Mechanisms of

Tolerance and

Dependence

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Mechanisms of Tolerance

and Dependence

Editor:

Charles Wm. Sharp. Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

NIDA Research Monograph 54

1984

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

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NB Associates, Health Sciences

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Alcohol and Drug Problems Association

of North America

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Cornell University Medical College

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JOSEPH V. BRADY, Ph. D.

The Johns Hopkins University School of

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Washington University School of

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National Federation of Parents for

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Langley Porter Neuropsychiatric Institute

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Yale University School of Medicine

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NIDA Research Monograph Series

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Parklawn Building, 5600 Fishers Lane, Rockville, Maryland 20857
Mechanisms of Tolerance

and Dependence
ACKNOWLEDGEMENT

This monograph is based upon papers presented at a technical review

which was held on November 11 and 12, 1983, in Boston,

Massachusetts. The meeting was sponsored by the Division of

Preclinical Research, National Institute on Drug Abuse.

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Alcohol, Drug Abuse, and Mental Health Administration

Printed 1984 Reprinted 1988

NIDA Research Monographs are indexed in the Index Medicus. They are

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Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

iv
Contents

Preface. . . . . . . . . . . . . . . . . . . .

In Memoriam: H. 0. J. Collier

Sydney Archer . . . . . . . . . . . . . . . . .

Mechanisms of Tolerance and Dependence: An Overview

Leo G. Abood . . . . . . . . . . . . . . . . .

Phenomenology and Theoretical Basis of Tolerance and

Dependence

William R. Martin. . . . . . . . . . . . . . . . 12

Contributions of Neuropharmacology to Understanding

Mechanisms of Tolerance and Dependence

Martin W. Adler and Ellen B. Geller. . . . . . . . . .

Various Factors Which Affect the Rate of Development of

Tolerance and Physical Dependence to Abused Drugs

William L. Dewey . . . . . . . . . . . . . . . .

Genetic Determinants of Responses to Drugs of Abuse:

An Evaluation of Research Strategies

Louis Shuster . . . . . . . . . . . . . . . . .

Opioid Tolerance and Dependence in Light of the

Multiplicity of Opioid Receptors

Rudiger Schulz and Albert Herz . . . . . . . . . . .

Sites and Mechanisms of Dependence in the Myenteric

Plexus of Guinea Pig Ileum

H. 0. J. Collier and J. F. Tucker . . . . . . . . . .

Biochemical Reactions Between Opiate Receptor Binding

and Inhibition of Neurotransmission

Doris H. Clouet and Norifumi Yonehara . . . . . . . . .

The Role of Adenyl Cyclase in Opiate Tolerance and Dependence

Werner A. Klee, Graeme Milligan, William S. Simonds,

and Bruno Tocque . . . . . . . . . . . . . . . .

ix

1

4

27

39

50

70

81

95

109

v
Mechanisms of Multiple Cellular Adaptation Processes in

Clonal Cell Lines During Chronic Opiate Treatment

Ping-Yee Law, Michael T. Griffin, and Horace H. Loh . . . . 119

The Opioid-Induced Desensitization (Tolerance) in

Neuroblastoma X Glioma NG 108-15 Hybrid Cells:

Results from Receptor Uncoupling

Michael Wuster and Tomnaso Costa . . . . . . . . . . . 136

Opiate Receptor Upregulation and Functional Supersensitivity

R. Suzanne Zukin, Ann Tempel, and Eliot L. Gardner. . . . . 146

The Role of Dynorphin in Narcotic Tolerance Mechanisms

Nancy M. Lee . . . . . . . . , . . . . . . . . 162

Post-Translational Processing of Neuropeptide Precursors

Richard E. Mains and Betty A. Eipper . . . . . . . . . 168

Opiate-Mediated Control of Luteinizing Hormone in the Male;

Role in the Development of Narcotic Tolerance and

Physical Dependence

Theodore J. Cicero . . . . . . . . . . . . . . . 184

Modulation of Opioid Physical Dependence During the

Hibernation Cycle

Alexander L. Beckman, Carmen Llados-Eckman,

Steven K. Salzman, and Toni L. Stanton . . . . . . . . . 209

Neurophysiological Investigations of Opiate Tolerance

and Dependence in the Central Nervous System

Steven Jon Henriksen . . . . . . . . . . . . . . . 239

Spinal Cord Tissue Culture Models for Analyses of

Opioid Analgesia, Tolerance and Plasticity

Stanley M. Crain . . . . . . . . . . . . . . . . 260

Experimental Approaches Toward Determining the Neuronal

Circuits Most Affected by Chronic Opiate Administration

Robert Elde . . . . . . . . . . . . . . . . . . 293

Effects of Opiates and Neuropeptides on Immune Functions

Robert E. Faith, N. P. Plotnikoff, and A. J. Murgo. . . . . 300

Changes in CNS Sensitivity to Cannabinoids with Repeated

Treatment: Tolerance and Auxoesthesia

R. Karler, L. D. Calder, and S. A. Turkanis . . . . . . . 312

Catecholamine-Independent Behavioral and Neurochemical

Effects of Cocaine in Rats

Karen Gale . . . . . . . . . . . . . . . . . . 323

Barbiturate Tolerance and Physical Dependence: Contribution

of Pharmacological Factors

Michiko Okamoto . . . . . . . . . . . . . , . . 333

vi
Behavioral and Biochemical Studies in Rats After Chronic

Exposure to Nicotine

Leo G. Abood, Susan Grassi, Maria Costanzo, and Jeffrey Junig . 348

Psychobiological Foundations of Behaviors Induced by Amphetamines

Philip M. Groves and David S. Segal . . . . . . . . . . 356

Index . . . . . . . . . . . . . . . . . . . . 367

List of NIDA Research Monographs . . . . . . . . . . . 371

vii

Preface

When the topic of mechanisms of tolerance/dependence was considered

by the National Institute on Drug Abuse for a major review, it

seemed at first to lack sufficient substance. Although there is

voluminous material on pharmacological dose response and on signs

and symptoms associated with the actions of opiates and other drugs,

only limited progress has been made on defining the basic mechanisms

involved.

For several reasons, progress has been slow in this area. As Eric

Simon stated at the symposium, progress has been encumbered by

several factors, some of which will eventually assist in defining

basic biochemical systems and subsequently the area of tolerance and

dependence. These include the discovery that there are several

types of opiate receptors, the discovery of at least three types of

endogenous pro-opioid peptides, the difficulties in measuring

dynamic biological turnover of these peptides and/or related

systems; the lack of an understanding of post-receptor events

(especially in vivo); and finally, the difficulty in establishing

any regulation of opiate receptors.

Yet progress has been made, and it is essential that we continue to

define the mechanisms of tolerance and/or dependence. This knowledge

is important in determining the underlying biological factors that

lead some individuals to abuse drugs. When we have more precise

knowledge of these processes we will be able to cope more

effectively with the ever increasing drug problem.

It took the quiet enthusiasm of Professor H.O.J. Collier during a

visit to NIDA in late 1982 to instigate this review. His confidence

in his colleagues and especially in his own work was contagious.

Once the seed had been planted with Institute Director Dr. William

Pollin and the rest of the NIDA staff, we undertook the task of

organizing this review of important recent findings defining the

mechanisms of tolerance and dependence.

We were buoyed by the interest and enthusiasm of those we spoke with

regarding a discussion of this topic--one that has been of major

concern to NIDA for a long time. Ever since the discovery of

endogenous opioids in 1975, the Institute has encouraged

investigators not only to identify the site of action of those

substances, but more importantly, to utilize these substances and

their systems to study the mechanisms through which tolerance and/or

dependence are mediated.

ix
Unfortunately, shortly before the meeting, Professor Collier died.

Thus, not only did the group miss his knowledgeable contributions,

we missed and will continue to miss his probing and articulate

discussions. In lieu of the original thoughts Professor Collier

would have contributed at the meeting, it is worth reflecting on his

most recent thoughts on the topic, which were published recently

following another symposium.1 We have drawn from this

illuminating manuscript pertinent and provocative excerpts which may

serve as guidelines for those interested in narcotic tolerance and

dependence.

Let us first consider what he thought to be important criteria for

defining tolerance and dependence:

To analyse cellular mechanisms of tolerance and

dependence, it is necessary to reproduce models of these

states in isolated or partly isolated preparations. It

is therefore important to have strict criteria of

tolerance and dependence with which to recognize them in

such models. Tolerance is generally agreed to be a

lessened response to a drug after its continued

application, and which may be observed in several ways:

1. Decreased response to the same dose of drug.

2. Increased dose of drug to yield the same response.

3. Lessened response to drug during continued exposure.

Dependence is, however, more difficult to define and

three criteria have largely been used:

1. "Spontaneous behavioural disturbance produced by

removal of drug and suppressed by its replacement.

2. Increased potency of the drug's specific antagonist.

3. Behavioural disturbance on precipitated withdrawal of

drug with its specific antagonist.

4. Heightened response to external stimulation after

withdrawal of inhibitory drug.....(p.109)

Another criterion, sometimes also used to measure the

intensity of dependence, is a heightened potency of the

drug's specific antagonist....A third criterion is also

sometimes used--that of increased excitability of the

preparation to external stimulation after withdrawal of

an inhibitory drug such as opiate....(p.109-110)

Whatever criteria of dependence we use, valid ones, in my

opinion, rely on withdrawing a drug from a preparation

exposed to it....We cannot measure tolerance, however,

without applying the drug. We therefore cannot measure

both tolerance and dependence in the same preparation at

the same time. This principle of uncertainty makes it

difficult to determine the relationship between opioid

tolerance and dependence, as may be illustrated by

experiments on the isolated ileum of the

guinea-pig....(p.11O)

Naturally, the uncertainty principle does not stop us

measuring tolerance and dependence in parallel

x
preparations at the same time, or in the same preparation at

different times. To validate such measurements, large numbers

of replicates are needed for which isolated preparations are

suitable....(p.111)

Based upon the above considerations, Professor Collier chose the

guinea-pig ileum as a model for exploring these areas of research.

Before he utilized the guinea pig ileum for these investigations, he

considered several important questions:

First, how closely does opioid dependence in the

preparation resemble that in experimental animals or man

in vivo? Second, can dependence on drugs other than

opioids be induced in the guinea pig ileum and, if so,

how are the different dependencies related? Third, what

neurone or combination of neurones of the myenteric

plexus participates in these dependencies? Fourth, what

are the cellular and molecular mechanisms of dependence

in the neurones involved? (p.116-117)

Furthermore, he considered the breadth of the problem:

That dependence occurs within the opiate-sensitive

neurone does not mean that factors outside this cell may

not also contribute to the effect. Among these factors,

which may be termed circumcellular, I include:

supersensitivity of a neurone or muscle cell downstream

of the opiate-sensitive neurone; and changes in the

production by adjoining cells of endogenous opioid

transmitter or of an enzyme destroying it. (p.115)

Thus, embarking on his inquest, he recently provided important

observations and conclusions to assist in the development of his

thoughts and hypothesis. These include:

...The same neurone can be made dependent on three

distinct types of agonist. When the neurone is dependent

on one of these agonists a withdrawal contracture cannot

be precipitated with specific antagonists of either of

the other two types of drug....Any one of the three

different types of agonist, however, readily suppresses

the withdrawal contracture of ileum made dependent on

either of the other two agonists. We have therefore

termed convergent those dependencies that begin through

activation of different recognition sites in the

cholinergic motor neurone and end in the common process

of liberating acetycholine at the terminal on withdrawal.

Since we have not yet observed any difference in

character between the three convergent dependencies in

this neurone, other than in the inducing drugs and their

specific antagonists, we provisionally assume that they

have a common mechanism, which must lie downstream of the

different recognition sites. (p.118)

xi
He further postulated:

That dependence occurs in the cholinergic neurone at a

point below the recognition site still leaves a number of

possible mechanisms for opioid dependence. The

likelihood that several of these operate is diminished by

the finding that withdrawal elicits a sharp increase in

impulse production recorded at the cell body...Hence the

most likely possibilities are that dependence occurs in

the post-recognition site part of the receptor apparatus

or in an associated second messenger system that

translates the message of site activation into the

language of cellular response. (p.120)

In regard to why progress in this area developed slowly, Collier

stated:

Until recently, there has been relatively little

opportunity to test these hypotheses in normal neurones,

but the discovery of convergent dependencies on

normorphine, clonidine and adenosine in the final

cholinergic motor neurone...now offers an opportunity to

do so. The general concept that dependence in normal

neurones arises in response to continued inhibition of

adenylate cyclase is supported by the fact that all of

the three agonists that induce dependence in the

myenteric plexus...have been shown to inhibit adenylate

cyclase in other biological preparations, as a result of

binding with their distinct and specific recognition

sites. Unfortunately, it has so far proved impossible to

test directly whether normorphine, clonidine and

adenosine each inhibits adenylate cyclase in the

cholinergic motor neurone of the myenteric plexus.

(pp.121-122)

Yet he remained optimistic that we were poised at the edge of a

major scientific breakthrough. He ended his paper with the

following statements those of us in the field should well remember.

1. The relationship between opiate tolerance and

dependence is not well understood...

2. The withdrawal syndrome of opiate dependence may

represent a physiological mechanism for rapid arousal

from inhibition induced by endogenous opioid.

3. The main site of opiate dependence is the opiatesensitive neurone...

4. The isolated ileum of the guinea-pig so far provides

the only model in which opiate dependence

and associated tolerance can be consistently induced and

measured in normal neurones in vitro....

xii
5. The characteristics of opiate dependence displayed by

the ileal model closely resemble those of dependence in

whole animals...

6. Distinct but convergent dependences on normorphine,

clonidine and adenosine can be separately induced in the

guinea-pig ileum in vitro.

7. ...The final cholinergic motoneurone of the myenteric

plexus participates in all three of these dependences.

8. With the cholinergic motor neurone these dependences

probably arise at a point below the separate recognition

sites and above the site of acetylcholine release at the

terminal.

9. A second transmitter, substance P, also participates

in dependences in the myenteric plexus. (p.123)

10. There is direct experimental evidence that opiate

dependence and associated tolerance in cultures of

neuroblastoma x glioma hybrid cells arises through an

hypertrophy of cellular adenylate cyclase.

FOOTNOTE

1. The quotations herein are taken from: Collier, H.O.J.

Cellular aspects of opioid tolerance and dependence. In:

Hughes, J.; Collier, H.O.J.; Rance, M.J.; and Tyers, M.B., eds.

Opioids, Past, Present and Future. London and Philadelphia:

Taylor & Francis 1984. pp. 109-125. Copyright 1984, Taylor &

Francis Ltd.

xiii

In Memoriam: H. O. J. Collier

On 29 August 1983, Harry Collier passed away, the victim of a

failed heart that for the past several years was sustained by a

pacemaker. To those who knew him as a friend as well as a

colleague, his death is a severe personal loss. This news was

particularly poignant in our household because on the very day he

died we received a letter from Harry confirming that he would visit

us right after the technical review on mechanisms of tolerance and

dependence on November 11-12, 1983. Unfortunately, the visit was

not to be. Instead, with a heavy heart and tearful eye, I have

prepared a eulogy for the man to whom the meeting and this

monograph are dedicated.

For this purpose, I have borrowed heavily from the obituary which

appeared in the Times of London on 2 September 1983. It was

written by Harry's son, Joseph Collier, and his former son-in-law,

Andrew Herxheimer. both of whom are pharmacologists.

Henry Oswald Jackson Collier was born on March 14, 1912, and

educated at the Royal Grammar School, Worcester, from where he went

to Trinity Hall, Cambridge. Here he graduated B.A. with First

Class Honors in the Natural Sciences Tripos (an honors examination)

in 1933 and went on to do research in the Department of Zoology,

being awarded his Ph.D. in 1938.

1
From 1937 to 1941 he was an assistant lecturer and demonstrator in

Comparative Physiology at the University of Manchester, and in 1941

he joined Imperial Chemical (Pharmaceuticals) Ltd. as a parasitologist. From here he was loaned to do research in chemotherapy at

Liverpool School of Tropical Medicine.

In 1945 he went to Allen and Hanbury's Ltd. at Mare to set up a new

pharmacology department for the firm. This he built up and ran

until 1958 when he left to join Parke-Davis at Hounslow as Director

of Pharmacological Research. Here he remained until 1969.

During this period, the natural materials known as prostaglandins

were discovered and recognized to be a novel kind of locally

produced and locally effective hormone, "the coinage of the body's

defenses," as Harry later described them.

In 1968, indeed, he even suggested that anti-inflammatory drugs

such as aspirin might function by interfering with the production

of naturally occurring hormones such as prostaglandins. Shortly

thereafter Harry became Director of the Stokes Court Laboratory of

Miles Laboratories, Ltd., and it was at this critical juncture of

his career that he did not have access to a laboratory wherein he

could test his hypothesis experimentally.

The demonstration that this is indeed what happens came 3 years

later, in Dr. J. R. Vane's Nobel Prize-winning study at the Royal

College of Surgeons.

In the latter part of his life his interests turned more

specifically to questions of the mechanism of tolerance and

dependence, and he published a number of papers on these and

kindred topics, many of which evoked considerable interest. This

work brought him international recognition as a leading authority

in the field.

In 1969 he and Hans Kosterlitz organized a satellite meeting on

opiates at the International Union on Pharmacology Congress in

Basel. This session, which attracted about 25 participants, was

the forerunner of the International Narcotic Research Conference

which has now a membership of over 500 scientists.

After his retirement from Miles in 1982, Harry Collier returned to

academic work as an Honorary Professor of London University at

Chelsea College, where he set up and directed a research unit to

study the mechanisms of drug dependence. There he showed that

adenosine, a substance which occurs naturally in the brain, can

interfere with the development of opiate dependence and its later

manifestations. This may well prove to be an important step

towards finding some means of preventing opiate dependence.

A highly literate man, Harry's interests outside his professional

concerns ranged widely over the cultural field, and he was

particularly fond of poetry and the theater.

2
His favorite poet was William Shakespeare, and it was altogether

fitting that the funeral service for Harry Collier closed with a

reading of the song from Cymbeline, the first six lines of which

are:

Fear no more the heat o'th' sun

Nor the furious winter's rages;

Thou thy worldly task has done,

Home art gone, and ta'en thy wages.

Golden lads and girls all must,

As chimney-sweepers, come to dust.

Sydney Archer, Ph.D.

School of Science

Rensselaer Polytechnic Institute

Troy, New York 12181
Mechanisms of Tolerance and

Dependence: An Overview

Leo G. Abood

Despite extensive knowledge of the mechanisms involved in the acute

action of drugs of abuse, relatively little is known of their chronic

effects and the mechanisms responsible for drug dependence and

tolerance associated with the compulsive use of drugs. The phenomena

of tolerance and dependence occur with a variety of psychotropic drugs

and are not unique to the opiates and other drugs of abuse. Throughout

the symposium, it was repeatedly emphasized that the problem of

physical dependence is highly complex, involving a series of adaptive

changes beginning with the diverse and widely scattered opioid receptors

and extending to complex arrays of autonomic and sensorimotor neural

networks; adaptive changes which are influenced by genetic, species, and

environmental factors.

The main emphasis of the symposium was opiate dependence and

tolerance, examining the biochemical, pharmacologic, and

electrophysiologic mechanisms in the central nervous and neuroendocrine

systems as well as various peripheral systems known to possess opioid

peptides and receptor subtypes. The most characteristic phenomenon

associated with opiate dependence is the abstinence syndrome following

drug withdrawal or administration of an opiate antagonist such as

naloxone. The phenomenon, which is manifested biochemically as well as

physiologically, involves virtually every system containing opioid

receptors, and it provides an index for assessing physical dependence in a

variety of animal species, organ systems, and cell cultures.

Martin stressed the fact that drug tolerance and dependence involves a

variety of adaptive mechanisms-both autonomic and sensorimotor-and

the necessity for model systems to be relevant to the problem of

tolerance and dependence in man as well as animals. Adler presented an

overview of neuropharmacologic procedures to assess drug tolerance and

dependence, including measurements of EEG, body temperature, brain

lesions, and behavioral paradigms. He pointed out that pharmacokinetic

factors, such as the dose schedule, route of administration, and lipid

solubility, influence the drug’s effectiveness in producing tolerance and

dependence. Dewey called attention to a recent report by Ternes

demonstrating tolerance and dependence with hydromorphone in rhesus

but not cynamologus monkeys and emphasized the differences in

4
tolerance and dependence liability among animal species as well as of

various classes of opiates. Pharmacokinetic factors must be considered

both in accounting for the differential effectiveness of classes of opiate

agonists and antagonists in producing tolerance and dependence within a

given species and when accounting for dependence liability of a given

drug among different species. He stated that -THC (intravenously) is

as potent as morphine in producing analgesia in rodents and that only

tolerance and no withdrawal effects (loss of body weight) are seen with

the drug.

Herz considered tolerance and dependence to opiates to be a function of

the multiplicity of receptors. By taking advantage of the fact that

tolerance development to prolonged opioid exposure in a given receptor

subtype is selective for that subtype, one can demonstrate the presence

of μ, -receptors in the mouse vas deferens and μ and

receptors in guinea pig ileum. It appears that -receptor ligands induce

sedation, are not self-administered, and do not suppress withdrawal. His

observation that the rewarding and analgesic properties of diprenorphine

(a multiple receptor-ligand) are mediated separately by a (possibly) and

μ receptor, respectively, poses a challenge to the prevalent notion that

both effects are mediated by the μ receptors. Herz described

experiments with the opiate-dependent ileum which indicated that

tolerance and dependence were not interdependent. With the use of the

mouse vas deferens made tolerant to opiates, it was not possible to

demonstrate cross-tolerance among any of the various receptor subtypes.

Conceivably, tolerance involves a modification of the receptor, while

dependence is related to adaptive changes at either the effector or

regulatory (adenylate cyclase) link. During the discussion period, it was

noted that physical dependence reflects a drug-seeking behavior aimed

at attenuating the aversive withdrawal effects of opiates.

Shuster addressed the problem of the genetic factors influencing

responsiveness to opiates and other drugs of abuse, employing inbred and

congenic lines of mouse. The naloxone-induced jumping after chronically

administered morphine was observed in C57 but not A/J mice; and,

although little correlation was observed between 3H-naloxone binding to

brain membranes and analgetic responsiveness to opiates, those strains

having the lowest receptor density were the least responsive. The

observed relationship between receptor density and analgetic

responsiveness appeared to involve the μ but not or receptors. Such

pharmacogenetic studies reveal the significance of genetic factors in

determining individual vulnerability to physical dependence on drugs of

abuse.

One of the most useful and interesting preparations for studying opiate

dependence in vitro is the isolated myenteric plexus of the guinea pig

after chronic opiate administration. Dependency in the ileum is

manifested by a naloxone-precipitated contracture which can be

overcome by morphine. Tucker described the recent studies of the late

Collier demonstrating the existence of three separate but convergent

systems impinging on the final cholinergic motorneurons responsible for

ileal contraction. The ileum exhibits a dependency on normorphine,

5
clonidine -norepinephrine agonist), and adenosine (an activator of

adenylate cyclase); furthermore, cyclohexyladenosine, an adenosine

receptor antagonist, is a potent inhibitor of the withdrawal response of

the ileum.

A number of speakers focused on the possible role of adenylate cyclase,

which is presumed to be one of the regulatory links between the receptor

and the effector components of the opioid system. Evidence for the

involvement of adenylate cyclase in the opioid system stems mainly from

the work of Klee and collaborators demonstrating that opioids inhibit

adenylate cyclase in a neuroblastoma-glioma cell line (NG 108-15).

There also exist receptors which stimulate adenylate cyclase, such as Cladenosine and prostaglandin El

, the response of the latter being inhibited

by opiates. Klee expanded on his recent report that the suppressive

action of opiates on adenylate cyclase resulted from stimulation of

GTPase, which regulates the cyclase by controlling the levels of the

essential cofactor GTP. Some discussion was devoted to pertussin toxin,

which, by promoting ribosylation of the Ni subunit, abolishes the

hormone inactivation of adenylate cyclase in a variety of inhibitory

systems. The toxin also decreases opiate receptor binding as well as

inhibition of the cyclase in the NG cell line. From studies showing that

the toxin had no effect on the acute action of normorphine on ileum,

while inhibiting the withdrawal response of the ileum, in vivo and in

vitro, Tucker proposed that the toxin was activating an inhibitory

regulator of adenylate cyclase and that physical dependence may involve

such a regulator.

A possible mechanism for drug tolerance and dependence would involve

an alteration in either the density or affinity of its receptor. The wellknown phenomenon of supersensitivity to dopamine (DA) agonists

following chronic exposure to the neuroleptic DA antagonists is

Attributable to an increase in the number of DA receptors. Attempts by

many investigators to modify the density or affinity of opioid receptors

by chronic administration of opiate agonists or antagonists have led to

variable results. Zukin reported that chronic treatment of rats with

naltrexone resulted in a two-fold increase in the brain density of opiate

receptors (

3H-etorphine binding), returning to normal levels after 6 days

without drug. Autoradiographic studies after chronic naltrexone

revealed that the density of μ and 6, but not and receptor, in

specific brain areas increased. Clouet viewed tolerance as any change

resulting from the occupancy of the receptor by an opiate and reviewed

changes in the neostriatum associated with acute and chronic exposure

to opiates. These included an increase in DA-stimulated adenylate

cyclase (acute and chronic), increase in DA turnover (especially with

agonists), a biphasic response in the Ca/calmodulin-dependent protein

kinase (acute morphine), and a suppression of the increased kinase

activity after chronic morphine. Protein phosphorylation, by regulating

neuro-transmitter turnover, adenylate cyclase, and ionic channels, may

be another mechanism associated with tolerance-dependence.

Loh described the use in his laboratory of the NG 108-15 cell line to

examine the phenomena of opiate desensitization and withdrawal.

6
Chronic exposure to etorphine produces a down-regulation of opiate

receptors and a naloxone-reversible inhibition of PGE1-stimulated

adenylate cyclase. He postulates that the down-regulation is due to

internalization of the receptor; while the rebound effect, which is

suppressed by Cl-adenosine and NaF, may involve a restoration of the

uncoupled inhibitory regulatory-coupling units of the cyclase complex. A

similar mechanism for opiate desensitization was proposed by Wuster,

who provided additional evidence for the dissociation of tolerance and

naloxone-induced withdrawal in the mouse vas deferens and guinea pig

ileum as well as the NG cell line. The opiate desensitization resulting

from pertussis toxin involves a suppression of the inhibitory coupling unit

(Ni) of the cyclase complex; and insofar as opioid receptors are also

decreased by the toxin, he proposed that Ni is needed to maintain the

receptors.

Based on studies with a variety of opioid peptides including -endorphin

and dynorphin agonist), Lee proposed that a balance of endogenous

opioid peptides was involved in analgesia, -endorphin serving as the

major effector and the others as modulatory. Dynorphin alone was not

analgetic, and whereas it potentiated opiate-induced analgesia in naive

mice, it prevented withdrawal in mice, monkeys, and man and suppressed

opiate binding in vivo.

Cicero discussed the mechanisms involved in opioid tolerance and

dependence in the rat hypothalamic-pituitary-luteinizing hormone (LH)

axis. The release of LHRH from the hypothalamus is modulated by

opioid receptors of unknown subtype; after acute or chronic morphine

treatment followed by naloxone, there is a dramatic increase (order of

magnitude) in serum LH levels. When morphine is given 31 days

following castration, there occurs an increase (instead of a suppression)

of LH along with a tolerance to opiate analgesia. The study is

illustrative of the multifarious nature of the opioid system and its

vulnerability to physical dependence.

Mains briefly reviewed the current knowledge of the precursors,

biosynthesis, and processing of neuropeptides, particularly those that are

pro-ACTH/endorphin-derived. With the use of mouse pituitary tumor

cells, he studied the process of -amidation, which results in the

formation of TRH, vasopressin, substance P, and many other

neuropeptides from the corresponding peptides with a terminus COOH.

With the use of the hibernating ground squirrel, Beckman was able to

demonstrate an absence of naloxone-induced withdrawal (vocalization

and shakes) following chronic exposure to morphine. Since DA turnover

decreased, while DA levels increased 10-fold in the hibernating animal,

he inferred that DA supersensitivity, which may be a withdrawal

mechanism, could not develop. Autoradiographic studies revealed a

decrease of opiate receptors in regional brain areas. Henriksen

presented a review of the neurophysiological literature concerned with

opioid tolerance and dependence, highlighting the use of slices of rat

hippocampus and locus coeruleus. When dynorphin is iontophoretically

applied to the CA3 pyramidal neurons of the hippocampal slice,
excitation occurs without acute desensitization; however, the application

of a synthetic agonist, U50-488H, produces inhibition. Herz reported

an increase in field potentials of hippocampal slices with exposure to

met-enkephalin, hut not with morphine.

Crain described the use of organotypic cultures of mouse spinal cord and

sensory ganglia in studying acute and chronic effects by

electrophysiologic analyses. Sensory-evoked synaptic responses from

dorsal, but not ventral, root ganglia are depressed in a naloxonedependent manner by morphine; and after exposure to morphine for 2

days, the depressant effect of opiates vanishes, but is restored by

naloxone. Proceeding on the assumption that drug tolerance should be

reflected in altered activity of regional brain areas, Elde measured

cytochrome oxidase histochemically in chronically morphinized mice.

The most apparent change was a decrease in the mammillary bodies.

With the use of the 14C-deoxyglucose method, others have reported a

decreased metabolism in frontal motor cortex and striatum and increases

in other brain areas. Because of the depressant effects of opiates in

respiration, it is difficult to determine whether the observed metabolic

changes are direct or indirect. With the demonstration of the existence

of opiate receptors in lymphocytes and other blood cells, interest has

centered on the possible functional role of the receptors in white cells.

Plotnikoff reviewed his and other findings showing that met-enkephalin

was an immunostimulant, which in mice increased the size of the

thymus, decreased the size of the spleen, increased lymphocytic

blastogenesis induced by mitogens, and enhanced the natural killer cell

activity on cancer cells.

The final session of the symposium was devoted to drugs of abuse other

than the opiates. Groves reported that since the stereotypic behavioral

effects of amphetamine are enhanced by long-term administration while

others are attenuated, multiple mechanisms must be involved in the

drug’s action. Apart from such mechanisms as increased release of

catecholamines and supersensitivity of postsynaptic and subsensitivity of

pre-synaptic DA receptors, there are widespread adaptive changes in

many brain areas affecting the integrative activity of brain in coping

with its disrupted environment. Karler found that increased sensitivity

to the cannabinoids develops concomitantly with tolerance, a

phenomenon that was first noted with cocaine. Furthermore, the

cannabinoids enhance the sensitization to convulsant drugs and

electroconvulsive shock, an effect which may endure for months.

Although some of the behavioral effects of cocaine may involve

activation of the catecholaminergic systems, many of the effects cannot

be accounted for by such mechanisms. In the search for alternate

mechanisms, Gale reported that chronic administration of cocaine or

cocaine + droperidol (a DA antagonist) resulted in a significant increase

in striatal glutamate decarboxylase, a concomitant increase in GABA,

and a decrease in 3H-GABA binding. The findings are indicative of

increased GABA turnover following chronic exposure to cocaine;

however, the neurotransmitter system directly involved in cocaine’s

action is still uncertain.

8
Okamoto reported that the dispositional (pharmacokinetic) tolerance to

barbiturates had a rapid onset and was of long duration, while the

functional (CNS depression) tolerance developed slowly and progressed

with treatment. By examining a variety of barbiturates, various dose

schedules, and pharmacokinetics, she demonstrated that tolerance and

dependence were dependent on the blood level of the barbiturate needed

to maintain CNS depression.

Abood described the behavioral and biochemical studies in rats given

nicotine chronically. No changes were observed in the density or affinity

of (-)-3H-nicotine binding sites to brain membranes or in the metabolism

of nicotine by isolated hepatocytes. Although tolerance develops to the

central effects of nicotine, the effect is transitory.

In addition to providing a number of conceptual and methodological

approaches towards understanding drug tolerance and dependence, the

symposium has attempted to better characterize the terms for specific

drugs of abuse. The mechanisms for drug tolerance, which are

comparable for most drugs and better understood than those for

dependence, generally involve adaptive biochemical mechanisms

affecting the rate of drug metabolism and elimination, the sensitivity of

receptors, and responsiveness of secondary messenger systems. On the

other hand, the phenomenon of dependence, which is more characteristic

of drugs of abuse, is highly complex and involves a variety of

biochemical and neurophysiological mechanisms. A number of

experimental models, ranging from neuronal tissue cultures to the whole

animals, are available for exploring the mechanisms of withdrawal of

opiates and, to a lesser extent, barbiturates and amphetamines. Such

models, which provide the basis for examining the underlying

mechanisms for dependence and withdrawal, have yet to be developed

for the cannabinoids, cocaine, nicotine, and other abused drugs. The

further development and refinement of such experimental models will

continue to be significant factors in elucidating such mechanisms.

There arises the question concerning the nature of the physiological

substrate that is mimicked and, presumably, replaced by the dependent

drug and the possible dependence of the organism on the endogenous

substrate. With respect to the opiates, we have some knowledge of the

endogenous substrates ( endorphin, dynorphin, and enkephalins) and

their receptors (μ, and respectively); and one might concur with

Collier that the organism is “physically dependent” on the endogenous

opioids. A similar argument holds for amphetamine which interacts with

catecholaminergic systems. The nature of the endogenous substrates for

barbiturates and other drugs of abuse is not known; however, many of the

drugs do appear to involve specific receptors. One of the important

problems for future research is to characterize the endogenous substrate

for the receptors and demonstrate its ability to mimic drug-induced

dependence and withdrawal.

By encompassing a wide spectrum of abused drugs and a number of

disciplinary approaches, the symposium, in its attempt to deal with

mechanisms of drug dependence, has highlighted some important issues

9
concerning the nature of the problem. If a drug’s action is receptormediated, and the receptor exists in multiple forms, can a unitary

molecular mechanism be involved? Are there common mechanisms

among different drugs of abuse? Is the development of dependence

associated with more than one subtype (e.g., the μ-form) of the opiate

receptor? If it is assumed that dependence is a complex process

involving a number of neural circuits, is it necessary for an adaptive

molecular mechanism to occur at the level of the receptor or the second

intermediary messenger?

Much of our information concerning the involvement of receptor-linked

mechanisms in drug tolerance and dependence derives from studies on

neuroblastoma-glioma cultures, where the second messenger appears to

be a prostoglandin-stimulated adenylate cyclase linked to a receptor.

How applicable is this model to the central nervous system which

contains μ and receptors as well? With the possible exception of the

nigrostriatum, attempts to demonstrate the involvement of adenylate

cyclase in opiate action in brain have been unsuccessful. Relatively

little progress has been made in demonstrating a primary action of

opiates on ionic conductances and neurotransmitter function and

turnover. Answers to these and other questions relating to the complex

problem of drug dependence must continue to come from a multidisciplinary approach, so well exemplified by this symposium.

Finally, there remains the problem of the criteria for determining

whether a drug of abuse is addictive. Some might argue that any drug or

substance which is abused, i.e., used compulsively, is addictive; in which

case the term would be applicable to innumerable drugs, substances, and

compulsive behavioral patterns. Since the degree or intensity of abuse

varies greatly among drugs and substances, this definition requires a

categorization in terms of their degree of addictive liability. The term

“habitual,” which is more generally used to describe compulsive

tendencies, would be synonymous with “addictive.” It may, however, be

useful to reserve the term “addictive” for abused agents and tendencies

exhibiting a “high degree” of compulsiveness. The problem would then be

to define the psychophysical parameters associated with compulsive

behavior and to decide arbitrarily when an agent is to be labeled

“addictive.”

