)*

1.68 ± 0.25 (12) 0.59 ± 0.18 (7)**

0.44 ± 0.21 (13) 0.05 ± 0.02 (9)

1.60 ± 0.39 (12) 0.83 ± 0.28 (7)

1.30 ± 0.32 (10) 0.60 ± 0.17 (7)

* Significantly different from group A, p<0.05. **Significantly

different from group A, p<0.02. values are expressed in μg/g (mean ±

S.E.M.); number of examined cases in parentheses. Data taken from ref. (9).

As to the efficacy of L-dopa as an

antiparkinson drug, a recent study

revealed that in the Parkinsonian

brain, L-dopa's metabolism basically

proceeds along the same pathways as

those established in the brain of

normal laboratory animals. Thus,

patients who received orally high

amounts of L-dopa until death, had 9-15

fold higher levels of DA and HVA in the

caudate nucleus and putamen than

non-dopa treated patients [15,26]. In

addition, low levels of dopa and higher

levels of 3-0-methyl-dopa and HVA were

measured throughout the brain. The

biochemical basis for the metabolic

transformations of L-dopa in the brain

of patients with Parkinson's disease is

indicated by the observations that (a)

although markedly reduced, enough

L-dopa decarboxylating activity remains

in the Parkinsonian striatum to account

for the formation of DA in these nuclei

[27]; (b) the L-dopa decarboxylase

activity in extrastriatal brain regions

shows much smaller changes; and (c)

both monoamine oxidase and

catechol-O-methyl transferase have a

widespread distribution in the brain,

with no significant alterations in

Parkinson's disease [26]. In this

context a significant observation is

that patients with a good response to

L-dopa therapy achieved striatal DA

levels several-fold higher than those

of poor responders [26]. This evidence

justifies the conclusion that in

patients with Parkinson's disease,

L-dopa acts as a DA replenishing drug.

Apart from the above observations

directly supporting the role of the DA

deficiency for the Parkinsonian

symptomatology, there is a large body

of (indirect) pharmacological evidence

related to this question. In

particular, it appears significant that

drugs which either deplete DA or block

DA release or its actions on the

specific receptors, produce catalepsy

in laboratory animals and in man a

neurological syndrome often indistinguishable from Parkinson's disease [for

ref. cf. 21,22].

The above observations describe in

neurochemical terms the most prominent

defect so far known in Parkinson's

disease. However, in order to

understand the functional, as well as

pathophysiological and

neuropharmacological implications of

this neurochemical alteration, it

appears profitable to ponder especially

four specific aspects. These aspects

can be regarded as being the direct

consequence of the neurochemical

changes typical of Parkinson's disease.

They are as follows: (a) the state of

reactivity of the DA receptors in the

Parkinsonian striatum; (b) the state of

activity of the remaining

nigro-striatal DA neurons; (c) the high

degree of divergence of the

dopaminergic innervation of the

striatum; and (d) compensatory brain

mechanisms and the pathophysiology of

Parkinson's disease.

State of reactivity of DA receptors in the

Parkinsonian striatum

Clinico-pharmacological correlations

have shown that there exists a positive

correlation between the severity of

akinesia and the patient's sensitivity

to L-dopa [9]. It could be shown that

patients with higher degrees of

akinesia, and correspondingly higher

degrees of striatal DA deficiency,

responded more sensitively to a single

i.v. test dose of L-dopa than patients

with mild akinesia. Considered

together with the observation that

therapeutic doses of chronically

administered L-dopa frequently produce

in Parkinsonian patients, but not in

control (=non-Parkinsonian) subjects,

16
dyskinetic, probably DA-mediated

side-effects, it can be concluded that

in Parkinson's disease the striatum

becomes supersensitive to DA. This is

not surprising as it has been known for

a long time that denervation of a given

tissue is often followed by the

development of the phenomenon of

"denervation supersensitivity" toward

the respective neurotransmitter.

Therefore the striatal receptors

deprived of their dopaminergic

innervation can be expected to develop

this type of supersensitivity to DA.

Obviously, the development of such a

"denervation supersensitivity" of the

striatal receptors would greatly

enhance any physiological effects of

the DA formed from L-dopa. Thus, this

phenomenon can be regarded as being

part of compensatory changes triggered

by the extensive loss of dopaminergic

innervation of the Parkinsonian

striatum. The physiological

significance of this receptor

supersensitivity for the efficacy of

L-dopa in Parkinson's disease can be

fully appreciated when considered

together with the phenomena of

compensatory overactivity of the

remaining DA neurons and divergence of

the dopaminergic innervation of the

striatum (see below).

Level of activity in the remaining nigrostriatal dopamine neutrons

There is evidence to show that in

Parkinson's disease the remaining

nigro-striatal DA neurons are in a

state of hyperactivity. Thus, the fact

that the ratio of striatal "DA: HVA" is

shifted in favour of the latter can be

interpreted as an indication of an

increased DA turnover (=increased rate

of synthesis and release) and a

corresponding decrease in the storage

capacity for DA in the functionally

intact neurons [21]. This

interpretation would explain the

additional observation that the levels

of tyrosine hydroxylase activity in the

Parkinsonian striatum were less severly

decreased than those of L-dopa

decarboxylase activity or DA

(manuscript in preparation). This may

indicate that there occurs in

Parkinson's disease an induction of the

rate-limiting enzyme activity in the

striatum. These changes can be

satisfactorily explained by assuming

that in Parkinson's disease the loss of

a large portion of the dopaminergic

innervation of the striatum triggers a

(receptor-mediated?) compensatory

feedback activation of the remaining

neurons. An analogous conclusion has

been recently drawn from animal

experiments with partial lesions of the

nigro-striatal DA pathway [1].

Concerning L-dopa's mechanism of

action the formation of large amounts

of DA in, and increased rate of release

from, the remaining overactive neurons

may play an important role in the

drug's therapeutic efficacy.

Divergence of nigro-stiatal DA neurons

It has been observed in the rat that

the dopaminergic innervation that the

striatum receives from the substantia

nigra displays a high degree of

divergence with one nigral DA fibre

establishing hundreds of thousand

synaptic contacts in the striatum [3].

The divergence of the dopaminergic

innervation of the striatum most

probably implies a high degree of

innervation overlap and, therefore, can

be taken to represent a considerable

safety factor; that is to say, only

loss of rather extensive portions of

this innervation will deprive the

striatal neurons of the critical

minimum innervation beyond which

functional compensation is impossible.

In conclusion, the combination of the

factors quoted in the preceding

paragraphs seems to ensure that (a)

there will be high amounts of DA formed

from L-dopa: (b) this DA will reach a

wide enough area of the diseased basal

ganglia; and (c) the newly formed DA

will act upon. supersensitive receptors.

As a consequence the dopaminergic

control of striatal function will be

restored.

Compensated and decompensated stages of

Parkinson's disease

Although there is a significant

positive correlation between the degree

of disturbance of striatal DA

metabolism and the symptoms of

Parkinson's disease, even mild,

clinically just detectable symptoms

were associated with a

disproportionately high degree of

striatal DA deficiency [9]. This

indicates that lesser degrees of DA

deficiency can be functionally

compensated by the striatum over longer

periods of time. The biochemical basis

for such a compensatory mechanism seems

to be provided by the observation

mentioned above, of a shifting in the

17
Parkinsonian striatum of the ratio "DA:

HVA" in favour of HVA indicating an

increase in DA turnover in the

remaining functionally intact

nigro-striatal DA neurons [21]. In the

case of postencephalitic Parkinsonism,

this compensatory mechanism may play a

part in the phenomenon of the usually

prolonged latency between the acute

encephalitic infection and

manifestation of the clinical

symptomatology. Thus, clinically

manifest Parkinsonism would appear to

represent the second, decompensated

stage of a process characterized by a

steadily progressing disturbance of the

striatal DA metabolism. From this, it

seems reasonable to postulate that the

main pharmacological feature of L-dopa

as a specific, but mainly symptomatic,

drug treatment of Parkinson's disease

is its ability to revert the

decompensated stage of the DA

deficiency syndrome to that of

functional re-compensation.

NOREPINEPHRINE

Apart from the severe cell loss in the

substantia nigra, pathological changes

(although less severe in degree) have

been observed in other

melanin-containing brain stem areas in

Parkinson's disease. The most

prominent of these areas is the locus

coeruleus, where loss of melanin and

formation of Lewy bodies have been

described. It has been shown

convincingly that the locus coeruleus

is an eminently noradrenergic area,

giving rise to norepinephrine

(NE)-containing neurons innervating

numerous structures at many levels of

the CNS. Therefore, changes in NE

levels in brain areas receiving inputs

from the locus coeruleus could a priori

be expected to occur in Parkinson's

disease. This is in fact the case.

Although no systematic studies have so

far been performed, the available data

indicate that the concentrations of NE

in the Parkinsonian hypothalamus as

well as the nuclei of the striatum, are

distinctly subnormal [8,16] (Table 3).

This observation is noteworthy because

recently evidence has been accumulated

showing that noradrenergic brain stem

mechanisms may be critically involved

in the control of locomotor activity

[17]. Similarly, pharmacological

evidence suggests that brain NE plays

an important auxiliary role in the

kinetic as well as the anti-akinesia

effectiveness of dopaminergic drugs

TABLE 3

Hyppothalamus 1.33 (2) 0.47 (5)

(1.15 - 1.53) (0.22 - 0.68)

Putamen 0.10 (3) 0.02 (6)

(<0.01 - 0.15) (<0.01 - 0.05)

Caudate nucleus 0.05 (2) 0.01 (6)

(0.04 - 0.06) (<0.01 - 0.02)

Glabus pallidus 0.02 (2) 0.01 (2)

(0.01 - 0.02) (<0.01 - 0.02)

Brain region

BRAIN NOREPINEPHRINE IN PARKINSON'S DISEASE

Norepinephrine

Controls Parkinson's disease

Values are expresed in μ/g wet tissue; number of cases examined

and range in parentheheses. Date taken from ref. (8).

[25,29,32]. Thus, it may be postulated

that the decrease of brain NE levels in

Parkinson's disease plays an

additional, but important, role in

producing the symptom of akinesia,

which is primarily related to striatal

DA deficiency. Since there is little

reason to doubt that NE is formed in

the CNS of patients receiving L-dopa,

it can be assumed that this NE will

contribute to L-dopa's effectiveness as

a specific anti-akinesia drug in

Parkinson's disease. This possibility

would help to explain why drugs acting

predominantly (or exclusively) on

dopaminergic mechanisms (such as

apomorphine or piribedil) are

considerably weaker than L-dopa as

antiparkinson agents.

SEROTONIN

In Parkinson's disease, the levels of

serotonin have been found to be

subnormal in several brain areas [7]

(Table 4). Most interesting presently

appears the fact that there was a

decrease in serotonin concentrations in

such areas as the central grey of the

midbrain and the hypothalamus. These

brain stem areas have been shown to

entertain numerous fibre connections

with the structures constituting the

forebrain limbic system. The latter

18
has been implicated in the control of

mood and affect. This relationship

assumes special significance if

considered in the context that (a) the

existence of a disturbance of brain

serotonin metabolism has been

postulated in mood disorders

especially the depressive illness [13],

and (b) mental depression is not

infrequent in Parkinson's disease, its

incidence in this disorder being as a

matter of fact above the statistically

expected average [2,10]. Therefore it

is tempting to speculate that this may

be related to the decreased levels of

brain serotonin (and possibly NE) found

in this disorder. This possibility

represents a challenge to future

research in the field of the

neurobiology of psychotic disorders.

TABLE 4

BRAIN SEROTONIN IN PARKINSON'S DISEASE

Brain region Serotonin

controls Parkinson's disease

Central grey of midbrain 0.53 (6) 0.36 (6)

(0.32 - 0.84) (0.09 - 0.85)

Hypothalamus 0.29 (6) 0.12 (5)

(0.14 - 0.51) (0.06 - 0.21)

Thalamus 0.26 (4) 0.13 (4)

(0.21 - 0.35) (0.08 - 0.18)

Globus pallidus 0.23 (6) 0.13 (5)

(0.19 - 0.29) (0.07 - 0.22)

Putamen 0.32 (6) 0.14(5)

(0.19 - 0.42) (0.08 - 0.16)

Caudate nucleus 0.33 (6) 0.12 (5)

(0.20 - 0.46) (0.11 - 0.15)

Values are expresed in μ/g wet tissue; number of cases examined and

range in parentheheses. Date taken from ref. (7)

REFERENCES

1. Agid, Y., F. Javoy and J.

Glowinski. Hyperactivity of remaining

dopaminergic neurones after partial

destrauction of the nigro-striatal

dopaminergic system in the rat. Nature

New Biology 245, 150-151, 1973.

2 . Ajuriaguerra, J.de, J .

Constantinidis, J.J. Eisenring, G.

Yanniotis and R, Tissot. Behaviour

disorders and L-dopa therapy in

Parkinson's disease. In: Parkinson's

Disease, vol. 1, edited by J.

Siegfried. Bern: Huber, 1972, PP.

202-217.

3. Andén N.E., K. Fuxe, B. Hamberger

and T. Hökfelt. A quantitative study

of the nigro-neostriatal dopamine

neuron system in the rat. Acta

physiol. scand. 67, 306-312, 1966.

4. Barbeau, A., T.L. Sourkes and G.

Murphy. Les catecholamines dans la

maladie de Parkinson. In: Monoamines

Et Systems Nerveux Central, edited by

J. De Ajuriaguerra, Georg, Geneva and

Masson, Paris, pp. 247-262, 1962.

19

5. Barolin, G.S., H. Bernheimer and O.

Hornykiewicz. Seitenverschiedenes

Verhalten des Dopamin

(3-Hydroxytyramin) in Gehirn eines

Falles von Hemiparkinson. Schweiz.

Arch. Neurol. Psychiat. 94: 241-248,

1964.

6. Bedard, P., L. Larochelle, A.

Parent and L.J. Poirier. The

nigrostriatal pathway: A correlative

study based on neuroanatomical and

neurochemical criteria in the cat and

the monkey. Exp. Neurol. 25: 365-377,

1969.

7. Bernheimer, H., W. Birkmayer and O.

Hornykiewicz. Verteilung des

5-Hydroxytryptamin (Serotonin) im

Gehirn des Menschen und sein Verhalten

bei Patienten mit Parkinson-Syndrom.

Klin. Wschr. 39: 1056, 1961.

8 . Bernheimer, H., W. Birkmayer and O.

Hornykiewicz. Zur Biochemie des

Parkinsonsyndroms des Menschen. Klin.

Wschr. 41: 564-569, 1963.
9 . Bernheimer, H., W. Birkmayer, O.

Hornykiewicz, K. Jellinger and F.

Seitelberger. Brain dopamine and the

syndromes of Parkinson and Huntington.

J. Neurol. Sci. 20, 415-455, 1973.

10. Birkmayer, W. Biochemistry of

depression in Parkinson's disease. In:

L-Dopa and Parkinsonism, edited by A.

Barbeau and F.H. McDowell,

Philadelphia: Davis, 1970, pp. 341-342.

20. Hornykiewicz, O. Die topische

Lokalisation und das Verhalten von

Noradrenalin und Dopamin

(3-Hydroxytyramin) in der Substantia

nigra des normalen und Parkinsonkranken

Menschen. Wien. Klin. Wschr. 73:

309-312, 1963.

21. Hornykiewicz, O. Dopamine

(3-hydroxytyramine) and brain function.

Pharmacol. Rev. 18: 925-964, 1966.

11. Birkmayer, W. and O. Hornykiewicz.

Der L-Dioxyphenylalanin (=L-DOPA)

Effekt bei der Parkinson-Akinese. 22. Hornykiewicz, O. Dopamine and its

Wien. Klin. Wschr. 73: 787, 1961. physiological significance in brain

function. In: The Structure And

12. Carpenter, M.B. and P. Peter. Function of Nervous Tissue, vol. VI,

Nigrostriatal and nigrothalamic fibers edited by G.H. Bourne. Academic Press,

in the Rhesus monkey. J. comp. Neurol. Ind. New York and London, 1972, pp.

144: 93-177, 1972. 367-415.

13. Coppen, A. Indoleamines and

affective disorders. J. Psychiat. Res.

9: 163-171, 1972.

14. Cotzias, G.C., M.H. Van Woert and

L.M. Schiffer. Aromatic amino acids

and modification of parkinsonism. New

Eng. J. Med. 276: 374, 1967.

15. Davidson, L., K. Lloyd, J. Dankova

and O. Hornykiewicz. L-dopa treatment

in Parkinson's disease: effect on

dopamine and related substances in

23. Hornykiewicz, O. Dopamine and

extrapyramidal function and

dysfunction. In: Neurotransmitters,

Res. Publ. A.R.N.M.D. vol. 50, edited

by I.J. Kopin. Baltimore: Waverly,

1972, pp. 390-412.

24. Hornykiewicz, O. Metabolism of

dopamine and L-dopa in human brain In:

Frontiers in catecholamine researach,

edited by E. Usdin and S.H. Snyder.

New York: Pergamon, 1973, PP.

1101-1107.

discrete brain regions, Experientia 27:

1048-1049, 1971. 25. Kuschinsky, K. and O.

Hornykiewicz. Effects of morphine on

16. Ehringer, H. and O. Hornykiewicz. striatal dopamine metabolism: Possible

Verteilung von Noradrenalin und Dopamin mechanism of its opposite effect on

(3-Hydroxytyramin) im Gehirn des locomotor activity in rats and mice.

Menschen und ihr Verhalten bei Europ. J. Pharmacol. 26: 41-50, 1974.

Erkrankungen des extrapyramidalen

Systems. Klin. Wschr. 38 1236-1239, :

1960.

26. Lloyd, K.G., L. Davidson and O.

Hornykiewicz. Metabolism of levodopa

in the human brain. In: Advances in

17. Forssberg, H. and S. Grillner. Neurology vol. 3, edited by D.B.

The Locomotion of the acute spinal cat Calne, New York: Raven Press, 1973,

injected with Clonidine i.v. Brin pp. 173-188.

Research 50: 184-186, 1973.

27. Lloyd, K. and O. Hornykiewicz.

18. Fuxe, K., T. Hökfelt and U. Parkinson's disease: Activity of L-dopa

Ungerstedt. Morphological and decarboxylase in discrete brain

functional aspects of central monoamine regions. Science 170: 1212-1213, 1970.

neurons. In: Internat. Rev.

Neurobiology. Vol. 13, edited by C.C. 28. Lloyd, K.G. and O. Hornykiewicz.

Pfeiffer and J.R. Smythies, New York, L-Glutamic acid decarboxylase in

London: Academic Press, 1970, PP. Parkinson's disease: effect of L-dopa

93-126. therapy. Nature 243: 521-523, 1973.

19. Hassler, R. Zur Pathologie der 29. Maj, J., H. Grabowska and E.

Paralysis agitans und des Mogilnicka. The effect of L-dopa on

postenzephalitischen Parkinsonismus. brain catecholamines and motility in

J. Psychol Neurol. (Lpz.) 48: 387-476, rats. Psychopharmacologia 22, 162-171,

197.

20
30. Moore, R.Y., R.K. Bhatnagar and A. 31. Riddell, D. and J.C. Szerb. The

Heller, Anatomical and chemical release in vivo of dopamine synthesized

studies of nigro-neostriatal

projection in the cat. Brain Res. 30:

from labelled precursors in the caudate

nucleus of the cat. J. Neurochem. 18:

119-135, 1971. 989-1006, 1971.

32. Strömberg, U. and T.H. Svensson.

L-Dopa induced effect on motor activity

in mice after inhibition of

dopamine-beta-hydroxylase.

Psychopharmacologia 19: 53-60, 1971.

AUTHOR

Oleh Hornykiewicz, M.D.: Department

of Pharmacology and Department of

Psychopharmacology, Clarke Institute of

Psychiatry, University of Toronto,

Toronto, Canada, M5T 1R8.

21

SUPERSENSITIVITY TO DOPAMINERGIC

AGONISTS INDUCED BY HALOPERlDOL

J.E. Thornburg, K.E. Moore.

INTRODUCTION

Prolonged disruption of nerve impulse

transmission at central dopaminergic

synapses with 6-hydroxydopamine

[16,20,21,22] or alpha-methyltyrosine

[2,6] leads to enhanced responses to

direct and indirect acting dopaminergic

agonists. Following chronic blockade

of dopaminergic receptors with

neuroleptics, the doses of dopaminergic

agonists required to produce

stereotyped behavior are reduced

[6,8,12,17,18,23]. In each case, the

enhanced responses to dompamine

agonists may be due to supersensitivity

of dopaminergic receptors.

The present study demonstrates that,

following prolonged administration of

haloperidol to mice, evidence of

supersensitivity of dopaminergic

receptors is manifested as increased

spontaneous locomotor activity,

increased apomorphine-stimulated motor

activity and increased sensitivity to

dopaminergic agonist-induced gnawing

behavior.

METHODS

Experiments were conducted in male

Swiss-Webster mice (Spartan Research

Animals, Haslett, Michigan), with

initial body weights of 20-25 g. These

mice were provided with water and a

diet of 20 g ground Wayne Lab Blox/4

mice/24 hrs (control diet) or the same

diet containing 0.005% haloperidol or

0.01% pimozide.

Locomotor activity of groups of 4 mice

was measured using Electronic Motility

Meters 40Fc (Motron Products,

Stockholm, Sweden) which have 40

infrared-sensitive photocells spaced 4

cm apart in the base and a light source

overhead. These units were placed in a

sound-proof, fan-ventilated chamber

(5'x5'x3'). For testing, the animals

were placed in a plexiglass cage

(18"x 10" and 5") which just covers the

photocell-sensitive area of the Meters.

In experiments in which nocturnal

activity was measured, the mice were

maintained continuously in the test

environment. Activity was recorded and

food presented from 1700 to 0800 hrs.

Hearing activity was determined using a

Vertical Movement Detector 5Fc (VMD,

Motron Products) attached to the

Motility Meter. The VMD consists of 5

photocells at one end of the cage, each

directly opposite a light source. The

photocells were positioned 2" above the

base, so that a standing or rearing

movement was necessary to activate the

photocell.

The ability of various dopamine

agonists and central stimulants to

induce gnawing or chewing behavior in

mice was determined as described by

Pedersen and Christensen [14].

Following an injection of drug, pairs

of mice were placed in a 4-walled

chamber (10-1/2"x5"x12") which rested

on a sheet of corrugated cardboard.

23
After one hour in the chamber, mice

were removed and the presence or

absence of gnawing on the cardboard was

noted. The characteristic drug-induced

gnawing must be distinguished from the

qualitatively different ripping or

shredding of the corrugated paper by

saline-treated control mice.

All motor activity data were analyzed

using Student's 't' test and gnawing

behavior data were analyzed using a Chi

square contingency test [9]. The level

of significance was chosen as P<0.01.

DRUGS

Haloperidol and pimozide, obtained from

Dr. John Kleis, McNeil Labs, Fort

Washington, Pa., were incorporated into

the diet of ground food. Apomorphine

HC1, purchased from Eli Lilly and Co.,

Indianapolis, Indiana, was dissolved in

0.1% sodium metabisulfite. Piribedil

(ET495, Trivastal), obtained from Dr.

J. Pearl Sterling-Winthrop Research

Laboratories, Hennselaer,

L-alpha-methyldopahydrazine (MK486),

obtained from Dr. C.A. Stone, Merck

liesearch Laboratories, West Point Pa.,

and L-DOPA, Nutritional Biochemicals,

Cleveland, Ohio, were suspended in 1%

methylcellulose. Benztropine mesylate,

obtained from Dr. C.A. Stone, Merck

Sharpe and Dohme Research Laboratories,

West Point, Pa., d-amphetamine Sulfate,

obtained from Dr. B.A. Kimes, Smith

Kline and French Laboratories,

Philadelphia, Pa., and methylphenidate

HCl, obtained from CIBA-Geigy

Corporation, Ardsley, N.Y., were

dissolved in saline.

RESULTS

Spontaneous Motor Activity During and

After Chronic Diets of Haloperidol and

Pimozide

Nocturnal activity of groups of 4 mice,

measured in their home environment, was

reduced by 60 to 75% during an 11 day

period in which 0.005% haloperidol was

added to their diet. Upon

reinstitution of a control diet,

activity increased to 150 to 175% of

pre-haloperidol activity (Fig. 1).

This increased activity was maintained

for 5 days after withdrawal of the

haloperidol diet. In a similar

experiment in which mice were fed a

diet containing 0.01% pimozide for 10

days nocturnal activity was reduced by

40 to 70% (Fig. 2). During the first 3

Figure 1. Spontaneous locomotor activity of mice during and after a chronic

diet containing haloperidol.

Activity of groups of 4 mice was recorded in the home environment during

the dark period (1700-0800 hr); a control diet or a diet containing 0.005%

haloperidol was presented during these

hours. Total nocturnal activity (bars

and vertical lines represent mean ± 1

S.E.) is expressed as percent of control of the mean activity for the two

nights preceding the addition of haloperidol to the diet. Activity was significantly increased on the first 5

days following the haloperidol diet

(P<0.01).

creased following the pimozide diet.

(P<0.01)

Figure 2. Spontaneous locomotor activity of mice during and after a chronic diet containing pimoaide. The

experimental protocol is described in

the legend to Fig. 1 except that mice

were fed a diet containing 0.01% pimozide. Activity was significantly decreased during and Significantly in-
days after withdrawal of pimozide,

activity increased to 150 to 200% of

control activity.

Relationship of the Duration of a Haloperidol Containing Diet to the Development of Withdrawal Hyperactivity

Groups of 4 mice were fed a diet

containing 0.005% haloperidol for

periods of 1,2,6 or 10 days and then

placed on a control diet. Activity

measured on the night of the second day

after withdrawal of the haloperidol

diet was significantly below control

after the 1 day diet and not

significantly different from control

after the 3 day diet (Fig. 3). Two

days after the 6 day diet of

haloperidol, activity was nearly 200%

of control. The degree of withdrawal

hyperactivity did not increase further

following cessation of a 10 day diet of

haloperidol.

Figure 3. Relationship of the duration

of feeding of a haloperido1 diet to the

development of withdrawal hyperactivity

Groups of 4 mice were fed a diet containing 0.005% haloperidol for periods

of l, 3, 6, 10 days and were then fed

a control diet. The circles represent

total nocturnal activity starting on

the second day after withdrawal of

haloperidol expressed as percent of

initial control activity (N=3). Solid

symbols indicate a significant difference from control at P<0.01.

Apomorphine-Induced Motor Activity and

Rearing Behavior Following Cessation of

Chronic Diets of Haloperidol

If the decrease of locomotor activity

during the administration of

haloperidol results from dopaminergic

receptor blocking actions of this drug,

then the observed increase in

spontaneous locomotor activity

following withdrawal of the

haloperidol-containing diet may be due

to enhanced sensitivity of central

dopaminergic neurons. If this is true,

the locomotor activity response to

apomorphine, a direct acting

dopaminergic agonist, should also

increase. To test this hypothesis,

mice were fed a diet of 0.005%

haloperidol for 10 days, and the

response to apomorphine or vehicle was

determined on the second day of

withdrawal (day 12). Apomorphine, at a

dose of 0.25 mg/kg, did not alter

activity in control mice but produced

significant locomotor stimulation in

the haloperidol-treated group (Table

1). The result is compatible with an

increased sensitivity of dopaminergic

receptors. At higher doses of

apomorphine, the degree of locomotor

stimulation was similar in the control

and the haloperidol-treated mice.

TABLE 1

Apomorphine-induced locomotor activity following a chronic

diet containing 0.005% haloperidol

Dose of Counts/30 min

Apomorphine

(mg/kg, s.c.) Diet: Control Haloperidol

0.25 -27O ± 78

0.50 1820±140

1.00 2728±135

1450± 94

2463± 85

2607±180

At 48 hours after withdrawal from a 10 day control

or haloperidol-containing diet the mice were accommodated to the test environment for 30 min prior to

receiving either 0.1% sodium metabisulfite or apomorphine S.C. The values represent apomorphine-stimulated activity (mean ± SEM for 6 determinations).

Underlined values indicate a significant difference

from control at P<0.01.

25
In another experiment, the effects of

apomorphine on rearing behavior were

determined on the third day following a

5 day diet of haloperidol. Mice

previously fed haloperidol exhibited

significantly greater rearing activity

in response to apomorphine at doses of

0.25 and 0.50 mg/kg, than did control

mice (Fig. 4).

Figure 4. Apomorphine-induced rearing

activity in mice following withdrawal

of a diet containing haloperidol.

Rearing activity of groups of 4 mice

was determined as described in METHODS.

At 3 days following 5 days of a control

diet ( ) or a diet containing 0.005%

halperidol ( ), the mice were accommodated to the test environment for 30

min prior to receiving either 0.1%

sodium metabisutphite or apomorphine

s. c. The data represent the number of

rears during the period of 25 to 30

minutes after injection (mean ± 1 S.E.,

N=6). Solid symbols indicate significant differences from control at

P<0.01.

6/6

6/6

6/6

5/6

6/6

2/6

3/6

6/8

4/7

3/5

6/6*

---

0/6

3/6

6/7

10/12

6/6

0/6

0/5

0/5

Effects of Dopaminergic Agonists on

Gnawing Behavior of Mice After Cessation of a Chronic Diet Containing

Haloperidol

piribedil and L-DOPA caused significant

gnawing only in the haloperidol-treated

mice. The gnawing responses to

d-amphetamine and methylphenidate were

more pronounced in the haloperidoltreated group. The enhanced sensitivity to d-amphetamine was also evidenced

by the fact that all mice previously on

the haloperidol-containing diet died

after 8 mg/kg of d-amphetamine while

there were no deaths in the control

group. Benztropine, which has dopamine

uptake-blocking and anticholinergic

properties, did not induce gnawing in

either control or haloperidolpretreated mice.

TABLE 2

Effects of dopaminergic agonists on gnawing behavior of mice

after a chronic haloperidol-containing diet

Treatment

(mg/kg)

Apomorphine HCl (s.c.)

2.5

5

10

2 0

30

Piribedil (s.c.)

1 0

2 0

4 0

d-Amphetamine sulfate (i.p.)

6

16

L-DOPA (i.p.)

400

800

Methylphenidate (i.p.)

30

Benztropine (i.p.)

20

40

Oiet

Control Haloperidol

0/6

2/6

0/6

1/6

5/6

0/6

0/6

0/8

0/7

1/5

3/6

6/6

0/6

0/6

1/7

2/18

3/7

3/6

0/5

0/5

The ability of various dopaminergic

agonists to produce gnawing was tested

in control mice and in mice 2 days

after a 5 day diet containing 0.005%

haloperidol. The results are

summarized in Table 2. A dose of 30

mg/kg apomorphine was required to

elicit gnawing in a majority of control

mice, while at all doses tested

apomorphine caused gnawing in mice

previously receiving a

containing diet.

haloperidolAt the doses tested,

26

Mice were placed on a diet containing 0.005% haloperidol for

5 days and then fed a control diet for 2 days. Pairs of mice

were then injected with the various dopaminergfc agonists,

placed in a box containing a corrugated cardboard floor for 1

hour. and the presence or absence of gnawing was recorded (see

METHODS and [14]. Data represent ratios of the number of pairs

tested. exhibiting gnawing'to the total number of pairs of mice

tested. Underlined ratios indicate those values that are significantly different from control (p<.01). Thirty minutes prior

to the injection of L-DOPA. 50 mg/kg of MK-486. a peripheral decarboxylase inhibitor, was administered (i.p.) to the mice.

*, all twelve mice died within 3 hours after the injection of

d-amphetamine.
DISCUSSION

Prolonged blockade of dopaminergic

receptors with neuroleptics appears to

cause development of supersensitivity

of the receptors; there is considerable

recent behavioral evidence to support

this view. Following withdrawal of

chronic treatment with neuroleptics,

dopamine agonists produce stereotyped

behavior at doses lower than required

in control animals [6,8,12,17,19,23].

The present study demonstrates that in

addition to stereotyped behavior,

several other behaviors in mice are

altered following cessation of chronic

diet of haloperidol. Such mice

exhibited increased spontaneous

locomotor activity, increased

apomorphine-stimulated locomotor

activity and rearing and increased

sensitivity to dopaminergic

agonist-induced gnawing behavior.

Asper et al. [l] and Møller-Nielsen et

al. [13] found that, after repeated

administration of haloperidol and other

neuroleptics, marked tolerance

developed toward the ability of these

drugs to inhibit apomorphine-induced

stereotyped behavior. The development

of tolerance may reflect receptor

supersensitivity before withdrawal of

the neuroleptic. The failure to

demonstrate development of tolerance in

the present study is possibly due to

administration of a receptor-saturating

dose of haloperidol.

The fact that sensitivity to

apomorphine is altered supports the

view that the major synaptic change

involved is at the receptors. This is

also consistent with earlier studies of

supersensitivity of central dopaminergic responses to apomorphine following

6-hydroxydopamine [16,20,21,22]. The

supersensitivity following chronic

alpha-methyltyrosine administration

[2,5] also likely involves a change at

the receptors, since the dose-response

curve for apomorphine-stimulated locomotor activity in mice after a chronic

diet of alpha-methyltyrosine is shifted

to the left [10].

Fjalland and Møller-Nielsen [6]

demonstrated that following chronic

administration of haloperidol,

methylphenidate induced gnawing at

lower doses than required in control

mice. The present study confirms and

extends this finding to include the

dopaminergic agonists, apomorphine,

piribedil, L-DOPA and d-amphetamine.

In rats, piribedil caused weak

stereotyped behavior only after prior

treatment with 6-hydroxydopamine [9], a

finding consistent with the effects of

piribedil in this study.

A transient, enhanced sensitivity to

dopaminergic agonists has been clearly

demonstrated upon withdrawal of chronic

treatment with neuroleptics in animals.

The increased spontaneous motor

activity in these animals may be due to

hyperactivity of dopaminergic neuronal

systems in the brain. Increased

activity of central dopaminergic

neuronal systems in humans after

termination of chronic neuroleptic

therapy may be reflected by a variety

of abnormal involuntary movements.

Klawans and Rubovits [12] and Gerlack

et al. [7] proposed that tardive

dyskinesias, which are observed in

patients on long term therapy with

neuroleptics after reductions in dosage

or discontinuation, may be due to

dopaminergic hypersensitivity. In

contrast to the often prolonged tardive

dyskineasias, the time course of the

enhanced motor responses in mice was

short-lived, correlating more closely

with the time course of mild,

reversible oral-facial dyskinesias in

adults following abrupt withdrawal of

phenothiazines or haloperidol [3,11],

and that of reversible choreiform-like

involuntary movements of the

extremities in children

withdrawal of neuroleptics [15].

after

ACKNOWLEDGMENT

The excellent technical assistance of

Nan Friedle is gratefully acknowledged.

(Supported by USPHS grant NS-090174.)

REFERENCES

1. Asper, H., M. Baggiolini, H.R.

Burki, H. Lauener, W. Ruch and G.

Stille. Tolerance phenomena with

neuroleptics: Catalepsy, apomorphine

sterotypy and striatal dopamine

metabolism in the rat after single and

repeated administration of loxapine and

haloperidol. Eur. J. Pharmacol.

22:287-294, 1973.

2. Beuthin, F.C., T.S. Miya, D.E.

Blake and W.F. Bousquet. Enhanced

sensitivity to noradrenergic agonists

and tolerance development to alphamethyltyrosine in rat. J. Pharmacol.

Exp. Ther. 181:446-456, 1972.

27
3. Crane, G.E. Persistent dyskinesia.

Brit. J. Psychiat. 122: 395-405, 1973.

4. Creese, I. Behavioral evidence of

dopamine receptor stimulation by

piribedil (ET495) and its metabolite

S584. Eur. J. Pharmacol. 28:55-58,

1974.

5. Dominic, J.A. and K.E. Moore.

Supersensitivity to the central

stimulant actions of adrenergic drugs

following discontinuation of a chronic

diet of alpha-methyltyrosine. Psychopharmacologia 15:96-101, 1969.

6. Fjalland, B. and I. Møller-Nielsen.

Enhancement of methylphenidate-induced

stereotypes by repeated administration

of neuroleptics.

34:105-109, 1974.

Psychopharmacologia

7 . Gerlack, J., N. Reisly and A.

Randrup. Dopaminergic hypersensitivity

and cholinergic hypofunction in the

pathophysiology of tardive dyskinesia.

Psychopharmacologia 34:21-35, 1974.

8. Gianutsos, G., R.B. Drawbaugh, M.D.

Hynes and H. Lal. Behavioral evidence

for dopaminergic supersensitivity after

chronic haloperidol. Life Sci. 14:

887-898, 1974.

9 . Goldstein, A. Biostatistics: An

Introductory Text, p. 272, MacMillan,

New York, 1972.

10. Gudelsky, G.A., J.E. Thornburg and

K.E. Moore. Blockade of alpha-methyltyrosine-induced supersensitivity to

apomorphine by chronic administration

of L-DOPA. Life Sci. (in press).

11. Jacobson, G., R.J. Baldessarini

and T. Manschreck. Tardive dyskinesia

associated with haloperidol (in press),

1974.

12. Klawans, H.L. and R. Rubovits. An

experimental model of tardive dyskinesia. J. Neural Trans. 33:235-246,

1972.

13. Møller-Nielsen, I., B. Fjalland,

V. Pedersen and M. Nymark. Pharmacology of neuroleptics upon repeated

administration. Psychopharmacologia

34:95-104, 1974.

14. Pedersen, V. and A.V. Christensen.

Antagonism of methylphenidate-induced

stereotyped gnawing in mice. Acta

Pharmacol. (Kbh.) 31:488-496, 1972.

15. Polizoes, P., D.M. Engelhardt and

S.P. Hoffman. CNS consequences of

psychotropic drug withdrawal in schizophrenic children. Psychopharmacol.

Bull. 9:34-35, 1973.

16. Schoenfeld, R.I. and N.J. Uretsky.

Altered response to apomorphine in

6-hydroxydopamine-treated rats. Eur.

J. Pharmacol. 19:115-118, 1972.

17. Tarsy, D. and R.J. Baldessarini.

Pharmacologically induced behavioral

supersensitivity to apomorphine.

Nature New Biol. 245:262-263, 1973.

18. Tarsy, D. and R.J. Baldessarini.

Behavioural supersensitivity to

apomorphine following chronic treatment

with drugs which interfere with the

synaptic function of catecholamines.

Neuropharmacology 13:927-740, 1974.

19. Thornburg, J.E. and K.E. Moore.

Supersensitivity to apomorphine ET495

(l-[3,4-(methylenedioxybenzyl)-4-(2-pyrimidyl]piperazine) and L-DOPA following intrastriatal 6-hydroxydopamine

administration. Pharmacologist 15:254

(ABS), 1973.

20. Thornburg, J.E. and K.E. Moore.

Supersensitivity to dopamine agonists

following unilateral 6-hydroxydopamineinduced striatal lesions in mice. J.

Pharmacol. Exp. Ther. 192:42-44, 1974.

21. Ungerstedt, U. Postsynaptic

supersensitivity after 6-hydroxydopamine-induced degeneration of the

nigro-striatal,dopamine system. Acta

Physiol. Scand. Suppl. 367:69-93, 1971.

22. Von Voigtlander, P.F. and K.E.

Moore. Turning behavior of mice with

unilateral 6-hydroxydopamine lesions in

the striatum: Effects of apomorphine

L-DOPA, amantadine, amphetamine and

other psychomotor stimulants. Neuropharmacology 12:451-463, 1973.

23. Von Voigtlander, P.F. Behavioral

and biochemical investigation of

dopamine supersensitivity induced by

chronic neuroleptic treatment. Fed.

Proc. 33:578 (ABS), 1974.

AUTHORS

J.E. Thornburg, Ph.D. and K.E. Moore,

Ph.D.: Department of Pharmacology,

Michigan State University, East

Lansing, Michigan 48824

28
BLOOD PLATELET MONOAMINE

OXIDASE ACTIVITY

IN ANERGIC SCHIZOPHRENICS

Edward G. Shaskan, Robert E. Becker

INTRODUCTION

Throughout the history of biological

research in schizophrenia there have

existed numerous claims for peripheral

(blood or urine) biochemical markers

for this disease [16]. A consistant,

albeit discouraging experience among

investigators working in this field has

been. the frequency, indeed the

predictability, by which so-called

biological markers of schizophrenia

have not stood up under replication

studies and which have often been

refuted [16].

Recent twin studies and adoptee studies

have provided data indicating that

between 25% to 38% of monozygotic twins

become concordant--i.e., both ill--for

schizophrenia at some time during the

period of high risk from age 15 to 45

[10]. Nine percent of children of

biologically schizophrenic parents

adopted-out near birth become

schizophrenic as adults [12]. This

data forms the core of the present

consensus that certain factors

predisposing to schizophrenia are

genetically, probably polygenetically,

determined.

In recent investigations from

laboratories at NIMH, a highly

significant reduction in the activity

of blood platelet monoamine oxidase

(MAO) has been reported for both

chronic and acute schizophrenic

patients as compared to controls

[7,14]. The reduction in platelet MAO

activity did not appear to be a product

of the schizophrenic illness nor

influenced by drug treatment [7,14; Dr.

Dennis Murphy, personal communication].

This reduced blood platelet MAO

activity may provide a genetic marker

for vulnerability to

[9,14].

schizophrenia

The widely quoted findings of Murphy

and Wyatt and their collaborators at

NIMH [7,14] provide enticing

implications for the understanding of

the etiology of schizophrenia, if a

relationship between peripheral and

central MAO activity can be

demonstrated. Preliminary

investigations, however, have not

suggested a reduced MAO activity in the

brains of schizophrenics at

[3,13].

autopsy

It has been suggested that an

excessively activated dopaminergic

system in the brain may be

etiologically important in some types

of schizophrenia [4,9]. Once again,

however, the finding that human

platelet and brain monoamine oxidases

are most dissimilar with respect to the

29
oxidation of dopamine [6,11] suggests

that reduction of platelet MAO activity

may have little relevance to the

oxidation of that biogenic amine most

relevant to schizophrenia [5].

Murphy (personal communication) now

questions the hypothesis that low blood

platelet MAO activity is a genetic

marker for vulnerability to

schizophrenia. He has modified his

original hypothesis by excluding acute

schizophrenic reactions (Carpenter,

Murphy and Wyatt, submitted for

publication). In this latest work,

however, they do not question the basic

relationship of low platelet MAO

activity found in their chronic

schizophrenic patients.

As part of a continuing

psychopharmacologic study of anergic

schizophrenia, we have now completed a

study of twenty-four depressed

schizophrenic outpatients treated with

combined antipsychotic and

antidepressant medication. As a

working hypothesis, we predicted that

lower MAO activity might sensitize the

schizophrenic patient to the induction

of psychotic symptoms by tricyclic

antidepressants [1]. As two criteria

for this effect of tricyclic

antidepressant drugs in schizophrenics,

we expected that those schizophrenics

who reacted adversely as a group would

show 1) an intensification of primary

and secondary schizophrenic symptoms

and 2) would have low MAO activity

significantly different from

non-reactors and/or controls. This

paper reports preliminary work which is

part of a larger study designed to

replicate the findings of Wyatt and

Murphy [7,14] and to test our

hypothesis that schizophrenic patients

with low platelet MAO activity will

show increased vulnerability to

induction of psychosis by tricyclic

antidepressants.

MATERIALS AND METHODS

Twenty-six anergic schizophrenic

outpatients were admitted to the study

at the University of Connecticut McCook

Hospital Psychiatric Service.

Diagnoses of schizophrenia with

depression were established before

referral and confirmed by one of us

(R.E.B.). All patients had a prior

hospitalization with a discharge

diagnosis of schizophrenic reaction and

had evidence of a thought disorder

recorded in the clinical record of

their hospitalization. All patients

had received antipsychotic medications

prior to admission into this study.

Ten hospitalized acute alcoholics free

of organic neurological sequelae and

seven laboratory personnel volunteered

as controls.

Patients were free of psychoactive

medication for two weeks and then

blindly treated with either

chlorpromazine (Thorazine, SKY)

100-1200 mg daily and imipramine

(Tofranol, Geigy) 150 mg daily or

thiothixine (Navane, Roerig) 5-60 mg

daily and placebo. Blood platelet MAO

activity was evaluated following the

drug-free period and once weekly for

four weeks on medication.

Platelet preparation from venous blood

collected by vacutainer into 18 ml

tubes anti-coagulated with

acid-citrate-dextrose (Becton-Dickinson

#4798) followed the method of Wyatt and

co-workers [15] with the following

modifications. Vacutainers containing

the blood were placed in crushed ice

and transferred to the laboratory

(maximum time on ice equalled two

hours) and the vacutainers were

centrifuged at 4" C for ten minutes at

170g. The platelet-rich plasma

supernatant was transferred to 10 ml

polypropylene centrifuge tubes using

plastic disposable pipettes. This

procedure was repeated at 380g and

680g. The polypropylene centrifuge

tube containing between 5 to 9 ml of

platelet-rich plasma (PRP) was

centrifuged again at 380g for 10

minutes to remove incidental

contamination of the PRP by red cells.

The PRP supernatant from the latter

centrifugation was then transferred to

a clean polypropylene centrifuge tube

and centrifuged at 10,000g for ten

minutes, after which the platelet-poor

plasma was discarded. The platelet

pellet was washed twice successively

with 2 ml of saline, drained, and

stored for up to 30 days at -60° C

without measurable loss of MAO

activity. Two-day old blood-platelet

concentrates (approximately 300 ml of

PRP) obtained from the American Red

Cross Blood Bank were pooled and

approximately 30 platelet pellets were

prepared in centrifuge tubes and stored

at -60°C as above.

Platelet pellets were sonicated in 0.1

ml of 0.05 M sodium-potassium phosphate

(Na-K-PO4) buffer, pH 7.2 per ml of

3 0
final PRP using a Kontes Cell Disrupter

with power setting at number 3 and

frequency setting between numbers 3 and

4. Complete disruption of platelet

membranes was effected in one minute,

as demonstrated by oil-immersion

light-microscopy. Using C

14-tryptamine

(1.25mC/mmole; 1.6 x 10-5 M, final

concentration) as substrate, platelet

MAO activity was linear for up to about

30 minutes and to about 0.4 mg of Lowry

protein when incubated at 37°C in 0.5

ml of 0.05 M Na-K-PO4 buffer, pH 7.2.

In preliminary experiments we found the

pH optimum value for human blood

platelet MAO activity to be

approximately pH 7.8. However, since

platelet MAO activity assayed at pH 7.2

was about 91% of the activity found at

pH 7.8 and since platelet MAO values

from other

assayed at pH 7.2, incubation

laboratories [7,14] were

conditions for MAO values reported here

were at pH 7.2, at 37°C, and for 20

minutes. Typically, reaction mixtures

contained platelet sonicates

representing between 0.1 to 0.2 mg'of

Lowry protein. The reaction was

stopped by injecting 0.1 ml of 30%

perchloric acid and the glass-stoppered

centrifuge tubes remained on ice for 20

minutes, after which time 5 ml of

toluene was added to each tube. The

glass-stoppered centrifuge tubes were

shaken for 10 minutes and centrifuged

for 10 minutes at 2000g to separate the

phases. Three ml of the toluene phase

was then transferred into scintillation

vials to which 10 ml of scintillation

cocktail was added. Radioactivity of

the samples was determined in

ISOCAP-300 (Searle Analytic, Inc.)

scintillation spectrometer with

efficiencies for our labelled-samples

ranging between 87 to 89%.

Red Cross platelet pellet samples were

routinely included in every assay as a

control for possible changes in MAO

activity due to incidental technical

causes. Boiling at l00°C for 10

minutes of the Red Cross blood platelet

samples prior to adding the radioactive

substrate served as the enzyme blank

values for the assay.

RESULTS

Of the twenty-six patients admitted to

the study, twenty-four completed the

two-week washout and twenty-two

patients completed the four weeks

on-drug period. The mean age of the

31

schizophrenic patients was 39.2 ± 3.8

years. This compared to a mean age of

44.6 ± 3.6 years for the alcoholic

patient controls and 34.9 ± 2.5 years

for the non-patient controls.

Blood platelet MAO activity values in

our sample of anergic schizohprenic

outpatients were bimodally distributed

and suggested a natural division into

two groups (Table 1). Arbitrarily,

those individuals having a mean

(minimum of three and maximum of five

weekly blood samples) platelet MAO

activity value of less than 2.99

mumoles/mg protein/hour are referred to

as the "LO-MAO" group, while those

individuals whose mean platelet MAO

activity was above this value are

included in the "HI-MAO" group (Table

1). Although the sample size for both

alcoholic and non-patient controls was

respectively smaller as compared to the

schizophrenics, a similar bimodal

distribution for these control subjects

could also be discerned (Table 1). No

striking correlation between subjects'

sex and the bimodal distribution of

blood platelet MAO activity was

apparent (Table 1).

TABLE 1

DISTRIBUTION OF PLATELET MONOAMINE

OXIDASE ACTIVITY IN SCHIZOPHRENICS

AND CONTROLS

Blood Platelet Numbers of Subjects (Sex)

MAO Values* Schizophrenics Alcoholics Non-patients

1.00 - 1.49 2 (M,F)

1.50 - 1.99 5 (4M,F)

2.00 - 2.49 5 (3M,2F)

2.50 - 2.99 1 (M)

3.00 - 3.49 4 (3M,F)

3.50 - 3.99 2 (M,F)

4.00 - 4.49 4 (M,3F)

Above 4.50 1 (F)

1 (F) 0

4 (4M) 2 (M,F)

1 (M) 1 (M)

1 (M)** 2 (2M)**

0 0

2 (M,F) 1 (M)

1 (F) 0

0 1 (F)

*Mumoles product formed per hour per mg Lowry Protein.

'*Values of 2.75 and below were assigned to the "low" group.