At the present state of our knowledge, the most characteristic psychophysical parameters associated with compulsive use of drugs are those

resulting from either their abrupt withdrawal or administration of an

appropriate antagonist. In the case of the opiates, barbiturates, amphetamines, and alcohol, the withdrawal signs are well-defined and severe;

whereas, with the cannabinoids and nicotine, they are not easily definable by physiological measures and tend to be far less severe. If the

term “addictive” is reserved for those drugs promoting severe, definable

withdrawal signs—often life-threatening in humans-the class of addictive drugs would be small; but, if the term is to be more generally applicable to abused drugs, it will be necessary to develop, in both animal and

human models, the psychophysical, pharmacologic, and biochemical

criteria for assessing their relative degree of compulsive liability.

10
AUTHOR

Leo G. Abood, Ph.D.

Center for Brain Research

Box 605

University of Rochester Medical Center

601 Elmwood Avenue

Rochester, New York 14642

11
Phenomenology and Theoretical

Basis of Tolerance and

Dependence

William R. Martin

I am pleased to dedicate this chapter on tolerance and dependence to

an acquaintance of many years, Harry O. J. Collier. Our paths first

crossed in the early 1960s when we both were in the process of developing our ideas concerning tolerance and dependence. Over the

years, Dr. Collier became concerned with the phenomenology of tolerance and dependence and its basis.

Those of us who work on drug abuse problems tend to view the drugs

as toxins, tolerance as a protective mechanism, and physical dependence as an aversive consequence of recruitment of these protective mechanisms. Yet another view is that dependence-producing

drugs are pleasure-giving and that physical dependence is the price

that is paid for their use; the view that if one dances one should

be expected to pay the piper. For several reasons included herein,

this chapter will deal with the underlying concepts of tolerance

and dependence. Both are fundamental biologic properties of most

animals and probably to some degree of all biological tissues.

They are intimately concerned with motivation, instincts, adaptive

behavior, survival, homeostasis, needs, drives, arousal and finally

with desires--concepts which have subtle but broad-reaching

distinctions and implications. Tolerance and dependence will be

approached here from a theoretical point of view as it relates to

these apparently diverse concepts.

Concepts of tolerance and dependence still plague us both conceptually and phenomenologically. Preoccupation with the phenomenology of tolerance and dependence and their molecular basis has

created problems that are new and important in their own right but

causes one to disregard important dimensions which relate these phenomena to psychopathology and to public health. In the end all of

these phenomena have to be viewed from the standpoint of evolution

and survival and are unique in that therapeutic chemostimuli (drugs

interacting with receptors) are being developed systematically.

12
It is important to remember that dependencies are needs. This

concept is frequently forgotten as we identify and use experimental

models which are presumed to be models of dependence.It will

serve a purpose to distinguish between two aspects of the concept

of needs. A general definition of need is a life requirement for

an environmental circumstance which is conducive to survival or

well-being. A more specific definition refers to the mechanisms

whereby congenial and hostile environments are recognized. It is

in this restricted meaning that needs will be used in the following

discussion.

figure 1 illustrates the simplest and presumedly most primitive

control mechanisms in organisms which have acquired the ability to

sense and to make integrated adaptive movements. In this model the

sensory system is the need system. We will assume for example that

the animal senses a nutrient need (environment) through a chemoreceptive mechanism and moves toward it. Thus the organism is

dependent on a nutrient chemical (need) and has a motor mechanism

for acquiring it (drive). An important control mechanism which

must have evolved early was negative feedback regulation (tolerance

to chemostimulation). This was necessary for motor control so that

the organism could (1) slow, stop, and make directional corrections

as it approached a food-rich environment and (2) stop feeding when

satiation occurred. Thus it is reasonable to conceive that

tolerance to chemostimulation evolved early in evolution as an

adaptive mechanism.

A second major principle evolved when organisms became more

complex, had to integrate the activity of different functional

systems, eventually to regulate internal environments, and finally

to develop new abstract needs such as aggression, power, and

developmental dependencies related to complex ecological and social

influences. These new needs as well as old ones became manifest by

the activity of neuronal nets which were stimulated by disequilibriums and hostile environments. This neuronal activity produced

discomforting feelings such as pain, fear, anxiety, suffering,

depression, and hypophoria. Thus the activity of these neuronal

nets is the sine qua non of internal needs and associated affective

states. Figure 2 is a scheme indicating this internalization of

need mechanisms which maintain homeostasis and enhance survival.

With this internalization, new systems of controls evolved which

involve reciprocal inhibition, a type of negative feedback

regulation. Here, too, negative feedback is necessary for central

need reduction and provides the mechanism whereby an organism can

integrate movements and recognize satiety. It is important to

recognize that all drugs of abuse diminish a variety of needs

(table 1), a relationship first emphasized by Hlmmelsbach (1943)

and Wikler (1952). An extensive body of research indicating that

these drugs also alter brain neurohumoral processes has a number of

implications, among which is the proposition that chemostimulation

continues to play an important role in need states.

13
FIGURE 1

BASIC IMPORTANCE OF TOLERANCE TO EXTERNAL

CHEMORECEPTIVE-NEED-DRIVE PROCESSES

FIGURE 2

PROPOSED PROCESSES RELATING NEEDS (DEPENDENCIES), AFFECT

AND NEGATIVE FEEDBACK MECHANISMS (TOLERANCE)

14
Drugs of Abuse Which Reduce Needs and Drives and Alter Homeostats,

and the Putative Neurotransmitters and Neuromodulators Involved

DA, NE

TABLE 1

Endorphines

Opiates Enkephalins

and Dynorphins

Food

C O 2

Pain

Sex

Temperature Regulation

Vasomotor Function

Hypophoria

Amphetamines

Food

Sleep

Pain

Hypophoria

Alcohol GABA

Pain

Hypophoria

Tobacco ACh

Sex

Pain

Food

Hypophoria

Marijuana

Pain

Hypophoria

Benzodiazepines GABA

Sleep

Anxiety

Sedative-Hypnotics GABA

Sleep

Pain

Hypophoria

LSD-Like Hallucinogens Tryptamine

Serotonin

Pain

Hypophoria

Tolerance and physical dependence in man have been explored most

extensively for the opiates. Himmelsbach (1943) conceptualized the

processes of tolerance and physical dependence to opiates as an

alteration of autonomic homeostasis and the recruitment of

contra-adaptive mechanisms. This hypothesis has been very

attractive; however, it has been rigorously tested only for the

regulatory system in man. There are many complexities in

conceptualizing tolerance and dependence and in devising measures

of these phenomena (cf. Martin and Sloan 1977). In nonhuman

studies there is evidence that this mechanism may be of importance

in the regulation of certain endocrine functions and in the

temperataure regulation of the dog Table 2 and figure 3

15
TABLE 2

The effects of morphine in nondependent, morphine-dependent (240

mg/day) and morphine-abstinent subjects on the responsivity (slope)

and sensitivity (intercept) of the respiratory system to CO2

(from Martin et al. 1968).

Slope ± S.E. Intercept ± S.E.

Preaddiction

Control 1.5 ± 0.1 51.7 ± 2.2

Morphine (30 mg/70 kg) 1.1 ± 0.1(0.05) 62.8 ± 2.6(0.05)

Late Dependence

Predrug 1.9 ± 0.2 62.1 ± 2.2

Morphine (60 mg/70 kg) 1.7 ± 0.2 63.4 ± 1.6

Early Abstinence (20 Hours)

3.2 ± 0.3(0.05) 52.7 ± 1.6

Protracted Abstinence

1.3 ± 0.2(0.25) 57.0 ± 1.9(0.005)

p values in parentheses when compared to control values

summarize the effects of morphine and morphine abstinence on the

responsivity and sensitivity of the respiratory center to CO,

(Martin et al. 1968). In the nondependent subject morphine shifted

the partial pressure of CO2-minute-volume stimulus-response

curve to the right and depressed its slope. In stabilized

dependent subjects the stimulus-response line remained shifted to

the right; however, its slope was steeper than that of the

stimulus-response line prior to the subject's becoming physically

dependent. Very large doses of morphine (60 and 120 mg/70 kg) had

no effect on the stimulus-response line, a not unexpected finding

since 240 mg/day of morphine probably produced maximum physical

dependence by occupying all of the morphine receptors (Martin et

al. 1972). When the subjects were abstinent 20 hours (mild to

moderate abstinence syndrome had developed), the slope of the

stimulus-response line was twice as steep as that of the control

stimulus-response line. Thus, even in this relatively simple

system, tolerance and dependence have complex dimensions. Morphine

continued to depress the respiratory center's sensitivity to CO,

throughout the period of chronic intoxication; however, chronic

intoxication enhanced the responsivity (increased the

16
FIGURE 3

Mean calculated regression lines (solid) for partial pressure

(pCO2)-minute volume (Va) response curves obtained during the

control period, after 15 mg/70 kg of morphine, and during early

withdrawal in subjects dependent on 240 mg/day of morphine. The

means for each parameter were determined from values obtained in 7

subjects. There was great variability in the first and last points

of the response curves. The top of the regression lines represents

the maximum Va obtained. A control ( l ), a 15 mg/70 kg dose of

morphine (x) and a withdrawal paCO2-Va response of one

subject (dashed lines) are presented to further illustrate these

changes. (From Martin et al. 1968.)

slope) of the respiratory center to CO2. Despite the persisting

depression, additional morphine was without effect. The respiratory studies provided further evidence about the relationship

between need and affective state. When alveolar ventilation

exceeded 10 minute, subjects experienced some discomfort which

increased as the minute-volume increased. In these studies the

intensity of the discomfort was indicated by hand signals. No

discomfort was reported by the subjects after they received single

doses of morphine (15 or 30 mg/70 kg) even when minute-volume

exceeded 10 minute. This may mear that subjective discomfort

associated with a need can be diminished without producing an equal

diminution of the drive mechanism.

17
Tolerance to and dependence on drugs of abuse are even more complicated phenomena when other parameters and drugs are considered.

This point is of sufficient importance to be emphasized, as much of

our effort, at the more basic levels, may be describing phenomena

that are not obviously related to the phenomena of tolerance and

dependence as seen in man with chronic drug administration. Figure

4 illustrates the effects of chronic intoxication to morphine (240

mg/day) on a variety of physiologic parameters. It is believed

that 240 mg/day of morphine saturates nearly all of the u receptors

and produces a maximal degree of physical dependence. Chronic

administration of morphine increases blood pressure, pulse rate,

and body temperature and decreases pupillary diameter and respiratory rate. Single doses of morphine in nondependent subjects

do not markedly alter blood pressure, pulse rate, or body

temperature. It is emphasized that these changes persist

throughout the period of chronic intoxication.

FIGURE 4

The effects of chronic administration of morphine on clinical

variables. Each point represents the weekly mean a.m. observation

for 7 subjects. The horizontal line represents the mean of control

determinations for 7 subjects. (From Martin and Jasinski 1969.)

18
Figure 5 illustrates changes that occurred in subjects who received

methadone in a dose of 100 mg/day chronically. A decrease in

pupillary diameter and respiratory rate and an increase in body

temperature was seen, changes similar to those produced by chronic

morphine. The chronic methadone syndrome differed from the chronic

morphine syndrome in that blood pressure and pulse rate were

decreased. Thus the chronic effects of closely related opioids

differ.

FIGURE 5

Changes in physiological indices and intensity of abstinence and

feeling state during cycle of methadone dependence. Horizontal

line in each graph represents mean of control observations made

three times daily (6 AM, 12 noon, and 6 PM) for 42 days in 5

patients. Each point represents mean weekly value. Triangles

indicate mean of observations obtained during last three weeks of

addiction cycle. (From Martin et al. 1973.)

19
Figure 6 shows the various signs of the morphine abstinence syndrome. Blood pressure, pulse rate, body temperatures, and respiratory rate are significantly increased. Although pupils return to

control value following withdrawal, they do not markedly dilate. A

similar abstinence syndrome was seen in methadone-dependent subjects

(figure 5). Thus the abstinence syndrome is not the mirror image

of the signs of acute or chronic intoxication with morphine and has

unique dimensions of its own. Figure 5 and table 2 illustrate

another important feature of the morphine abstinence syndrome. The

acute abstinence syndrome persists for about a month and then a new

syndrome develops which is characterized by a mild hypotension,

bradycardia. hypothermia, miosis, and hyposensitivity of the

respiratory center to CO2. This syndrome persists for several

months and was still evident when the studies were terminated. It

is important to emphasize that there are both an acute and a

protracted abstinence syndrome which are different and indicate

FIGURE 6

Changes seen during early and protracted abstinence. Each point

represents the mean weekly a.m. values for 6 subjects. The first

point of each curve represents the mean value for the last 7 weeks

of addiction. The horizontal line represents the mean control

value for the 6 subjects. One subject was withdrawn from the study

near the end of the chronic intoxication phase because of episodes

of acute cholecystitis. (From Martin and Jasinski 1969.)

20
that chronic administration of opioids alters neuronal excitability

in diverse ways and probably through several mechanisms.

Morphine-like drugs produce feelings of well-being (euphoria or

anti-hypophoria) in abstinent addicts. It is not generally

recognized or attended to that chronic opiate-intoxicated patients

become hypochondriac, develop feelings of tiredness, inefficiency,

unpopularity, and become withdrawn (table 3). These symptoms are

more marked during early withdrawal and remained exacerbated during

protracted abstinence (Martin et al. 1973). This constellation of

symptoms has been called hypophoria and their disappearance,

euphoria (Martin et al. 1977). All of these negative feelings are

decreased in a dose-related way by opiates, amphetamines (Martin et

al. 1971), and barbiturates (Martin et al. 1974; Jasinski 1977).

Although systematic studies have not been done, anecdotal accounts

indicate that they are also probably reduced by nicotine, alcohol,

marijuana, and the LSD-like hallucinogens. Thus the effect of

chronic opioid administration is not merely the loss of its ability

to produce feelings of well- being but the reduction of hypophoria

and withdrawal from social interactions. These feelings are

exacerbated during both early and protracted abstinence.

It is known that opiates in single doses decrease all types of

sleep (Kay et al. 1969). Figure 7 illustrates changes in different

types of sleep seen during a cycle of methadone dependence. As can

be seen, sleep is increased during both the stabilization phase of

dependence and protracted abstinence (Martin et al. 1973). Thus

the effect of chronic opioid treatment on sleep is not simply the

reduction of the insomniac action in the presence of opioids. It

also produces hypersomnia in protracted abstinence.

Whereas a single dose of opioids can reduce a number of needs,

chronic administration may both decrease (e.g.. CO2 elimination)

and increase (e.g., hypophoria, sleep) needs. Although many need

states are enhanced during early abstinence, some may be decreased

(e.g., sleep). Perhaps, more importantly, during protracted

abstinence some needs are increased (hypophoria and sleep) and

others decreased (responsivity to CO2) (see table 4).

CONCLUSION

Most, if not all, drugs of abuse are intimately involved in needs

and need reduction. These needs are dependencies which have

inherent tolerance mechanisms necessary for the control of need

reduction. With internalization of need states and the evolution

of cognitive processes, affect has become a fundamental need

mechanism. Chronically administered drugs of abuse interact with

and change need states. The characterization of these changes in

terms of conventional definitions of tolerance and dependence

greatly oversimplifies the impacts of chronic drug administration. Indeed this oversimplification of the concepts of tolerance

and dependence and the development of methods for assessing them

21
TABLE 3

Changes in certain ARCI and MMPI scale scores during a cycle of addiction to methadone

Controlled Addiction Early Protracted

(4 and 10 Weeks) Abstinence Abstinence

ARCI Scale Score Negative Feelings

PCAG

Weak

Tired

Social

Withdrawal

48 55 73** 47

42 51 72** 47

44 61** 73** 47**

56 56 58** 61

MBG 55

Efficiency 58

ARCI Scale Score Positive Feelings

53 38**

48** 24**

MMPI Scale Scores

48

51**

Hs 54 71* 75* 53

Hy 53 66* 71* 58

SC 62 70** 74* 74*

PCAG=(Pentobarbital, Chlorpromazine, Alcohol Group Scale) - Apathetic Sedation

MBG=(Morphine, Benzedrine Group Scale) - Euphoria - Antihypophoria

Hs=(Hysteria); Hy=(Hypochondriasis); Sc=(Schizophrenia); * P<0.05; ** P<0.0l
FIGURE 7

Number of minutes during various phases of the addiction cycle of

total sleep, B-bursts, REM sleep, and -sleep. Each point

represents the mean obtained for 5 patients. Three control

observations were obtained (Cl, C2, C3) as well as observations

during the ascending (A) and stabilization (S) phases and during

withdrawal (numbers in weeks). Asterisks indicate values are

significantly different from control values: *P<0.1; **P<0.05.

(From Martin et al. 1973.)

23
TABLE 4

Respiration

Mood

Sleep

Summary of the Effects of a Cycle of Opioid Dependence on Three Needs

Acute Stabilization

Effects (Tolerance)

Early

Abstinence

Protracted

Abstinence

Decreased

sensitivity

and

responsivity

to CO2

Decreased sensitivity Increased Increased

and increased responsivity and decreased

responsivity to CO2 and probably sensitivity

sensitivity

Decreased

hypophoria

(euphoria)

Increased

hypophoria

Increased

hypophoria

Increased

hypophoria

Decreased all

sleep stages

Increased total

sleep and

bursts

Increased and

total sleep
may result in the study of phenomena which may not be related to

the clinically relevant changes produced by chronic administrations

of opioids and perhaps of other drugs.

Chronically administered opiates clearly produce long-persisting

changes in need states and affect, enhancing some and depressing

others, and may induce new needs which can only be met with drugs

which may have the potential for abuse. The changes which are

induced are enormously complex, probably involving multiple

mechanisms such as homeostasis at molecular, cellular and tissue

levels, supersensitivities, up- and down-regulations of receptors

and their endogenous agonists and recruitment of redundant

mechanisms. The enormous amount of research which has been done

with chronic opiate administration has shown that these drugs

produce long-lasting changes. Our own work continues to show that

individuals with drug problems have exaggerated needs and

associated affective disorders (hypophoria).

Drug-induced tolerance and dependence still have major clinical

importance. Many patients with excessive needs and hypophoria

attempt to cope with these discomforting subjective states by using

drugs which possess antihypophorlc properties. Most of these drugs

interact with need states and alter them. Some of these

alterations may cause long-persisting changes and worsen existing

psychopathologic processes. Since one of the alternative

strategies for treating drug abusers is chemotherapy, an

understanding of the basic mechanisms of phenomena designated as

tolerance and dependence is important, as these phenomena may alter

brain functions related to needs and affect. Thus the search for

new chemotherapeutics for the treatment of drug dependence presents

a formidable challenge, for we will be interceding in functional

systems which are intimately concerned with basic motivation.

Interventions, because of the complexities of need systems, may

have unexpected outcomes.

REFERENCES

Himmelsbach, C.K. Morphine with reference to physical

dependence. Fed Proc 2:201-203, 1943

Jasinski, D.R. Clinical evaluation of sedative-hypnotic for abuse

potential. In: Thompson, T., and Unna, K., eds. Predicting

Dependence Liability of Stimulants and Depressant Drugs.

Baltimore: University Park Press, 1977. pp. 285-289.

Kav. D.C., Eisenstein. R.B.. and Jasinski. D.R. Morphine effects

on human REM state, waking state and NREM sleep. Psychopharm

14:404-416, 1969.

Martin, W.R.; Gorodetzky, C.W., and Thompson, W.O. Receptor

dualism: Some kinetic implications. In: Kosterlitz, H.W.,

Collier, H.O.J., and Villarreal, J.E., eds. Agonist and

Antagonist Actions of Narcotic Analgesic Drugs. Macmillan:

New York, 1972. pp. 30-44.

Martin, W.R.. Hewett, B.B., Baker, A.J., and Haertzen. C.A.

Aspects of the psychopathology and pathophysiology of

addiction. Drug Alc Depend 2:185-202, 1977.

25
Martin, W.R., and Jasinski. D.R. Physiological parameters of

morphine dependence in man--tolerance, early abstinence,

protracted abstinence. J Psychiatric Res 7:9-17, 1969.

Martin, W.R., Jasinski, D.R., Haertzen, C.A., Kay, D.C., Jones,

B.E., Hansky, D.A. and Carpenter, R.W. Methadone: A

re-evaluation. Arch Gen Psychiatry 28:286-295, 1973.

Martin, W.R., Jasinskl, D.R., Sapira, J.D., Flanary, H.G., Kelly,

O.A., Thompson, A.K.. and Logan, C.R. The respiratory effects

of morphine during a cycle of dependence. J Pharmacol Exp Ther

162:182-189, 1968.

Martin, W.R. and Sloan, J.W. Neuropharmacology and neurochemistry

of subjective effects, analgesia, tolerance and dependence

produced by narcotic analgesics. In: Martin, W.R., ed. Drug

Addiction I. Handbook of Experimental Pharmacology, Vol. 45,

Heidelberg: Springer-Verlag. 1977. pp. 43-158.

Martin, W.R., Sloan, J.W., Sapira, J.D., and Jasinski, D.R.

Physiologic, subjective and behavioral effects of amphetamine,

methamphetamine, ephedrine, phenmetrazlne and methylphenidate in

man. Clin Pharmacol Ther 12:245-258, 1971.

Martin, W.R., Thompson, W.O., and Fraser, H.F. Comparison of

graded single intramuscular doses of morphine and pentobarbital

in man. Clin Pharmacol Ther 15:623-630, 1974.

Wikler. A. Opiate Addiction. Psychological and

Neurophysiological Aspects in Relation to Clinical Problems.

Springfield: Charles C. Thomas, 1952.

AUTHOR

William R. Martin, M.D.

Department of Pharmacology

University of Kentucky

Lexington, Kentucky 40506

26
Contributions of Neuropharmacology to Understanding

Mechanisms of Tolerance and

Dependence

Martin W. Adler and Ellen B. Geller

INTRODUCTION

Neuropharmacological research has played a major role in the

formulation of our concepts regarding tolerance and dependence

to abused drugs. As our knowledge about these phenomena has

increased, certain ideas have been reevaluated and modified. To

compile a comprehensive review of all the studies in this field

which have contributed to our present state of knowledge about

tolerance and dependence for all drugs of abuse is a formidable

task and one which will not be attempted here. By way of merely

indicating the diversity of research in this area, some of the

techniques and endpoints used in studying the neuropharmacology

of drugs of abuse appear in table 1. The necessity of having so

many approaches available to us has become more apparent as we

have gradually moved away from the notion of a single, global

mechanism of tolerance and dependence. Only a short time ago,

for example, the view that there must be one discrete locus in

the brain responsible for opiate dependence was generally

accepted. However, as a result of research involving brain

lesions (e.g., Linseman 1976; Adler et al. 1978; Calvino et al.

1979) and modification of neurotransmitter systems (review,

Takemori 1974), this idea has been largely abandoned. With the

recognition of the multiplicity of both chemical and anatanical

systems that are involved in tolerance and dependence to drugs

of even a single class, the value of examining a variety of

drug-induced responses is readily understood. Furthermore, in

light of the broad spectrum of drug classes to which tolerance

and/or dependence may develop, the need for such an array of

techniques and endpoints becomes even more obvious.

27
TABLE 1. Neuropharmacological studies of drugs of abuse

Techniques Endpoints

EEG

Microiontophoresis

Operant Techniques

Brain Lesions

Intraventricular Cannulation

Selective Agonist & Antagonist

Drugs

Drugs to Modify Neurotransmitter

systems

Electrically & Chemically Induced

Seizures

In-vivo pA Determinations

Binding Studies

Isolated Tissue Studies

Drug Infusion Methods

Body Temperature

Body Weight

Antinociceptive

Thresholds

Pupil Size

Seizure Thresholds

Neuronal Firing Patterns

Neurotransmitter Levels

& Turnover

Locomotor Activity

Behavior

Drug Discrimination

Cross-Tolerance

Cross-Dependence

TOLERANCE

Despite the lack of a clear understanding of the mechanisms

responsible for the phenomenon we call tolerance, we can define

the term in an operational sense. Tolerance can be defined

either as the reduced effect of the same dose of a drug on subsequent administrations, or as a need to increase the dosage in

order to maintain the same level of effect. This simple definition allows for a variety of pharmacokinetic, pharmacodynamic,

and other mechanisms to be implicated in the tolerant state.

Although our definition of tolerance has not changed, many of

our concepts relating to it have. For example, a belief held by

most workers in this field until recently was that "tolerance to

the depressant but not to the excitant effects of narcotics

develops at the spinal cord level, just as elsewhere in the

cerebrospinal axis" (Goodman and Gilman 1955). It is now known

that tolerance can be demonstarted to the classical excitant

effects, namely, pupillary response and gastrointestinal tract

activity. Both the miotic effect of heroin in humans (Tress et

al. 1978) and the mydriatic effect of morphine in mice (Adler et

al. 1980) and rats (Adler et al. 1981) are subject to tolerance

development. Similarly, tolerance has been found to the effects

of morphine on intestinal motility in dogs (Weisbrodt et al.

1980) and rats (Cowan et al. 1977). Although the degree of

tolerance may not be equivalent for all of the actions of an

opioid, at least a partial tolerance is demonstrable for most

effects. One exception may be found in the effect of morphine

on lowering reward threshold. Not only does no tolerance develop on repeated administration, but an increased effect has been

reported (Kornetsky and Bain 1982).

Another commonly accepted idea in the recent past was that

tolerance develops to all of the effects of amphetamine. While

it is true that one sees marked tolerance to some of the actions

of this drug (e.g., anorexic, cardiovascular), reverse

28
tolerance, or increased responsiveness with repeated administration, occurs with some stereotypies (Rebec and Segal 1980).

Interestingly, no tolerance to its effect against narcolepsy

seems to occur Weiner 1980). Marijuana, on the other hand, is

an example of a drug to which no tolerance was thought to develop. In fact, it was felt by many that increased sensitivity

occurred with repeated administration. Experiments such as

those by Domino (1971), however, have shown conclusively that

tolerance does develop to at least some of marijuana's effects.

While it was once believed that tolerance development requires

repeated administrations of a drug, in recent years the

occurrence of single-dose tolerance to morphine has been

reported (e.g., Cochin and Kornetsky 1964; Kornetsky and Bain

1968; Huidobro et al. 1976). In some cases, tolerance can be

demonstrated within hours of a single priming dose; in other

instances intervals of days are necessary.

The examples cited above serve to illustrate how a more detailed

look at tolerance has resulted in a recognition of new elements

involved in that phenomenon. Examination of these factors has

yielded important insights into the mechanisms behind tolerance.

Although the terms tolerance and dependence are inextricably

woven into the fabric of our thinking about several classes of

drugs, there is recent in vivo evidence with opiates, for

instance, that the two phenomena, though linked, are not inseparable (Cochin and Mushlin 1976; Dafny 1982). Other types of

studies have also indicated the separability of the two phenomena. One must be careful, however, to consider other explanations, such as differences in dose or duration of drug

administration, that may be required for the demonstration of

each. Furthermore, if a drug acts on a particular receptor type

to produce a relatively irreversible or slowly reversible

canplex, tolerance may become apparent without concomitant

signs of physical dependence. Only careful evaluation can

determine if the tolerance exists without physical dependence.

There are also examples of drugs showing relatively low degrees

of tolerance but marked physical dependence (barbiturates), and

vice versa

DEPENDENCE

Traditionally, dependence has been defined in terms of the physiological and psychological symptoms that appear when the drug

is withdrawn or antagonized. As our knowledge about dependence

has grown, acceptance of such a simple definition has waned.

Although the term may be used in an operational sense to denote

the entire series of events associated with chronic abuse of a

drug, dependence really comprises two components: the abuse

liability of a drug (potential for abuse) and its dependence

potential (potential for behavioral and/or physical signs of

abstinence). The "abstinence syndrome"can be reversed by readministering the drug or another one with which it is crossdependent. Each class of drugs, however, has its own dependence

29
characteristics, and thus it may be better to describe dependence in term of a particular class. In fact, newer developments in pharmacology have even produced drugs of the same class

which show different dependence characteristics. Examples of

this situation can be found with the mixed agonist/antagonist

opioids. Although it was originally contended that these

opioids would not produce dependence, unfortunately, this has

turned out not to be the case for most drugs of this type. For

example, our view of pentazocine as a non-dependence-inducing

opioid has undergone radical change, and dependence to pentazocine is now well known (Jaffe and Martin 1980).

Buprenorphine seems to have little, if any, dependence associated with it, as judged by the virtual absence of a withdrawal

syndrome. However, the weak withdrawal syndrome is probably the

result of the stability of the drug-receptor complex and its

slow dissociation, since some abstinence signs can be

demonstrated under certain conditions (Dum et al. 1981).

Amphetamine was at one time considered a nonaddicting substance.

Yet, both physical and psychological signs appear after cessation of drug administration following chronic use (Jaffe 1980).

Even more controversial is the dependence developed to nicotine.

Recent studies have documented both physical and psychological

withdrawal symptoms on cessation of cigarette smoking (Shiffman

1979). Although additional cases and more details could be

cited, the above examples serve to illustrate how our conception

of dependence has evolved. With each advance in our thinking,

we discover more about the mechanisms involved.

PHARMACOLOGICAL PRINCIPLES

One of the problem encountered in the evaluation of research in

this area is that too often the basic principles of pharmacology

are forgotten or ignored. What happens to a drug after administration is a critical determinant of its actions and this,

in turn, can influence interpretation of data on tolerance and

dependence. Obviously, the levels of drug in plasma and brain

can be markedly affected by first-pass effects through the liver

and by drug metabolism. Moreover, route of administration can

dramatically alter both free and bound drug levels, as in the

case of morphine (table 2). Peak plasma levels of free morphine

following a dose of 4 mg/kg were approximately three times

higher after subcutaneous (SC) than after intraperitoneal (ip)

injections, and brain levels were about four times higher. With

the 64 mg/kg dose, however, plasma and brain levels of free

morphine rose to a higher level and fell more rapidly after ip

than after sc administration.

The effect of route of administration on the amounts of pharmacologically active morphine is shown in table 3. These studies are discussed in more detail by Cerletti et al. (1980). An

additional factor to consider when dealing with centrally acting

drugs is the blood-brain barrier (BBB). What many investigators

30
TABLE 2. Concentration of free morphine in rat plasma and

brain after injection of 4 mg/kg or 64 mg/kg morphine

sulfate sc or ip

Min Post- Plasma

Injection ng/ml ± S.E.M.

SC ip

4 mg/kg

Brain

ng/ml ± S.E.M.

SC ip

5 569±24 189±18* 29±3 12±1*

10 642±36 196±22* 50±5 20±2*

15 712±43 242±30* 64±5 21±2*

30 460±26 123±22* 87±3 17±4*

60 260±25 84±ll 53±11 19±2*

120 64±9 38±8** 35±1 13±3*

180 33±9 34±14 15±2 6±l*

64 mg/kg

15

15

4941±570 7202±819** 394±66 654±117

4823±395 1053±2777 562±43 1249±129*

60 10897±1416 12722±4228 1762±170 1980±374

120 6610±1276 5788±3065 1634±232 990±315

180 2831 1849 ± 860 815 469±42

*p < 0.01 vs. sc; **p < 0.05 vs. sc

fail to take into account, though, is that even when the BBB is

circumvented by intracerebroventricular (icv) administration,

the drug must still leave the ventricular compartment and reach

specific areas of the brain in order to be effective.

Differences in drug distribution to various brain parts probably

contribute to the disparity in qualitative as well as quantitative responses often seen with systemic vs. central administration. For example, differences can be seen on seizure

threshold. Table 4 shows that although morphine and etorphine

are anticonvulsant after both SC and icv administration, pentazocine and meperidine are proconvulsant by the SC route but

anticonvulsant by the icv route. Normeperidine is proconvulsant

irrespective of the route of administration. Even sensitivity

to naloxone blockade may be altered by the route used (e.g.,

meperidine). Further details may be found in a paper by

Tortella et al. (1984, in press).

Another example may be seen with body temperature (figure 1).

Although morphine exhibits the well-known dual response after sc

administration, only a dose-related hyperthermia is produced in

rats when morphine is administered icv at an ambient temperature

of 20°C (Adler et al. 1983; Geller et al. 1983). The examples

31
TABLE 3. Morphine in rat plasma following injection of 4 or 64

mg/kg morphine sulfate sc or ip. Glucuronide

percentage is calculated as (total- free

morphine) (total morphine)

Min Post- Total Morphine % Morphine

Injection ng/ml ± S.E.M. as Glucuronide

SC ip SC ip

4mg/kg

5 695± 31 283± 37* 18.1 33.2

10 726± 52 400± 51* 11.6 51.0

15 796± 74 748± 253 10.6 67.6

64mg/kg

15 6700± 894 19580±1190* 26.3 63.2

60 1559O±3649 24520±5186 30.1 48.1

*p < 0.01 vs. sc.

Substance and Alcohol Actions/Misuse, 1:65-70, 1980.

© 1980, Pergamon Press, Ltd. Reprinted by permission.

TABLE 4. Effect of route of administration on seizure threshold

changes induced by opioids in rats

Drug Route of Change in Flurothyl Antagonized

Administration Seizure Threshold by Naloxone

Morphine sc

icv

Etorphine sc

icv

Pentazocine sc

icv

Meperidine sc

icv

Normeperidine sc

icv

Yes

Yes

Yes

Yes

No

No

No

Yes

No

No

(Table courtesy of Dr. F.C. Tortella, Walter Reed Army Institute of

Research)

32
cited above serve to demonstrate that comparisons of efficacy

and potency between drugs given by different routes are invalid

and can lead to erroneous interpretation of data and false

conclusions.

FIGURE 1 Effect of morphine on body temperature: icv vs sc

administration. Subjects were male Sprague-Dawley

rats.

Another basic pharmacological principle relates to receptor

occupancy. Whether or not a receptor is continuously occupied

by a drug may well determine the development of tolerance and

dependence and the degree to which they occur. A study by

Cochin and Mushlin (1976) showed that tolerance to morphine can

be blocked by the concurrent administration of naloxone.

Further work fran this laboratory has shown that tolerance can

be blocked even if naloxone is administered an hour or more

after morphine. This is illustrated in figure 2. As can be

seen, if a single dose of naloxone (6 mg/kg, SC) is administered

to mice at various periods of time after 15 q/kg morphine

33
sulfate, tolerance to morphine is partially blocked when sensitivity to morphine is tested 3 days later. The shorter the

time interval between the first dose of morphine and the adminisration of naloxone, the more effective the blockade. It

appears from this and other work by Cochin and his colleagues

that tolerance and cross-tolerance can be attenuated if the

drug-receptor complex is broken even after the agonist effect of

FIGURE 2 Results of hot-plate assay performed in mice 3 days

after initial drug treatment. The ordinate shows the

mean area derived from multiplying the number of minutes naloxone is given after morphine by the number of

seconds on the hot plate. (Figure courtesy of Dr. J.

Cochin, Boston University School of Medicine.)

34
morphine has occurred. Work in our laboratory demonstrated that

the severity of the morphine abstinence syndrome depends on

whether the drug is administered continuously by pellet or

intermittently via injection (Cerletti et al. 1976). If the

morphine is given so that equivalent levels of drug are measured

in plasma over 72 hours, the pelleted animals display a more

pronounced withdrawal syndrome even though abstinence signs are

qualitatively identical.