Of the twenty-four schizophrenics who

completed the washout and at least two

weeks of the on-drug study, thirteen

could be assigned to the LO-MAO group

and eleven were assigned to the HI-MAO

group (Table 2). The control subjects

were also arbitrarily assigned on the

basis of their platelet MAO activity to

LO-MAO or HI-MAO groups. Thus, six
alcoholics and three non-patient

volunteers had mean platelet MAO

activity not significantly different

from the mean platelet MAO activity of

schizophrenics assigned to the LO-MAO

group (Table 2). Similarly, four

alcoholics and four non-patient

volunteers had mean platelet MAO

activity not significantly different

from mean values for schizophrenic

patients who had been assigned to the

HI-MAO group (Table 2).

TABLE 2

MEAN BLOOD PLATELET MAO ACTIVITY

IN OUTPATIENT ANERGIC SCHIZOPHRENICS

AND CONTROLS

Monoamine Oxidase Activity

(mμmoles product/hr/mg protien)

Subjects LO MAO HI MAO P Values

Anergic

Schizophrenics 1.99±0.42(13) 3.78±0.65(11) <0.001

Alcoholics 1.73±0.21(6) 3.62±0.69(4) <0.001

Non-Patient

-controls 2.04±0.30(3) 3.68±0.93(4) <0.1

RELATINSHIP OF SELECT PATIENT-VARIABLES

AT BASELINE TO MAO GROUPS

Table 2: Values represent the mean ±

standard error of the mean for the

number of individuals expressed in

parentheses. One blood sample from

each individual was assayed for MAO

activity in triplicate. Mean MAO

values for all subjects studied are

not significantly different from each

other within the LO-MAO or HI-MAO

groupings.

Mean Score Qualitative

Variable LO-MAO HI-MAO F-Value Importance

Katz-Hyperactivity 3.6 2.1 12.7 Overhelming

Score

HDS*Sleep Disturbance 1.8 1.0 5.0 Strong

Score

Sex 9 male 5 male 4.0 Marginal

4 male

HDS Apathy Score 2.3 2.9 3.9 Marginal

Age 5 aged<35 6 aged<35 3.2 Weak

8 aged<35 5 aged<35

*HDS - Hamilton Depression Scale

It has been reported that blood

platelet MAO activity correlates highly

with increasing age in humans [8] and

has a tendency towards higher

activities in women than in men [8].

Also, platelet MAO activity has been

shown to vary over the menstrual cycle

in women with a mean variation of about

23% [2]. In this pilot sample our

results suggest that neither. sex nor

age appear to significantly correlate

with the mean MAO activity for either

the LO- or HI-MAO groups of

schizophrenics (Table 3). Similarly,

neither sex (Table 1) nor age appeared

to be correlated with this high-low

dichotomy in control subjects.

Since it has been suggested that blood

platelet MAO activity might be a

genetic marker for vulnerability to

schizophrenia [7, 14], one might

reasonably expect that primary symptoms

of schizophrenia (thought disorder and

flatness of affect) and secondary

symptoms (hallucinations and delusions)

would correlate with low platelet MAO

activity. However, in our population

of schizophrenics, no measures relating

to primary schizophrenic symptomatology

discriminated the LO- or the HI-MAO

groups at either the beginning or the

end of the study period. In a

discriminate function analysis the

hyperactivity score the

Adjustment Scale (KAS) significantly

distinguished between the LO- and the

HI-MAO groups, with the mean value in

the HI-MAO group being significantly

(F=12.7) lower, thus less

hyperactivity, than the mean value for

the LO-MAO group (Table 3). The sleep

disturbance score on the Hamilton

Depression Scale (HDS) also

discriminated at baseline between these

blood platelet MAO groups (Table 3).

TABLE 3

Psychological testing was again

performed on the twenty-two patients

who completed the 4-week on-drug study,

and analyses of covariance were run for

some 66 psychological test variables as

a two-factor analysis of covariance.

The two factors were drug combination

administered and platelet MAO group.

As found upon discriminate analysis of

primary schizophrenic symptoms at

baseline, once again at the completion

of the study, items such as

"bizarreness" (KAS) and "thinking

disorder"

Rating Scale

the Brief Psychiatric

(BPRS) did not

discriminate between LO- and HI-MAO

groups (Table 4). The thinking

disorder scores (BPRS) had a

significant interaction with drug

group, with chlorpromazine/imipramine

better than thiothixene/placebo

32
(p<0.01, Table 4). Schizophrenic

patients in the LO-MAO group, as

compared to patients in the HI-MAO

group, appeared significantly worse

(lower score values) on the following

items that are usually not considered

to comprise primary or secondary

schizophrenic symptomatology: HDS

sleep disturbance (p<0.10), HDS

somatization (p<0.05), BPRS anergia

(p<0.05), and KAS hyperactiviy

(p<0.05).

Factor

Hamilton Depression Scale

Sleep Disturbance

Somatization

"Psychotomimetic" Cluster

Imipramine was maintained at a constant

dosage of 150 mg per day, while

thiothixene and chlorpromazine dosages

could be adjusted by the psychiatrist

in charge (R.E.B.) as per clinical

management of the patients. No

significant differences in

between LO- and

dosage

HI-MAO groups were

observed for either thiothixene or

chlorpromazine, although there was an

apparent trend towards prescribing more

chlorpromazine to patients in the LOas compared to the HI-MAO groups.

TABLE 4

RELATIONSHIP BETWEEN SELECT CLINICAL VARIABLES

AND PLATELET MAO ACTIVITY OR DRUG THERAPY

Adjusted Final Score

Thiothixene Chlorpromazine/Imipramine

LO MAO (N=7) HI MAO (N=5) LO MAO (N=5) HI MAO (N=5) Statistical Significance

1.3 0.9 1.5 0.4

1.1 0.5 1.3 0.4

3.2 3.5 4.7 1.7

p<0.10 in favor of HI MAO

groups.

p<0.05 in favor of HI MAO

groups.

p<0.01 interaction between

CPZ/Imip. and MAO

groups

Brief Psychiatric Rating Scale

Depression 2.3 2.6 2.8 1.9 None

Thinking Disorder 3.1 2.3 1.5 1.9 p<0.01 CPZ/Imip. significantly better than

thiothixene.

Anergia 1.8 1.3 1.9 1.0 p<0.05 in favor of Hl MAO

groups

Katz Behavior Inventory Scale

Hyperactivity 3.7 2.4 2.9 2.4 p<0.05 in favor of HI MAO

Bizarreness 3.3 3.6 2.5 3.6

groups

None

Table 4: "Psychotomimetic" cluster on

the Hamilton Depression Scale is arbitrarily represented here as the mean

sum of the following factors:

Insight (#17), Diurnal Variation (#18),

Depersonalization and Derealization

(#19), Paranoid Symptoms (#20) and

Obsessional and Compulsive Symptoms

(#21).

DISCUSSION

The studies

[7,14]

by Murphy and colleagues

reporting a reduced blood

platelet MAO activity in schizophrenic

patients as compared to normal controls

could not be replicated in a group of

twenty-four schizophrenic outpatients.

In our study individuals have either

low or high platelet MAO activity,

Analyses for a total of 66 factors

were run as a two factor analysis of

covariance, the two factors being drug

and MAO group. Final scores (at completion of study) were adjusted

according to baseline (drug washout)

scores.

scales,

On all of the above rating

the lower the score the

"better" the patient on any factor.

which is stable over a four-week

period. The platelet MAO activity is

independent of the diagnosis of

schizophrenia. Low blood platelet MAO

activity reported for chronic

schizophrenics [7,14] may be an effect

of chronic schizophrenic illness,

hospitalization or a physiological

33
characteristic of some individuals

independent of the schizophrenic

illness per se, but disposing toward a

chronic outcome. Primary and secondary

schizophrenic symptomatology did not

discriminate between LO- and HI-MAO

groups within our schizophrenic

outpatient population. Symptoms that

relate more closely with affective

states such as sleep

hyperactivity, anergia, and

disturbance,

somatization may provide directions

toward understanding the interaction of

platelet MAO activity and schizophrenic

illness (Tables 3 and 4). In an

essentially replicative study we are

presently evaluating blood platelet MAO

activity and clinical symptomatology in

a group of inpatient anergic

schizophrenics. We are also performing

a more systematic study using these

ACKOWLEDGMENTS

The authors gratefully acknowledge the

technical assistance of Mr. Kevin

Norige and Ms. Gloria Grillo and thank

Dr. Dennis Murphy and Ms. Cindy

Donnelly for their helpful suggestions

on the monoamine oxidase assay and

interpretations.

REFERENCES

1. Becker, R.E. Evaluation of an

amitriptyline-perphenazine combination

in chronic schizophrenia. Amer. J.

Psychiat. 127:827-831, 1970.

2 . Belmaker, R.H., D.L. Murphy, R.J.

Wyatt and D.L. Loriaux. Human platelet

monoamine oxidase changes during the

menstrual cycle. Arch. Gen.

Psychiatry. 31:553-556, 1974.

3. Domino, E.F., R.R. Krause and J.

Bowers. Various enzymes involved with

putative neurotransmitters. Arch. Gen.

Psychiat. 29:195-201, 1973.

4. Matthysse, S. Antipsychotic drug

actions: a clue to the neuropathyology

of schizophrenia? Fed. Proc. 32:

200-205, 1973.

5. Meltzer, H.Y. and S.M. Stahl.

Platelet monoamine oxidase activity and

substrate preferences in schizophrenic

patients. Research Communications in

Chemical Pathology and

7(2):419-431, 1974.

Pharmacology.

variables on a larger group of

non-patient volunteers and hospitalized

alcoholics.

Triyclic antidepressants have been

shown to induce schizophrenic symptoms

in some anergic schizophrenic patients

[1]. We postulated that low MAO

activity patients would show an

intensification of primary and

secondary schizophrenic symptoms from

treatment with a tricyclic

antidepressant. Thinking disorder

scores (BPRS), bizarreness scores (KAS)

and secondary symptomatology did not

appear to be worsened by tricyclic

antidepressant (i.e . , imipramine)

treatment of our anergic schizophrenic

patients (Table 4). We therefore do

not have any direct evidence supporting

this hypothesis.

We appreciate the help of Alfred J.

Katz, M.D., and A. Matthew Gottlieb

M.A., of the Connecticut Red Cross

Blood Program,

Farmington,

209 Farmington

Connecticut.

Ave.,

6. Murphy, D.L. and R. Weiss. Reduced

monoamine oxidase activity in blood

platelets from

patients.

bipolar depressed

Amer. J. Psychiat. 128:

1351-1357, 1972.

7 . Murphy, D. and J. Wyatt. Reducted

monoamine oxidase activity in blood

platelets from schizophrenic patients.

Nature. 238:225-226, 1972.

8 . Robinson, D.S., J.M. Davis, A.

Nies, C.L. Ravaris and D. Sylwester.

Relation of sex and aging to monoamine

oxidase activity of human brain,

plasma, and platelets. Arch. Gen.

Psychiat. 24:536-539, 1971.

9. Snyder, S.H., S.P. Banerjee, H.I.

Yamamura and D. Greenberg. Drugs,

neurotransmitters, and schizophrenia.

Science, 184:1243-1253, 1974.

10. Stabenau, J. and W. Pollin.

Experiential differences for

schizophrenics as compared with their

34
non-schizophrenic siblings: twin and

family studies. In: (eds.) Roff, M.

and D. Ricks Life Historv Research in

Psychopathology. The University of

Minnesota Press, Minneapolis, 1970.

11. Tipton, K.F. Some properties of

monoamine oxidase. Adv. Biochem.

Psychopharm. 5:11-24, 1972.

12. Wender, P. The role of genetics

in the etiology of the schizophrenias.

Am. J. Orthopsychiatry.

1969.

39:447-458,

13. Wise, C.D. and L. Stein.

Dopanine-beta-hydroxylase deficits in

the brains of schizophrenic patients.

Science. 181:344-347, 1973.

AUTHORS

14. Wyatt, R.J., D.L. Murphy, R.

Belmaker, S. Cohen, C.H. Donnelly and

W. Pollin. Reduced monoamine oxidase

activity in platelets: a possible

genetic marker for vulnerability to

schizophrenia. Science. 179:916-918,

1973.

15. Wyatt, R.J., J.M. Saavedra and J.

Axelrod. A dimethyltryptamine-forming

enzyme in human blood.

Psychiatry. 130:754-760, 1973.

Am. J.

16. Wyatt, R., Termini and J. Davis.

Biochemical and sleep studies of

schizophrenia: A review of the

literature - 1960-1970 - I. Biochemical studies. Schizophrenia Bulletin.

4:10-45, 1971.

Edward G. Shaskan, Ph.D., Robert E.

Becker, M.D.: Department of

Psychiatry, University of Connecticut

School Medicine

Connecticut 06032 (U.S.A.)

Farmington,

35

THE ROLE OF SEROTONIN AND

NOREPINEPHRINE IN SLEEP-WAKING ACTIVITY

Peter J. Morgane, Warren C. Stern

INTRODUCTION

A wide variety of studies in the past

decade have indicated that both the

serotonergic neurons of the raphé

system and catecholaminergic neurons,

in particular those of the dorso-latera1 pontine tegmentum, seem to play an

essential, but by no means exclusive,

role in the regulation of the sleepwaking cycle. These chemical systems

regulate the vigilance continuum probably by modulating the activity of

other neuronal systems and by interacting, directly or indirectly, with each

other at several integrative levels in

the brainstem. The view most widely

put forward is that serotonin-containing nuclei and pathways play a more

dominant role in regulating slow-wave

sleep while activity in noradrenergic

systems is involved in the generation

of REM sleep. Since, however, these

two chemical systems interact at several levels in the brain we will not only

consider the evidence relating them

individually to the states of sleep but

also summarize their possible reciprocations in regulating the oscillations

between the different states of vigilance.

THE ROLE OF CENTRAL MONOAMINE

NEURONS IN THE REGULATION

SLEEP AND WAKING:

EVIDENCE AND OVERVIEW

The discovery of direct, probably

monosynaptic, serotonergic and noradrenergic pathways to the cerebral cortex

from the reticular formation of the

brainstem [5,18,87,63] has greatly

stimulated research on the role of

monoamine neurons in sleep. An

additional important factor has been

the work of Jouvet [36,37,39], who

discovered that monoaminergic drugs and

lesions of the catecholamine and

serotonin-containing nerve cell nuclei

greatly interfere with particular

facets of the sleep-wakefulness cycle.

In order to define the role of

monoamines in sleep mechanisms the

following three types of studies have

usually been undertaken: (1) The

effects of drugs (usually psychoactive)

that alter monoamines on the cortical

EEG; (2) The effects of specific

lesions of the monoamine pathways to

the telencephalon and diencephalon on

the sleep-wakefulness cycle. This

latter, presumably more specific,

lesion approach has been made possible

due to the introduction of 6-hydroxydopamine which, when injected into

central catecholaminergic pathways,

causes rather selective destruction of

these fibers [85,87]. Also, 5,6-dihydroxytryptamine has been shown to

exert somewhat selective actions on

central serotonin pathways [6,33] and

much research is now underway using

these chemical lesioning tools in the

evaluation of the relative roles of

serotonin and norepinephrine neurons in

sleep. However, the specificity of

both of these neurotoxic agents is

still in considerable doubt [69,16,75];

37
(3) Finally, studies have been made on

the effects of sedative-hypnotic drugs

and psychostimulant drugs on the

relation of the sleep states to amine

turnover in various dopamine,

noradrenaline and serotonin pathways in

the brain. Each of these three major

categories of studies will be

elaborated upon in this paper so as to

typify the relations between biogenic

amines and the sleep-waking states.

The definitive role of serotonin-containing neurons in the induction of

slow-wave sleep and in the "priming" of

REM sleep has been suggested predominantly by Jouvet's group [38] and is

based on several varieties of converging experimental evidence. The following findings are most strongly persuasive in this regard: (1) The inhibition

by para-chlorophenylalanine (PCPA) of

the first step of serotonin synthesis

results in total insomnia and this can

be reversed to "physiological" sleep by

subsequent injection of the serotonin

precursor 5-hydroxytryptophan (5-HTP),

which bypasses the inhibition of

synthesis; (2) Lesions of the

serotonin-containing neurons of the

rostral and middle raphé system

(especially the nucleus raphé dorsalis,

nucleus centralis superior of Betcherew

and nucleus raphé medialis) induce

insomnia which is proportional to the

extent of ablation of the serotonincontaining perikarya and to the

decrease of serotonin in the forebrain

at the level of the neuron terminals--

this type of insomnia is not reversed

by injection of 5-hydroxytryptophan;

(3) Biochemical, and presumably selective, lesions of the serotonin terminals produced by the intraventricular

injection of 5,6-dihydroxytryptamine

result in a significant decrease of

both slow-wave and REM sleep.

Our goal here is to discuss primarily

the role of serotonin in sleep-waking

mechanisms but, quite clearly, this

cannot be done in isolation from the

catecholinergic systems of the brain

since there are obviously complex

interactions occurring between these

systems. Some of the evidence favoring

the involvement of catecholaminergic

systems in regulation of the vigilance

states is as follows: (1) The

inhibition of catecholamine synthesis

at the level of tyrosine hydorxylase by

alpha-methylparatyrosine has been shown

to reverse the arousal and insomnia

following the destruction of the

rostral raphé system, thereby resulting

in a state of sedation with cortical

synchronization; (2) The destruction of

the dorsal noradrenergic bundle in the

mesencephalon is followed by a significant decrease of noradrenaline in the

telencephalon and diencephalon and by a

disturbance in arousal mechanisms both

in waking and REM sleep; (3) Stirmula

tion of catecholamine systems by d-amphetamine results in arousal.

The dopamine nigrostriatal pathways

from the substantia nigra are not

significantly involved with cortical

EEG activity since quantitatively

normal levels of polygraphic sleep and

waking are seen after almost total

ablation of the substantia nigra,

following which there is practically

complete depletion of striatal dopamine. The substantia nigra appears,

however, to be concerned with the

maintenance of waking activity since

its destruction is followed by akinesia

[34].

THE SEROTONIN-SLEEP HYPOTHESIS

We should now elaborate on the evidence

for the role of serotonin in the

regulation of the sleep states. The

main experimental results which relate

to the so-called serotonergic hypothesis of sleep have been the subject of

several extensive reviews by Jouvet

[37,38,40] and by Morgane and Stern

[56]. The serotonergic hypothesis of

sleep is an integral part of the general "monoamine theory" of the regulation

of the states of vigilance. Its essence can be summarized as follows

based, in large part, on Jouvet's earlier findings. Two antagonistic histochemically identified sleep and waking

systems, whose cell bodies are located

in the brainstem and whose terminals

are dispersed widely in the brain, are

involved in the alternation of the

sleep-waking cycle. Serotonin-containing neurons are responsible for slowwave sleep and for "priming" REM sleep,

whereas ponto-mesencephalic catecholamine-containing neurons are involved

in the "executive" mechanisms of REM

sleep and in the maintenance of both

tonic behavioral and EEG arousal. Each

level of integration of the central

nervous system is submitted to the

synaptic action of these

neurotransmitters, with the behavioral

and EEG aspects of the sleep-waking

cycle thus depending upon the

organization of the post-synaptic

efferent neurons. This monoamine

38
theory of sleep also states explicitly

that REM sleep depends upon some

"priming" serotonergic mechanism, and

that slow-wave sleep and REM sleep must

be in some way correlated. The theory

emphasizes the crucial importance of

the brainstem for the modulation of the

sleep-waking cycle but also allows for

the fact that other structures,

primarily in the hypothalamus and in

the basal forebrain area, are also

intimately involved in the regulation

of sleep-waking activity. Some supportive evidence for the hypothesis may be

succinctly summarized as follows: (1)

Inhibition of synthesis of serotonin

with PCPA induces a secondary insomnia

which correlates well with the depletion of brain serotonin. Further, this

insomnia can be immediately reversed

and "physiological" sleep is induced

by the injection of a small dose of

5-HTP, which is rapidly converted into

serotonin as shown by the subsequent

relative increase of serotonin and

5-HIAA [8]; (2) The surgical destruction of serotonin-containing neurons of

the midbrain and pontine raphk system

induce a marked and protracted insomnia

that is proportional to the decrease of

serotonin in the terminal areas of the

serotonin fiber pathways in the

forebrain. This tybe of insomnia is

not reversible by "secondary injection

of a small dose of 5-HTP. no doubt due

to the fact that the 5-HTP cannot be

metabolized into serotonin since the

serotonin cell bodies are destroyed and

the terminals are secondarily degenerated. Both of these lines of data

indicate quite clearly that neuropharmacological and neurophysiological alterations of serotonin synthesis lead

to the same overall result, i.e., a

significant decrease of sleep. There

is reason to believe that pharmacological approaches, largely due to their

non-specific effects, cannot give more

precise information concerning the

topography of the serotonin neuron

systems involved in sleep control. As

a result, several experimenters have

recently chosen some more presumably

specific techniques to alter the regional metabolism of serotonin neurons,

either by chemically lesioning them

with central injections of 5,6-dihydroxytryptamine or by indirectly

increasing their metabolism with lesions of the dorsal noradrenergic

pathway.

Jouvet, in particular, has marshaled

evidence that groups of serotonincontaining neurons, located mostly in

the rostra1 part of the raphé system,

are responsible for the induction of

sleep-- probably by releasing serotonin

in strategic forebrain regions. Among

these regions there is considerable

evidence that preoptic serotonin terminals may Play a cardinal role in the

induction of the post-synaptic events

leading to cortical synchronization.

Jouvet feels that this concept may

reconcile, on both histochemical and

biochemical bases, the two main

theories that are currently proposed

for the explanation of sleep, i.e., the

"ascending" hypothesis related to the

role of the serotonin-containing cells

of the raphé [39] and the "descending"

hypothesis related to the role of the

preoptic area [59]. Any selective

inactivation of serotonin neurons

bearing upon the cell bodies located in

the raphé, the serotonin

pathways in

ascending

the medial fore-brain

bundle, or the serotonin terminals

located in the preoptic area itself

might, therefore, be expected to lead

to a significant decrease of sleep.

The activation of serotonin-containing

neurons can theoretically be achieved

through the direct stimulation of the

serotonin cell bodies, as shown by the

subsequent increase of 5-hydroxyindoleacetic acid in the terminals (2). However, this approach has given somewhat

equivocal results upon the vigilance

states, either increasing sleep in one

study [48], or increasing waking in

others [70,31]. Such opposing results

might be due to the fact that electrical stimulation may activate other nonserotonin neurons in the raphé or may

provoke the release of serotonin in

some unphysiological manner. Overall,

however, the bulk of the evidence

favors the generation of arousal

following raphé stimulation.

Jouvet has attempted to shed additional

light on this problem by carrying out

the activation of serotonin-containing

neurons using neuropharmacological

techniques. Thus, he has shown that

the inactivation of catecholamine

neurons with alpha-methylparatyrosine

induces an increased turnover in

serotonin neurons. In such instances

the decrease of endogenous catecholamine is accompanied in the cat brainstem by an increase in the conversion

of labeled tryptophan into labeled

serotonin and by a significant increase

of 5-HIAA [43]. If the hypothesis that

catecholamine neurons inhibit serotonin

neurons is true, then the destruction

39
of some known catecholamine pathways

should induce some activation of the

serotonin system.

Jouvet's group first approached this

problem in the cat by a select lesion

of the dorsal noradrenergic bundle

[52]. Jones et al. [35] had shown

rather conclusively that this bundle

was involved in the maintenance of

tonic cortical arousal. haeda et al.

[52] found that bilateral lesioning of

the dorsal noradrenergic bundle at the

level of the isthmus immediately

adjacent to the periaqueductal gray

matter and to the rostral raphé system

induced a most dramatic hypersomnia.

They found an increase of up to 400% of

REM sleep which was observed during the

first day after the lesion. This

remarkable degree of hypersomnia, at

first characterized by a selective

increase REM sleep and, secondarily, by

an increase of both states of sleep,

lasted for 8-10 davs. after which there

was a slow return to baseline levels.

Control lesions more dorsally in the

colliculi or in the rostral midbrain

did not induce any significant

alterations of sleep. Only the caudal

extension of the lesion into the cell

groups of the locus coeruleus and locus

subcoeruleus totally impaired this

phenomenon by suppressing REM sleep.

The mechanism of this most characteristic hypersomnia produced by far, the

most pronounced increase of REM sleep

yet observed experimentally and was

analyzed by neuropharmacological and

biochemical means, the results strongly suggesting that the serotonergic

neurons of the raphé were involved.

Thus, the following points are most

relevant: (1) The pretreamtment of cats

with PCPA, which induces an almost

total insomnia , prevents the subsequent

hypersomnia which normally follows the

destruction of the dorsal noradrenergic

bundle; (2) When the biosynthesis of

serotonin is measured during the

maximum period of hypersomnia, i.e., 24

hours after the lesion, there is a

significant increase in the conversion

of labeled tryptophan into labeled

serotonin in both the cerebral cortex

and the raphé, while no alteration of

endogenous serotonin or catecholamine

can be detected; (3) Specific alterations of endogenous levels of monoamines have been detected in another

series of experiments in which cats

were sacrificed 8-10 days after the

lesion [66]. In these experiments

there was a decrease of norepinephrine

in the forebrain (mostly in the

cortex), which was explained by the

destruction of the fibers essential to

the maintenance of norepinephrine in

these areas, i.e., the dorsal noradrenergic bundle. This decrease in norepinephrine was accompanied by a significant increase of both tryptophan

and 5-HIAA levels in the brain, while

serotonin did not change significantly.

It would appear from these results that

the turnover of serotonin neurons was

still elevated 8-10 days after these

lesions.

The specific mechanisms by which the

serotonin-containing neurons of the

raphé complex are activated have not

yet been discovered. A possible

explanation is that components of

noradrenergic fiber systems ascending

from the rostral part of the locus

coeruleus might tonically control the

activity of the serotonin-containing

cell bodies in the raphé complex.

Fluorescence mapping studies have shown

that many catecholamine-containing

terminals terminate close to the

serotonin cell bodies in this area

[12,51], but all criteria for a direct

noradrenergic-serotoin synapse have not

yet been met. If such a control

exists, it is possible that lesions of

noradrenergic fibers would release the

activity of serotonin neurons as is

suggested by the two biochemical

controls used by the Jouvet group

above.

We have recently [57] obtained preliminary electrophysiological evidence

supporting the view that catecholamine

neurons directly inhibit the activity

of the anterior raphé cells. It was

observed that electrical stimulation Of

the locus coeruleus in anesthetized

rats produced a long-lasting inhibition

of spontaneous raphé unit activity

(Fig. 1). Control recordings of units

1 mm lateral to the nucleus raphk

dorsalis did not show this inhibition

following locus coeruleus stimulation.

Such a mechanism would provide much

plasticity and flexibility in the

influence of norepinephrine neurons

(which are strongly implicated in the

control of cortical arousal) upon the

serotonin neurons involved in sleep.

In studies on the role of central

serotonin neurons in sleep the

relations to certain drugs affecting

sleep should be mentioned. These can

be summarized in general as follows:

Barbiturates in sedative-hypnotic doses

40
FIGURE 1

INTER - SPIKE INTERVAL OF RAPHÉ UNIT FIRINGS AFTER

BRAINSTEM STIMULATION

Figure 1.: Histograms of single unit activity in three raphé dorsalis

neurons of rats in response to single pulse stimulation of the locus

coeruleus (LC) or the adjacent reticular formation (non-LC stimulation).

Rats were anesthetized with chloral hydrate, 350 mg/kg, i.p. and the LC

was given a biphasic pulse of 20 A, 500 sec duration once every 30 sec

for at least 5 min. In the base-line condition the interspike intervals

(ISI) are clustered in the 0.5-1.0 sec. range, characteristic of raphé

dorsalis spontaneous discharge rates. Following LC stimulation, however,

there was a marked overall depression in the number of unit firings,

illustrated by a decrease in the short ISIs and an increase in the long

ISIS. The non-LC stimulation control produced no change in the distribution of raphé ISIs. (From Morgane, Stern and Berman, 57).

41
appear to decrease serotonin turnover

in both ascending cortical and

sub-cortical serotonin pathyways. It

should be remembered that the ascending

sub-cortical serotonin pathways to the

hypothalamus originate mainly from the

serotonin cell groups B7 and B8

(nucleus raphé dorsales and nucleus

raphé medianus), whereas the cortical

serotonin pathways run more laterally

in the mesencephalon and appear to

originate partly from cell group B9

[33]. The organization of the ascending serotonin neurons is therefore

similar to that of the ascending norFIGURE 2

epinephrine neurons, with distinct cortical and sub-cortical pathways distinguishable (Fig. 2). Barbiturates

appear to have a general depressant action, whereas minor tranquilizers may

selectively depress activity in the

cortical serotonin pathways which, as

noted, are distinctly separated from

the sub-cortical serotonin pathways.

The turnover changes probably reflect

changes in nervous activity in the

pathways, since no direct action of

these compounds on serotonin uptake and

release have been discovered.

Figure 2.: Schema showing the principal monoaminergic pathways in the

brain. Many of the projections of these aminergic systems terminate in

subcortical areas such as the hypothalamus, striatum, limbic system and

basal forebrain areas. Other projections pass diffusely into several

zones of the neocortex. The "A" and "B" coding systems on the diagram

are the cell groups indicated in Dahlström and Fuxe (12). Abbreviations

are as follows: MED: medulla; MES: mesencephalon; MM: mammillary nuclei;

3V: third ventricle; NC: neocortex; SEP: septum; LFA: limbic forebrain

area; STR: striatum; GP: globus pallidus; DA: dopamine; NA: norepinephrine;

5-HT: serotonin; LHA: lateral hypothalamic area; IP: interpeduncular

nucleus; IT: internal capsule.

42
As reviewed above, Jouvet [42] has

postulated that the ascending serotonin

neurons are of critical importance

especially for slow-wave sleep. The

turnover studies of Lidbrink [50],

revealing a dose-dependent decrease in

the activity of the serotonin pathways

by barbiturates and minor tranquilizers, do not, however, favor this view.

It would seem that if the hypothesis of

Jouvet is correct, the decrease in activity of the serotonin neurons would

have to be visualized as a compensatory

feedback response. Thus, the barbiturates and minor tranquilizers might

induce slow-wave sleep by an action on

or beyond the serotonin receptors and

activity in the serotonin neurons might

no longer be needed to induce slow-wave

sleep. These results have been described in relation to changes in the

firing rate of the raphé serotonin

nerve cells [50].

THE ROLE OF CENTRAL CATECHOLAMlNERGlC NEURONS IN SLEEP

The role of the catecholaminergic

systems in relation to sleep-waking

behavior can be outlined under three

major headings:

1. The Effects of Monoamine-Modulating

Psycho-Active Drugs on the Cortical

EEG. Most monoamine-modulating drugs

interfere with both dopamine and

noradrenergic synapses. However, a few

neuropharmacological tools have been

developed which make it possible to

interfere almost exclusively with

activity in noradrenergic pathways.

Additional drugs are known to block

only dopamine and not noradrenergic

receptors. By appropriate combinations

of these agents Lidbrink et al. 1501

found that a decrease in wakefulness

coincides with a maximal depletion of

brain norepinephrine. They also speculated that newly synthesized norepinephrine is of particular importance for

the maintenance of REM sleep. In other

studies they showed that amphetamine

caused EEG arousal and that the desynchronization produced by amphetamine

was not blocked by dopamine receptor

blocking agents such as spiroperidol,

whereas haloperidol (which blocks both

central dopamine and norepinephrine

receptor sites) reduced the action of

amphetamine on the cortical EEG. Their

results were interpreted as underlining

the important role of the coerulo-cortical (dorsal) noradrenergic neuron

system as an arousal system in the

brain in that they demonstrated that

the effects of sedative-hypnotic drugs,

such as barbiturates and minor

tranquilizers, are mediated partly via

depressing activity in this specific

pathway.

With regard to psycho-stimulant drugs,

amphetamine and related compounds have

been found to cause release of dopamine

and norepinephrine [19]. The cortical

noradrenergic nerve terminals, however,

appear to be particularly sensitive to

these drugs. Furthermore, cortical

norepinephrine turnover is increased

and the decrease of cortical norepinephrine turnover found after barbiturates is counteracted by amphetamine.

In view of the fact that amphetamine

reduces hexobarbital sleeping time

these results further demonstrate the

important role that these cortical noradrenergic networks play in

wakefulness.

2. The Effects of 6-HvdroxvdopamineInduced Lesions of the Dorsal Noradrenernic Pathway. 6-Hydroxydopamine was injected into the cortical

noradrenergic bundle (dorsal noradrenergic pathway) at the level of the

dorsomedial tegmentum of the caudal

mesencephalon of rats by Lidbrink et

al. [50]. This neurotoxic treatment

resulted in interruption of nerve

impulse flow in the dorsal

noradrenergic pathway following which

histochemical analysis showed an almost

complete disappearance of cortical

noradrenergic nerve terminals 15 days

after the lesion. The interruption of

the cortical noradrenergic pathway by

6-hydroxydopamine in these studies

resulted in a 20% decrease of waking

during 8 days of recording. REM sleep

was also suppressed by 18% whereas

there was no significant change in

percent of slow-wave sleep after

6-hydroxydopamine. Thus, the effects

appear to be transient, being most

pronounced in the beginning of the

period following 6-hydroxydopamine

administration. No changes in waking

and sleep were found in animals 25-30

days after chemical lesions of the

dorsal (coerulo-cortical) noradrenergic

pathway.

These studies of Lidbrink et al. [50]

agree generally with the conclusions

reached by Jouvet's group (see below).

The latter group have underlined the

role of the catecholamine-containing

43
group A8 in the ventro-lateral midbrain

tegmentum in waking mechanisms. These

findings were based on the results of

lesions in this area but, since these

lesions also damaged part of the

ascending noradrenergic pathways, they

could also be explained partly on the

basis of interruption of these pathways. Lidbrink's group demonstrated

the importance of the anterior dorsolateral portion of the locus coeruleus

(so-called principal locus coeruleus),

since this portion of the nucleus gives

rise to the cortical noradrenergic

pathway which was damaged in their

experiments. Also, the recent studies

of Henley and Morrison [26] showed that

after bilateral lesions in the pontine

tegmentum there resulted episodes of

REM sleep in which the atonia of that

state was absent. They concluded that

the hypotheses, stating that the locus

coeruleus or other isolated nuclei of

the pons are specifically concerned

with the initiation of REM sleep, are

not supported by their findings. Their

histological data appeared to show

lesions largely confined to the locus

coeruleus area.

3. The Effect of Psychoactive Drugs on

Norepinephrine Turnover. With regard

to the role of sedative-hypnotic drugs

and psycho-stimulant drugs on amine

turnover in various chemical pathways,

it has been shown that barbiturates and

minor tranquilizers cause, in sedativehypnotic doses, a selective decrease in

noradrenergic turnover in the coerulocortical noradrenergic pathway [11,50].

These experiments showed that the subcortical noradrenergic nerve terminals,

having a different origin, are unaffected unless the noradrenergic turnover is accelerated by stress. Under

circumstances of stress in rats these

drugs slow down the turnover: of

norepinephrine both in the hypothalamus

and in the cerebral cortex. In studies

of neuroleptics Lidbrink found that the

sedation caused by these drugs is

related to noradrenergic receptor

blockage, which results in increased

norepinephrine turnover [9,3,62].

These drug studies all show the

importance of an intact neurotransmission in

for

central noradrenergic

neurons the maintenance of

wakefulness.

The literature on the effects of

catecholamines on sleep is confusing

because of two separate paradoxes or

contradictions. First, there is the

question of whether catecholamines in

the brain produce sleep or arousal.

There is evidence that direct brain

application of norepinephrine

epinephrine produce sleep or coma [53],

yet a great deal of indirect evidence

using amphetamines, precursors, etc.,

suggest that catecholamines have a

definite role in waking. This

disagreement might best be resolved at

the moment in favor of the second

alternative. It is clear that the

reticular activating System, and

catecholamine-containing neurons in it,

are essential for waking, and that by

increasing or decreasing catecholamine

levels in a relatively physiological

way, i.e., producing increases where

they normally occur, this tends to

induce wakefulness after increases, and

drowsiness or sleep after decreases.

The data suggesting that catecholamines

produce sleep involve either direct

brain application or administration of

large doses to chicks or other immature

organisms with poorly developed

blood-brain barriers. Probably the

results can best be explained as local

and unphysiological effects of large

quantities of amines entering the

central nervous system. In fact, what

is produced by direct application of

the catecholamines is seldom "sleep" by

EEG criteria, but rather behavioral

inactivity or coma. Thus, it would

appear that brainstem catecholamines

play a major role in insuring and

maintaining wakefulness.

There is another controversy about the

role of catecholamines, in the two

gualitativelv distinct states of sleep,

synchronized and

Jouvet's

desynchronized.

group, as noted, has

suggested, on the basis of indirect

evidence, that norepinephrine is a

neurohumor that produces or releases

REM sleep. Hartmann [23] and we

ourselves [82] have presented evidence

for the opposite view, i.e., that low

catecholamines are related to high REM

time. This view has led to a theory of

the function of REM sleep, one that

accounts for both the relationship of

catecholamines to REM and their effects

in maintaining arousal. Hartmann [24]

and Stern and Morgane [82] suggest that

one of the biological functions of REM

sleep is to maintain or restore the

functional integrity of the catecholamine-dependent neuronal systems. The

following arguments are cogent in this

regard: It is important to consider

the effects on sleep of drugs and other

conditions associated with increases

and decreases in brain catecholamine

44
levels. In human studies it seems

clear that drugs such as amphetamines,

monoamine oxidase inhibitors and the

tricyclic anti-depressants, which are

thought to produce their effects by increasing levels or availability of

brain norepinephrine, all induce sharp

decreases in REM time. Hartmann [23]

notes a wide variety of drugs which,

when first administered, produce a

small decrease in REM time, but in his

human studies the effects of these

anti-depressants is far more marked.

Furthermore, a great decrease in REM

time is produced with very little or

delayed rebound, as though, for a time

at least,the drugs were "substituting"

for REM time. The same effect has been

shown for electroconvulsive shock in

animals (also an "anti-depressant"

situation associated with increased

available catecholamines), i.e., greatly reduced REM time with little or no

rebound. Also, in the clinical

syndrome of mania (thought to be

associated with highly available

catecholamines) there was found to be a

very low REM time, low even as a

percent of the total of the lowered

sleep. Thus, in many situations it

seems that high central catecholamine

levels appear to be associated with low

REM time.

We can summarize the role of catecholaminergic systems in sleep-waking activity as follows:

A. Waking Mechanisms. The intervention of catecholaminergic mechanisms in

the control of tonic cortical arousal

in the cat is suggested by converging

experimental evidence which is as

follows: (1) The increase of cerebral

catecholamine (after L-dopa) induces a

long-lasting arousal in the cat; (2)

The inhibition of the synthesis of

catecholamines with alpha-methylparatyrosine decreases waking in normal

cats and totally suppresses the

behavioral and EEG arousal which

normally follows the injection of

d-amphetamine.

Norepinephrine, but not dopamine, seems

to be involved in the maintenance of

tonic cortical arousal for the

following reasons: (1) The destruction

of the substantia nigra, which decreases telencephalic and diencephalic

(including striatal) dopamine levels by

more than 90%, does not interfere significantly with cortical EEG arousal but

strongly alters behavioral waking; (2)

The destruction of the dorsal noradrenergic pathway in the mesencephalon, or

of cell group A8 in the mesencephalic

reticular formation, strongly reduces

telencephalic and diencephalic norepinephrine and increases cortical synchronization. There is a significant

correlation between the decrease of

cortical arousal and the decrease of

norepinephrine in the mesencephalon and

the forebrain.

B. REM sleep. The intervention of

catecholamine- and/or acetylcholinecontaining neurons of the latero-dorsal

pontine tegmentum in the "executory

mechanisms" of REM are suggested by a

variety of neuropharmacological and

neurophysiological findings: (1)

Reserpine-pretreated cats receiving

dopa exhibit REM much earlier (2-4 hrs)

than animals not receiving dopa. This

suggests that the refilling of some

pools in the catecholamine terminals

could be a condition for the

reappearance of REM. It is noteworthy

that in our experiments in cats we were

not able to observe any dopa-induced

increase in REM time [81]; (2) Certain

alpha-receptor blocking drugs, which

cross the blood-brain barrier of the

rat, were found to suppress REM; (3)

The administration of alpha-methyldopa,

which results in the synthesis of the

false transmitter alpha-methylnorepinephrine, selectively suppresses REM in

the cat for 12 to 16 hrs and decreases

waking; (4) In Jouvet's studies, and in

those of Henley and Morrison [26], the

destruction of the caudal third of the

nucleus locus coeruleus suppressed the

motor inhibition, but not other

components of the REM state, which

occur during REM sleep. After such a

lesion Jouvet's cats presented,

periodically during sleep, some

peculiar hallucinatory-like behavior

(rage., defense against some imaginary

enemy, etc.). Such behavior is good

evidence that REM sleep may be

accompanied by oneiric activity in the

cat. The total destruction of the

caudal part of the locus coeruleus and

of the nucleus sub-coeruleus selectively suppressed all the central and

peripheral components of REM sleep including PGO waves [39]. On the other

hand Henley and Morrison [26] continued

to observe what they consider to be REM

sleep episodes for up to six months

following locus coeruleus lesions. In

chemical lesion studies selective suppression of REM was obtained secondarily after micro-injections of 6-hydroxydopamine which was thought to selectively destroy catecholamine neurons in

45
the latero-dorsal pontine tegmentum

[30,67,90]. However, Panksepp et al.

[64] found a significant elevation of

REM following administration of this

agent into this same approximate

region.

ON THE ROLE OF CENTRAL DOPAMINE

NEURONS IN SLEEP

Barbiturates and minor tranquilizers in

hypnotic doses cause a decrease in

turnover of dopamine in the neostriatum

and the limbic forebrain areas [11].

Another drug known to induce sleep is

gamma-hydroxybutyric acid [7] and this

effect has been related to an abrupt

rise of the dopamine levels in the

neo-striatum [73]. This marked rise is

similar to that seen after interruption

of the dopamine pathway [4,61] and may,

therefore, represent a dramatic decline

in dopamine release, resulting in

increased dopamine storage due to

continued dopamine synthesis. It is

also well known that the nigro-neostriatal dopamine pathway is essential for

behavioral arousal [18,87]. It is,

therefore, likely that the observed

decreases in dopamine activity in the

forebrain found after treatment with

sedative-hypnotic drugs can mediate the

behavioral depression found after these

drugs are administered. It is possible

that these effects are mediated by the

meso-limbic dopamine pathways. Harner

and Dorman [25] have shown that dopamine may be responsible for cortical

activation and reported a consistent

behavioral and electrographic arousal

to result from L-dopa administration.

Of course, in the normal state coordination must exist between the sleepwakefulness cycle and behavioral activity and this involves, in all likelihood, complex interactions between different chemical systems in the brain.

It would, thus, appear that the action

of the sedative-hypnotic drugs may primarily be on the nervous pathways subserving sleep and wakefulness. When

sleep is favored, the behavioral arousal system, such as the dopamine system

to the neostriatum, would then be turned off. Similarly, when arousal is

favored by activity in the dorsal noradrenergic pathway, the serotonin cells

in the raphé may be inhibited.

RELATIONSHIP OF THE BIOGENIC AMINES

TO PHASIC EVENTS AND BEHAVIOR

In summarizing the various results

above, it should be noted that: (1)

The cortical noradrenergic pathway from

the principal locus coeruleus appears

to be important for the maintenance of

wakefulness and REM sleep and that the

effects of sedative-hypnotic drugs,

such as barbiturates and minor tranquilizers, are partly mediated by a depressing activity on this particular

pathway; (2) The activity of the cortical and subcortical serotonin pathway are also affected, directly or indirectly, by sedative and hypnotic

drugs such as barbiturates and minor

tranquilizers; (3) The activity of the

ascending dopamine neurons, which are

involved in behavioral and probably EEG

arousal, is depressed by barbiturates

and minor tranquilizers. This effect

may be responsible for the behavioral

depression seen after administration of

these compounds. A suppression of the

nigro-neostriatal dopamine neurons may

be the way in which the neural

substrate for sleep-wakefulness control

can insure behavioral depression during

sleep. Of course, many of these

formulations are still tentative, but

enough models of chemical neuron

interactions have been derived to begin

to test specific hypotheses regarding

their role in the vigilance states and

in drug action.

Dement and his co-workers [13] have

gathered data suggesting that serotonin

is somehow involved in PGO discharge

control. They have pointed out that in

Jouvet's original investigations on

serotonergic neurons he achieved pharmacological effects on

systems

serotonernic

with acute administration o f

PCPA and reserpine and obtained

anatomical specificity with stereotaxic

lesions. An example of the reserpine

effect on PGO occurrence in the cat is

shown in Fig. 3. Both the pharmacological and stereotaxic approaches have

led to insomnia or a great reduction in

total sleep time and the emergence of

PGO activity into the waking state.

Dement's group [14] focused on the

chronic effects of PCPA administration

and found that this regimen does not

appear to produce a direct pharmacological, effect, i.e., no behavioral

response was observed in the immediate

post-injection period. In their dose

regimen PCPA did reduce serotonin

concentrations in all parts of the

brain to 0 to 10% of controls by the

fifth PCPA-treatment day.

During the first 24 hrs after the

beginning of PCPA administration, they

46
FIGURE 3

Figure 3: Polygraph write-out illustrating the effects of reserpine

(Serpasil) 0.25 mg/kg, i.p. upon the occurrence of PGO spikes in the

lateral geniculate nucleus (LGN) of a cat during waking, slow-wave and

REM sleep. Cortical, neck and eye tracings are taken in order to

identify the vigilance state. Note the large increase in the occurrence

of monophasic PGO spikes in waking and slow-wave sleep following

reserpine administration. (From Stern and Morgane, 81).

found that REM time generally stayed for limited periods, the minima being

the same or slightly increased. Slow- generally seen on the fifth day of PCPA

wave sleep was somewhat reduced, drop- treatment. After two or three days of

ping more rapidly on the second and very low sleep values a marked recovery

third PCPA days. Near the end of the in total sleep time then began and

third day, total sleep time decreased included both REM and slow-wave sleep.

precipitously and often reached zero Total sleep time (both REM and slow47
wave sleep) reached approximately 70%

of the baseline values even during

continued administration of PCPA. At

the point where total sleep time began

to increase, reorganization in the

temporal sequencing of the sleep states

become apparent. Normally in the

intact cat, there is a marked tendency

for the order of the sleep states to be

wakefulness, followed by slow-wave

sleep, then REM sleep. In the chronic

PCPA cat, the order observed by

Dement's group was most typically

wakefulness, followed by REM sleep, and

then slow-wave sleep.

The earliest changes in phasic activity

during PCPA administration noted by

Dement was an increase in the rate of

PGO spikes during REM sleep and a

decrease in the number of spikes that

commonly precede REM periods. These

changes become evident within 8-12 hrs

after the first dose of PCPA. Later on

they observed an emergence of PGO

activity throughout slow-wave sleep and

the waking state and such activity

could be seen at each point in the

pons-geniculate-occipital cortex circuitry. As this activity appeared in

the waking state, the overall rate of

spike discharge began to drop in REM

sleep. Another change in the chronic

PCPA cat was an increase in the

intensity of muscular twitching.

Finally, behavioral effects began

accompanying PGO waves in the waking

state, e.g., orienting behavior occurred

in association with bursts of PGO

waves.

During the period when PGO waves were

discharging in the waking state, other

inhibitory effects on these waves

became apparent. Behavioral arousal or

intense stimulation caused suppression

of PGO waves. If the animal became

frightened, the waves would temporarily

disappear. In addition, L-dopa or

amphetamine would totally suppress the

PGO waves for variable lengths of time

and 5-HTP totally reversed the release

of PGO waves during wakefulness.

Thus, if it is assumed that a primary

effect of PCPA is inhibition of serotonin turnover, then the earliest and

possibly the most basic effect of this

change is on the regulation of PGO

spike discharge. Even after sleep

returned PGO spike regulation was still

disrupted. Furthermore, with respect

to the neurophysiology of PGO control,

McGinty et al. [55],

strained, chronically

using unrerecorded cats,

studied unit activity in the raphé

nuclei in relation to PCPA-induced PGO

waves. They found that thirty percent

of the units in the dorsal raphé exhibited regular and slow firing which was

stable during a wide variety of waking

behaviors and stimulus conditions, such

regularity even continuing in slow-wave

sleep. Clear cessations in the firing

of these units occurred only during REM

sleep and in slow-wave sleep preceding

LGN waves. The specificity and consistency of these findings strongly

suggests that the release of serotonin

from the dorsal raphé neurons normally

suppresses LGN waves of REM sleep.

In further attempts to confirm these

findings, Dement's group has observed

the effects of electrical stimulation

of the raphé nuclei in cats with

electrodes implanted in the nuclei

raphé dorsalis, pontis and magnus.

Stimulation of the nucleus raphé

dorsalis during REM sleep caused

complete suppression of geniculate PGO

waves during the period of stimulation.

These same stimuli were ineffective in

suppressing PGO waves when delivered to

the nuclei raphé pontis and raphé

magnus. Stimulation of the nucleus

raphé dorsalis in the waking state

produced little or no discernable

behavioral effects and, when delivered

to the sleeping animal, did not result

in waking from slow-wave sleep or from

REM sleep. However, concurrent with

stimulation were some EEG changes that

might indicate some type of subbehavioral changes in arousal level.

One interpretation of these data is

that stimulation of the nucleus raphé

dorsalis in some manner blocks PGO

activity as a secondary result of subbehavioral arousal. However, these

data are not inconsistent with the

notion that nucleus raphé dorsalis

neurons might exert tonic inhibitory

control over PGO waves and possibly

other classes of phasic events, except

of course, during REM sleep.

NEUROCHEMICAL BASES OF

THE PGO WAVE

Jouvet and his colleagues [39,40] have

stressed that PGO waves depend upon

catecholamines. One piece of evidence

for this is that alpha-methyldopa,

which is metabolized to the false

transmitter alpha-methylnoradrenaline

and displaces noradrenaline from

stores, suppresses PGO spike activity.

There is also some supporting evidence

48
from electrolytic and chemical lesions

in the locus coeruleus. The most

damaging evidence against this notion

are the several studies [46,80] with

alpha-methylparatyrosine, which selectively inhibits tyrosine hydroxylase and leads to a depletion of

noradrenaline and dopamine. In these

studies REM time increased and the

total amount of PGO spikes were

increased. High doses of this compound

are nephrotoxic, however, and it is

difficult to achieve total catecholamine depletion. In an effort to overcome this objection, Henriksen and

Dement [27] administered alpha-methylparatyrosine to cats by intravenous

drip in a dosage calculated to be

supra-maximal and saw, as have several

other investigators, a modest increase

in the amount of slow-wave sleep,

associated with a small decrease in the

amount of wakefulness and, secondly, an

increase in the amount of HEM sleep.

Thus, they feel it is difficult to

attribute PGO spike discharge to catecholaminergic neurons. Furthermore,

Haefely et al. [20] have provided several lines of pharmacological evidence

that catecholamines are actually involved in the inhibition of PGO spike

activity.

Various investigators have pointed out

that REM sleep is acutely susceptible

to disruption by pharmacological intervention in the normal animal. Of

course, this complicates the study of

the neurochemical bases of any single

component of the REM state. If a drug

treatment blocks tonic muscle inhibition, produces nausea or discomfort, or

raises the arousal level, it would

block the occurrence of REM sleep and,

therefore, secondarily could prevent

the appearance of PGO waves. Dement's

group has studied the PCPA-treated cat

in which waking PGO waves can be studied without concern about the other REM

components. They regard waking PGO

waves as homologues to or displaced

from REM sleep for several reasons,

namely: (1) As the number of waking

PGO waves increases after PCPA treatment, the number of PGO waves decreases

per REM sleep epoch; (2) Following the

discharge of a number of PGO waves

during a REM sleep period in a PCPAtreated cat, waking waves only slowly

reappear in the subsequent waking

state; (3) both waking and REM sleep

PGO waves frequently precede eye

movements, while in the normal awake

animal, lateral geniculate waves

associated with eye movements always

follow eye movements; (4) Finally, both

the amplitude of waking and REM sleep

PGO waves are recorded unattenuated

from leads in visual cortex when the

animal is placed in the dark, while the

amplitude of cortical waves associated

with waking eye movements in normal

cats diminishes in the dark.

Using these findings, Dement's group

waited until PGO waves appeared in the

waking state as the result of

pre-treatment with DL-PCPA (150 μg

subcutaneously) and then administered

the following presumptive receptor

blockers: pimozide; phentolamine (alpha-adrenergic blocker) and propranalol (beta-adrenergic blocker). Their

results were unequivocal in that none

of the catecholamine receptor blockers

had a significant effect on the rate of

PGO wave discharge.

Dement et al. [28,32] also investigated

the possible role of cholinergic

mechanisms in PGO spike activity and,

in order to study these, administered

atropine to chronic PCPA-treated cats.

They found that this compound totally

blocked the occurrence of PGO waves in

low doses and that this effect was

partially reversed by subsequent injection of eserine. Evidence that the

atropine effect was a direct one on the

central nervous system derives from the

fact that equivalent doses of atropine

methylbromide, which does not cross the

blood-brain barrier in significant

quantities, had no effect on discharge

rate of PGO waves. An observation of

note in these studies was that the

lowest dose of atropine which did suppress PGO spikes, did not produce the

well-known EEG synchronization during

the period of spike suppression.

Atropine's blocking of PGO activity was

further substantiated in acute experiments. Unimplanted cats were pretreated with PCPA for five days and

were then anesthetized and deep electrodes were positioned for recording

PGO activity from both the pons and

lateral geniculate nucleus. Once the

level of anesthesia was adjusted for

optimal PGO discharge, drugs were then

administered and evaluated. Effects of

PGO waves of atropine, atropine methylbromide, and eserine in these acute

experiments were similar to findings in

chronic animals regardless of where PGO

activity was recorded. In view of the

work of Gadea-Ciria and Jouvet [21],

atropine-induced slow-waves and spindles in the neocortex might well be

49
assumed to have a secondary effect on

PGO waves. However, since the atropine

effect was obtained in both the lightly

and deeply barbiturized animal, the

non-specific effect of diminished

arousal does not appear to be a likely

explanation of the atropine suppression

of PGO waves.

INTERACTIONS BETWEEN THE

CATECHOLAMINERGIC SYSTEM

AND THE SEROTONERGIC SYSTEM

The nature of interplay between these

two major chemical systems in the brain

is based on the following lines of

experimental findings: (1) Lesions of

the rostral third of the locus

coeruleus or the ascending dorsal

noradrenergic pathway at the level of

the junction of the pons and midbrain

in the cat induces a striking

hypersomnia with increase in both

slow-wave and HEM sleep, e.g., up to

300% in Jouvet's studies [39], for a

period of 5-10 days. In these studies

biochemical analysis showed a decrease

of norepinephrine in the forebrain and

midbrain and a significant increase of

both tryptophan and 5-HIAA. These data

indicate that damage to noradrenergic

fibers arising from the anterior

(rostral third) components of the locus

coeruleus results in an increased

serotonin turnover in the rostral raphé

as demonstrated by an increase of

5-HIAA in the forebrain. In the

nucleus raphé dorsalis region Loizou

[51] has demonstrated many catecholamine terminals which are in a

strategic position to affect, directly

or indirectly, the activity of serotonin neurons. It is relevant to note

that the increase of serotonin turnover is also accompanied by an increased level of tryptophan since this

indicates that the uptake of tryptophan may play an influential role in

the regulation of biosynthesis of brain

serotonin; (2) In the rat destruction

of catecholamine-containing neurons

with 6-hydroxydopamine (injected intraventricularly or intracisternally) induces a highly significant increase in

the synthesis of central serotonin as

indicated by the increase of 5-HIAA and

by an increase of labeled serotonin

synthesized from labeled tryptophan

injected intracisternally, whereas

endogenous serotonin levels do not

change. This phenomenon is observed as

soon as 40 hrs after the injection of

6-hydroxydopamine and lasts as long as

three weeks [45]. In the cat, however,

direct injection of 6-OHDA into the

central nervous system has been

repeatedly found to lower brain serotonin and 5-HIAA levels [64,90,30].

Hence, important species differences in

effects of this agent should be

emphasized when attempting to interpret

data across species; (3) In the cat the

inhibition of catecholamine synthesis

with alpha-methylparatyrosine induces,

after approximately 8 hours, a significant increase of endogenous 5-HIAA

and of labeled serotonin synthesized

from labeled tryptophan in the lower

brainstem (pons and medulla). Thus,

there is a large body of evidence indicating interplay between the serotonergic and catecholaminergic systems

in the brain.

The effects on the vigilance states

obtained by surgical destruction of the

catecholaminergic neurons have been

partially verified by Jouvet [41,42] in

the cat by the use of intraventricular

6-hydroxydopamine. These results can

be summarized as follows: (1) About 15

days after intraventricular injection

of 6-hydroxydopamine a decrease of

catecholamine synthesis is observed in

all structures of the brain as shown by

a decrease of endogenous norepinephrine

and dopamine and by a substantial

decrease of labeled catecholamine

(40-90%) and O-Methyl-3H-catecholamine

(25-90%) synthesized from exogenous

3

H-tyrosine. Under the same conditions, a significant decrease of the

concentration of endogenous serotonin

and 5-HIAA has been observed [41]; (2)

During the period after 6-hydroxydopamine injection, when there are marked

depletions in both serotonin and catecholamines, there is no significant

alteration of the quantitative level of

the cortical synchronization. However,

cortical synchronization does, on occasion, appear during behavioral waking

[42]. In these studies REM sleep is

diminished according to the dose of

6-hydroxydopamine injected. The frequency of PGOs during REM sleep was

shown to be permanently decreased by

about 50% and this decrease affected

mostly the bursts of PGO. However, in

chlorimipramine-pretreated cats 6-hydroxydopamine produces several interesting findings: (a) No alteration of

cerebral indoleamines while cerebral

catecholamines are decreased and (b)

significant (50%) and permanent

increases of cortical synchronization

with REM sleep returning to normal

levels after about six days. but

50
decreasing thereafter; and (c) no

alteration of the rate of PGO activity

during REM sleep. Taken together these

results all suggest that cortical

synchronizing and desynchronizing

mechanisms which act during slow-wave

sleep and waking are under the

regulation of antagonistic serotonin

and catecholaminergic systems.

REM sleep appears to involve, according

to Jouvet and others, some serotonin

"priming" mechanisms which appear to

be located in the pontine group of

nuclei of the raphé system, i.e., the

nucleus raphé pontis and nucleus raphé magnus, whereas its so-called

"executive" mechanisms may partly

depend upon catecholaminergic, and

possibly cholinergic, neurons located

in the nuclear complex of the nucleus

locus coeruleus and subcoeruleus. In

this regard, the ponto-geniculo-occipital (PGO) activity, which is highly

characteristic of REM sleep, has been

selectively suppressed in the lateral

geniculate nucleus by extensive lesions

of the locus subcoeruleus or by lesions

of a pathway originating from this

region, ascending in the mesencephalon, and coursing in the supraoptic decussation [49].

Some complex interactions exist between

the serotonergic systems responsible

for sleep and the catecholaminergic

system responsible for waking, while

there appears to be agonistic

interaction between the serotonin and

the noradrenergic mechanisms which are

involved in REM sleep. The intimate

nature of these regulations is not yet

understood but they may explain the

interactions which exist between the

increased waking and the following

rebounds of both slow-wave sleep and

REM. The increased waking which

follows the destruction of the anterior

nuclei raphé is most probably mediated

by catecholaminergic mechanisms, as

shown by the following experiments

carried out by Jouvet and his group:

An injection of 200 mg/kg of alpha-methylparatyrosine was given to

cats with raphé destruction at a time

when behavioral and EEG waking was

almost permanent (2-6 days after the

lesion). The animals then showed cessation of running movements, miosis

appeared, and there was an almost

continuous cortical synchronization.

This lasted for approximately 24 hrs.

after which time there was a rapid

return to behavioral and EEG insomnia.

This experiment provides some neuropharmacological evidence that the

almost permanent arousal which follows

the destruction of the rostral group of

the serotonin-containing neurons of the

raphé system might be related to the

increased turnover in central catecholaminergic neurons. From these results

it is possible to conclude that, under

normal conditions, serotonin neurons

might exert some tonic inhibitory

action, directly or indirectly, upon

some catecholamine-containing neurons

at the onset of sleep. The nature of

the interconnecting circuitries that

mediate these interactions is now under

intensive investigation.

The hypothesis that the serotonin-containing neurons play a role

in the "priming" of REM is founded on

the following facts:

that there must

(1) It appears

exist a critical

minimum of slow-wave sleep in order for

REM to appear (after either sub-total

lesion of the raphé system or

inhibition of the serotonin synthesis

with PCPA). Indeed, Jouvet reports

that a statistical correlation exists

between the amount of slow-wave sleep

and REM sleep. For example, in the cat

the percent of REM sleep equals percent

of (slow-wave sleep - 16) / 3.2. This

holds true for cats subjected to

lesions of the raphé system (with

decrease of serotonin turnover) and for

hypersomniac cats subjected to lesions

of the dorsal noradrenergic bundle

(with increase of serotonin turnover).

Thus, it appears that a minimum of 16%

of daily slow-wave sleep, which is

roughly one-third of the normal amount,

is necessary to "actuate" REM sleep

mechanisms. Since there is some

relationship between the amount of

slow-wave sleep and serotonin turnover,

this would suggest that any important

decrease of serotonin turnover, induced

either by a lesion or drug, would

decrease or suppress REM, although

other interpretations are possible,

such as the suppression of deaminated

metabolites of serotonin (5-hydroxyacetaldehyde or 5-hydroxytryptophol)

which may play some physiological role

[74]. These findings might explain why

the inhibitors of monoamine oxidase,

which decrease the serotonin turnover,

are very potent suppressors of REM

sleep.

Another possible mechanism for the

"priming" or actuating of REM sleep by

the prior occurrence of slow-wave sleep

involves the possible role of growth

hormone secretion. Since we [82] have

51
recently reported that injection of

growth hormone into cats increases the

occurrence of REM sleep, it may be that

the prior release of growth hormone

during slow-wave sleep is the reason

why REM always follows slow-wave sleep.

However, the large release of growth

hormone during slow-wave sleep has so

far only been reported in primates

[84].

SOME REMARKS ON THE

SEROTONIN-SLEEP HYPOTHESIS

The cellular mechanisms by which either

serotonin cells of the raphé complex or

neurons of the locus coeruleus are

activated are not known, although there

is now ample evidence that noradrenergic fibers from the locus coeruleus

synapse on serotonergic cells of the

nucleus raphé dorsalis and are in ideal

position to regulate activity in this

part of the raphé complex. As noted

we [57] have shown that electrical

stimulation of the locus coeruleus

inhibits cell firing in the nucleus

raphé dorsalis. What are now needed

are studies of the correlations between

cell firing in specific brainstem

areas, such as the raphé and locus

coeruleus, and amine release. It might

be assumed that cells of the raphé

nuclei would discharge more rapidly in

slow-wave sleep and those of the locus

coeruleus more rapidly in REM sleep.

Sheu et al. [79] showed, on the contrary, that cells of the nucleus raphé

medianus and nucleus raphé magnus fire

more slowly in slow-wave sleep than in

waking or REM sleep. McGinty et al.

[55] likewise showed that cells of the

nucleus raphé dorsalis fire much slower in slow-wave sleep and REM sleep as

compared to waking. It has

stressed by McGinty [54] that

been

the

slowing of raphé unit firing during

sleep contradicts current theories

suggesting a facilitating role for

serotonin in sleep and these studies do

indeed constitute gathering evidence

against a direct serotonin-raphé- slowwave sleep hypothesis.

al. [29] showed

Hishikawa et

that with imipramine

there is a great increase in slow-wave

sleep in the cat but during this

slow-wave sleep the raphé is not

showing an increase in firing at the

unit level as indicated by the studies

of Aghajanian et al. [1]. On the other

hand, Chu and Bloom [10] did show that

norepinephrine-containing cells of the

locus coeruleus fire fastest in REM

sleep, thus supporting the view that

increased activity in the locus

coeruleus is associated with an

increase in REM sleep. Some indirect

evidence in favor of the raphéserotonin-slow-wave sleep theory comes

largely from pharmacological experiments. The most convincing of these

shows that when tryptophan hydroxylase

is blocked with PCPA, serotonin synthesis decreases, levels of serotonin

in the brain are reduced, and insomnia

of several days duration ensues.

Circumventing this block with 5-HTP

restores both serotonin levels and

sleep. However, when the depletion

experiments are performed chronically,

animals recover their sleep within

several days even though serotonin is

maintained at extremely low levels by

repeated injections of PCPA. This

latter finding does place in some doubt

at least the apparent uniqueness of the

role of serotonin in sleep.

In vivo studies of the turnover of the

biogenic amines in relation to natural

sleep and waking states are also

greatly needed. Presently it is still

only possible to conclude that the

biogenic amines appear to co-vary with

experimentally-induced changes in sleep

and waking and vice versa. Normal

functioning of their metabolic and

neuronal pathways would then appear to

be necessary for the occurrence of

normal sleep., However, the nuclei,

chemical pathways and circuits cannot

yet be justifiably identified as a

"sleep system" or "sleep center," nor

can the amines themselves be tagged as

hypnogens or sleep substances. They

may serve as actuator agents but their

role in maintenance of a particular

vigilance state is highly questionable.

The fact that the amine-containing

neurons lie in brainstem areas that are

known to be important for normal sleep

makes the cellular approach in relation

to amine release the one that might

eventually prove to be the most

informative about mechanisms. The idea

of a "sleep substance"

the demonstration of

long antedates

neurohumoral

synaptic transmission in the central

nervous system. Thus, Piéron [68] performed experiments to test his hypothesis that a "hypnotoxin" gradually

accumulated during waking and then,

reaching a critical level, depresses

the brain, resulting in sleep. In

these experiments the cerebrospinal

fluid of sleep-deprived dogs was found

sometimes to produce somnolence when

52
injected into the ventricles of normal

recipients. However, various unphysiological factors were later found to be

involved in this approach by Schnedorf

and Ivy [77]. Thus, some recipient

animals were found to become feverish,

possibly owing to contamination of the

transferred cerebrospinal fluid. Some

of the technical limitations have been

overcome in the recent studies by

Pappenheimer and his group [65] who

have been able to obtain large amounts

of cerebrospinal fluid from goats and

re-inject extracts of these into

smaller animals under completely

sterile and hydrodynamically controlled

conditions. However, in most of their

reported work they have used "inactivity" of the recipients as a measure of

sleep. So far, the work of Fencl et

al. [17], as well. as that of Monnier et

al. [58,78] dealing with humoral factors presumably responsible for the

induction of sleep, have not specifically isolated the substance although

the search still goes on.

Regardless, no conclusive evidence has

been gathered up to this time to

consider any of the biogenic amines

themselves, or even their primary

metabolites, as the sleep-inducing

substance. An extreme example of this

attempt was the proposal by Koella [47]

to re-label serotonin as "somnotonin",

with the observation that serotonin is

the (or one component of) "hypnotoxin"

responsible for sleep. In our studies

we have shown conclusively that the

tryptophols (metabolites of serotonin)

are not themselves sleep-inducing

agents. Indeed, they actually produce

increased waking in the cat (Fig 4).

FIGURE 4

EFFECTS OF TRYPTOPHOL ON SLEEP AND WAKING IN CATS

Mean (+S.D.) percent of total time

TRYPTOPHOL (mg/kg)

BASELINE TWEEN-80 100-200 400

n=13 n=4 n=6 n=4

WAKING 3 8 2 6 4 6 71

(±5) (±19) (±15) (±10)

SWS 5 0 6 3 4 9 2 9

(±6) (±13) (±11) (±11)

REM SLEEP 13 12 5 1

(±3) (±4) (±4) (±1)

TOTAL SLEEP 63 74 5 4 3 0

(±6) (±19) (±19) (±10)

REM / T.S. 2 0 14 8 2

(±4) (±8) (±3) (±2)

Figure 4. Effect of intra-peritoneal administered (suspended in

Tween-80) tryptophol on the vigilance states in the cat. Note a

dose-related decrease of both slow-wave sleep and REM sleep

following tryptophol administration and a corresponding increase

in waking behavior.

53
How can we now view the serotonin-sleep

hypothesis if brain serotonin can be

reduced by up to 90% while producing

sleep losses only in the range of 0 to

35%? In the cat, Jouvet [37] reported

that a single injection of PCPA

produces an almost total insomnia

lasting from approximately the 30th to

60th hour following injection. Agreed

that this appears to be a support for

the serotonin-sleep hypothesis, two

considerations suggest a need for

cautious interpretation. Both Jouvet

[37] and Dement [15] have noted that

during the PCPA-induced insomnia there

is a striking and almost continuous

appearance of the PGO waves which

normally occur mostly within REM sleep.

If, then, this is not a "normal" state

of wakefulness, it does raise the issue

of whether serotonin depletion can

account for physiological wakefulness.

Serotonin might very well play a

determinant role in sleep, but since we

are also probing into the origin of

normal wakefulness, we will very likely

have to consider its genesis as more

"actively" derived and not simply due

to a lowering of brain serotonin at

some critical site in the brain.

Secondly, Dement [15] has studied cats

who were chronically administered PCPA

and insomnia developed in these animals

in a manner similar to that described

by Jouvet. By the end of the first

week of drug administration, however,

sleep was returning to approximately

70% of baseline values, although brain

serotonin was almost totally depleted

at this point in time. A parallel

phenomenon was also seen in the rat.

Further, data of Mouret et al. [60]

show that the recovery of brain

serotonin after PCPA administration

lags considerably behind the recovery

of sleep, e.g., by the 10th day

following PCPA administration slow-wave

sleep had returned to approximately 85%

of baseline values, whereas brain

serotonin had only reached 55% of

baseline values.

Several possible counter-arguments to

the lack of temporal correspondence

between serotonin recovery and sleep

recovery are possible. It might be

that sleep recovery precedes serotonin

recovery because of the fatigue of

waking mechanisms during the period of

insomnia [72]. But if we assume

additional determinants of sleep, such

as a "fatiguing" of wakefulness, which

itself is not dependent on serotonin,

in effect this argument diminishes the

importance of serotonergic-induced

sleep mechanisms.

It has also been argued that the actual

occurrence of sleep is determined by

two largely independent factors, a

sleep need and a sleep mechanism.

According to this view, serotonin

depletion might interfere with the

sleep mechanism, but the accumulation

of the need during the period of the

insomnia might drive the impaired sleep

mechanism to perform to its limit. As

noted by Rechtschaffen et al. [72],

this is not an unreasonable position,

but, again, it postulates a nonserotonergic sleep determinant, which,

in turn, diminishes the role of serotonergic factors and brings into question some major foundations of the

monoaminergic theory of the vigilance

states.

With respect to other species, Weitzman

et al. [88] have shown modest sleep

decrements with moderate PCPA-induced

serotonin decrements in monkeys and

this data appears to be consistent with

the serotonin-sleep hypothesis. In

humans Wyatt et al. [89] found that

PCPA produced a significant decrease in

the amount of REM sleep in four

carcinoid patients. In three of the

patients there was no change in the

amount of non-REM sleep while in the

fourth there was a small but

significant increase in non-REM sleep.

Such negative findings have generally

not been taken very seriously, presumably because massive brain serotonin

decrements cannot be produced in humans

with oral PCPA. Yet there is considerable evidence that sleep and wakefulness vary spontaneously within each

day without overwhelming oscillations

in brain serotronin. Why, then, are

such tremendous serotonin changes

needed in order to produce effects on

sleep? It could be that serotonin is

acting at a specific receptor site in

the brain and that significant elevations of serotonin are needed to perfuse the. brain adequately and get at

the target zone in critical amounts

sufficient to "trigger" the necessary

mechanisms for sleep induction.

Along these same lines, the work of

Hartmann [22] shows that neither a

tryptophan-free diet nor a tryptophanrich diet significantly affects total

sleep in the rat. The failure of a

tryptophan-free diet, which decreases

brain serotonin by some 40%, or a tryp54
tophan-rich diet to affect significantly total sleep time in rats appears to

be contrary to the serotonin-sleep

hypothesis. Such evidence should not

be neglected simply because the brain

serotonin changes are not as large as

those produced by serotonin inhibitors

and precursors. Hartmann's experiments

do have the advantage at least of mimicking physiological routes of serotonin change, and it would presumably

be by such routes that sleep and wakefulness might be normally regulated by

serotonin.

Radulovacki [71] has also assembled

some evidence that can be considered

contrary to the serotonin-sleep hypothesis. He implanted cannula into the

cisterna magna of cats so as to be able

to draw cerebrospinal fluid repeatedly

without disturbing a sleeping or waking

cat. He failed to find a correlation

between the concentration of 5-HIAA in

the cisternal cerebrospinal fluid as a

function of sleep and waking states.

In many samplings from cats during

wakefulness, slow-wave sleep and REM

sleep he found no significant differences in mean values of 5-HIAA concentration.

On the whole there appears to be no

suitable assembly of hard evidence

which makes the acceptance of the

serotonin-sleep hypothesis obligatory.

For most types of evidence that have

been offered in its favor there is

either contradictory evidence or certainly reasonable alternative interpretations. Some of these alternative

interpretations result from problems

intrinsic to the determination of the

physiological role of any single chemical agent in regulating a total or

global behavior. Studies which

correlate spontaneous changes in brain

chemistry with spontaneous changes in

behavior have the advantage of

following physiological variations, but

as to questions of cause and effect,

these latter must be approached by a

variety of experimental manipulations.

In experimentally manipulating the

biochemical events of the brain we can

never be certain that we are mimicking

the natural course of events. At

present there simply is not a wholly

adequate accumulation of supporting

evidence for the serotonin-sleep

hypothesis and now there is

considerable evidence being gathered to

the contrary. However, this evidence

does not rule out serotonin as playing

some role in sleep mechanisms or that

norepinephrine-serotonin interactions

are not critical parts of the chemical

oscillations involved in transitions

from one vigilance state to another.

However, this raises the question as to

exactly what is meant by the monoamine

theory of sleep-waking behavior. It

does not imply a simple relationship

such as how saturated the brain is with

serotonin or norepinephrine and how

much of each type of sleep is present.

The evidence is quite clearly against

such simple direct relations. Examining the PCPA data on rats, for example, how can we explain the fact of

moderate sleep decrements with up to

90% depletion in brain serotonin, while

Scheving et al. [76] report that brain

serotonin levels fluctuate spontaneously only an approximately 18% range

during any 24 hour period? Quite

clearly, the normal rat would not

likely require a 90% depletion of brain

serotonin to maintain wakefulness.

Something much more complex than gross

serotonin or norepinephrine levels, or,

for that matter, gross serotonin or

noreinephrine turnover, is involved in

controlling sleep and the oscillations

between the states of vigilance. Some

aspect of serotonin or norepinephrine

activity is likely to be varying

spontaneously at some specific receptor

site in the brain, probably in interaction with other chemical systems, to

affect neuronal discharge patterns

which, in turn, contribute to specific

aspects of sleep-waking control. As

Rechtschaffen et al. [72] and others

have pointed out, by varying the whole

brain chemistry, we are testing only

the rather crude hypothesis, i.e., one

amine effect on one sleep state.

Hopefully, by careful use of more

specific chemical lesioning agents

placed in particular chemoanatomical

pathways and by studying local

variations in brain amine turnovers in

relation to the vigilance states we may

move nearer to unraveling the role of

the monoamines in sleep and other

behaviors. At least it appears we are

well beyond the "one amine--one

behavior" stage of reasoning. Neuron

firing in relation to each state of

vigilance and in relation to amine

release is now beginning to provide

valuable insights at the cellular

level. Studies of interactions between

chemical systems in the brain appears

to be one of the most profitable

approaches for future studies. Once

enough is known about the chemical

55
scaffolding in the brain the proper

experiments can be designed to test the

interactions among these systems.

Massive manipulation of brain chemistry

by pharamacological or lesioning

methods has so far provided us with

some general and informative insights

into the role of monoamines in

sleep-waking activity but these methods

have not yet identified the chemical

agent(s) responsible for actuating or

maintaining each state of vigilance or

shed much light on how the several

chemical systems interact to generate

behavior. Obviously, the uniqueness of

any chemical agent in behavior is still

very much in doubt. At the present

time we can probably agree that the

biogenic amines serotonin and norepinephrine appear to be involved in

some aspects of generation and maintenance of the vigilance continuum.

However, by no means can we, at

ACKNOWLEDGMENT

Supported by National Science

Foundation grant GB 43529x and by

grants MH 10625 and MH 02211, National

Institute of Mental Health.

REFERENCES

1. Aghajanian, G.K., B.S. Bunney and

M.J. Kuhar. Use of single unit

recording in correlating transmitter

turnover with impulse flow in monoamine

neurons. IN: New Concepts in

Neurotransmitter Regulation, A.J.

Mandell, Ed., New York, Plenum Press,

1973, pp 115-145.

2. Aghajanian, G.K., J.A. Rosecrans

and M.H. Sheard. Serotonin: Release

in the forebrain by stimulation of

midbrain raphé. Science 156: 402-403,

1967.

3. Andén, N.E., H. Corrodi, K. Fuxe

and T. Hokfelt. Increased impulse flow

in bulbospinal NA neurons by

catecholamine receptor blocking agents.

Europ. J. Pharmacol. 2: 59-64, 1967.

4 . Andén, N.E., H. Corrodi, K. Fuxe

and U. Ungerstedt. Importance of

nervous impulse flow for the

neuroleptic induced increase in amine

present, accept the view that these

biogenic amines are themselves physiological sleep-inducing substances.

Jouvet [44] has recently stressed that

we should no longer ask the question as

to whether serotonin neurons play a

role in sleep mechanisms but rather we

should ask how do serotonin neurons

regulate sleep? Although we cannot at

the present time state, with any degree

of certainty, how the monoamines regulate sleep we have at least taken some

stock of the field and brought several

lines of research and their interpretation under critical scrutiny. We have

reviewed some of the various evidence

for and against monoamine regulation of

the states of vigilance and hinted at

lines of future investigation. It is

clear that there is still a long way to

go before we are on solid ground in

relating chemistry of the brain to

behavior.

turnover in central dopamine neurons.

Europ. J. Pharmacol. 15: 193-199, 1971.

5. Andén N.E., A. Dahlström, K. Fuxe,

K. Larsson, L. Olson and U. Ungerstedt.

Ascending monoamine neurons to the

telencephalon and diencephalon. Acta

Physiol. Scandf. 67: 313-326, 1966.

6. Baumgarten, H.G., A. Bögrklund, L.

Lachenmayer, A. Nobin and U. Stenevi.

Long-lasting selective depletion of

brain serotonin by 5,6-dihydroxytryptamine. Acta Physiol. Scand.

Suppl. 373: 1-15, 1971.

7. Benda, P. and A. Perlés. Étude

expérimentale de l'abaissement de la

vigilance par la gamma-butyrolactone.

Comp. Rend. Soc. Biol 251: 1312-1313,

1960.

8 . Bobillier, P., J.L. Proment, S.

Seguin and M. Jouvet. Effets de la P.

Chlorophenylalanine et du 5-hydroxy56
tryptophane sur le sommeil et le métabolisme central des monoamines et des

protéines chez le chat. Biochemical

Pharmacology 1974 (in press).

9 . Carlsson, A. and M. Lindqvist.

Effect of chlorpromazine or haloperidol

on formation of 3-methoxytyramine and

normethanephrine in mouse brain. Acta

Pharmacol. Toxicol. 20: 140-144, 1963.

10. Chu, N. and F.E. Bloom.

Norepinephrine-containing neurons:

Changes in spontaneous discharge

patterns during sleeping and waking.

Science 179: 908-910, 1973.

11. Corrodi, H., K. Fuxe, P. Lidbrink

and L. Olson. Minor tranquilizers,

stress and central catecholamine

neurons. Brain Res. 29: 1-16, 1971.

12. A. Dahlström and K. Fuxe.

Evidence for the existence of

monoamine-containing neurons in the

central nervous system. Acta Physiol

Scand. 62: 1-55, 1964.

13. Dement, W.C., S. Henriksen, B.

Jacobs and M.M. Mitler . Biogenic

Amines - Phasic events and behavior.

IN: Pharmacology and the Future _of

Man. Basel, Switzerland, Karger, 1973,

pp 74-89.

14. Dement, W.C., M.M. Mitler and S.

Henriksen. Sleep changes during

chronic administration of parachlorophenylalanine. Rev. Can. Biol. 31:

239-246, 1972.

15. Dement, W.C., V. Zarcone, J.

Ferguson, H. Cohen, T. Pivik and J.

Barchas. Some parallel findings in

schizophrenic patients and serotonin

depleted cats. IN: Schizophrenia:

Current Concerts and Research

Siva Sankar, Ed., Hicksville, N.Y., PJD

D.V.

Publications LTD., 1969, pp 775-811.

16. Descarries, L. and G. Saucier.

Disappearance of the locus coeruleus in

the rat after intraventricular

6-hydroxydopamine. Brain

310-316, 1972.

Res. 37:

17. Fencl, V., G. Koski and J.R.

Pappenheimer. Factors in cerebrospinal

fluid from goats that affect sleep and

activity in rats. J. Physiol. 216

565-589.

:

18. Fuxe, K., T. Hökfelt and U.

Ungerstedt. Morphological and functional aspects of central monoamine

neurons. Int. Rev. Neurobiology 13:

93-126, 1970.

Histochemical

19. Fuxe, K. and U. Ungerstedt.

biochemical and functional studies on central monoamine

neurons after acute and chronic

amphetamine administration. IN:

Amphetamine and Related Compounds.

E . Costa and S. Garattini. Eds.. New

York, Raven Press, 1970, pp 257-288.

20. Haefely, W., M. Jalre and M.A.

Monachon. NE-neurons and phasic sleep.

Biochemical Pharmacology Supp.,

638-640, 1974.

21. Gadea-Ciria, E. and M. Jouvet.

Corticofugal control of pontine PGO

activity in the cat. Psychophysiology

9: 129, 1972.

22. Hartmann, E. The effect of LSD

and L-tryptophane on the sleep-dream

cycle in the rat. Psychonomic Science

8: 295-296, 1967.

23. Hartment, E. The biochemistry and

pharmacology of the D-state (dreaming

sleep). Experimental Medicine and

Surgery 27: 105-120, 1969.

24. Hartmann, E. The D-state and

norepinephrine-dependent systems. IN:

Sleep and Dreaming, E. Hartmann, Ed.,

Boston, Little Brow; and Co., 1970, PP

308-328.

25. Harner, R.N. and R.M. Dorman. A

role for dopamine in control of sleep

and wakefulness. Trans. Amer.

Neurological Assn. 95: 252-254, 1970.

26. Henley, K. and A.R. Morrison. A

re-evaluation of the effects of lesions

of the pontine tegmentum and locus

coeruleus on phenomena of paradoxical

sleep in the cat. Acta Neurobiol. Exp.

34: 215-232, 1974.

27. Henriksen, S. and W. Dement.

Effects of chronic intravenous administration of 1-alpha-methylparatyrosine

on sleep in the cat: A preliminary

investigation. IN: Sleep Research

Vol. 1, M. Chase, W. Stern, and P.

Walter, Eds. Brain Information

Sevice/Brain Research Institute,

U.C.L.A., Los Angeles, 1972, p 55.

28. Henriksen, S.J., B.L. Jacobs and

W.C. Dement. Dependence of REM sleep

PGO waves on cholinergic mechanisms.

Brain Res. 48: 412-416.

57
29. Hishikawa, Y., K. Nakai, I.

Hidenobu and Z. Kaneko. The effect of

imipramine, desmethylimipramine and

chlorpromazine on the sleep-wakefulness

cycle of the cat. Electroenceph. clin.

Neurophysicol. 19: 518-521, 1965.

30. Howard, J.L. and G.R. Breese.

Physiological and behavioral effects of

centrally-administered 6-hydroxydopamine in cats. Pharmacol. Biochem. and

Behavo. 2: 651-661, 1974.

31. Jacobs, B.L., R. Asher and W.C.

Dement. Electrophysiological and

behavioral effects of electrical

stimulation of the raphé nuclei in

cats. Physiol. and Behavior 11:

489-495, 1973.

32. Jacobs, B.L., S.J. Henriksen and

W.C. Dement. Neurochemical bases of

the PGO wave. Brain Res. 48: 406-411,

1972.

33. Jonsson, G., K. Fuxe and J. Daly.

Intracerebral injections of 5,6-dihydroxytryptamine (5,6-HT). Evidence for

selective degeneration central

5-hydroxytryptamine (5-HT) neurons.

Acta Pharmacol. Toxicol. 31: Suppl. 1,

24, 1972.

34. Jones, B.E., P. Bobillier and M.

Jouvet. Effets de la destruction des

neurones contenant des catécholamines

du mésencéphale sur le cycle

veille-sommeils du chat. Compt. Rend.

Soc. Biol. 163: 176-180, 1969.

35. Jones, B.E., P. Bobillier, C. Pin

and M. Jouvet. The effect of lesions

of catecholamine-containing neurons

upon monoamine content of the brain and

EEG and behavioral waking in the cat.

Brain Res. 58: 157-177, 1973.

36. Jouvet, M. Mechanisms of the states

of sleep: A neuropharmacological

approach. IN: Sleep and Altered

States of Consciousness. S. Kety, E.

Evarts and H. Williams, Eds.

Baltimore, The Williams and Wilkins

co., Vol. 45, 1967, pp 86-126.

37. Jouvet, M. Biogenic amines and

the states of sleep. Science 163:

32-41, 1969.

38. Jouvet, M. Some monoaminergic

mechanisms controlling sleep and

waking. IN:

A.G.

Brain and Human Behavior,

Karczmar and J.C. Eccles, Eds.,

New York, Springer Verlag, 1972, PP

131-161.

39. Jouvet, M. The role of monoamines

and acetylcholine-containing neurons in

the regulation of the sleep-waking

cycle. Ergebnisse der Physiologie 64:

166-307, 1972.

40. Jouvet, M. Serotonin and sleep in

the cat. IN: Serotonin and Behavior

J . Barchas and E. Usdin, Eds., New

York, Academic Press, 1973, pp 385-400.

41. Jouvet, M. Monoaminergic regulation of the sleep-waking cycle. IN:

Pharmacology and the Future of Man.

Basel, Switzerland, Karger, 1973, pp.

103-107.

42. Jouvet, M. The role of monoaminergic neurons in the regulation and

function of sleep. IN: Basic Sleep

Mechanisms. O. Petre-Quadens and D.J.

Schlag, Eds., New York, Academic Press,

1974, pp 207-236.

43. Jouvet, M. and J.F. Pujol. Les

médiateurs chimiques--Leur role dans la

physiopathologie de la motricite de la

vigilance et du comportement. Etude

neurophysiologique et biochimique.

Revue Neurologique 127: 115-138, 1972.

44. Jouvet, M. and J.F. Pujol.

Effects of Central alterations of

serotoninergic neurons upon the

sleep-waking cycle. IN: Advances in

Biochemical Psychopharmacology.

Gessa and M. Sandler

E .

Costa, G.L. Eds.

Vol. 11 New York, Raven Press, 1974, pp

199-209.

45. Juge, A., F. Sordet, M. Jouvet and

J. Pujol. Modification du métabolisme

de la sérontonine cérébrale aprés

6-hydroxydopamine chez le rat. Comp.

Rnd. Acad. Sci. 274: 3266-3268, 1972.

46. King, C.D. and R.E. Jewett. The

effects of alpha-methyltyrosine on

sleep and brain norepinephrine in cats.

J. Pharmacol. Exp. Ther. 177: 188-195,

1971.

47. Koella, W.P. Discussion of

insomnia and decrease of cerebral

5-hydroxytryptamine after destruction

of the raphé system in the cat. IN:

Advances in Pharmacology S. Garattini

and P.A. Shore, Eds., Vol.. 6, Part B,

New York, Academic Press, 1968, pp

280-282.

48. Kostowski, W., E. Giacalone, S.

Farattini and L. Valzelli. Electrical

stimulation of midbrain raphé: Biochemical behavioral and bioelectrical

58
effects. European J. Pharmacol. 7:

170-175, 1969.

49. Laurent, J.P., R. Cespuglio and M.

Jouvet. D&limitation des voies

ascendantes responsables de. l'activaté

ponto-géniculo-occipitale chez le chat.

Experientia 28: 1174-1175, 1972.

50. Lidbrink, P., H. Corrodi, K. Fuxe

and L. Olson. On the role of central

monoamine neurons in sleep mechanisms.

IN: Pharmacology and the Future of

Man. Basel, Switzerland, Karger, 1973,

pp 90-102.

51. Loizou, L.A. Projects of the

nucleus locus coeruleus in the albino

rat. Brain Res. 15: 563-566, 1969.

52. Maeda, T., C. Pin, D. Salvert, M.

Ligier and M. Jouvet. Les neurones

contenant des catécholamines du

tegmentum pontique et leurs voies de

projection chez le chat. Brain Res.

57: 119-152, 1973.

53. Mandell, A.J. and C.E. Spooner.

Psychochemical research studies in man.

Science 162: 1442-1453, 1968.

54. McGinty, D.J. Neurochemically

defined neurons: Behavioral correlates

of unit activity of serotonin

containing cells. IN: Brain Unuit

Activity During Behavior. M.I.

Phillips, Ed., Springfield, Chas. C.

Thomas, 1973, pp 244-267.

55. McGinty, D.J., R.M. Harper and

M.K. Fairbanks. 5-HT-containing neurons: Unit activity in behaving cats.

IN: Serotonin and Behavior, J. Barchas

and E. Usdin, Eds., New York, Academic

Press, 1973, pp 267-279.

56. Morgane, P.J. and W.C. Stern.

Chemical anatomy of brain circuits in

relation to sleep and wakefulness. IN:

Advances in Sleep Research, E. Wietzman,

Ed., New York, Spectrum Publications,

1974, pp 1-131.

57. Morgane. P.J., W.C. Stern and E.

Berman. Inhibition of unit activity in

the anterior raphé by stimulation of

the locus coeruleus. Anatomical Record

178: 421, 1974.

58. Monnier, M. and E. Schoenenberger.

Neuro-humoral coding of sleep by the

physiological sleep factor delta. IN:

Neurohumoral Coding of Brain Function.

R.D. Myers and R. Drucker-Colin,

Eds., New York, Plenum Press, 1974, pp

207-232.

59. Moruzzi, G. The sleep-waking

cycle. Ergebnisse der Physiologie 64:

1-165, 1972.

60. Mouret, J., P. Bobillier and M.

Jouvet. Insomnia following parachlorophenylalanin in the rat. European J.

Pharmacol. 5: 17-22, 1968.

61. Nyback, H. Effect of brain

lesions and chlorpromazine on

accumulation and disappearance of

catecholamines formed in vivo from

14C-tyrosine. Acta Physiol. Scand. 84:

54-64, 1972.

62. Nyback, H. and G. Sedvall.

Further studies on the accumulation and

disappearance of catecholamines formed

from tyrosine- 14C in mouse brain.

Effect of some phenothiazine analogues.

European J. Pharmacol. 10: 193-205 ,

1970.

63. Olson, L. and K. Fuxe. On the

projections from the locus coeruleus

noradrenaline neurons: The cerebellar

innervation. Brain Res. 28: 165-171,

1971.

64. Panksepp, J., J.E. Jalowiec, P.J.

Morgane, A.J. Zolovick and W.C. Stern.

Noradrenergic pathways and sleep-waking

states in cats. Experimental Neurology

41: 233-245, 1973.

65. Pappenheimer, J.R., T.B. Miller

and C.A. Goodrich. Sleep-promoting

effects of cerebrospinal fluid from

sleep-deprived goats. Proc. Nat.

Acad. Sci. 58: 513-517, 1967.

66. Petitjean, F. and M. Jouvet.

Hypersomnie et augmentation de l'acide

5 hydroxy-indolacétique cérébral par

lésion isthmique chez le chat. Comp.

Rnd. Acad. Soc. 164: 2288-2293, 1970.

67. Petitjean, F., R. Laguzzi, F.

Sordet, M. Jouvet and J.F. Pujol.

Effets de l'injection intraventriculaire de 6-hydroxydopamine. I. Sur les

monoamines cérébrales du chat. Brain

Res. 48: 281-293, 1972.

68. Piéron, H. Le Probleme Physiologique du Sommeil. Paris, Masson,

1913.

69. Poirier, L.J., P. Langelier, A.

Roberge, R. Boncher and A. Kitsikis.

59
Non-specific histopathological changes

induced by the intracerebral injection

of 6-hydroxy-dopamine (6-OH-DA). J.

Neurol. Sci. 16: 401-416, 1972.

70. Polc, P. and M. Monnier. An

activating mechanism in the

ponto-bulbar raphé system of the

rabbit. Brain Res. 22: 47-61, 1970.

71. Radulovacki, M. 5-Hydroxyindoleacetic acid in cerebrospinal fluid:

measurements in wakefulness, slow-wave

and paradoxical sleep. Brain Res. 50:

484-488, 1973.

72. Rechschaffen, A., R.A. Lovell,

D.X. Freedman, W.E. Whitehead and M.

Aldrich. The effect of parachlorophenylalanine on sleep in the rat:

Some implications for the

serotonin-sleep hypothesis. IN:

Serotonin and Behavior, J. Barchas and

E . Usdin, Eds., New York, Academic

Press, 1973, pp 401-418.

73. Roth, R.H. and Suhr, Y. The

mechanism of the gamma-hydroxybutyrate-induced increase in brain

dopamine and its relationship to

'sleep'. Biochem. Pharmacol. 19:

3001-3012, 1970.

74. Sabelli, H., W. Giardina, S.

Alivisatos, P. Seth and F. Ungar.

Indoleacetaldehydes: Serotonin-like

effects on the central nervous system.

Nature (London) 223: 73-74, 1969.

75. Saner, A., L. Pieri, J. Moran, M.

DaPrada and A. Pletscher. Decrease of

dopamine and 5-hydroxytryptamine after

intracerebral application of

5,6-dihydroxytryptamine. Brain Res.

76: 109-117, 1974.

76. Scheving, L., W. Harrison, P.

Gordon and J. Pauly. Daily fluctuation

(circadian and ultradian) in biogenic

amines of the rat brain. American J.

Physiology 214: 166-173, 1968.

77. Schnedorf, J.G. and A.C. Ivy. An

examination of the hypnotoxin theory of

sleep. American J. Physiology 125:

491-505, 1939.

78. Schoenenberger, G.A., L.B. Cueni,

A.M. Hatt and M. Monnier. Isolation

and physical-chemical characterization

of a humoral, sleep inducing substance

in rabbits (factor 'delta'). Experientia 28: 919-922, 1972.

79. Sheu, Y., J.P. Nelson and F.E.

Bloom. Discharge patterns of Cat raphé

neurons during sleep and waking. Brain

Res. 73: 263-276, 1974.

80. Stern, W.C. and P.J. Morgane.

Effects of alpha-methyltyrosine on REM

sleep and brain amine levels in the

cat. Biological Psychiatry 6:

301-306, 1973.

81. Stern, W.C. and P.J. Morgane.

Effects of reserpine on sleep and brain

biogenic amine levels in the cat.

Psychopharmacologia (Berlin) 28:

274-286, 1973.

82. Stern. W.C. and P.J. Morgane.

Theoretical view of REM sleep function:

Maintenance of catecholamine systems in

the central nervous system. Behav.

Biol. 11: 1-32, 1974.

83. Stern, W.C., J.E. Jalowiec, H.

Shabshelowitz and P.J. Morgane.

Effects of growth hormone on sleepwaking patterns in cats. Hormones and

Behavior 6: 189-196, 1975.

84. Takahashi, Y., D. Kipnis and W.

Daughaday. Growth hormone secretion

during sleep. J. Clin. Invest. 47:

2079-2090, 1968.

85. Ungerstedt, U. 6-Hydroxy-dopamine

induced degeneration of central monoamine neurons. European J. of Pharmacology 5: 107-110, 1968.

86. Ungerstedt, U. Adipsia and

aphagia after 6-hydroxydopamine induced

degeneration of the nigro-striatal

dopamine system. Acta Physiol. Scand.

Suppl. 367: 95-122, 1971.

87. Ungerstedt, U. Histochemical

studies on the effect of intracerebral

and intraventricular injections of

6-hydroxydopamine on monoamine-containing neurons in the CNS. IN:

6-Hydroxydopamine and Catecholamine

Neurons. T. Malmfors and H. Thoenen

Eds., Amsterdam, North-Holland Publish:

ing Co., 1971, pp 1-48.

88. Weitzman, E., M. Rapport. P.

McGregor and J. Jacoby. Sleep patterns

of the monkey and brain serotonin

concentration: Effect of p-chlorophenyl-alanine. Science 160: 1361-

1363, 1968.

60
89. Wyatt, R., A. Sjoerdsma and F.

Snyder. Effects of parachlorophenylalanine on sleep in man. EEG. Clin.

Neurophysiology 27: 529-532, 1969.

90. Zolovick, A.J., W.C. Stern, J.E.

Jalowiec, J. Panksepp and P.J.

Morgane. Sleep-waking patterns and

brain biogenic amine levels in cats

after administration of 6-hydroxydopamine into the dorsolateral pontine

tegmentum. Pharmac. Biochem. and

Behavior 1: 557-567, 1973.

AUTHORS

Peter J. Morgane, Ph.D., and Warren C.

Stern, Ph.D.: Laboratory of Neurophysiology and Psychopharmacology, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545

61

A ROLE FOR DOPAMINE

IN THE PSYCHOPHARMACOLOGY

OF ELECTRICAL SELF-STIMULATION

Barrett R. Cooper, George R. Breese

INTRODUCTION

The electrical self-stimulation

phenomenon, discovered nearly two

decades ago by Olds and Milner [21],

has received considerable experimental

attention in the area of

psychopharmacology. In this field, use

of self-stimulation was prompted by the

possibility that drug-induced changes

in responding might provide a means of

relating the effects of various

centrally acting compounds to

biochemical changes in structures and

pathways stimulated at the tip of the

electrode. Application of this

strategy, which employed many classes

of centrally acting drugs, resulted in

the evolution of what has been referred

to as the "catecholamine hypothesis" of

self-stimulation [16].

The development of a catecholamine

hypothesis of self-stimulation can be

traced to several studies demonstrating

that monoamine oxidase inhibitors [24],

amphetamine [25,29], and other drugs

that increase central adrenergic tone

resulted in increased rates of

self-stimulation of the lateral

hypothalamus. Conversely, administration of drugs that could be considered

to decrease central adrenergic tone

such as reserpine [28],

alpha-methyltyrosine [3,25], disulfiram

[35], or chlorpromazine [22] was found

to decrease lateral hypothalamic

self-stimulation. Since

intraventricular infusion of

norepinephrine reversed the depression

of self-stimulation produced by

alpha-methyltyrosine and disulfiram

[35], attention was focused on the

importance of this catecholamine for

the maintenance of self-stimulation

behavior. Furthermore, several

investigators have noted the striking

overlap of the anatomical maps of

structures and pathways that support

self-stimulation behavior with maps of

the ascending catecholamine neural

systems. From this relationship, it

was proposed that noradrenergic neural

pathways in brain may mediate, or serve

as the anatomical substrate of the

reinforcing properties of brain

stimulation [30].