SUMMARY

In summary, many of our once generally accepted ideas about

tolerance and dependence have been revised as the result of

neuropharmacological research. Included among these concepts

are the following: (1) Although tolerance and dependence are

usually linked, they appear to be separable phenomena in some

instances. Further exploration into each should be profitable.

(2) Although chronic administration of a drug is usually

required to produce tolerance and dependence, even a single dose

can result in these effects under certain conditions. That fact

may provide us with clues as to the neuroadaptive changes

occurring during the development of tolerance and dependence.

(3) A fruitful line of research emanates from the findings that

tolerance may be pharmacokinetic or neuronal and that time of

receptor occupancy appears to be a vital factor in neuronal

tolerance. (4) It is now known that tolerance does not develop

equally to all actions of a drug and reverse tolerance

(increased responsiveness) can occur. Further studies along

these lines are needed to correct many false assumptions about

this issue and to shed new light on the receptors and

transmitter systems involved. (5) We now accept the notion that

a drug usually exerts effects on more than one receptor type or

subtype. Consequently, the actions of drugs on the various

receptors and the responses to receptor blockade promise to

yield significant new knowledge about the mechanisms involved in

tolerance and dependence to a wide variety of drugs. (6) We

have become increasingly aware of species differences and genetic determinants of responses to drugs of abuse, after both

acute and chronic dosing. Other papers in this volume will

focus on this topic.

Our current state of knowledge about tolerance and dependence

suggests a number of questions whose answers will undoubtedly

lead to a much better understanding of these phenomena. First,

we have learned much about the endogenous opioid peptides and

opiate receptors in the last few years. But what is the role of

these chemicals in tolerance and dependence? Moreover, are they

continuously released and, if so, why doesn't naloxone precipitate an abstinence syndrome when administered to otherwise

untreated subjects? As they appear to be released when opioid

drugs are administered, what role does this release play in the

development of tolerance and dependence to opioids? Second,

since we live in a society where polydrug abuse is the rule and

not the exception among drug abusers, what are the interactions

35
among the drugs of abuse? Right now we know very little about

these interactions per se, let alone the mechanisms that may be

involved. Third, is there a commonality of mechanisms for

tolerance and dependence among the drugs of abuse? Although

such hypotheses have been advanced, little scientific support

for these ideas has been forthcoming. Fourth, what are the

correlations between binding data and pharmacological effects?

This is a crucial question, for both acute and chronic administration of drugs, that must be addressed to a greater degree

than it presently is. Lastly, what are the interactions between

transmitter systems that have been implicated in tolerance and

dependence, both in cases where pharmacological receptors have

been identified (e.g., opioids) and in cases where they have not

(e.g., cocaine, marijuana)?

A little over 10 years ago, Harry Collier, to whose memory

this symposium is dedicated, wrote a paper entitled "A

Pharmacological Analysis of Drug-Dependence" (1972). In it he

stated that the neuropsychological properties of a drug help to

determine its abuse liability. He felt that since drugs produce

dependence, other drugs should be able to reverse or ameliorate

it. He proposed that "we should now try to develop drugs especially to help combat dependence. To do this effectively and to

make progress in the pharmacotherapy of drug abuse, we need to

know more about the basic mechanisms whereby dependencies arise

and about the interactions of other drugs with these

mechanisms." We have made good progress in elucidating the

mechanisms involved. Nevertheless, much more basic research

must be carried out before we can design specific drugs to

affect specific receptors in specific loci and before we can

develop drugs to correct any underlying deficiencies that may

play a role in drug dependence.

REFERENCES

Adler, C.H., Keren, O., and Korczyn, A.D. Tolerance to the

mydriatic effects of morphine in mice. J Neural Transm,

48:43-47, 1980.

Adler, C.H., Robin, M., and Adler, M.W. Tolerance to morphineinduced mydriasis in the rat pupil. Life Sci, 28:2469-2475,

1981.

Adler, M.W., Geller, E.B., Beeton, P.B., and Gildenberg, P.L.

Inability of acute or chronic thalamic, limbic, or cortical

lesions to alter narcotic dependence and abstinence in rats. In:

Van Ree, J.M., and Terenius, L., eds. Characteristics and

Function of Opioids. Amsterdam: Elsevier/North-Holland, 1978.

pp. 51-52.

Adler, M.W., Hawk, C., and Geller, E.B. Comparison of intraventricular morphine and opioid peptides on body temperature of

rats. In Lomax, P. and Schonbaum, E., eds. Environment, Drugs,

and Thermoregulation. Basel: Karger, 1983. pp. 90-93.

Calvino, B., Lagowska, J., and Ben-Ari, Y. Morphine withdrawal

syndrome: Differential participation of structures located

36
within the amygdaloid complex and striatum of the rat. Brain

Res, 177:19-34, 1979.

Cerletti, C., Keinath, S.H., Reidenberg, M.M., and Adler, M.W.

Chronic morphine administration: Plasma levels and withdrawal

syndrome in rats. Pharmacol Biochem Behav, 4:323-327, 1976.

Cerletti, C., Keinath, S.H., Tallarida, R.J., Reidenberg, M.M.

and Adler, M.W. Morphine concentrations in the rat after intraperitoneal or subcutaneous injection. Subst Alcohol Actions

Misuse, 1:65-70, 1980.

Cochin, J., and Kornetsky, C. Development and loss of tolerance

to morphine in the rat after single and multiple injections.

J. Pharmacol Exp Ther, 145:1-10, 1964.

Cochin, J., and Mushlin, B.E. Effect of agonist-antagonist

interaction on the development of tolerance and dependence. In:

Vesell, E.S., and Braude, M.C., eds. Interactions of Drugs of

Abuse. Ann NY Acad Sci, Vol. 281. New York: NY Academy of

Sciences, 1976. pp.244-251.

Collier, H.O.J. A pharmacological analysis of drug dependence.

In: van Pragg, H.M., ed. Biochemical and Pharmacological Aspects

of Dependence and Reports on Marijuana Research, Haarlem: Bohn,

1972. pp. 23-45.

Cowan, A., Boardman, S., and Robinson, T. Buprenorphine and

intestinal motility: A pharmacological analysis of the biphasic

dose-response curve. Fed Proc, 36:994, 1977.

Dafny, N. The hypothalamus exhibits electrophysiologic evidence

for morphine tolerance and dependence. Exp Neurol, 77:66-77,

82.

Domino, E.F. Neuropsychopharmacologic studies of marijuana:

Some synthetic and natural THC derivatives in animals and man.

In: Singer, A.J., ed. Marijuana: Chemistry, Pharmacology, and

Patterns of Social Use. Ann NY Acad Sci, Vol 191. New York: NY

Academy of Sciences, 1971. pp. 166-191.

Dum, J., Blasig, J., and Herz, A. Buprenorphine: Demonstration

of physical dependence liability. Eur J Pharmacol, 70:293-300,

1981.

Geller, E.B., Hawk, C., Keinath, S.H., Tallarida, R.J., and

Adler, M.W. Subclasses of opioids based on body temperature

change in rats: Acute subcutaneous administration. J Pharmacol

Exp Ther, 225:391-398, 1983.

Goodman, L.S., and Gilman, A. The Pharmacological Basis of

Therapeutics. 2nd edition. New York: Macmillan, 1955. p. 223.

Huidobro, F., Huidobro-Toro, J.P., and Way, E.L. Studies on

tolerance development to single doses of morphine in mice. J

Pharmacol Exp Ther, 198:318-329, 1976.

Jaffe, J.H. Drug addiction and drug abuse. In: Gilman, A.G.,

Goodman, L.S., and Gilman, A., eds. Goodman and Gilman's The

Pharmacological Basis of Therapeutics. 6th edition. New York:

Macmillan, 1980, pp. 535-584.

Jaffe, J.H., and Martin, W.R. Opioid analgesics and antagonists.

In: Gilman, A.G., Goodman, L.S., and Gilman, A., eds. Goodman and

Gilman's The Pharmacological Basis of Therapeutics. 6th edition.

New York: Macmillan, 1980, pp. 494-534.

Kornetsky, C., and Bain, G. Biobehavioral bases of the reinforcing properties of opiate drugs. In: Verebey, K., ed. Opioids

in Mental Illness. Ann NY Acad Sci, Vol 398. New York: NY

37
Academy of Sciences, 1982. pp. 241-259.

Kornetsky, C., and Bain, G. Morphine: single-dose tolerance.

Science, 162:1011-1012, 1968.

Linseman, M.A. Effects of lesions of the ventromedial hypothalamus on naloxone-induced morphine withdrawal in rats.

Psychopharmacologia (Berl.), 45:271-276, 1976.

Rebec, G.V., and Segal, D.S. Apparent tolerance to some aspects

of amphetamine stereotypy with long-term treatment. Pharmacol

Biochem Behav, 13:793-797, 1980.

Shiffman, S.M. The tobacco withdrawal syndrome. In: Krasnegor,

N.A., ed. Cigarette Smoking as a Dependence Process, National

Institute oh Drug Abuse Research Monograph 23. DHEW Pub. No.

(ADM) 79-800. Washington, D.C: Supt. of Docs., U.S. Govt.

Print. Off., 1979. pp. 158-184.

Takemori, A.E. Biochemistry of drug dependence. Annu Rev

Biochem, 43:15-33, 1974.

Tortella, F.C., Cowan, A., and Adler, M.W. Studies on the excitatory and inhibitory influences of icv opioids on seizure

threshold in rats. Neuropharmacology, 1984, in press.

Tress, K.H., El-Sobky, A.A., Aherne, W., and Piall, E. Degree of

tolerance and the relationship between plasma morphine concentration and pupil diameter following intravenous heroin in

man. Br J Clin Pharmacol, 5:299-303, 1978.

Weiner, N. Norepinephrine, epinephrine, and the sympathomimetic

amines. In: Gilman, A.G., Goodman, L.S., and Gilman, A., eds.

Goodman and Gilman's the Pharmacological Basis of Therapeutics.

6th edition. New York: Macmillan, 1980. pp. 138-175.

Weisbrodt, N.W., Thor, P.J., Copeland, E.M., and Burks, T.F.

Tolerance to the effects of morphine on intestinal motility of

unanesthetized dogs. J Pharmacol Exp Ther, 215:515-521, 1980.

ACKNOWLEDGEMENTS

Drs. Joseph Cochin and Frank Tortella provided some of the data

presented in this paper. Work in the authors' laboratory was

supported in part by Grant DA 00376 fran the National Institute

on Drug Abuse.

AUTHORS

Martin W. Adler, Ph.D.

Ellen B. Geller, M.A.

Department of Pharmcology

Temple University School of Medicine

3420 N. Broad Street

Philadelphia, PA 19140

38
Various Factors Which Affect the

Rate of Development of Tolerance

and Physical Dependence to

Abused Drugs

William L. Dewey

A recent study by Ternes and his colleagues (1983) showed that

tolerance and physical dependence developed to the chronic

treatment of hydromorphine in rhesus monkeys but not in

cynomolgus monkeys. This report prompted us to reevaluate the

factors which are involved in the differences which have been

reported for the rates of development of tolerance and physical

dependence in various species. It is clear that even in the same

animal tolerance develops to different effects of opiates and

other drugs at different rates. This observation rules out the

possibility of an alteration in drug absorption, metabolism,

excretion, etc., as a basis for the tolerance. There are certain

effects to which tolerance does not develop. For instance, most

reports indicate that although tolerance develops to many of the

effects of the opiates, miosis appears to be resistant to

tolerance development. Apparently, the drug receptors on the

cells of different tissues become less sensitive to the drug at

different rates, whereas pupil changes are always sensitive to

opiates. These observations support the hypothesis generated

from other types of data that many different types of opiate

receptors exist. The number might exceed by far the seven or

eight types reported to date. There is no question that

metabolic tolerance does occur with some drugs. When this is the

only factor involved in the development of tolerance, tolerance

would be expected to develop to all the effects of the drug at

the same rate. It is important to differentiate and quantitate

the contribution of metabolic and pharmacodynamic factors in the

development of tolerance. The discussion that follows will be

devoted to pharmacodynamic tolerance.

Aceto et al. (1977) have shown that the subcutaneous

injection of 3 mg/kg of morphine sulfate every 6 hrs for 30

days to rhesus monkeys did not render them dependent as

measured by the ability of naloxone or nalorphine to induce

39
vomiting. Vomiting as a withdrawal sign is an indication of

severe dependence of the morphine type in rhesus monkeys. Some

other less severe signs of dependence were observed following the

30 days of treatment. The ED50 for naloxone required to induce

vomiting was higher following 60 days of morphine treatment than

following 90 days of treatment. These data support the

hypothesis that the level of dependence, like that of tolerance,

increases as the time of treatment is extended. These results

were confirmed further by the observation that nalorphine was

able to induce vomiting in rhesus monkeys only following 90 days

of treatment. These results along with those of Ternes et al.

(1983) make another important point concerning the development of

tolerance and dependence to drugs. That is, the end point

measured should be very carefully stipulated and monitored.

Ternes et al. (1983) reported that in the rhesus monkey

dependence and to some extent tolerance and withdrawal developed

following chronic treatment with hydromorphine. This was not

observed following more frequent and longer treatment with

morphine in rhesus monkeys (Aceto et al. 1977).

For a number of years, we have been investigating the effects of

chronic opiate treatment of rats using the ip infusion technique

of Teiger (1974). Animals are infused constantly, 24 hrs a day,

with the test drug. The animals are freely moving, with access

to food and water ad libitum. One of the advantages of this

procedure is that after the end of the morphine infusion,

spontaneous withdrawal symptoms are observed. These symptoms are

characterized by a pronounced loss in body weight of 20% or more

within 24 hrs after the infusion of the opiate. Other signs of

the opiate withdrawal previously described, including

hyperexcitability, irritability, chewing, scratching, wet dog

shakes and diarrhea, are quantitated at various times throughout

the first 96 hrs of withdrawal. We have utilized this technique

to study the importance of a number of variables on the

development of tolerance and physical dependence in the rat. We

first studied the alteration of the duration of morphine infusion

to determine the time required to induce physical dependence in

rats. We compared the infusion schedules, as shown, in the table

below:

TABLE 1

Study Dose of Morphine Infused (mg/kg/day)

1 200 for one day (on day 1)

2 100 on day 1

200 on day 2

3 50 on day 1

100 on day 2

200 on day 3

4 50 on day 1

100 on day 2

200 on day 3, 4, 5 and 6

40
PERCENT LOSS IN BODY WEIGHT

FOLLOWING ONE DAY MORPHINE

INFUSION

FIGURE 1. Loss in Body Weight Following Schedule 1

PERCENT LOSS IN BODY WEIGHT

FOLLOWING TWO DAY MORPHINE

INFUSION

FIGURE 2. Loss in Body Weight Following Schedule 2

41
PERCENT LOSS IN BODY WEIGHT

FOLLOWING THREE DAY MORPHINE

INFUSION

FIGURE 3. Loss in Body Weight Following the 3-Day Schedule

There was no loss in body weight after 1 day of infusion (figure

l), whereas weight loss did occur as early as 6 hrs after 2 days

of infusion (figure 2); an even greater loss in body weight was

seen when the animals were infused on the 3-day schedule (figure

3). The 6-day infusion schedule presented in table 1 is used to

produce the greatest level of physical dependence to morphine

(figure 4).

As can be seen to the left of the dotted vertical line, the rats

in each group either maintained their body weight or gained

slightly during the last 2 days of the infusion period. The data

from the right of the vertical line indicates that rats infused

with morphine for 6 days and then given saline lost more than 20%

of their body weight during the first 24 hrs after infusion.

This was a greater weight loss than was presented in figure 3 for

rats which had been infused with morphine on the 3-day schedule.

This decrease in body weight persisted throughout the 96-hr

observation period. Signs, such as a loss in body weight, were

most pronounced 24 hrs after opiate infusion.

In similar studies measuring a longer period of withdrawal,

following the end of chronic morphine infusion, rats do gain

weight, but not to the degree of controls up to 20 days after

withdrawal. The results of the 6-day experiment indicated

infusion of saline or naloxone did not induce reduction in body

weight following the end of the infusion period. Also, the

exposure to naloxone with morphine completely blocks the loss in

body weight and other signs of opiate withdrawal in these rats.

42
FIGURE 4. Loss in body weight following schedule 4. Placebo or

naloxone (75 mg) pellets were implanted immediately prior to the

start of the infusion. The end of the 6-day infusion period is

represented by the dotted vertical line indicated on the abscissa

as 0 hrs. All percent changes in body weight are calculated

based on the body weight of each rat at the end of the infusion.

In general, the rate of appearance of the other signs of

withdrawal correlate with the decrease in body weight.

It is our hypothesis that the constant infusion of naloxone

competitively inhibited access of the morphine to the opiate

receptors, thus blocking the initial step required for the

development of physical dependence, Naloxone has a shorter

duration of action than morphine and must be given in a fashion

similar to this in order to maintain proper levels at the opiate

receptors. This portion of the experiment also shows that

tolerance did not develop to the antagonistic effect of the

narcotic antagonist. These observations and hypotheses have

interesting implications. We are proposing that morphine causes

physical dependence and tolerance (see below) during constant

infusion by constantly or at least repeatedly interacting with

opiate receptors. When naloxone is infused simultaneously with

morphine, it too has an effect at the opiate receptors. That is,

it blocks the ability of morphine to induce physical dependence.

Tolerance does not develop to naloxone but does develop to the

constant exposure of another drug, morphine. Does this mean that

tolerance at the receptor level is specific for different drugs?

These data, like considerable data generated from binding assays

43
and other in vitro experiments, are explained by the purported

lack of efficacy of naloxone at the opiate receptor.

Patrick et al. (1978) reported that during the induction of

tolerance and physical dependence on morphine using the 6-day

infusion procedure described in table 1, antinociceptive activity

correlated quite well with brain levels of morphine. The

antinociceptive activity, as well as the level of morphine in

brain following the 6-day infusion (that is, the 4th day of the

200 mg/kg/day dose) was significantly less than after the first

day of infusion of the 200 mg/kg/day dose of morphine. These

authors also reported that the brain level of morphine fell

precipitously after the end of its ip infusion. Yet tolerance to

subsequent injections of morphine remained for many hours beyond

the time they could measure levels of morphine in whole brain.

It was apparent from these results that the duration of the

phenomenon of tolerance far exceeded the presence of morphine in

the brain. Two possibilities exist to explain these results.

Either the analytical technique used in these experiments was not

sensitive enough to measure the low levels of morphine in the

brain at the end of the infusion, or morphine is not required in

the brain to manifest tolerance.

In other experiments using mice, the brain level of morphine

correlated closely with the antinociceptive activity as measured

by the tail-flick test after the implantation of a 75 mg morphine

pellet (Patrick et al. 1975; Way et al. 1969). Again, the

antinociceptive activity somewhat paralleled the brain level of

morphine as described in the rat experiments above; the peak

analgesic activity preceded peak brain levels in mice. When the

pellet was removed 72 hrs after implantation, the level of

morphine fell dramatically in mice as it did at the end of the

ip infusion of morphine in rats. Again, tolerance to an

injection of morphine lasted well beyond the time we could

measure levels of morphine in mouse brain. Taken together, the

data generated suggest that there is a temporal component to

tolerance development in animals and that once tolerance has

formed the duration of the phenomenon outlasts detectable levels

of morphine in brain.

In a further attempt to investigate the characteristics of the

temporal phenomena involved with tolerance development, we

injected a dose of 160 mg/kg of morphine or saline in many groups

of mice and determined the ED50 values for subsequent injections

of morphine in the mouse tail-flick test at 16, 24, 48 hrs and at

4 and 8 days after the initial injection. Morphine injected in

the saline-treated mice served as controls for the animals that

were pretreated with 160 mg/kg morphine. The ED50 values for

these groups were not significantly different at any time,

showing that tolerance did not develop in these experiments.

It can be inferred from these data that the pharmacokinetics of

opiates are such that one injection of a very high dose does not

induce tolerance. However, when we injected mice with 10 mg/kg

of morphine every 12 hrs for 10 days and then determined an ED50

44
TABLE 2

Activity in the Mouse Tail-Flick Test

Injection schedule of

10 mg/kg morphine S.C.

1 injection

Percent analgesia

Group 1 - 83%

Group 2 - 83%

every 12 hrs for 10 days Group 1 - 10%

Group 2 - 10%

every 12 hrs for 10 days

(each given 10 mg/kg morphine, S.C.

10 min after 1 mg/kg naloxone, s.c.)

Group 1 - 59%

Group 2 - 56%

value for morphine in the tail-flick test, we found pronounced

tolerance to the antinociceptive action of morphine in mice.

These data are presented in table 2. The results obtained in

these mouse experiments were similar to those in the rat infusion

experiments, in that pretreatment with naloxone blocked the

development of tolerance to the chronic effects of morphine.

Thus, a total dose of 200 mg/kg morphine injected at regular

intervals over a 10-day period induced tolerance, whereas an

acute dose of 160 mg/kg did not. These data suggest the

necessity for a temporal relationship for the development of

tolerance to the antinociceptive effect of morphine in mice. It

is important to point out that these data do not indicate the

best temporal rate of injection of morphine to produce tolerance

to the antinociceptive activity of morphine in other species nor

do they tell us anything about the frequency of injection of

morphine to demonstrate the development of tolerance to any

effect of morphine in mice other than antinociception. Clearly,

tolerance develops to the same effect in various species at

different rates and also develops at different rates to different

effects in the same species.

In the next series of experiments, we compared the development of

tolerance and dependence to two other narcotic analgesics, methadone

and 1 acetylmethadol (LAAM). The antinociceptive potencies of

these drugs in mice and rats are presented in table 3.

TABLE 3

The Potency of Methadone and LAAM in Tail-Flick Test

Species

Mouse 10.8 (6.3-18.5)

ED50 in mg/kg

Methadone LAAM

10.8 (5.0-23.6)

Rat 14 2

45
As can be seen in this table, methadone is equally potent to LAAM

in mice but 1/7 as potent as LAAM in rats. Rats were injected

chronically with oral doses of LAAM and methadone according to

the dosage schedule presented in table 4.

TABLE 4

Oral Dosage Schedule for Methadone and LAAM in Rats

Methadone (every 0-12 hrs or 2 times/day)

Days

1 - 3

4-7

8 - 12

LAAM (every 0-24 hrs or once daily)

Days

1 -3

4-7

8 - 12

Dose (mg/kg)

14

28

56

Dose (mg/kg)

2

4

8

Considerable tolerance developed to the antinociceptive activity

of LAAM, whereas tolerance did not develop to the same effect of

methadone following the injection schedule presented in table 4.

Methadone was given twice a day, whereas LAAM was injected only

once a day. Tolerance developed to LAAM much more rapidly than

to methadone. We next investigated the characteristics of the

temporal phenomena involved in the ability of these two agents to

produce physical dependence, using the oral administration

schedule in table 5.

TABLE 5

Oral Dosage Schedule for Mouse Jumping Test (mg/kg)

Day Methadone* LAAM

1 20 2

2 40 4

3 80 4

4 160 8

*Methadone was administered at these doses twice a day and

LAAM was administered once a day.

46
The dose of naloxone required to produce jumping in 50% of the

mice was used as an index of physical dependence. The lower the

dose of naloxone required to induce jumping, the greater the

level of physical dependence. The results of these experiments

are presented in table 6.

TABLE 6

Naloxone ED50 (s.c.) in the Mouse Jumping Test

Methadone LAAM

74.5 mg/kg

(58.9-94.2)

0.47 mg/kg

(0-16-1.36)

(Potency Ratio = 158)

Methadone-treated mice appeared normal, LAAM-treated mice lost

weight and some died.

Physical dependence was more pronounced with LAAM than with

methadone in these experiments. There appears to be a

correlation between the temporal aspects of the development of

tolerance and the development of physical dependence for these

two substances. An explanation for this similarity becomes quite

apparent when one looks at the data obtained on the duration of

antinociceptive action of methadone and LAAM in mice and rats.

Rats treated orally with 6 mg/kg LAAM showed significant

analgesic activity up to 14 hrs after medication. The duration

of antinociceptive action of 16 mg/kg methadone given orally was

less than 2 hrs. These experiments were repeated in mice.

Methadone at a oral dose of 25 mg/kg produced significant

analgesia, but its duration of action was less than 2 hrs. The

duration of action of LAAM exceeded 12 hrs. These data suggest a

similar mechanism is involved in both species, and it seems

reasonable to suggest that this is so. In order to induce

tolerance, methadone must be in contact with brain receptors for

a longer period of time than is possible with this treatment

schedule. Equianalgesic injections of LAAM given half as

frequently as the injections of methadone are sufficient to

develop tolerance and physical dependence.

Since tolerance and dependence can develop to a particular effect

at different rates with two drugs in the same class, it is not

surprising that they can develop at different rates for a number

of drugs of abuse from different classes. We have previously

reported (Dewey et al. 1972, McMillan et al. 1971, 1972) several

characteristics of tolerance to THC. Tolerance is pronounced,

develops rapidly, and lasts for many days after the drug is

withdrawn. It is also important to note that withdrawal symptoms

do not develop following the cessation of chronic treatment with

cannabinoids in many species. Tolerance develops to the

cannabinoids even if the injections are given at infrequent

intervals. Tolerance develops to most of the effects of

cannabinoids that have been studied, and cross-tolerance among a

number of cannabinoids has been reported.

47
The major portion of this article has been devoted to the

characteristics of tolerance and physical dependence that occur

with opiates. Also mentioned are some of the most important

aspects of tolerance and dependence to the cannabinoids. The most

important point is that tolerance and dependence develop to

different effects of drugs at different rates. Tolerance and

dependence are not interchangeable. The terms tolerance and/or

dependence should only be used to describe the state of an animal

when both characteristics have been quantitated in that animal.

An animal may be tolerant to a specific effect of an opiate and

not be physically dependent. Conversely, an animal may be

physically dependent and not be tolerant to a specific effect of

the drug. This is even more obvious for non-opiate drugs.

REFERENCES

Aceto, M.D., Flora, R.E., and Harris, L.S. The effects of

naloxone or nalorphine during the development of morphine

dependence in rhesus monkeys. Pharmacol, 15:1-9, 1977.

Dewey, W.L., Jenkins, J., O'Rourke, T., and Harris, L.S. The

effects of chronic administration of trans -tetrahydrocannabinol on behavior and cardiovascular system of dogs.

Arch Int Pharmacodyn, 198(1):118-131, 1972.

of tetrahydrocannabinol tolerance. Ann NY Acad Sci,

McMillan, D.E., Dewey, W.L., and Harris, L.S. Characteristics

191:83-100, 1971.

McMillan, D.E., and Dewey, W.L. On the mechanism of tolerance to

THC. In: Lewis, M.F., ed. Current Research in Marijuana.

New York and London: Academic Press, 1972 pp.97-114.

Patrick, G.A., Dewey, W.L., Spaulding, T.C., and Harris, L.S.

Relationship of brain morphine levels to analgesic activity in

acutely treated mice and rats and in pellet-implanted mice.

J Pharmacol Exp Ther, 193:876-883, 1975.

Patrick, G.A., Dewey, W.L., Huger, F.P., Daves, E.D., and Harris,

L.S. Distribution of morphine in chronically infused rats:

Relationship to antinociception and tolerance.

J Pharmacol Exp Ther, 205:556-562, 1978.

Teiger, D.G. Induction of physical dependence on morphine,

codeine, and meperidine in the rat by continuous infusion.

J Pharmacol Exp Ther, 190:408-415, 1974.

Terries, J.W., Ehrman, R., and O'Brien, C.P. Cynomolgus monkeys

do not develop tolerance to opioids.

Behav Neurosci, 97(2):327-330, 1985.

Way, E.L., Loh, H.H., and Shen, F.H. Simultaneous quantitative

assessment of morphine tolerance and physical dependence.

J Pharmacol Exp Ther, 167:1-8, 1969.

ACKNOWLEDGEMENTS

The work reported in this article was supported in part by USPHS

grants DA-01647, DA-00490, and T32 DA-07027 from the National

Institute on Drug Abuse.

48
AUTHOR

William L. Dewey, Ph.D., Department of Pharmacology, Medical

College of Virginia, Virginia Commonwealth University, MCV

Station, Box 613, Richmond, Virginia 23298-0001

49
Genetic Determinants of Responses to Drugs of Abuse: An

Evaluation of Research Strategies

Louis Shuster

The use of certain genetically defined animal models can be very

helpful in studies of drugs of abuse. The choice of model will be

influenced by the aims of the study. This overview is intended to

indicate the kind of information that can be obtained and some of

the strategies that can be employed.

GOALS OF GENETIC ANALYSIS

1. To explain individual variations in response. About 25% of

post-operative patients do not experience appreciable pain relief

when injected with a standard dose of morphine sulfate. Another

35% obtain substantial relief from a placebo injection of saline

(Lasagna and Beecher 1954). Sorting out the relative contributions of environment and heredity to such variability is very difficult to accomplish with human subjects. However, it is now

widely appreciated that genetic determinants can affect the responses of animals to psychopharmacologic agents in many ways,

ranging from altered metabolic inactivation to variations in population of neurons and neuronal receptors. Differences between

inbred strains when all are raised and tested in the same environment are assumed to indicate the contribution of a genetic component. For example, Brase et al. (1977) measured five different

responses to morphine in six inbred strains of mice and found

variations of as much as 12-fold in some of them, such as the

ED50 of naloxone for precipitated abstinence. The heritability or

relative genetic contribution to a drug response can be calculated

by testing the offspring of a few crosses and comparing them to

the parents (Falconer 1960).

There are still some workers who continue to document drug responses obtained with "rats" or "white mice" or "laboratory mice."

A larger number persist in using random-bred (outbred) populations

which may exhibit wide ranges of response. Such animals, e.g.

Swiss-Webster, may give results which vary with the supplier (Way

et al. 1969), and compound the difficulty of reproducing values

obtained in other laboratories. In general, however, it is widely

appreciated that responses to ethanol, morphine or cocaine can

vary with the genetic background of the test animal. Establishing

50
this fact should no longer by itself be a major goal of pharmacogenetic investigations.

2. To separate different components of the response to a given

drug. By using different inbred strains of mice it can be shown

that motor stimulation by morphine can be dissociated from the

analgesic response (Castellano and Oliverio 1975). One can also

separate some manifestations of physical dependence, such as naloxone-induced withdrawal jumping, from the development of tolerance (Brase et al. 1977).

Comparison of different rat strains has revealed similarities between some actions of morphine and ethanol on the central nervous

system which are not shared by barbiturates (Mayer et al. 1983).

Genetic evidence has also 'been advanced from correlations between

the number of D2 dopamine receptors in the brain and several drug

responses (Helmeste et al. 1981).

3. To develop models that differ quantitatively in a given drug

example, strain F344 rats and Buffalo rats differ in responses to

response. These models may be two or more inbred strains. For

dopaminergic drugs (Helmeste 1983); low doses of ethanol stimulate

the motor activity of BALB/c and DBA mice, but not C57B1/6 mice

(Kiianmaa and Tabakoff 1983); morphine stimulates the motor activity of C57B1/6 mice but not DBA mice (Oliverio and Castellano

1974).

An alternative approach is to breed selectively high-responding

and low-responding lines from a heterozygous foundation stock--for

example, mice that sleep for a longer or a shorter time after a

given dose of ethanol (McClearn and Anderson 1979).

4. To establish neurochemical mechanisms of drug action. A common

aim is to develop meaninqful correlations between a drug response

and variables such as the amount or turnover of a neurohormone or

the number of receptors. For example, F344 rats, which exhibit

greater spontaneous locomotor activity and more inhibition of

locomotion by apomorphine than Buffalo rats, also have greater

numbers of D2-dopamine receptors in the striatum and olfactory

tubercle (Helmeste 1983). There are also striking differences

between these strains in the epinephrine content, rate of epinephrine synthesis, and number of alpha adrenergic receptors in

certain brain regions (Perry et al. 1983).

5. To identify the genetic determinants of particular responses.

Does a single gene control narcotic analgesia, or are there multiple genes? Are they dominant, recessive, autosomal? Which

chromosomes and which particular loci are involved? Do the same

genes modulate the responses to several different classes of

drugs? Can useful linkage be established between certain drug

responses and pleotropic markers such as coat color? For instance, there is a report that the albino locus in mice is associated with decreased responsiveness to morphine (Katz 1980).

51
STRATEGIES

Several useful strategies are now available to investigators who

are interested in the goals described here.

1. Selective breeding. A defined drug response, such as analgesia, sleep time, motor activity, etc. is chosen, and a program

is developed to produce strains characterized by the two extremes

of this response. The foundation stock should exhibit considerable genetic variability. McClearn started by intercrossing eight

purebred strains of mice. Selective breeding of high-responding

and low-responding individuals has continued for over 25 generations. This selection has produced a long sleep (LS) line of

mice, which sleep several hours, and a short sleep (SS) line of

mice, which sleep several minutes, in response to a test dose of

ethanol (McClearn and Anderson 1979).

Ideally, up to several hundred animals should be started in such a

selection program in order to avoid premature inbreeding, and the

entire process, including maintenance of a control line, should be

replicated. A recent example of properly executed selective

breeding is that carried out by Crabbe et al. (1983a) for the

severity of withdrawal from ethanol.

The difference between LS and SS lines of mice seems to reflect a

difference in the sensitivity of the central nervous system rather

than a difference in ethanol metabolism (Heston et al. 1974).

Cerebellar Purkinje cells from LS mice are 10-20 times more

sensitive to depression by ethanol than those from SS mice

(Sorennsen et al. 1980). The latter's cells also develop tolerance to ethanol much more readily. The fluidizing effect of ethanol on synaptic membranes from LS mice is more pronounced than on

those from SS mice (Goldstein et al. 1982).

The main advantage of selective breeding is that it provides animals which were chosen specifically because of an inherited difference in a particular drug response. All other strategies yield

animals which were found to differ in a drug response only after

they had been selected or bred for other qualities such as coat

color or susceptibility to tumor growth. However, this method

also has some serious disadvantages. Selective breeding is cumbersome, expensive and slow. Repeating or comparing experiments

is almost impossible until homozygosity has been achieved--a matter of 40 generations or about 15 years.

Selective breeding for only a few generations in order to reveal

that a given drug response has a genetic component is usually not

worth the effort. For example, showing that mice could be selectively bred on the basis of their running response to levorphanol

(Judson and Goldstein 1978) did not reveal any more than had

already been established by comparing inbred lines (Oliverio and

Castellano 1974). Correlations which develop during the first few

generations of selection can be misleading. When Wimer and Wimer

(1982) bred mice for high and low brain weight, the initial

52
parallelism between brain weight and ability to achieve certain

learning tasks disappeared by generation 14.

2. The Use of Inbred Strains. A large number of inbred (purebred)

strains of mice are now available commercially. They are essentially homozygous, being derived from over 40 generations of

brother-sister matings (Heiniger and Dorey 1980). The choice of

which strains to compare can be based on a genetic similarity

matrix (Taylor 1972). As can be seen from figure 1, C57 Black

mice and related strains differ in a large number of loci from

BALB/c, A/J and DBA/2 mice. Furthermore, there are already

numerous examples in the literature of drug-related differences

between C57 and the other three strains.

Selections can also be made on the basis of neurochemical differences. For example, C57B1/6 mice have more choline acetyltransferase and tyrosine hydroxylase, but less glutamate decarboxylase,

in certain brain regions than A/J mice (Waller et al. 1983). They

also have fewer dopamine receptors in the corpus striatum and

olfactory tubercle (Boehme and Ciaranello 1981). Crabbe et al.