More recently, however, attention has

shifted to the possibility that

dopamine neural systems may also serve

some function in self-stimulation of

the brain. Evidence favoring a role

for dopamine is based on findings that

high rates of self-stimulation can be

obtained from electrodes placed in the

substantia nigra and that an increased

rate of dopamine turnover accompanies

63
reinforcing stimulation of this site

[2,16]. It has also been shown that

haloperidol and pimozide, drugs thought

to block dopamine receptors in brain,

decrease self-stimulation of the

lateral hypothalamus [19,34], while

phentolamine, a noradrenergic blocking

drug, does not [19]. Finally, Phillips

and Fibiger [23] have related the

different biochemical effects of d- and

l-amphetamine with regard to

norepinephrine or dopamine uptake into

synaptosomes [23] to differential

effects on self-stimulation of

noradrenergic and dopaminergic sites in

brain.

In view of the data reviewed above, a

current evaluation of a catecholamine

hypothesis of self-stimulation would

have to include possible roles for both

norepinephrine and dopamine neural

systems in brain in the mediation of

some aspect of self-stimulation

behavior. In this paper, we will

describe our work with 6-hydroxydopamine and other centrally acting

drugs in which we sought to examine

further the functional significance of

central catecholamine neural systems in

the psychopharmacology of

self-stimulation of the brain.

EFFECTS OF 6-HYDROXYDOPAMINE

ON ELECTRICAL SELF-STIMULATION

OF THE LATERAL HYPOTHALAMUS

Following the discovery that

6-hydroxydopamine possessed a

neurocytotoxic action on catecholamine

containing fibers in brain [5], Stein

and co-workers [31] and Breese et al.

[9] reported that an acute depression

of self-stimulation of the lateral

hypothalamus occurred in pargyline

treated rats after intraventricular or

intracisternal administration. An

additional treatment with 6-hydroxydopamine, which resulted in a greater

destruction of catecholamine neural

processes [5], chronically depressed

self-stimulation of the lateral hypothalamus [9], providing further support

for the view that catecholamine neural

systems were required for the

expression of self-stimulation

behavior.

The development of procedures to obtain

a relatively selective, or

"preferential" depletion of either

norepinephrine or dopamine content of

brain with 6-hydroxydopamine [6] led to

the application of these procedures to

the study of self-stimulation [7,8,11].

Table 1 summarizes the behavioral

effects of these different

6-hydroxydopamine treatments. Acute

TABLE 1

Effects of 6-hydroxydopamine on self-stimulation

of the lateral hypothalamus (a)

Electrode (b) 6-OHDA (c)

Placement treatment

Control

Lateral

Hypothalamus N E

DA

P+6-OHDA

Days After 6-OHDA Treatment (c)

2 4 8 12

87 ± 10 103 ± 8 100 ± 7 105 ± 10

93 ± 9 98 ± 7 104 ± 8 103 ± 9

22 ± 11** 39 ± 13** 88 ± 11 92 ± 8

35 ± 11** 58 ± 15** 74 ± 13* 71 ± 13*

(a) All values are expressed as the mean S.E.M. percent of pretreatment responding for

each group. Rats were tested for 15 min a day at a current level which maintained

moderate to high rates of self-stimulation. A constant current sine wave stimulus

of 0.3 sec duration was delivered for each bar press. There are at least 5 animals

in each 6-hydroxydopamine treatment group.

(b) The coordinates for electrode placements are described in Cooper et al. (11).

(c) The 6-hydroxydopamine treatments are described in text and in other publications (11).

See Table 3 for catecholamine content obtained after treatment.

** p <.01 Dunnet's t-test. Means within each placement group were compared.

64
depression of self-stimulation was

observed in rats pretreated with

pargyline prior to 6-hydroxydopamine to

reduce both catecholamines (P+60HDA)

and in rats treated with desipramine

prior to 6-hydroxydopamine to reduce

primarily dopamine in brain ( DA: Table

1). Reduction of brain norepinephrine

by giving repeated small injections of

6-hydroxydopamine (NE : Table 1) had

little effect on responding. In

addition, this experiment was found to

replicate the earlier finding that

treatment with pargyline plus

6-hydroxydopamine (P+60HDA: Table 1)

chronically decreased self-stimulation

of the lateral hypothalamus. Rats

treated to deplete dopamine eventually

recovered pretreatment levels of

responding. Characteristic depletions

of catecholamines produced by the

different 6-hydroxydopamine treatments

can be seen in Table 3.

EFFECTS OF ALPHA-METHYLTYROSINE, TABLE 2

U-14, 624 AND d-AMPHETAMINE

ON SELF-STIMULATION OF THE

LATERAL HYPOTHALAMUS AFTER

6-HYDROXYDOPAMINE TREATMENT

(b)

Effects of Methyltyrosine (25 mg/kg) or U-14,624 (50 mg/kg) on

self-stimulation of 6-hydroxydopamine treated rats with

electrodes in the lateral hypothalamus (a)

(6-OHDA) (b) Percent of Pretreatment Responding (c)

Placement Group -MT U-14,624

Lateral Control 89 ± 5 102 ± 6

Hypothalamus

N E 87 ± 9 103 ± 3

DA 50 ± 6** 99.3 ± 4

P+6-OHDA 22 ± 7** 101 ± 8 

(a) Values are expressed as the mean S.E.M. percent of pretreatment

responding determined during a 15 minute session on the day 

to drug treatment. Drug were given in balanced order 1 week apart

beginning 16-18 days after 6-hydroxydopamine treatment. At least

6 rats were used In each group.

(b) 6-hydroxydopamine (6-OHDA) treatments are described in Table 1.

(c) MT refers to a 25 mg/kg dose of L methyltyrosine given 4 hours

before testing, U-14,624 (50 mg/kg) was given 6 hours before testing.

** p< .01 Dunnet's t-test. Means within each placement group were

compared.

Although the previous study indicated

that the acute behavioral depression

accompanying catecholamine depletion

produced by 6-hydroxydopamine appeared

to be related to the reduction of

dopamine but not norepinephrine, it did

not provide the necessary evidence to

determine which of the catecholamine

systems were implicated in chronic

reduction of self-stimulation observed

when both catecholamine containing

neural systems were destroyed [9].

Previous studies of the behavioral

effects of catecholamine depleting

drugs in rats treated with

6-hydroxydopamine indicated that damage

to catecholamine neural systems

resulted in an enhanced sensitivity to

the behavioral depressant effects of

alpha-methyltyrosine or reserpine

[13,14,15]. The above finding

suggested that this pharmacological

approach could be used to determine the

functional significance of these amines

for the maintenance of

self-stimulation, since a differential

sensitivity to additional catecholamine

reduction would presumably relate to a

functional role in this behavior.

Thus, studies were initiated to

determine the effects of the tyrosine

hydroxylase inhibitor, alphamethyltyrosine, or the dopamine-betahydroxylase inhibitor, U-14,624, on

self-stimulation of the lateral

hypothalamus in 6-hydroxydopamine

treated rats. The results of this

experiment (Table 2) indicated that

rats with prior depletion of brain

dopamine, but not norepinephrine,

showed an enhanced reduction in lateral

hypothalamic self-stimulation after

alpha-methyltyrosine treatment [8,11].

No effect on self-stimulation of the

lateral hypothalamus was seen after

U-14,624 administration, in spite of a

large reduction of norepinephrine

content in both control and

6-hydroxydopamine treated rats (Table

3). The finding that U-14,624 did not

affect self-stimulation of rats treated

to deplete both catecholamines in brain

(P+60HDA) was of particular interest,

since the data concerning the

biochemical effects of U-14,624 in such

animals indicated a 95% reduction of

whole brain norepinephrine content at

the time of testing (Table 3).

While the previous experiment suggested

the importance of dopamine neural

pathways for the maintenance of lateral

hypothalamic self-stimulation, failure

to obtain evidence that would implicate

norepinephrine prompted examination of

the effects of d-amphetamine in

6-hydroxydopamine treated rats. Since

facilitation of lateral hypothalamic

self-stimulation produced by

d-amphetamine has been proposed to be

related to indirect release of newly

synthesized norepinephrine [29,30], it

was anticipated that destruction of

noradrenergic terminals would attenuate

the facilitation of self-stimulation

produced by d-amphetamine. Figure 1

shows the results of these experiments.

65
TABLE 3

Catecholamine content of 6-hydroxydopamine treated rats

after methyltyrosine (25 mg/kg) or U-14,624 (50 mg/kg)

6-OHDA Untreated Rats methyltyrosine U-14,624

Treatment NE DA NE DA NE DA

Control 100 ± 7 100 ± 5 61 ± 7* 55 ± 7* 27 ± 6* 117 ± 19

N E 39 ± 4 86 ± 7 21 ± 1* 53 ± 6* 8 ± 2* 91 ± 11

DA 87 ± 7 30 ± 5 52 ± 4* 18 ± 4* 27 ± 3* 26 ± 3

P+6-OHDA 26 ± 3 22 ± 2 11 ± 4* 7 ± 3* 5 ± 1* 25 ± 8

(a) Values are expressed as the mean ±S.E.M. percent of catecholamine content of untreated control rats.

Catecholamines were determined 4 hours after a-methyltyrosine or 6 hours after U-14.624 to correspond

to behavioral testing (See table 1). There are at least 6 rats in each group.

* < P .01 when compared with control rats of the same control or 6-hydroxydopamine treatment condition.

FIGURE 1

Figure 1: Effects of d-amphetamine on

lateral hypothalamic self-stimulation

after treatments with 6-hydroxydopamine

which reduce norepinephrine (NE+) or

dopamine (DA ) in brain. A current

intensity which just maintained responding was determined for each rat

prior to treatment with 0.25 mg/kg

d-amphetamine sulfate.

P<.05 when compared with control.

When the 6-hydroxydopamine treated rats

with lateral hypothalamic electrode

placements were given d-amphetamine and

tested at an intensity that just

maintained responding, d-amphetamine

sulfate (0.25 mg/kg) failed to

facilitate responding of those animals

in which brain dopamine was reduced

(Fig 1). Rats with brain

norepinephrine content depleted by

6-hydroxydopamine, however, displayed

significant facilitation of responding

which did not differ from control (Fig

1).

In addition to the use of

6-hydroxydopamine, another approach to

test the significance of newly

synthesized catecholamines for the

action of d-amphetamine on

self-stimulation was used. Taking a

procedure adopted from Hanson's earlier

work [17], rats were treated with

reserpine (2.5 mg/kg) to eliminate

stores of amines and then, 24 hours

later, given alpha-methyltyrosine (25

mg/kg) to block synthesis of newly

formed norepinephrine. A 1 mg/kg dose

of d-amphetamine sulfate was given 1 hr

after the synthesis inhibitors. Figure

2 shows that d-amphetamine restored the

bar-press response of rats treated with

reserpine alone and of rats given

66
FIGURE 2

Figure 2: Effects of d-amphetamine on

self-stimulation of reserpinized rats

with electrodes placed in the lateral

hypothalamus. Reserpine (2.5 mg/kg

s.c.) was given 24 hours before treatment with either vehicle, U-14, 624

(50 mg/kg) or l-alpha-methyltyrosine

(25 mg/kg). D-amphetamine sulfate

(1 mg/kg, ip) was given 1 hour after

the synthesis inhibitors. Whole brain

catecholamine content of rats with

similar treatment was determined at the

time of amphetamine administration and

is given below as a percent of untreated control values: Reserpine =

1.6 + 0.3% norepinephrine; 11.7 ± 2.5%

dopamine; Reserpine + U-14, 624 =

0.7 ± 0.01% norepinephrine; 6.1 ± 1.1%

dopamine; Reserpine + alpha-MT =

1.1 ± 0.3% norepinephrine; 2.6 ± 1.1%

dopamine.

DISCUSSION

reserpine plus U-14,624. In contrast,

alpha-methyltyrosine completely

eliminated the ability of d-amphetamine

to reinstate lateral hypothalamic

self-stimulation in the reserpinized

rat. Thus, these results, and those of

the previous studies, suggested that

dopamine neural systems are implicated

in self-stimulaton of the lateral

hypothalamus, as well as in the

pharmacological alterations of lateral

hypothalamic self-stimulation produced

by alpha-methyltyrosine, 6-hydroxydopamine, and d-amphetamine.

67

STUDIES OF THE EFFECT OF BIOGENIC

AMINE DEPLETION ON SELF-STIMULATION OF THE LOCUS COERULEUS

Although the work described thusfar was

consistent with the hypothesis that

dopamine plays a significant role in

self-stimulation of the lateral

hypothalamus, we were perplexed by the

failure to obtain a definite indication

that self-stimulation of the lateral

hypothalamus involved a noradrenergic

component. In order to evaluate

further the possible contributions of

noradrenergic and dopaminergic pathways

to self-stimulation, rats were

implanted with electrodes aimed at the

locus coeruleus and the effect of a 40

mg/kg dose of alpha-methyltyrosine was

compared with the response following a

50 mg/kg dose of U-14,624 [4,12]. When

tested 4 hours after alpha-methyltyrosine or 6 hours after U-14,624,

only those animals receiving

alpha-methyltyrosine displayed reduced

responding. In these experiments,

U-14,624 reduced norepinephrine by 73%,

and alpha-methyltyrosine depleted

norepinephrine by 50% and dopamine by

60% suggesting that the reduction of

dopamine produced by alphamethyltyrosine was responsible for the

depression of the locus coeruleus

self-stimulation [4,12].

The results of the experiments

described in this paper add to the

increasing number of reports concerning

a significant role for dopamine in

electrical self-stimulation of the

brain [16,19,23,34]. Alteration of

functional integrity of the dopamine

neural systems would appear to be of

significance for changes in

self-stimulation responding produced by

6-hydroxydopamine, alpha-methyltyrosine

and d-amphetamine--even when electrodes

are placed in the region of the locus

coeruleus. Since the locus coeruleus

contains no reported dopamine cell

bodies, axons, or terminals [33],

self-stimulation of the locus coeruleus

must involve an indirect activation of

dopamine systems. That is to say, the

assumption that changes in

self-stimulation of a particular site

following drug treatment may be related

to an action of the drug on the

structures stimulated at the tip of the

electrode cannot be freely applied in

this instance.
It is difficult, at this time, to

speculate on how dopamine is involved

in self-stimulation. Certainly, the

functional significance of the dopamine

systems are indicated in many of the

behaviors frequently employed in

psychopharmacology to evaluate drug

effects [7,8,10]. In this context, our

current results with self-stimulation,

even of the locus coeruleus, are not

unique to this measure. Furthermore,

the clinical association of Parkinson's

disease with reduced content of

dopamine in the striatum [l8] make it

tempting to speculate that the role of

dopamine in behavior, as indicated by

applications of catecholamine depleting

drugs such as 6-hydroxydopamine,

alpha-methyltyrosine, or reserpine, may

reflect a disturbance in an

extra-pyramidal motor control or an

integrative system responsible for

performance of the self-stimulation

response. Such a view, taken to

extreme, would suggest that much of the

animal work with these agents using

operant measures over the past decade

may be of primary theoretical relevance

clinically to Parkinson's disease

and/or related extrapyramidal

disorders. There is, however,

increasing evidence that the mesolimbic

dopamine pathways may be of

significance to functions less easily

related to extrapyramidal motor control

(15,20]. One area for future research

in the field of self-stimulation would

be to try to associate the deficits

produced by dopamine depletion with the

nigrostriatal and/or mesolimbic

pathways.

If it is difficult to assess the role

of dopamine in the maintenance of

self-stimulation, it is even more

difficult using the data presented in

this manuscript to speculate about a

role for norepinephrine. Our work,

even with locus coeruleus electrode

placements did not give a clear

indication that this amine participates

in the mediation of self-stimulation of

the brain. The treatments that

preferentially depleted norepinephrine

produced no change in responding even

for stimulation of the locus coeruleus

placement. Finally, no apparent role

for norepinephrine neural systems could

be defined in the facilitation of

self-stimulation produced by

d-amphetamine. However, it may be

premature to conclude that

norepinephrine is not involved in

self-stimulation of the brain. With

regard to our own experimental

procedures, it is quite feasible that

6-hydroxydopamine did not destroy a

sufficient number of noradrenergic

processes in the appropriate area of

brain to produce a clear effect.

Problems may also exist in the use of

dopamine-beta-hydroxylase inhibitors.

Recently, evidence has been reported

suggesting that these compounds may

result in the release of dopamine from

central noradrenergic neurons [1]. It

is possible, therefore, that dopamine

released from noredrenergic neurons

could activate noradrenergic receptors

to maintain self-stimulation after

U-14,624. Such a possibility, however,

would not explain why intraventricular

infusions of norepinephrine facilitate

self-stimulation while intraventricular

infusions of dopamine do not [30,35].

Finally, it is most difficult to

understand why there exists such a

close correspondence of selfstimulation loci in brain with the

central noradrenergic pathways (16,26],

if norepinephrine has little, if any,

significance for self-stimulation.

Nevertheless, the anatomical

correlation of self-stimulation with

noradrenergic pathways might not

necessarily reflect

relationship for this

functional

amine to

responding. Clearly, the need for

additional research in this area is

indicated.

ACKNOWLEDGMENTS

The authors wish to acknowledge the

assistance of Gregory Ervin, Joseph

Farmer, Susan Hollister, and Edna

Edwards. This work was supported by

USPHS grants MH-16522, MH-25573 and HD03110. George R. Breese is a USPHS Career Development Awardee (HD-24585).

REFERENCES

1. Anden, N.E., C.V. Atack and T.H.

Svensson. Release of dopamine from

central noradrenaline and dopamine

nerves induced by a dopamine-betahydroxylase inhibitor. J . Neural

Transmission 2:92-100, 1973.

2 . Arbuthnott, G.W., T.J. Crow, K.

Fuxe, L. Olson and U. Ungerstedt.

Depletion of catecholamines induced by

electrical stimulation of central

monoamine pathways. Brain Res.

24:47l-483, 1974.

68
3. Black, W.C. and B.R. Cooper.

Reduction of electrically rewarded

behavior by interference with monoamine

synthesis. Physicol. Behav.

1409, 1970.

5:1405-

4. Breese, G.R. and B.R. Cooper.

Relationship of dopamine neural systems

to the maintenance of self-stimulation.

In: Neurotransmitters and Autocoid

Balances Regulating behavior. In

Press, 1975.

5 . Breese, G.R. and T.D. Traylor.

Effect of 6-hydroxydopamine on brain

norepinephrine and dopamine: evidence

for selective degeneration of

catecholamine neurons. J. Pharmacol.

Exp. Ther. 174:413-420, 1970.

6. Breese, G.R. and T.D. Traylor.

Depletion of brain noradrenaline and

dopamine by 6-hydroxydopamine. Brit.

J. Pharmacol. 42:88-99, 1971.

7. Breese, G.R., B.R. Cooper and A.S.

Hollister. Relationship of biogenic

amines to behavior. J. Psychit. Res.

11:125-133, 1974.

8. Breese, G.R., B.R. Cooper and R.D.

Smith. Biochemical and behavioral

alterations following 6-hydroxydopamine

administration into brain. In:

Frontiers in Catecholamine Research, E.

Usdin and S. Snyder, editors. Pergamon

Press, N.Y., pp. 701-706, 1973.

9. Breese, G.R., J. Howard and P.

Leahy. Effect of 6-hydroxydopamine on

electrical self-stimulation of brain.

Brit. J. Pharmacol. 43:255-257, 1971.

10. Cooper, B.R. and G.R. Breese.

Relationship of dopamine neural systems

to the behavioral alterations produced

by 6-hydroxydopamine administration

into brain. In: Neuropsychopharmacology of Monoamines and Their

Regulatorv Enzymes, E. Usdin, editor.

Raven Press pp 35-368, 1974.

11. Cooper, B.R., J.M. Cott, and G.R.

Breese. Effects of

catecholamine-depleting drugs and

amphetamine on self-stimulation of

brain following various

6-hydroxydopamine treatments.

Psychopharmacologia (Berl.) 37:235-248,

1974.

12. Cooper, B.R., G.N. Ervin and G.R.

Breese. A role for dopamine in the

stimulant effects of amphetamine on

locus coeruleus self-stimulation.

Pharmacologist 16:249 abs, 1974.

13. Cooper, B.R., L.D. Grant and G.R.

Breese. Comparison on the behavioral

depressant effects of neuroleptic and

biogenic amine depleting agents

following various 6-hydroxydopamine

treatments. Psychopharmacologia

3l:95-109, 1973.

14. Cooper, B.R., G.R. Breese, J.L.

Howard and L.D. Grant. Enhanced

behavioral depressant effects of

reserpine and alpha-methyltyrosine

after 6-hydroxydopamine treatment.

Psychopharmacologia 27:99-110, 1972.

15. Cooper, B.R., J.L. Howard, L.D.

Grant, R.D. Smith and G.R. Breese.

Alteration of avoidance and ingestive

behavior after destruction of central

catecholamine pathways with

6-hydroxydopamine. Pharmacol.

Biochem. Behav. 1:639-649, 1974.

16. Crow

Catecholamine-containing

T.J.

neurons and

electrical self-stimulation I. A

review of some data.

2:414-421, 1972.

Psychol. Med.

17. Hanson, L.C.F. Evidence that the

central action of amphetamine is

mediated via catecholamines.

Psychopharmacol. 9:78-80, 1966.

18. Horneykeiwicz, O. Parkinson's

disease: From brain homogenate to

treatment. Fed. Proc. 32:183-190,

1973.

19. Lippa, A.S., S.M. Antelman, A.E.

Fisher and D.R. Canfield. Neurochemial

mediation of reward: A significant role

for dopamine? Pharmacol. Biochem.

Behav. 1:23-28, 1973.

20. Matthysse, S. Schizophrenia:

Relationship to dopamine transmission,

motor control and feature extraction.

In: The Neurosciences. Third Annual

Studv Program, F. Schmitt and F.G.

Worden, editors. MIT Press, Cambridge,

Mass., pp. 733-737, 1964.

21. Olds, J. and P. Milner. Positive

reinforcement produced by electrical

stimulation of septal area and other

regions of rat brain. J. Comp.

Physiol. Psych. 47:419-427, 1954.

22. Olds, J. and R.P. Travis. Effects

of chlorpromazine, meprobamate,

pentobarbital and morphine

self-stimulation. Pharmacol. Exp.

Ther. 128:397,404, 1960.

69
23. Phillips, A.G. and H. Fibiger, 30. Stein, L. Chemistry of reward and

Dopaminergic and noradrenergic punishment. In: Psychopharmacologv.

substrates of positive reinforcement: A Review of Progress (1957-1967)

Differential effects of d- and l- Efron, editor,

D:

PHSP No. 1836. U.S.

amphetamine. Science 179:575-577,

1973.

24. Poschel, B. and F. Ninteman.

Excitatory (antidepressant) effect of

monoamine oxidase inhibitors on the

reward system of the brain. Life Sci.

3:903-910, 1964.

25. Poschel, B. and F. Ninteman.

Hypothalamic self-stimulation: Its

suppression by blockade of

norepinephrine biosynthesis and

reinstatement by methamphetamine. Life

Sci. 5:11-16, 1966.

26. Hitter S. and L. Stein.

Self-stimulation of noradrenergic cell

group (A6) in locus coeruleus of rats.

J . Comp. Physiol. Psych. 85:443-452,

1973.

27. Skinner, J.E.: Neuroscience: A

Laboratory Manual. Saunders, London,

1971.

28. Stein, L. Effects and interactions of imipramine, chlorpromazine,

reserpine, and amphetamine on selfstimulation: Possible

neurophysiological basis of depression.

Rec. Adv. Biol. Psychiat. 4:288-309,

1962.

29. Stein, L. Self-stimulation of the

brain and the central stimulant action

of amphetamine. Fed. Proc. 23:826,

1964.

AUTHORS

Barrett R. Cooper, Ph.D., and George R.

Breese, Ph.D.: Departments of

Psychiatry and Pharmacology, Biological

Sciences Research Center of The Child

Development Institute, University of

North Carolina School of Medicine,

Chapel Hill, N.C. 27514

Government Printing Office, Wash., D.C.

PP. 105-123, 1968.

31. Stein, L. and C.D. Wise. Possible

etiology of schizophrenia: Progressive

damage to the noradrenergic reward

system by 6-hydroxydopamine. Science

171:1032-1036, 1971.

32. Taylor, K.M. and S.H. Snyder.

Differential effects of d- and

l-amphetamine on behavior and on

catecholamine disposition in dopamine

and norepinephrine containing neurons

of rat brain. Brain Res. 28:295-309,

1971.

33. Ungerstedt, U. Stereotaxic mapping of monoamine pathways in the rat

brain. Acta Physiologica.

Scandanavica. Suppl. 367:l-48, 1971.

34. Wauquier, A . and C.J.E.

Neimegeers. Intracranial self-stimulation in rats as a function of various

stimulus parameters. II. Influence of

haloperidol, pimozide, and pipamperone

on medial forebrain bundle stimulation

with monopolar electrodes.

Psychopharmacologia. (Berl.)

27:191-202, 1972.

35. Wise, C.D. and L. Stein.

Facilitation of brain self-stimulation

by central administration of norepinephrine. Science 163:299-301, 1969.
AGGRESSION AND THE BRAIN MONOAMINES:

WHAT ARE THE ANSWERS,

BUT OF MORE IMPORTANCE,

WHAT ARE THE QUESTIONS... ?

1,2

Bruce Kenneth Bernard

INTRODUCTION

The study of aggressive behavior and

the biochemical mechanisms which may

underlie it is not a new endeavor.

Comparatively speaking, however, the

investigation of the Functional

significance of brain monoamines in

relation to aggression is of recent

vintage. Several discoveries in the

early 1950's and 60's, including the

introduction of antipsychotic drugs

such as the phenothiazines and the

development of sensitive analytical

techniques for detecting nanogram

quantities of postulated neurotransmitters, led to the voluminous brain

biogenic amine research experienced

during the past 10 years. Yet, the

importance of these papers has been

diminished by the endless variety of

correlations and factors which make

generalizations across studies seemingly impossible.

Although there may be many reasons for

the apparent lack of cross-study

validity? this paper will explore one

possibility, non-specificity in questioning. That is, although the various

methodologies being employed may provide valid and reliable data, these

data may not be generalizable due to

basic dissimilarities in the models

themselves. Thus, the function of the

brain amines may vary across models of

aggressive behavior and only after

careful evaluation of tne behavioral

models themselves can one expect

similar relationships to occur. In

recent years, workers in our

laboratories have investigated a number

of models of aggression and their

relationship to brain monoamine functioning. These include: isolationinduced aggression [11], natural

muricide in the wild rat [10], shockinduced conspecific and ranacide aggression [9,8], hormonal manipulation

of natural ranacide [5,4] septal

lesion-induced hyperirritability [6]

and age related conspecific aggression

in mice [7]. It is the expressed

purpose of this paper to employ several

of these model systems and demonstrate

not ohly behavioral similarities and

dissimilarities but also similarities

and dissimilarities in relation to the

brain amines.

Models of aggressive behavior can be

compared using a number of criteria.

For the purposes of this paper, I will

employ two classificatory schematas,

71
the "eliciting stimulus" and the

"neurochemical" models. In the first

system, Moyer [28] divides aggressive

behavior into six separate models based

upon differences in eliciting stimuli:

predatory, inter-male, irritable,

territorial defensive, maternal and

instrumental. In a more recent

publication, he reduces this number to

five by eliminating territorial defense

[27]. In addition to the eliciting

stimuli, the behavior is characterized

on the limited neurological and

endocrinological data available at the

time. Thus for example, predatory

aggression is thought to be

non-androgen dependent and have a

neurological basis in the lateral

hypothalamus. Intermale aggression,

however, appears to involve the septal

region and requires the presence of

testosterone.

The second schemata, suggested by Reis

[30,31] stresses a pharmacological and

neurological distinction between two

sub-groupings of aggressive behavior,

predatory and affective. Predatory

aggression may be characterized as: 1)

involving the ventral medial tegmentum

[15], 2) having no autonomic activation

components, and 3) being inhibited by

drugs which increase catecholamine

(norepinephrine and dopamine) or

serotonergic activity, and enhanced by

drugs which increase cholinergic

functioning [30]. The second subclass

of agonistic behavior (affective) can

be characterized as: 1) involving the

brain central gray, 2) having quite

obvious autonomic activation components

and 3) being enhanced by drugs which

stimulate catecholamine activity and

inhibited by drugs which enhance

cholinergic or serotonergic activity.

Furthermore, as demonstrated in recent

articles [4,5] predatory aggression is

probably not androgen dependent whereas

affective aggression probably is [8].

Using the above classifications, three

distinct models of aggressive behavior

have been chosen From the numerous ones

employed in our laboratories: these

are shock-induced fighting, ranacide

behavior and septal lesion-induced

hyperirritability. As will be reported

later in this paper, shock-induced

fighting is both androgen-dependent and

has autonomic activation components

associated with it whereas ranacide is

neither androgen dependent nor involves

autonomic components. Thus, we have

examples of the two classically

different models. The third model,

hyperirritability, is a mixture of the

above two in that it contains autonomic

components but is not androgen dependent. Employing these 3 behaviorally

distinct models, we will investigate

some of the possible relationships

which exist between aggression and the

brain monoamines.

SHOCK-INDUCED FIGHTING

Fighting between pairs of animals

induced by aversive stimulation is one

of the more frequently employed models

of aggressive behavior cited in the

literature. It is a type of

"irritable" agonistic behavior as

defined by the eliciting stimulus model

and "affective" aggression in the

neurological classification. Both

classificatory schematas suggest

androgen dependency for this type of

behavior [28,5] and there is supporting

evidence for that view [20]. Other

reports [16], however, indicate that

shock-induced fighting is not

manipulable via the hormonal route.

Similar discrepancies appear in the

literature with regard to hormonal

control of brain monoamines. Thus, for

example, Stefano and Donoso [35] and

others [l7] reported an increased

hypothalamic NE concentration following

castration whereas Sandler [32] and

others [21] reported no effect. It

occurred to us3 that variations in the

castration-testing interval may be the

cause of these apparently contradictory

findings. Furthermore, if both

shock-induced aggression and the brain

amines are androgen dependent, a

time-related covariance of these

parameters might provide some insight

into a possible causal relationship.

Method and Results

Adult male, Sprague-Dawley rats,

170-190 gms. at the start of the

experiment, were employed as subjects.

Animals were castrated under light

ether anesthesia. Control subjects

(sham-castrates) underwent identical

surgical manipulations as castrated

animals; however, the testicles were

not removed. Three or six weeks

following surgery, animals underwent a

series of behavioral tests followed 24

hours later by biochemical analyses of

brain amines. The behavioral testing

consisted of: a) 3 emotionality

testing sessions, 24 hours apart, in an

open-field apparatus [18], b) a test

72
for shock-induced flinch-thresholds

[29,3] and c) a test for shock-induced

aggressive behavior between pairs of

rats [16].

Behavior

The effect of castration on open-field

emotionality is shown in Figure 1. A

repeated measures analyses of variance

[38] showed that castrate subjects

traversed significantly more squares

than sham subjects but only at the 3

week interval (p< 0.025). Analysis of

rearing behavior resulted in a pattern

identical to that seen in Figure 1 and

therefore is not shown. Three other

measures of emotionality, latency to

enter the first square, boli and

grooming behavior were unaltered by

castration and there were no

significant interactions with either

time or trials. This alteration in

open-field behavior at the 3 week

interval was unrelated to any change in

shock-induced aggressive behavior

FIGURE 1

Figure 1: Adult male rats were castrated or sham-castrated. Three or 6

weeks later, the mean number of squares

transversed during 3 separate emotionality (habituation) trials spaced 24

hours apart was recorded. Data were

analyzed using analyses of variance.

Castration had a significant effect

only at the 3 week interval (p<0.025).

Both groups were significantly decreased over time (p<0.025). Each

point represents the mean value obtained from 12 rats.

(Figure 2). In the latter behavioral

paradigm, castration Failed to alter

the fighting pattern at the 3 week

interval but did have an effect at the

6 week time period. Analyses of other

aggression measures such as number of

trials until first fight or First two

consecutive fights gave the same

results, namely that shams fought

significantly more than castrates and

took less trials to do so but only at

the 6 week interval (p< 0.05). These

findings could not be attributed to an

alteration in Foot-shock sensitivity

since (as shown in Figure 3) castration

failed to alter flinch-thresholds at

either interval.

Brain Amines

Rat brains were separated into

hypothalamus (H) and whole-brain minus

hypothalamus (WB-H) and simultaneously

analyzed for brain levels of serotonin

(5-HT), 5-hydroxyindoleacetic acid

(5-HIAA), norepinephrine (NE) and

dopamine (DA) using a modification of

previously reported methods [1,14,24].

In addition, the instantaneous rate

constants (k), turnover times (TT) and

utilization rates (K) for the

FIGURE 2

Figure 2: Adult male rats were castrated or sham-castrated. Three or 6

weeks later, animals were tested in

pairs for shock-induced aggressive

behavior at three shock-intensities

(0.5, 1.3 and 2.5 ma). Castration has

a significant effect only at the 6

week interval (p<0.001). Time significantly increased the aggressiveness

of the shams but not the castrate

animals (p<0.00l). Each bar represents

the mean value obtained from 12 rats,

vertical lines represent ± one standard

error.

73
FIGURE 3

TIME-DEPENDENT EFFECTS OF CASTRATION

ON HYPOTHALAMIC NOREPPINERHINE DINAMICS

Initial concentration Rate constant, k Turnover Time Utilization rate

(ng/g) (h-1) (TT) (h) (k) (ng/g/h)

3 weeks

SHAM 1201 ± 38 0.107 ± 0.022 9.35 129 ± 46

(3) [7.75 - 11.76]

CASTRATE 1558 ± 142 0.187 ± 0.025a 5.36

(4)a 291 ± 222

[5.00 - 6.17]

6 weeks

SHAM 1335 ± 37 0.209 ± 0.041b 4.79

(3)b 279 ± 50b

[4.00 - 5.95]b

CASTRATE 1693 ± 81 0.239 ± 0.039 4.19 40 ± 138 (4)c

[3.60 - 5.00]

Number of animals in parenthesis.

a Approaches p < 0.05 (sham vs castrate)

b Approaches p < 0.05 (3 vs 6 week)

c p < 0.01 (sham vs castrate)

[ ] 67 per cent confidence interval

Figure 3: Adult male rats were

trated or sham-castrated. Three or 6

weeks Later, animals were tested for

shock-induced flinch thresholds.

Analysis of variance indicated that

neither castration nor time altered

this measure. Each bar represents the

mean value obtained from 12 rats,

vertical bars represent ± one standard

error.

cas -

TABLE 1

TIME-DEPENDENT EFFECTS OF CASTRATION ON

WHOL.E BRAIN MINUS HYPOTHALAMUS LEVELS OF 5-HT AND 5-HIAA Number of animals in parenthesis.

a Approaches p < 0.05 (sham vs castrate)

b Approaches p < 0.05 (sham vs castrate)

c p < 0.01 (sham vs castrate)

d p < 0.05 (3 vs 6 week)

[ ] 67 per cent confidence interval

CASTRATION-DECAPITATION INTERVAL

3 WEEK 6 WEEK

5-HT 5-HIAA 5-HT 5-HIAA

SHAM 497 ± 7 478 ± 12 535 ± 20 383 ± 13

(10) (12) (17) (17)a

CASTRATE 527 ±12 560 ±12 697 ±16

(11)b

413 ± 12

(12)c

(17)a

Concentration expressed in ng/g tissue; Mean ± S.E.

Number of animals in parenthesis.

a p < 0.0001 (3 vs 6 weak)

b p < 0.05 (sham vs castrate)

c p < 0.0001 (sham vs castrate)

TABLE 2

TABLE 3

334 ± 13 0.257 ± 0.044 3.90 86 ± 12

(3) [3.32 - 4.96]

TIME-DEPENDENT EFFECTS OF CASTRATION

ON HYPOTHALAMIC DOPAMINE DINAMICS

Initial concentration Rate constant, k Turnover Time Utilization rate

(ng/g) (h-1) (TT) (h) (k) (ng/g/h)

3 weeks

CASTRATE 431 ± 30 0.386 ± 0.034a 2.59b 166 ± 18c

(5)a [2.38 - 2.84]

6 weeks

SHAM 608 ± 77 3.15

(4)d 0.317 ± 0.088 191 ± 52

[2.47 - 4.36]

CASTRATE 575 ± 43 0.283 ± 0.054 3.53

(5)d

163 ± 29

[2.97 - 4.36]

catecholamines, NE and DA, were

obtained using the methods of Brodie et

al., [13] and Bernard and Paolino [9],

and that data analyzed by analysis of

variance [38]. As can be observed in

Table 1, brain levels of 5-HIAA were

significantly higher in castrate

subjects compared to controls only at

the 3 week interval while 5-HT levels

were significantly greater in castrate

animals with the magnitude of the

difference increasing in a time-dependent manner. These effects were not

seen in the hypothalamus, where castration failed to alter 5-HIAA or 5-HT

levels at either time period.

74
Region specific alterations were also

observed for NE dynamics in that

castration failed to alter the levels,

k, TT or K for this amine in WB-H, yet

effected, albeit transitively, NE

dynamics in the hypothalamus. As seen

in Table 2, both levels and k were

increased in the castrate animals at

the 3 week interval and although the

magnitude of the difference in levels

increased at 6 weeks, the rate

constants were no longer different. A

similar effect on hypothalamic DA rate

constants can be seen in Table 3 where

castrate subjects had higher levels and

rate constants at the 3 week interval

but were the same as sham subjects by

the 6 week time period. These results

are opposite to the findings for WB-H

DA analyses, which revealed no

alterations in k, TT or K at either

interval but did find a significant

decrease in DA levels at the 6 week

interval (960 ± 29 ng/gm vs 771 ± 43

ng/gm; p< 0.005).

Discussion

The results of this study provide

evidence that the effect of castration

on both brain monoamine dynamics and

shock-induced aggressive behavior is

time-dependent in nature. Thus,

castration altered aggressive behavior

at the 6 but not the 3 week interval

thereby independently confirming

apparently contradictory literature

reports [20,16]. Furthermore, these

behavioral effects could not be

attributed to alterations in general

emotionality or altered foot-shock

sensitivity since the latter did not

change and the former was altered only

at the 3 week interval. Similarly,

alterations in hypothalamic NE are

clearly time-dependent. Thus, experiments which employed castrationanalyses intervals of 14 and 21 days

[32,21] and reported no change in NE

are not necessarily in conflict with

those using an interval of 20 and 30

days [35,17] and reporting increased

NE. This is further supported by the

fact that the results of the 21 day

study were just shy of significance at

the p< 0.05 level.

At first glance, one might conclude

that castration led to a time-dependent

decrease in aggressive behavior and

levels of WB-H DA. Careful analyses

however revealed that in both

instances, castration per se had no

effect on either measure (3 vs 6 week).

Rather, in both cases age-dependent

increase was observed in the sham

subjects. It would appear therefore

that the effect of castration was the

inhibition of this natural, maturational process, thereby fixating the

animal at the level achieved at the

time of castration. These data suggest

a definitive pattern of covariance

between decreased aggressive behavior

and: 1) increased brain 5-HT and 2)

decreased brain DA. An additional

covariance pattern was observed between

increased open-field activity and brain

levels of 5-HIAA and DA. This sequence

of events may be interpreted in several

ways, one of which is a simplistic

causal relationship between brain

amines (where 5-HT has inhibitory and

NE and DA excitatory functions) and

affective behavior. A more complex,

yet justified conclusion relates these

behaviors to relative aminergic

dominance, where a single brain

aminergic neuronal system is of

functional importance only when

considered in relation to the other

brain amine systems. These interpretations will be more fully explored in

the GENERAL DISCUSSION section of this

paper.

RANACIDE BEHAVIOR

Ranacide, or the killing of frogs, is a

second type of aggressive behavior but

one which has anatomical and

pharmacological properties which are

quite distinct from shock-induced

fighting. It has been classified as

"predatory" aggression in both

classificatory schemes in that: 1) it

is a very stereotyped behavior pattern

elicited by a limited range of stimuli

[2], and 2) the hehavior is facilitated

by cholinergic stimulation of the

lateral hypothalamus (see Ref. 2).

Furthermore, it has been reported that

rats will seek out, kill and eat frogs

under natural conditions

Eibesfeldt, I., pers. comm., (EiblPredatory aggression is described as

non-androgen dependent [28,5] and

although there is no evidence relating

this question to ranacide one might

expect the behavior to be

non-manipulable via the hormonal route.

The purposes of the following

experiments were two-fold: First, to

determine if ranacide aggression was

androgen dependent in either the adult

75
male or female rat and second, to

determine what if any, relationship

might exist between the effects of

testosterone on ranacide behavior and

the brain monoamines.4

Method and Results

Adult male and female Wistar rats,

70-80 days of age at the beginning of

the experiments, were employed as

subjects. In the first series of

experiments, male rats were screened

for ranacide behavior in 2 separate, 24

hour testing sessions spaced 24 hr.

apart. Animals which killed frogs

within 1 min. or less on both trials

were defined as killers (K) while

animals which failed to kill during

both 24 hrs. tests were defined as

non-killers (NK). The killers were

then separated into 2 experimental and

two control groups; some animals

received alternate day subcutaneous

injections of testosterone propionate

(200 ug in sesame oil) for 30 days (K +

T) or sesame control injections (K + S)

while others were castrated (K +

castrate) or sham-castrated (K + sham).

The non-killing male animals were

divided into two groups and received

either testosterone (NK + T) or vehicle

injections (NK + S) as described for

the killers. Thirty-one days following

the beginning of hormonal manipulations, all subjects were given 3

additional ranacide screening sessions

24 hr. apart.

ALTERATION IN THE MEDIAN LATENCY TO KILL AS A

FUNCTION OF TESTOSTERONE ADMINISTRATION

GROUPS TRIAL 1 2 3 4 5

Killers 45* 42 40 30 32**

+ (50-35) (65-30) (70-15) (65-10) (55-15)

Sesame Oil 10*

Killers 45 40 40 32 35**

+ (60-25) (80-20) (300-15) (85-10) (65-15)

Testosterone

* Median latency to kill (seconds)

** .1 > p > .05 Trial 1 vs 5

( ) Range of values

+ Number of subjects/group

In the second series of experiments,

female rats were screened for ranacide

on the basis of a single 30 minute

session. Subjects which attacked or

killed during this period were defined

failed to do so were called

as agressors (A) while animals which 

non-aggressors (NA). Aggressors were

divided into two groups; those which

received the above regimen of

testosterone (A + T) or vehicle control

injections (A + S). A similar division

of the NA group resulted in subjects

which received testosterone (NA + T)

and their controls (NA + S).

Thirty-one days following the+ first

injections, all subjects received an

additional 30 min. testing session.

Following this screening trial, all

animals were continued on their

appropriate hormonal treatment.

Seventy-two hours later, animals were

decapitated and their brains assayed

for monoamine content.

Behavior

The failure of testosterone

administration to significantly alter

the median latency to kill in adult

male killer rats can be seen in Table

4. Analyses of these data using a

Wilcoxon Matched-Pairs Sign-Rank Test

[34] indicated a tendency for the

latencies of both groups (K + S and K +

T) to decrease when Trial 1 was

compared to 5 (0.1>p> 0.05). However,

a comparison of the trends between the

two groups using a Mann-Whitney U test,

revealed no significant differences.

Identical results were obtained when

comparing the latencies of the killer +

sham and killer + castrate groups

(Table 5). Once again there was a

tendency for the latencies of both

groups to decrease over trials, yet

there was no difference in trends

between groups. Thus, neither

castration nor testosterone administration per se altered the latency to

kill in adult male killer rats.

TABLE 4

TABLE 5

ALTERATION IS THE MEDIAN LATENCY TO KILL AS A

FUNCTION OF CASTRATION

GROUPS TRIAL 1 2 3 4 5

Killers 40* 37 30 30 27

+ (55-20) (85-20) (50-15) (65-15) (70-15)

Sham-Castration 10†

Killers 40 35 37 27 30

+ (60-15) (140-15) (300-15) (90-10) (80-5)

Castration 20†

* Median latency to kill (seconds)

( ) Range of values

+ Number of subjects/group

76
The results of the administration of

testosterone to adult male non-killer

(NK) provided additional evidence for

the non-androgencity of this behavior.

Of the 40 rats which failed to kill in

the pre-injection trials (1 and 2), not

a single animal killed in

post-injection trials. The conclusions

from these studies are similar to those

obtained using adult, female

non-aggressive and aggressive rats

(Table 6). Although some subjects from

both the NK + S and NK + T did kill

(2/5 and 4/16, respectively) or attack

(5/5 and l2/16, respectively) following

the injection sequence, there was no

difference between the groups on these

measures or the final median latency to

attack (6.33 vs 9.53 min.,

respectively). Similar behavioral

analyses showed that adult, female

naturally aggressive rats could not be

differentiated following either

testosterone (A + T) or sesame (A + S)

administration (Table 6).

TABLE 6

* Data expressed as ng amine/gm tissue; Mean ± standard error

Amine levels were analyzed using ANOVA

FAILURE OF TESTOSTERONE ADMINISTRATION TO ALTER ATTACK ( ) Number of animals/group

OR KILL BEHAVIOR IN AGGRESSIVE & NON-AGGRESSIVE FEMALE RATS

LATENCY TO ATTACK *

BEHAVIOR TREATMENT TRIAL 1 TRIAL 2

SESAME OIL [5] 30.00 6.33

(13-2)

NON-AGGCRESSIVE Behavior Treatment

TESTOSTERONE [16] 30.00 9.53 Sesame Oil Testosterone

(30-.3)

SESAME OIL [9] 10.25 9.251

AGGRESSIVE (29-5) (14-2)

TESTOSTERONE [8] 9.58 2.422

(15-1) (27-1)

* Median latency to attack (min)

( ) Range of values (min)

[ ] Number of animals/group

1 p < 0.02 Trial 1 vs 2

2 p < 0.035 Trial 1 vs 2

Brain Amines

* Data expressed as ng amine/gm tissue; Mean @ standard error

( ) Number of animals/group

1 p < .01 Non-agg. test. vs non-agg. sesame

p < .05 Non-agg. test. vs agg. sesame

2 p < .05 Agg. test. vs non-agg. sesame

3 p < .05 Agg. ses. vs. Non-agg. ses.

4 p < .001 test. vs sesame

The effect of the above hormonal

manipulations on whole-brain levels of

5-HT and DA in these aggressive and

non-aggressive adult female rats is

shown in Table 7. Data were analyzed

using two way analyses of variance.

The results indicated no significant

effect for behavior, treatment groups

or their interaction for either brain

5-HT, or DA. This was not the case for

brain levels of NE (Table 8) which was

analyzed in a similar manner.

Testosterone injected subjects had

significantly higher brain NE levels

than sesame treated controls (p<

0.001); analysis by behavioral group

(non-aggressive vs aggressive) revealed

no significant differences.

TABLE 7

FAILURE OF TESTOSTERONE TO ALTER

WHOLE-BRAIN SEROTONIN AND DOPAMINE

LEVELS IN AGGRESSIVE AND NON-AGGRESSIVE FEMALE RATS

BRAIN MONOAMINE LEVELS*

GROUPS SEROTONIN DOPAMINE

NON-AGGRESSIVE + 558.9

SESAME OIL ± 34.07 (5)

TESTOSTERONE 573.7

± 24.0 (4)

AGGRESSIVE + 529.7

SESAME OIL ± 15.7 (3)

TESTOSTERONE 515.0

± 19.4 (5)

1382.8

± 23.59 (5)

1424.0

± 47.4 (4)

1460.9

± 17.02

1320.3

± 53.3 (5)

TABLE 8

BRAIN NOREPINERPHINE LEVELS IN ADULT FEMALE RATS AS

A FUNCTION OF BEHAVIOR AND HORMONAL TREATMENT

Non-Aggressive 270.3 ± 5.84* 305.5 ± 6.181

(5)

285.9 ± 7.36

(4) (9)

Aggressive 287.1 ± 3.433293.1 ± 2.232 290.1 ± 2.34

(5) (5) (10)

278.7 ± 4.25 298.6 ± 3.674

(10) (9)

Discussion

The results of the above series of

experiments provides evidence in adult

male and female, aggressive and

non-aggressive, testosterone treated

and castrated rats, that ranacide

behavior is a non-androgen dependent

model of aggressive behavior.

77
Testosterone was neither an initiator

of ranacide behavior in non-aggressive

subjects nor was it a promoter of this

behavior in naturally

Conversely,

aggressive

animals. testosterone did

not inhibit aggressive behavior in

animals so inclined. These results are

further supported by the fact that in

the shock-induced aggression study

previously reported, castrated rats

which were less aggressive than their

sham controls at the 6 week interval

(Figure 2), did not differ from each

other with respect to ranacide at that

same time period (Figure 4).

FIGURE 4

Figure 4: Adult male rats were castrated or sham-castrated and tested for

ranacide behavior 3 or 6 weeks later.

Data were analyzed using analysis of

variance. Neither castration nor time

altered either group with respect to

this behavior.

SEPTAL LESION INDUCED

HYPERIRRITABILITY

The third and final model of aggression

to be discussed involves the

hyperirritability syndrome which can be

induced by stereotaxically placed

lesions of the septal region of the

brain. The behavior is characterized

by increased responsiveness to

inanimate object presentation, and an

increased likelihood of attack, biting

and vocalization during handling

[12,6]. This rather stereotyped

pattern of the behavior together with

its autonomic components is very

similar to that described

"affective" aggression in the

neurochemical classification and

"irritable" aggression in the eliciting

stimuli model. Hyperirritability

differs from shock-induced aggression,

however, in that it does not appear to

be androgen dependent [26,33]. Thus,

hyperirritability has characteristics

which are common to both shock-induced

and predatory aggression and yet

differs in other ways from these same

categories.

The purpose of this experiment, as with

the previous ones, was to determine if

this type of behavior could be

correlated with brain monoaminergic

changes. Secondly, if the behavior

could be related to monoaminergic

changes, were these changes similar to

that seen in shock-induced aggression

or ranacide behavior?

Method and Results

Adult male Wistar rats were employed as

subjects in the following experimental

paradigm. Animals were screened for

emotional reactivity [22] on 3

consecutive days. The final ratings

were employed to form 2 behaviorally

equivalent groups for surgery. One

group received stereotaxically placed,

electrolytically induced bilateral

lesions of the septum while the other

group underwent the same surgical

procedure but no current was placed.

Forty-eight hours following surgery,

animals were again screened for

emotional reactivity and then subjected

to biochemical procedures designed to

evaluate brain monoamine levels and

dynamics.

78
Behavior

The effects of bilateral electrolytic

lesions of the septum on emotional

reactivity can be observed in Table 9.

As previously stated, groups were

behaviorally matched prior to surgery

(4.97 vs 5.13). The surgical procedure

by itself did not effect the

postoperative behavior (5.66 vs 5.13,

p> 0.1), however, the animals which

received septal lesions were more than

3 times as reactive as their previous

scores (15.15 vs 4.97, p< 0.01) and

significantly greater than the sham

controls (p< 0.01).

TABLE 9

THE EFFECT OF BILATERAL ELECTROLITIC

SEPTAL LESIONS ON EMOTIONALITY RATINGS1

Emotionality Ratings2

Treatment Final Preoperative Postoperative

Septal Lesion 4.97 ± 0.24 15.15 ± 0.013

Sham Control 5.13 ± 0.16 5.66 ± 0.24

Brain Amines

The effect of septal lesions on brain

monoamine levels can be seen in Table

10. Alterations were observed only in

the hypothalamus and limbic system

where NE levels were significantly

reduced in the former (p< 0.01) and DA

levels reduced in both (p< 0.05 and p<

0.01, respectively). No alterations

were observed in either the cortex or

the pons-medulla. Furthermore, there

were no changes in the indoleamine

system (5-HT and 5-HIAA) in any of the

brain parts under consideration.

Analysis of NE and DA rate constant

(k), turnover time (TT) and utilization

rate (K) data revealed no alteration

for either catecholamine in any of the

brain parts under consideration.

Discussion

The results of this experiment clearly

demonstrate the striking behavior

effects of septal lesions. The

importance of the biochemical profile,

that is decreased hypothalamic and

limbic DA and hypothalamic NE, is

uncertain due to the failure of these

lesions to alter catecholamine dynamics

(k, TT or K). Furthermore, these

biochemical effects are not only

anatomically localized, but also

confined to the catecholaminergic as

opposed to the indoleaminergic neuronal

1 Data analyzed using a two way, repeated measures ANOVA followed by

Norman-Keule analysis for pairs of means. Date expressed as Mean

± Standard Error (N = 15 per treatment).

2 Table values are for ratings taken 24 hr prior to and 48 hr following

surgery

3 p < 0.01 (Preop. septals vs postop. septals, postop. septals vs postop.

shams)

TABLE 10

THE EFFECTS OF SEPTAL LESIONS ON

MONOAMINE LEVELS IN DISCRETE BRAIN REGIONS

Nonrepinephine* Dopamine Serotonin 5-Hydroxyindoleacetic acid

Septal Sham Septal Sham Septal Sham Septal Sham

Limbic 307 374 2689 3317 513 497 521 491

(783) ± 16 ± 26 ±50 l

± 158 ± 24 ± 33 ± 49 ± 74

Hypothalamus 1353 1532 934 1214 846 939 921 870

(69) ± 64l ± 56 ±301 ± 83 ± 81 ± 24 ± 67 ± 28

Cortex 274 325 698 676 924 896 253 303

(551) ± 19 ± 13 ± 138 ± 121 ± 48 ± 51 ± 16 ± 21

Pane-Medulle 456 510 176 167 692 754 518 483

(205) ± 27 ± 38 ±19 ± 8 ± 23 ± 20 ± 40 ± 52

Whole-Brain 358 426 1574 1924 691 685 446 442

(1608) ± 202 ± 24 ± 762 ± 123 ± 35 ± 37 ± 37 ± 51

* Data expressed as ng/g tissue; Mean ± Standard Error (4 N 5); Weight of brain tissue (mg )in parentheses.

1 p < 0.01 (septal vs sham)

2 p < 0.05 (septal vs sham)

79
systems. The combination of

biochemical effects did however alter

brain catecholamine to indoleamine

ratios, the importance of which will be

discussed under GENERAL DISCUSSION.

GENERAL DISCUSSION

The above series of experiments were

chosen to demonstrate some of the basic

differences between models of

aggressive behavior. As stated in the

introduction, these behaviors were

selected because of their differences

in androgen dependency and autonomic

components. The importance of this

Paper is not, however, these behavioral

differences, but rather the divergent

effects the treatments had on the brain

monoamines in relation to the

behaviors.

It has been suggested that the

importance of the brain monoamines lay

not in the individual neuronal systems,

but in the balance between the

excitatory and inhibitory transmitters

[37,9]. This has been expressed in

terms of brain NE/5-HT or DA/5-HT

ratios where it is suggested that 5-HT

is an inhibitory and NE and DA are

excitatory transmitters. When the

biochemical alterations in the above

experiments are analyzed in this

manner, a very interesting pattern

emerges. Table 11 summarizes the

hypothalamus and whole brain minus

hypothalamus monoamine concentrations

at 3 and 6 weeks following castration.

In addition, the percent change in

NE/5-HT and DA/5-HT ratios are shown.

The hypothalamic values at the 3 week

interval are initially negative

(indicating a relative catecholaminergic

dominance) but are greatly reduced at

the 6 week interval. In contrast, the

whole-brain minus hypothalamus is

dominated by the serotonergic system at

both intervals. Thus, castration which

is associated with a less aggressive

animal (shock-induced) as compared to

controls, is also associated with an

increase in serotonergic dominance.

These data would agree with the

postulate inhibitory role for 5-HT and

excitatory role for NE and DA.

TABLE 11

ALTERATIONS IN THE NE/5-HT AND DA/5-HT RATIOS IN SAT

HYPOTHALAMUS AND WHOLE BRAIN MINUS HYPOTHALAMUS 3 AND 6

WEEKS FOLLOWING CASTRATION

Time

(Weeks Following

Castration)

Tissue Amine

Concentration

Changes After

Castration*

NE (Increase)

Hypothesized Transmitter

% Changes in Ratios produced

by castration**

NE/5-HT DA/5-HT

DA (Increase)

3 5-NT (No change) -77.2 -21.1

Hypothalamus

NE (Increase)

6 DA (No Change) -21.1 - 0.3

5-HT (No Change)

NE (No Change)

3 DA (No Change) +21.0 +13.2

Whole Brain 5-HT (Increase)

(minus hypothalamus)

NE (No Change)

6 DA (Decrease) +13.9 +68.9

5-HT (Increase)

* Comparing Sham Castrate at the same time interval.

* Calculated by substracting the Sham from the Castrate ratio at the same time interval.

80
If the biochemical data obtained in the

ranacide experiments are analyzed in

terms of monoamine ratios, the

resulting pattern can be seen in Table

12. As was observed in the

TABLE 12

ALTERATIONS IN WHOLE-BRAIN NOREPINEPHRINE/SEROTONIN

RATIOS IN AGGRESSIVE & NON-AGGRESSIVE FEMALE RATS

AS A FUNCTION OF TESTOSTERONE ADMINISTRATION

Behavior* Treatment

Sesame Testosterone

Non-Aggressive .463 ± .010

(4)

.535 ± .030 .499 ± .020

(4) (8)

Aggressive .545 ± .014 .572 ± .023 .562 ± .0l6

(3) (5) (8)

.498 ± .017l .556 ± .019

(7) (9)

* Data expressed as Mean ± standard error

( ) Number of animals/group

1 p < .05 Sesame vs Testosterone

2 p < .02 Aggressive vs Non-aggressive

shock-induced aggression study, the

aggressive animals had significantly

higher NE/5-HT ratios than the

non-aggressive ones. Likewise, the

subjects receiving exogenous

testosterone had significantly higher

ratios than those receiving sesame oil

control injections. These two factors,

aggression and testosterone, appear to

be of equal importance inasmuch as the

ratios for the aggressive sesame

treated animals were the same as the

non-aggressive testosterone treated

subjects and both values were located

between the other two groups. These

results may be interpreted as

indicating no functional relationship

between the changes in aminergic ratios

and ranacide behavior since the former

was altered and the latter remained

unchanged.

DIFFERENCES IN BRAIN NE/5-HT RATIOS

FOLLOWING SEPTAL LESIONS*

While this interpretation may in fact

be valid, there exist at least two

other possible explanations. First,

although the change in NE/5-HT ratios

was qualitatively the same in both the

shock-induced aggression and ranacide

studies, the alterations were not

quantitatively the same. The changes

observed in the former study were of

greater magnitude than those observed

in the latter experiment. Thus, in

spite of the fact that the changes in

the second study were statistically

significant, they may not have been

large enough to be of functional

significance. This of course implies

the existence of a minimal change in

the NE/5-HT ratios which is required

prior to any possible effect on the

behavior. The second alternative

interpretation involves the means by

which the change in ratios occurs

rather than the size of the change.

The NE/5-HT ratios of the shock-induced

castrated animals were lower than the

sham-castrated controls due to

differences in 5-HT concentrations, not

NE. This was not the case in the

ranacide study, where changes in

NE/5-HT ratios resulted from NE rather

than 5-HT alterations. Thus, although

the concept of a necessary balance

between the brain aminergic systems may

be valid, the value of this concept may

depend upon changes induced by 5-HT

alterations, whereas small changes in

NE may be relatively unimportant.

The third and final experiment

described herein related the behavioral

alterations (hyperirritability) induced

by septal-lesions to the monoaminergic

effects at the same 2 day post-surgery

time period. The results of analyzing

the data (in terms of ratios) as just

described, can be seen in Table 13. It

should be noted that the only

alteration observed was in the limbic

system NE/5-HT ratio where the septal

animals were significantly lower, more

serotonergic, than the sham controls.

This result is opposite to that which

might have been expected since a more

reactive animal might conceivably have

been more catecholaminergic

(excitatory) in nature as compared to

to the controls. Furthermore, this

change in NE/5-HT was induced by NE

rather than 5-HT alterations.

TABLE 13

Brain Region

NE/5-HT DA/5-HT

Septal Sham Septal Sham

Lesion Control Lesion Control

Limbic System 0.591 ± 0.0381 0.755 ± 0.039 5.29 ± 0.27 6.69 ± 0.57

Hypothalamus 1.64 ± 0.11 1.63 ± 0.06 1.15 ± 0.13 1.30 ± 0.11

* Data are expressed as Mean ± Standard Error (N = 5)

1 p < 0.05 (sham vs septal)

81
In light of the above, what can we

conclude about the basic question,

"What is the functional significance of

the brain monoamines with regard to

aggressive behavior?" Surely, if these

experiments have demonstrated anything,

it is that the question has become

naive. Its generality is obsolete,

rendered so by the progressive

discrimination of subcategories of

aggressive behavior. It is one of

those starting points from which

scientific inquiry works away, towards

a more specific and definitive plateau

of understanding. Its major weakness

is not in what it asks, but in what it

fails to ask due to the generality of

its underlying assumption, that

aggression is an undifferentiated mode

of behavior with which brain monoamine

function might be expected to

correlate. To be of value, the

question must be more particular: "How

do the brain amines relate ...to

ranacide, muricide and irritable

aggression, ...to predatory, irritable

and spontaneous aggression, ...or even

to differences within a single

(irritable) aggression?" These

questions must be asked and indeed are

now being asked [25, 19 and 36,

respectively]. These studies and the

ones reported herein suggest that a

more fruitful approach to the study of

the brain amine aggressive behavior

hypothesis may lie in the careful

elaboration of the characteristics of

the behavioral model being employed and

determinations of how alterations in

these basic characteristics relate to

the different relationships with the

brain amines.

FOOTNOTES

1. This paper was presented at the

tnirteenth annual meeting of the

American College of Neuropsychopharmacology, in San Juan, Puerto Rico, 1974.

2 . Parts of this work were supported

by the following agencies: University

of Connecticut Research Foundation,

National Institutes of Mental Health,

and Public Health Service.

3. The shock-induced aggression study

described herein was co-authored with

Dr. Ronald M. Paolino, present address:

Department of Psychiatry and Behavioral

Sciences, University of Oklahoma

Sciences Center, Oklahoma City,

Oklahoma, 73190.

4. The ranacide study in female rats

was performed with the technical

assistance of Miss Beverly Cocrane and

Mr . David Furlano.

5. The septal lesion study was

co-authored with Dr. David A. Yutzey

and Mr. James R. Berchek, present

address: Department of Psychology,

University of Connecticut, Storrs,

Connecticut 06268.

REFERENCES

1. Ansell, G.B. and M.F. Beeson. A

rapid and sensitive procedure for the

combined assay of noradrenaline,

dopamine and serotonin in a single

brain sample. Analyt. Biochem. 23:

196-206, 1968.

2. Bandler, R. Cholinergic synapse in

the lateral hypothalamus for the

control of predatory aggression in the

rat. Brain Res. 20: 409-424, 1970.

3. Beatty, W. and P. Beatty. Hormonal

determinants of sex differences in

avoidance behavior and reactivity to

electric shock in the rat. J. Comp.

Physical. Psychol. 73: 446-455, 1970.

4. Bernard, B.K. Testosterone

manipulations: effects on ranacide

aggression and brain monoamines in the

adult female rat. Submitted for

publication.

5. Bernard, B.K. Frog killing

(ranacide) in the male rat: lack of

effect of hormonal manipulations.

Physiol. Behav. 12(3): 405-408, 1974.

6 . Bernard, B.K., J.R. Berchek and

D.A. Yutsey. Alterations in brain

monoaminergic functioning associated

with septal lesion induced hyperreactivity. Pharmac. Biochem. Behav. 3:

121-126, 1975.

82
7. Bernard, B.K., E.R. Finkelstein and

G. M. Everett. Alterations in mouse

aggressive behavior and brain monoamine

dynamics as function of age.

Physiol. Behav., In press.

8. Bernard, B.K. and R.M. Paolino.

Temporal effects of castration on

emotionality and shock-induced

aggression. adult male rats.

Physiol. Behav., 14: 201-206, 1975.

9 . Bernard, B.K. and R.M. Paolino.

Time-dependent changes in brain

biogenic amine dynamics following

castration in male rats. J. Neurochem.

22: 951-956, 1974.

10. Bernard, B.K. and R.M. Paolino.

The use of biochemical correlates for

the prediction of muricidal behavior in

the wild rat. Fed. Proc., 32(3): 785,

1973.

11. Bernard, B.K. and R.M. Paolino.

Brain norepinephrine levels and

turnover rates in castrated mice

isolated for 13 months.

(2): 221-222, 1973.

Experientia a

12. Brady, J.V. and W.J.H. Nauta.

Subcortical mechanisms in emotional

behavior: affective changes following

septal forebrain lesions in the albino

rat. J. Comp. Physiol. Psychol. 46:

339-346, 1953.

13. Brodie, B., E. Costa, A. Dlabac,

N.H. Neff and W.H. Smookler.

Application of steady state kinetics to

the estimation of synthesis rate and

turnover time of tissue catecholamines.

J. Pharmac. Exp. Ther. 154: 493-498,

1966.

14. Chang, C.C. A sensitive method

for spectrophotofluorometric assay of

catecholamines. Int. J. Neuropharmac.

3: 643-649, 1964.

15. Chi, C. and J. Flynn. Neural

pathways associated with

hypothalamicallv elicited

behavior in cats.

attack

703-705, 1971.

Science 171:

16. Conner, R.L. and S. Levine.

Hormonal influences on aggressive

behavior. In: Aggressive Behavior,

edited by S. Garrattini and E. Sigg.

New York: Wiley, 1969, pp. 150-163.

17. Coppola, J.A. The apparent

involvement of the sympathetic nervous

system in the gonadotrophin secretion

of female rats. J. Reprod. Fert. 4:

35-45, 1968.

18. Denenberg, V.H. Open-field

behavior in the rat: what does it

mean. Ann. N.Y. Acad.

852-859, 1969.

Sci. 159:

19. Eichelman, B.S. and N.B. Thoa.

The aggressive monoamines. Biol.

Psychiat. 6(2): 143-164, 1973.

20. Hutchison, R., R. Ulrich and N.

Azrin. Effects of age and related

factors on the pain aggression

reaction. J. Comp. Physiol. Psychol.

59: 365-369, 1965.

21. Hyyppa M. and

effect of castration

on the content of

M. Valavaara. The

and hypophysectomy

noradrenaline and

serotonin in the hypothalamus of the

rat. Experientia 15: 193-194, 1970.

22. King, F.A. Effects of septal and

amygdaloid lesions on emotional

behavior and condition avoidance

responses in the rat. J. Nerv. Ment.

Dis. 126: 57-63, 1958.

23. Knutson, J.F. and M.T. Hyman.

Predatory aggression and irritable

aggression: shock-induced fighting in

mouse-killing rats. Physiol. Behav.

11: 113-115, 1973.

24. Maickel, R., R. Cox, J. Saillant

and F. Miller. A method for the

determination of serotonin and

norepinephrine in discrete areas of rat

brain. Int. J. Neuropharmac. 7:

275-281, 1968.

25. McLain, W.C., B.T. Cole, R.

Schrieber and D.A. Powell. Central

catechol- and indolanine systems and

aggression. Pharnacol. Biochem.

Behav. 2: 123-126, 1974.

26. Montgomery, R.L., M.K. Berkut,

E.F. Grubb and D.L. Westbrook.

Hormonal influence on behavior in brain

lesioned male rats. Physiol. Behav. 7:

107-111, 1971.

27. Moyer, K.E. Territorial

aggression: is this concept useful?

Totus Homo, 4(3): 104-117, 1972.

28. Moyer, K.E. Kinds of aggression

and their physiological basis. Behav.

Biol. 2(A): 65-87, 1968.

83
29. Pare, W.P. Age, sex and strain

differences in the aversive threshold

to grid shock in the rat. J. Comp.

Physiol. Psycho1 69: 214-218, 1969.

30. Reis, D.J. Central neurotransmitters in aggression. Aggression.

Res. Publ. Ass. Res. Nerv. Ment. Div.,

52: 119-148, 1974.

31. Reis, D.J. The relationship

between brain norepinephrine and

aggressive behavior. Res. Publ. Ass.

Res. Nerv. Ment. Dis. 50: 266-297,

1972.

32. Sandler, R. Concentration of

norepinephrine in the hypothalamus of

the rat in relation to the estrous

cycle.

1968.

Endocrinology, 83, 1383-1386,

33. Seggie, J. Effect of adrenalectomy or gonadectomy on affective

behavior changes following septal

lesions in the rat. J. Comp. Physiol.

Psychol. 74: 11-19, 1971.

AUTHOR

Bruce Kenneth Bernard, Ph.D.: Division

of Pharmacology and Toxicology,

University of Connecticut, Storrs,

Connecticut 06268

34. Siegal, S. In: Nonparametric

Statistics For the Behavioral Sciences,

New York: McGraw-Hill, 1956.

35. Stefano, F. and A. Donoso.

Norepinephrine levels in the rat

hypothalamus during the estrous cycle.

Endocrinology 81: 1405-1406, 1967.

36. Stolk, J.M., R.L. Conner, S.

Levine and J.D. Barchas. Brain

norepinephrine metabolism and

shock-induced fighting behavior in

rats: differential effects of shock

and fighting on the neurochemical

response to a common foot shock

stimulus. J. Pharmacol. Exp. Ther.

190: 193-209, 1974.

37. Tagliamonte, A., P. Tagliamonte,

G . Gessa and B. Brodie. Compulsive

sexual activity induced by

p-chlorophenylalanine in normal and

pinealectonized rats. Science 166:

1433-1435, 1969.

38. Winer, B.J. In: Statistical

Principles in Experimental Design. New

York: McGraw-Hill, 1971.

8 4
PHARMACOGENETIC STUDIES

OF THE SEROTONERGIC SYSTEM

IN ASSOCIATION WITH

CONVULSIVE SEIZURES IN MICE

Benson E. Ginsburg, Paul Y. Sze

INTRODUCTION

The effects of genetic differences on

behavioral capacities can be

demonstrated in a variety of ways. If

we confine our interest to mammals,

including man, such differences have

been demonstrated in inbred strains of

mice and rats for behaviors as diverse

as sexual performance [11,14,21],

learning [1], aggression [7,13,15,25],

alcohol preference [6,20], alcohol and

drug tolerance [2,17,28], maternal

behavior [23], susceptibility to

seizures [10,13,19], various forms of

ataxia [3], reactions to stress [29],

differential effects of identical

lesions [19], and many others [26].

These researches depend upon finding

strain differences in behavioral

parameters and demonstrating, by means

of appropriate genetic analyses based

on crosses among strains that differ in

these parameters, that the differences

are genetically based. Selection

experiments in genetically variable

populations can accomplish the same

thing --namely, the demonstration that

there is a genetic underpinning for the

behavior. In human pedigrees, the

major techniques used to establish a

genetic etiology include genealogical

analyses, twin studies, sibship

analyses, and karyotyping.

Once the demonstration of a genetic

basis is achieved, its further

characterization in genetic terms, far

from being an exercise in genetic

esoterica, becomes relevant for the

understanding of the mechanisms

involved, and thereby of the

possibility and direction of

pharmacological intervention. The same

behavioral or morphological phenotype

often occurs in a given species on a

variety of genotypic

Each genetic

bases [9,13].

route to the common

phenotype may represent a different

link in a chain of causal mechanisms,

as would be the case with a series of

enzymatic reactions where the product

of one becomes the substrate for the

second. Another possibility is that of

distinct alternative mechanisms. A

single phenotype may, therefore,

include a diversity of underlying

mechanisms, which if parsed

genetically, could be distinguished for

purposes of differential treatment.

85
One of our seizure-prone mouse mutants,

for example, responds well to

pre-treatment with monosodium

glutamate, while another does not [8].

Both share a common phenotype.

Aggressive behavior in male mice is

influenced by a number of genetically

distinguishable predispositions.

Regimes that will change this behavior

in one strain will not necessarily

produce a similar change in another

[12,15]. Even where a given gene can

be shown to affect a process and both

the genetic substitution and the

associated process are highly

correlated with a behavior, the

associated mechanism may not be causal

for the behavior, but may represent one

of several pleiotropic actions of the

gene that may be producing its effect

on the behavior via another action

[19]. Since we will be dealing with

such data in this presentation, the

burden of proof that a mechanism, in

this case serotonergic, that is

associated with both genetic and

behavioral differences, is involved in

the mediation of the genetic

differences and does, in fact,

represent an essential mechanism in the

development of the behavior, devolves

upon us.

In the light of massive data

demonstrating genetic control of

enzymatic processes, it becomes

reasonable to hypothesize that genetic

effects on behavioral capacities might

be mediated, in part, by genetic

variation in enzyme systems involved in

the synthesis and degradation of the

major neural transmitters. In

investigating this possibility, it must

be recognized that key variations in

transmitter characteristics can be

localized both temporally (to a

particular period in development) and

spatially (to a particular CNS

structure). Such genetically based

developmental differences have been

described in our laboratory for

glutamic acid decarboxylase in relation

to the production of GABA, and for

tryptophan hydroxylase, the ratelimiting enzyme in the synthesis of

serotonin [4,16,27]. These enzyme

differences are correlated with genetic

differences in predisposition to

audiogenic seizures and with aggressive

behavior in male mice. No similar

relationships involving tyrosine

hydorxylase were found. In addition,

seizures induced by the administration

of alcohol during early development and

seizures induced by alcohol withdrawal

in older mice, are both associated with

serotonergic effects [17,28]. The

effect of chronic alcohol treatment on

tryptophan hydroxylase activity could

not be demonstrated with adrenalectomized mice, but was restored by the

injection of corticosterone.

Glucocorticoids are similarly required

for the effects of ethanol withdrawal

seizures to occur [28].

MATERIALS AND METHODS

Animals

The following mouse stocks were used in

these studies: DBA/l/Bg, C57BL/6/Bg,

C57BL/l0/Bg and CD1 (Swiss albino).

Audiogenic seizures

Seizure tests were conducted in an

insulated chamber 38 cm. x 43 cm. x 38

cm. high in which an Edwards-Lungen

house bell (7.6 cm. in diameter) was

used to provide a sound level of 95-105

dB (in re, 2 x 10-4 dyne/sq. cm.) at

the floor of the chamber. The mouse

was placed in the chamber and its

activity, defecation and urination were

recorded for one minute. The bell was

then activated for 90 seconds.

Convulsive seizures occurring during

the test period were recorded.

Seizures were scored for severity,

latency scores were recorded, and

seizure incidences were calculated as

the percent of mice exhibiting

convulsive seizures out of the total

number of mice tested. Incidences in

Table 1 represent the percent of mice

that convulsed when tested on four

consecutive days beginning at 28 days

of age. Other incidences are based on

a single test.

Ethanol administration

Ethanol was administered in the

drinking water (10% v/v) of mice on ad

lib normal diets in those experiments

where effects on the offspring while in

utero and/or during lactation were

under investigation [11]. For studies

of withdrawal seizures of adult mice,

male CD1 and C57BL/l0/Bg mice, each

bred in our own colony, were housed in

groups of five. The CD1 mice were 70 ±

7 days of age, and the C57BL/10/Bg mice

were 66 ± 8 days of age on the first

day of ethanol treatment. All mice

were fed a liquid diet as the only

86
source of food and water. The liquid

diet for the ethanol groups consisted

of 60% Shape-Metracal (Mead-Johnson &

Co.), 6% (v/v) ethanol, 0.9% NaCl, and

water. The average daily consumption

of liquid diet containing ethanol was

7-9 ml. per mouse, an ethanol intake of

approximately 340-440 mg./day. Control

mice received the identical liquid diet

containing an isocaloric amount of

sucrose instead of ethanol. CD1 mice

received ethanol for 21 days, and

C57BL/10/Bg mice received it for 14

days, since these periods result in

maximal seizure induction after

withdrawal for each strain. At the end

of the experimental period, ethanol was

withdrawn at 9 a.m. and the control

diet was substituted. Seven hours

later, the mice were tested for

audiogenic seizures (28).

Aggression test

Litters were weaned at 29 days of life

and males were individually isolated

until days 50-52 post partum, when they

were tested for intermale,

intragenotype aggressive behavior

[24,25]. Isolation-induced aggression

was measured in dyadic encounters on

three consecutive days. Male pairs

were usually littermates and partners

remained constant over the three

trials. Aggression testing occurred in

a "neutral" cage (16 x 26.5 x 11.5

cm.), divided in half by an opaque

partition and covered at the top by a

6.4 mm. wire mesh. No attempt was made

to eliminate odors from the test

apparatus or room between pairs.

Testing took place between 8 a.m. and 6

p.m. in a room used only for that

purpose. Overhead fluorescent lighting

delivered 94 ftc at the level of the

test cage floor. One member of each

pair was tail-marked with a

MarkerR

 so

Magic

that the observer could

readily distinguish them. Animals were

handled with padded forceps throughout

the testing period. At 52 days of

life, each mouse was weighed to the

nearest 0.5 grams. Pairs differing in

weight by more than 3.0 g were dropped

from the sample.

Subject animals were placed one on each

side of the partition and the partition

removed after an initial five minute

adaptation period. Behavioral records

were kept per 20 second interval for a

test duration of ten minutes.

Agonistic acts scored consisted of tail

rattling, flank biting, wrestling,

chasing and full attack. Latency

values from the time the partition was

lifted to the first agonistic act and

to the first full-blown attack were

also recorded. Composite "any

aggression" scores were determined for

each pair by Dr. M. Selmanoff [24] by

counting "1" for each 20 second

interval during which any agonistic act

upon an opponent occurred. If both

members of a pair exhibited one or all

of these behaviors in an interval, the

score was "2” for that interval.

Hence, with thirty 20-second intervals

Per day, the maximum pair score was 180

for three days. Scores for each pair

were summed on three trials, combined

with those of the other pairs for each

genotype and averaged.

Enzyme assays

Tryptophan hydroxylase was assayed

using the method of Ichyama et al.

[18]. Tyrosine hydroxylase was

measured using the method of Nagatsu

and co-workers [22]. Enzyme activities

were determined in whole brain

homogenates.

RESULTS

Tryptophan hydroxylase activity was

measured from the fourth day of life

post partum until the thirty-third day

in the C57BL/10/Bg and the DBA/l/Bg

strains by Dr. J.A. Diez, working in

our laboratory [43]. The results are

illustrated in Figure 1, where the

solid lines represent the enzyme

activity of the C-57 strain and the

broken lines that of the DBA/1 strain

[4,5]. Measurements were made in whole

brain homogenates of males only. The

C57BL/10/Bg mice exhibited a higher

level of enzyme activity throughout the

test period than that shown by the

DBA/1/Bg males. They also exhibited a

faster rate of increase in enzyme

activity during development than did

the DBA/1's. Since the brain

concentrations of tryptophan precursor

were not different in the two strains,

our data, indicate that these C57BL mice

have a greater capacity to synthesize

serotonin than do their DBA/1

counterparts. This activity difference

is most likely due to the amount of

enzyme present rather than to a

structural difference in the enzyme

molecule which would affect its

activity, since the Km of the enzyme

for tryptophan is identical in the two

87
FIGURE 1

Figure 1: Developmental changes of

brain tryptophan hydroxylase activity

in C57BL/10/Bg ( ) and DBA/1/Bg mice

( ). (From: Dies, J. A., P. Y. Sze

and B. E. Ginsburg, 1975. Difference

in brain tryptophan hydroxylase activity in C57BL and DBA mouse genotypes.

Behav. Genet. 5:94-95). (See, also,

reference 25).

FIGURE 2

Figure 2: Apparent Km of tryptophan

hydroxylase for tryptophan in brain

homogenates for C57BL/10/Bg ( ) and

DBA/1/Bg ( ) mice.

(From: Diez, J. A., P. Y. Sze and

B. E. Ginsburg, 1975. Difference in

brain tryptophan hydroxylase activity

in C57BL and DBA mouse genotypes.

Behav. Genet. 5:94-95). (See, also,

reference 251.

strains at 30 days of age (see Figure

2). The progressive increase in the

straine difference is also consistent

with this interpretation [4,5 op.

cit.].

As Diez (op. cit.) pointed out, the

results in Figure 1 could also be

attributed to an excess of tryptophan

hydroxylase activator in brains of the

C57DL/10/Bg mice, or, conversely, to an

enzyme inhibitor contained in the

brains of the DBA/1/Bg animals. If the

first alternative were true, a mixture

of C57 and DBA brain homogenates would

be expected to provide a higher level

of enzyme activity than the algebraic

average of the two strains. If the

DBA's had an enzyme inhibitor, a

mixed-strain homogenate would have

lower activity than the algebraic

average of the two strains. When such

homogenates were actually mixed, the

expected algebraic average was

obtained, suggesting that the strain

difference is not due to either an

excess of activators or inhibitors [19

and Table 2]. Table 1 summarizes the

results obtained for tryptophan

hydroxylase activity at 30 days of age

for male mice of the strains already

mentioned, as well as those of the

C57BL/10/Bg strain and for the various

F1 crosses among them. These results

are arranged in ascending order

relative to their enzyme activities.

Differences between genotypes separated

by a dashed line are statistically

significant. The right hand column

represents the incidence of audiogenic

seizures in these strains and crosses,

where the tests were carried on four

consecutive days beginning at day 28

post partum. The highest enzyme

activity was exhibited by the two C57BL

strains, which did not differ

significantly from each other. When

these were crossed, however, the

resulting F1 had a significantly lower

incidence than either parental strain.

F1's between DBA/1/Bg and either C57BL

strain were significantly lower than

those obtained from the high parental

strains or the F1 between them and were

significantly higher than those

obtained from DBA/1/Bg. All were

somewhat lower than the expected

mid-parental value, suggesting

possible partial dominance of the DBA/1

characteristic. Reciprocal F1's

between the C57BL/10/Bg and the

DBA/1/Bg strains were not appreciably

different from each other. There is,

therefore, no suggestion of sex linkage

or of a maternal effect [4,5].

88
TABLE 1 FIGURE 3

Ginsburg and Sze

Correlation Between Brain Tryptophan

Hydroxylase (TPH) Activity and Audiogenic Seizures1

Genotype*

DBA/1

Audiogenic

Seizuresc

(pmole/hr/mg prot.)b (% Incidences)

4.17 ± 0.05 (16) 81%

B6D1F1 4.54 ± 0.08 ( 6) 48%

BIODIF1 4.66 ± 0.06 ( 8) 57%

DIBIOFl 4.67 ± 0.07 ( 8) 57%

BlOB6Fl 5.00 ± 0.09 (16) 0%

C57BL/1O 5.40 ± 0.07 (16) 0%

C57BL16 5.44 ± 0.08 (16) 0%

a

 All mice were males, one month of age.

b

 Mean ± S.E.M. with number of nice in parentheses.

Values separated by dotted lines are significantly

different (p< O.05) from adjacent values.

c

 Each value from at least 300 mice tested.

1

 From: Diez, J.A., P.Y. Sze and B.E. Ginsburg, 1975.

Difference in brain tryptophan hydroxylase activity in

C57BL and DBA mouse genotypes. Behav. Genet. 5:94-95.

Figure 3: Developmental changes of

brain tyrosine hydroxylase activity in

C57BL/10/Bg ( ) and DBA/1/Bg ( ) mice.

(From: Dies, J. A., P. Y. Sze and

B. E. Ginsburg, 1975. Difference in

brain tryptophan hydroxylase activity

in C57BL and DBA mouse genotypes.

Behav. Genet. 5:94-95). (See, also,

reference 25).

TABLE 2

Effect of Mixing DBA/1/Bg and C57BL/10/Bg Brain Homgenates

Homogenate

Source

DBA/1/Bg

C57BL/10/Bg

DBA + C57

TRYPTOPHAN HYDROXYLASE 5-HTP DECABBOXYLASE

Triplicate Triplicate

Mean Values Mean Values

1043 (1012, 1094, 1024) 22.6 (22.8, 22.7, 22.5)

1344 (1341, 1359, 1331) 21.6 (21.7, 21.6, 21.5)

1174 (1202, 1156, 1165) 22.4 (22.5, 22.2, 22.6)

Calculated Mean

of DBA and C57 1193 22.1

Tryptophan hydroxylase activity is DPM/100 mg tissue.

5-HTP decarboxylase activity is (DPM/100 mg tissue) x 10-3.

Eight brains from each strain were pooled and homogenized in 4.0

volumes of 0.3 M sucrose. Triplicate assays were performed on

each homogenate and a 1:1 mixture of the two (DBA + C57).

Enzyme activity was determined as described under Methods, but the

5-HTP decarboxylase substrate did not contain exogennnous factor.

1 From: Diez, J. A., P. Y. Sze and B. E. Ginsburg, 1975. Difference

in brain tryprophan hydroxylase activity in C57BL and DBA mouse genotypes.

Behav. Genet. 2:94-95. (see, also, reference 25).

89
Figure 3 summarizes the data we have

obtained on a developmental basis

measuring tyrosine hydroxylase

activities for the DBA/1/Bg and

C576L/10/Bg strains over the same time

period studied for tryptophan

hydroxylase [19]. Only male mice were

used for these assays, which were

performed with whole brain homogenates.

No significant differences in tyrosine

hydroxylase activity were found. By

comparison with tryptophan hydroxylase,

the developmental pattern is different.

In both strains, the increase in

tryptophan hydroxylase was essentially

linear and began at approximately the

eighth day of life. By contrast,

tyrosine hydroxylase activity remained

low until about day 12 and increased

sharply to day 16 after which it

remained approximately level. The two

enzymes, therefore, appear to be

regulated quite differently, both with

respect to the levels of enzyme

activity between the two strains and

the pattern of developmental change

occurring within the strains. At 30

days of age, no significant differences

in tyrosine hydroxylase activity were

seen between the two strains in

question and the C57BL/6/Bg strain or

the Ft's between the DBA/1/Bg and

either C57BL/10/Bg or

strains (Fig. 3).

C57BL/6/Bg

TABLE 3

Ginsburg and Sze

Correlation Between Brain Tryptophan Hydroxylase

(TPH) Activity and Aggression1

TPH Activityb

Aggression

Genotypea (pmole/hr/mg prot.) Score2

DBA/1 4.17 ± 0.05 (16) 35.4

B6D1F1

4.54 ± 0.08 ( 6) 5.2

B10D1F1 4.66 ± 0.06 ( 8) 19.8

D1B10F1

4.67 ± 0.07 ( 8) 10.7

B10B6F1

5.00 ± 0.09 (16) 7.1

a All mice were males, one month of age

b Mean ± S.E.M. with number of mice in parenthesis.

Values separated by dotted lines are significantly

different (p<0.05) from adjacent values.

1 From : Diez, J.A., P.Y. Sze and B.E. Ginsburg,

1975. Difference in brain ryptophan hydroxylase

activity in C57BL and DBA mouse genotypes. Behav.

Genet. 5:94-95.

2 From: Selmanoff, M.K., J.E. Jumonville, S.C.

Maxson and B.E. Ginsburg, 1975. Evidencce for a

Y chromosomal contribution to an aggressive

phenotype in inbred mice. Nature 253:529-530.

So far as the behavioral measures are

concerned, neither seizures (Table 1)

nor aggression (Table 3) differences

between the strains tested and their Ft

hybrids are related to tyrosine

hydroxylase activities since these were

the same in all cases tested. With

respect to both audiogenic seizures and

aggression scores, there appears to be

an inverse correlation with tryptophan

hydroxylase activity. These correlations suggest that the serotonergic

system may be involved in both of these

behaviors. Since the behaviors are not

independent of each other, it is not

surprising that similar correlations

with the serotonergic systems are

obtained for both.

In addition to investigating the

relationship between tryptophan

hydroxylase activity, tyrosine

hydroxylase activity, and genetic

susceptibility to audiogenic seizures,

the relationship of tryptophan

hydroxylase activity to audiogenic

seizures resulting from withdrawal from

ethanol was investigated using CD1 mice

and C57BL/10/Bg mice [28]. The results

are summarized in Figures 4-7. Under

the conditions of our test, neither of

these strains exhibits convulsive

seizure activity when exposed to sound.

After withdrawal from ethanol, however,

both strains are markedly susceptible

to sound induced seizures. The

susceptibility to withdrawal seizures

is, however, dependent upon

corticosterone, since adrenalectomized

mice of either strain exhibit only a

small fraction of the withdrawal

seizures that would be expected were

their adrenals intact. If, however,

the adrenalectomized mice are given

daily subcutaneous injections of

corticosterone (0.5 mg/mouse) during

the period of chronic ethanol

administration, the seizure incidence

is restored to that of the intact

animal. Tryptophan hydroxylase activities occurring in brain homogenate and

brain P fractions obtained from mice

undergoing ethanol treatment with and

without adrenalectomy shows a similar

dependence on the intact adrenal or on

substituted corticosterone. Thus, the

association between this aspect of the

Serotonergic system and the associated

behavior is either causal, in the sense

that the glucocorticoids affect the

seizure incidence via the serotonergic

system, or dependent upon a common

mechanism involving glucocorticoids

[28].

90
FIGURES 4 and 5

Figure 4 and Figure 5: Incidence of

audiogenic seizures during withdrawal

in CD1 (Fig. 4) mice and C57BL/10

(Fig. 5) mice receiving various treatmen ts. Control: intact mice without

ethanol administration; ETOH: intact

mice with chronic ethanol administration; ETOH + AdX: adrenalectomized

mice with chronic ethanol administration; ETOH + AdX + CCS: adrenalectomised mice with chronic ethanol administration and daily subcutaneous

injections of corticosterone (0.5 mg/

mouse). Seizure incidence is expressed

as percent of mice exhibiting convulsive seizures in the total number of

mice tested (indicated by the number in

each bar). Significance of differences:

*p<0.05, ***p<0.001, as compared with

the control group; † p<0.05, †† p<0.01,

††† P<0.001, as compared with the ETOH

+ AdX group. (From: P. Y. Sze, J. Yanai and B. E. Ginsburg, 1974. Adrenal

glucocorticoids as a required factor in

the development of ethanol withdrawal

seizures in mice. Brain Research 80:

155-159.

FIGURES 6 and 7

Figure 6 and Figure 7: Tryptophan

hydroxylase (TPH) activities in (Fig.

6) brain homogenate and (Fig. 7) brain

P2 fraction from mice under various

treatments. Control: intact mice

without ethanol administration: ETOH:

intact mice with chronic ethanol

administration for 2 weeks; ETOH + AdX:

adrenalectomized mice with ethanol

administration for 2 weeks: ETOH +AdX

+ CCS: adrenalectomized mice with

chronic ethanol administration and

daily subcutaneous injection of corticosterone (0.5 mg in 0.1 ml sesame oil/

mouse). The enzyme activity is

expressed as counts/min/h/mg protein.

Each value is the mean from 6-8

animals ± S.E.M. * p<0.01 as compared with the control, ** p<0.001 as

compared with the control. (From:

P. Y. Sze and L. Neckers, 1974.

Requirement for adrenal glucocorticoid in the ethanol-induced increase

of tryptophan hydroxylase activity in

mouse brain. Brain Research 72:

375-378).

91
The association between sound induced

seizures and the serotonergic system is

further strengthened by the data

summarized in Figure 8. Here, seizures

were induced in C57BL/10/Bg mice by

administering ethanol to them via the

mothers during intrauterine development

and the first two weeks of lactation.

Using a total of 124 mice, 22% were

found to be seizure susceptible when

tested at 30 days of age by comparison

with control samples showing no seizure

activity. Figure 8 summarizes the

effects of aminergic drugs in modifying activity.

FIGURE 8

Figure 6: Effects of drugs on ethanolinduced audiogenic seizures in mice.

All mice were offspring from C57BL/10/

Bg parents receiving 10% ethanol in

drinking water throughout the entire

period of pregnancy and 14 days postpartum. Only controls (CON) were not

exposed to ethanot. Audiogenic seizures were tested starting at 29 days

of age. Reserpine (RES): 2 mg/kg at

2.5 hr before testing; DOPA: 200 mg/kg

at 0.5 hr; 5-hydroxytryptophan (5-RTP):

200 mg/kg at 0.5 hr; alpha-methyl-ptyrosine (alpha-MT): 200 mg/kg at 6

hr; alpha-chlorophenylalanine (PCPA):

300 mg/kg at 24 hr. Saline (SAL) was

given to ethanol controls. Numbers in

each bar indicate the number of mice

in each group. ** p<0.01, *** p<0001

for the deviation from the saline

injected ethanol control. (From:

J. Yanai,

1375.

P. Y. Sze and B. E. Ginsburg,

Effects of aminergic drugs

and glutamic acid on audiogenic seiazures induced by early exposure to

ethanol. Epilepsia 16: 67-71).

the ethanol induced susceptibility to

these seizures. When reserpine was

injected 2-1/2 hours prior to testing,

the seizure incidence increased from

22%, observed in alcohol treated saline

controls, to 48%. The injection of

DOPA one-half hour prior to testing did

not change the incidence of the

seizures in the reserpine treated

animals. If, however, 5-HTP was

injected one-half hour before testing

in the reserpine treated group, the

seizures were completely prevented.

Alpha-MT injected at six hours prior to

testing did not modify the seizure

incidence induced by early ethanol

administration. A single injection of

PCPA administered 24 hours after the

first test increased the seizure

incidence to 42%, a level that was not

statistically different from that

obtained with reserpine. These results

suggest that the serotonergic system is

very likely associated with the

production or the expression of

seizures induced by alcohol

administered to mouse pups during early

development. They also failed to

demonstrate any association between

these events and the catecholaminergic

system [17].

DISCUSSION

The biogenic amines (serotonin,

dopamine and norepinephrine) have been

implicated in the regulation of a

number of behavioral measures for which

the DBA and C57BL strains of mice

differ. Since the biogenic amines may

be functionally regulated at several

levels (including synthesis, storage,

release, post-synaptic action,

re-uptake and catabolism), genetic

variation in any of these processes

could prove to be a mechanism by which

differential gene action might regulate

behavioral function. We chose to study

the regulation of synthesis using as

endpoints the activities of the

rate-limiting biosynthetic enzymes

--tryptophan hydroxylase (for

serotonin) and

(for the

tyrosine hydroxylase

catecholamines). Since an

enzyme is a gene product, genetic

variations in enzyme activity could

reflect differences in the gene's

coding for the enzyme, or in regulatory

genes. They might also be due to other

metabolic differences, such as

hormones, which could affect gene

expression or enzyme activity. The

association between genes, behavior,

and biogenic amines were here studied

developmentally. Such an approach

92
should provide information useful in

identifying critical or sensitive

periods for biochemical and behavioral

differentiation that involve

genetic regulation. In general, and in

pharmacological studies, it is assumed

that changes in neurotransmitter

turnover rates have a functional

significance. Measures of in vivo

synthesis rates are often taken as

indicators of turnover. Since C57BL

mouse brains have the capacity to

synthesize more serotonin than do

DBA/1's, we would predict that the

amine has a higher turnover rate in the

C57. In attempting to interpret the

functional significance of this

difference, one must consider the

possibility that the C57BL mice may

require a higher turnover to maintain

the same functional levels that DBA/1

mice achieve with a lower rate of

turnover. It should also be noted that

the lack of genetic differences in

whole brain tyrosine hydroxylase

activity reported here does not

preclude the possibility of significant

differences in particular brain

regions.

With respect to the ethanol induced

increase of tryptophan hydroxylase

activity, this was shown to be

accompanied by a marked increase in the

turnover rate of serotonin. If changes

of synaptic transmitter systems are the

molecular basis for the rebound

hyperexcitability resulting in

audiogenic seizure susceptibility after

prolonged neuronal depression by

alcohol, the case for the involvement

of the serotonergic system as a

component in the development of

withdrawal seizures is strengthened.

With respect to seizures induced by

alcohol administered transplacentally

and via the mother's milk, it would

appear from our results that these are

associated with the serotonergic system

and not with possible catecholamine

differences. Reserpine, which enhances

such seizures, decreasees both brain

serotonin and catecholamines,

presumably by depleting the presynaptic

storage depots. The fact that

administration of 5-HTP, the precursor

of serotonin, not only reversed the

effect of reserpine, but completely

prevented the occurrence of seizures,

further supports the idea that a

serotonergic mechanism is involved in

the ethanol induced audiogenic

seizures. This argument is

strengthened by the fact that the

administration Of PCPA (a drug that

depletes brain serotonin by inhibiting

tryptophan hydroxylase activity) also

enhanced the seizure incidence to a

level comparable to that obtained with

reserpine. By contrast, drugs acting

on the catecholaminergic system were

ineffective in modifying either the

effect of reserpine or the seizure

incidence. The administration of DOPA,

viewed here in its role as precursor of

catecholamines, did not reverse the

effect of reserpine, as did 5-HTP. The

blocking of catecholamine biosynthesis

by alpha-MT, an inhibitor of tyrosine

hydroxylase activity, similarly

produced no effects on the seizure

incidence.

While the mechanism by which the

serotonergic system acts in the

production of susceptibility to

audiogenic seizures is not identified

in these experiments, its essential

role in the seizure mechanism is

indicated by its association with

genetic variation in seizure incidence,

with ethanol withdrawal seizures, with

the effects of adrenalectomy and

corticosterone substitution on ethanol

withdrawal seizures, and by its

association with seizures induced by

the administration of ethanol during

early development.

With respect to aggressive behavior, we

have demonstrated a significant

negative correlation between tryptophan

hydroxylase activity and the aggression

scores of male mice of various

genotypes. The genetic basis for

differences in aggressive behavior is

complex and other factors, such as male

hormone have been implicated in the

expression of aggressive behavior, as

well as associated with the genetic

differences in these mouse strains

[5,6,7,8,22,26]. It has also been

shown that there are possible causal

interactions between aggressive

behavior and seizures and that they may

even have overlapping genetic

predetermination. It is possible,

therefore, that the serotonergic

mechanism is a component of both

behaviors and that those genetic

factors that are common to both exert

their effect via the serotonergic

system.

93
ACKNOWLEDGMENT

The authors gratefully acknowledge the

support of NIAAA grant AA 00297-03, PHS

grant RR-00602, a grant from The Grant

Foundation, Inc., and a grant from The

Connecticut Research Foundation.

REFERENCES

1. Bovet, D., F. Bovet-Nitti and A.

Oliviero. Genetic aspects of learning

and memory in mice. Science 163:

139-149, 1969.

2 . Catz. C. and S.J. Yaffe. Strain

and age variation in hexobarbital

response.

155: 152-156,

Pharmacol. Exp. Ther.

1967.

3. Coggeshall, R.E., C.J. D'Amato,

M.A. Brodbine and S.P. Hicks.

Developmental neuropathology of genetic

mutant mouse (paralytic). Fed. Proc.

20: 330, 1961.

4. Diez, J.A. Genetic variation and

the role of adrenal function in the

regulation of serotonin and catecholamine synthesis in developing and

adult mouse brain. Ph.D. dissertation,

Department of Biobehavioral Sciences,

The University of Connecticut, 1973.

5. Diez, J.A., P.Y. Sze and B.E.

Ginsburg. Difference in brain

tryptophen hydroxylase activity in

C57BL and DBA mouse genotypes. Behav.

Genet. 5: 94-95, 1975.

6 . Fuller, J.L. Measurement of

alcohol preference in genetic

experiments. Comp.

Psychol. 51: 85-88 1964.

Physiol.

7 . Ginsburg, B.E. and W.C. Allee.

Some effects of conditioning on social

dominance and subordination in inbred

strains of mice. Physiol. Zool. 15:

485-506, 1942.

8 . Ginsburg, B., S. Ross, M.J. Zamis

and A. Perkins. Some effects of

1(+)-glutamic acid on sound-induced

seizures in mice. J. Comp. Physiol.