(1981) found that C57B1/6 mice have twice as much beta endorphin

in their pituitaries as BALB/c or DBA/2 mice. The striatum of

CBA/J mice is 20% smaller than that of BALB/cJ mice. There is a

corresponding difference in the activities of striatal tyrosine

hydroxylase and choline acetyltransferase (Baker et al. 1980).

BALB/cJ mice have half as many benzodiazepine receptors in the

brain as C57B1/6J mice (Robertson 1979). A compilation of neurochemical values for different mouse strains has been published by

Ingram and Corfman (1980).

Extensive comparisons of the pharmacologic and biochemical responses of C57B1/6, BALB/C, and DBA/2 mice to morphine has been

carried out by Castellano, Oliverio and their colleagues. Morphine produces analgesia in both strains, but increases the locomotor activity of only the C57B1/6 mice (Castellano and Oliverio

1975). Diallel analysis of crosses and backcrosses suggests that

the C57 trait is dominant (figure 2). Morphine increases the release of dopamine from the striatum and limbic forebrain in C57

mice but not in DBA mice (Racagni et al. 1979).

Reggiani et al. (1980) found that C57 mice have more receptors in

the striatum for enkephalins than DBA mice. On the other hand,

contraction of the vas deferens of DBA mice is more readily inhibited by normorphine than that of C57Bl mice (Szerb and Vohra

1979). Another interesting difference between these two strains

is that it takes much less naloxone to produce withdrawal jumping

in DBA mice than in C57 mice (Brase et al. 1977).

An additional response to morphine that displays interesting

strain differences is sensitization, or reverse tolerance to motor

stimulation. C57Bl/6J mice are sensitized more than A/J mice, and

the F1 hybrid gives an intermediate response (Shuster et al.

1975a) (figure 3).

53
FIGURE 1. Positions of 27 inbred mouse strains in a 2-dimensional genetic

similarity matrix. Reproduced, by permission, from Taylor 1972. Copyright 1972,

by the American Genetic Association.
FIGURE 2. Genetic triangles depicting the observed and expected

population means for the running activity, in response to morphine, of BALB/cJ, C57B1/6J, and DBA/2J mice and their crosses.

The corner points of the triangle represent the mean values for

the parental lines. The expected mean for the F1 generation (M)

is halfway between the parental values. The expected means for

backcrosses of F1 to each parent (B1 and B2) are halfway between

the parental and F1 values. Observed values are indicated by

filled circles, with arrows pointing to the expected values. B

and C and D refer to BALB/cJ, C57B1/6J and DBA/2J respectively.

Reproduced, by permission, from Castellano and Oliverio (1975).

Copyright 1975, Springer Verlag New York, Inc.

55
FIGURE 3. Changes in the running response of C57B1/6J, A/J, and

the B6AFl/J hybrid male mice to repeated weekly i.p. injections of

morphine sulfate, 25 mg/kg. Each point represents 10 mice ± SEM.

Reproduced, by permission, from Shuster, Webster, and Yu (1975).

Copyright 1975, American Society for Pharmacology and Experimental

Therapeutics.

56
Crabbe et al. (1983b) have examined several responses to ethanol

in 19 different inbred strains of mice. They found that the

severity of withdrawal was inversely correlated to the initial

sensitivity and the degree of tolerance to ethanol hypothermia.

C57B1/6 mice are less sensitive to the activating and hypnotic

effects of ethanol than BALB/c or DBA/2 mice. (Kiianmaa and

Tabakoff 1983). Low doses of ethanol release less dopamine from

the striatum of C57B1/6 mice than from the striatum of the other

two strains (ibid).

It is unlikely that the kinds of differences and correlations described here can lead to the identification of the genetic determinants of the responses to morphine or ethanol. The inbred

strains that are compared in these studies may differ in hundreds

or thousands of genes. However, they can suggest which neurochemical mechanisms are worth pursuing. They also provide readily

available animal models which can yield consistent, reproducible

results in different laboratories.

3. Recombinant-Inbred and Congenic Lines. Even elementary genetic

analysis of differences in drug responses between inbred lines

requires time-consuming crossing and backcrossing. The purpose of

these crosses is to shift around the genetic determinants in order

to establish which ones might be associated with the drug response. Bailey (1971) has developed a method for randomizing and

fixing the genes from two inbred lines in a replicable, homozygous

form. A battery of recombinant-inbred (RI) lines is derived by

repeated sib matings for 40 generations or more from the F2 generation of the original cross. Because the distribution of histocompatibility loci among the lines has been defined by skin-grafting, it is possible to ascribe linkage of drug responses to these

loci on the basis of a pattern of similarity to one or other of

the progenitor strains. Every time a different drug is tested,

the drug response itself becomes a new linkage marker for a given

chromosome region.

As the number of markers increases, so does the probability of

linking new characteristics. The seven RI lines derived from

C57B1/6 and BALB/c have been typed for about 80 loci, so that the

probability of detecting linkage with these lines is 0.37. For

the 24 RI lines derived from C57B1/6 and DBA/2. the identification

of 80 markers would bring this probability to about 0.7 (Taylor

1978).

The availability of a test battery of congenic lines enhances the

usefulness of the RI lines (Bailey, 1975). Repeated backcrossing

from the F1 generation to one progenitor strain (A) produces animals in which only a small portion of the genome is derived from

the other progenitor (B). This portion can be identified by histocompatibility testing. If the histocompatibility follows the

same distribution pattern across the R1 lines (e.g. AABABBA) as a

drug response, then one can identify the genetic locus which

governs that response.

57
RI analysis is most useful when a large difference in response

mexists between the two progenitor strains, and this difference is

controlled by a single gene. If more than one gene is involved,

then the phenotype of some RI lines may be unlike that of either

progenitor. This situation seems to describe running and analgesia in response to morphine (Shuster et al. 1975b; Oliverio et al.

1975). Furthermore, the strain distribution pattern for analgesia

was different with the hot-plate assay than with the tail-flick

assay (Shuster 1975).

Other responses that have been examined in RI mice include the

development of narcotic tolerance (Oliverio and Castellano 1974),

acute naloxone-induced withdrawal jumping (figure 4) and the motor

response to d-amphetamine (Oliverio et al. 1973). There are interesting differences in the elimination of d-amphetamine (figure

5). We have observed marked differences in cocaine-induced liver

damage (table 1). The strain distribution pattern varies with sex

as well as with the inducer which serves to stimulate the conversion of cocaine to a hepatotoxic metabolite. At least part of the

FIGURE 4. Naloxone-precipitated jumping in morphine-treated

recombinant-inbred mice. Twelve mice of each RI line were

injected daily for 5 days with 12.5 mg/kg morphine sulfate i.p.

On the first day and on the fifth day they were injected 2 hours

later with naloxone-HCl. 10 mg/kg i.p. The number of jumps was

counted during the first 9 minutes after naloxone injection. Each

bar represents the mean value for 12 mice. Stippled bars - first

day; solid bars - fifth day. B,C,BF1 and CF1 represent progenitors C57B1/6By, BALB/cBy and the 2 reciprocal hybrids,

respectively.

58
FIGURE 5. Rate of disappearance of tritium-labeled d-amphetamine

from the blood of RI mice. Mice were injected i.p. with 5 mg/kg

of generally labeled tritiated d-amphetamine sulfate (10 microcuries per mouse). At 15-minute intervals during the following 2

hours each mouse was warmed under an infrared lamp for 1 minute,

and 25 microliters of blood was collected from the tail into a

heparinized micropipette. The blood was transferred to 1 ml of 1

M K2HPO4 in a scintillation vial. Fifteen ml of toluene-based

scintillation fluid was added and the labeled amphetamine was extracted into the scintillation fluid for counting. The rate of

disappearance of d-amphetamine from the blood between 15 and 120

minutes after injection followed first order kinetics. Each point

represents the mean value for from 4 to 6 mice. The groups

separated by vertical lines showed a statistically significant

difference in half-time.

TABLE 1

HEPATOTOXICITY OF COCAINE IN R.I. MICE

STRAIN SGOT, K.U./ML X l03

Pine-Induced Phenobarbital-Induced

Male Male Female

BALB/cBy 0.2 ± 0.02 0.1 ± 0.03 4.6 ± 1.6

C57B1/6By 16.8 ± 9.8* 10.9 ± 1.4* 24.0 ± 1.7*

CXB D 8.2 ± 06

3.5 ± 1.7

2 2 ± 0.4 8.1 ± 11

E

G 28.7 ± 13.0*

12.0 ± 3.9* 9.l ± 1.6

3.6 ± 1.0 16.1 ± 0.8*

H 31.1 ± 6.6* 7.2 ± 1.4* 19.0 ± 1.1*

I 23.3 ± 10.6* 8.8 ± 3.9* 12.4 ± 1.9*

J 2.6 ± 1.6 7.2 ± 1.3 2.7 ± 0.7

K 3.0 ± 1.6 3.3 ± 1.0 12.6 ± 2.4*

Microsomal enzymes were induced by exposing the mice to pine bedding for at least 7 days, or by giving phenobarbital in drinking

water (0.8 g/l). The mice were injected with 50 mg/kg cocaine HCl

i.p. and bled from the tail after 18 hrs. Each value is the mean

± S.E. for a group of 5 mice (on pine) or from 5-19 mice (phenobarbital induced).

*Significantly different from BALB/c progenitor, p < .05

59
difference between the progenitor strains can be attributed to

differences in some of the metabolic steps involved (Thompson et

al. 1984).

RI analysis has turned up some interesting differences in narcotic

receptors. One of the Bailey lines, CXBK, has a decreased number

of naloxone binding sites in the brain associated with a decreased

analgesic response to morphine (Baran et al. 1975), to acupuncture

(Pomeranz 1978). D-amino acids (Cheng and Pomeranz 1979) and to

stress (Miczek et al. 1982). The binding of several ligands at a

single concentration to brain membranes from the same Rl lines was

examined by Reith et al. (1981). The lowest binding of naloxone,

dihydromorphine and ethyl ketocyclazocine was obtained with CXBK

membranes, and the highest with progenitor BALB/cBy. There were

no significant genetic differences in the binding of D-ala2-Dleu5

-enkephalin, which was assumed to be specific for delta

receptors.

Where a drug response shows a clear-cut difference between two

progenitor strains, it should be possible to define a chromosomal

locus by surveying all of the existing congenic lines. If these

lines each contain only a small chromosomal segment from progenitor A, most of them will respond like strain B. The exception,

which responds like strain A, can be defined as having a genetic

determinant for the drug response located in a known fragment of a

single chromosome. However, sufficient numbers of congenic mice

for testing are not readily available. Furthermore, there is

still a 50% chance that the chromosome segment which has been isolated in any given congenic line may contain more than one histocompatibility locus (Johnson 1981).

4. Defined Mutants. The use of defined point mutations avoids any

uncertainties which may be encountered with congenic lines. A

large number of such mutants are available on a C57B1/6J background (Heiniger and Dorey 1980; Green 1981). A survey of some of

these mutants has revealed at least three which differ in one or

more responses to morphine from the progenitor strain. The mutant

Pallid shows more running activity and hypothermia (Katz and Doyle

1980). We have found that mutants Sepia and Gunmetal display more

analgesia and running activity (figures 6 and 7). There was much

less naloxone-precipitated withdrawal jumping of Gunmetal (Gm)

mice than either C5781/6J or Sepia (figure 8). A comparison of

heterozygotes and homozygotes revealed dominance in some cases.

Other mutants which we tested resembled C57B1/6J. These included

Brown, Pearl, Viable Dominant Spotting, Black and Tan 33J, Ragged

and Ruby Eye (Heiniger and Dorey 1980).

It is intriguing that the mutations which affect the responses to

morphine do not seem to be directly related. The respective chromosomes bearing the mutations Pallid, Gunmetal and Sepia, are 2,

14 and 1 (Heiniger and Dorey 1980). These mutations are assumed

to be pleiotropic, because they have been identified by a change

in coat color, or some other characteristic unrelated to drug responses. The Pallid mutant is known to have a defect in manganese

60
FIGURE 6. Analgesic response to morphine sulfate of C57BL/6J mice

and homozygous and heterozygous single gene mutant mice. Sepia/+

and Gm/+ refer to heterozygotes. Bars indicate mean ± SEM change

in tailflick latency 30 min after injection of morphine. The cutoff was 8.0 sec. The number (n) of mice in each group is indicated in parentheses below the bars. A star indicates significant

difference from C5781/6J, p<.05.

metabolism (Erway et al. 1970). Mutants which exhibit defective

development of the central nervous system may display multiple

abnormalities (Nussbaum et al. 1969). This situation makes it

difficult to interpret the decreased responsiveness of Jimpy mice

to morphine as proof that cerebroside sulfate is an important constituent of narcotic receptors (Law et al. 1978).

5. Sublines of Inbred Strains. The maintenance of separate

colonies in different laboratories has given rise to sublines that

differ in a small number of genes from the original breeding

stock. These differences can be attributed either to new mutations or to residual heterozygosity. According to Bailey (1978)

sublines that have been maintained for 50 generations (20 years)

after branching should have only 14 gene differences due to new

61
FIGURE 7. Running response to morphine sulfate of C57B1/6J mice

and homozygous and heterozygous single gene mutants. Bars indicate mean number of photocell interruptions accumulated during a

2-hour test session following the injection of morphine. Each

mouse was tested individually. Mice were allowed to habituate to

the test cage for 1 hour prior to the injection of morphine. The

number of mice in each group is indicated in parentheses. A star

indicates significant difference from C57B1/6J, p<.05.

mutations. Because the original lines have been inbred for 40

generations or more, the extent of residual heterozygosity is also

very low--for example, 14 gene differences after 30 generations.

We have encountered clear-cut differences in drug response between

the Bailey (By) subline of C57B1/6 mice and the original Jackson

(J) line. The By mice were separated from the J line after the

latter had undergone 75 generations of inbreeding. The lines have

been separate for 62 generations and differ in less than 50 gene

pairs (Bailey 1978). In an animal possessing 30,000 structural

genes (Green 1981), it is extremely unlikely that as much as 1% of

these would be associated with one particular trait such as the

analgesic response to morphine. Hence, any difference between

the two sublines with respect to this trait can reasonably be attributed to a single gene difference.

62
FIGURE 8. Naloxone-precipitated acute withdrawal jumping in

C57B1/6J mice and homozygous and heterozygous single gene C57B1/6J

mutant mice. Sepia/+ and Gm/+ refer to heterozygotes. Bars reflect mean ± SEM number of jumps per mouse observed during a

10-min period after the injection of naloxone. Naloxone was injected 2 hrs after an injection of morphine sulfate, 12.5 mg/kg

i.p. A star indicates significant difference from C57B1/6J,

p<.05; two stars, p<.01. The number of mice in each group is

indicated in parentheses.

The By subline shows a greater analgesic response to morphine than

J mice (Shuster 1982). Analysis of dose-response curves by the

method of Litchfield and Wilcoxon (1949) gave ED50 values of 2.5

mg/kg morphine sulfate for By and 8.0 mg/kg for J mice. The By

mice also showed more analgesia by the hot plate assay. Experiments with tritiated morphine showed no difference between the two

lines in the rate of elimination of morphine from the blood.

Whole brain homogenates from the two sublines were used to measure

the specific binding of several ligands. Scatchard analysis revealed that brain membranes from each subline had the same number

of low affinity receptors for dihydromorphine, but a marked difference in the number of high affinity receptors (22.5 ± 3 vs.

12.0 ± 2 fmoles/mg protein for By vs J, p=.0l). There was no difference in KD (about 0.6 nM). When other tritiated ligands were

used, both sublines had similar Bmax's and KD's for D-ala2-metenkephalinamide, ethyl ketocyclazocine and naloxone (Cremins and

63
Shuster 1982). The Bmax for dihydromorphine binding to brain

homogenates from F1 mice was not significantly different from that

of the By parent, and the analgesic response was also inherited as

dominant trait (Shuster and Cremins, in preparation).

These findings suggest that there is a single gene difference between By and J mice which controls both the analgesic response to

morphine and the number of high affinity mu receptors in the

brain. The situation in other parts of the body may be different.

Our preliminary experiments indicate that the By subline may actually have fewer mu receptors in the spinal cord than J mice.

We also examined the running response of By and J mice to morphine, and found that J mice showed a greater response (Shuster

1982). The narcotic receptors which mediate the running response

appear to be different from the mu receptors which are believed to

mediate analgesia. An indication of the nature of these receptors

comes from the work of Reggiani et al. (1980). They compared the

binding of several ligands in different brain regions from C57/Bl

(high running response to morphine) and DBA/2 mice (no running response). They found a greater number of binding sites for tritiated D-ala2

-met-enkephalin and leu-enkephalin in the striatum of

C57 mice, with no difference in affinity. The binding of dihydromorphine and naloxone was the same in both strains. These observations suggest that delta receptors may be involved in the running response.

The subline differences in narcotic receptors also appear to affect the responses of mice to endogenous opiates. One kind of

stress-induced, naloxone-reversible analgesia which we have examined is that seen when one mouse is attacked and defeated by

another mouse (Miczek et al. 1982). Here, too, By mice show more

analgesia than J mice (figure 9).

By mice also have more fatty liver damage after high doses of morphine than J mice (Needham et al. 1981). There are indications

that a good part of this response can be attributed to morphineinduced release of epinephrine from the adrenals. Here, also,

genetic comparisons suggest that mu receptors may be involved.

The Jackson subline of C57B1/6 mice shows a greater stimulant

response to amphetamine than the Bailey subline. In this case,

too, the By gene is dominant (Moisset 1977).

Additional subline differences await closer investigation. For

instance, the vasa deferentia from the Biobreeding subline of

C57B1/6 mice are about 3 times as sensitive to inhibition by normorphine or by met-enkephalin as those from the Jackson line

(Szerb and Vohra 1979). Balb/cJ mice have higher levels of enzymes for synthesizing epinephrine than the NIH subline

(Ciaranello et al. 1974). Moisset (1978) found that BALB/cBy mice

sleep over twice as long after an injection of ethanol, four g per

kg, as mice of the Jackson subline.

64
FIGURE 9. Defeat-induced analgesia in mice of 6 different

strains. Bars indicate mean ± SEM change in tailflick latency

following 70 bites. An asterisk indicates significant difference

from C57B1/6J, p<.05.

CONCLUSIONS

Genetic differences in responses to drugs of abuse can be found if

one looks for them. The particular model system which is most useful will depend to a great extent on the goals of pharmacogenetic

analysis. There is still a great need for systematic surveys of

the drug responses of available inbred strains, sublines and

mutants. These surveys should also extend to the assay of drug

receptors, drug metabolism, and the mapping of brain proteins by

two-dimensional gel electrophoresis. The heroic labors of selective breeding also pose a tremendous challenge. Eventually one

should be able to identify specific genes that determine drug responses and relate these to the mechanism of action.

REFERENCES

Bailey, D.W. Recombinant inbred strains. Transplantation,

11:325-327 1971.

Bailey, D.W. Genetics of histocompatibility in mice. I. New loci

and congenic lines. Immunogenetics, 2:249-256 1975.

65
Bailey, D.W. Sources of subline divergence and their relative

importance for sublines of six major inbred strains of mice.

In: Morse, H.C., III, ed. Origins of Inbred Mice, New York,

Academic Press, 1978. pp. 197-215.

Baker, H., Joh, T.H., and Reis, D.J. Genetic control of number of

midbrain dopaminergic neurons in inbred strains of mice: Relationship to size and neuronal density of the striatum. Proc Nat

Acad Sci USA, 77:4369-4373, 1980.

Baran, A. Shuster, L., Eleftheriou, B.E., and Bailey, D.W.

Opiate receptors in mice. Genetic differences. Life Sci,

17:633-640. 1975.

Boehme, R.R., and Ciaranello, R.D. Dopamine receptor binding in

inbred mice: Strain differences in mesolimbic and nigrostriatal

dopamine binding sites. Proc Nat Acad Sci USA, 78:3255-3259,

1981.

Brase, D., Loh, H.H., and Way, E.L. Comparison of the effects of

morphine on locomotor activity, analgesia and primary and protracted physical dependence in six mouse strains. J Pharmacol

Exp Ther, 201:768-374, 1977.

Castellano, C., and Oliverio, A. A genetic analysis of morphineinduced running and analgesia in the mouse. Psychopharmacol,

41:197-200. 1975.

Cheng, R.S.S., and Pomeranz, B. Correlation of genetic differences

in endorphin systems with analgesic effects of D-amino acids in

mice. Brain Res, 177:583-587, 1979.

Ciaranello, R.D., Hoffman, H.J., Shire, J.G.M., and Axelrod, J.

Genetic regulation of catecholamine biosynthetic enzymes. II

Inheritance of tyrosine hydroxylase, dopamine hydroxylase and

phenylethanolamine methyl transferase. J Biol Chem,

249:4520-4536, 1974.

Crabbe, J.C., Jr., Allen, R.G., Gaudette, N.D., Young, E.,

Kosobud, A., and Stack, J. Strain differences in pituitaryendorphin and ACTH content in inbred mice. Brain Res,

219:219-223, 1981.

Crabbe, J.C., Kosobud, A., and Young, E.R. Genetic selection for

ethanol withdrawal severity: Differences in replicate mouse

lines. Life Sci, 33:955-962, 1983a.

Crabbe, J.C., Jr., Young, E.R., and Kosobud, A. Genetic correlations with ethanol withdrawal severity. Pharmacol Biochem

Behavior, 18:541-547, 1983b.

Cremins, J., and Shuster, L. A genetically controlled difference

in morphine analgesia and narcotic receptors in mice. Fed

Proc, 41:1314, 1982.

Erway, L., Hurley, L.S., and Fraser, A.S. Congenital ataxia and

otolith deficits due to manganese deficiency in mice. J

Nutrit, 100:643-654, 1970.

Falconer, D.S. Introduction to Quantitative Genetics. New York:

Ronlad Press, 1960

Goldstein, D.B., Chin: J.H., and Lyon, R.C. Ethanol disordering

of spin-labeled mouse brain membranes: correlation with

genetically determined ethanol sensitivity of mice. Proc Nat

Acad Sci USA, 79:4231-4233, 1982.

Green, M.C. Genetic Variants and Strains of the Laboratory Mouse.

Stuttgart, Gustav Fischer Verlag, 1981.

66
Heiniger, H.J., and Dorey, J.J., eds. Handbook on Genetically

Standardized JAX Mice, 3rd edition. Bar Harbor, Me: The

Jackson Laboratory, 1980.

Helmeste, D.M., Seeman, P., and Coscina, D.V. Relation between

brain catecholamine receptors and dopaminergic stereotypy in

rat strains, Eur J Pharmacol, 69:465-470, 1981.

Helmeste, D.M. Spontaneous and apomorphine-induced locomotor

changes parallel dopamine receptor differences in two rat

strains. Pharmacol Biochem Behavior, 19:153-155, 1983.

Heston, W.D.W., Erwin, V.G., Anderson, S.M., and Robbins, H. A

comparison of the effects of ethanol on mice selectively bred

for differences in ethanol sleep time. Life Sci, 14:365-370,

1974.

Ingram, D.K., and Corfman, T.P. An overview of neurobiological

comparisons in mouse strains. Neurosci Biobehav Rev,

4:421-435, 1980.

Johnson, L.L. At how many histocompatibility loci do congenic

mouse strains differ? Probability estimates and some implications. J Hered, 72: 27-31, 1981.

Judson, B.A., and Goldstein, A. Genetic control of opiate-induced

locomotor activity in mice. J Pharmacol Exp Ther, 206:56-60,

1978.

Katz, R.J. The albino locus produces abnormal responses to opiates in the mouse. Eur J Pharmacol, 68:229-232, 1980.

Katz, R.J., and Doyle, R.L. Enhanced responses to opiates produced by a single gene substitution in the mouse. Eur J

Pharmacol, 67:301-303, 1980.

Kiianmaa, K., and Tabakoff, B. Neurochemical correlates of tolerance and strain differences in the neurochemical effects of

ethanol. Pharmacol Biochem Behavior, 18, Suppl. 1: 383-388,

1983.

Lasagna, L., and Beecher, H.K. The optimal dose of morphine. J

Am Med Assoc, 156:230-234, 1954.

Law, P.Y., Harris, R.A., Loh, H.H., and Way, E.L. Evidence for

the involvement of cerebroside sulfate in opiate receptor

binding: Studies with Azure A and Jimpy mutant mice. J

Pharmacol Exp Ther, 207:458-468, 1978.

Litchfield, J., and Wilcoxon, F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther, 96:99-113,

1949.

Mayer, J.M., Khanna, J.M., Kim, C., and Kalant, H. Differential

pharmacological responses to ethanol, pentobarbital and morphine in rats selectively bred for ethanol sensitivity.

Psychopharmacol, 81:6-9, 1983.

McClearn, G.E., and Anderson, S.M. Genetics and ethanol

tolerance. Drug Alcohol Depend, 4:61-76, 1979.

Miczek, K.A., Thompson, M.L., and Shuster, L. Opioid-like analgesia in defeated mice. Science, 215:1520-1522, 1982.

Moisset, B. Genetic analysis of the behavioral response to damphetamine in mice. Psychopharmacology 53:263-267 (1977).

Moisset, B. Subline differences in behavioral responses to pharmacological agents. In: More, H.C. III ed. Origins of Inbred

Mice, New York, Academic Press, 1978. pp. 483-484.

67
Needham, W.P., Shuster, L., Kanel, G.C., and Thompson, M.L. Liver

damage from narcotics in mice. Toxicol Appl Pharmacol,

58:157-170, 1981.

Nussbaum, J.L., Nescovic, N., and Mandel, P. A study of lipid

components in the brain of the "Jimpy" mouse, a mutant with

myelin deficiency. J Neurochem, 16:927-934, 1969.

Oliverio, A., and Castellano, C. Genotype-dependent sensitivity

and tolerance to morphine and heroin: dissociation between

opiate-induced running and analgesia in the mouse.

Psychopharm, 39:13-22, 1974.

Oliverio, A., Eleftheriou, B.E., and Bailey, D.W. Exploratory

activity: Genetic analysis of its modification by scopolamine

and amphetamine. Physiol Behav, 10:893-899. 1973.

Oliverio, A., Castellano, C., and Eleftheriou, B.E. Morphine

sensitivity and tolerance. A genetic investigation in the

mouse. Psychopharmacologia, 42:219-224, 1975.

Perry, B.D., Stolk, J.M., Vantini, G., Guchhart, R.B., and

U'Prichard, D.C. Strain differences in rat brain epinephrine

synthesis: Regulation of alpha-adrenergic receptor number by

epinephrine. Science, 221:1297-1299, 1983.

Pomeranz, B. Do endorphins mediate acupuncture analgesia? Adv in

Biochem Psychopharm, 18:351-360, 1978.

Racagni, G., Bruno, F., Juliano, E., and Paoletti, R.

Differential sensitivity to morphine-induced analgesia and

motor activity in two inbred strains of mice: Behavioral and

biochemical correlations. J Pharmacol Exp Ther, 209:111-116,

1979.

Reggiani, A., Battaini, F., Kobayashi, H., Spano, P., and

Trabucchi, M. Genotype-dependent sensitivity to morphine: Role

of different opiate receptor populations. Brain Res,

189:289-294, 1980.

Reith, M.E.A., Sershen, H., Vadasz, C., and Lajtha, A. Strain

differences in opiate receptors in mouse brain. Eur J

Pharmacol, 74:377-380, 1981.

Robertson, H.A. Benzodiazepine receptors in "emotiona1" and

"non-emotional" mice: Comparison of four strains. Eur J

Pharmacol, 56:163-166, 1979.

Shuster, L. Genetic analysis of morphine effects: Activity,

analgesia, tolerance and sensitization. In: Eleftheriou,

B.E., ed. Psychopharmacogenetics, New York: Plenum Press, 1975.

pp. 73-98.

Shuster, L., Webster, G.W., and Yu, G. Increased running response

to morphine in morphine-pretreated mice. J Pharmacol Exp Ther,

192:64-72. 1975a.

Shuster, L., Webster, G.W., Yu, G., and Eleftheriou, B.E. A genetic analysis of the response to morphine in mice: analgesia

and running. Psychopharmacol, 42:249-254, 1975b.

Shuster. L. A pharmacogenetic approach to the brain. In:

Lieblich, I., ed. Genetics of the Brain. Amsterdam, Elsevier

Biomedical Press, 1982. PP. 157-176.

Sorenssen, S., Palmer, M., Dunwiddie, T., and Hoffer, B.

Electrophysiological correlates of ethanol-induced sedation in

differentially sensitive lines of mice. Science,

210:1143-1145, 1980.

68
Szerb, J.C., and Vohra, M.M. Potencies of normorphine and

kephalin in the vas deferens of different strains of mi

Life Sci, 24:1983-1988, 1979.

Taylor, B.A. Genetic relationships between inbred strains

J. Heredity, 63:83-86, 1972.

Taylor, B.A. Recombinant-inbred strains: Use in gene mappi

Morse, H.C. III, ed. Origins of Inbred Mice. New York

Academic Press, 1978. pp 423-438.

met-ence.

of mice.

ng. In:

Ihompson, M.L., Shuster, L., and Casey, E. Sex and strain differences in response to cocaine. Biochem Pharmacol, in press.

Waller, S.B., Ingram, D.K., Reynolds, M.A., and London, E.D. Age

and strain comparisons of neurotransmitter synthetic enzyme

activities in the mouse. J Neurochem, 41:1421-1428, 1983.

Way, E.L., Loh, H.H., and Shen, F.H. Simultaneous quantitative assessment of morphine tolerance and physical dependence. J

Pharmacol Exp Ther, 167:1-8, 1969.

Wimer, R.E., and Wimer, C.C. A geneticists map of the mouse

brain. In: I. Lieblich, ed. Genetics of the Brain. Amsterdam,

Elsevier Biomedical, 1982, pp. 395-420.

ACKNOWLEDGMENTS

The research described here was supported by grants DA-01626 and

DA-01885 from the National Institute on Drug Abuse. Guy

Webster, Peter H. Short, Allison Wood, and Michell Hoffman

assisted with breeding and pharmacological assays. Drs. Earl

L. Green, Don W. Bailey, and Michael L. Thompson provided

helpful discussions.

AUTHOR

Louis Shuster, Ph.D.

Department of Biochemistry and Pharmacology

Tufts University School of Medicine

Boston, MA 02111

69
Opioid Tolerance and Dependence

in Light of the Multiplicity

of Opioid Receptors

Rüdiger Schulz and Albert Herz

Opioid tolerance and dependence are the consequence of chronic

activation of opioid receptors. To explain these phenomena,

several theories have been advanced to account for the respective

adaptational mechanisms (Collier 1980, Shuster 1961, Goldstein and

Goldstein 1968). A common postulate of these is the appearance of

tolerance and dependence in a unitary manner. They, thus, take into consideration the well-known observations in man and animals

that tolerance and dependence appear in parallel. Within this

framework, the existence of cross-tolerance has been exploited as

an essential criterion for the characterization of opioid agonists.

That is, tolerance to one opioid is accompanied by cross-tolerance

to another opioid.

In recent years, these concepts have been seriously challenged. It

has become clear that the absence or the presence of cross-tolerance is by no means a prerequisite to judge whether or not a drug is

a narcotic. These findings are closely associated with the concept

of the multiplicity of opioid receptors, as originally proposed by

Martin et al. (1976). Indications for the existence of different

types of opioid receptors have come from experiments with intact

animals (Martin et al. 1976) but also from studies with isolated

tissues (Lord et al. 1977). Investigations with the isolated

mouse vas deferens and guinea-pig ileum, which have been employed to

study the development of tolerance and cross-tolerance Wüster et

al. 1981a), proved very successful.

The mouse vas deferens represents an isolated tissue preferentially employed to study tolerance and cross-tolerance (Schulz et al.

1980). This preparation seems to be unique as it contains at least

each of the classical opioid receptors, such as the μ-,

type. It was the aim of several investigations to test whether

chronic stimulation of, for example, the μ-receptors is associated

with an adaptation of other receptor types. In other words, is

tolerance to a specific receptor type associated with crosstolerance to a different type of opioid receptor? Mice were treated

with an opioid, e.g., a μ-agonist, for several days, using osmotic

minipumps for the application of the drug. The vasa deferentia of

these animals here set up in vitro for electrical stimulation

70
in the presence of the opioid applied in vivo. This procedure

allows for maintenance of tolerance in vitro. According to this

schedule, vasa deferentia were rendered tolerant in vivo to μ-,

receptor agonists. The preparations were challenged in

vitro for the degree of tolerance and cross-tolerance.

FIGURE 1: Tolerance and cross-tolerance studies of opioids on the

isolated mouse vas deferens. Vasa deferentia were rendered tolerant

in vivo to sufentanil (SUF), ethylketazocine (EK) and D-Ala2, D-Leu5-

enkephalin (DADL), respectively. The degree of tolerance, measured

by calculation of the quotient (Q) of the IC50 values of tolerant

arid naive preparations is plotted on the ordinate. Q=1 indicates

lack of tolerance. The druq chronically applied is indicated in

each panel. Abscissa: opioids tested for tolerance and crosstolerance. Each column represents the quotient of mean IC50 values.

For details see Wüster et al. (1981a).

Figure 1 depicts the deqree of tolerance and cross-tolerance of

vasa deferentia rendered tolerant to the μ-agonist sufentanil (SUF),

the agonist ethylketazocine (EK), and the agonist D-Ala2

-D

Leu5-enkephalin (DADL), respectively. Apparently, preparations

highly tolerant to SUF display an almost uncharged sensitivity to

the or the agonist. Analogous results were obtained with preparations rendered tolerant to EK or DADL.

These data clearly reveal the existence of a state of tolerance

and non-tolerance in the same vas deferens preparation. In principle identical results were obtained with the isolated guineapig ileum (Wüster et al. 1981a) and in experiments with intact animals (Schulz et al. 1981). Thus, these results do not favor the

notion that cross-tolerance between opioids is an essential criterion to classify a druq as an opioid.

This demonstration of "selective tolerance" does not-in itselfconflict with theories claiming a "unitary mechanism" for tolerance and dependence. The question arises, however, whether those

highly "tolerant receptors" also display a high degree of dependence, and whether those receptors lacking tolerance also lack dependence. If such assumptions are correct, each bindinq site for a

specific opioid should be firmly linked to a subsequent system and

such an entity should function independently from other opioid

71
FIGURE 2: Naloxone-induced contracture (twitch tension) of longitudinal muscle-myenteric plexus preparations taken from a morphine

tolerant/dependent guinea pig. The preparations were kept in

Krebs-Ringer solution containing normorphine (200 nM), except

during the time indicated by the hatched field. Washes were conducted in the presence and absence of the opiate every 5-10 min.

Naloxone was added to the bath l min after electrical stimulation

was turned off. The ordinate represents tension (contracture)

expressed as percent of the preceding electrically induced twitches.

For details see Schulz and Herz (1976).

receptor entities. A different situation would occur if one adapts

the "mobile-receptor hypothesis" (Cuatrecasas 1974) to the functioning of opioid receptors. In this case, the binding site is

suggested not to be linked to a single transducer system. That is,

the different opioid bindinq sites would have access to a common

subsequent system floating in the membrane. One may assume, thus,

that even those receptors which were not chronically exposed to an

opioid in a tolerant preparation should display dependence. This

argumentation remains vague, as the definition for "the receptor" is

still under discussion. It is generally accepted that the receptor

consists of a recognition site for its ligand and a subsequent

transducer system. While the interaction of the recognition site

with its ligand is well described, the function of the "subsequent

transducer system" is not. Since opioid tolerance and dependence

may find their expression at different sites of the receptor

entity, an understanding of the molecular mechanism is highly desired. Basically, an association of tolerance and dependence development with the "opioid receptor" does not exclude adaptational

mechanisms down-or upstream-from the neuron primarily affected by

the opioid.