Psychol. 44: 134-141, 1951.

9. Ginsburg, B.E. Genetics as a tool

in the study of behavior. Persp.

Biol. Med. 1: 397-424, 1958.

1O. Ginsburg, B.E. and D.S. Miller.

Genetic factors in audiogenic seizures.

In: Psychophysiologie, Neuropharmacologie et Biochimie de la Crise

Audiogene. Paris: Centre National de

la Recherche Scientifique, No. 112,

1963, pp. 217-225.

11. Ginsburg, B.E. Coaction of

genetical and nongenetical factors

influencing sexual behavior. In: Sex

and Behaviour, edited by F. Beach. New

York: John Wiley and Sons, 1965, pp.

53-75.

12. Ginsbrug, B.E. All mice are not

created equal -- recent findings on

genes and behavior. Soc. Serv. Rev.

40: 121-134, 1966.

13. Ginsburg, B.E. Genetic parameters

in behavior research. In: BehaviorGenetic Analysis, edited by J. Hirsch.

New York: McGraw-Hill, 1967, PP.

135-153.

14. Ginsbrug, B.E. Breeding structure

and social behavior of mammals: a

servo-mechanism for the avoidance of

panmixia. In: Genetics. Biology and

Behavior Series, edited by D. Glass.

New York: Rockefeller University Press

and Russell Sage Foundation, 1968, PP.

117-128.

15. Ginsburg, B.E. Genotypic factors

in the ontogeny of behavior. Science

and Psychoanalysis 12: 12-17, 1968.

16. Ginsburg, B.E., J.S. Cowen, S.C.

Maxson and P.Y. Sze. Neurochemical

effects of gene mutations associated

with audiogenic seizures. In:

Progress in Neuro-Genetics, edited by

A . Barbeau and J.R. Brunette.

Amsterdam: Excerpta Medica, 1969, pp.

695-701.

17. Ginsburg, B.E., J. Yanai and P.Y.

94
Sze. A developmental genetic study of

the effects of alcohol consumed by 24. Selmanoff, M.K. Genetic variation

parent mice on the behavior and and the role of testosterone in the

development of their offspring. In: development and adult expression of

Proceedings of the Fourth Annual aggression in inbred mice. Ph.D.

Alcoholism Conference of the National

Alcouhol Abuse and

dissertation, Department of

Institute on

Alcoholism, in press.

Biobehavioral Sciences, The University

of Connecticut, 1974.

18. Ichyama, A., S. Nakamura, Y.

Nichizuka and 0. Hayaishi. Tryptophan5-hydroxylase in mammalian brain. Adv.

Pharmacol. 6A: 5-17, 1968.

19. Maxson, S.C. The effect of

genotype on brain mechanisms involved

in audiogenic seizure susceptibility.

Ph.D. dissertation, Department of

Psychology, University of Chicago,

1966.

20. McClearn, G.E. and D.A. Rodgers.

Differences in alcohol preferences

among inbred strains of mice. Quart.

J. Stud. Ale. 20: 691-695, 1959.

21. McGill, T.E. Sexual behavior in

three inbred strains of mice.

Behaviour 14: 341-350, 1962.

22. Nagatsu, T., B.G. Levitt and S.

Udenfriend. Tyrosine hydroxylase: the

initial step in

biosynthesis. J. Biol. Chem. 238:

norepinephrine

2910-2917, 1964.

25. Selmanoff, M.K., J.E. Jumonville,

S.C. Maxson and B.E. Ginsburg.

Evidence for a Y chromosomal contribution to an aggressive phenotype in

inbred mice. Nature 253: 529-530,

1975.

26. Sprott, R.L. and J. Staats.

Behavioral studies using genetically

defined mice--a bibliography. Behav.

Genet. 5: 27-82, 1975.

27. Sze. P.Y. Neurochemical factors

in auditory stimulation and development

of susceptibility to audiogenic

seizures. In: Physiological Effects

of Noise, edited by B.L. Welch and A.S.

Welch. New York: Plenum Press, 1970,

pp. 259-269.

28. Sze, P.Y. and L. Neckers.

Requirement for adrenal glucocorticoid

in the ethanol-induced increase of

tryptophan hydroxylase activity in

mouse brain. Brain Research 72:

375-378, 1974.

23. Paschke, R.E., V.H. Denenberg and 29. Treimar, D.M., D.W. Fulker and S.

M.X. Zarrow. Mice reared with rats: Levine. Interaction of genotype and

An interstrain comparison of mother and environment as determinants of

"aunt" effects. Behaviour 38: 315-331, antiesteriol response to stress.

1971. Develop. Psychobiol. 3: 13l-l40, 1970.

ATUHORS

Benson E. Ginsburg, Ph.D., and Paul Y.

Sze, Ph.D.: Behavioral Genetics

Laboratory, Department of biobehavioral

Sciences, The University of

Connecticut, Storrs, Connecticut 06268

95

A MODEL FOR THE NEUROBIOLOGICAL

MECHANISMS OF ACTION INVOLVED

IN LITHIUM PROPHYLAXIS

OF BIPOLAR AFFECTIVE DISORDER

Arnold J. Mandell, Suzanne Knapp

INTRODUCTION

At a recent conference sponsored by the

Neurosciences Research Program of MIT,

Mogens Schou challenged the basic

researchers who are investigating the

neurobiological actions of lithium by

listing some requirements he felt must

be met in order for their findings to

be relevant to the drug's clinical

actions. He pointed out the necessity

of using doses that produce blood

levels of drug reasonably comparable to

clinically effective levels. He noted

the necessity of differentiating acute

and chronic effects because efficacious

treatment requires adaequate time as

well as adequate drug. His most

interesting caveat, from our point of

view, relates to the clinical work

[1,2] showing that following sufficient

treatment (sometimes many months)

lithium is equally prophylactic against

the recurrence of manic and depressive

states: Any proposed mechanism of

action involving a single transmitter

system (e.g. stabilization of membrane

reactivity [3,4]; inhibition of

catecholamine release [5]; or

alterations in serotonin biosynthesis

[6-8]) would necessarily be capable of

affecting transmitter function in both

directions, i.e. increasing and

decreasing neurotransmission. In this

paper we describe our work on the acute

and chronic effects of lithium

administration on the serotonin

biosynthetic apparatus in the brains of

rats and include a model by which our

results may be understood. In brief,

we introduce the possibility that

lithium treatment results in reduced

adaptive capacities in the serotonergic

biosynthetic system in brain that

nonetheless allow the system to

continue to function within a

restricted but normal range. We also

discuss the relationship between our

animal model and the recently reported

data about serotonergic metabolites in

the CSF of manic-depressive patients

treated with lithium which appear to

mirror our findings with regard to time

course and directions.

In our first report on the effects of

short- and long-term lithium administration on the serotonergic biosynthetic systems in rat brain [9], we

said that lithium initially stimulated

high affinity synaptosomal uptake of

radioactive tryptophan in striatum,

which apparently increased the rate at

which radioactive tryptophan was

converted to serotonin by the striate

synaptosomes. These changes were

followed by a decrease in soluble

tryptophan hydroxylase activity in the

97
midbrain, and a delayed return of the

synaptosomal conversion rate to control

levels while the high affinity uptake

of tryptophan continued at its

accelerated rate. We speculated that a

feedback-regulated decrease in intrasynaptosomal enzyme originated in the

serotonergic cell bodies of the

midbrain and was transported by

axoplasmic flow to the nerve endings of

the striate cortex. The artificial

pteridine cofactor that we used in

those experiments (2-amino-4-hydroxy-6,

7-dimethyl-5,6,7,8-tetrahydropteridine,

DMPH4) was relatively inefficient, and

we could not directly measure

solubilized tryptophan hydroxylase

activity in striate synaptosomes. Now,

with the cofactor 2-amino-4-hydroxy6-methyl-5-,6,7,8-tetrahydropteridine

(6-MPH.; [10]), the sensitivity of our

assay has increased [11] and we have

measured the effects of short- and

long-term administration of lithium

chloride on solubilized synaptosomal

enzyme from striate cortex, confirming

that lithium induces and maintains a

delayed increase in intrasynaptosomal

tryptophan hydroxylase activity,

countering the maintained stimulation

of tryptophan uptake into synaptosomes

[9,12] and returning the conversion

measure to control values.

METHODS

Materials

L-[l-14C]-Tryptophan (15 mCi/mmol) and

L-[3-l4C] -Tryptophan (50 mCi/mmol)

were purchased from New England Nuclear

Corporation, Boston, Mass. L-[2,3-3

H]-

tyrosine (12 Ci/mmol) was purchased

from Amersham-Searle, Arlington

Heights, Ill., and DMPH4 and 6-MPH4

were purhcased from Calbiochem, La

Jolla, Ca. Adult male Sprague-Dawley

rats (150 to 200 g each). obtained from

Hilltop Lab Animals, Inc., Scottsdale,

Pa., were individually caged and given

free access to food and water. At

specified times prior to sacrifice rats

were given subcutaneous injections of

various doses of lithium chloride

(Mallinckrodt Chemical Works, St.

Louis, MO.) or sodium chloride crystals

dissolved in water. Although daily

doses of 5 to 10 meq of lithium

chloride per kg are reportedly toxic to

rats weighing 300 to 400 g [13], within

the limits of our paradigm the smaller

rats behaved normally and showed no

neurological abnormalities.

Immediately after decapitation, brain

regions were dissected freehand

according to Craigie's Neuroanatomv of

the Rat [14]. The striate cortex (wet

weight, 80 mg) included most of the

caudate and putamen and some globus

pallidus, and the midbrain sample (130

m g extended rostrally up to the

mamillary bodies.

Regional subcellular fractionation

Individual regions were homogenized in

25 volumes of ice-cold 0.32 M sucrose

in a Thomas glass-Teflon homogenizer

with clearance of 0.025 cm. Because of

the limited amount of tissue from a

single animal, in some experiments like

regions were pooled from animals that

had received identical treatment.

After centrifugation at 1,000g the

pellets (nuclei and cell debris) were

discarded, and the supernates were

centrifuged at 12,000g for 20 minutes

[5]. After those supernates were

transferred to clean tubes, the pellets

were resuspended in the original

homogenization volume of 0.32 M

sucrose. Without delay, tryptophan

hydroxylase activity was measured in

the supernates and synaptosomal

conversion of tryptophan to serotonin

was measured in the pellets. In some

experiments we homogenized (with a

clearance of 0.01 cm) the midbrain and

striate tissue in 0.01 M Tris-acetate

buffer (pH 7.4) and centrifuged the

homogenates at 35,000g for 20 minutes.

Practically all the measurable enzyme

activity in both regions appeared in

the supernates. This hynotonic soluble

preparation we distinguish from both

solubilized synantosomal and isotonic

soluble preparations, which are

described below.

Our conversion measure requires maintenance of isotonicity to preserve the

integrity of the synaptosomes. To

measure the enzyme activity component

of the conversion measure we

resuspended the 12,000g pellet in

0.0001 M Tris-acetate buffer (pH 7.4) and

centrifuged it at 35,000g for 30

minutes. The resulting pellet contained

negligible enzymatic activity, and we

measured the solubilized synaptosomal

enzyme activity in the supernate in the

presence of 6-MPH4.

To measure the pharmacological effects

of lithium chloride on tryptophan

hydroxylase activity, we assayed the

98
soluble fraction from midbrain and the

solubilized synaptosomal fraction from

striate cortex, in view of observations

that soluble enzyme activity is

correlated with serotonergic cell body

regions and synaptosomal conversion

activity is correlated with

serotonergic nerve ending regions

[11,16]. We also measured synaptosomal

conversion of tryptophan to serotonin

in striate tissue homogenized in 0.32 M

sucrose and fractionated as described

above.

The assays

The assay for tryptophan hydroxylase

developed by Ichiyama et al. [17,18]

couples tryptophan hydroxylase with

aromatic amino acid decarboxylase (EC

4.1.1.26) and is possible because the

decarboxylase manifests distinctly

different affinities for tryptophan (Km

= 1 mM) and 5-hydroxytryptophan (Km =

20 μM). L-[1-14C]-tryptophan was

dissolved in 0.1 M Tris-acetate buffer

(pH 7.4], and impurities were removed

by lyophilization. Our optimal

incubation mixture contained 40 μmol

Tris-acetate buffer (pH 7.4), 400 nmol

6-MPH4, 700 nmol dithiothreitol, 5 to

10 units of aromatic L-amino acid

decarboxylase from rat kidney, 100 to

200 μ1 enzyme preparation (0.3 to 0.6

mg protein, and 4 to 6 nmol L-[1-14C]-

tryptophan (15 mCi/mmol) in a final

volume of 700 μ1. Our preparation of

decarboxylase from rat kidney [9] was a

modification of the method of

Christenson et al. [19]. In this assay

the highest usable substrate

concentration is 10 μM, and in the

presence of 6-MPH4 rather than DMPH4

measurable enzyme activity increased

more than eight fold; the enzyme's

affinity for cofactor doubled; and its

affinity for substrate more than

tripled [10,ll].

In the assay for conversion of

tryptophan to serotonin the optimal

incubation mixture contained 40 umol

Tris-acetate buffer (pH 8.1), 100 to

200 μ1 enzyme preparation (0.1 to 0.3

mg protein), and 4 to nmol

L-[l-14C]-tryptophan (15 mCi/mmol) in a

final volume of 700 μl.

For either assay, blanks contained

boiled enzyme or the appropriate

buffer. Before incubation the mixtures

were sealed in 15-ml tubes with rubber

caps from which were suspended plastic

wells (Kontes Glass Company, Vineland,

N.J.) containing 100 μl of NCS

(Nuclear-Chicago Corporation, Des

Plaines, Ill.) for the collection of

14CO2. All samples were incubated with

shaking for 45 minutes at 37° C, and the

reactions were stopped by the injection

of 500 μl of 2 N perchloric acid

through the caps with an automatic

syringe. The 14CO2 accumulated in the

NCS over three more hours of incubation

at 37° C in the shaker. The wells were

then removed and placed directly into

counting vials containing 10 ml of a

mixture of toluene phosphor (80 ml

toluene containing 3.4 g PPO and 0.41 g

POPOP) and absolute ethanol in a ratio

of 4 to 1. Radioactivity was counted

in a Beckman LS-250 liquid

scintillation spectrophotometer with

external standard quench correction.

The assays for both tryptophan

hydroxylase activity and synaptosomal

conversion were linear with time for as

long as 45 minutes and with protein

over the range of protein concentration

examined. Products of the conversion

assay were analyzed by TLC [ll].

Synaptosomal uptake of tryptophan

Aliquots of

samples to

taken served

studies of

chloride on

the pellets from which

measure conversion were

as the tissue source for

the effect of lithium

substrate uptake. The

incubation medium was Krebs-Ringer

phosphate buffer containing 100 mM

NaCl, 5 mM KCl, 2 mM KH2PO4 , 2 mM

MgSO4 7H20, 3.1 mM NaHC03, 1.5 mM NaPO4

buffer (0.1 M, pH 7), 5.5 mM sodium

fumarate, 5 mM sodium pyruvate, 5 mM

sodium glutamate, and 12 mM D-glucose

[20]. In total volumes of 600 to 700

μ1 there were 0.2 to 0.3 mg protein,

and substrate concentrations were

varied from 10 to 53 μM, within the

range of the high affinity uptake

system for tryptophan [21]. Uptake was

stopped by dilution with 0.32 M

sucrose, and the synaptosomes were

collected on Millipore filters (25 mm

diameter, 0.65 μ pores) with a

Millipore multiple-sampling manifold.

Each filter was washed immediately with

2 ml of 0.32 M sucrose, and then

radioactivity was counted in 10 ml of

toluene phosphor/ethanol in a Beckman

LS-200 liquid scintillation counting

system. The uptake of L-[3-14C]-

tryptophan (50 mCi/mmol) was linear

with time for 4 minutes at 37° C and

with protein concentration from 0.06 to

0.4 mg.

99
Synaptosomal uptake of tyrosine

For comparison we measured the effects

of lithium chloride on the uptake of

radioactive tyrosine in tissue from the

same source. The incubation constituents were the same except for

substrate, which varied in

concentration from 8 to 42 μM.

Incubations proceeded and radioactivity

was measured as above, except that the

counting solution was 10 ml Aquasol

(New England Nuclear, Boston, Mass.).

Synaptosomal uptake of L-[3H]-tyrosine

was linear for 4 minutes at 37° C.

RESULTS AND DISCUSSION

Stimulation of high affinity tryptophan uptake

Administration of lithium chloride (5

to 10 meq/kg/day) stimulated the uptake

of radioactive tryptophan by striate

synaptosomes, the latency of the

stimulation varying with the dose.

With 10 meq the Vmax of the uptake

reaction was increased within three

days (Figure 1). With 5 meq there were

FIGURE 1

Figure 1: Velocity is plotted as nmoles

of L-(3-1 4C)-tryptophan retained per mg

per 4 minutes. Substrate concentrations ranged from 15 to 53 μM. Vmaz

and Km ± SE are estimated according to

the method of Wilkinson (44).

no changes by the third day, but at 21

augmentation of V max reflects

days the Vmax was augmented. Although

conditions consistent with an increase

in the uptake of radioactive

tryptophan, the Km for the uptake was

either unchanged or increased only

slightly from control values.

Our index of uptake is the

radioactivity retained after incubation

of the synaptosomes with radioactive

substrate. However, lithium treatment

has reportedly inhibited catecholamine

release from nerve endings [5]. so, to

rule out both similar inhibition of

serotonergic release and facilitated

exchange of endogenous for exogenous

tryptophan, we preincubated synaptosomes from rats treated with lithium

chloride or sodium chloride for 5

minutes with L-[3-14C]-tryptophan (10

μM) and found that over 10 minutes the

efflux of radioactivity from the

preparations from lithium-treated rats

was essentially the same as that from

sodium-treated rats.

Stimulation of the uptake of

radioactive tryptophan by striate

synaptosomes in vitro is relatively

specific when compared to the effect on

the uptake of radioactive tyrosine, and

also relatively dependent on the level

of lithium present. When synaptosomes

were incubated with 0 to 50 mM lithium

chloride or sodium chloride, high

affinity uptake of radioactive

tryptophan was stimulated by 10 mM

lithium chloride and stimulated more by

50 mM lithium chloride. The

reaction did not change, but the Vmax

of the

rose 45 percent with 50 mM lithium

chloride.

radioactive

The Km for the uptake of

tyrosine was appreciably

higher than that for radioactive

tryptophan under control conditions at

equimolar concentrations of substrate

(50 μM versus 200 μM) but stimulation

of the uptake of radioactive tyrosine

in the presence of 50 mM lithium

chloride was only 20 percent (versus 45

percent for tryptophan). We chose

these relatively high absolute levels

of lithium for their efficiency in

vitro and did not try to reflect

possible mechanisms in vivo where dose

and time interact.

Stimulation of conversion of tryptophan to serotonin

Both time and dose are related to the

effects of lithium chloride on the

conversion of radioactive tryptophan to

100
serotonin by striate synaptosomes

(Figure 2). High doses of lithium in

in vivo resulted in 30 to 40 percent

increases in conversion as well as

uptake (Figures 1 and 2). Serum levels

FIGURE 2

Figure 2: Controls received equal doses

of NaCl. Animals were sacrificed 24

hours after the final daily subcutaneous

drug administration. Control activity:

200 pmoles per mg per 45 minutes.

* p<0.01.

[23,241, or a relatively low net

hydroxylation because of the efflux of

unconverted tryptophan.

Effects on tryptophan hydroxylase

activity in the midbrain

The cell bodies of the serotonergic

system are in the raphé nuclei of the

brain stem (B7, B8, and B9; [25]), a

region dominated by soluble tryptophan

hydroxylase [12,16,24]. From

homogenization of midbrain tissue in

0.32 M sucrose and centrifugation at

12,000g for 20 minutes, we obtain an

isotonic soluble preparation, which is

distinct from the hypotonic soluble and

solubilized synaptosomal preparations

characterized above. With 10 meq/kg of

lithium chloride a progressive

decrement in midbrain isotonic soluble

tryptophan hydroxylase activity began

on the first day (Figure 3). A maximum

FIGURE 3

of lithium in the rats at the time of

sacrifice ranged from 0.8 to 1.0 meq

per liter [22]. In vitro much higher

levels of lithium were required to

produce comparable increments in either

uptake or conversion. At substrate

concentrations of about 10 μM and

identical doses of lithium chloride,

the specific activity of conversion was

between 5 and 7 pmol product per mg

protein per minute, while that of high

affinity uptake was between 350 and 450

pmol. In spite of the large disparity

in the absolute values, we have

elsewhere demonstrated similar

proportional changes in conversion and

uptake with other treatments [9,11,21].

Such a phenomenon might be explainable

by the presence of a preferential

pathway for uptake and synthesis, a

high affinity uptake for substrate in

cells other than serotonergic neurons

Figure 3: Controls received equal doses

of NaCl. Animals were sacrificed 24

hours after the final daily subcutaneous

drug administration. Control activity:

500 ± 15 pmoles per mg per 45 minutes.

* p<0.002; ** p<0.01.

decrement of about 30 percent was

reached on the third day, and was still

present on the fifth and seventh days.

With a lower dose (5 meq/kg) ten days

of drug administration were required

for a significant decrement to appear,

although the maximum decrement with the

lower dose also reached 30 percent. As

with uptake and conversion activities

(Figures 1 and 2), interactions between

dose and time are implicit in the

101
effects of lithium treatment on soluble

tryptophan hydroxylase activity (Figure

3), although, in vitro, concentrations

of drug from 1 to 100 mM had no effect

on the activity of midbrain isotonic

soluble enzyme activity.

The decrease in enzyme activity

observed after treatment with lithium

chloride could be due to a decrement in

the number of available enzyme sites or

even molecules because the kinetics of

the reduced enzyme activity with

respect to cofactor showed a decrease

ln Vmax rather than a change in Km, and

the decrement appears to move by fast

axonal flow from the raphé nuclei to

the striate cortex. It is not clear

whether the change induced by lithium

chloride might be secondary to

decreased synthesis or increased

turnover of tryptophan hydroxylase.

Simultaneous measurement of tryptophan hydrozylase activity and synaptosomal conversion of tryptophan to

serotonin

We monitored simultaneously the effects

of short-term daily administration of

lithium chloride (10 meq/kg) on (1)

striate synaptosomal uptake of

radioactive tryptophan, (2) conversion

of tryptophan to serotonin by striate

synaptosomes, (3) midbrain tryptophan

hydroxylase activity, and (4)

solubilized striate synaptosomal

tryptophan hydroxylase activity (Figure

4). Both the uptake and the conversion

of radioactive tryptophan to serotonin

began to rise within the first day of

treatment. Soluble enzyme activity

from the serotonergic cell bodies in

the midbrain and from the lysed striate

synaptosomes showed no change the first

day. On the second day the increases

in uptake and conversion did not

continue, but the enzyme activity from

the cell bodies and nerve endings began

to decrease. On the third day the

uptake of substrate and its conversion

to transmitter increased further.

After 21 daily treatments with lithium

chloride

cell body

(5 meq/kg) the decrement in

and nerve ending enzyme

activity was still present, as was the

increase in the uptake of radioactive

tryptophan; conversion of radioactive

tryptophan to serotonin had returned to

control levels (Figure 5). As we

speculated before [9,12], the values

for conversion after 21 days of

treatment seem to represent the net

effect of the lithium-stimulated

increase in uptake (and conversion) and

a decrease in intraneuronal ratelimiting biosynthetic enzyme activity.

FIGURE 4

Figure 4: Simultaneous effects of 3

daily lithium injections (10 meq/kg)

on four measures related to serotonin

synthesis. Control velocities:

o-o high affinity uptake of (

1 4 C)-

tryptophan (10μM),-1220 ± 50.pmol/mg/

4 min; striate synaptosomal conversion activity (8μM substrate),

250 ± 12 pmol/mg/45 min; midbrain

soluble enayme activity (8μM substrate),

525 ± 20 pmol/mg/45 min; striate

sotubilized enzyme activity (8μM substrate), 150 ± 5 pmol/mg/45 min. Each

day uptake and conversion activities

were increased (p<0.05). At 2 and 3

days the enayme activities were decreased (p<0.05).

102
FIGURE 5

THE EFFECT OF LONG TERM (3WKS) LITHIUM TREATMENT

ON RAT BRAIN SEROTONERGIC SYSTEMS

Figure 5: Simultaneous effects of 21

daily lithium injections (5 meq/kg)

on four measures related to serotonin

synthesis. Control velocities:

midbrain soluble enzyme activity,

475 ± 20 pmol/mg/45 min (* p<0.05;

striate synaptosomal conversion activity, 200 ± 10 pmol/mg/45 min; striate

Stimulation of conversion of radioactive tryptophan to serotonin after

tryptophan loads

Perez-Cruet el al.

that treating

[26] have shown

rats with lithium

carbonate increases serum and brain

levels of tryptophan. To rule out

increased extraneuronal tryptophan as

an intervening variable in our

experiments, we dissolved progressively

larger amounts of L-tryptophan

hydrochloride in saline and

administered it to rats subcutaneously,

and then determined the effects on

conversion activity. Within the range

of tryptophan loads tested the

dose-response relationship was linear.

The addition of cold tryptophan would,

if anything, dilute the radioactivity

of the product, so our results may even

underestimate the increase in

conversion. To determine whether a

load-induced increase in uptake of

radioactive substrate preceded the

load-induced increase in conversion, we

preincubated striate synaptosomes with

100 μM L-tryptophan and examined the

solubilized enzyme activity, 160 ± 5

pmol/mg/45 min (** p<0.001). High

affinity uptake velocity (12 to 53 μM

substrate) is plotted as nmoles of

L-(3-1 4C)-tryptophan retained per mg

per 4 minutes. Vmax and Km ± S.E.

are estimated according to the method

of Wilkinson (44).

uptake kinetics. The Vmax increased,

but the Km for tryptophan did not,

which suggests that the tryptophan load

increased the uptake of radioactive

tryptophan by increasing the number of

uptake sites. We cannot tell whether

this substrate-induced increase in

uptake (and conversion) accounts for

the initial effects of lithium because

the concentration of tryptophan in vivo

is obviously critical, and we do not

know whether tryptophan loads can bring

substrate levels within the appropriate

range at the relevant sites. Attempts

to determine regional and subcellular

levels of tryptophan after the

administration of exogenous tryptophan

[26-28] are not conclusive.

SIGNIFICANCE

One of the intriguing aspects of

psychotropic drug action is latency to

action. In the case of lithium,

treatment for mania may require five to

10 days to reach efficacy, and

prophylaxis for both mania and

depression may not achieve full

103
effectiveness for months or longer than

a year [29-32]. The most conservative

explanation of such temporal parameters

is that the doses used to initiate

treatment do not readily achieve

effective levels in the blood or brain.

Schou [33-35] and others have reported

that in animals brain levels of lithium

do not achieve a steady state for the

first five to seven days of daily drug

administration. However, Schou

(personal communication) has also found

that large initial doses of lithium

reduce the latency to action very

little while magnifying the clinical

side effects greatly. Those studies

and our own clinical experience lead us

to believe that plasma or brain blood

levels, while perhaps accounting for

some latency to effectiveness, do not

account for it all. When we consider

the sequence of events observed in the

laboratory over a human time base, the

possibility arises that some aspects of

clinical efficacy may be due to the

secondary or tertiary adaptive

mechanisms set in motion by the primary

drug effects [36]. Generally,

conventional "tolerance" fails to

develop to psychotherapeutic drugs,

although the many so-called side

effects do diminish or disappear with

chronic administration. Nonetheless,

the prophylactic and antimanic effects

of lithium (as well as the

antipsychotic effects of the

neuroleptics and the antidepressant

effects of the thymoleptics) do not

usually require progressively greater

doses; the same dose may actually

become more efficacious over time.

Moreover, these treatments tend to

retain their clinical efficacy over

months, years, and even decades. It

might be said that tolerance mechanisms

induced by such drugs as morphine,

alcohol, or amphetamine simply overcome

their primary actions very quickly

[21,37,38]. Furthermore, we suggest

that treatment with lithium (as well as

neuroleptics and

could be

thymoleptics [39])

de facto, treatment by the

induction of tolerance mechanisms.

The almost perfect symmetry of lithium

prophylaxis against both manic and

depressive components of affective

disorder must be the keystone in any

construction of a neurobiological model

of the drug's action. For example, to

explain the antimanic effects of

lithium by saying that it inhibits the

release of norepinephrine, even if that

were proved to be true, would be

inadequate for two reasons. Our recent

study [40] has shown that tolerance

develops within eight days to

lithium-induced suppression of

amphetamine-induced hyperactivity, and

that it is associated with increases in

tyrosine hydroxylase activity in the

nigro-striatal pathway. Secondly, what

would be said about its antidepressant

effects? The sequence of events

induced in central serotonergic

biosynthetic nmechanisms by the chronic

administration of lithium hints at

adaptive systems capable of increasing

serotonin svnthesis as well as

decreasing it: The relationships among

serotonin synthesis, levels, turnover,

and metabolism and behavioral states in

animals and man are extremely complex,

and it is possible that serotonin

serves a regulatory role rather than a

direct role with regard to behavior.

For example, tryptophan has been

administered in conjunction with both

families of antidepressant medication,

thymoleptics and monoamine oxidase

inhibitors, with varying degrees of

success and failure. There have been

suggestions that 5-hydroxytryptophan

may be antimanic, and brain stem

5-hydroxyindoleacetic acid has been

found to be low in patients who

committed suicide, yet Coppen and his

coworkers [41] have found low

5-hydroxyindoleacetic acid in the CSF

of patients with affective disorder

when they were sick or well. Although

no tidy hypothesis relating increases

or decreases in serotonin synthesis to

one or another pole of the affective

continuum is possible at present, our

findings do suggest another

possibility.

The control levels of nerve ending

serotonin synthesizing capacity after

chronic lithium treatment represent the

net effect of two maintained changes

that were induced by the drug, i.e. the

increase in the high affinity uptake of

tryptophan and the decrease in

intrasynaptosomal rate-limiting enzyme

activity. Although each of these

changes can be induced separately by

other agents --tryptophan loads activate

substrate transport on a dose-response

basis, and amphetamine decreases

intraneuronal tryptophan hydroxylase--

neither the increased uptake nor the

decreased intrasynaptosomal enzyme

activity can be extended after lithium

treatment. It is as though the uptake

were elevated and stabilized at a

ceiling, the intraneuronal enzyme

activity were reduced and stabilized at

the floor, and the bi-directional

104
adaptive capacity were "used up,"

having returned the overall measure Of

nerve ending biosynthetic capacity, the

conversion rate, to baseline.

Perhaps lithium's efficacy in

preventing or dampening both manic and

depressive episodes without changing

many control-level or normal behaviors

in the intersymptomatic intervals is

related to the way lithium influences

the serotonergic system. It was

gratifying to learn of an almost point

for point correlation between CSF

metabolites in man and this sequence of

neurobiological events in rats.

Goodwin et al. [42] report a biphasic

response in 5-hydroxyindoleacetic acid

turnover in the CSF of depressive

patients after five and 21 days of

clinical doses of lithium. After

probenecid blockade of metabolite

egress from the CSF, 5-hydroxyindoleacetic acid was elevated after five

days of lithium carbonate

(corresponding to the increase in

tryptophan uptake and conversion to

serotonin by synaptosomes) and then

back to or below control level after 21

days (corresponding to the state of

affairs we illustrate in Figure 5).

An implication of this model for the

action lithium in treatment is the

possibility that daily or regular

tryptophan loads might be prophylactic

in bipolar affective disorder. The

ACKNOWLEDGMENT

This work is supported by United States

Public Health Service Grants MH-14360,

DA-00046, and DA-00265.

REFERENCES

1. Angst, J., P. Weis, P. Grof, P.

Baastrup and M. Schou. Lithium

prophylaxis in recurrent affective

disorders. Br. J. Psychiat. 116:

604-614, 1970.

2. Coppen, A., R. Noguera, J. Bailey

et al. Prophylactic lithium .

affective disorders. Lancet 1971-II:

275-279, 1971.

3. Skou, J.C., Enzymatic aspects of

active linked transport of Na+

and K

+

through the cell membrane. Prog.

Biophys. Mol. Biol. 14: 133-166, 1964.

increased circulating tryptophan might

increase uptake of substrate into the

nerve endings by mass action rather

than stimulation of the uptake

mechanism, as with lithium, leading to

increased transmitter synthesis and a

reflexive decrease in tryptophan

hydroxylase, which would return nerve

ending conversion of tryptophan to

serotonin to normal. Thus uptake and

conversion might be pushed to their

ceiling and tryptophan hydroxylase

activity pushed to the floor, narrowing

the range of potential adaptive changes

without changing the control level of

function. Recently it has been

reported that daily tryptophan loads

can substitute for lithium as effective

prophylaxis in affective disease in

patients who show marked intolerance to

lithium (N. Kline, personal communication). Murphy et al. [43] have

recently reported both antidepressant

and antimanic effects from tryptophan

given to patients with bipolar

disorder.

Much more work needs to be done at

basic and clinical levels before it

will be clear whether the clinical

action of lithium can, in fact, be

explained by this kind of

"cross-tolerance" for psychopathological amounts of neurotransmitter. For

now, it may serve heuristically to help

us explore the complex adaptive

capacity of the serotonergic system in

brain.

4. Whittam, R. and M.E. Ager.

Vectorial aspects of adenosinetriphosphatase activity in erythrocyte

membranes. Biochem. J. 93: 337-348,

1964.

5 . Katz, R.I., T.N. Chase and I.J.

Kopin. Evoked release of norepinephrine and serotonin from brain

slices: inhibition by lithium. Science

162: 466-467, 1968.

6 . Corrodi, H., K. Fuxe and M. Schou.

The effect of prolonged lithium

administration on cerebral monoamine

105
neurons in the rat.

643-651, 1969.

Life Sci. 81:

7. Sheard, M.H. and G.K. Aghajanian.

Neuronally activated metabolism of

brain serotonin: effect of lithium.

Life Sci. 911: 285-290, 1970.

8.

R.J.

Ho, A.K.S., H.H. Loh, F. Craves,

Hitzemann and S. Gershon. The

effect of prolonged lithium treatment

on the synthesis rate and turnover of

monoamines in brain regions of rats.

Eur. J. Pharmacol. 10: 72-78, 1970.

9 . Knapp, S. and A.J. Mandell.

Short-and long-term lithium

administration: effects on the brain's

serotonergic systems. Science

645-647, 1973.

180:

10. Friedman, P.A., A.H. Kappelman and

S. Kaufman. Partial purification and

characterization of tryptophan

hydroxylase. Biol. Chem. 247:

4165-4173, 1972.

11. Knapp, S., A.J. Mandell and M.A.

Geyer. Effects of

regional

amohetamines on

tryptophan hydroxylase

activity and synaptosomal conversion of

tryptophan to 5-hydroxytryptamine in

rat brain. J. Pharmacol. Exp. Ther.

189: 676-689, 1974.

12. Knapp, S. and A.J. Mandell.

Effects of lithium chloride

parameters of biosynthetic capacity for

5-hydroxytryptamine in rat brain. J.

Pharmacol. Exp. Ther. 193: 812-823,

1975.

13. Schildkraut, J.J. Pharmacology--

the effects of lithium on biogenic

amines. In: Lithium: Its Role in

Psvchiatric Research and Treatment

edited by S. Gershon and B. Shopsin,

Plenum Press, New York, 1973.

14. Zeman, W. and J.R.M. Innes.

Craigie's Neuroanatomy of the Rat,

Academic Press, New York, 1963.

15. Gray, E.G. and V.P. Whittaker.

The isolation of nerve endings from

brain: an electron microscopic study

of cell fragments derived by

homogenization and centrifugation. J.

Anat. 96: 71-88, 1962.

16. Knapp, S. and A.J. Mandell.

Parachlorophenylalanine: its three

phase sequence of interactions with the

two forms of brain tryptophan

hydroxylase. Life Sci. 11I: 761-771,

1972.

17. Ichiyama, A., S. Nakamura, Y.

Nishizuka and O. Hayaishi. Tryptophan

hydroxylase in mammalian brain. Adv.

Pharmacol. 6A: 5-17, 1968.

18. Ichiyama, A., S. Nakamura, Y.

Nishizuka and O. Hayaishi. Enzymic

studies on the biosynthesis of

serotonin in mammalian brain. J. Biol.

Chem. 245: 1699-1709, 1970.

19. Christenson, J.G., W. Dairman and

S. Udenfriend. Preparation and

properties of a homogeneous aromatic

L-amino acid decarboxvlase from hog

kidney. Arch. Biochem. Biophys. 141:

356-367, 1970.

20. Dawson, R.M.C., D.C. Elliott, W.H.

Elliott and K.M. Jones, editors. Data

for Biochemical Research, Oxford

University Press, Cambridge, 1969.

21. Knapp, S. and A.J. Mandell.

Narcotic drugs: effects on serotonin

biosynthetic systems of the brain.

Science 177: 1209-1211, 1972.

22. Zettner, A., K. Rafferty and H.

Jarecki. Determination of lithium in

serum and urine by atomic absorption

spectroscopy. Atomic Absorption

Newsletter 7: 32-34, 1968.

23. Kuhar, M.J., R.H. Roth and G.K.

Aghajanian. Synaptosomes from

forebrains of rats with midbrain raphe

lesions: selective reduction of

serotonin uptake. J. Pharmacol. Exp.

Ther. 181: 36-45, 1972.

24. Mandell, A.J., S. Knapp and L.L.

Hsu. Some factors in the regulation of

central serotonergic synapses. Life

Sci. 14: 1-17, 1974.

25. Dahlstrom, A. and K. Fuxe.

Evidence for the existence of

monoamine-containing neurons in the

central nervous system. Acta Physiol.

Scand. 62: suppl. 232: 1-55, 1964.

26.

P. Tagliamonte and G.L. Gessa.

Perez-Cruet, J., A. Tagliamonte,

Stimulation of serotonin synthesis by

lithium. Pharmacol. Exp. Ther.

178: 325-330, 1971.

27. Fernstrom, J.D. and R.J. Wurtman.

Brain serotonin content: physiological

dependence on plasma tryptophan levels.

Science 149: 149-152, 1971.

28. Grahame-Smith, D.G. Studies in

vivo on the relationship between brain

tryptophan, brain 5-HT synthesis, and

106
hyperactivity in rats treated with a

monoamine exidase inhibitor and

L-tryptophan. J. Neurochem. 18:

1053-1066, 1971.

29. Cade, J.F.J. Lithium salts in the

treatment of psychotic excitement.

Med. J. Australia 36: 349-352, 1949.

30. Schou, M., N. Juel-Nielsen, E.

Stromgen and H. Volby. The treatment

of manic psychoses by the

administration of lithium salts. J.

Neurol. Psychiat. 17: 250-260, 1954.

31. Baastrup, P.C. and M. Schou.

Lithium as a prophylatic agent: its

effect against recurrent depressions

and manic-depressive psychosis. Arch.

Gen. Psychiat. 16: 162-172, 1967.

32. Freyhan, F.A. Lithium treatment:

prophylactic or compensatory? Am. J.

Psychiat. 128: 122, 1971.

33. Schou, M. Biology and pharmacology of the lithium ion. Pharmacol.

Rev. 9: 17-58, 1957.

34. Schou, M. Possible mechanisms of

action of lithium salts: approaches and

perspectives. Biochem. Soc. Trans. 1:

81-87, 1973.

35. Schou, M. Prophylactic lithium

maintenance treatment in recurrent

endogenous affective disorders. In:

Lithium: Its Role in Psychiatric

Research and Treatment, edited by S.

Gershon and B. Shopsin, Plenum Press,

New York, 1973.

36. Mandell, A.J. Neurobiological

mechanisms of presynaptic metabolic

adaption and their organization:

implications for a pathophysiology of

the affective disorders. In:

Neurobiological Mechanisms of Adaption

and Behavior, edited by A.J. Mandell,

Raven Press, New York 1975.

AUTHORS

37. Mandell, A.J., D. Segal, R.

Kuczenski and S. Knapp. Some

macromolecular mechanisms in CNS

neurotransmitter pharmacology and their

psychobiological organization. In:

Chemistrv of Mood, Motivation, and

Memory, edited by J. McGaugh, Plenum

Press, New York, 1972.

38. Rogawski, M.A., S. Knapp and A.J.

Mandell. Effects of ethanol on

tryptophan hydroxylase activity from

striate synaptosomes. Biochem.

Pharmacol. 23: 1955-1962, 1974.

39. Segal, D.S., R. Kuczenski and A.J.

Mandell. Theoretical implications of

drug-induced adaptive regulation for a

biogenic amine hypothesis of affective

disorder. Biol. Psychiat. 9: 147-159,

1974.

40. Segal, D.S., M. Callaghan and A.J.

Mandell. Alterations in behavior and

catecholamine synthesis induced by

lithium. Nature 254: 58-59, 1975.

41. Coppen, A.J. The chemical

pathology of the affective disorders.

Sci. Basis Med. Ann. Rev. 179-198, 1970.

42. Goodwin, F.K., R.L. Sack and R.M.

Post. Clinical evidence for

neurotransmitter adaption in response

to antidepressant therapy. In:

Neurobiological Mechanisms of Adaption

and Behavior, edited by A.J. Mandell,

Raven Press, New York, 1975.

43. Murphy, D.L., M. Baker, F.K.

Goodwin. H. Miller. J. Kotin and W.E.

Bunney, Jr. L-Tryptophan in affective

disorders: indoleamine changes and

differential clinical effects.

Psychopharmacologia 34: 11-20, 1974.

44. Wilkinson, G.N. Statistical

estimations in enzyme kinetics.

Biochem. J. 80:324-332, 1961.

Arnold J. Mandell, Ph.D., and Suzanne

Knapp, Ph.D.: Department of

Psychiatry, School of Medicine,

University of California, San Diego, La

Jolla, California, 92037.

107

EFFECTS OF DISULFIRAM ON THE

AMPHETAMINE-INDUCED BEHAVIORAL SYNDROME

IN THE CAT AS A MODEL OF PSYCHOSIS

A. Sudilovsky

INTRODUCTION

The use of psychoactive drugs as experimental tools holds a fascination

for those interested in psychiatric

research because it offers possibilities for a better understanding of the

correlations between the psychopathological manifestations of functional

psychoses and their underlying mechanisms. Consequently, an effort has

been made in the past decades to alter

pharmacologically the behavior of animals or humans in the direction of

psychosis. Focus on the mechanisms of

action of psychotogenic and psychotropic compounds has produced convergent lines of evidence that the biogenic amines of the brain are involved

in the modulation of disturbed

behavior. Furthermore, in recent years

a "dopamine hypothesis" of schizophrenia, i.e. central dopaminergic

overactivity, has been advanced; it

seems useful, as a frame of reference,

therefore, to provide here its general

outline.

The administration to experimental

animals of amphetamine*, a sympathomimetic amine that potentiates the

*Throughout this paper the expressions ‘amphetamine’ and ‘methamphetamine’ are used interchangeably.

effect of catecholamines, results in

the stimulation of certain psychomotor

components of behavior and the concurrent reduction or suppression of

others (70,72,78). Typically, a decrease in locomotion and the development of a hyperaroused hyperkinetic

state, including species-specific

stereotyped compulsive motor activity,

and the abolition of ordinary goalseeking motivated behavior are observed

after single or repeated administration

of moderate doses of the drug (73). The

stereotyped activity is a conspicuous

and reproducible phenomenon apparently

obtainable in all mammals and is

currently used 'in the preclinical

screening of new compounds for potential antipsychotic properties (50).

Blockade of stereotypy has highly

predictive value, and has been helpful

to detect many of the effective antischizophrenic drugs now available (22).

There exists extensive evidence from

pharmacologic, neuroanatomic ablation,

and micro-injection studies that

dopamine plays an important role in the

mediation of amphetamine-induced

stereotypy (36,72,73). Consistent with

this evidence is the 'finding of a lack

of stereospecificity for the d and 1

isomers of amphetamine, both in

inhibiting catecholamine uptake in

109
synaptosomes of dopamine-containing

areas of the rat brain (striatum) and

in inducing stereotyped behavior, as

contrasted with a tenfold stereospecificity for the d isomer in inhibiting catecholamine uptake in synaptosomes of norepinephrine-containing

areas and in enhancing locomotor

activity (89).

In human subjects, amphetamine elicits

stereotyped behavior comparable to that

seen in animals (29,73,76), but may

also lead to the occurrence of

psychotic episodes closely resembling

paranoid schizophrenia (6,18,41)

irrespective of the pre-existence of

the disease or of schizoid tendencies

(6,7,10). The results of experiments

in animals (28,72,73) as well as of

those in humans (7,l0) suggest the

involvement of dopamine in the elicitation of this stimulant psychosis. For

example, comparison of the doseresponse relationships of d and 1

amphetamine in producing psychosis has

shown their relative potencies to vary

from 1:1 to 2:1, making it im- probable

that norepinephrine events play a

crucial role and suggesting that

dopaminergic or other, as yet unknown,

non-stereospecific mechanisms are

critical. These results, as well as

the frequent misdiagnosis of

amphetamine psychosis as paranoid

schizophrenia (13,18,52), the specific

activation of schizophrenic symptoms by

small doses of the drug (20,48), and

the effectiveness of antischizophrenic

agents in the treatment of amphetamine intoxication (30) and

psychosis (8), argue strongly for a

relationship between the mechanisms

underlying amphetamine stereotypy and

psychosis and those underlying certain

forms of schizophrenia.

On the other hand, there is also

evidence that overactivity in the

dopaminergic system or some of its

pathways might contribute to the

pathogensis of schizophrenia: the high

degree of correlation between the

therapeutic activity of neuroleptics in

man and both the antistereotypy effect

and the blocking activity of such

compounds on dopamine receptors

(45,49,75) or on the presynaptic

impulse-coupled release of dopamine

(79). Such a notion is consistent with

the lack of therapeutic activity of

agents that are structurally related to

the neuroleptics but do not block

dopamine receptors (64), the ability of

antipsychotic agents to evoke Parkinsonian side effects that are regarded

as manifestations of reduced dopaminergic activity in the nigrostriatal

system (47), the worsening of schizophrenics or the occurrence of psychotic

reactions in Parkinsonian patients

after the administration of the

dopamine precursor L-Dopa (9), the

infrequent occurrence of schizophrenia

in patients suffering from Parkinson's

disease (which is characterized by low

concentrations of dopamine in the

nigro-striato-pallidal system), and the

amelioration of psychotic symptomatology in schizophrenics affected with

encephalitis lethargica (75). Further

support for this line of reasoning lies

in the recent demonstration of dopamine

nerve terminals in the limbic cortex of

the rat (441, since a cortical dopamine

innervation in man (still unproved

could be related to certain symptoms in

schizophrenia, i.e., thought disorders,

impairment of goal-directed behavior,

disturbances of affect, and

hallucinations, the implied cortical

dysfunction occurring in addition to

the involvement of subcortical areas.

A number of excellent reviews of work

on catecholamine neuronal pathways, as

well as on other significant findings

that either support or weaken the

dopamine hypothesis, can be found in

the literature (20,25,29,34,56,62,64,

75,82). The indirect and inferential

nature of the evidence, which does not

preclude the participation of other

neurotransmitter systems, has permitted

invoking a multiplicity of mechanisms

as the basic alteration. These postulated mechanisms include exaggerated

release of the transmitter,

supersensitivity of the receptor,

defective feedback control of dopamine

pathways, imbalance of the interaction

with other neurotransmitter systems,

i.e. cholinergic, noradrenergic,

serotonergic, or GABAergic, central

overload of dopamine from decreased

conversion to norepinephrine, or

formation of abnormal psychotogenic

metabolites. In addition, at the

anatomical level, hyperinnervation of

dopamine areas or plastic compensatory

growth of dopamine terminals into areas

previously devoid of them, as a result

of a primary lesion in other pathways,

should be considered.

In light of the above, the relevance of

the amphetamine intoxication process

for psychiatric research becomes

apparent; indeed, of all drugs amenable

to use in the production of a model

110
psychosis, amphetamine is now recognized as the most valuable (22,80).

During the past several years, we have

stressed the need for employing direct

observational techniques in the

assessment of animal behavior (28,84)

This approach permits the evaluation of

the entire behavioral spectrum, including qualitative as well as quantitative aspects, and might lend itself

to the establishment of correlates of

human symptomatology, thereby adding a

psychopathologically meaningful

dimension to the paradigm.

We have previously reported that, from

a phenomenological standpoint, the

behavioral manifestations of cats

chronically intoxicated with amphetamine parallel the evolution of the

paranoid psychosis induced by the drug

in humans (29). However, a group of

manifestations in the cat, such as

frozen postures, disjunctive behaviors

and postures, cataleptoid phenomena,

obstinate progression, and loss of

righting reflex, did not appear to be

consistent with the phenomenology of

the paranoid psychosis.

Since treatment of schizophrenic

patients with disulfiram (tetraethylthiuram disulfide), an inhibitor

of norepinephrine synthesis that acts

at the level of the enzyme dopamine

beta-hydroxylase thereby leading to

increased dopamine concentrations, had

been found to profoundly exaggerate

psychotic symptomatology (43), the

current experiments were undertaken to

examine the amphetamine behavioral

syndrome in the cat as it is modified

by pretreatment with disulfiram. It

was hypothesized that the end-stage

behaviors characteristic of chronic

amphetamine intoxication would develop early in the animals pretreated

with disulfiram, and that a more

critical differentiation of psychomotor

alterations relevant to the model

psychosis could be obtained through

possible activation of given behaviors.

If a differential enhancement of

certain aspects of behavior occurred,

it might then be assumed that such

aspects bear a relationship to a

stimulated dopamine system in the

relative absence of norepinephrine

action, whereas those not affected or

affected only later are based on other

mechanisms.