These considerations prompted investigations into opioid dependence

and cross-dependence, extendinq, thereby, studies of tolerance and

72
cross-tolerance. Since highly tolerant vasa deferentia of mice fail

to display any sign of dependence (Gillan et al. 1979), the studies

were conducted with the isolated longitudinal muscle-myenteric

plexus preparation of the guinea pig ileum. This preparation has

been widely used to study both opioid tolerance and dependence.

The demonstration of dependence in tolerant preparations requires

the displacement of the agonist (Schulz and Herz 1976). Thus, administration of the narcotic antagonist naloxone may uncover dependence; that is, it elicits a withdrawal contracture. A serious problem relates to the fact that naloxone covers the different types of

opioid receptors with only minor differences in affinity. This implies that naloxone alone cannot be employed as a drug to study

dependence associated with a specific type of receptor. Since a

withdrawal sign can be precipitated by naloxone only by displacing

an agonist, dependence and cross-dependence were therefore studied

by use of highly selective μ- or agonists. A withdrawal contracture caused by naloxone under these conditions would reveal dependence of the specific opioid receptor occupied by the agonist.

In the light of the above considerations, the experiments were designed as follows: The ilea of guinea pigs, which had been chronically treated with low concentrations of morphine or EK, were set

up in vitro for electrical stimulation. The preparations were withdrawn by washing in order to unmask all opioid receptors. A successful removal of the agonist from its receptor is manifested in the

failure of naloxone to precipitate a withdrawal contracture. Thereafter, the strips ware re-exposed for 30 min to a specific opioid

receptor agonist of a very low concentration. Subsequently,

naloxone was added to the bath to test for dependence (figure 2).

Figure 3, upper panel, displays the results obtained with preparations rendered in vivo dependent on the μ-agonist morphine (10 and

100 μg/h, 6 days). After the preparations were withdrawn, they were

re-exposed to either the K-agonist normorphine (NORM) or to the

agonists dynorphin Al-13 (DYN1-13) or neoendorphin1-8(ANE1-8).

Tests for dependence revealed a similar intensity of withdrawal

contracture in all preparations, regardless of whether a μ- or

agonist was employed for reexposure. Essentially identical results

were obtained with preparations taken from guinea pigs chronically

treated with EK (lower panel, 5 and 50 μg/h, 6 days). Taken together, dependence cannot be allocated to a specific opioid receptor type. The preparations chronically exposed to a μ-agonist

display dependence, but a sign of dependence can equally well be

demonstrated after the μ-agonist has been exchanged with a

agonist. This phenomenon may be called cross-dependence.

However, any overlap of the agonist employed for reexposure in

vitro to a different receptor type may affect the interpretation

of these above-mentioned results. Thus, the aim of further experiments was to rule out the possibility that the naloxone-induced.

withdrawal contracture could relate to more than a single receptor

population. To accomplish this aim, the irreversible μ-receptor

antagonist ß-funaltrexamine (ß-FNA) (Ward et al. 1982) was employed

in these studies. The experimental design followed that described

above with the exception that the withdrawn preparations were ex73
FIGURE 3: Opioid dependence and cross-dependence in the isolated

guinea pig ileum. The preparations were rendered dependent by infusing guinea pigs with morphine or ethylketazocine (EK). After

withdrawal of the preparations, they were reincubated with opioids

with a high receptor selectivity such as normorphine (NORM) for ,

μ-receptors, and EK, dynorphin Al-13 (DYN1-13) and neo-endorphin1-8 (ANE1-8) for receptors. The reincubation concentrations

of the drugs are indicated on the abscissa and the elicited strength

of the withdrawal interaction is given on the ordinate. For details

see Schulz et al. (1982).

posed to ß-FNA (Seidl and Schulz 1983). This treatment provides

preparations containing receptors only. The intensity of the

withdrawal contractions of preparations exposed in vivo to fentanyl

(FEN) and treated in vitro with ß-FNA are given in figure 4. The

left panel (A) illustrates the data of preparations exposed in

vitro to different concentrations of FEN. In preparations not exposed to ß-FNA, the contracture evoked by naloxone amounts in all

tissues to about 50 percent of the electrically evoked contractions.

74
In the presence of ß-FWA, the response was greatly reduced, although the intensity of the contractions increased as the concentration of FEN increased. The fact that ß-FNA-exposed preparations

responded to naloxone in the presence of FEN most likely relates

to an incomplete inactivation of μ-receptors upon ß-FNA treatment.

Under analogous experimental conditions, but exposing the preparations in vitro to the agonist EK instead to FEN, the withdrawal

contractions were of similar intensity in both the ß-FNA-exposed

and non-exposed preparations (panel B). Thus, the studies employing

the irreversible antagonist ß-FNA, which interacts selectively with

μ-receptors, confirm previous results. They indicate the development of dependence at μ- and K-receptors (cross-dependence), although only a single type of receptor has been chronically activated

in vivo.

A different issue relates to the assumption that tolerance and

dependence are phenomena of a common mechanism. It would follow

that these phenomena should both be displayed together under appropriate conditions. Applying these considerations to the isolated

muse vas deferens, opioid tolerance has never, in fact, been

FIGURE 4: Withdrawal contractions precipitated by naloxone in

isolated guinea pig ileum preparations rendered dependent to fentanyl (FEN) in vivo. Open columns are data from preparations not

exposed to ß-FNA in vitro; hatched columns indicate data from

preparations exposed in vitro to ß-FNA. The ordinate reflects the

tension of the withdrawal contracture. The abscissa reflects the

concentrations of FEN and ethylketazocine (EKC) employed to expose

the preparations in vitro. For details see Seidl and Schulz (1983).

75
demonstrated to be associated with any sign of dependence. Although

electrophysiolcgical data have been reported indicating that the

neuronal firing rate increases in the tolerant mouse vas deferens

upon naloxone exposure (North and Vitek 1980), the question under

discussion remains whether or not this observation, in fact, represents a sign of dependence or an overshoot reaction subsequent to

the disinhibition of the opioid effect. In contrast to the mouse

vas deferens, both tolerance and dependence are commonly observed

in the isolated guinea-pig ileum. However, a detailed analysis of

the development of both parameters casts doubts on the principle

of a simultaneous and parallel appearance.

FIGURE 5: Correlation between tolerance and dependence in the

isolated guinea pig ileum. Guinea pigs were treated with different

dosages of morphine. The isolated preparations taken from these

guinea pigs were maintained in vitro at different concentrations

of normorphine. Each dot represents data from a single strip which

was used once to assay tolerance and dependence. The dotted line

indicates the tolerance/dependence curve obtained by best-fit

computer analysis. The solid lines indicate the regression lines

for the degree of tolerance (Q) ranging from to 10 and from 50-

200. bl and b2 reflect the respective regression coefficients.

Abscissa: degree of tolerance (Q) calculated from IC50 values of

tolerant and naive preparations. Ordinate: intensity of withdrawal

contracture expressed as percent of the preceding electrical stimulation. For details see Schulz et al. (1982).

In order to investigate this matter, guinea pigs were infused

chronically with different amounts of morphine (10 to 100 μg/h,

6 days). The strips were set up in vitro at corresponding concentrations of morphine in plasma and were tested for tolerance to

morphine and for dependence by addition of naloxone to the organ

76
bath. Figure 5 correlates the degree of tolerance with the intensity of withdrawal contracture (dependence). Apparently, a very

steep slope of the curve at low degrees of tolerance is followed by

an extremely flat slope at high degrees of tolerance. In fact,

dependence was observed already in preparations which did not

display a measurable degree of tolerance. From these experiments,

it became obvious that the degree of tolerance and the degree of

dependence do not appear in a parallel fashion in the isolated

quinea pig ileum.

While the aforementioned experiments do not always demonstrate a

separation between tolerance and dependence, a clear-cut dissociation of these phenomena can be obtained by experimental manipulations. Cholera toxin, a neurotoxin which interferes with the

receptor mechanism, specifically with the guanyl regulatory unit

(Rodbell 1980), has been shown to reduce the expression of dependence in the isolated guinea-pig ileum without changing the deqree

of tolerance to opioids (Lux and Schulz 1983). This result, thus,

represents a further hint for a dissociation of tolerance from

dependence.

CONCLUSIONS

Theoretical considerations have intimately linked opioid tolerance

with dependence and provided the conceptual basis for a postulated

"unitary mechanism" underlying these phenomena. Several recent

findings are not in correspondence with this notion. Similarly, it

has become clear that cross-tolerance between opioids is not a

criterion for the classification of a drug as an opioid.

The studies with the mouse vas deferens reviewed here exemplify

the above-mentioned issues in two respects. First, a highly opioidtolerant vas deferens fails to display any sign of dependence.

Second, a lack of cross-tolerance between different opioid receptor agonists has been convincingly demonstrated. A plausible explanation of these phenomena may be derived from studies of adaptational mechanisms observed in neuroblastoma cells. These cultured

hybrid cells respond to chronic opioid exposure with a "down regulation" of the binding sites for opioids (Chang et al. 1982, Law

et al. 1982). Thus, assuming a sufficiently high degree of "down

regulation," the tolerance phenomenon could be explained by this

mechanism. On the other hand, an uncoupling of the opioid binding

site from its subsequent system has already been suggested (Rubini

et al. 1982) as being responsible for tolerance. Such a mechanism

may, in fact, occur in neuroblastoma hybrid cells (Wüster et al.

1983, Wüster and Costa, this volume). Both mechanisms would imply

a disruption of the receptor function and could, thus, explain

tolerance. In contrast, however, the expression of dependence

demands a functioning opioid receptor. Since the highly tolerant

mouse vas deferens fails to display any sign of dependence,

the mechanisms underlying tolerance in the neuroblastoma cells and

the vas deferens may be similar. Thus, both tolerance and dependence may dissociate in the vas deferens. This would suggest that

only tolerance develops in the nerve terminals of the mouse vas

deferens. Attempts, however, to demonstrate alterations of opioid

77
binding sites in the tolerant mouse vas deferens have not, as yet,

proven successful (Rubini et al. 1982). In contrast to the cellular

development of tolerance (Collier 1980), adaptive processes may

occur in transmission between neurons, that is, subsequent to the

primary opioid-affected cell (Williams and North 1983).

An analysis of the findings with the isolated ileum would suggest

multiple adaptational mechanisms. Since dependence has been demonstrated in all preparations chronically exposed to an opioid, a

functional opioid receptor must be assumed to exist under these

conditions. However, there appears to be a discrepancy between

the degree of tolerance and the intensity of dependence. A maximal

withdrawal sign is observed already at a low degree of tolerance.

As tolerance gradually increases, dependence does not develop to

the same degree. Apparently, two adaptive mechanisms maybe involved. One involves tolerance and dependence, the second mainly

the development of tolerance. If the second mechanism is due to

a disturbance of the function of the opioid receptor, only a

certain fraction may be affected. The remaining receptors must be

intact since a withdrawal sign is still precipitated under these

conditions. One may argue that the extremely high degree of tolerance seen in this preparations may be due to mechanisms similar

to those assumed to account for the tolerant mouse vas deferens,

or those manifested in neuroblastoma cells (Chang et al. 1982,

Law et al. 1982, Wüster et al. 1983). Then the discrepancy mentioned above between tolerance and dependence could be explained.

Promising tools for the further investigation of these mechanisms

seem to be neurotoxins, such as pertussis toxin and cholera toxin.

Both toxins have already been employed for studies of opioid

tolerance/dependence in the guinea-pig ileum (Collier et al.

1983, Lux and Schulz 1983).

Of considerable interest is the observed cross-dependence between

different opioid receptors in the isolated guinea-pig ileum

(Schulz et al. 1982, Seidl and Schulz 1983). This finding may

imply that all receptor binding sites are somehow linked to a

common transducer system which adapts to chronic stimulation. A

theoretical basis for such a mechanism would be subsumed in the

"mobile receptor theory" (Cuatrecasas 1974).

ACKNOWLEDGEMENT

This work has been supported by Deutsche Forschungsgeme inschaft,

Bonn.

REFERENCES

Chang, K.-J., Eckel, R.W., and Blanchard, S.G. Opioid peptides

induce reduction of enkephalin receptors in cultured neuroblastoma cells. Nature, 296:446-448, 1982.

Collier, H.O.J. Cellular site of opiate dependence. Nature, 283:

625-629, 1980.

78
Collier, H.O.J., Plant, N.T., and Tucker, J.F. Pertussis

vaccine inhibits the chronic but not acute action of normorphine

on the myenteric plexus of guinea-pig ileum. Europ J Pharmacol,

91:325-326, 1983.

Cuatrecasas, P. Membrane receptors. Ann Rev Biochem, 43:169-214,

1974.

Gillan, M.G.C., Kosterlitz, H.W., Robson, L.E., and Waterfield,

A.A. The inhibitory effects of presynaptic adrenoceptor

agonists on contractions of guinea-pig ileum and mouse vas

deferens in the morphine-dependent and withdrawn states produced in vitro. Br J Pharmacol, 66: 601-608, 1979.

Goldstein, D.B., and Goldstein, A. Enzyme expansion theory of

drug tolerance and physical dependence. Assoc for Res of

Nervous and Mental Diseases, Res Publications, 46:265-267,

1968.

Law, P.Y., Hom, D.S., and Loh, H.H. Loss of opiate receptor

activity in neuroblastoma x glioma NG108-15 hybrid cells

after chronic opiate treatment. A multiple-step process. Mol

Pharmacol, 22:1-4, 1982.

Lord, J.A.H., Waterfield, A.A., Hughes, J., and Kosterlitz, H.W.

Endogenous opioid peptides: Multiple agonists and receptors.

Nature, 267:495-499, 1977.

Lux, B., and Schulz, R. Cholera toxin selectivity affects the

expression of opioid dependence in the tolerant myenteric plexus

of the guinea-pig. Europ J Pharmacol, 96:175-176, 1983.

Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E., and

Gilbert, P.E. The effect of morphine- and nalorphine-like

drugs in the non-dependent and morphine-dependent chronic

spinal dog. J Pharmacol Ther 197:517-532, 1976.

North, R.A., and Vitek, L.V. The effect of chronic morphine

treatment on excitatory junction potentials in the mouse vas

deferens. Br J Pharmacol, 68:399-405, 1980.

Rodbell, H. The role of hormone receptors and GTP-regulatory

proteins in membrane transduction. Nature, 284:17-22, 1980.

Rubini, P., Schulz, R., Wüster, M., and Herz, A. Opiate receptor binding studies in the mouse vas deferens exhibiting tolerance without dependence. Naunyn-Schmiedeberg's Arch Pharmacol, 319:142-146, 1982.

Schulz, R., and Herz, A. Aspects of opiate dependence in the

myenteric plexus of the guinea-pig. Life Sci, 19:1117-1128,

1976.

Schulz, R., Wüster, M., Krenss, H., and Herz, A. Lack of crosstolerance on multiple opiate receptors in the mouse vas

deferens. Mol Pharm, 18: 395-401, 1980.

Schulz, R., Wüster, M., and Herz, A. Differentiation of opiate

receptors in the brain by the selective development of tolerance. Pharmacol Biochem Behav, 14:75-79, 1981.

Schulz, R., Seidl,E., Wüster, M., and Herz, A. Opioid dependence and cross-dependence in the isolated guinea-pig ileum.

Europ J Pharmacol, 84:33-40, 1982.

Seidl, E., and Schulz, R. Selective opiate tolerance in the

guinea-pig ileum is not associated with selective dependence.

Life Sci, 33:357-360, 1983.

79
Shuster, L. Repression and depression of enzyme synthesis as a

possible explanation of sure aspects of drug action. Nature,

189:314-315, 1961.

Ward, S.J., Portoghese, P.S., and Takemori, A.E. Improved assays

for the assessment of properties of opioid ligands.

Europ J Pharmacol, 85:163-170, 1982.

Williams, J.T., and North, R.A. Tolerance to opiates in locus

coeruleus neurons. Life Sci, in press, 1983.

Wüster, M., and Gramsch, Ch. Uncoupling of receptors is

essential for opiate-induced desensitization (tolerance) in

neuroblastoma x glioma hybrid cells NG 108-15. Life Sci,

in press, 1983.

Wüster, M., Rubini, P., and Schulz, R. The preference of putative pro-enkephalins for different types of opiate receptors.

Life Sci 29:1219-1227, 1981a.

Wüster, M., Schulz, R., and Herz, A. Multiple opiate receptors

in the peripheral tissue preparation. Biochem Pharmacol, 30:

1883-1887, 1981b.

AUTHORS

Dr. R. Schulz

Prof. Dr. A. Herz

Max-Planck Institut für Psychiatrie, Department of Neuropharmacology, Am Klopferspitz 18A, D-8033 Martinsried, FRG.

80
Sites and Mechanisms of Dependence in the Myenteric Plexus

of Guinea Pig Ileum

H. O. J. Collier and J. F. Tucker

INTRODUCTION

The understanding of drug dependence mechanisms has probably

reached its highest point so far with opioids. This is due to

several factors. including: (1) the availability of a specific

opiate antagonist, naloxone; (2) the feasibility of inducing and

measuring opiate dependence in two in vitro preparations (a) neuroblastoma-glioma hybrid cells in culture and (b) guinea pig ileum;

and (3) a better understanding of the complex processes regulating

adenylate cyclase activity.

The isolated guinea pig ileum has been used frequently in the study

of the long-term effects of opiates (Paton 1957; Ehrenpries et al.

1972; Goldstein and Schultz 1973; Hammond et al. 1976; Schultz and

Herz 1976; Villarreal et al. 1977; Lujan and Rodriquez 1978;

Gintzler 1979; Collier et al. 1981b). Tolerance and dependence in

this tissue and in the whole animal have many characteristics in

common (table 1).

Two recent developments have enabled the analysis of the problem of

opiate dependence to be carried forward using the guinea pig ileum

model. One is the demonstration that dependence on adenosine can

be induced in the isolated ileum (Collier and Tucker 1983). The

other is the demonstration that pertussis vaccine, through its

content of islet activating protein, can selectively interfere with

the inhibitory regulation of adenylate cyclase (Katada and Ui 1980,

1981; Hildebrandt et al. 1983).

SITE OF DEPENDENCE IN THE ILEUM

Subsequent to the demonstration that prolonged in vivo or in vitro

exposure of the guinea pig ileum to opiates results in dependence,

it has been shown that a similar phenomenon is associated with both

clonidine (Collier et al. 1981a) and adenosine (Collier and Tucker

1983). Exposure of guinea pig ileum to either clonidine (10-6M)

or adenosine (4 x 10-6M) for 18-24 hr induces in the tissue a

dependence, the withdrawal from which is expressed as a contracture

81
TABLE 1. Characterization of Opiate Dependence in Both Guinea Pig

Isolated Ileum and Experimental Animals (based on work in Collier

et al. 1981 a,b)

1. Dependence does not appear immediately, but requires a period

of continued exposure to opiate.

2. Dependence is accompanied by tolerance to the acute effects of

opiate.

3. Withdrawal of opiate elicits a "spontaneous" behavioral

disturbance that can be suppressed by retreatment with opiate.

4. Withdrawal disturbance can be precipitated with a specific

antagonist, the potency of which is related to the degree of

dependence.

5. Induction of dependence requires the activation of a specific

and stereospecific opiate receptor.

6. Precipitation of withdrawal requires a specific and

stereospecific opiate antagonist.

7. Clonidine inhibits the opiate withdrawal disturbance.

Collier 1984. Copyright 1984, by Taylor Francis Ltd.

in response to adrenoceptor antagonists or adenosine receptor

antagonists respectively.

Although normorphine, clonidine, and adenosine each induce

dependence in the ileum, there are reasons for believing that the

dependencies are distinct. Firstly, the three agents induce

dependence by interaction with different receptors, activation of

which inhibits the release of acetylcholine from neurons of the

myenteric plexus (table 2).

Secondly, withdrawal from these drugs can only be precipitated with

a selective antagonist of the dependence-inducing agent (Collier et

al. 1981a; Collier and Tucker 1983). For example, neither

yohimbine nor naloxone will produce a response in tissues

previously incubated for 18-24 hr in adenosine, but challenge with

the adenosine antagonist 8-phenyltheophylline produces a marked

withdrawal contracture (figure 1).

All the evidence to date suggests that the site of dependence in

the ileum is within neurons of the myenteric plexus. This is

illustrated by the fact that normorphine, clonidine, and adenosine

withdrawal contractures can all be blocked with tetrodotoxin

(Collier et al. 1981a,b; Collier and Tucker 1983). Additionally,

North and Karras (1978) have demonstrated that after continued

82
Table 2. Selective Agonists and Antagonists at Specific Receptors

Inhibiting Acetylcholine Release From the Final Cholinergic

Motoneuron of Guinea Pig Ileum

Receptor Agonist

example

Opioid, Mu Normorphine

Kappa Ethylketocyclazocine

dynorphins

Adrenoceptor, Clonidine,

alpha2 noradrenaline

adrenaline

Purinoceptor, Adenosine,

Al (Ri)

2-chloroadenosine

Antagonist

example

Naloxone

---

Yohimbine

8-Phenyltheophylline

Reference

Lord et al. 1977

Schultz et al. 1981

Chavkin & Goldstein

1981

Paterson et al. 1983

Kosterlitz et al. 1970

Tanaka & Starke 1979

Malta et al. 1981

Paton 1981

Collier 1984. Copyright 1984, by Taylor Francis Ltd.

exposure to morphine, a burst of impulse production can be elicited

with naloxone in neurons of the myenteric plexus. In this last

study, electrical activity was recorded from cell bodies via

suction electrodes, suggesting that dependence develops in the soma

of neurons of the myenteric plexus rather than in the axon or nerve

terminal.

MEDIATORS OF THE WITHDRAWAL CONTRACTURE

Early studies of the withdrawal response in the guinea pig ileum

supported the contention that the consequence of neuronal firing,

occasioned by naloxone-precipitated morphine withdrawal, was

acetylcholine release. Recently it has been shown that a second

transmitter, first observed by Gintzler (1980) and later by Tsou et

al. (1982), also participates in the normorphine withdrawal

contracture. Experiments with capsaicin, densensitization to

substance P (SP), and SP antagonists have shown that this second

transmitter is SP. Chahl (1983) has shown that the relative

contributions by acetylcholine and SP to the [Met]enkephalin

withdrawal contracture in the guinea pig ileum vary depending on

the duration of exposure to enkephalin. Short periods of exposure

83
FIGURE 1. Lack of Responsiveness to Yohimbine (YO) and Naloxone

(Nx) of 8-Phenyltheophylline (8-PT)-Sensitive Guinea Pig Ileum.

Pieces of ileum were incubated in 4 x 10-6M adenosine for 18-24

hr at 19-21° and for 30-40 min at 37°C.

appear to be associated with a withdrawal contracture due to both

acetylcholine and SP, whereas the withdrawal contractures associated with longer periods of contact are predominantly due to SP

alone.

We have extended these observations and shown that the same neuron

or neurons that are involved in opiate withdrawal in the ileum are

also likely to be involved in adenosine and clonidine withdrawal

contractures. That is, in the ileum, withdrawal from these agents

can only be completely antagonized by abolishing responses to both

acetylcholine and SP. For example, figure 2 shows that after

adenosine withdrawal, a hyoscine-resistant component of the

withdrawal contracture remains which can be abolished by SP

desensitization. Similar results have been obtained by using a

combination of hyoscine and the SP antagonist D Pro2-D Trp7,9

SP.

CONVERGENT DEPENDENCE IN THE ILEUM

Since the dependencies on normorphine, clonidine, and adenosine

appear not to differ in character other than in the nature of the

84
FIGURE 2. Prevention by Substance P (SP) [Desensitization] of the

Hyoscine (Hy)-Resistant 8-Phenyltheophylline (8-PT)-Precipitated

Adenosine Withdrawal Contracture. Upper strip shows effect of Hy

alone; lower strip shows effect of Hy and SP-desensitization.

Details as in figure 1.

FIGURE 3. Suppression by N6-Cyclohexyladenosine (CHA) of

Withdrawal Contracture Precipitated With Naloxone (Nx). Pieces of

ileum were incubated in 10-6M normorphine for 18-24 hr at 4°C

and for 30-40 min at 37°C. ES: electrical stimulation with 0.5

ms pulses at 0.1 Hz; ACh: acetylcholine (Tucker et al. 1983).

85
inducing drug and its selective antagonist, these dependencies have

been termed convergent (Collier 1983). That is, dependence can be

induced through binding of a particular drug with a specific

recognition site on the neuron, but beyond this point the

mechanisms converge, so that withdrawal of all three is expressed

through liberation of the same transmitters, and any one of the

three agonists can suppress withdrawal of the other two (Collier et

al. 1981a,b; Collier and Tucker 1983). For example, activation of

purinoceptors with N

6

-cyclohexyladenosine (CHA), a stable derivative of adenosine, can abruptly terminate naloxone- precipitated

normorphine withdrawal contractures in the ileum (figure 3).

A possible useful consequence of convergent dependence is that not

only clonidine but also adenosine derivatives might be useful in

inhibiting opiate withdrawal symptoms in man. We have previously

shown that 2-chloroadenosine (2-CA), a stable derivative of

adenosine, potently suppresses morphine withdrawal signs in the

mouse (Tucker et al. 1983). Recently we have compared CHA, with

clonidine with respect to inhibition of jumping, diarrhea, and

weight loss in naloxone-precipitated morphine withdrawal in mice.

Mice were made dependent by implanting one 50 mg pellet of morphine

subcutaneously under ether anesthesia. Three days later, mice were

treated ip with CHA, clonidine, or saline; 25 min later, withdrawal

was precipitated with 1 mg/kg ip naloxone.

TABLE 3. Inhibition with N6-Cyclohexyladenosine (CHA) and

Clonidine (CLON) of Withdrawal Effects Precipitated With Naloxone

in Morphine-Dependent Mice

Drug Dose Jumps/l5 min Incidence of Weight Loss

(mg/kg) n ± SE Diarrhea (mg ± SE)

Control 58 177 ± 10 78 743 ± 55

CHA 0.1 17 91 ± 11* 24* 468 ± 62*

0.5 6 77 ± 25* 0* 143 ± 76*

3.125 6-7 35 ± 12* 0* 63 ± 32*

CLON 0.05 10 121 ± 20* 10* 441 ± 80*

0.2 11 125 ± 11* 0* 325 ± 9O*

* P < 0.01 for significance of difference from control value

From table 3 it is evident that clonidine is about equipotent with

CHA in suppressing naloxone-precipitated diarrhea and weight loss

but less potent in suppressing jumping. This may be explained by

van der Laan's finding (1983) that at higher doses than those shown

here clonidine can in fact potentiate naloxone-precipitated

morphine withdrawal jumping.

86
The demonstration that, in the ileum at least, dependence can

develop to adenosine has important consequences. It has recently

been demonstrated in mice that interference with adenosine uptake

or antagonism of its effects can modify the action of acutely and

chronically administered alcohol [Dar et al. 1983). This raises

the possibility that adenosine might mediate some of the effects of

alcohol and might be involved in the dependence phenomenon

associated with its chronic use.

There is as yet no evidence that chronic administration of

adenosine or its derivatives induces dependence in the whole

animal, but after repeated administration of CHA to mice, tolerance

to some of its actions develops (unpublished observation).

Adenosine has also been implicated as a mediator of some of

morphine's actions (Sawynok and Jhamandas 1976; Stone 1981).

Additionally, in NG 108-15 cells, enhanced sensitivity of adenylate

cyclase to the excitatory action of adenosine has been reported to

occur after prolonged incubation with morphine (Griffin et al.

1983). Moreover, adenosine has been identified as the cytosolic

component responsible for the enhanced activity of adenylate

cyclase in morphine-dependent NG 108-15 cells. It is difficult to

equate the work with NG 108-15 cells with that in the ileum, as in

the latter it is believed that adenosine inhibits rather than

stimulates adenylate cyclase activity.

CYCLIC AMP HYPOTHESIS

The adenylate cyclase/cAMP system and its involvement in the

mechanisms of drug dependence has been the subject of extensive

work over the past 10 years. Collier and Roy (1974) showed that

opiates specifically inhibit prostaglandin El (PGE1) stimulated

CAMP production and proposed that opiate tolerance and dependence

represent a compensating hypertrophy of a part of the inhibited

PGE1/cAMP mechanism. At about the same time, Sharma et al.

(1975) demonstrated that cultivation of NG 108-15 cells in the

presence of morphine slowly increases PGE1-stimulated and basal

adenylate cyclase activity. Their observations led them to propose

that late positive regulation of adenylate cyclase counteracts the

inhibitory action of morphine and is responsible for narcotic

dependence and tolerance. Recently, further evidence supporting

this theory has come from work with pertussis toxin and NG 108-15

cells (Wuster et al. 1983).

Until recently it has been difficult to test the CAMP hypothesis of

dependence in normal neurons, but the demonstration of dependence

on normorphine, clonidine, and adenosine in the ileum now makes

that possible.

The general concept that dependence arises in normal neurons in

response to inhibition of adenylate cyclase is supported by the

fact that all three agonists that induce dependence in the ileum

have been shown to inhibit adenylate cyclase in other biological

preparations as a consequence of binding with their distinct

recognition sites (table 4).

87
TABLE 4. Inhibition of Adenylate Cyclase by Activation of Various

Receptors

Receptor Agonist Site Reference

Opioid, mu

kappa

delta

Adrenoceptor,

alpha2

Purinoceptor,

Al (Ri)

Morphine

EthylketocyclazocineRat brain

(Leu)5- NG 108-15

enkephalin cells

Clonidine,

noradrenaline

Adenosine

2-chloroadenosine

Mammalian

brain

rat brain

Thrombocytes

Adipocytes

NG 108-15

cells

Adipocytes

brain

membranes

Brain glial

cells in

culture

Collier & Roy 1974

Collier 1980

Barchfeld et al. 1982

Pay & Bhoola 1983

Barchfeld et al. 1982

Lampert et al. 1976

Brandt et al. 1976

Law et al. 1981

Garcia-Sainz

& Fain 1982

Burns et al. 1982

Limbird 1983

Sabol & Nirenberg

1979

Londos et al. 1980

Bruns et al. 1980

van Calker et al. 1979

Collier 1984. Copyright 1984, by Taylor Francis Ltd.

Because of the difficulty of separating nervous from muscular

tissue in the ileum, it has so far proved impossible to test

directly whether normorphine, clonidine, and adenosine inhibit

adenylate cyclase in the cholinergic neurons of the myenteric

plexus. Recently, however, some indirect evidence relevant to this

matter has been obtained.

88
Katada and Ui (1980, 1981) and Hildebrandt et al. (1983)

demonstrated that pertussis vaccine through the islet-activating

protein-toxin can inactivate an inhibitory regulator of adenylate

cyclase. More recently, Costa et al. (1983) have shown that

pertussis toxin can reduce opiate inhibition of adenylate cyclase

in NG 108-15 cells, possibly due to an uncoupling of the opiate

receptor and the inhibitory regulator (Ni) of adenylate cyclase.

We have explored the CAMP hypothesis of dependence by determining

the effect of pertussis vaccine on the actions of normorphine on

the myenteric plexus of the ileum (Collier et al. 1983). Initially

we looked at the effect of pertussis vaccine on the acute effects

of normorphine.

The IC50 values for normorphine in vaccine-treated tissues and

nontreated tissues were not significantly different, being 36 and

37 nM respectively for in vitro treatment, and 21 and 23 nM respectively for in vivo treatment. However, in normorphine-dependent

ileum, treatment with vaccine affects contraction (table 5).

TABLE 5. Inhibition by Pertussis Vaccine of Normorphine Dependence

in the Guinea Pig Isolated Ileum

Vaccine treatment n Mean response to Nx ± SEM

Treated Control

In vitro 14 ± 14 19.2 ± 2.7* 36.1 ± 5.7

In vivo 5 ± 5 21.1 ± 4.9** 49.7 ± 4.9

Responses expressed as per cent maximal response to acetylcholine.

Nx, 0.3 x 10-6M Naloxone.

*p < 0.02; **P < 0.01 for difference from control.

Collier et al. 1983. Copyright 1983, Elsevier Science Publishers BV.

In vitro treatment with vaccine reduced the withdrawal response by

about 47%. In ilea from guinea pigs treated with vaccine, the

withdrawal contracture was reduced by nearly 60% when compared to

control. In both sets of determinations, responses to electrical

stimulation and acetylcholine were unaffected by vaccine treatment. We have also shown that pertussis vaccine has similar

inhibitory effects on adenosine dependence in the ileum.

We conclude from this that although pertussis vaccine does not

change the acute potency of normorphine it profoundly inhibits the

dependence mechanism, possibly by inactivating the inhibitory

regulator of adenylate cyclase which opiates and adenosine normally

activate.

89
It has recently been reported that cholera toxin, a nonspecific

activator of adenylate cyclase, inhibits the expression of opioid

dependence in the myenteric plexus of the guinea pig (Lux and

Schulz 1983). This complements the work with pertussis vaccine.

The latter prevents the prolonged inhibition of adenylate cyclase

by morphine by inactivating the inhibitory regulator of adenylate

cyclase (Katada and Ui 1980, 1981; Hildebrandt et al. 1983), and

cholera toxin opposes the prolonged inhibition of adenylate cyclase

by direct activation of its excitatory regulator (Gill and Meren

1978). Thus both agents prevent the prolonged inhibition of

adenylate cyclase which is proposed to be central to the

development of dependence on opiates.

SUMMARY

The isolated guinea pig ileum provides a model in which drug

dependence can be induced in normal neurons.

The characteristics of opiate dependence in the ileum closely

resemble those of dependence in whole animals.

Convergent dependence on normorphine, clonidine, and adenosine can

be separately induced in the ileum in vitro.

Use of selective antagonists indicates that both acetylcholine and

substance P participate in the withdrawal response associated with

all three of these dependencies.

The demonstration that adenosine derivatives suppress opiate

withdrawal in the guinea pig ileum and in mice raises the

possibility that they might act similarly in man.

The point at which the dependencies on normorphine, clonidine, and

adenosine converge is probably below their separate recognition

sites and is possibly at the level of adenylate cyclase

regulation.

REFERENCES

Barchfeld, C.C., Maassen, Z.F., and Medzihradsky, F.

Receptor-related interactions of opiates witn PGE-induced

adenylate cyclase in brain. Life Sci, 31:1661-1665, 1982.

Brandt, M., Fischer, K., Moroder, L., Wunsch, E., and Hamprecht,

B. Enkephalin evokes biochemical correlates of opiate tolerance

and dependence in neuroblastoma X glioma hydrid cells. FEBS

Lett, 68:38-40, 1976.

Bruns, R.F., Daly, J.W., and Snyder, S.H. Adenosine receptors in

brain membranes: Binding of N6-cycle-hexyl (3H) adenosine

and 1,3-diethyl-8-(3H) phenylxanthine. Proc Natl Acad Sci

USA, 77:5547-5551, 1980.

Burns, T.W., Langley, P.E., Bylund, D.B. and Forte, L.R. Alpha-2

adrenergic activation inhibits forskolin-stimulated adenylate

cyclase activity and lipolysis in human adipocytes. Life Sci,

31:815-821, 1982.

90
Chahl, L.A. Contracture of guinea-pig ileum on withdrawal of

methionine5-enkephalin is mediated by substance P. Br J

Pharmacol, 80:741-751, 1983.

Chavkin, C., and Goldstein, A. Demonstration of a specific

dynorphin receptor in guinea pig ileum myenteric plexus. Nature

Lond, 291:591-593, 1981.

Collier, H.O.J. Cellular site of opiate dependence. Nature Lond,

283:625-629, 1980.

Collier, H.O.J. Cellular aspects of opioid tolerance and dependence. In: Hughes, J., Collier, H.O.J., Rance, M.J., and Tyers,

M.B., eds. Opioids, Past, Present and Future. London: Taylor

Francis Ltd., 1984. pp. 109-125.

Collier, H.O.J., Cuthbert, N.J., and Francis, D.L. Clonidine

dependence in the guinea-pig isolated ileum. Br J Pharmacol,

73:443-453, 1981a.

Collier, H.O.J., Cuthbert, N.J., and Francis, D.L. Model of opiate

dependence in the guinea-pig isolated ileum. Br J Pharmacol,

73:921-932, 1981b.

Collier, H.O.J., Plant, N.T., and Tucker, J.F. Pertussis vaccine

inhibits the chronic but not acute action of normorphine on the

myenteric plexus of guinea-pig ileum. Eur J Pharmacol,

91:325-326, 1983.

Collier, H.O.J., and Roy, A.C. Morphine-like drugs inhibit the

stimulation by E prostaglandins of cyclic AMP formation by rat

brain homogenate. Nature Lond, 248:24-27, 1974.

Collier, H.O.J., and Tucker, J.F. Novel form of drug-dependence -

on adenosine in guinea pig ileum. Nature Lond, 302:618-621,

1983.

Costa, T., Aktories, G., Schultz, G., and Wuster, M. Pertussis

toxin decreases opiate receptor binding and adenylate cyclase

inhibition in a neuroblastoma x glioma hybrid cell line. Life

Sci, 33:219-223, 1983.

Dar,M.S., Mustafa, S.J., and Wooles, W.R. Possible role of

adenosine in the CNS effects of ethanol. Life Sci,

33:1363-1374, 1983.

Ehrenpries, S., Light, I., and Schonbuck, G.H. Use of the

electrically stimulated guinea pig ileum to study potent

analgesics. In: Singh, J.M., Miller, L.H., and Lal, H., eds.

Drug Addiction: Experimental Aspects. New York: Futura, 1972.

pp. 319-342.

Garcia-Sainz, J.A., and Fain, J.N. Regulation of adipose tissue

metabolism by catecholamines: Roles of alpha1, alpha2 and

beta-adrenoceptors. TIPS, 3:201-203, 1982.

Gill, D.M., and Meren, R. ADP - ribosylation of membrane proteins

catalyzed by choleratoxin: Basis of the activation of adenylate

cyclase. Proc Natl Acad Sci USA, 75:3050-3054, 1978.

Gintzler, A.R. Serotonin participation in gut withdrawal from

opiates. J Pharmacol Exp Ther, 211:7-12, 1979.

Gintzler, A.R. Serotonin-substance-P pathway in the manifestation

of gut withdrawal. In: Way, E.L., ed. Endogenous and Exogenous

Opiate Agonists and Antagonists. Oxford: Pergamon, 1980. pp.

497-500.

Goldstein, A., and Schultz, R. Morphine-tolerant longitudinal

muscle strip from guinea-pig ileum. Br J Pharmacol 48:644-666,

1973.

91
Griffin, M.T., Law, P.Y., and Loh, H.H. Modulation of adenylate

cyclase activity by a cytosolic factor following chronic opiate

exposure in neuroblastoma X glioma NG 108-15 hybrid cells. Life

Sci, 33:365-369, 1983.

Hammond, M.D., Schneider, C., and Collier, H.O.J. Induction of

opiate tolerance in isolated guinea pig ileum and its

modification by drugs. In: Kosterlitz, H.W., ed. Opiates and

Endogenous Opioid Peptides. Amsterdam: Elsevier, 1976. pp.

169-176.

Hildebrandt, J.D., Sekura, R.D., Codina, J., Iyengar, R., Manclark,

C.D., and Birnbaumer, L. Stimulation and inhibition of adenyl

cyclases mediated by distinct regulatory proteins. Nature Lond,

302:706-708, 1983.

Katada, T., and Ui, M. Slow interaction of islet-activating

protein with pancreatic islets during primary culture to cause

reversal of alpha-adrenerqic inhibition of insulin secretion. J

Biol Chem, 255:9580-9588,-1980.

Katada, T., and Ui, M. Islet-activating protein: A modifier of

receptor-mediated regulation of rat islet adenylate cyclase. J

Biol Chem, 256:8310-8317, 1981.

Kosterlitz. H.W.. Lydon, R.J., and Watt, A.J. The effects of

adrenaline, noradrenaline and isoprenaline on inhibitory alpha

and beta-adrenoreceptors in the longitudinal muscle of the

guinea-pig ileum. Br J Pharmac, 39:398-413, 1970.

Lampert, A., Nirenberg, M., and Klee. W.A. Tolerance and

dependence evoked by an endogenous opioid peptide. Proc Natl

Acad Sci, USA, 73:3165-3167, 1976.

Law, P.Y., Wu, J., Koehler, J.E., and Loh, H.H. Demonstration and

characterization of opiate inhibition of the striatal adenylate

cyclase. J Neurochem 36: 1834-1836, 1981.

Limbird, L.E. Alpha2-adrenergic systems: Models for exploring

hormonal inhibition of adenylate cyclase. TIPS, 4:135-138,

1983.

Londos, C., Cooper, D.M.F., and Wolff, J. Subclasses of external

adenosine receptors. Proc Natl Acad Sci USA, 77:2551-2554,

1980.

Lord, J.A.H., Waterfield, A.A., Hughes, J., and Kosterlitz, H.W.

Endogenous opioid peptides: Multiple agonists and receptors.

Nature Lond, 267:495-499, 1977.

Lujan, M., and Rodriquez, R. Further characterization of opiate

physical dependence in the isolated guinea-pig ileum.

Pharmacologist, 20:268, 1978.

Lux, B., and Schulz, R. Cholera toxin selectively affects the

expression of opioid dependence in the tolerant myenteric plexus

of the guinea-pig. Eur J Pharmacol, 96:175-176, 1983.

Malta, E., Raper, C., and Tawa, P.E. Pre- and postjunctional

effects of clonidine- and oxymetazoline-like compounds in

guinea-pig ileal preparations. Br J Pharmacol, 73:355-362,

1981.

North, R.A., and Karras, P.J. Opiate tolerance and dependence

induced in vitro in single myenteric neurones. Nature Lond,

272:73-75, 1978.

Paterson, S.J., Robson, L.E. and Kosterlitz, H.W. Classification

of opioid receptors. Br Med Bull, 39:31-36, 1983.

92
Paton, D.M. Structure-activity relations for presynaptic

inhibition of noradrenergic and cholinergic transmission by

adenosine: Evidence for action on Al receptors. J Auton

Pharmacol, 1:287-290, 1981.

Paton, W.D.M. The action of morphine and related substances on

contraction and on acetylcholine output of coaxially stimulated

guinea-pig ileum. Br J Pharmacol, 12:119-127, 1957.

Pay, S., and Bhoola, K.D. Inhibition of rat striatal membrane

adenylate cyclase by opiates. 5th Int. Conf. Cyclic Nucleotides

and Protein Phosphorylation, Milan, 1983. Abstract, in press.

Sabol, S.L., and Nirenberg, M. Regulation of adenylate cyclase of

neuroblastoma X glioma hybrid cells by alpha-adrenergic

receptors. J Biol Chem, 254:1913-1920, 1979.

Sawynok, J., and Jhamandas, K.H. Inhibition of acetylcholine

release from cholinergic nerves by adenosine, adenine

nucleotides and morphine: Antagonism by theophylline.

J Pharmacol Exp Ther, 197:379-390, 1976.

Schultz, R., and Herz, A. Aspects of opiate dependence in the

myenteric plexus of the guinea-pig. Life Sci, 19:1117-1128,

1976.

Schultz, R., Wuster, M., Rubini, P., and Herz, A. Functional

opiate receptors in the guinea-pig ileum: Their differentiation

by means of selective tolerance development. J Pharmacol Exp

Ther, 219:547-550, 1981.

Sharma, S.K., Klee, W.A., and Nirenberg, M. Dual regulation of

adenylate cyclase accounts for narcotic dependence and

tolerance. Proc Natl Acad Sci, USA, 72:3092-3096, 1975.

Stone, T.W. The effects of morphine and methionine-enkephalin on

the release of purines from cerebral cortex slices of rats and

mice. Br J Pharmacol, 74:171-176, 1981.

Tanaka, T., and Starke, K. Binding of 3H-clonidine to an

a-adrenoceptor in membranes of guinea-pig ileum.

Naunyn-Schmiedebergs Arch Pharmacol, 309:207-215, 1979.

TSOU, J., Louie, G., and Way, E.L. Manifestations of gut opiate

withdrawal contracture and its blockade by capsaicin. Eur J

Pharmacol, 81:377-383, 1982.

Tucker, J.F., Plant, N.T., von Uexkull, A., and Collier, H.O.J.

Inhibition by adenosine analogs of opiate withdrawal effects.

In: Harris, L.S., ed. Problems of Drug Dependence, 1983.

National Institute on Drug Abuse Research Monograph 49. DHHS

Pub. No. (ADM) 84-1316. In press. pp. 85-91.

van Calker, D., Muller, M., and Hamprecht, B. Adenosine regulates

via two different types of receptors, the accumulation of cyclic

AMP in cultured brain cells. J Neurochem, 33:999-1005, 1979.

van der Laan, J.W. Potentiation of jumping in precipitated

morphine withdrawal by alpha2-agonists: Adrenergic and

dopaminergic mechanisms. International Narcotic Research

Conference, Garmisch Partenkirchen, 1983. Abstract.

Villarreal, J.E., Martinez, J.N., and Castro, A. Validation of a

new procedure to study narcotic dependence in the isolated

guinea-pig ileum. In: Harris, L.S., ed. Problems of Drug

Dependence, 1977. Washington, D.C.: National Academy of

Sciences, 1977. pp. 305-314.

93
Wuster, M., Costa, T., and Gramsch, C.H. Uncoupling of receptors

is essential for opiate induced desensitization (tolerance) in

neuroblastoma x glioma hybrid cell NG 108-15. Life Sci,

33:341-345, 1983.

ACKNOWLEDGEMENT

The Committee on Problems of Drug Dependence, Inc., provided

financial support.

AUTHORS

The late Harry O.J. Collier, B.A., Ph.D., Sc.D., F.I.Biol.

John F. Tucker, B.Sc., Ph.D.

Department of Pharmacology, Chelsea College, University of London,

Manresa Road, London, SW3 6LX, U.K.

94
Biochemical Reactions Between

Opiate Receptor Binding and

Inhibition of Neurotransmission

Doris H. Clouet and Norifumi Yonehara

An account of the mechanisms by which opiate tolerance develops at

the molecular level should begin with two caveats: there is no reason to assume (1) that all opioids act through the same mechanisms

in all neural tissues (although a general response of a depression

of neuronal firing when an opioid is applied to a cell suggests a

commonality of mechanisms) or (2) that all subtypes of opioids produce the same states of biochemical tolerance (the lack of crosstolerance among the different subtypes suggests that there are

differences in the tolerant state).

One aspect of this review will be to note similarities and differences in the molecular events leading to tolerance among opioid

subtypes and also among various nervous tissues. The studies from

this laboratory have been carried out exclusively in the rat nigrostriatal system, both because the relationship between behavior and

the dopaminergic system has been examined rigorously and because

opiate receptors and endogenous opioids have a relatively enriched

distribution in rat striatum. In the nigrostriatal dopaminergic(DA)

pathways, the cell bodies, located in the substantia nigra, have

long axons with extensive terminal arborization in the caudate nucleus of the striatum. The very numerous nerve terminals make synapses with GABA, acetylcholine and probably enkephalin-containing

neurons, leading to the presence of large numbers of pre- and postsynaptic DA-responsive neurons. Important neurochemical effects induced by acute or chronic opioid treatment that have been found in

neuronal cells in culture, in peripheral nerve, or in brain areas

other than striatum, will be included in this review.

EFFECTS OF ACUTE OPIOID TREATMENT

The binding of an opioid agonist to a presynaptic receptor on the DA

terminal leads to a sequence of biochemical events that are disrupted by the administration of an opioid. In table 1, some of the biochemical steps between opiate receptor binding and impulse transport

between striatal neurons are listed, together with the effects produced at these steps by acute opioid administration. The inhibition

95
Biochemical

hibition of

TABLE 1

Reactions Between Opiate Receptor Binding and the InNeurotransmission in Rat Striatum: Responses to Acute

Opiate Administration

Biochemical Step Responses

Binding of Ligand to * Coupling of signal from binding to

Receptor amplifier impaired (Childers et al. 1983)

Formation of cAMP

GTP-binding * Decreased GTP binding due to enhanced

GTPase activity (Koski and Klee 1981;

Parenti et al. 1983)

Adenylate cyclase * Inhibition of adenylate cyclase (Collier

and Roy 1974; Shanna et al. 1977;

Barchfeld et al. 1982; Gentleman et al.

1983)

Stimulation by DA * DA binding inhibited (Gentleman et al.

1983)

* Levels of CAMP and cyclase activity in

striatum back to normal by 120 minutes

(Clouet et al. 1975).

Ion Fluxes

Influx of K+, Ca++

Efflux of Na+

Depolarization

Retention of intraterminal Ca++ and increased Ca++ dep. K+ conductance

(North and Williams 1983)

Hyperpolarization and inhibition of

firing of most neurons

(Zieglgansberger and Fry 1978)

* Increased firing of nigrostriatal DA

neurons (Iwatsubo and Clouet 1977)

Protein Phosphorylation * Increased phosphorylation of some membrane proteins (Williams & Clouet 1982)

* Increases in calmodulin-Ca++ dep. protkinase activity (phosphorylation of

tyrosine hydroxylase, Ca++ATPase, etc.)

(Williams and Clouet 1982)

Calmodulin * Movement from cytosol to membranes increased (Clouet and Williams 1982b)

* Inhibition "in vitro" by opioids of Ca++-

dep. kinase not due to interaction

between calmodulin and opioid

(Clouet et al. 1983)

Synthesis and Release of Generally inhibited by opioids

Neurotransmitters

* DA biosynthesis increased (Clouet and

Ratner 1970; Gauchy et al. 1973)

* Delta inhibition of DA release (Wood et

al. 1980; Yonehara and Clouet, in press)

* Effect shown in rat striatum

96
of adenylate cyclase activity by an opiate was first reported by

Collier and Roy (1974), who found that prostaglandin-stimulated

adenylate cyclase activity was inhibited by morphine in brain homogenates. In neuroblastoma-glioma (NG) hybrid cells in culture, Klee

and his colleagues reported that DA-stimulated adenylate cyclase

activity was inhibited by ß-endorphin (Sharma et al. 1977). Later,

the inhibition of cyclase was shown to be related to a destruction

of the GTP required in the coupling of opiate receptor and adenylate

cyclase by an increased GTPase activity induced by opioids (Koski

and Klee 1981). The same changes in DA-dependent adenylate cyclase

and GTPase activities have been found in rat striatal membranes

(Gentleman et al. 1983; Parenti et al. 1983). Both cyclase activity

and CAMP levels in striatum are restored to control levels 30 to 120

minutes after opiate administration (Clouet et al. 1975).

Ion fluxes change during the passage of a nerve impulse from neuron

to neuron: the influx of calcium ions and potassium ions is increased while the efflux of sodium ions is also increased. Opiate

treatment can produce a hyperpolarization that prevents subsequent

firing of the cell in most neuronal systems (Zieglgansberger and Fry

1978). The nigrostriatal DA system is one that is highly regulated

by both presynaptic and feedback controls, so that neuronal firing

is increased by opiates due to the inhibition of an inhibitory neuron

(Iwatsubo and Clouet 1977). Thus, there are opposing forces regulating DA release during acute opioid treatment, an increased propagation of the nerve impulse to the nerve terminals that promotes neurotransmitter release, and presynaptic opiate effects that promote

decreased neurotransmitter release. In many species (cat, mouse) DA

release in striatum is stronger than any presynaptic inhibitory

effect and opioids induce DA release (Chesselet et al. 1981; Wood et

al. 1980). In rats, the presynaptic inhibitory effect predominates

and there is no release of DA when opiopeptides or narcotic drugs

are administered (Yonehara and Clouet, in press). In these studies,

the delta opiopeptides had greater effect on DA turnover than the

mu opiopeptides.

The two "second messengers" are involved in opiate actions. The

formation of CAMP is decreased when an opioid is introduced into a

neuron, or a NG cell in culture, in an "in vitro" tissue preparation

or into an animal. All of the consequences of a limited CAMP supply

come into play, including decreased Ca++ influx into nerve terminals,

decreased phosphorylation by CAMP-dependent protein kinases, etc.

The role of the other "second messenger" calcium, in impulse conduction is well known. Other sites of calcium activity involve a number

of enzymes that are phosphorylated by Ca++/calmodulin-dependent protein kinases or Ca++/phospholipid-dependent protein kinases (Greengard 1978; Takai et al. 1979). We have found that the increased phosphorylation of striatal SPM proteins following the acute administration of an opioid had a protein pattern after gel electrophoresis

that was very similar to that produced by Ca++/calmodulin stimulation

of phosphorylation, in terms of increased radioactivity in certain

protein bands after phosphorylation with ATP32 (Williams and Clouet

1982). We examined the levels and intracellular site of calmodulin

after opioid administration to see whether calmodulin availability

97
was the cause of the opioid-induced effect. Calmodulin is a Ca++

binding protein primarily localized in nervous tissue. While localized in the cytosol, it is relatively inactive. Its movement into

membranes seems to be related to increased cellular activity in many

forms: a capping of cell wall prior to endocytosis (Salisbury et al.

1981); an activation of the red cell calcium pump (Hinds et al. 1978);

and the binding of soluble proteins to membranes (Geisow and Burgoyne

1983). The acute administration of either etorphine or met-enkephalin to rats resulted in a significantly higher level (60-80% above

control) of calmodulin in both striatal synaptic membranes and in

synaptosomal cytosol, with the ratio of cytosolic to membranal calmodulin remaining at about 3:7 (Clouet and Williams 1982b). It is

possible that the excess calmodulin was sequestered so effectively in

control animals that it was not removed by homogenization in detergent, but was mobilized by increasing levels of Ca++ in the neurons.

The acute effects of opioids in increasing the endogenous phosphorylation of striatal membrane proteins could be ascribed completely to

the increased calmodulin available in the preparation to activate

Ca++-calmodulin-dependent protein kinases found in SPM preparations.

EFFECTS OF CHRONIC OPIOID TREATMENT

A common consequence of repeated stimulation of many neurohormone

receptors is a down-regulation that decreases the number of active

receptors. Such a phenomenon has been demonstrated for the opiate

receptor after chronic opioid exposure. Hippocampal slices incubated

with d-ala2d-leu5enkephalin for 4 hours had decreased numbers of

delta (d-ala2,d-leu5enkephalin) receptors, but no changes in the number of mu receptors (Dingledine et al. 1983). The NG hybrid cells

and fetal brain cells exposed to opiates or opiopeptides similarly

showed a down-regulation of opiate receptors (Lenoir et al. 1983;

Wuster et al. 1983). The levels of enkephalins in the striatum seem

to be fairly stable and resistant to change during opiate treatment

when examined at time periods from five to 180 minutes after opioid

treatment. It is possible that the response to opiate receptor occupation related to enkephalin biosynthesis may be dependent temporally

on the initial binding of opiates to receptors rather than on the

period of pharmacological responses. Enkephalin levels are, however,

responsive to treatment with other types of neuroactive drugs. Striatal enkephalin levels are increased by the administration of either

benzodiazepines or neuroleptics (Duka et al. 1980; Hong et al. 1978).

Long-term chronic treatment of rats with opiates did decrease both

leu- and met-enkephalin levels in the striatum (Przewlocki et al.

1979). The chronic administration of an opiate antagonist, naltrexone, to rats produced an up-regulation of both delta and kappa receptors (Zukin et al. 1982) (table 2).

Adenylate cyclase activity was normal in brain areas of opiate-tolerant animals (Clouet et al. 1975). The enzyme, however, was supersensitive to both DA (Clouet and Iwatsubo 1975) and to GTP (Parenti

et al. 1983). The increase in GTPase activity found after acute

opioid administration was no longer seen in tolerant tissues including

striatal synaptic plasma membranes (Parenti et al. 1983). The large

increases in rat striatal DA turnover produced by an initial dose of

an opioid no longer occur as tolerance develops. The release of DA in

98
TABLE 2

Biochemica

Inhibition

l Reactions Between Opiate Receptor (O.R.) Binding and the

of Neurotransmission in Rat Striatum: Responses to Chronic

Opiate Administration

Biochemical Step

Binding of Ligand to

Receptor

Responses

Down-regulation of O.R. in hippocampus

or NG cells (Parenti et al. 1982)

Up-regulation of O.R. after chronic

antagonist treatment

(Zukin et al. 1982)

Formation of CAMP * GTPase decrease (Parenti et al. 1983)

GTP binding

Adenylate Cyclase * Basal activity normal

Stimulation * Supersensitivity to DA

(Clouet and Iwatsubo 1975)

* Supersensitivity to GTP

(Parenti et al. 1983)

Ion Fluxes

Influx of K+ Ca++ * Ca++ fluxes normal

Efflux of Na+

Depolarization * Some tolerance to hyperpolarization

(Zeiglgansberger and Fry 1978)

Protein Phosphorylation * Decreased phosphorylation of some membrane proteins

(O'Callaghan et al. 1979)

Calmodulin * Calmodulin in cytosol

(Clouet and Williams 1982b)

* Chronic trifluperazine blocked effect

of morphine on calmodulin levels

(Williams et al. in press)

Synthesis and Release Generally normal in tolerant tissue

of

Neurotransmitters * No increase in DA turnover

* DA-receptor supersensitivity

(Parenti et al. 1983)

* Effect shown in rat striatum

99
areas other than rat striatum is no longer inhibited.

In the striatum of morphine-tolerant rats, the levels of endogenous

phosphorylation of SPM proteins are greatly reduced (O'Callaghan

et al. 1979). When separated by SDS-gel electrophoresis, the many

specific protein bands in which phosphorylation is reduced by chronic

opioid treatment are the same as those in which phosphorylation is

increased by acute opioid treatment or by Ca++-calmodulin stimulation.

To remedy the defect in phosphorylation, supplementation of the protein kinase assay with calmodulin was examined. In contrast to the

situation in acutely treated animals, when calmodulin supplementation

was able to restore phosphorylation, extra calmodulin was never able

to restore the rates of phosphorylation to those in the striata of

placebo-treated rats (Clouet and Williams 1982a). A transfer of calmodulin from membranal to cytosolic sites was also seen in rat striata

from morphine-tolerant rats (Clouet and Williams 1982a).

FIGURE 1. Effect of supplementation with calmodulin on the phosphorylation of striatal synaptic plasma membrane (SPM) proteins measured

in densitometer units for autoradiographs.

Calmodulin (CaM) was added as units described in Clouet and Williams,

1982b. Optimal Ca++ levels were maintained in all assays except the

zero control.

a= Acute etorphine vs. Saline-control; b = Acute methadone vs. Salinecontrol; c= Chronic morphine vs.. Placebo pellets; d= Etorphine added

to assay vs. Same preparation without drug.

100
FIGURE 2. Phosphorylation of two striatal SPM proteins.

Supplementation of striatal SPM proteins from morphine-tolerant

(stippled) or control (clear) rats with Ca++ and/or CaM in assays

of protein kinase activity. The proteins have aparent M.W. in 

SDS-gel of 54 kilodaltons (left panel) or 63 kilodaltons (right

panel).

The effect of calmodulin supplementation in the protein kinase assay

is shown in figure 1. As described above, control levels of total

phosphate incorporated into protein can be brought to the increased

levels found 10 minutes after the injection of etorphine by adding

calmodulin (1a). The decreased levels found 30 minutes after methadone administration were not brought to control levels by adding

calmodulin (lb), nor were the levels found in SPM proteins from morphine-tolerant rats (1c). The effect of an opioid in reversing the

inhibition of Ca++-calmodulin stimulation is shown in 1d. It should

be noted that different effects of calmodulin supplementation can be

seen between individual protein bands (figure 2). After SDS gel

electrophoresis of SPM proteins from the striata of tolerant rats, a

protein band with an apparent M.W. of 54 kilodaltons was increasingly phosphorylated with the addition of Ca++, calmodulin or both to

the assay, maintaining a constant ratio with a sample from placebotreated rats (left panel). A protein with a M.W. of 63 kilodaltons

was phosphorylated more in response to calmodulin than to Ca++, although the protein from placebo-treated rats responded to both cofactors (right panel). Yamauchi and Fujisawa (1980) attributed the

phosphorylation of the 63K protein to protein kinase III, that acts

101
on only two proteins in brain. One is believed to be a protein kinase

subunit. The band at 54 K is probably a mixture of proteins such as

a tubulin subunit, an adenylate cyclase subunit, etc. Four soluble

Ca++-calmodulin-dependent protein kinases have been found in rat

cerebral cortex (Yamauchi and Fujisawa 1980) and membranal kinases

are also activated by the same cofactors. These enzymes may be unequally affected by opioid treatment.

A phenomenon that we noted several years ago is a reversal of the

stimulation of protein kinase activity by Ca++-calmodulin by adding

opioids to the assay tubes (Clouet et al. 1978). Using a rapid flow

multi-mixer to conduct short-time assays of protein kinase activity,

we found that the inhibitory effect of opioids was apparent in less

than 200 milliseconds (Clouet and Williams 1982a). The possibility

that opioids, like neuroleptics, interact directly with calmodulin,

removing it from the enzyme assay, was examined. The stimulation of

endogenous phosphorylation of striatal SPM proteins by Ca++-calmodulin is inhibited by etorphine, methadone, beta-endorphin and d-ala2

met enkephalin with KiS of 5x10-7M (Clouet et al. 1983) (figure 3).

The constant ratio for inhibition of calmodulin to opioid suggested a

direct interaction. As shown in figure lc, additional calmodulin can

reverse the opioid effect. However, when a calmodulin-free purified

preparation of phosphodiesterase was examined in the same paradigm,

FIGURE 3. The effect of opioids on CaM-dependent enzyme activity

The basal (lower curves) and Ca++-CaM stimulated (upper curves)

activity of protein kinasesin striatal SPM preparations and of a

purified phosphodiesterase are shown. The dose-dependent effects of

(a) beta- endorphin, (b) d-ala2met enkephalin, (c) methadone and (d)

etorphine are depicted as inhibition of stimulated enzyme activity.

102
stimulated phosphodiesterase activity was inhibited by beta-endorphin

and etorphine with Kis of 2x10-4M and not inhibited by methadone or dala2

met enkephalin. Thus, a direct interaction of calmodulin and opioids may be ruled out as obligatory for this "in vitro" inhibition of

Ca++-calmodulin dependent enzyme, although it may play a role in some

effects of beta-endorphin. The importance of changes in the state of

phosphorylation of proteins, particularly enzymes, may be assessed by

tentatively identifying some of the proteins in a striatal SPM preparation that are phosphorylated by Ca++-calmodulin-dependent protein

kinases, and are affected by opioid treatment. Needless to say, a protein can not be identified by M.W. in SDS gel alone, but a consensus

is beginning to be formed concerning certain brain proteins (table 3).

Some protein kinases are relatively non specific as to substrate preference (e.g. , protein kinase II (table 3). Others like protein kinase

III have only two substrates that seem to be subunits of another protein kinase (Yamauchi and Fujisawa 1980). In depolarized neurons or in

synaptosomal preparations from depolarized cells, Ca++ uptake into the

cell stimulated both neurotransmitter release and phosphorylation of

two proteins in synaptic vesicles (DeLorenzo et al. 1979). These proteins of 55 and 62K M.W. are believed to participate in the endocytotic process. Synapsin I, a neuron-specific protein, also participates

TABLE 3

Molecular weights and possible identities of brain proteins of their

subunits that are phosphorylated by Ca++-calmodulin dependent protein

kinases

Approximate M.W. Protein References

**185 Clathrin A

96 Ca++ATPase SU B

** 87-82 Synapsin I B

74 Protein Kinase SU C

68 Neurofilament SU B

***63 Protein kinase A

62 Synaptic vesicle SU C

60 Tyrosine hydroxylase B

55 Synaptic vesicle protein C

54 Adenylate cyclase SU D

**52 Tublin SU D

50 Protein kinase SU A

**45 (Substrate) A

**37 A

*21 A

18 Myelin basic protein B

**17 A

16.5 GABA modulin B

***15 A

14 Myeline basic protein B

Proteins may be phosphorylated by Ca++-calmodulin-dependent protein

kinases I (*), II (**) or III (***) SU=Subunit. References are to:

A= Yamauchi and Fujisawa 1980; B= Nestler and Greengard 1983;

C= DeLorenzo et al. 1979; D= Burke and DeLorenzo 1981.

103
in endocytosis since its phosphorylation decreases its affinity to proteins on the outer wall of the vesicles (Forn and Greengard 1976). The

total amount of Synapsin I is regulated by various hormones, and also

by morphine (Nestler and Greengard 1983). Also, in depolarized neurons,

the biosynthesis of neurotransmitter is accelerated by the activation

of tyrosine hydroxylase through a Ca++-calmodulin-dependent process

(Mestikawy et al. 1983). The phosphorylation of GABAmodulin decreases

its affinity for the GABA receptor leaving the receptor free to bind

GABA (Wise et al. 1983).

Interconnections in the striatum between the DA system and other neurotransmitter systems make it possible for opioid effects to be extended to other systems. Both morphine and ß-endorphin injections into rat

striatum cause a decreased turnover of GABA in the caudate nucleus and

an increased turnover in the globus pallidus (Moroni et al. 1979).

Since an activation of caudate DA neurons by electrical stimulation or

by apomorphine does not change GABA turnover, it seems that enkephalinergic neurons may impinge directly on GABA cells (Oertel et al. 1983).

Similarly, a direct correlation between opioid effects on cholinergic

neurons and on DA in the striatum has been ruled out by studies in

which acetylcholine turnover and binding were not affected by opioid

treatment (Schurr et al. 1983). In other areas of the central and

peripheral nervous system opioid effects on GABA and acetylcholine

have been shown.

CONCLUSIONS

In the striatum a variety of responses to chronic opioid exposure have

been discovered. Some phenomena are merely reversals of activations

or inhibitions that were produced as part of the response to the initial exposure to the opioid; e.g., the reversal of GTPase activation

in samples from tolerant rats, thereby increasing the GTP supply for

opiate receptor binding and adenylate cyclase activation. Other molecular effects of chronic opioid exposure involve changes in the sensitivity of receptors; e.g.; the down- and up- regulation of opiate

receptors in response to chronic treatment with opioid agonists or

antagonists, respectively; or the supersensitivity of DA receptors to

DA in tolerant rat striatum.

The reversal during tolerance to the initial responses to opioid treatment in the phosphorylation of striatal SPM proteins does not seem to

be simple, since the acute effects are reversed by the addition of

calmodulin to the assay, suggesting a lack of active calmodulin, while

the lack of complete activity after supplementation of the assay with

calmodulin and Ca++ in assays of protein kinase activity in striatal

samples from tolerant animals suggests a decreased amount of active

enzymes. Calmodulin is active as a Ca++ cofactor when at least two

out of four Ca++ binding sites are occupied by Ca++. The same Ca++

binding is required for calmodulin to be localized on the inner surface

of neuronal membranes or the outer side of synaptic vesicles (Means

and Dedman 1980). Thus, the availability of Ca++ regulates calmodulin

effectiveness and determines its intracellular site to some extent. The

104
movement of calmodulin to SPM after acute opioid treatment and to the

cytosol during chronic treatment is probably a consequence of opioid

effects on the levels and localization of Ca++.

We conclude, in common with other participants in this symposium, that

the biochemical mechanisms of opiate tolerance are diverse. The biochemical effects produced by an initial exposure of nervous tissue to

opioids are largely specific to each tissue, although a common response

is inhibition of neurotransmission. Neurochemical changes following

chronic opioid exposure tend to result in a diminution of disturbance

of neuronal cell function with succeeding doses of drug by an available mechanism (change in number or sensitivity of receptors, in activation or inhibition of biosynthetic enzymes or in cellular localization of cofactors, etc.). Thus, the usual ways in which biochemical

events are regulated are evoked during the development of tolerance to

enable the nervous system to limit the aberrations produced by opioid

exposure. It is possible that in largely unexplored systems psychic dependence may result from a series of biochemical responses tending to

limit the wide mood swings due to sequential euphoria and withdrawal.

REFERENCES

Barchfeld, C.C. Maassen, F.A. and Medzihradsky, F. Receptor-related

interactions of opiates with PGE-induced adenylate cyclase in brain.

Life Sci 31:1661-1665, 1982

Burke, B.E. and DeLorenzo, R.J. Ca++ and calmodulin-stimulated endogenous phosphorylation of neurotubulin. Proc Natl Acad Sci 78:

991-995, 1981.

Chesselet, M.F. Cheramy, A. Reisine, T.D. and Glowinski, J. Morphine

and delta opiate agonists locally stimulate in vivo dopamine release

in rat caudate nucleus. Nature 291:320-322, 1981.

Childers, S.R. Lambert, S.M and LaRiviere, G. Selective alterations

in guanine nucleotide regulation of opiate receptor binding and

coupling with adenylate cyclase. Life Sci 33:215-218, 1983.

Clouet, D.H. and Iwatsubo, K. Dopamine-sensitive adenylate cyclase

of the caudate nucleus of rats treated with morphine. Life Sci 17:

35-40, 1975.

Clouet, D.H. and Ratner, M. Catecholamine biosynthesis in the brains

of rats treated with morphine. Science 168:854-856, 1970.

Clouet, D.H. and Williams, N. The role of calmodulin in opioid-induced changes in the phosphorylation of rat striatal synaptic membrane proteins. Neurochem Res 7:1135-1148, 1982a.

Clouet, D.H. and Williams, N. fhe effect of opioids on striatal calmodulin levels. Life Sci 31: 2283-2286, 1982b.

Clouet, D.H. Gold, G.J. and Iwatsubo, K. Effects of narcotic analgesic drugs on the cyclic AMP- adenylate cyclase system in rat brain.

Br J Pharmac 54: 541-548, 1975.

Clouet, D.H. Williams, N. and Yonehara, N. Is a calmodulin-opiopeptide interaction related to the mechanism of opioid action?

Life Sci 33:727-730, 1983.

105
Clouet, D.H., O'Callaghan, J.P. and Williams, N. The effect of opiates

on calcium-stimulated phosphorylation of synaptic plasma membrane

proteins in intact synaptosomes from rat striatum. In: VanRee, J and

Terenius L.eds. Characteristics and Functions of Opioids, Amsterdam,

North-Holland, 1978, pp. 351-352

Collier, H.O.J., and Roy, A.C. Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain

homogenate. Nature 248:24-27, 1974.

DeLorenzo, R.J. Freedman, S.D. Yohe, W.B. and Maurer, S.C. Stimulation of Ca++-dependent neurotransmitter release and presynaptic

nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles. Proc Natl Acad

Sci 76:1838-1842, 1979.

Dingledine, R. Valentino, R.J. Bostock, E. King, M.E. and Chang,

K-J. Down regulation of delta but not mu opioid receptors in hippocampal slice associated with loss of physiological response. Life

Sci 33: 333-336, 1983.