METHODS

Twenty-six female cats, 2.5 to 3.5 kg,

were used. All animals were housed in

individual cages and fed ad libitum

throughout the experiment. Under

sodium pentobarbital anesthesia (36

mg/kg i.p.), stainless steel electrodes

were implanted stereotaxically

implanted in various nuclei of the

forebrain and midbrain to permit the

recording of electrical activity. The

purposes and results of these

recordings have been reported elsewhere

(23,26,27,85,86). At least two weeks

were allowed for recovery after

surgery. Each cat was then assigned to

one of three groups for treatment with:

(A) oral disulfiram (N = 10); (B)

intraperitoneal amphetamine (N = 6); or

(C) a combination of disulfiram and

amphetamine (N = 10) .

The subjects in Group B received daily

intraperitoneal injections of normal

saline (1 ml) for 5 days before the

administration of amphetamine was

begun. Data collected on the fifth day

were used as a control for changes

induced by handling during injection Or

by the injection itself. Treatment in

this group consisted of two injections

of methamphetamine hydrochloride daily,

one at 9 AM and one at 3 PM. The

dosage was increased from 15 mg/kg per

day (7.5 mg/kg per injection) on the

first day to 28 mg/kg on the eleventh

day. No drug was administered on Days

6 and 7. All behavioral data for Group

B animals were collected after the

first injection on any given day.

The animals in Groups A and C were

given disulfiram (in tablet form*) at 9

AM daily for 12 days. Daily dosage of

disulfiram for each animal ranged from

150 to 180 mg/kg, depending on

variations in its weight. In Group C,

methamphetamine hydrochloride was also

injected intraperitoneally each day at

3 PM, beginning on the second day of

disulfiram treatment. In previous

dosage trials, deaths caused by

methamphetamine had been found to

increase in frequency in animals

pre-treated with disulfiram, particularly when the dosage range of

*The oral route of administration was used because repeated subcutaneous or intraperitoneal

injections of disulfiram have been found to

cause local irritative effects that distort

quantitative behavioral data (68).

111
amphetamine reached 15 to 30 mg/kg.*

Lewander (58) has shown that there is

no development of tolerance for the

motor effects of amphetamine. Since

our primary interest was to study such

effects in Group C, the dosage of

amphetamine in these animals was

maintained well below the lethal level.

Dosages of amphetamine used were 7.5

mg/kg on the first day and 10 mg/kg on

subsequent days. On what would have

been Days 6 and 7 of amphetamine

administration, only disulfiram was

given to cats in Group C, the

amphetamine cycle being interrupted for

two days as in Group B. The six-hour

interval between the administration of

disulfiram and that of amphetamine

allowed time for the norepinephrinedepleting effects of disulfiram to

develop.

Twenty minutes before the beginning of

an experimental session, each animal

was placed in a metal cage commercially

available measuring 17.5 x 24 x 19.5

inches, to which a wooden floor and a

Plexiglas door and walls had been

attached. Data were gathered through

direct observation of the cat's

behavior and by 3-minute videotape

recordings that were made at the

following times: (a) on the two days

prior to the administration of any

drug; (b) six hours after the first

dose of disulfiram; and (c) just before

the daily injection of methamphetamine

and 10,20,30 and 90 minutes after its

administration. Thus, each subject

served as its own control.

The animal's behavior was rated later

after careful scrutiny of the videotape

recordings, each 3-min recorded period

being divided into 18 observational

intervals, 10 seconds each. Scoring

for each interval was made on a

multidimensional rating chart, using a

catalogue of elementary motor,

postural, and autonomic phenomena that

had been defined and coded as units of

behavior in previous observations of a

large number of cats before and after

*Fuller and Snoddy (35) reported increased

sensitivity to the effects of exercise and

lethality (within 3 hours) in rats treated

with disulfiram and placed in a rotary drum

turning at 8 rev/min, a situation not more

exhausting than amphetamine-induced hyperactivity. This phenomenon in rats may be

analogous to the increased lethality observed

in our initial trials with increasing doses

of methamphetamine.

amphetamine treatment. A detailed

presentation of the catalogue of

definitions and of our extensive rating

system has been made elsewhere (87).

(For the reader's benefit, the

definitions of special terms to be used

through this paper are given in the

Appendix.) Briefly, 27 categories of

behavior are listed in the rating

chart, including position and

orientation of the animal in the cage,

attitude, arousal, orientation and

quality of the animal's gaze, presence

of any grooming, sniffing, autonomic

reactions, ataxia, dystonia or

disjunction, movement speed, position

and movements of the various body

segments (head, trunk, forelegs,

hindlegs, and tail) as well as modality

and relatedness of any head stereotypy

to that of other regions of the body.

Within each category, the rater chooses

from the catalogue those units that are

most representative of the animal's

actual behavior during the 10-sec

interval. The separate rating of

postures and movements for each region

of the animal's entire body allows a

detailed description and accurate

quantitation of the drug effects on

motor activity.

Movements and alterations of motor

function were scored as present if they

occurred at any time during an

observational interval; postural

features and attitudes, on the other

hand, were considered present only when

maintained for the major portion of an

interval or for a longer period than

other features and attitudes occurring

within the same interval. Thus, when a

cat had maintained the same posture or

the same attitude for most of a 10-sec

observational interval, the appropriate

rating was assigned.

The psychomotor profiles of the animals

in Groups A, B and C, as constructed

from the evaluation of their attitudes

and motor activities, are reported

below.

RESULTS

Effects Of Disulfiram Administration

(Group A):

A slight but consistent reduction in

total spontaneous activity, as compared

with that of the controls, was observed

as early as 6 hours after the first

dose of disulfiram (Fig. 1, top). The

major decrease of movements was

localized at the head-neck region and

112
involved orienting activity. As a

correlate, the attitudinal scales (Fig.

1, bottom) demonstrate that the

FIGURE 1

Fig. 1: Initial effects of disulfiram treatment on the amount of spontaneous activity

and Occurrence of the observed attitudes.

Control- before disulfiram treatment.

Day 1+6 hr.- 6 hours after the first administration of disulfiram. Day 2+6 hr.- 6 hours

after the second administration of disulfiram.

Interested and Investigative attitudes

remained stable after the initial dose

of disulfiram, but that the incidence

of the Indifferent attitude was doubled

on the first day, and was increased in

frequency even further on the second

day. On that day, however, spontaneous

alerting responses in the form of

startle reactions appeared*; an animal

lying on the floor of the cage in an

Indifferent attitude might suddenly,

and for no apparent reason, raise its

head and perform one or more fast

orienting movements while adopting a

tense Interested attitude for a short

*The spontaneous occurrence of these reactions

is not consistent with the sedative effect

usually attributed to disulfiram and suggests

thatits action is of a more complex nature

than that of sedative agents, such as the

barbiturates. In the same vein, Wise and

Stein (91) described "disinterest in etectrical reinforcement" concurrent with the

suppression of medial forebrain bundle selfstimulation in rats treated with acute doses

of disulfiram, and differentiated this aspect

of the animal's behavior from a sedative effect

of the drug, since sedation after large doses

of barbiturates, for example, does not necessarily interfere with self-stimulation and rats

may respond at normal or supernormal rates.

113

With continued drug administration, all

subjects developed a sleepy pattern of

behavior that was manifest during the

observational periods, or when the

animal was left on the floor undisturbed. On these occasions, there was

a marked preference for lying on one

side, and an effort to regain the

preferred side if tne animal's position

was changed manually. By the end of

the first week, somnolence and prolonged episodes of sleep from which the

animals could be easily aroused, were

predominant. Such episodes were, however, punctuated by not infrequent

hyperactive startle reactions triggered

by the occurrence of muscle twitches

about the legs or body. Enhanced

dopamine activity at the level of

norepinephrine receptors subserving

proprioceptive reflex mechanisms might

explain the hyperactive quality of the

response. both sleepiness and muscle

twitches constituted early indications

of neurotoxicity. In fact, hypotonia

and weakness of the hind limbs were

noted in most cats by the fourth day

and were consistent with the ensuing

manifestations (muscle wasting, ataxia,

paresis) of an ascendant, irregularly

progressive neuropathy. Impairment and

subsequent loss of the righting reflex

occurred between the fifth and tenth

days. Recovery from all neurological

disturbances occurred within 10 days

after cessation of treatment, except in

the case of one cat that died on the

tenth day.

period. This startle activity is

accounted for in the Abnormal

Interested attitude shown in the graph.

Similarly, it contributed to the scores

for head-neck activity on the second

day of treatment, which would otherwise

have been expected to decrease because

of the increase in Indifferent

attitude.

Comparative Effects of Methamphetamine

Alone* And Combined with Disulfiram

(Groups B and C):

Observations after a single dose of

amphetamine: Despite some differences

in degree, the immediate responses to

amphetamine were similar in nature in

*A detailed account and phenomenological characterization of the behavioral effects of

acute and chronic administration of amphetamine in the cat have been reported elsewhere

(84). Therefore, reference will be made only

to those aspects relevant to observations in

animals pretreated with disulfiram.
the animals in Groups B and C. A

reduction in locomotor movements,

coupled with increased muscular

tension, arousal, and transient

orienting responses of the head,

occurred at the outset. In some

animals, in either group, especially in

those pretreated with disulfiram,

postural fixation (i.e., maintenance of

a single posture and the absence of any

transient movement for a protracted

period) with lack of orienting

responses was a remarkable initial

feature. Such inertia of the motor

apparatus was revealed in responsive

animals by particular signs of hypokinesia: maintenance of awkward postures due to lack or insufficiency of

postural adjustments; drooling in the

absence of altered deglutitory

mechanisms (usually in the earlier

periods of a recording session);

suppression of automatic expressive

movements of the face, e.g., reduced

eye mobility, blinking activity, or

changes in size of the palperbral

fissures. The lack of mimicry was

frequently associated with wide-open

eyes and protracted visual fixation

(staring response) resulting in a

characteristic spaced-out physiognomy

not unlike the "glazed expression"

reported in cats treated with

bulbocapnine and described as catatonic

(21), augmented by a progressive

bilateral mydriasis with diminished

reaction to light. These manifestations of hypokinesia were, in general,

more pronounced in animals pretreated

with disulfiram. These same animals

not infrequently displayed disparities

in pupillary size through a full range

of diameters, not only when the pupils

were dilated. Although diminished

reaction of the pupils to light was

common to Groups B and C, unilateral

failure to react was observed only in

Group C (Fig. 2).

FIGURE 2

Fig. 2: Staring response and spaced-out facial expression following combined treatment. Unilateral autonomic dysfunction is illustrated by disparities in pupilltary size and expansion of the

nictitating membrane.

114
The initial reduction in spontaneous

activity provided a flat background

against which the emergence of

stereotyped behavior stood out sharply;

such behavior occurred in all subjects

within 15 minutes after the injection

of amphetamine and consistently

involved the head region. Fig. 3 shows

the occurrence of total and regional

stereotyped activity on the first day

of amphetamine administration both in

Group B and in Group C. The maximum

level of stereotypy in the animals

treated with amphetamine alone was

reached at 30 minutes; eventually,

during the last recording period,

stereotyped activity involving the

trunk (hunching or pivoting movements)

supervened. Under combined treatment,

peak stereotypy levels were reached

earlier (at 20 minutes), and the early

appearance of stereotyped movements in

the trunk and leg regions (already

present 20 minutes after the amphetamine injection) clearly contributed to

the enhancement of the total stereotyped activity. Activation of dopamine

systems in the relative absence of

coordinating and inhibiting effects of

norepinephrine systems, as a result of

its depletion by disulfiram, could

indeed be related to these observaFIGURE 3

Fig. 3: Presence of stereotyped activity in

each region of the body and the sum of the

regional occurrences (total) on the first day

of methaphetamine administration in Groups

B and C. P=immediately preceding the first

methamphetamine injection. 10, 20, 30, 90=

10, 20, 30, 90 minutes after the first methamphetamine injection. 115

tions. Interestingly, in the first

10-minute recording period, stereotypy

did not occur as frequently as it did

in the animals receiving amphetamine

alone, a feature obviously related to

more frequent initial postural fixation

in pretreated cats.

Stereotyped head movements were

performed in a relatively fixed

sequence and developed into the motor

components of exploratory patterns of

looking and/or sniffing modalities.* On

those occasions in which the pattern of

stereotypy was interrupted by a pause

or by transient head movements, it was

noticed that these elements might be

incorporated into the sequence of

movements, changing the stereotypy into

a new configuration or a combined

modality, just as the imperceptible

turn of a kaleidoscope causes a derived

pattern to take shape. Later on,

either the incorporated or the initial

component of the pattern might be

dropped out, the original modality

being conserved or changed. Eventually, in animals treated with amphetamine alone, the head-neck stereotypy most frequently evolved into a

constricted sniffing pattern ("minutiae

search"), with a pecking-like oscillation of the head.

Stereotyped head movements, grouped

according to their pattern modality,

occurred under both experimental

conditions, as illustrated in Figure 4

(left panel). In the animals receiving

amphetamine alone, the looking modality

was the predominant response in the

earlier recording periods (10 and 20

minutes), but its incidence decreased

markedly in the last one (90 minutes).

In contrast, the sniffing modality

showed a late increase of incidence,

with highest levels at 90 minutes.

Disulfiram pretreatment differentially

affected the occurrence of both

head-pattern modalities. Thus, while

the looking modality was a sustained

response in the last recording period,

the sniffing modality showed a higher

early incidence (at 20 minutes) and a

marked decrease at 90 minutes. A

differential participation of

anatomical substrates, modulated by

varying activity of neurotransmitters

*The qualification of a stereotypic pattern as

of the looking or sniffing modality does not

imply an afferent triggering stimulus; it

merely suggests that the motor components of

the pattern are similar to those observable

in either of these behaviors.
FIGURE 4

Fig. 4: Stereotyped and transient head movements in Groups B and C on the first day of methamphetamine administration. P=immediately preceding the first methamphetamine injection. 10,

20, 30, 90= 10, 20, 30, 90 minutes after the first methamphetamine injection. The summed

incidence of both modalities does not reflect the total stereotyped activity of the head-neck

region, since these activities may occur as a combined pattern of stereotypy (minutiae-side-toside, for example).

involved in the elicitation of the in pretreated animals (see below). In

stereotypy patterns, might be reflected both cases, a significant increase in

in these changes. frequency, as well as in speed and

suddenness, of tne transient activity

The time course of the concurrent occurred at 90 minutes, when most Cats

transient looking movements remained were highly aroused and hyperactive.

unmodified under combined treatment

(Fig. 4, right panel), but the The selectivity of the amphetamine

movements were more frequent in effect on muscle tone of the entire

pretreated animals during all periods, body was usually expressed by

including the pre-injection period. differences in local patterns of

This increase appeared to be related to involvement; muscles acting on either

the occurrence of the previously side of a joint appeared to be

described startle reactions induced by unequally affected by increase in tone,

disulfiram. The sustained occurrence resulting in the development of an

of transient looking movements observ- awkward position for that body region.

able at 30 minutes under combined Such manifestations of regional

treatment, as opposed to the slight dystonia were also more intense and

decrease after treatment with am- appeared earlier in pretreated animals

phetamine alone, was correlated with than in those receiving only amphetathe development of a Reactive attitude mine. Most intense and persistent was

116
a cataleptoid extensor leg posturing

that we have called "abortive grooming"

because it represents the initial component of a never completed grooming

behavior. Thus, not infrequently, the

haunch of a sitting cat would slide to

a lying position (supported by a side

on the floor) with both hind limbs

simultaneously extended, one of them

being raised and placed behind the

animal's head and the other between the

forelegs. On other occasions, in an

animal leaning against a wall of the

cage, a foreleg might also be involved

in such extensor response (Fig. 5).

The resulting contorted postures were

usually preserved in spite of the

concurrent transient or stereotyped

activity of the head, and constituted a

clear expression of dissociation

between the animal's postural base and

the superimposed movements and

attitude, as observed on later days

under treatment with amphetamine alone.

Similarly, the typical propped-up

posture ("camel posture") usually seen

in Group B at the height of intoxication in subsequent days occurred in

most subjects in Group C on Day 1

during the periods of hyperkinesia and

maximum arousal. This standing posture

consists of a stiff appearance, widebased hyperextended hind legs, arched

back, and tail showing a waxy immobility (analogous to the Straub's

tail-raising phenomenon in mice and

rats given opiates or L-Dopa), and it

was sustained even while the cats were

peering about the environment and

actively moving the head and extremities. The peculiar plastic rigidity

of the tail (Fig. 6) was a long-lasting

feature.

FIGURE 5

Fig. 5: Extensor leg posturing (abortive grooming) involving both hind- and fore-legs in a

disulfiram pretreated animal.

1l7
FIGURE 6

Fig. 6: Preservation of abnomal posture of

the tail (waxy immobility), seen to last more

than 20 minutes, 2 hours after the amphetamine

injection in a cat pretreated with disutfiram.

Another manifestation of behavioral

disjuntion that appeared earlier under

combined treatment than with

amphetamine alone was akathisia, which

was expressed in both sitting and

standing postures by restless leg

repositioning or stepping and a

vacillating maintenance of posture, as

if a change of the animal's location

within the cage might occur imminently.

As illustrated in Fig. 7, akathisia was

displayed only by pretreated animals on

the last recording period of the first

day. Dystonia, ataxia, and disjunction

also reached a peak in these animals

much earlier than in those treated only

with amphetamine. Localized dyskinetic

phenomena, such as myoclonic jerks,

orofacial dyskinesias, or paw-shaking

movements, occurred, on the other hand,

later in the intoxication cycle under

both experimental conditions.

FIGURE 7

Fig. 7: Occurrence of ataxia, dystonia, disjunction and akathisia on the first day of methphetamine administration in Groups B and C.

injection. 10, 20, 30, 90= 10, 20, 30, 90 minutes after the first amphetamine injection.

P= immediately preceding the first amphetamine

118
FIGURE 8

Fig. 8: Effect of disulfiram pretreatment

(Group Cl on the distribution of attitudes on

the first day of methamphetamine administration. P= immediately preceding the first

methamphetamine injection. 10, 20, 30, 90=

10, 20, 30, 90 minutes after the first methamphetamine injection.

BEHAVIOR MEASURED

INTERESTED ATTITUDE

INVESTIGATIVE ATTITUDE

REACTIVE ATTITUDE

STEREOTYPY

TRANSIENT MOVEMENTS

AKATHISIA

DISJUNCTION

DYSTONIA

ATAXIA

The general psychomotor pattern of

response, as represented in the

attitudinal scales, is shown in Fig. 8.

An abnormal quality of the animal's

attitude was detectable shortly after

the injection of amphetamine, with the

attitude scores being similar for

Groups B and C during the 10-minute

recording period. In pretreated cats,

however, the prevailing Indifferent

attitude induced by disulfiram (preinjection period) was still present 10

minutes after the administration of

amphetamine and paralleled the already

mentioned decreased occurrence of the

sterotyped motor response. At variance

with the observations on animals

treated with amphetamine alone, in

which the Investigative attitude became

predominant during later recording

periods, was the sustaining of the

Interested attitude scores through all

periods under combined treatment. More

importantly, a Reactive attitude

(usually the outcome of repeated

injections over several days in cats

receiving only amphetamine) was noted

in pretreated animals at 30 and 90

minute after the first injection,

manifested as exaggerated sensitivity

toward the environment, with frequent

attentional shifting, over-reactive

movements and signs of hallucinatory

behavior (visual tracing, aimless paw

TABLE 1

GROUP A GROUP B

DISULFIRAM ALONE AMPHETAMINE ALONE

10-30 Min. 90-Min.

<.01 <.01 NS

NS <.01 <.01

NS NS NS

NS <.01 <.01

<.01 NS NS

(decrease)

GROUP C

DISULFIRAM AND AMPHETAMINE

10-30 Min.

<.01

<.01

NS

90-Min.

<.01

<.01

<.01

NS NS NS

NS <.05 <.01

NS <.01 <.01

NS NS <.01

<.01

<.01

(increase)

NS

<.01

<.05

<.05

<.01

<.01

(increase)

<.01

<.01

<.01

<.01

Statistical significance of recorded behavioral changes. All changes (except where indicated)

represent an increase in the occurrence of the behavior in the treated animals from that in

saline-treated controls. NS= no significant difference. Group A: 6 hours after the second dose

of disulfiram; Group B: 10-30 minutes and 90 minutes after a single dose (7.5 mg/kg, ip) of

methaphetamine; Group C: 10-30 minutes and 90 minutes after the first injection of methamphetamine in pretreated animals.

119
movements, or eluding jumps apparently

related to non-existent objects in the

environment).

A summary of the statistical

significance of various behavioral

changes observed on Day 1 under the

three experimental conditions is

presented in Table I. The figures are

based on comparisons with control data

collected on the fifth day of the

initial period of treatment with saline

and on the pre-drug periods.

Observations after repeated doses of

amphetamine: Generally, a comparable

evolving pattern of behavior was

observed in animals in Groups B and C.

Consistent trends connected with the

acute effects of amphetamine were

apparent for most behavioral responses

in any recording

intoxication

session during the

cycle. The cumulative

effects of the drug differed from those

in the naive animal, i.e., there was a

shorter latency of the induced

manifestations and an increase of

transient activity, including adventitious movements bearing little or no

relationship to the main vector of

behavior.

However, the combined use of amphetamine and disulfiram resulted both in

an accelerated development of the

neuropathy caused by repeated administration of disulfiram alone, as

described above for the animals in

Group A, and the appearance, in several

animals, of electrographic or generalized seizures. Interestingly,

whereas manifestations of neurologic

derangement were evident in the early

days during the periods between

amphetamine injection, or after its

acute effects had worn off, the

consequence of muscle weakness and

progressive wasting could be detected

during the recording sessions by the

evaluation of certain motor activities

that usually are increased under

treatment with amphetamine alone (Fig.

9). Movements that require the active

FIGURE 9

Fig. 9: Effects of developing neuropathy on specific motor units during continued treatment with

disulfiram and methamphetamine (Group C). Day 1, 3, 11= first, third and eleventh days of methamphetamine administration. P= immediately preceding the first methamphetamine injection of the

day. 10, 20, 30, 90= 10, 20, 30 90 minutes after the first methamphetamine injection of the day.

120
support of the hind limbs (hunching and

pivoting), although present on Day 1,

did not appear thereafter. On the

contrary, hindleg and foreleg

repositioning, movements that require

much less support than do hunching and

pivoting, were still performed on the

eleventh day when the disulfiraminduced paresis had already become obvious. No comparison of the quantitative aspects of behavior in Groups

B and C can legitimately be made for

any day after the first. However,

certain features induced by treatment

with amphetamine alone were accentuated

or affected qualitatively by pretreatment with disulfiram and deserve

further comment from a phenomenological

standpoint.

In contrast to the animals in Groups A

and B, all subjects in Group C had

impairment of the righting reflex about

the third day of amphetamine injection

(after four doses of disulfiram), and

the reflex was completely lost within

the first week. In many of these

animals, as in those in Group A,

unilateral postural preferences and

resistance to attempts to change the

laterality of the posture were noted

during neurologic examination.

Following the injection of amphetamine

during the recording sessions, unilateral preference could be appreciated

by the dominant

right

direction (left or

for a particular

stereotyped

cat) of

circling locomotion, if

any.

Within the initial recording periods of

each successive day, marked

deficiencies of postural

as well as other signs

adjustments,

indicative of

alteration of the motor initiative,

were particularly noticeable in Group

C . For example,

amphetamine

animals receiving only

might occasionally lean

against the cage wall while executing a

repetitive skating movement in which a

foreleg slides outwards along the floor

and is repositioned after considerable

spread has occurred. Not only did this

activity occur more frequently in pretreated animals, but it was obtunded,

since the adjusting repositioning movement of the foreleg occurred only with

maximum spread and after a protracted

period (Fig. 10). When the animal was

in locomotion, sudden and transient

"freezing" of movement in mid-air could

be observed; a cat might suddenly stop

a circling locomotor movement with a

hind leg already lifted for the next

step remaining frozen in the air. On

FIGURE 10

Fig. 10: Maintenance for a 15-minute period of

abnormal posture in the absence of postural

adjusting movement, observed in a pretreated

animal (Day 3, 10-min period). Note spacedout facial expression and drooling.

Videotape picture.

other occasions, a foreleg would reach

the ground crossed in front of the

other one and the animal would freeze

in that position for a while.

Analogous phenomena recorded during

experimental catatonia in cats and

monkeys and in catatonic patients have

been described as "barrage" of the

psychomotor initiative (21).

Manifestations of hypokinesia were also

observed in the later periods of

hyperkinesia, intermingled with

reactive motor activity; a cat might

retain uncomfortable postures that

resulted from overreactive jumps

apparently triggered by non-existent

stimuli while still responding to the

"imaginary" object (Fig. 11).

The tendency to adopt and retain a

crouching posture in which the forelegs

were spread out, a manifestation of

regional dystonia only rarely observed

in animal's given amphetamine alone,

developed further under combined

treatment. Toward the final days of

the experiment, six of the ten pretreated cats demonstrated a locking of

the proximal joints of the forelegs in

such an abnormal range that the legs

lay flat on the floor in a position

almost perpendicular to the longtitudi121
FIGURE 11 FIGURE 12

Fig. 11: Maintenance of abnormal posture concurrent with reactive movement toward "Imaginary" object during the hyperkinetic stage in

an animal pretreated with disulfiram (Day 3,

65 minutes after the injection of amphetamine).

Note facial expression and drooping of

whiskers.

Fig. 12: Crucifixion posture following treatment with disulfiram and amphetamine (Day 11

75 minutes after the injection of amphetamine).

Note piloerection and clawing.

122
nal axis of the body (Fig. 12). This

bizarre posture persisted for at least

two hours after the injection of

amphetamine and resembled the

"crucifixion posture" described in

bufotenine or LSD-intoxicated monkeys

(31) and in experimental and human

catatonia (21).

Most dramatic, however, was the effect

of chronic drug combination on the

characteristic motor response to

amphetamine, the stereotyped activity

of the animal's head. An outstanding

feature of this response during the

heigt of amphetamine intoxication is

the compulsion attached to the performance of repetitive movements, as

shown by the cats' undistractibility

and failure to respond to environmental

stimuli that would normally attract

their attention (e.g., noise, light,

catnip, rat). Athough the stereotypy

was prolonged, its increased intensity

in pretreated animals was manifest

through augmentation of its compulsivity, rather than by a higher

frequency. Thus, a smoother flow and

cohesion of the sequential movements

within patterned stereotypies, with

incorporation of otherwise interfering

The particularly high level of

compulsivity of the last days could

also be appropriated by the occurence,

in several animals in Group C, of

episodes of forced locomotor propulsion

comparable to the obstinate progression

syndrome reported by Bailey and Davis

(12). Although none of these cats

could, at this stage, correctly

maintain its stance and all showed

stagger and swaying of the body, they

would thrust the head forward blindly,

seemingly unable to turn or to back up

after having walked into a corner of

the cage. Strong propulsive movements

of the legs would immediately follow, Fig. 13: Stereotyped side-to-side movement of

until exhaustion caused their transi- the head concurrent with crucifixion posture

tory interruption as the hind limbs in an animal pretreated with disulfiram (Day

remained spread out on the floor. If 11, 70 minutes after the injection of amphetamine).

FIGURE 13

transient movements and subsequent

development of combined constricted

patterns, such as minutiae-searchside-to-side or up-and-down-minutiaesearch, were frequently observed. In

no other circumstance under amphetamine

alone was the compulsivity as striking

as was the sight, in the final days of

the combined condition, of an animal in

a crucifixion posture struggling to

perform the stereotypy, while feebly

dragging its nose over the floor in a

side-to-side movement (Fig. 13).

123
the cat were placed in the center of

the cage, it would keep its head

forward and start pushing against the

same corner or a different one. The

appearance of obstinate progression

was, at all times, concomitant with a

highly aroused stupor-like state.

Finally, two animals in Group C

demonstrated on the last day of the

experiment a similar phenomenon to a

"flashback": immediately after being

placed in the observational cage and

prior to the injection of amphetamine,

these cats became hyperaroused and

over-reactive. Apparently attempting

to escape from some non-existent

object, they made repeated hyperkinetic

eluding jumps and locomotor movements

until exhaustion made further activity

impossible. The episode was accompanied by panting, tachycardia, unilateral mydriasis, urinary incontinence,

and a facial expression connoting

intense panic.

DISCUSSION

If dopamine is considered to be involved in the pathogenesis of schizophrenia, as suggested by the results of

pharmacological, biochemical and

clinical investigations referred to in

the Introduction, then disulfiram meets

several theoretical desiderata for an

agent that can underline those effects

of amphetamine intoxication germane to

the endogenous psychosis.

1) There is evidence that disulfiram

synergizes the potentiating effects of

amphetamine on dopamine. Amphetamine

is known to displace norepinephrine and

dopamine from the neuronal stores and

to inhibit their uptake into the nerve

terminals (5,37). The inhibition of

dopamine uptake, with consequent

enhancement of dopamine activity, is

greater at the level of the corpus

striatum than in norepinephrine areas

(19). In addition, the behavioral

effects of amphetamine are closely

related to the release of newly

synthesized dopamine and norepinephrine

(15). Disulfiram, on the other hand,

via its inhibitory action on dopamine

beta-hydroxylase, is effective in

reducing the concentration of endogenous norepinephrine and increasing

that of endogenous dopamine in the

norepinephrine-containing regions of

the brain (38,391. by decreasing the

amount of norepinephrine relative to

that of dopamine, disulfiram administration, in addition to potentiating

dopamine activity in dopamine neurons,

may cause the replacement of dopamine

as a modulator of activity in norepinephrine neurons, since it is known

that norepinephrine nerve terminals

store and release dopamine when dopamine beta-hydroxylase is inhibited

(4,46). These effects of disulfiram

might be enhanced by using the drugs in

combination. The blocking action of

disulfiram on the synthesis of

norepinephrine would reduce the

availability of this neurotransmitter

and, conversely, the releasing action

of amphetamine would be expected to

hasten the depletion of norepinephrine

after disulfiram administration.

2) It is known that the absorption of

disulfiram after its administration in

vivo is followed at once by chemical

reactions leading to complete reduction

of the disulfide (38,83). Both

disulfiram (28,61) and its main

metabolites, diethyldithiocarbamate

(65,74) and carbon disulfide (60), have

been shown to increase the intensity,

the duration, or both, of amphetamineinduced stereotypy (a proposed model

psychosis per se): In addition the

possibility exists that disulfiram may

produce stereotypy, since this effect

has been produced by FLA-63, an inhibitor of dopamine beta-hydroxylase which

is structurally related to disulfiram

(3).

3) Disulfiram may induce schizophreniclike psychotic episodes. Such episodes on occasion clinically indistinguishable from schizophrenia (11),

have been reported as a secondary

development of the use of disulfiram in

the treatment of chronic alcoholic

patients (40,59,63,90). Although the

risk of activating psychotic conditions

is acknowledged by the contraindication

of the use of disulfiram in their

presence,

exacerbate

the ability of the drug to

schizophrenic

especially of the paranoid type, gentendencies,

erally contraindicates its use even if

behavioral adjustments are

"borderline" (40).

only

4 ) Disulfiram has been shown to

exaggerate profoundly the

symptomatology

typical

in

patients (43).

schizophrenic

Additionally, because of its inhibiting

action on dopamine beta-hydroxylase,

disulfiram may mimic state of

dopamine beta-hydroxylase a deficit that

has been implicated as a mechanism for

324
the involvement of either dopamine (56)

or norepinephrine in schizophrenia, the

latter on the basis of experiments with

6-hydroxydopamine (81) and of findings

in postmortem brain specimens

schizophrenic patients (92). Further

indirect support for these implications

iS provided by the enhancement of

psychotic manifestations in manic

patients treated with fusaric acid, a

blocker of dopamine beta-hydroxylase

that possesses a relatively high degree

of specificity (77).

Before the inhibiting action of

disulfiram on dopamine beta-hydroxylase

had been demonstrated, Heath et al.

(43) studied the effects of the drug,

given at an average dose of 1 g/day

during 14 to 17 days, in nine chronic

schizophrenic patients and nine

prisoner-volunteers lacking personal or

familial history of nervous or mental

disease. Whereas all the subjects

displayed, at the time of maximum

effect (14 days), symptoms of

neurotoxicity such as lethargy, reduced

stream of thought, abbreviated memory

span, and impaired ability to calculate, no alteration of thought processes and only increased emotional

ability were recorded in the control

group*. All the schizophrenic patients, on the other hand, experienced

severe withdrawal and exaggeration of

all schizophrenic symptoms. Thus, although secondary symptoms were particularly affected (auditory hallucinations, for example, developed de novo

in eight patients and were accentuated

in the remaining one), the primary ones

were also heightened. Autism, inappropriate affective responses to the

environment, and disturbance of thought

processes gradually became more prominent, the latter becoming bizarre and

with marked defects of association that

precluded communication with the

psychiatrists and ward personnel; total

loss of previously intact orientation

occurred in four of these patients, and

akathisia was manifest in three others.

In addition, schizophrenic patients

complained of tightness of the legs

after medication**.

*One exception was a subject with temporal lobe

abnormality, as demonstrated etectroencephalographically before medication, who developed

fill-blown psychotic symptoms toward the end

of the treatment period.

**Such complaint was also recorded in the VOlunteer with temporal lobe dysfunction.

Whereas disulfiram is a non-specific

inhibitor of dopamine beta-hydroxylase

and causes multiple metabolic changes,

the lack of development of schizophrenic-like or psychotic symptoms in

the control subjects, as well as the

accentuation of primary and secondary

symptoms in schizophrenics, particularly the occurrence of thought

disorders in contrast to their absence

in the control group, clearly suggest

that whatever alteration in the

metabolism of catecholamines was

provided by disulfiram, it was highly

significant regarding the basic

mechanisms of the disease. (Stronger

support for a link between deficient

activity of dopamine beta-hydroxylase

and exacerbation of psychosis has

recently been provided by the study

with fusaric acid mentioned above.)

Consideration of this background suggested the current study to determine

whether given end-stage components of

the amphetamine-induced behavioral

syndrome could be influenced by

blocking the synthesis of norepinephrine. Table II presents our

triple-layered model, with amphetamine

psychosis serving as a model of

functional paranoid psychosis, and

changes of behavior induced by chronic

amphetamine intoxication in the cat

serving as a model of amphetamine

psychosis. In the early days of

intoxication, the restrictedness and

directedness of the animals' behavior

is reflected in their

"postural-attitudinal" set, as well as

in the stereotypic responses. As

intoxication progresses, however, there

is an increasing dissociation or

"disjunction" between the animal's

underlying postural base and the

superimposed movements and attitudes.

Disjunction, as we have defined it in

our observations, represents a given

body segment taking on an autonomous

movement or position which does not

seem to have a purpose or any relatedness to other body segments, e.g.,

repetitious repositioning of the

forelegs occurring while a sniffing or

looking pattern of stereotypy of the

head develops, but lacking any apparent

relationship to this pattern. Thus,

the performance of movements unrelated

to the main vector of behavior or the

maintenance of a posture inappropriate

for the activity being displayed

characterizes the bizarre, disjunctive

behavior-posture relationship of this

stage of the intoxication cycle. Not

125
TABLE 2

ANIMAL MODEL PSYCHOSIS

Chronic Amphetamine

Intoxication

Abnormal

lnvestigatory

Attitude with

Repetitious

Activity

HUMAN MODEL PSYCHOSIS

Chronic Amphetamine

Intoxication

Repetitious

Examining

Curiosity Searching

Sorting

Behavior

FUNCTIONAL

PARANOID

PSYCHOSIS

Paranoid

Tendencies

Paranoid

Psychosis

Sustained Looking for

Meanings

Restricted

Pleasurable

Suspiciousness

Repetitious

Minutiae

Activity

Ideas of Fearful

Reference Panic Stricken

Persecutory Agitated Reactive

Delusions and Over-reactive Attitude

Hallucinations

Triple-layered model of chronic amphetamine-induced bekavior as a model of functional paranoid

psychosis.
infrequently, akathisia-like repositioning and stepping movements that

have a starting-and-stopping quality

similar to that noted in chronic

schizophrenia or as part of the tardive

dyskinesia syndrome are manifest in the

later stages of intoxication. The

final state is best illustrated by the

animal's Reactive attitude, in which

hypersensitivity to minor or inapparent

stimuli in the environment results in

over-reactive orienting responses or

eluding jumps that, in turn, may

trigger increasingly hyperkinetic

reactions. At this point, the composite picture is one of an agitated

state of increased muscular tension in

which the breakdown of behavior is

accompanied by marked hyperarousal and,

in many animals, signs of hallucination.

In the current experiments, a faster

development of certain end-stage

components of the amphetamine behavioral syndrome was obtained by

pretreating amphetamine-intoxicated

animals with disulfiram. Thus, on the

first day of combined treatment, an

earlier and more prominent behavioral

disjunction was recorded than had been

obtained with amphetamine alone. The

behavioral disjunction represents an

increased compartmentalization or

autonomy of body regions in regard to

both posture and movement, and was

manifest in a greater incidence and

intensity of extensor leg posturing,

frequent display and maintenance of a

camel posture, occurrence of akathisia,

and early and relatively pronounced

occurrence of trunk and legs stereotyped activity on Day 1. On that day,

toward the end of the recording

session, several pretreated animals

developed a Reactive attitude, one of

the more prominent end-stage components

of the syndrome in cats receiving

repeated doses of amphetamine. In

contrast with the facilitation of these

behaviors, was the absence of dyskinesias and hyperreflexia on Day 1. It is

possible that development of receptor

supersensitivity is necessary for the

appearance of localized dyskinetic

phenomena and exaggerated reflex

activity.

The early loss of the righting reflex

in the animals receiving combined

treatment would be in keeping with an

accentuated depletion of norepinephrine

in and around the vestibular nuclei,

following release of norepinephrine by

amphetamine (24) and blocking of its

synthesis by disulfiram, or with a

functional alteration at the level of

the basal ganglia which are known to

mediate gravity-oriented labyrinthine

reflexes (57). Manifestations of unilateral preference, on the other hand,

might be due to accentuation of an

intrinsic neurochernical imbalance of

the nigrostriatal system (51), with

greater release from one side than from

its contralateral counterpart.

Several other features that were either

more evident or delayed under combined

treatment can be related to manifestations in animals treated with

bulbocapnine or different psychotogenic

compounds. Thus, loss of motor initiative, as represented by lack of

mimicry, a "barrage" phenomenon, and

decreased adjusting postural movements

have been reported in experimental

catatonia induced by bulbocapnine. The

camel posture in our animals is

reminiscent of the one transitorily

adopted by cats in a stressful

situation and is not unlike the

"kangaroo posture" described by Adey

(1) in cats treated with LSD. It is

also suggestive of the "crooked back"

posture of cats receiving various

mescaline derivatives (21). Underlying

interactions with neurotransmitters

such as serotonin, which is known to be

released by administration of

amphetamine (36), might explain these

commonalities.

As a correlate, Tatetsu (88) has

described amphetamine addicts who

manifested catatonic and paranoid

syndromes in which the facial expression appeared slightly stiff and

apathetic and the motor activity was

dull and retarded, with absence of

associated expressive movements of the

hands in talking or arm movements while

walking. Tatetsu described as a

characteristic feature of amphetamine

intoxication in these patients when

they were seated a peculiar rigidity of

the trunk in extended position

accompanied by head, arms, and leg

movements; this same feature was frequently part of the prodromal phase of

the catatonic state (most frequently a

mixture of stupor and hyperkinesia)

that appeared under heavy intoxication.

Another catatonic-like phenomenon in

amphetamine addicts, similar to the

postural fixation that was prominent in

the animals given the combined

treatment, is the "overamping", state,

a state of hyperaroused immobility that

may be elicited by a single large

intravenous dose of amphetamine (55).

127
Because of the occasional occurrence of

catatonic symptoms or catatonic forms

of amphetamine psychosis and the

findings of disjunctive behavior, loss

of motor initiative,

reflex, and pupillary changes, there is

loss of righting

a need to resolve the "paranoid model"

in any amphetamine model of psychosis

to fit those bizarre motor behaviors.

In a Previous publication we suggested,

on the basis of results from the

experiments described herein and of

clinical data from the literature that

manifestations not consonant with the

phenomenology of the paranoid psychosis

reflect a "catatonic" phase of the

amphetamine intoxication process (25)

which is reminiscent of human catatonic

behavior. Certainly, many points of

commonality exist between catatonic and

paranoid schizophrenia, especially

since both conditions may appear in

different phases in the course of an

individual's illness (54). In

examining the catatonic manifestations

and their parallels with the paranoid

manifestations, stereotypy may be a

common mode in both forms of schizophrenia.

Bleuler (14) listed a variety of motor

disorders noted in catatonic patients

that also appeared in other forms of

schizophrenia. Under stereotypies,

Bleuler included posture, attitude,

thinking, and movement. He also

mentioned stereotypies of drawing,

writing, music, and speech (verbigeration), and described stereotypies

of posture showing ma persistence. For

example, catatonics may stare at the

same spot for weeks at a time. In a

manner similar to that of our chronic

amphetamine cats, stereotypies of

position are expressed in two ways,

viz. the patients always select the

same corner of the room and always go

to the same place to carry on a

particular activity. Other signs of

motor disorder mentioned by Bleuler are

muscular tension or rigidity, loss of

spontaneous movement or initiative,

negativistic attitude, automatic

obedience, hyperkinesia, relative

autonomy of single muscular groups,

general lack of coordination of body

segments (for example, arm and leg

movements that do not relate in a

synchronous fashion during walking, or

having feet step irregularly in time

and space), and stupor. A unilateral

increase in tendon reflexes has been

noted. Differences in size of the two

pupils and the absence of pupillary

reactions to both light and accommodation are frequent. Also,

oculomotor reaction (nystagmus) to

vestibular stimulation is reduced in

schizophrenics. This inhibition is

seen primarily in catatonic patients

and is related either to caloric or

galvanic stimulation (17,32). In

addition, postural reactions to

vestibular stimulation found to be

inhibited in schizophrenia and the

finding of significantly less swaying

induced by rotation in schizophrenics

suggest reduced activity of the

ventibulo-spinal mechanisms (33,69).

Ornitz (71) postulated that pathological vestibular mechanisms would be

manifested in an inability to initiate

and carry out purposeful actions that

are ordinarily automatically regulated.

This inability would lead, in adult

schizophrenics, to the psychomotor

slowing and catatonic episodes that

have been so eloquently described and

related to vestibular and proprioceptive disturbance by McGhie and

Chapman (66) and Chapman (16).

Loss or inability of initiative might

well be a common denominator that cuts

across many of the symptoms, including

frozen postures and stereotypies. An

amphetamine-induced distortion of the

mechanisms that regulate the continuous

flow of behavior might be the factor

underlying not only stereotypy but also

the preservation of perceptualattitudinal sets characteristic of

chronic amphetamine intoxication in

both man and animals (29).

In viewing the amphetamine-induced

syndrome in this way, the induced

constraint or restriction of attitude

or perception would have the same under lying mechanism as exists in a

postural-motor stereotypy. Similarly,

thinking itself could become repetitious, secondary to perseveration

in one of the same systems subserving

perception and cognition. Thus a

beginning or incipient paranoid

schizophrenic or an individual in the

beginning phase of the amphetamine

psychosis might start with an

all-pervasive attitude of curiosity,

suspiciousness or both and the

content of this attitude would become

increasingly constricted. Under the

influence of increasing arousal,

agitation, or both, the individual's

behavior might then evolve into a

paranoid state. Alternately, taking

off from the same branching point of

either stereotypy or restricted

attitude, the catatonic pathway may

128
have a predilection to move in a motor

or postural form because of a more

intensive arousal or a more marked

regression to lower levels of

organization.

Although the current study may be

criticized because of the neurotoxic

effects of repeated administration of

disulfiram, the fact remains that the

over-reactive end-stage behaviors of

the latter days of chronic amphetamine

intoxication did occur under combined

treatment, as represented by Reactive

attitude on Day 1 (a time at which no

sign of neurotoxicity was apparent in

either Groups A or C), and that certain

behaviors induced in the animals in

Group B (amphetamine alone) were, at

least qualitatively, enhanced by

pretreatment with disulfiram in a

direction consistent with comparable

features in experimental catatonia and

the catatonic form of schizophrenia.

The more rapid development of end-stage

behaviors in amphetamine-intoxicated

animals pretreated with disulfiram can

be explained as resulting from a

stimulating effect of the drug

combination in the relative absence of

norepinephrine similar to that seen in

chronic amphetamine intoxication, where

there is also an imbalance in the ratio

of norepinephrine to dopamine (42).

There is an interesting parallel

between the accelerated development of

peripheral neuropathy and the early

occurrence of akathisia in animals

pretreated with disulfiram, on the one

hand, and the occurrence of tightness

of the legs (a possible early

manifesta- tion of peripheral

neuropathy) and akathisia found in

schizophrenic patients studied by Heath

and asso- ciates, on the other. In

addition to a possible greater

vulnerability of these patients to the

neurotoxic effects of the drug*, a

correlation between the animal and the

human conditions is suggested by the

absence of the aforementioned symptoms

in the control subjects.

The possibility that a correlation

between induced behavioral manifestations in animals and a psychopathological condition in humans could

provide new insights into the nature of

mental derangements is an important

one, and had been foreshadowed by T. S.

Eliot, who wrote:

That Cats are much like you and me

And other people whom we find

Possessed of various types of mind

For some are sane and some are mad.

(from "The Ad-dressing of small Cats"

in Old Possum's Book of Practical Cats

by T.S. Eliot

by permission of Harcourt Brace Jovanovich, Inc.)

*One might speculate that this increased vulnerability may be related to abnormalities

of musculature in schizophrenic patients which

were first reported many years ago. Kraepelin

(54), for example, refers to the not infrequent idiopathic muscular swellings concomitant with heiahtened sensibility of the

muscles to percussion, and cites-Ajello's

interpretation of peculiar muscle reactions

to electrical stimuli in prospective catatonic

patients as due to heightened irritability of

the sarcoplasm. More recently, Meltzer (67)

has postulated that changes in skeletal

muscles occurring in psychotic patients are

of mixed myopathic and neuropathic origin.

129
The assessment of behavioral

alterations by means of direct

observational technique requires

concrete and specific definition of the

units to be recorded. A limited

meaning should, therefore, be assigned

to terms that might, outside of this

context, have various common usages.

Following are the definitions not given

in the text for such terms, as used in

the current presentation.

Ataxia: Inadequate control of the

range and precision of movements with

or without quivering of flexed and/or

extended regions of the body. It is as

well applied to postural

manifestations. Thus, while ataxia

might be represented by marked motor

inooordination, so that the animal

tends to fall or is grossly unable to

coordinate all four legs in order to

walk or to maintain a standing posture,

staggering or swaying movements in an

animal keeping a certain posture are

also indicative of its occurrence.

Attitude: Attentional mode and

involvement of the animal, as

manifested by its postural appearance

and motor activity in relation to the

surroundings and to its own body

(environment). Whereas posture

represents the endpoint of a movement

or action, attitude reflects the

quality of the animal-environment

relationship. Classifications are

Asleep: self-explanatory; Indifferent:

lack of manifest attentional

involvement with the environment;

Interested: directed attentional

contact with the environment or a part

relationship. Classifications are

Asleep: self-explanatory; Indifferent:

lack of manifest attentional

involvement with the environment;

Interested: directed attentional

contact with the environment or a part

of it, but without definite physical

approach; Investigative: selected

attentional contact and active reaching

out or approaching and examining of one

or more parts of the environment. In

the Interested attitude, the animal may

single out aspects of the environment,

but in the Investigative attitude an

active interaction with those aspects

is achieved through a selected approach

response. Whenever these attitudes

have a nervous, tense, or apprehensive

quality, they are termed Interested

APPENDIX

Abnormal and Investigative Abnormal,

respectively. The latter represents

the persistent "compulsive" searching

out of restricted "stimuli", often

occurring even when the animal may have

broken from a patterned (investigatory)

motor stereotypy. Reactive: an

abnormal attitude characterized by

shifting attentional contact manifested

in increased peering about the

environment, frequent postural

readjustments, and sudden inadequate or

apparently unmotivated movements having

a tense, agitated, jumpy quality.

Disjunction: Lack of proper

relationship among postures or

movements in two or more body regions,

resulting in a fragmentation of the

static or kinetic configuration of the

body.

Dystonia: Abnormal postures or

movements characterized by hyperflexion

or hyperextension of body regions,

resulting in an awkward appearance.

Hunching: Transitory elevation of the

back forming a momentary hump or

exaggerating an existing one. It may

appear as a result of active backing up

of forelegs against fixed hypertonic

hindlegs or as compensatory movements

associated with the execution of a

stereotypy of the head.

Pivoting: Oscillating movement of the

body around a fixed point, usually the

hindquarter. Most frequently, the

cage floor, with or without shifting of

the body weight and without

modification of the animal's place and

orientation.

Stereotypy: Motor activity consisting

of sequential repetition of movements

describing a path of displacements

recognizable as a kinetic

configuration. Frequently observed

stereotypic configurations that have

been mentioned above are:

Minutiae-search, pecking-like movements

of the head executed by rotation around

the interauricular axis; Side-to-side,

horizontal head-neck movements around

the base of the neck or around the

occipito-atlantoid articulation;

Up-and-down, vertical head-neck

movements (smooth or jerky) around the

base of the neck or around the

occipito-atlantoid articulation.

130
ACKNOWLEDGMENTS

This work was supported in part by NIMH

Grants 5 TO1 MH 07073 and MH 18904 and

by Veterans Administration Funds. I

thank Dr. E.H. Ellinwood, Director of

the Behavioral Neuropharmacology

Section, Duke University Medical

Center, for providing the materials and

physical facilities for this study. My

thanks are also due to Linda Nelson and

Nancy Wagoner for their expert

technical assistance, and to Dr. David

Frost for editing this manuscript.

REFERENCES

1. Adey, W.R., F.R. Bell and J.B.

Dennis: Effects of LSD-25, psilocybin

and psilocin on temporal lobe EEG

patterns and learned behavior in the

cat, Neurology, 12: 591-602, 1962.

2. Adams, R.N.: Stein and Wise theory

of schizophrenia: a possible mechanism

for 6-hydroxydopamine formation in

vivo. Behav. Biol., 7:861-866, 1972.

3 . Ahlenius, S. and E. Jörgen:

Effects of a dopamine

(DA)- -hydroxylase inhibitor on timing

behavior; Psychopharmacologia, 24:

243-246, 1972.

4 . Anden, N.E., C.V. Atack and T.H.

Svensson: Release of dopamine from

central noradrenaline and dopamine

nerves induced by a dopamine

-hydroxylase inhibitor. J. Neuronal.

Trans., 34:93, 1973.

5. Anden, N.E., A. Carlsson, J.

Haggendal: Adrenergic mechanisms.

Annu. Rev. Pharmacol., 9:119:134. 1969.

6. Angrist, B. and S. Gershon: The

phenomenology of experimentally induced

amphetamine psychosis-preliminary

observations. Biol. Psychiat.,

2:95-107, 1970.

79 Angrist, B. and S. Gershon: A

pilot study of pathogenic mechanisms in

amphetamine psychosis utilizing

differential effects of d and

l-amphetamine. Pharmakopsychiat.,

Neuro-Psychopharmakol. 4:64-75, 1971.

8. Angrist, B., H.K. Lee and S.

Gershon: The antagonism of

amphetamine-induced symptomatology by a

neuroleptic. Am. J.

817-819, 1974.

Psychiat., 131:

9. Angrist, B., G. Sathananthan, s.

Wilk and S. Gershon: Behavioral and

biochemical effects of L-Dopa in

psychiatric patients. Biochem.

Pharmacol., suppl. (part 2), pp.

822-825, 1974.

10. Angrist, B., B. Shopsin and S.

Gershon: Comparative psychotomimetic

effects of stereoisomers of

amphetamine. Nature, 234: 152-153,

1971.

11. Angst, Von J.: Zur Frage der

Psychosen bei Behandlung mit Disulfiram

(Antabus): Literatur über Sicht und

Kasuistischer Beitrag. Schweiz. Med.,

88: 1304-1306, 1956.

12. Bailey, P. and E.W. Davis:

Effects of lesions of the

periaqueductal gray matter in the cat.

Proc. Soc. exp. Biol. Med., 51:

305-306, 1942.

139 Beamish, P. and L.G. Kiloh:

Psychoses due to amphetamine

consumption. J. Meni. Sci.,

337-343, 1960.

106:

14. Bleuler, E.: Textbook of

Psychiatry, New York, MacMillian, 1924.

131
15. Carlsson, A.: phetamine and

brain catecholamines. in: Amphetamine

and Related Compounds, E. Costa and S.

Garattini (Eds.), Raven Press, New

York, 1970, pp. 289-300.

16. Chapman, J.: Visual imagery and

motor phenomena in acute schizophrenia.

Brit. J. Psychiat., 113: 711-778, 1967.

17. Claude, H., H. Baruk and M.

Aubry: Contribution a l'étude de la

demence precoce catatonique:

Inexcitabilité labyrynthique au cours

de la catatonfe. Rev. Neurol., 1:

976-980, 1927.

18. Connell, P.H.: Amphetamine

Psychosis. Maudsley Monograph No. 5,

Oxford University Press, London, 1958.

19. Coyle, J.T. and S.H. Snyder:

Antiparkinsonian drugs: inhibition of

dopamine uptake in the corpus striatum

as a possible mechanism of action.

Science, 166: 899-901, 1969.

20. Davis, J.M. and D.D. Janowsky:

Amphetamine and methylphenidate

psychosis. Biochem. Pharmacol., suppl.

(part 2), pp. 811-815, 1974.

21. DeJong, H.: Experimental Catatonia,

Williams and Wilkins CO., Baltimore,

1945.

22. Domino, E.F.: Is the indole

hallucinogen model worthwhile pursuing?

In: Predictability in Psychopharmacolonv-Clinical and Preclinical Correlations, A. Sudilovsky, S. Gershon and

B. Beer (Eds.), Raven Press, New York

1975, pp. 247-268.

132

23. Ellinwood E.H.: Behavioral and

EEG Changes in the Amphetamine Model of

Psychosis. In:

Neuropsychopharmacologv of Monoamines

and Their Regulatorv Enzymes, E. Usdin

(Ed.),Raven Press, New York, 1974, pp.

281-197.

24. Ellinwod, E.H. and O. Escalante.

Behavioral and histopathological

findings during chronic amphetamine

intoxication. Biol. Psychiat., 2:

27-39, 1974.

25. Ellinwood, E.H. and A. Sudilovsky:

Chronic amphetamine intoxication:

behavioral model of psychoses. In:

Psychopathology and Psyhopharmacology

J.O. Cole, A.M. Freedman and A.J.

Friedhoff, (Eds.), The Johns Hopkins

University Press, Baltimore, 1973, pp.

51-70.

26. Ellinwood, E.H. and A. Sudilovsky:

Arousal and EEG changes in the

amphetamine model of psychosis. J.

Pharmacologic, 5: 27, 1974.

27. Ellinwood, E.H., A. Sudilovsky and

R. Gabrowy: Olfactory forebrain

seizures induced by methamphetamine and

disulfiram. Biol. Psychiat., 7: 89-99,

1973.

28. Ellinwood, E.H., A. Sudilovsky and

L. Nelson: Behavioral analysis of

chronic amphetamine intoxication.

Biol. Psychiat., 4: 215-230, 1972.

29. Ellinwood, E.H., A. Sudilovsky and

L. Nelson: Evolving behavior in the

clinical and experimental amphetamine

(Model) psychosis. Am. J. Psychiat.

130:1088-1093, 1973.

30. Espelin, D.E. and A.K. Done:

Amphetamine poisoning - effectiveness

of chlorpromazine, N. Eng. J. Med.,

278: 1361-1365, 1968.

31. Evarts, E.V.: Some effects of

bufotenine and lysergic acid

diethylamide on the monkey. A.M.A.

Arch. Neurol Psychiat., 75: 49-53,

1956.

32. Fitzgerald, G. and E. Stengel:

Vestibular reactivity to caloric

stimulation in schizophrenics.

Ment. Sci., 91: 93-100,

J.

1945.

33. Freeman, H. and E.H. Rodnick:

Effect of rotation on postural

steadiness in normal and in

schizophrenic subjects. Arch. Neurol

Psychiat., 48: 47-53, 1942.

34. Friedhoff A.J. Biogenic Amines

and Schizophrenia in Biological

Psychiatry, J. Mendels [Ed), John Wiley

& Sons, New York, 1973, pp. 113-129.

35. Fuller, R.W. and H. Snoddy:

Inhibition by Disulfiram of the

accelerated turnover of catecholamines

in the adrenal glands of exercised

rats. J.

156-157, 1968.

Pharm. Pharmacol., 20:

36. Fuxe, K. and U. Ungerstedt:

Histochemical, biochemical and

functional studies on central monoamine
neurons after acute and chronic

amphetamine administration. In:

Amphetamine and Related Compounds, E.

Costa and S. Garattini (Eds.), Raven

Press, New York, 1970, pp. 257-288.

37. Glowinski, J. and J. Axelrod:

Effect of drugs on the uptake, release

and metabolism of H3-norepinephrine in

the rat brain. J. Pharmacol. Exp.

Ther., 149: 43-49, 1965.

38. Goldstein, M.: Inhibition of

norepinephrine biosynthesis at the

dopamine-beta-hydroxylation stage.

Pharmacol. Rev., 18: 77-82, 1966.

39. Goldstein, M. and K. Nakajima:

The effect of disulfiram on

catecholamine levels in the brain. J.

Pharmacol. Exp. Ther., 157: 96-102,

1967.

40. Gottesfeld, H.B., M.L. Lasser,

E.J. Conway and N.M. Mann: Psychiatric

implications of the treatment of

alcoholism with tetraethylthiuram

disulfide, Quart. J. Stud. Alco., 12:

184-205, 1951.

41. Griffith, J.J., J. Oates and J.

Cavanaugh: Paranoid episodes induced

by drug J. Amer. Med. Assoc., 205: 39,

1968.

42. Gunne, E., T. Lewander: Long-term

effects of some dependence-producing

drugs on the brain monoamines. In:

Molecular Basis of Some Aspects of

Mental Activity. Wahaas, O. (Ed),

Academic Press, New York, 1967, Vol 2,

pp. 75-81.

43. Heath, R.G., W. Nesselhof, M.P.

Bishop and L.W. Byers: Behavioral and

metabolic changes associated with

administration tetraethylthiuram

disulfide (Antabuse). Dis. Nerv.

Syst., 26: 99-105, 1965.

44. Hökfelt, T., K. Fuxe, 0. Johansson

and A- Ljungdahl:

Pharmaco-histochemical evidence of the

existence of dopamine nerve terminals

in the limbic cortex. Eur. J. Pharmac,

25: 108-112, 1974.

45. Horn, A.S. and S.H. Snyder:

Chlorpromazine and dopamine:

conformational similarities that

correlate with the antischizophrenic

activity of phenothiazine drugs. Proc.

Natl. Acad. Sci. USA, 68: 2325-2328,

1971.

46. Hornykiewicz. O.: Dopamine

(3-hydroxytryptamine) and brain

function. Pharmacol. Rev., 18:

925-964, 1966.

47. Hornykiewicz, 0.: Dopamine: its

physiology, pharmacology and

pathological neurochemistry. In:

Biogenic Amines and Physiological

Membranes in Drug Therapy, part B, J.H.

Biel and L.G. Abood (Eds), M. Dekker

Inc., New York, 1971, pp. 173-258.

48. Janowsky, D.S., M.K. El-Yousef,

J.M. Davis and H.J. Sekerke:

Cholinergic antagonism of

methylphenidate induced stereotyped

behavior. Psychopharmacologia,

27:295-302, 1972.

49. Janssen, A.J.: The evolution of

the butyrophenones haloperidol and

trifluperidol from meperidinelike-4-phenylpiperidines. Int. Rev.

Neurobiol., 8: 221-263, 1965.

50. Janssen, P., C. Niemergeers, K.

Schellekens and F. Lanaerts: Is it

possible to predict the clinical

effects of neuroleptic drugs (major

tranquillizers) from animal data.

Arzneim-Forsch., 17: 841-854, 1967.

51. Jerussi, T.P. and S.D. Glick:

Apomorphine-induced rotation in normal

rats and interaction with unilateral

caudate lesions.

40: 329-334, 1975.

Psychopharmacologia,

52. Kallant, O.J.: The amphetamines -

Toxicity and Addiction. University of

Toronto Press, Toronto, 1966.

53. Kety, S.S.: Toward hypotheses for

biochemical component in the

vulnerability to schizophrenia. Semin.

Psychiat., 4: 233-238, 1972.

54. Kraepelin, E.: Dementia Praecox

and paraphrenia. Translated by R.M.

Barclay and G.M. Robertson: Robert

Krieger Publishing Company, Huntington,

N.Y., 1971.

55. Kramer, J.C., V.S. Fischman and

D.C. Littlefield: Amphetamine

abuse-pattern and effects of high doses

taken intravenously.

305-309, 1967.

Amer. Med.

Assoc., 201:

56. Lamprecht, F., R.J. Wyatt, R.

Belmaker, D.L. Murphy and W. Pollin:

Plasma dopamine beta-hydroxylase in

identical twins discordant for

133
schizophrenia. Biochem. Pharmacol.,

suppl. (part 2), 937-940, 1974.

57. Langworthy, O.R.: The Sensory

Control of Posture and Movement. A

review of the studies of Derek

Denny-Brown. Williams and Wilkins Co.,

Baltimore, 1970.

58. Lewander. T.: On the accumulation

of p-Hydroxynorephedrine in

noradrenaline neurons during chronic

administration of amphetamine in the

rat in relation to amphetamine

tolerance. Psychiatr. Neurol

Neurochir., 75: 215-218, 1972.

59. Liddon, S. and R. Satran:

Disulfiram (Antabuse) psychosis. Am.

J. Psychiat., 123: 1284-1289, 1967.

60. Magos, L.: Amphetamine reduced

stereotypy in carbon disulphide-exposed

and diethyldithiocarbamate-treated male

rats, In: The Correlation of Adverse

Effects in Man with Observations in

AnimUals, S.B. De C. Baker (Ed.),

Exerpta Medica, Amsterdam, 1971, pp.

24-27.

61. Maj., J. and E. Przegalinski:

Disulfiram and the effect of

catecholamines on neuroleptic-induced

catalepsy in mice and rats. J. Pharm.

Pharmacol., 19: 68-76, 1967.

62. Mandell, A.J. and D.S. Segal: The

psychobiology of dopamine and the

ethylated indoleamines, with

particular reference to psychiatry.

In: Biological Psychiatry J. Mendels

(Ed). John Wiley & Sons, New York,

1973, pp. 89-112.

63. Mann, N.M, E.J. Conway, H.B.

Gottesfeld and M.L. Lasser, coordinated

approach to antabuse therapy. J. Amer.

Med. Assoc., 149:40-46, 1952.

64. Matthysse, S.: Antipsychotic drug

actions: a clue to the neuropathology

of schizophrenia? Fed. Proc.,

32:200-205, 1973.

65. Mayer, 0. and V. Eybl: The effect

of diethyldithiocarbamate on

amphetamine induced behavior in rats.

J. Pharm. Pharmacol., 23, 894-896,

1971.

66. McGhie, A. and J. Chapman:

Disorders of attention and perception

in early schizophrenia. Brit. J. Med.

Psychol., 34: 103-116, 1961.

67. Meltzer, H.Y.: Central core

fibers in an acutely psychotic patient.

Evidence for a neurogenic basis for the

muscle abnormalities in the acute

psychoses. Arch. Gen. Psychiat., 27:

125-132, 1972.

68. Moore, K.E. and R.H. Rech:

Behavioral and norepinephrine-depleting

effects of disulfiram in

reserpine-pretreated rats. Arch. Int.

Pharmacodyn. Ther., 180: 413-422,

1969.

69. Myers, S., D. Caldwell and G.

Purcell: Vestibular dysfunction in

schizophrenia. Biol. Psychiat., 7:

255-261, 1973.

70. Norton, S.: The effects of

psychoactive drugs on cat behavior.

Ann. N.Y. Acad.

1969.

Sci., 159: 915-927,

71. Ornitz, E . Vestibular

dysfunction in schizophrenia and

childhood autism. Compreh. Psychiat.,

11: 159-173, 1970.

72. Randrup, A. and I. Munkvad:

Biochemical, anatomical and

psychological investigations of

stereotyped behavior induced by

amphetamines, in Amphetamines and

Related Compounds, E. Costa and S.

Garattini (Eds.), Raven Press, New

York, 1970, pp. 695-713.

73. Randrup, A. and I. Munkvad:

Pharmacology and physiology of

stereotyped behavior. J. Psychiat.

Res., 11: l-10, 1974.

74. Randrup, A. and J. Scheel-Kruger:

Diethyldithiocarbamate and amphetamine

stereotyped behaviour. J . Pharm.

Pharmacol., 18: 752, 1966.

75. Rossum, J.M. van: The

significance of dopamine-receptor

blockade for the action of neuroleptic

drugs, in Neuropsychopharmacology, H.

Brill (Ed.) Excerpta Medica

Foundation, The Hague,- 1967, pp.

321-329.

76. Rylander, G. and M. Holmberg:

Stereotype behaviour in man following

amphetamine abuse. Symposium on

Addiction and Tolerance

Amphetamines. A. Liljestrand (Ed),

Uppsala, 1970, pp: 258-262.

77. Sack, R.L. and F.K. Goodwin:

Inhibition of dopamine beta-hydroxylase

134
in manic patients, a clinical trial

with fusaric acid. Arch. Gen.

Psychiat., 31: 649-654, 1974.

78. Schirring, E.: Amphetamine

induced selective stimulation of

certain behavior items with concurrent

inhibition of others in an open field

test with rats. Behaviour, 39: l-17,

1971 l

79. Seeman, P. and T. Lee:

Antipsychotic drugs: direct

correlation between clinical potency

and presynaptic action on dopamine

neurons. Science, 188: 1217-1219,

1975.

80. Snyder, S.H.: Amphetamine

Psychosis: a "Model" schizophrenia

mediated by catecholamines. Am. J.

Psychiat., 130: 61-67, 1973.

81. Stein, L. and C.D. Wise: Possible

etiology of schizophrenia: progressive

damage to the noradrenergic reward

system by 6- hydroxydopamine. Science,

171: 1032-1036, 1971.

82. Stevens, J.R.: An anatomy of

schizophrenia? Arch. Gen. Psychiat.,

29: 177-189, 1973.

83. Strömme, J.H.: Metabolism of

disulfiram and diethyldithiocarbamate

in rats with demonstration of an in

vivo ethanol-induced inhibition of the

glucuronic acid conjugation of the

thiol, Biochem. Pharmacol. 14:

393-410, 1965.

84. Sudilovsky A.: Disruption of

behavior in cats by chronic amphetamine

intoxication, Int. J. Neurol. 10:

259-275, 1975.

85. Sudilovsky, A. and E.H. Ellinwood:

Unpublished results in Discussion,

AUTHOR

Current Concepts of Amphetamine Abuse,

E.H. Ellinwood and S. Cohen (Eds.).

U.S. Government Printing Office,

Washington, D.C., 1972.

86. Sudilovsky, A. and E.H. Ellinwood:

EEG changes and motor impairment after

disulfiram administration to cats.

Fed. Proc., 32: 791, 1973.

87. Sudilovsky A., E.H. Ellinwood, F.

Dorsey and L. Nelson. Evaluation of

the Duke University behavioral rating

inventory for drug-generated

(BRIDGE). In: Predictability in

Psychopharmacology - Preclinical and

Clinical Correlatiotions, A. Sudilovsky,

S. Gershon and B. Beer (Eds.), Raven

Press, New York 1975, pp-189-212.

88. Tatetsu, S., A. Goto and T.

Fujiwara: The Methamphetamine

Psychosis. Igaku Shoin Ltd.,

Tokyo 1956, pp. 76 and 80.

89. Taylor, K.M. and S.H. Snyder:

Differential effects of d- and

l-amphetamine on behavior and on

catecholamine disposition in dopamineand norepinephrine-containing neurons

of rat brain, Brain Research 28:

295-309, 1971.

90. Wallerstein, R.S.: Hospital

Treatment of Alcoholism, Basic Books,

New York, 1957.

91. Wise C.D. and L. Stein:

Facilitation of brain self-stimulation

by central administration

norepinephrine. Science, 163:

209-211, 1969.

92. Wise, C.D. and L. Stein: Dopamine

-hydroxylase deficits in the brains of

schizophrenic patients. Science, 181:

344-347, 1973.

Abraham Sudilovsky, M.D.: E.R. Squibb

and Sons, P.O. Box #4000, Princeton,

New Jersey 08540.

135

THE EFFECTS OF HEROIN

ON CATECHOLAMINE METABOLISM

IN MAN

Joseph J. Schildkraut,

Roger E. Meyer,

Paul J. Orsulak,

Steven M. Mirin

Mark Roffman

Patricia A, Platz,

Edwin Grab,

Mary E. Randall

Mark McDougle

INTRODUCTION

Since the observation by Vogt (34) that

morphine decreases the level of

norepinephrine in cat hypothalamus and

midbrain, numerous studies have

examined the effects of morphine on

catecholamines in the brain. In recent

years many investigators have studied

the effects of morphine on the turnover

of catecholamines either in animal

brain or in brain regions using several

different methods including: the

synthesis and accumulation of radioactively labeled norepinephrine and

dopamine from radioactively labeled

tyrosine; the rate of decline of

intracerebrally administered radioactively labeled norepinephrine; the rate

of decline of endogenous dopamine and

norepinephrine after synthesis inhibition by alpha-methylparatyrosine; the

accumulation of homovanillic acid (the

principal metabolite of dopamine); and

we have recently examined the effects

of morphine on the accumulation of

3-methoxy-4-hydroxy-phenylglycol (MHPG)

sulfate (the major metabolite of norepinephrine in rat brain (27).

Animal Studios

In a number of studies, acutely

administered morphine has been found to

increase the turnover of dopamine in

whole rodent brain as well as in the

striatum and other brain regions, as

determined by the conversion of

radiolabeled tyrosine to radiolabeled

dopamine (4,6,11,15,17,31,32), the rate

of depletion of dopamine after inhibition of its synthesis (13,22,33), and

the accumulation of homovanillic acid

(1,10,16). After chronic administration, tolerance to the effects of morphine on the turnover of dopamine in

brain has been observed in several

studies (6,13,26,32), but not in all

(4,15). During withdrawal from chronic

morphine, the turnover of dopamine in

brain appears to be decreased (13,26).

In several studies, after acute

administration of morphine, an increase

in norepinephrine turnover has been

observed both in whole mouse brain and

137
in mouse cortex, cerebellum, brain stem

and diencephalon as well as in rat

hypothalamus as determined by the

synthesis of "C-norepinephrine from

4

C-tyrosine (15,31,32), and in rat

pons-medulla but not hypothalamus as

determined by the rate of depletion of

norepinephrine after synthesis

inhibition (33). However, in several

other studies, acutely administered

morphine did not appear to alter the

turnover of norepinephrine in brain as

determined by the synthesis of

4

C-norepinephrine from 4C-tyrosine

(4,5,6,) or the rate of depletion of

norepinephrine after synthesis

inhibition (13).

In our studies, the acute

administration of morphine produced an

increase in norepinephrine turnover in

whole rat brain as determined by the

levels of MHPG-SO, in brain (24,25).

After chronic treatment with morphine,

tolerance developed to this morphineinduced increase in the levels of

MHPG-SO4 in rat brain (24,25). These

findings support the observations of

another group of investigators (26,32)

who used other techniques to demonstrate tolerance to the morphineinduced increase in norepinephrine

turnover in mouse brain. However, a

persistent increase in norepinephrine

turnover in either whole rat brain or

rat brain regions has been observed

after chronic administration of morphine in other studies (4,15).

After spontaneous withdrawal from

chronic morphine, Rosenman and Smith

(26) observed a decrease in

norepinephrine turnover in mouse brain

with return to normal by 66 hours after

withdrawal, while Neal (21) reported no

change in norepinephrine turnover in

rat brain 60 hours after morphine

withdrawal. In our studies, the level

of MHPG-SO4 in brain was significantly

reduced 16 hours after withdrawal of

morphine from tolerant rats and

returned to control values by 24 hours

after withdrawal In contrast,

after nalorphine. precipitated

withdrawal, Gunne and his associates

(13) reported an increase in

norepinephrine turnover in rat brain

(which they attributed to stress).

These differences in findings among the

various studies of the effects of acute

or chronic morphine administration or

its withdrawal on the turnover of

norepinephrine in brain may be related

to differences in doses of drug,

schedules and routes of administration,

the techniques used to determine

norepinephrine turnover, and the times

these measurements were performed in

relation to the schedule of drug

administration.

Clinical Studios

Several investigators have examined the

urinary excretion of catecholamines and

metabolites during a cycle of morphine

addiction and withdrawal in man (9,35).

In one of these studies (involving two

subjects) the urinary excretion of

norepinephrine, epinephrine, dopamine,

3-methoxy-4-hydroxy-mandelic acid (VMA)

normetanephrine (in one subject) and

metanephrine (in one subject) increased

at the beginning of the addiction phase

when the dose of morphine was increasing. Tolerance appeared to develop to

some of these changes (particularly the

increase in epinephrine, norepinephrine

and metanephrine excretion) during the

course of continued morphine

administration. During the withdrawal

phase, excretion rates of all of these

substances returned to near normal

levels (35).

In another study (involving seven

subjects), urinary excretion of

norepinephrine and epinephrine, but not

dopamine increased during the first two

weeks of the addiction cycle when the

dose of morphine was increasing.

Initially, the increase in epinephrine

excretion was greater than that of

norepinephrine; however, epinephrine

decreased during the latter part of the

ascending dose phase. During the

coarse of continued morphine

administration (at a stable dose),

tolerance was observed to the increase

in epinephrine excretion -i.e.,

epinephrine did not differ from

preaddiction values while norepinephrine tended to be higher than preaddiction levels but not as high as during the ascending dose phase. During

withdrawal of morphine, urinary norepinephrine, epinephrine and dopamine

levels were within the preaddiction

range. Urinary epinephrine was elevated when examined 7 and 17 weeks

after withdrawal (9). In a subsequent

study, Hoeldtke and Martin (14) showed

that these changes in urinary cate138
cholamines could not be ascribed to the

alterations in urine volume that occurred during morphine administration

and withdrawal.

The increase in urinary norepinephrine

and epinephrine excretion during

administration of ascending doses of

morphine in human subjects is

consistent with the results of

comparable studies in animals (12,30).

However, in contrast to the studies in

animals that showed increased

norepinephrine and epinephrine

excretion during withdrawal of morphine

(12,30) such increases were not

observed during withdrawal in the

clinical studies (9,35), possibly due

to the lower doses and slower schedules

of withdrawal.

In ongoing collaborative studies, we

have been studying the urinary

excretion of catecholamines and

metabolites in human subjects

participating in a series of

experimental protocols that involved

heroin administration. This paper

reports the initial findings of these

studies.

administration varied from subject to

subject. After the tenth day of heroin

administration, methadone was substituted for heroin. A large battery of

psychiatric and behavioral assessments

were performed during the course of

this study, but these data will not be

reported here.

This study was carried out in the

context of a program designed to

evaluate narcotic antagonist drugs in

the rehabilitation of opiate addicts.

The details of informed consent and

citizen's review procedures involved in

this work have been described elsewhere

(19).

Throughout this study, daily 24-hour

urine collections were obtained.

Urinary catecholamines and metabolites*

including norepinephrine, epinephrine,

normetanephrine, metanephrine,

3-methoxy-4-hydroxymandelic acid (VMA),

and 3-methoxy-4-hydroxy-phenlyglycol

(MHPG) were determined (2,8,29,36) on

the urine specimens obtained during the

third and second day before heroin

administration (Baseline), the day

immediately before heroin administration (BH), the first three days of

* Urinary norepinephrine is thought to derive

METHODS mainly from the peripheral sympathetic nervous

system, while urinary epinephrine comes from

the adrenal medulla. Urinary normetanephrine,

In our initial biochemical study of the the O-methylated metabolite of norepinephrine,

effects of heroin administration on is also thought to derive mainly from the peUrinary catecholamines and metabolites, ripheral sympathetic nervous system, and may

we examined these measures in nine best reflect the level of norepinephrine

subjects during an initial drug-free

baseline period, a ten day period of

present extroneuronally and available to

interact with receptors. Urinary metanephrine,

heroin administration, and a subsequent

detoxification.

the O-methylated metabolite of epinephrine,

period of methadone may provide a better index of the adrenal

All subjects in this study had a histo- medullary output of epinephrine than does unry of at least two years of heroin use changed urinary epinephrine since the urine

with two documented treatment failures contains considerably more metanephrine than

prior to study. The schedule of heroin epinephrine. Urinary VMA, the deaminated

administration allowed the patient to O-methylated metabolite of both norepinephrine

receive a maximum of 6 mg of heroin on

with an increase of 6 mg/day

and epinephrine, is the major urinary metabDay 1, olite of these catecholamines in man. Most

over each of the 10 days such that the VMA comes from peripheral sources and relativemaximum dose on Day 10 was 60 mg of ly 1ittle urinary VMA is thought to derive

heroin. Within each 24 hour period, from the brain. Norepinephrine originating in

the dosage of heroin was regulated the brain is largely excreted in the urine as

according to a complex schedule that the deaminated O-methylated metabolite MHPG,

permitted the patient maximum freedom although some urinary MHPG may also come from

in choosing the schedule of peripheral sources. Although the exact

administration, while at the same time fraction of urinary MHPG that derives from the

preventing the patient from taking more brain remains problematic (with estimates in

than one quarter of the maximum daily non-human primates ranging from 30% to 60%),

dose in any six hour period. it has been suggested that MHPG may be the

Throughout the 10 day period, virtually urinary metabolite of norepinephrine that best

all patients administered the maximum reflects the synthesis and metabolism of

dose each day, although the schedule of norepinephrine in the brain (3,18,28).

139
heroin administration (Hl,H2,H3), the

last three days of heroin

administration (H8,H9,H10), and the

first three days of methadone

detoxification (D1,D2,D3).

In order to analyze the effects of

heroin administration on these

measures, change scores were computed.

The values obtained on the third and

second day before heroin administration

(which were taken as the baseline

values for each subject) were averaged;

and for each subsequent day we computed

individual change scores expressed as a

percent of the average baseline value

for each subject. From these

individual change scores on each

subject we then computed an overall

mean value of the change score for each

day for the entire group of nine

subjects. Matched t-tests were used to

determine the statistical significance

of these daily change scores.

RESULTS

The mean baseline values for the group

of nine subjects are shown in Table 1.

The change scores for subsequent days

are shown in Figure 1. Urine volume

increased during the course of heroin

administration, with statistically

significant increases observed during

the last three days of heroin

administration and on the third day of

methadone detoxification. In contrast

to the increase in urine volume

observed with heroin administration,

creatinine excretion was not

meaningfully altered during the period

of heroin administration (Fig. 1).

TABLE 1

BASELINE VALUES FOR URINARY CATECHOLAMINES

AND METABOLITES

Measure (Units) Mean ± SEM

Urine Volume (ml/24hrs) 1134 ± 101

Creatinine (mg/24hrs) 1724 ± 93

Norepinephrine (μg/24hrs) 43 ± 6

Epinephrine (μg/24hrs) 9±2

Normetanephrine (μg/24hrs) 312 ± 40

Metanephrine (μg/24hrs) 205 ± 22

VMA (μg/24hrs) 4113 ± 378

MBPG (μg/24hrs) 1817 ± 119

FIGURE 1

URINARY CATECHOLAMINES AND METABOLITES

DURING HEROIN ADMINISTRATION

Figure 1. Urinary catecholamines and metabo- pressed as a percent of the average baseline

lites during heroin admninistration. Baseline value for each of the nine subjects were

values for each measure were obtained by tak- computed. From these individual change scores

ing the average of the values for the third the overall mean change score for each day for

and second day before heroin administration. the entire group of nine subjects was computed.

For each subsequent day, change scores ex- *p<0.05 (matched t-test).

140
Figure 1 shows that all of the measures

of urinary catecholamines and

metabolites tended to be increased over

baseline values on the first day of

heroin administration, with the largest

percentage increases occurring in

noerpinephrine, epinephrine and

normetanephrine. However, during the

subsequent course of heroin

administration, markedly different

patterns of change emerged for each of

these measures.

As shown in Figure 1, norepinephrine

tended to be increased throughout the

course of heroin administration. In

contrast, epinephrine, which was

increased during the early phase of

heroin administration, returned to

baseline values during the later period

of heroin administration.

Normetanephrine excretion was markedly

increased throughout the period of

heroin administration. Highest values

of normetanephrine occurred during the

latter phase of heroin administration,

and there was some return toward

baseline values during methadone

detoxification (Fig.1).

In contrast to this persistent increase

in normetanephrine excretion, after the

first day of heroin administration

metanephrine excretion decreased with

statistically significant decrements

noted during the last three days of

heroin administration. During

methadone detoxification, metanephrine

excretion returned toward baseline

values (Fig.1).

After increasing on the first day of

heroin administration, VMA returned to

approximately baseline values (with the

exception of an elevation on the ninth

day of heroin administration that was

not statistically significant and may

be an artifact). Since VMA is the

major deaminated-O-methylated metabolite of both norepinephrine and epinephrine in the periphery, the lack of

change in VMA excretion during heroin

administration may be explained by the

finding that normetanephrine was increased but metanephrine was decreased

(Fig. 1).

In these studies, we were particularly

interested in the urinary excretion of

MHPG, since (as noted above) an

appreciable fraction of this metabolite

may be derived from norepinephrine

originating in the brain. As shown in

Figure 1, MHPG excretion tended to be

elevated throughout the course of

heroin administration with statistically significant increases observed on

Days 8 and 10. During methadone detoxification, there was a return toward

baseline values.

Further analysis of the data on MHPG

excretion (Fig. 21, indicated that a

meaningful increase in MHPG excretion

was not consistently observed in all of

the subjects but only in a sub-group

(four of the nine subjects). This is

in contrast to the increase in

normentanephrine excretion and the

decrease in metanephrine excretion that

was observed in the entire group of

nine subjects. Moreover, it appeared

FIGURE 2

Figure 2. MHPG excretion before and during

heroin administration in subjects with and

without increased MHPG excretion during heroin

administration. Individual values of MHPG

excretion were averaged across subjects for

each of the following periods: baseline (i.e.,

the third and second day before heroin administration); day before heroin; early heroin

(i.e., the first three days of heroin a&inisration--H1,H2,H3); late heroin (i.e., the

last three days of heroin administration--H8,

H9,H10); and detoxification (i.e., the first

3 days of methadone detoxification).

141
as if the increase in MHPG excretion

began on the day prior to the

administration of heroin in the

subgroup of patients with increased

MHPG excretion during heroin

administration (Fig. 2A).

DISCUSSION

In the present study of the effects of

heroin administration on catecholamine

metabolism in man, norepinephrine

tended to be increased throughout

heroin administration while epinephrine

was increased above baseline only

during the early phase of heroin

administration. These findings are in

agreement with the results of earlier

studies (9,35) as reviewed above. The

increase in urine volume observed

during heroin administration in this

study also agrees with earlier reports

of increased urine volume during

morphine administration (9,35).

The results of the present study showed

a significant increase in urinary

normetanephrine throughout heroin

administration. Metanephrine which

increased on the first day of heroin

administration, decreased during the

subsequent days of heroin

administration. A decrease in

phenylethanolamine-N-methyl-transferase

(PNMT) activity in the adrenal gland of

the rat has been described during

chronic morphine administration (23),

and such a decrease in adrenal PNMT

activity might account for this

decrease in metanephrine excretion

during heroin administration.

During heroin administration, an

increase in MHPG excretion was observed

in a subgroup of four of the nine

subjects studies. Since we have

observed that tolerance develops to the

morphine-induced increase in

in

MHPG-SO4

rat brain, it is conceivable that

persistence of, or development of,

tolerance might account for the failure

to observe an increase in MHPG

excretion in all of the subjects.

As reported elsewhere physiological and

behavioral data suggested that subjects

in the subgroup with increased MHPG

excretion during heroin administration

manifested a greater response to

injected heroin than did the remaining

subjects (20). Three of the subjects

in this subgroup elected to administer

heroin more frequently, particularly in

the latter phase of administration, and

tended to have higher plasma-morphine

levels. (However, the total amount of

heroin administered, which was limited

by the design of this study, was not

different in this subgroup). All four

of the subjects in this subgroup showed

higher scores on measures of opiate

intoxication and nodding behavior as

well as the most pronounced respiratory

depression (after each dose)

particularly during the latter phase of

heroin administration. Further studies

will be required to confirm and extend

these observations.

The observation that the increase in

MHPG excretion began on the day prior

to the administration of heroin, in the

subgroup of patients with increased

MHPG excretion during heroin administration, suggests the possibility of an

anticipatory or conditioned response in

these subjects, with the anticipation

of heroin administration producing an

increase in MHPG excretion. The subsequent administration of heroin in

these subjects may then be required to

maintain MHPG excretion at this level,

and this might conceivably be related

to the finding that these subjects

elected to administer heroin more

frequently. Further observations on a

larger number of subjects will be

required to confirm this suggestion of

an anticipatory increase in MHPG

excretion prior to heroin administration; and correlative studies in animals should help to determine whether

opiate-induced increases in norepinephrine turnover (as reflected by MHPG

levels) in brain can, in fact, be conditioned.

As described above, in our studies in

animals, using levels of endogenous

MHPG-SO4 in brain as an index of

norepinephrine turnover, we have found

that one of the neuropharmacological

effects of acute administration of

morphine is to increase the turnover of

norepinephrine in the brain (24,25).

with continued administration of

morphine, tolerance appears to develop

to this effect on norepinephrine

turnover and our findings further

suggest that under these conditions the

normal level of functioning of

noradrenergic neuronal systems in brain

may be dependent upon the continued

administration of maintenance doses of

morphine. Similar conclusions have

been reported by Smith and his

associates (31,32) who used a different

technique to assess norepinephrine

turnover in the brain.

142
Self-administration of morphine in

animals can be attenuated by inhibition

of norepinephrine biosynthesis (7).

Thus, it is possible that the acute

effects of morphine on norepinephrine

turnover may help to account for the

initial reinforcing properties of this

drug, while its longterm effects on

noradrenergic neurons may help to

account for opiate-seeking behavior in

addicted subjects. Further basic and

clinical studies will be required to

explore this possibility.

ACKNOWLEDGMENT

This work was supported, in part, by

Grant No. DA 00257 from the National

Institute on Drug Abuse.

REFERENCES

1. Ahtee, L. and I. Kaariainen. The

effects of narcotic analgesics on the

homovanillic acid content of rat

nucleus caudatus. Eur. J. Pharmacol.

22: 206-208, 1973.

2. Anton, A.H. and D.F. Sayre. A

study of the factors affecting the

aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. 138: 360-375,

1962.

3. Axelrod, J. Noradrenaline: fate

and control of its biosynthesis.

Science 173: 598-606, 1972.

4 . Clouet, D.H. and M. Ratner.

Catecholamine biosynthesis in brains of

rats treated with morphine. Science

168: 854-855, 1970.

5. Clouet, D.H., J.C. Johnson, M.

Ratner, M. Williams and G.J. Gold. The

effect of morphine on rat brain

catecholamines: turnover in vivo and

uptake in isolate synaptosomes. In:

Frontiers of Catecholamine Research, E.

Usdin and S. Snyder, Eds., Pergamon,

New York, pp. 1039-1042, 1973.

6 . Costa, E., A. Carenzi, A. Guidotti

and A. Revuelta. Narcotic analgesics

and the regulation of neuronal

catecholamine stores. In: Frontiers

in Catecholamine Research, E. Usdin and

S . Snyder, Eds., Pergamon, New York,

pp. 1003-1010, 1973.

7. Davis, W. and S . Smith.

Noradrenergic basis for reinforcement

associated with morphine action in

non-dependent rats. In: Drug

Addiction Vol. 3, Neurobiologv and

Influences on Behavuior, J.M. Singh and

H . Lal, Eds., Symposia

Miami, 1974.

Specialists,

Psychiat. Res; 7: 19-28, 1969.

a. Dekirmenjian, H. and J.W. Maas. An

improved procedure of 3-methoxy4-hydroxyphenylglycol determination by

gas-liquid chromatography. Anal.

Biochem. 35: 113-122, 1970.

9 . Eiseman, A.J., J.W. Sloan, W.R.

Martin, D.R. Jasinski and J.W. Brooks.

Catecholamine and 17-hydroxycorticosteroid excretion during a cycle of

morphine dependence in man. J.

10. Fukui, K. and H. Takagi. Effects

of morphine on the cerebral contents of

metabolites of dopamine in normal and

tolerant mice: its possible relation

to analgesic action. Brit. J.

Pharmacol. 44: 45-51, 1972.

11. Gauchy, C., Y. Agid, J. Glowinski

and A. Cheramy. Acute effects of

morphine on dopamine synthesis and

release and tyrosine metabolism in the

rat striatum. Eur. J. Pharmacol. 22:

311-314, 1973.

12. Gunne, L.M. Catecholamines and

5-hydroxytryptamine in morphine

tolerance and withdrawal. Acta

Physiol. Scand. 58: Supplementum 204,

1963.

13. Gunne, L.M., J. Jonsson and K.

Fuxe. Effects of morphine intoxication

on brain catecholamine neurons. Eur.

J. Pharmacol. 5: 338-342, 1969.

14. Hoeldtke, R.D. and W.R. Martin.

Urine volume and catecholamine

excretion. J. Lab. Clin. Med. 75:

166-174, 1970.

15. Johnson, J.C., M. Ratner, G.J.

Gold and D.H. Clouet. Morpbhine effects

on the levels and turnover of

catecholamines in rat brain. Res.

Comm. Chem. Path. Pharmacol. 9: 41-53,

1974.

16. Kuschinsky, K . and 0.

Hornykiewicz. Effects of morphine on

striatal dopamine metabolism: possible

mechanism of its opposite effect on

locomotor activity in rats and mice.

Eur. J. Pharmacol. 26: 41-50, 1974.

143
17. Loh, H.H., R.J. Hitzemann and E.L.

Way. Effects of acute morphine

administration on the metabolism of

brain catecholamines. Life Sci. 12:

33-41, 1973.

18. Maas, J.W., H. Dekirmenjian, D.

Garver. D.E.

Landis: Catecholamine

Redmond. Jr., and H.D.

metabolite

excretion following intraventricular

injection of 6-OH-dopamine. Brain.

Res. 41: 507-511, 1971.

19. Meyer, R.E., Mirin, J.L.

paradigm for the evaluation of narcotic

antagonists. Arch. Gen. Psychiat. (in

press).

Altman and H.B. McNamee. Behavioral

20. Meyer, R.E., J.J. Schildkraut,

S.M. Mirin, P.J. Orsulak, M. Randall,

M. McDougle, E. Grab and P. Platz.

Catecholamine excretion, behavior and

mood in opiate addiction. In

preparation.

21. Neal, M.F. Failure of morphine

dependence in rats to influence brain

noradrenlin turnover. J. Pharm.

Pharmacol. 20: 950-953, 1968.

22. Puri, S.K., C. Reddy and H. Lal.

Blockade of central dopamine receptors

by morphine: effect of haloperidol,

apomorphine and benztropine. Res.

Comm. Chem. Path. Pharmacol. 5:

389-401, 1973.

23. Reis, D.J., P. Hess, E.C. Azmitia,

Jr. Changes in enzymes subserving

catecholamine metabolism in morphine

tolerance and withdrawal in rat. Brain

Res. 20: 304-312, 1970.

24. Hoffman, M., T. Reigle, P.

Orsulak, J . Hardin and J.J.

Schildkraut. Effects of morphine on

the turnover of norepinephrine in rat

brain. The Pharmacologist 16: 456

(ABS), 1974.

25. Roffman, M., T. Reigle, P. Orsulak

and J.J. Schildkraut. The effects of

acute and chronic morphine administration on the levels of 3-methoxy4-hydroxyphenylglycol sulfate in rat

brain. Res. Comm. Chem. Path. Pharmacol. 10: 403-417, 1975.

26. Rosenman, S.J. and C.B. 14C-catecholamine synthesis in mouse

Smith.

brain during morphine withdrawal.

Nature 240: 153-155, 1972.

27. Schanberg, S.M., J.J. Schildkraut,

G.R. Breese and I.J. Kopin. Metabolism

of normetanephrine-H. in rat brain:

identification of conjugated 3-methoxy4-hydroxyphenylglycol as the major

metabolite. Biochem. Pharmacol. 17:

247-254, 1968.

28. Schildkraut, J.J., Neuropsychopharmacology and the Affective

orders Little, Brown & Co.,

DisBoston,

1970.

29. Schildkraut, J.J. E.K. Gordon and

J. Durell. Catecholamine metabolism in

affective disorders I. Normetanephrine

and VMA excretion in depressed Patients

treated with imipramine. J. Psychiat.

Res. 3: 213-228, 1965.

30. Sloan, J.W. and A.J. Eisenman.

Long persisting changes in

catecholamine metabolism following

addiction to and withdrawal from

morphine. Ass. Res. Nerv. Ment. Dis.

46: 96-105, 1968.

31. Smith, C.B., J.E. Villarreal, J.H.

Bednarczyk and M.I. Sheldon. Tolerance

to morphine-induced increases in

..C-catecholamine synthesis in mouse

brain. Science 170: 1106-1107, 1970.

32. Smith, C.B., M.I. Sheldon, J.H.

Bednarczyk and J.E. Villareal.

Morphine-induced increase in the

incorporation of ..C-dopamine and

..C-norepinephrine in the mouse brain:

antagonism by naloxone and tolerance.

J. Pharmacol. Exp. Ther. 180: 547-557,

1972.

33. Sugrue, M.F. The effects of

acutely administered analgesics on the

turnover of noradrenlin and dopamine in

various regions of the rat brain.

Brit. J. Pharmacol. 52: 159-165, 1974.

34. Vogt, M. The concentration of

sympathin in different parts of the

central nervous system under normal

conditions and after the administration

of drugs. J. Physiol. 123: 451-481,

1954.

35. Weil-Malherbe, H., E.R.B. Smith,

A.J. Eisenman and H.F. Fraser. Plasma

catecholamine levels and urinary

excretion of catecholamines and

metabolites in two human subjects

during a cycle of morphine addiction

and withdrawal. Biochem. Pharmacol. 14:

1621-1633, 1965.

144
36. Wilk, S., S.E. Gitlow, M.

Mendlowitz, N.J. Franklin, H.E. Carr

and D.D. Clark. A quantitative assay

for vanillylmandelic acid (VMA) by

gas-liquid chromatography. Anal.

Biochem. 13: 544-551, 1965.

AUTHORS

Joseph J. Schildkraut, M.D., Roger E.

Meyer, M.D., Paul J. Orsulak, Ph.D.,

Steven M. Mirin, M.D., Mark Hoffman,

Ph.D., Patricia A. Platz, B.S., Edwin

Grab, A.M., Mary E. Randall, M.A., Mark

McDougle, B.A.

Neuropsychopharmacology Laboratory,

Massachusetts Mental Health Center,

Boston, Massachusetts 02115;

Alcohol and Drug Abuse Research Center,

McLean Hospital, Belmont, Massachusetts

02178; and

Department of Psychiatry, Harvard

Medical School, Boston, Massachusetts

02115

145

CONCLUSION

The purpose of this monograph was to bring together

in a single volume papers describing different lines

of current research in the area of brain amine

functioning. Such a broad approach is necessary for

the evaluation of the entire state of the art at this

time.

Several conclusions derive from these papers. First,

there ore several hypotheses of behavior, not all of

which are complementary or consistent. Secondly, these

hypotheses vary greatly with regard to presently

available supporting data and the breadth of behavior

each hypothesis encompasses. Thirdly, with only a

few exceptions, these theoretical constructs concerning the roles of the brain monoamines are based

upon correlations obtained from highly specific experimental manipulations, the results of which are difficult to generalize to other aminergic research.

The problem, as suggested in the Introduction and

several of these papers, probably lies in some assumptions until recently underlying aminergic research

and the techniques being employed. For example, it

appears doubtful that results of experiments measuring whole-brain or even large section amine levels

can in any functional way be related to alterations

in specific behavioral syndromes in anything but the

crudest manner. Futhermore, there exists a basic

problem with the dependent variables we seek to

measure. Variables such as changes in brain amine

levels, ratios or turnover rates even in very

limited brain areas, may in fact have little functional

relationship to the behaviors under investigation.

Additional basic research is required to determine

which measurements are reliable and valid in terms of

the functioning of brain neuronal systems. New

approaches to the problem, new methodologies and even

entirely new questions are needed.

The advent of new dissection and separation techniques

together with more sensitive analytical tools such as

the gas chromatograph-mass spectorscopy combination

may provide some of these much needed answers. Other

techniques such as the electrical identification of

single cell units which selectively fire in response

to highly specific visual stimuli but not to other

visual stimulation, the use of an imunological approach to selectively bind hypothesized receptors in

147
specific brain regions and a number of as yet untried

approaches, may aid in providing the needed impetus

to research in this area.

As our techniques become increasingly refined and

new methodologies and approaches are employed, our

understanding of functional neuronal dynamics will

increase. With this will come on increased ability

to quantify and evaluate the relevance of a multiplicity of dependent variables with more precision.

As that occurs, so will our ability increase to see

relationships across behaviors and ultimately our

ability to generalize from them. Thus, by a kind of

paradox must we progress through ever greater intellectual and technical specificity in order to

achieve greater conceptual generality.

This publication was not meant to be comprehensive.

There are additional areas of amine research which

are not covered in this volume. Investigations into

the biochemical mechanisms of alcoholism, narcotic

addiction and drug abuse have significant social

importance. Yet, advances in these fields hove also

been hindered by the above mentioned problems.

It is my hope that the questioning put forth in this

monograph, the growing concern of my fellow researchers for the generalizability of their work, the new

approaches to these problems and the greater sophistication of older techniques, together with an increased scientific and social impetus to understand

and deal with these areas, will culminate in a

renewed and sustained drive towards understanding

the workings of the central nervous system and in

particular the functioning of the brain monoamines.

Bruce K. Bernard

Storrs, Connecticut

1975

148
U. S. GOVERNMENT PRINTING OFFICE : 1975 632-513/1084
DEPARTMENT OF

HEALTH, EDUCATION, AND WELFARE

PUBLIC HEALTH SERVICE

ALCOHOL, DRUG ABUSE, AND

MENTAL HEALTH ADMINISTRATION

5600 FISHERS LANE

ROCKVILLE, MARYLAND 20852

OFFICIAL BUSINESS

Penalty for private use, $300

POSTAGE AND FEES PAID

U.S, DEPARTMENT OF H.E.W

HEW 389

NOTICE OF MAILING CHANGE

Check here if you wish to discontinue receiving this type of publication.

Check here if your address has changed and you wish to continue receiving this

type of publication. (Be sure to furnish your complete address including zip code.)

Tear off cover with address label still affixed and send to:

Alcohol, Drug Abuse, and Mental Health Administration

Printing and Publications Management Section

5600 Fishers Lane (Rm. 6-105)

Rockville, Maryland 20852

DHEW Publication No. (ADM) 76-295

Printed 1975

U.S. DEPARTMENT OF HEALTH. EDUCATION. AND WELFARE

PUBLIC HEALTH SERVICE

ALCOHOL, DRUG ABUSE, AND MENTAL HEALTH ADMINISTRATION