Duka, T., Wuster, M., and Herz, A. Benzodiazepines modulate striatal

enkephalin levels via a GABAergic mechanism. Life Sci 26:771-776,

1980.

Forn, J. and Greengard, P. Regulation by lipolytic and antilipolytic

compounds of the phosphorylation of specific proteins in isolated

intact fat cells. Arch Biochem Biophys 176(2): 721-733, 1976.

Gauchy, C. Agid, Y. Glowinski, J. and Cheramy, A. Acute effects of

morphine on dopamine synthesis and release and tyrosine metabolism

in the rat striatum. Eur J Pharmacol 22:311-319, 1973.

Geisow, M.J. and Burgoyne R.D. Recruitment of cytosolic proteins

to a secretory granule membrane depends on Ca++-calmodulin.

Nature 301: 432-435, 1983.

Gentleman, S. Parenti, M. Neff, N.H. and Pert, C.B. Inhibition of

dopamine-activated adenylate cyclase and dopamine binding by opiate

receptors in rat striatum. Cell Mol Biol 3:17-26, 1983.

Greengard, P. Phosphorylated proteins as physiological effectors.

Science 199:146-152, 1978.

Hinds, T.R. Larsen, F.L. and Vincenzi. F.F. Plasma membrane Ca2++

transport: Stimulation of soluble proteins. Biochem Res Commun

81(2): 455-61. 1978.

Hong; J.S. Yang; H-Y-T. Fratta, W. and Costa, E. Rat striatal methionine-enkephalin content after chronic treatment with cataleptogenic and non-cataleptogenic antischizophrenic drugs. J Pharm Exp

Therap 202: 141-147, 1978

Iwatsubo, D. and Clouet, D.H. Effects of morphine and haloperidol

on the electrical activity of rat nigrostriatal neurons. J Pharm

Exp Therap 202: 429-436, 1977.

Koski, G. and Klee, W.A. Opiates inhibit adenylate cyclase by stimulating GTP hydrolysis. Proc Natl Acad Sci 78: 4185-4189, 1981

Lenoir, D. Barg, J. and Simantov, R. Down regulation of opiate

receptors in serum-free cultures of aggregating fetal brain cells.

Life Sci 33: 337-340, 1983.

Means, A.R. and Dedman, J.R. Calmodulin-an intracellular calcium

receptor. Nature 285: 73-77, 1980.

106
Mestikawy, S.El., Glowinski, J. and Hamon, M. Tyrosine hydroxylase

activation in depolarized dopaminergic terminals-involvement of

Ca++ dependent phosphorylation. Nature 302: 830-832, 1983.

Moroni, F. Peralta, E. Cheney, D.L. and Costa, E. On the regulation of gamma aminobutyric acid neurons in caudatus, pallidus and

nigra, Effects of opioids and dopamine agonists. J Pharm Exp

Therap 208: 190-194. 1979.

Nestler, E.J. and Greengard, P. Protein phosphorylation in the brain.

Nature 305: 583-588, 1983.

North, R.A. and Williams J.T. Opiate activation of potassium conductance inhibits calcium action potential in rat locus coeruleus.

Br J Pharm 80: 225-228, 1983

O'Callaghan, J.P., Williams, N. and Clouet, D.H. The effect of morphine-on the endogenous phosphorylation of synaptic plasma membrane

proteins of the rat. J Pharm Exp Therap 208: 96-106, 1979

Oertel, W. Riethmuller, G. Mugnaini, E. Schmechel, D.E. Weindl, A.

Gramsch, E. and Herz, A. Opioid peptide like immunoreactivity

localized in GABAergic neurons of rat neostriatum and central amygdaloid nucleus. Life Sci 33: 73-76, 1983

Parenti, M. Gentleman, S. Olianas, M.C. and Neff, N.H. The dopamine

receptor adenylate cyclase complex: Evidence for a post recognition site involvement for the development of supersensitivity.

Neurochem Res 7:115-124, 1982.

Parenti, M. Gazzotti, G. Tirone, F. and Groppetti, A. Opiate tolerance and dependence is associated with a decreased activity of

GTPase in rat striatal membranes. Life Sci 33: 345-348, 1983

Przewlocki, R. Hollt, V. Duka, T. Kleber, G. Gramsch, C. Haarmann,

I. and Herz, A. Long time morphine treatment decreases endorphin

levels in rat brain and pituitary. Brain Res 174: 357-361, 1979

Salisbury, J.L. Condeelis, J.S. Maihle, N.J. and Satir, P. Calmodulin localization during capping and receptor-mediated endocytosis.

Nature 294: 163-166, 1981

Sharma, S.K. Klee, W.A. and Nirenberg, M. Endorphin from pituitary

inhibits cyclic AMP formation in homogenates of neuroblastoma x

glioma hybrid cells. Nature 265: 362-363, 1977

Schurr, A. Rigor, B.M. Strong, R. and Gottesfeld, A. The cholinergic system in rat striatum during morphine tolerance and dependence

Life Sci 33: 2521-2525, 1983.

Takai, Y. Kishimoto, A. Iwasa, Y. Kawahara Y. Mori, T. and

Nishizuka, Y. Calcium-dependent activation of a multifunctional

protein kinase by membrane phospholipids. J Biol Chem 254:

3692-3695, 1979.

Williams, N. and Clouet, D.H. The effect of acute opioid administration on the phosphorylation of rat striatal membrane proteins.

J Pharmacol Exp Therap 220: 278-286, 1982.

Williams, N. Yonehara. N. and Clouet. D.H. Synergistic effect of

trifluoroperazine on analgesia and-turnover-of dopamine responses

induced by morphine in rats. Ann NY Acad Sci (in press).

Wise, B.C. Guidotti, A. and Costa, E. Phosphorylation induces a

decrease in the biological activity of the protein inhibitor

(GABA-modulin) of gamma-aminobutyric acid binding sites.

Proc Natl Acad Sci 80: 880-890, 1983.

107
Wood, P.L. Stotland, M. Richard, J.W. and Rackham, A. Actions of

mu, kappa, sigma, delta and agonist/antagonist opiates on striatal

dopamine function. J Pharmacol Exp Therap 215:697-703, 1980.

Wuster, M., Costa, T. and Gramsch, Ch. Uncoupling of receptors is

essential for opiate-induced desensitization (tolerance) in

neuroblastoma x glioma hybrid cells:NG 108-15. Life Sci 33:341-

344, 1983.

Yamauchi, T. and Fujisawa, H. Evidence for the distinct forms of

calmodulin-dependent protein kinases from rat brain. FEBS Lett

116: 141-144. 1980.

Yonehara, N. and Clouet, D.H. The effects of delta and mu opiopeptides on the turnover and release of dopamine in rat striatum.

J Pharmacol Exp Therap (in press).

Zieglgansberger, W. and Fry, J.P. In: Developments in Opiate Research.

Herz, A. ed. New York: Marcel Dekker, 1978, pp 192-299.

Zukin, R.S. Sugarman, J.R. Fitz-Syage, M. Gardner, E.L, Zukin, S.R.

and Gintzler, A.R. Naloxone-induced opiate receptor supersensitivity. Brain Res 245:285-297, 1982.

ACKNOWLEDGEMENT

The experimental work was supported in part by Research Grant DA00087

from the National Institute on Drug Abuse.

AUTHORS

Doris H. Clouet, Ph.D.

New York State Division of Substance Abuse

Services Research Laboratory

80 Hanson Place

Brooklyn, New York 11217 and

Professor of Psychiatry

Downstate Medical Center

Brooklyn, N.Y. 11203

Norifumi Yonehara, Ph.D.

New York State Division of Substance Abuse

Services Research Laboratory

80 Hanson Place

Brooklyn, New York 11217

now at

Osaka University School of Medicine

Osaka, Japan

108
The Role of Adenyl Cyclase in

Opiate Tolerance and Dependence

Werner A. Klee, Graeme Milligan, William F. Simonds,

and Bruno Tocque

The topic of this paper is one which was very important to H.O.J.

Collier because it concerns an area of research in which he and

his colleagues made pioneering contributions. It was with great

sadness that I delivered it at a symposium dedicated to his memory

rather than in his presence as originally planned. With Harry

Collier's death, some of us have lost a good friend, and all of us

have lost a clarifying and civilizing influence in our work. Harry

Collier's contributions to the understanding of the relationships

between the acute and chronic actions of opiates and the production

of cyclic AMP are relatively well known and will be referred to in

the body of this paper. His contributions to the understanding of

the mechanisms of drug receptor action in general go much deeper

and are, perhaps, even more profound and original. In 1972, at the

International Pharmacology Congress in San Francisco, he predicted

the existence of the endogenous opiates, long before most of the

rest of us were thinking in such terms: "... the receptor for a

foreign drug is really the receptor for a humoral substance, with

which the foreign molecule also interacts. . . . . we do not yet know

the natural function of the macromolecule with which morphine

specifically interacts." (Collier 1973). In only 3 years the discovery of the enkephalins proved the correctness of this speculation

(Hughes et al. 1975). If we were all to emulate the precision in

thought and in language that Harry Collier demanded so successfully

of himself, our studies of tolerance and dependence would undoubtedly progress much faster.

One well-established biochemical mechanism by which opiates exert

their acute effects is by inhibition of adenylate cyclase in neuronal cell membranes. This action of opiates was first described

by Collier and Roy (1974), and has since been demonstrated in a

variety of systems including both cultured cells (Sharma et al.

1975), and, more recently, brain membranes (Wilkening et al. 1976;

Law et al. 1981; Neff et al. 1981; Cooper et al. 1982). The immediate result of opiate-mediated inhibition of adenylate cyclase is a

decrease in cellular CAMP levels. As a direct consequence of this

109
decrease, the activity of CAMP-dependent protein kinase is diminished, so that many cellular proteins whose phosphorylation is

effected by this kinase will remain unphosphorylated. Thus, opiate

receptor occupancy is the first step in a cascade of processes

leading to a panoply of effects on neuronal metabolism and excitability. The existence of such a cascade provides a powerful

amplification mechanism in terms both of amounts and of rates.

A mechanism for tolerance and dependence is implicit in the above

formulation of acute opiate actions, as Collier and Roy realized in

their very first publication on the subject in 1974. Because

opiates inhibit adenylate cyclase and lower CAMP levels in their

acute actions, the homeostatic response of the cell to the chronic

presence of opiates might well be to see to it that adenylate

cyclase activity levels increase. Evidence that such a response

actually occurs in neuroblastoma x glioma hybrid, NG 108-15, cells

was supplied by the work of Sharma et al. (1975b) and of Traber et

al. (1975). The NG 108-15 cell line is richly endowed with opiate

receptors (Klee and Nirenberg 1974), and the adenylate cyclase

activity of such cells is inhibited by up to about 50% when these

receptors are occupied by opiate agonists (Sharma et al. 1975a).

After culture of these cells for 12 or more hours in the presence

of opiate agonists such as morphine (Sharma et al. 1975b) or enkephalin (Lampert et al. 1976), adenylate cyclase activity is increased. In these experiments enzyme activity was measured in the

presence of naloxone to insure reversal of inhibition by any opiates

remaining in the membranes of the washed cells. Interestingly, the

increase in activity found after culture in the presence of morphine

was such that, even though functional opiate receptors are present

in undiminished amount, CAMP levels in the cells were normal. Thus,

the cells had become tolerant to morphine, because they exhibited

normal metabolism, growth, and morphology in the presence of amounts

of morphine which greatly diminish the CAMP levels of control cells.

They are also dependent upon morphine in the sense that addition of

naloxone causes a dramatic rise in CAMP levels, a biochemical

analogy of the withdrawal syndrome.

The increased levels of adenylate cyclase activity of chronically

exposed cells are, in large part, reflections of changes in the

state of the enzyme rather than in its amount. Thus, when the

adenylate cyclase activities of control and opiate-treated cell

membranes are compared in the presence of sodium fluoride, or of

nonhydrolyzable GTP analogues, no differences are found (Sharma et

al. 1977). Because much, if not all, of the regulatory apparatus

for controlling adenylate cyclase activity is completely activated

under these conditions, changed levels of the catalytic component

of the enzyme should have resulted in changes in activity here too.

Furthermore, it was found that simultaneous treatment of the cells

with morphine and the protein synthesis inhibitor, cycloheximide,

still allows the expression of opiate-induced increases in adenylate cyclase activity. The detailed nature of the regulatory

110
changes taking place is an intriguing mystery, awaiting solution.

Some experiments aimed at clarifying this mechanism will be reported

below.

Important advances in the understanding of the mechanisms of receptor control of adenylate cyclase activity have been made in the

past few years. It had been known since the work of Rodbell et al.

(1971), that the presence of GTP is essential for hormone stimulation of adenylate cyclase. It is now clear that the GTP binding

protein Ns (also called Gs or G/F), which is the substrate for

cholera toxin catalyzed ADP-ribosylation, is the mediator of such

stimulation (Ross and Gilman 1980). Agonists coupled to the appropriate receptors, such as ß- adrenergic and glucagon, for example,

facilitate the interaction of GTP and Ns, and this complex (or a

component of it) activates the catalytic subunit of adenylate

cyclase. A second GTP binding protein Ni (sometimes called Gi)

mediates receptor-coupled inhibition of the enzyme. This protein

is the substrate for pertussis toxin catalyzed ADP-ribosylation, a

process which greatly diminishes the ability of inhibitory hormones, including opiates, to attenuate adenylate cyclase activity

(Katada and Ui 1979, 1982a, 1982b). These recent advances have laid

the foundation for a deeper understanding of opiate actions at the

molecular level.

Among the earliest observations made with NG 108-15 membranes was

the lack of opiate inhibition when measured in the presence of the

nonhydrolyzable GTP analogue, GppNHp (Sharma et al. 1975a). These

observations have recently been repeated with highly purified membranes. In such a system it was also possible to show that opiate

inhibition of adenylate cyclase is actually the reversal of GTP

stimulation. Such observations led to the inference that GTP

hydrolysis is a step necessary for opiate inhibition of adenylate

cyclase. Cassel and Selinger (1977) had already demonstrated that

a low Km-GTPase is associated with the adenylate cyclase of turkey

erythrocyte membranes. Using their methodology, Koski and Klee

(1981) showed that opiates stimulate a low Km-GTPase in NG 108-15

membranes. The GTPase stimulation is receptor mediated since it is

blocked by naloxone, is observed only in cells known to posess

opiate receptors, and the potencies of a series of opiate-stimulators of GTPase match exactly the same drugs inhibition of adenylate

cyclase. Perhaps more tellingly, even the efficacies (intrinsic or

maximal activities) of the opiates in the two types of assay match

very well (Koski et al. 1982). Thus, the relative amounts of GTPase

stimulation and of adenylate cyclase inhibition observed at saturating concentrations of opiates are very close to the same over the

range from apparently full agonists such as dalamid (D-Ala2, Met5 -

enkephalin amide) to the pure antagonist, naloxone (Table 1).

111
TABLE 1

Relative Efficacies of Opiates as Inhibitors of Adenylate Cyclase

and as Stimulators of GTPase in NG 108-15 Membranes*

OPIATE ADENYLATE CYCLASE GTPase

Dalamid

Etorphine

Morphine

Levorphanol

Diprenorphine

Nalorphine

Buprenorphine

Naltrexone

Naloxone

100

99

79

79

61

50

35

21

0

100

95

60

67

52

23

18

12

0

*Efficacy was determined by measuring the inhibition of adenylate

cyclase, or stimulation of GTPase produced by each opiate at concentrations high enough to be saturating. Data are expressed relative

to those of dalamid. Linear regression analysis of the data results

in a correlation coefficient of 0.94. Taken from Koski et al. (1982).

Opiate inhibition of adenylate cyclase requires the presence of both

Na+ and GTP as first shown by Blume et al. (1971). Importantly therefore, opiate stimulation of GTPase activity was also found to exhibit

a similarly absolute requirement for Na+ (Table 2). Surprisingly,

perhaps, the effect of Na+ is to inhibit GTPase (in the presence of

Mg2 + ) and opiates simply reverse this inhibition. These data are

strongly supportive of the proposal that opiate inhibition of adenylate cyclase is the direct result of its stimulation of a GTPase

component of the adenylate cyclase complex. Thus, in diagramatic

fashion: adenylate cyclase shuttles between two states, an active

one with bound GTP, and an inactive one with bound GDP. Stimulatory

receptors facilitate exchange of GTP for bound GDP, whereas inhibitory

exchange

Rs

CYCLASE-GTP CYCLASE-GDP

(active) Ri

(inactive)

hydrolysis

112
receptors, including those for opiates, stimulate the GTPase activity

which converts GTP, bound to the adenylate cyclase-N protein complex,

to GDP.

TABLE 2

Effects of Na+ and Mg2 +on Basal and Dalamid Stimulated GTPase

in NG 108-15 Membranes*

Salt Added GTPase Activity

Basal 1 μM Dalamid

none 64.8 ± 2.5 67.9 ± 3.4

1OOmM NaCl 63.1 ± 2.5 66.0 ± 3.6

5mM MgCl2 51.0 ± 2.5 63.0 ± 0.7

lOOmM NaCl + 5mM MgCl2 30.0 ± 1.5 64.8 ± 4.4

*GTPase is expressed as pmols of GTP hydrolyzed/min/mg protein.

Data are means ± S.E. for triplicate determinations. Taken from

Koski et al. (1982).

Because GTPase is an essential part of the adenylate cyclase mechanism and opiates stimulate its activity, it was natural to wonder

whether chronic exposure of cells to opiates modifies the activity

of the enzyme. Answering this question experimentally has been a

surprisingly difficult undertaking because of the unfortunate fact

that optimum conditions for the study of the GTPase require a relatively highly purified membrane. However, the stimulatory effects

of opiate pretreatment upon adenylate cyclase activity are largely

lost during the long times required to prepare such membranes.

Table 3 shows data obtained with membranes prepared from control

and opiate-treated NG 108-15 cells using a rapid preparative procedure to preserve some of the opiate-induced increase in adenylate

cyclase activity. As shown in the table, treatment of cells with

etorphine led to both increased adenylate cyclase activity and decreased GTPase activity. Neither of the structurally related

partial agonists tested affected either enzyme activity. These

data suggest that the chronic effects of opiates include a modification of Ni which lowers its GTPase activity in a manner analagous

to that of pertussis toxin (Klee et al. 1984). Interestingly, only

relatively pure agonists affect either adenylate cyclase or GTPase

activities.

113
TABLE 3

Chronic Opiate Effects on Adenylate Cyclase and GTPase*

Adenylate Cyclase GTPase

Control l00 ± 4 100 ± 3

Etorphine 132 ± 7 84 ± 9

Diprenorphine 111 ± 6 98 ± 8

Buprenorphine 111 ± 13 112 ± 17

*NG 108-15 cells were cultured for 48 hours in the presence of 1 μM

drug. Membrane suspensions were assayed in the presence of 100 μM

naloxone. Data are expressed as per cent of control ± S.E. of quadruplicate determinations.

Until recently, little attention has been paid to the quantitation

of opiate agonist efficacy or to the question of the relationship

between efficacy in acute experiments and the ability of a substance

to produce tolerance and dependence. However, the precipitation of

abstinence by partial agonists such as nalorphine, for example,

clearly indicates a need for a more quantitative approach to the

question. As already seen in Table 1, measurements of adenylate

cyclase inhibition (or GTPase stimulation) by opiates at saturating

concentrations of drug provide a reliable quantitative index of

agonist efficacy. Because of the greater precision of adenylate

cyclase assays, we prefer to base efficacy determinations on them.

One immediately striking aspect of the data presented in Table 1 is

that morphine is clearly a partial agonist, with less than 80% the

efficacy of dalamid. In assessing the significance of this fact,

it is important to remember that efficacy is defined so as to be

independent of potency (the concentrations at which drugs bind to

receptors or exert other actions) since it is based upon measurements made at saturating levels of the drugs.

Because morphine is a partial, rather than full, opiate agonist,

much of the literature in which it was used as the inducer of

tolerance and dependence may need to be reexamined. As a case in

point, consider the effects of chronic opiate treatment upon receptor numbers. It had been quite well established that chronic morphine treatment has no measureable effect upon receptor numbers or

specificity, both in animals (Klee and Streaty 1974; Hollt et al.

1975) and in NG 108-15 cells (Sharma et al. 1975a; Simantov et al.

1982). Recently it has become clear that agents such as the

114
enkephalins and etorphine effect dramatic decreases in the number

of opiate receptors present in neuroblastoma cells (Simantov et al.

1982) and that chronic naloxone administration can result in an

increased number of opiate receptors in rat brain (Zukin, this

volume). Clearly, opiate receptor numbers can be regulated by the

prolonged presence of some agonists but not by others. We asked

whether there is a relationship between agonist efficacy and receptor down-regulation in NG 108-15 cells by exposing them to each of

a series of opiates of defined efficacy for 48 hours and subsequently measuring receptor numbers. Following the opiate treatment, the

cells were washed and homogenized and the membranes were washed

further after exposure to GppNHp and Na+ at 37° in order to maximize

dissociation of the bound opiate. Receptor numbers were then measured by studying 3

[H]-dalamid binding over a range of concentrations between 1 and 40 nM. Scatchard analysis of the data gave the

results listed in Table 4. The results show that opiates with

efficacies greater than 90% that of dalamid, and which are therefore

considered to be full agonists, lead to marked receptor down-regulation. Enkephalin is apparantly somewhat weak in this respect, but

its concentration is greatly diminished during the experiment by

proteolysis (Lampert et al. 1976). Opiates, including morphine,

with efficacies less than 80% that of dalamid do not produce significant down-regulation. Because chronic morphine administration

leads to an increased adenylate cyclase activity and no receptor

down-regulation, the two may be completely independent manifestations of chronic drug treatment.

Opiate

None

Dalamid

Phenazocine

Etorphine

Enkephalin

Butorphanol

Naloxone

Diprenorphine

Morphine

Buprenorphine

TABLE 4

Opiate Regulation of Receptor Numbers

Receptor Numbers Remaining

% of control

100 (9)

9 (2)

18 (2)

21 (4)

55 (4)

76 (1)

83 (2)

Efficacy

%

100

96

100

100

72

0

96 (4) 58

96 (4) 79

123 (4) 35

115
It is of some interest that chronic morphine administration to

animals does not lead to a loss of receptors. The implication of

these findings is that the partial agonist character of morphine is

not limited to the delta receptors of NG 108-15 cells, but applies

to both the mu and delta populations of rat brain as well.

REFERENCES

Although we have progressed in our understanding of the roles of

CAMP metabolism in both the acute and chronic actions of opiates,

much more remains to be done. The detailed mechanisms involved in

these actions are not yet completely defined, at least in part because of gaps in our understanding of the structure and function of

the adenylate cyclase complex. Furthermore, evidence is mounting

to show that several mechanisms may be operating in parallel during

the development of tolerance and dependence. Both the efficacy and

potency of the drug may determine which of the pathways is predominantly followed.

Much of this paper has been concerned with tolerance and dependence

in a cultured cell line of neuronal origin. To what extent are

these results and concepts applicable to animals and man? This

question does not have a simple answer. There is no reason to

believe that the changes seen with NG 108-15 cells subsequent to

opiate treatment do not also occur in brain. In fact some observations made with rats suggest that just as with NG 108-15 cells:

"...adenylate cyclase activity is enhanced in opiate dependence."

(Collier at al. 1975).

Blume, A.J., Lichtshtein, D., and Boone, G. Coupling of opiate receptors to adenylate cyclase: requirement for Na+

 and GTP. Proc

Natl Acad Sci USA 76:5626-5630, 1979.

Cassel, D., and Selinger, Z. Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes. Biochim Biophys Acta

452:538-551, 1976.

Collier, H.O.J. Pharmacological mechanisms of drug dependence. In:

Cochin, J., and Way. E.L., eds. Proc 5th Int Congr Pharmacol

San Francisco, Vol. 1. Karger, Basel, 1973. pp 65-76.

Collier, H.O.J., and Roy, A.C. Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain

homogenate. Nature 248:24-27, 1974.

Collier, H.O.J., Francis, D.L., McDonald-Gibson, W.J., Roy, A.C.,

and Saaed, S.A. Prostaglandins, cyclic AMP and the mechanism of

opiate dependence. Life Sci 17:85-90, 1975.

Cooper, D.M.F., Londos, C., Gill, D.L., and Rodbell, M. Opiate receptor-mediated inhibition of adenylate cyclase in rat striatal

plasma membranes. J. Neurochem 38:1164-1167, 1982.

Hollt, V., Dum, J., Blasig, J., Schubert, P., and Herz, A. Comparison of in vivo and in vitro parameters of opiate receptor binding

in naive and tolerant/dependent rodents. Life Sci 16:1823-1828,

1975.

116
Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L., Morgan,

B., and Morris, H.R. Identification of two related pentapeptides

from the brain with potent opiate agonist activity. Nature

258:577-579, 1975.

Katada, T., and Ui, M. Islet-activating protein: enhanced insulin

secretion and cyclic AMP accumulation in pancreatic islets due

to activation of native calcium ionophores. J Biol Chem 254:469-

479, 1979.

Katada, T., and Ui, M. ADP-ribosylation of the specific membrane

protein of C6 cells by islet-activating protein associated with

modification of adenylate cyclase activity. J Biol Chem 257:

7210-7216, 1982.

Katada, T., and Ui, M. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci USA 79:

3129-3133, 1982.

Klee, W.A., and Nirenberg, M. A neuroblastoma x glioma hybrid cell

line with morphine receptors. Proc Natl Acad Sci USA 71:3474-

3477, 1974.

Klee, W.A., Koski, G., Tocque, B., and Simonds. W.F. On the mechanism

of receptor-mediated inhibition of adenylate cyclase. Advances in

Cyclic Nucleotide and Phosphoprotein Research 17:153-159, 1984.

Klee, W.A.. and Streaty. R.A. Narcotic receptor sites in morphinedependent rats. Nature 248:61-63, 1974.

Koski, G., and Klee, W.A. Opiates inhibit adenylate cyclase by

stimulating GTP hydrolysis. Proc Natl Acad Sci USA 78: 4185-

4189, 1981.

Koski, G., Streaty, R.A., and Klee, W.A. Modulation of sodium-sensitive GTPase by partial opiate agonist: an explanation for the

dual requirement for Na+ and GTP in inhibitory regulation of

adenylate cyclase. J Biol Chem 257: 14035-14040, 1982.

Lampert, A., Nirenberg, M., and Klee, W.A. Tolerance and dependence

evoked by an endogenous opiate peptide. Proc Natl Acad Sci USA

73:3165-3167, 1976.

Law, P.Y., Wu, J., Koehler, J.E., and Loh, H.H. Demonstration and

characterization of opiate inhibition of the striatal adenylate

cyclase. J Neurochem 36:1834-1846, 1981.

Neff, N.H., Parenti, M., Gentleman, S., and Olianas, M.C. Modulation

of dopamine receptors by opiates. In: Gessa, G.L. and Corsini

G.U. eds. Apomorphine and Other Dopaminomimetics. Vol 1. New

York: 1981. pp 193-200.

Rodbell, M., Birnbaumer, L., Pohl, S.L., and Krans, H.M. The

glucagon-sensitive adenyl cyclase system in plasma membranes of

rat liver. V. An obligatory role of guanyl nucleotides in

glucogon action. J Biol Chem 246:1877-1882, 1971.

Ross, E.M., and Gilman A.G. Biochemical properties of hormonesensitive adenylate cyclase. Ann Rev Biochem 49:533-564, 1980.

Sharma, S.K., Nirenberg, M., and Klee, W.A. Morphine receptors as

regulators of adenylate cyclase activity. Proc Natl Acad Sci

USA 72:590-594, 1975a.

117
Sharma, S.K., Klee, W.A., and Nirenberg, M. Dual regulation of

adenvlate cyclase accounts for narcotic dependence and tolerance.

Proc Natl Acad Sci USA 72:3092-3096, 1975b.

Sharma, S.K., Klee, W.A., and Nirenberg, M. Opiate-dependent modulation of adenylate cyclase Proc Natl Acad Sci USA 74:3365-3369,

1977.

Simantov, R., Baram, D., Levy, R., and Nadler, H. Enkephalin and

adrenergic receptors: evidence for both common and differentiable regulatory pathways and down-regulation of the enkephalin

receptor. Life Sci 31:1323-1326, 1982.

Traber, G., Gullis, R., and Hamprecht, B. Influence of opiates on

the levels of adenosine 3':5' cyclic monophosphate in neuroblastoma X glioma hybrid cells. Life Sci 16:1863-1868, 1975.

Wilkening, D., Mishra, R.K., and Makman, M.M. Effects of morphine

on dooamine-stimulated adenylate cvclase and on cyclic GMP

formation in primate brain amygdaloid nucleus. Life Sci 19:

1129-1138, 1976.

AUTHORS

Werner A. Klee, Ph.D.

Graeme Milligan, Ph.D.

William F. Simonds, M.D.

Bruno Tocque, Ph.D.

Laboratory of General and Comparative Biochemistry

National Institute of Mental Health

Bethesda, MD 20205

118
Mechanisms of Multiple Cellular

Adaptation Processes in Clonal

Cell Lines During Chronic Opiate

Treatment

Ping-Yee Law, Michael T. Griffin, and Horace H. Loh

Among the various in vitro models for studies of opiate action,

the clonal cell line, neuroblastoma x glioma NG 108-15 hybrid

cells, represents the simplest model of brain origin in which the

molecular basis of opiate action is being investigated. The

hybrid cells possess many neuronal properties, such as having

excitable membrane containing light and dense core vesicles;

also the hybrid cells can be synapsed onto striatal muscle cells

(Hamprecht 1977). Furthermore, the NG 108-15 cells possess a

homogeneous opiate delta receptor system (Chang et al. 1978) and

can synthesize opioid peptides (Glaser et al. 1983). The opiate

receptor is coupled to a well-defined second messenger system,

the adenylate cyclase. Opiate agonist inhibits the adenylate

cyclase activity (Sharma et al. 1975) and the agonist inhibition

requires the presence of Na+ and GTP (Blume et al. 1979). For

this delta receptor system, enkephalin and its analogs possess the

greatest potencies in regulating the adenylate cyclase activity

(Law et al. 1983). Most interestingly, the hybrid cells exhibit

adaptation processes in response to the chronic presence of opiates. Continuous exposure of the NG 108-15 cells to the agonists

resulted in a gradual loss of opiate ability to inhibit adenylate

cyclase activity. Eventually, the intracellular CAMP level in

the presence of opiate returns to that in the control cells when

the opiates are absent. At this point, addition of naloxone

results in an increase in adenylate cyclase activity. This

increase in the CAMP level in the hybrid cells could be the

biochemical correlate for the proposed increase in cyclic nucleotide's metabolism during opiate withdrawal in the animal. Hence,

the hybrid cells represent a good model for studies of the chronic opiate effect at the cellular level. In the current review,

we will examine the multiple cellular adaptation processes which

occur in the chronic presence of opiate agonists. The possible

mechanisms for these adaptation processes will be discussed.

MULTIPLE CELLULAR RESPONSES TO CHRONIC OPIATE TREATMENT

The ability of naloxone to increase the adenylate cyclase activity

in NG 108-15 cells after chronic opiate treatment led Sharma et al.

(1977) to conclude that the loss of opiate ability to inhibit aden119
ylate cyclase activity was due to the concomitant increase of

the enzyme’s activity. The opiate receptor remained coupled to

the adenylate cyclase after chronic agonist treatment. This hypothesis suggested that there was no desensitization of the

opiate receptor or uncoupling of receptor from adenylate cyclaee.

Loss of opiate activity, or tolerance to the opiate drugs in

NG 108-15 cells, was simply due to the increase in production of

CAMP. In this model, the heterologous desensitization of other

receptors which inhibit the adenylate cyclase activity in NC 108-

15 cells is predicted, i.e., chronic exposure of the hybrid cells

to opiate agonist would eliminate or, at least, attenuate the

ability of muscarinic or alpha2-adrenergic agonist to inhibit the

adenylate cyclase activity. However, when such experiments were

carried out, it could be shown that opiate receptor desensitization after chronic opiate treatment was homologous only (table

1). The ability of carbachol and norepinephrine to inhibit the

adenylate cyclase after 24 hrs of 10 nM etorphine treatment remained intact, even though the ability of etorphine to inhibit

the adenylate cyclase activity was completely abolished. This

observation does not support the notion of concomitant increase

in adenylate cyclase activity as the cause of the observed loss of

opiate activity in NC 108-15 cells.

There is other evidence to support the hypothesis that the opiate

receptor is indeed desensitized during chronic drug treatment.

If the model proposed by Sharma et al. (1977) is correct, then

treatment of an opiate drug would produce the loss of all opiate

ligands’ ability to regulate adenylate cyclase. As shown in

figure 1, treatment of the hybrid cells with opiate ligands with

various intrinsic activities and potencies produces dissimilar

responses. Treatment of the NG 108-15 cells with enkephalin or

its analogs or an agonist such as etorphine results in a complete

loss of D-Ala2-Met5-enkephalin’s (DAME) ability to inhibit adenylate cyclaee activity. However, treatment of the NG 108-15

cells with a partial agonist, such as cyclazocine or morphine,

though completely abolishing the partial agonist’s ability to

regulate the adenylate cyclase, did not completely abolish DAME’s

activity. In all these treatments, the magnitude of adenylate

cyclase activity increase was the same. Furthermore, concentration-dependent studies revealed that the concentration of drugs

required to elicit these two effects, the loss of opiate inhibition and the increase of adenylate cyclase activity, was not

identical. The concentration of etorphine to produce 50% loss of

opiate inhibition after 24 hrs of chronic treatment was determined to be 14.3±5.0 nM. This was considerably higher than the

concentration required to elicit the increase of adenylate cyclase activity. As shown in figure 2, at 1 nM etorphine, the

increase of adenylate cyclase after chronic treatment for 24 hrs

was already at its maximum.

The separation of the loss of opiate receptor activity and the

increase in the adenylate cyclase activity after chronic treatment could be further demonstrated by the use of another cell

120
TABLE 1. Chronic Etorphine Treatment on Receptors' Activities

in Neuroblastoma x Glioma NG 108-15 Hybrid Cells

Ligand-receptor binding

Receptor

Opiate,

5.9 nM 3H-diprenorphine

Muscarinic,

3H-QNB, 3.6 nM

2

-Adrenergic,

3H-Rauwolscine, 1.5 nM

Prostaglandin,

3H-PGE1

, 1.2 nM

Regulation of 3H-cAMP level

-Adrenergic,

norepinephrine

IC50, uM

n H

Maximal inhibition, %

Muscarinic, carbachol

IC50 uM

nH

Maximal inhibition, %

Prostaglandin, PGE1

EC50, uM

n H Maximal activity,

cpm/mg prot/20 min

Specific binding, fmole/mg protein

Control Chronic etorphine

596.0±18.7 200.0±12 (-66.3)

38.6±1.3 34.5±1.2 (-10.2)

59.9±6.5 62.6±4.6 (+7.9)

7.3±0.2

Control

6.8±0.4 (-6.5)

Chronic etorphine

2.08±0.36

1.50±0.17

50.0±l.9

1.92±0.50

1.40±0.16

41.0±2.3a

0.99±0.31

1.55±0.16

49.3±2.3

2.12±0.04a

0.90±0.10a

35.9±2.3a

0.103±0.018

1.15±0.23

0.108±0.022

1.16±0.16

2.96±0.04 x 1052.85±0.02 x 105

Neuroblastoma x glioma NC 108-15 hybrid cells were treated with 10

nM of etorphine for 24 hrs. Receptor binding was carried out with

membrane preparations from the hybrid cells. Carbachol- or norepinephrine-inhibition of PGE1

-stimulated production in chronically

treated cells was measured with 10 nM of etorphine present in

incubation mixture to prevent the spontaneous increase of cAMP due

to the absence of opiate agonist. The values presented are the

average ± S.E.M. of determinations from 3 cell samples. The

numbers in parentheses are the % changes in receptor binding after

chronic etorphine treatment.

a = p values <0.05 when compared with control.

121
FIGURE 1. The relationship between the intrinsic activity of

opiate ligands and opiate receptor desensitization in NG 108-15

cells. PGE1 (5 uM)-stimulated cAMP production in suspensions of

hybrid cells were inhibited by various concentrations of D-Ala2,

Met 5-enkephalin (DAME) in control cells (-0-0-) or in cells

treated chronically with opiates Hybrid cells were

treated for 24 hrs with (a) 5 nM DAME, (b) 1.0 uM Sandoz FK33824,

(c) 100 nM cyclazocine or (d) 1.0 uM naloxone. Control activities in the absence of added opiates were 2.3±0.09 x 10 cpm/

mg protein/ 20 min (n = 9).

line, neuroblastoma N 18TG2. In these neuroblastoma cells, one

of the parents of NG 108-15 hybrids, opiate agonist also inhibits

adenylate cyclase activity (Law et al. 1982). Chronic treatment

of this cell line with etorphine also produced loss of etorphine's ability to inhibit adenylate cyclase. However, addition

of naloxone after chronic treatment did not elicit an increase of

the intracellular CAMP level (figure 3). Hence, in this cell -

line, the loss of opiate activity after chronic treatment clearly

is not due to the concomitant increase of adenylate cyclase

activity.

During the chronic incubation of the neuroblastoma x glioma NG108-15 hybrid cells with opiates, a third cellular adaptation

process occurs. Incubation of the hybrid cells with opiate

122
FIGURE 2. Etorphine concentration-dependent desensitization of

the opiate receptor in NG 108-15 cells. Hybrid cells, cultured on

17 mm plates, were treated with various concentrations of etorphine for 24 hrs. PGE1

 (5 uM)-stimulated 3H-cAMP production was

determined in the presence of 100 uM naloxone (curve I) or in the

presence of 1 uM etorphine (curve II). Values represent the average ± standard error of the mean of 3 cell samples. Quadruplicate 17 mm plates were used in each treatment. The control value

for PGE -stimulated 3H-cAMP production was 1.52±0.06 x 105 cpm/mg

protein/ 10 min.

agonist resulted in a decrease in detectable opiate binding

sites from the cell's surface, i.e., receptor down-regulation.

It can be demonstrated that receptor desensitization is not due

to receptor down-regulation, for both opiate agonists and partial agonists could desensitize the receptor. However, only

opiate agonist could elicit receptor down-regulation. As shown

in table 2, after treating the hybrid cells with approximately

5 X the Kd value of various ligands, the decrease in the 3Hdiprenorphine binding, a reflection of receptor density, was

observed with ligands with intrinsic activities greater than

0.9. Some metabolically labile peptides did not elicit downregulation in normal growth medium, which contains fetal calf

serum. Replacement of the serum with chemically defined medium resulted in down-regulation by all opioid peptides following

chronic treatment. Opiate partial agonists, such as cyclazocine,

morphine, levorphanol and, and diprenorphine did not cause a reduction in 3H-diprenorphine binding. Cyclazocine, morphine and levorphanol could attenuate diprenorphine's ability to regulate

adenylate cyclases. Furthermore, concentration-dependent studies

123
FIGURE 3. Effect of the concentration of etorphine during preincubation (2.5 hrs) on PGE -stimulated cyclic AMP production. (a)

NG 108-15 cells (2.3 x 105 cells) were treated with various concentrations of etorphine for 2.5 hrs. Sixty min before the

assay, (

3H)adenine was introduced to the media containing varying

concentrations of etorphine. The amounts of radioactivity being

taken up by the cells in the presence and absence of etorphine were

identical. During the enzyme assay (10 min at 37°C), after

removal of (

3H)adenine from the preincubation mixture, 10 uM PGEl

+ 1 uM etorphine (curve I) or 10 uM PGE1 + 100 uM naloxone (curve

II) were added. (b) Neuroblastoma N 18TG2 cells were pretreated

with etorphine as described in (a). The number of cells present

in each assay was 9.5 x 105. Curve I represents the amount of 3Hcyclic AMP formed during the 10 min assay in the presence of 10

uM PGE1 + 1uM etorphine. Curve II represents the 3H cyclic AMP

formed in the presence of 10 uM PGE1 + 100 uM naloxone.

also revealed the differences in the concentration of drug required to elicit these two responses. The concentrations of

drugs to promote down-regulation are analogous to the drugs’ Ki

values while the concentrations to promote desensitization are

similar to the Kd values. Time-dependent studies also suggested

differences in receptor desensitization and receptor down-regulation.

Receptor desensitization occurs prior to the appearance of receptor down-regulation. Reappearance of opiate regulation of

adenylate cyclase activity precedes the reappearance of opiate

binding. Cycloheximide and tunicamycin both can prevent the

reappearance of the binding sites after chronic treatment, but

neither inhibitor prevents the desensitization of the opiate

regulation of the adenylate cyclase. Therefore, it can be concluded that the receptor desensitization and receptor down-regulation are two separate cellular adaptation processes during chronic opiate treatment. It can be concluded, also, that there are

multiple cellular processes in response to the presence of the

opiate agonist.

124
TABLE 2. Chronic Treatment of NG 108-15 Hybrid Cells with Opiate

Alkaloids and Opiate Peptides: Relationship Between Intrinsic

Activity and Ability to Down-Regulate the Opiate Receptor

Opiate Alkaloids Intrinsic % Control

Incubation Concentration Activity a Binding

Etorphine, 10 nM 1.00 41.0±0.7

Ethylketocyclazocine. 500 nM 0.96 41.3±1.3

GPA1657, 500 nM 0.96 64.9±7.9

Cyclazocine, 50 nM

Diprenorphine, 20 nM

Levorphanol, 300 nM

Metazocine, 600 nM

Morphine, 100 uM

N-allylnormetazocine, 200 nM

Nalorphine, 300 nM

Oxymorphone, 400 nM

0.62 91.8±2.9

0.70 95.7±3.7 108±8.0*

0.65 96.8±6.7

0.69 105±3.4

0.69 98.2±6.3 92.6±4.7*

0.40 100±10 93.3±4.8

0.18 103±2.9*

0.68 98.1±2.9

GPA2163, 500 nM 0 98.7±7.7

Naloxone, 20 uM 0 100±5.7 95.8±4.1*

Opioid Peptides

Leu5-enkephalin, 20 nM

Met5-enkephalin, 20 nM

D-Ala2,Met5-enkephalin, 10 nM

D-Ala2,Met5-enkephalinamide.

15 nM

D-Ala2,D-Leu5-enkephalin, 5 nM

Sandoz FK33824

N-CH3-D-Ala2,D-Met5-enkephalin,

500 nM

N-CH3-D-Ala2,D-Leu5-enkephalin,

5 00 nM

Dynorphin 1-13, 300 nM

-Neo-endorphin, 100 nM

Bh-endorphin, 100 nM

0.94

0.95

0.98

0.98

0.92

1.01

0.94

0.94 52.7±4.7

0.96 83.9±1.9 65.1±2.7*

0.97 27.9±2.6*

0.99 65.2±3.2*

50.6±2.4*

47.6±5.8*

78.8±3.5 50.6±2.6"

53.3±8.9

51.5±8.9

41.4±3.7

50.4±4.9

NG108-15 hybrid cells were incubated with various opiates or opioid peptides for 24 hrs at 37°C. Concentrations of ligands were

approximately 5 x the Kd values for each ligand. Before assay,

identical concentrations of opiate ligands were added to control

plates and incubated at 37°C for 5 min, then washed free of ligands and incubated with 3H-diprenorphine to measure binding.

Control binding was 497.0±12.4 fmole/mg protein (n=39). Values

are the average ± standard error SEM from three cell samples.

* Denotes incubations were carried out in Sato medium (Law et al.

1983). a Intrinsic activities of opiate ligands were from Law et

al. 1983.

125
POSSIBLE MECHANISM OF RECEPTOR DESENSITIZATION

As discussed in the previous section, loss of the opiate agonist's ability to regulate adenylate cyclase activity after

chronic opiate treatment is due to the homologous desensitization of the opiate receptor. To facilitate understanding the

mechanism involved, possibly involving the uncoupling of the

binding sites from the effector, it is necessary to investigate

agonist binding properties in the control membrane. In other

receptor systems, in which the agonist regulates adenylate cyclase activity (e.g., the beta-adrenergic receptor) the ability of

agonist to induce the receptor to change to a high affinity state

is a prerequisite for activity. The intrinsic activities of various beta-adrenergic agonist correlates with the percent of the

receptor in the high affinity states after agonist binding (Lefkowitz et al. 1980). Because this high affinity state is a

measurement of coupling, desensitization of the receptors would

result in the decrease in the percent of agonist-induced formation of the high affinity states (Lefkowitz et al. 1980). This

results in an overall reduction of the agonist affinity for the

binding sites.

Apparently, opiate receptor desensitization follows such a pathway. Computer analysis of competition binding data indicated

that opiate binding sites in the NG 108-15 cells could undergo

changes which represent multiple affinity states (table 3). In

the presence of Na+ and guanine nucleotides, the two components

which are required for coupling, DADL could change the receptor

from a single affinity state in the presence of Mg+2 to show at

least two affinity states. The Kd of the high affinity sites

compare favorably with that of the Ki value of DADL to inhibit

adenylate cyclase. If this altered affinity is related to receptor coupling, then it should be agonist specific. Opiate antagonist naloxone competes with 3H-diprenorphine binding in a

single affinity manner, regardless of the condition of the binding

assays. As in the case of B-adrenergic receptor desensitization,

chronic opiate treatment of the NC 108-15 cells reduces the

percent of agonist-induced high affinity states. After exposing

the hybrid cells to 100 nM DADL for 3 hrs at 24°C, when there was

minimal receptor down-regulation but significant receptor desensitization, competition binding studies with DADL in the presence

of Na+ revealed a reduction of the percent of binding sites in

the high affinity states (figure 4). There was a decrease from 75%

of the total binding sites in the high affinity states (Kd = 2.5

nM) to 45% in the DADL treated membrane. This resulted in an

overall reduction of the DADL affinity constant from 12.5 nM to

269 nM. Thus, chronic treatment of hybrid cells with opiate

agonists results in the uncoupling of the binding sites from the

effector systems. What causes this uncoupling remains to be

elucidated.

RECEPTOR INTERNALIZATION AS THE CAUSE FOR RECEPTOR DOWN-REGULATION

Another cellular response to the chronic opiate treatment is the

disappearance of detectable binding sites from the cell surface.

126
In other polypeptide hormone receptor systems (e.g., insulin,

light-density-lipoprotein and epidermal growth factor), down-regulation of the receptor is due to the internalization of the

receptor (Brown et al. 1983). Receptor internalization usually

is accompanied by the internalization of the ligand. As a matter

of fact, Chang and his coworkers (Chang et al. 1982), using

neuroblastoma N4TG1, reported the accumulation of 3H-DADL within

the cells during chronic treatment with the radioactive peptides.

However, chronic treatment of the cell with 10 nM 3H-DADL did

TABLE 3. DADL Affinities for Opiate Receptor in NG 108-15 Cells

under Various Conditions.

DADL affinities were determined by computer analysis of competition binding experiments carried out with P2P3

 membrane preparations of NG 108-15 hybrid cells. Membranes equivalent to 0.25 mg

3H-diprenorphine, 2 nM. Binding assays were carried out at 24°C

protein were used per assay. Opiate receptor was labelled with

for 90 min.

not result in the accumulation of radioactivity. Rather, there

was a time-dependent decrease in the 3H-DADL associated with

the cells, which paralleled that of the 3H-diprenorphine binding

decrease. These observations suggested that either down-regulation of opiate receptors in NG 108-15 cells did not involve

receptor internalization or that internalization occurred without

accumulation of the opiate ligands. These two possibilities were

resolved by carrying out the chronic peptide exposure in the presence of chloroquine, a lysosomotropic agent. In the presence of

0.1 mM chloroquine, which inhibited the degradation process within

the lysosomes, accumulation of 3H-DADL within the lysosomes was

observed during chronic treatment. The magnitude of increase in

3H-DADL accumulated corresponded to the magnitude of receptor

down-regulated. The intracellular location of the isotope could

127
FIGURE 4. DADL affinities' alteration after chronic opiate

treatment. NG 108-15 cells treated with 100 nM DADL for 3 hrs at

24°C were harvested and excess DADL was removed by repeated washing of membrane preparations. 3H-Diprenorphine binding was carried out in the presence of various concentrations of DADL. Computer analysis of binding data was then carried out.

represents binding data with control membranes and represents chronic opiate-treated membrane.

KH = Kd for high affinity state

KL = Kd for low affinity state

RH = receptor in high affinity state

RL = receptor in low affinity state

be demonstrated by insensitivity toward trypsin treatment, inability of excess diprenorphine to displace the accumulated 3H-DADL,

and by the Percoll gradient fractionation of the subcellular particles. There was a time-dependent translocation of the radioactivity from the plasma membrane-enriched fractions to the lysosome-enriched fractions in the Percoll gradients (figure 5). The

translocation could be prevented by lowering the incubation

temperature during chronic treatment. The accumulated 3H-DADL

can be demonstrated to be associated with macromolecules by the

detergent extraction of the 3H-DADL-macromolecular complexes and

resolution of free ligand from the bound form using Sephadex G-50

chromatography. The accumulated 3H-DADL was released into the

128
medium upon removal of chloroquine and exhaustive washing of the

cells. Hence, it is concluded that during chronic opiate agonist

treatment, internalization of the ligand-receptor complexes occur

with the delivery of the complexes to the lysosomes, where degradation of the receptors occurs. The free ligands are then released into the medium.

The mechanism for this overall decrease of receptor level in

the cell surface is unknown. Although our chloroquine studies

indicated that the receptor internalizes, our cycloheximide and

tunicamycin studies indicated that the degradation, synthesis

and processing of the receptor were not affected by the presence

of agonist. Chronic opiate treatment of the hybrid cells must

then interfere with the normal recycling process of the opiate

receptor in order to elicit the observed down-regulation. At

which step of the life cycle

act remain to be determined?

of the receptor does the agonist

FIGURE 5. Time-dependent translocation of 3H-DADL from the plasma membrane to the lysosomes of NG 108-15 cells. Hybrid cells were

incubated with 10 nM 3H-DADLE in the presence of 0.1 mM chloroquine

for (a) 5 min, (c) 1 hr, (d) 2 hrs, (e) 4 hrs, and (f) 12

hrs. Percoll gradient fractionation of the homogenates (minus

nuclei) was carried out. Then, 0.65 ml fractions were collected

and radioactivity in each fraction was determined. In (e) and

(f), represents the Percoll gradient profile of the

homogenates from the hybrid cells treated with 10 nM 3H-DADL

chronically in the absence of chloroquine. In (b), the cells

were treated with 0.1 mM chloroquine for 4 hrs and exposed to

3H-DADL for 5 min at 37°C.

129
INVOLVEMENT OF CYTOSOLIC FACTORS IN THE INCREASE OF ADENYLATE

CYCLASE ACTIVITY

It has been suggested by Nirenberg and his coworkers that the

increase in adenylate cyclase activity after chronic opiate

treatment was not due to the de novo synthesis of adenylate

cyclase molecules, but rather due to the activation of the existing enzymatic molecule. Wilkening and Nirenberg (1980) suggested

that serum lipids or linoleic acid were required for the full

expression of this increased activity since the culturing of the

hybrid cells in delipidized serum reduced the magnitude of the

adenylate cyclase response, an effect which was reversed by the

addition of linoleic acid. We can demonstrate that selective

hydrolysis of the phospholipids with phospholipase C, but not

phospholipase A2, can attenuate the increase of the adenylate cyclase activity during chronic treatment. However, culturing hybrid cells in a serum-free chemically-defined medium did not

alter the magnitude of the increased response. Hence, it can be

concluded that the acyl side chains of the phospholipids did not

play a role in the increase of the adenylate cyclase activity

during chronic opiate treatment.

If an increase in membrane fluidity cannot account for the positive regulation of adenylate cyclase activity, then what cellular components could stimulate adenylate cyclase activity during

chronic treatment? One such candidate is calmodulin. It has

been reported that opiates can acutely decrease the amount of

calmodulin bound to the plasma membrane (Nehmed et al. 1982) of

the hybrid cells. Since the ability of calmodulin to stimulate

adenylate cyclase is well known (Brostrom et al. 1978), it is

possible that during chronic treatment, there is a retranslocation of the calmodulin. There is evidence to suggest that the

naloxone-induced increase in adenylate cyclase activity following

chronic opiate treatment may be calcium dependent. When the

experiments with intact cells were carried out in calcium free

medium, in the presence of EGTA, it could be demonstrated that

the increase in adenylate cyclase is attenuated. Furthermore,

addition of La+3, the calcium antagonist, completely abolished the

increase in adenylate cyclase activity (figure 6). When membrane

fragments are prepared from hybrid cells chronically treated with

opiates in the presence of EGTA, a reduction in the magnitude of

the increased adenylate cyclase activity is observed. Upon combining the cytosol preparations, there was a potentiation of the

increased activity (table 4). Intermixing of membranes isolated

from both control cells and opiate treated cells with cytosols

from these same two conditions revealed that maximal stimulation

of the adenylate cyclase was achieved when the chronic opiatetreated cytosol was added with the membranes isolated from cells

treated with opiates. Resolution of the cytosol preparations

with a Bio-Gel P6DG desalting column revealed two components

which stimulated adenylate cyclase. One component, which is at

the void volume of the column, representing a M.W. greater than

130
FIGURE 6. Effect of La+3 and Other Cations on Naloxone-Induced

Increase in Adenylate Cyclase Activity.

5,000, is calcium dependent (figure 7). The other component,

which has a molecular weight approximately equal to 300, is not

calcium dependent. We have determined that a second component

which stimulates adenylate cyclase activity is adenosine, since

its activity is inhibited by 1) an adenosine antagonist, 2)

desensitization of the cells to adenosine and 3) adenosine deaminase. During chronic opiate treatment, there was an apparent

increase in the adenosine level within the hybrid cells. Furthermore, the activity of adenosine and the first component, possibly

calmodulin, is synergistic in cells chronically treated with an

opiate. Hence, a model for the increase of the adenylate cyclase

could be formulated to involve these two components. One can

postulate that after chronic opiate treatment there is an increase

in the efficiency of coupling between stimulatory receptors such

as adenosine, and an increase in binding of calmodulin to the

adenylate cyclase molecules. Since phospholipase C could attenuate the increase, and phospholipase C could hydrolyze the polar

head groups which bind calcium, and since there are also reports

of alteration in ganglioside metabolism during chronic opiate

treatment (another calcium binding component), the increase in

131
the adenylate cyclase activity could be due to an alteration in

the membrane-bound calcium level in the plasma membrane. These

events must remain under the control of the opiate receptor,

since the increase in adenylate cyclase activity is only observed

upon the removal of opiate agonist. This hypothesis is currently

under investigation in our laboratory.

TABLE 4. Effect of Cytosolic Factors on Adenylate Cyclase

Activities Following Acute and Chronic Etorphine Treatment.

Adenylate Cyclase Activity (pmole/min/mg protein)

% Increase % Increase

Additions Over Control Chronic Over Control

to Membrane Control Membranes Etorphine Membranes

None 3.35±0.10 4.88±0.43 ± 45.8

Peak I

(control)

6.22±0.76 ± 85.9 8.89±0.76 ± 165.6

Peak II

(control)

9.01±1.05 ± 169.5 10.08±0.35 ± 201.0

Peak I + II 11.56±0.85

(control)

± 245.1 16.14±1.04 ± 381.9

Peak I 8.08±0.20 ± 141.2 10.85±0.65 ± 223.9

(etorphine)

Peak II 10.47±0.88 ± 212.8 12.74±0.05 ± 280.3

(etorphine)

Peak I ± II 180.2±1.96

(etorphine)

± 437.9 25.81±1.84 ± 670.7

Following vehicle or chronic etorphine treatment (100 nM, 16

hrs), cells were lysed and separated into membrane (control and

etorphine) and cytosol (control and etorphine) preparations.

Cytosol was further separated by column chromatography into two

fractions (Peak I and Peak II). Adenylate cyclase activity was

assayed in the presence of 100 nM etorphine + 400 uM naloxone and

contained equal equivalents of cytosol and membrane protein.

132
FIGURE 7. Adenylate cyclase activity profiles of cytosol from

chronic etorphine-treated cells following gel chromatography on a

Bio-Gel P-6DG desalting column (exclusion limit 6,000 daltons).

Hybrid cells were treated with 1 uM etorphine for 4 hrs and separated into cytosolic and membrane fractions. Following chromatography, equal portions of cytosol fractions were added back to

membrane fragments and adenylate cyclase was assayed in the

presence and absence of 0.5 mM Ca++

.

CONCLUDING REMARKS

From our current studies, it is clear to us that multiple cellular adaptation processes occur during chronic opiate treatment.

Thus far, we have identified three: receptor desensitization,

receptor down-regulation and the increase in adenylate cyclase

activity. This does not mean that these three processes are the

only ones. Because of the complexity of the cyclic nucleotide's

systems, it is possible that other cellular reactions which

involve the cyclic nucleotides, e.g., protein phosphorylation,

are also altered during chronic opiate treatment. A possible

mechanism for receptor desensitization, i.e., the uncoupling of

the receptor from the effector system, could be caused by a

phosphorylation or dephosphorylation of the receptor molecules.

Other cellular adaptation processes remain to be identified.

133
If one assumes that receptor desensitization and increase in

adenylate cyclase activity after chronic treatment are the biochemical correlates of tolerance development and the dependence

behavior (hyperactivity of the system), then from our current

data, it could be suggested that these two events are separate

cellular adaptation processes. The mechanisms involved are different and it is possible to induce one process without inducing

the other. Hence, probably, tolerance and dependence behavior in

animals are also two separate processes.

Although the neuroblastoma x glioma NG 108-15 hybrid cells have

proven to be an excellent model in studies of the molecular

basis of chronic opiate effects, one must realize its limitation. Unfortunately, most of the observations reported with

NG 108-15 cells have not been repeated with brain preparations.

Conceivably, because the hybrid cells are transformed cells,

there might be some unusual expression of chromosomal material.

However, one must also realize that the complexity of the cell

types in brain might mask some of the effects. For example,

not until recently, because of the relatively small inhibition

(20%), opiate inhibition of brain striatal adenylate has not been

reported. Thus, the failure to observe opiate effects in the

brain similar to those in the hybrid cells does not necessarily

invalidate the model. On the other hand, interpretation of the

results from the model should be restrained, also. If such

guidelines are followed, the studies with neuroblastoma x glioma

NG 108-15 hybrid cells can provide us with some excellent insights

into the molecular basis of chronic opiate action.

REFERENCES

Blume, A.J., Lichtstein, D., and Boone, G. Coupling of opiate

receptors to adenylate cyclase: requirement for Na+ and GTP.

Proc Natl Acad Sci USA 76:5626-5630, 1979.

Brostrom, M.A., Brostrom, C.O., Breckenridge, B.M. and Wolff,

D.L. Calcium-dependent regulation of brain adenylate cyclase.

Adv Cyclic Nucl Res, 9:85-99, 1978.

Brown, M.S., Anderson, R.G.W., and Goldstein, J.L. Recycling

receptors: the round-trip itinerary of migrant membrane proteins. Cell, 32:663-667, 1983.

Chang, K.J., Eckel, R.W., and Blanchard, S.G. Opioid peptides

induce reduction of enkephalin receptors in cultured neuroblastoma cells. Nature, 296:446-448, 1982.

Chang, K.J., Miller, R.J., and Cuatrecasas. P. Interaction

of enkephalin with opiate receptors in intact cells. Molec

Pharmacol, 14:961-970, 1978.

Glaser, T., Hubner, K., and Hamprecht, B. Neuroblastoma x

glioma hybrid cells synthesize enkephalin-like opioid peptides. J Neurochem, 39:59-69, 1983.

Hamprecht, B. Structural, electrophysiological, biochemical and

pharmacological properties of neuroblastoma x glioma cell hybrids in cell culture. Int Rev Cytol, 49:99-170, 1977.

134
Law, P.Y., Horn, D.A., and Loh, H.H. Opiate regulation of adenosine 3',5'-monophosphate level in neuroblastoma x glioma

NG108-15 hybrid cells - relationship between receptor occupancy and effect. Molec Pharmacol, 23:25-35, 1983.

Law, P.Y., Koehler, J.E., and Loh, H.H. Comparison of opiate

inhibition of adenylate cyclase activity in neuroblastoma

N 18TG2 and neuroblastoma x NG 108-15 hybrid cell lines. Molec

Pharmacol, 21: 483-491, 1982.

Lefkowitz, R.J., Wessels, M.R., and Stadel, J.M. Hormones,

receptors and cyclic AMP: their role in target cell refractoriness. Topics in Cellular Regulation, 17:205-230, 1980.

Nehmed, R., Nadler, H., and Simantov, R. Effects of acute and

chronic morphine treatment on calmodulin activity of rat brain.

Molec Pharmacol, 22:389-394, 1982.

Sharma, S.K., Klee, W.A., and Nirenberg, M. Opiate-dependent

modulation of adenylate cyclase. Proc Natl Acad Sci USA, 74:

3365-3369, 1977.

Sharma, S., Nirenberg, M., and Klee, W. Morphine receptor as

regulators of adenylate cyclase activity. Proc Natl Acad Sci

USA, 72:590-594, 1975.

Wilkening, D., and Nirenberg. M. Lipid requirement for long-lived

morphine-dependent activations of adenylate cyclase of neuroblastoma x glioma hybrid cells. J Neurochem, 34:321-326,

1980.

ACKNOWLEDGEMENTS

Supported in part by Grants DA-00564 and DA-01696 from the National Institute on Drug Abuse. H.H.L. is a recipient of a NIDA

Research Scientist Career Award, 5-K02-DA-70554.

AUTHORS

Ping-Yee Law, Ph.D., Michael T. Griffin, Ph.D., and Horace H.

Loh, Ph.D., Departments of Psychiatry and Pharmacology, School of

Medicine, University of California, San Francisco, CA 94143,

USA

135
The Opioid-Induced Desensitization

(Tolerance) in Neuroblastoma X

Glioma NG 108-15 Hybrid Cells:

Results from Receptor Uncoupling

Michael Wüster and Tommaso Costa

The phenomena of tolerance and physical dependence arising from

chronic opiate exposure have attracted the interest of numerous

investigators for many years, and several concepts for the underlying biochemical processes have been advanced (Collier 1980;

Goldstein and Goldstein 1968; Martin 1968; Shuster 1961). The

different theories, though diverging with respect to the precise

nature of the adaptive mechanisms, are in agreement that the

developnent of both tolerance and dependence is due to the same

substrate.

Investigations from this laboratory on the development of opioid

tolerance and dependence in isolated tissue preparations rejected

the idea of a common mechanims between these two phenomena (see

Schulz and Herz,this volume). The isolated mouse vas deferens,

offering a system where extremely high degrees of opioid tolerance

can be developed, does not show any sign of dependence, as evidenced by the absence of withdrawal signs after discontinuing opioid action, as well as the lack of increased sensitivity towards

excitatory compounds under these conditions (Gillan et al. 1979,

Schulz et al. 1980). Another classical tissue preparation used to

study opioid effects, the isolated guinea pig ileum preparation,

exhibits both tolerance and dependence on chronic opioid treatment. It is obvious, however, that a quantitative correlation of

both phenomena is missing; indeed, in the mouse vas deferens most

of the tolerance development occurs without a concomitant generation of dependence (Schulz et al. 1982, Wiister et al. 1982).

Electrophysiological investigations in the central nervous system

have revealed that the locus coeruleus slices, obtained from morphine-pretreated animals, display a significant degree of opioid

tolerance; again, no signs of dependence are detectable (Andrade

et al. 1983). Taken together, there are increasing indications

that opioid tolerance and dependence may be generated by distinct

adaptive processes; the underlying biochemical mechanisms for

these events, however, are still obscure.

These difficulties are due mainly to the extreme complexity of

neuronal networks in both the central and peripheral nervous system, which severely limits any investigation into the cellular

processes bywhichopioidreceptors exert a control on neuronal

136
activity. It is evident that such studies are test perfomed on in

vitro systems such as cultured cell clones; their availability has

facilitated numerous studies on the cellular regulation of the

intracellular second messenger system, adenylate cyclase. The

classical cell clone for investigations of opioid-receptor interactions is the mouse neuroblastoma x rat glioma hybrid cell line

NG 108-15 which has been extensively employed to study the cellular mechanisms of opioid-receptor mediated inhibitory effects

(see also W. Klee and H.H. Loh, this volume). Apart from the opioid

receptors, there are other inhibitory receptors (adrenergic,

cholinergic) that control cAMP-synthesis in these cells (Traber

et al. 1975a, Hamprecht 1977). Also, excitatory receptor systems

(prostaglandin, adenosine) have been demonstrated in this cell

line (Hamprecht 1977). Thus, a single pool of adenylate cyclase

is under multireceptor control (Sabol and Nirenberg 1979).

The experiments described here focus on the problem of whether

opioid-induced tolerance and dependence result from a common biochemical process within the opioid receptor system. There are a

number of questions of particular interest: 1. Is tolerance development towards opioid agonists associated with desensitization

towards the other prototype inhibitory compounds acting via different receptor types? The recent concept of receptor functioning

supports the existence of a common intermediate for excitatory

as well as inhibitory hormone receptors (N-unit, G/F protein),

(Rodbell 1980), but their significance for the adaptive processes

has not yet been investigated. 2. Is the development of subsensitivity towards opioids necessarily correlated with supersensitivity of adenylate cyclase towards excitatory hormones? Earlier investigations employing this cell clone have suggested that an increase

in adenylate cyclase activity as a consequence of chronic opioid

receptor activation is the common biochemical mechanism underlying

both tolerance and dependence (Sharma et al. 1975; Traber et al.

1975b). 3. What is the significance of the recent investigations

that revealed a dam-regulation of opioid binding sites in neuroblastma x glioma hybrid cells as a consequence of prolonged treatment with particular opioid agonists (Chang et al. 1982; Law et

al. 1982)? Receptor down-regulation could eventually explain a

phenomenon such as drug tolerance but not dependence.

Mouse neuroblastoma x rat glioma hybrid cells NG 108-15 were kindly

donated by Dr. B. Hamprecht, Würzburg, FRG, and cultured as described (Hamprecht 1977). Chronic drug treatment was performed

over a 24-hr period by the addition of different concentrations of

D-Ala2,D-Leu5-enkephalin (DADL), morphine or other opioid agonists

to the cell medium. Thereafter, cells were harvested and divided

into aliquots to test for the acute effects of DADL and other inhibitory compounds upon the PGEl-stimulated cAMP-formation. Care

was taken to maintain during the acute testing period the drug

concentration which was employed for the chronic treatment, in

order not to disturb the newly established equilibrium. Details

of the methods and materials employed are given elsewhere (Wüster

et al. 1983).

137
Figure 1. Effect of chronic DADL pretreatment upon the inhibition

of PGEl-stimulated CAMP accumulation by an acute DADL challenge.

Values are given as percent of the respective cAMP content obtained in the absence of the acute DADL. Each point is the mean

of three experiments performed in duplicate. SEM was generally

less than 10%.

DADL inhibits the PGEl-stimlated cAMP accumulation in control

cells by 75%. Pretreatment of cells for 24 hr with increasing

doses of DADL resulted in a dose-dependent decrease in the inhibitory effect of a subsequent acute opioid challenge (figure 1). It

was apparent that the pretreatment solely affected the maximal inhibition obtainable: the doses needed for a half-maximal effect

were not significantly different from controls. These findings

are at variance with results obtained in the intact animal and in

isolated tissue preparations such as the guinea pig ileum and the

mouse vas deferens, where drug tolerance is characterized by a

parallel shift of the dose-effect curve to the right. Drug tolerance has been explicitly defined as a state of the organism, where

"an increased dosage will again elicit the typical drug response"

(Goldstein 1974). The findings with the opioid-pretreated hybrid

cells obviously do not fit such a definition; it is appropriate,

therefore, to employ the term "desensitization" rather than "tolerance" for the observed effect. However, the correlation of the

desensitization observed in hybrid cells to the tolerance phenomena in intact nervous tissues is indicated by the fact that

138
careful analyses of tolerance phenomena have, in fact, revealed

that similar noncompetitive mechanisms also occur in the isolated

guinea pig ileum (Schulz, unpublished) and intact animals (Bläsig

et al. 1979).

Complete desensitization of NG 108-15 cells by DADL occurred with

a treatment of 100 nm DADL for 24 hr. Thus, doses which induce

a maximal inhibitory effect in naive cells also produce a maximal

desensitization after long-term exposure. This close correlation

between the dose necessary to produce maximal acute inhibition

and maximal desensitization was also confirmed using other types

of opioid agonists. Morphine and dynorphin A produced results

qualitatively identical with those of DADL; a concentration of

100 μM morphine and l μM dynorphin A induced a maximal inhibition

and a maximal desensitization. These experiments furthermore revealed a complete cross-desensitization between the different

prototypes of opioid ligands, supporting the notion that NG 108-15

cells contain a homogeneous opioid receptor population (Chang

et al. 1978; McLawhon et al. 1981).

Opioid-desensitized cells were also tested for dependence, as revealed by the ability of the opiate antagonist naloxone to precipitate a "withdrawal sign" in the form of elevated cAMP-levels.

However, these "withdrawal signs" were either highly variable or,

in highly desensitized preparations, not apparent. Indeed, a naloxone challenge affected only preparations which had not yet regained their initial (naive) cAMP levels, as the doses of DADL

or morphine were too small to induce a complete desensitization

within 24 hr. Thus, the activity of naloxone was attributed to

the reversal of a still persisting agonistic effect in these

preparations. A minor (< 20%) net overshoot (rebound effect) of

cAMP formation over control data was observed in some of these

experiments.

The specificity of opioid desensitization was investigated using

non-opioid compounds by testing their inhibitory effect on cAMPformation. The adrenergic agonist noradrenaline and cholinergic

agonist carbachol retained their potencies, irrespective of the

degree of desensitization which was displayed towards opioids

(figure 2). Thus, the opioid-induced desensitization is "specific"

and does not occur with a change of the inhibitory effects mediated via adrenergic or muscarinic receptors. Accepting that the

different inhibitory receptors control adenylate cyclase activity

through a uniform transducer component (Ni), these findings therefore reveal that the adaptive process is localized neither at the

level of the catalytic unit nor at the Ni-component.

Down-regulation of opioid binding sites within the cell membrane

has been demonstrated to occur after long-term DADL administration

(Chang et al. 1982; Law et al. 1982) and could explain an opioidspecific desensitization; however, the present data are at variance

with such a hypothesis. In contrast to effects seen with DADL,

chronic morphine treatment does not affect receptor density in

the cells (Chang et al. 1982), yet, the present investigation reveals an identical desensitization process. Furthermore, the DADL139
FIGURE 2. Effect of maximal inhibitory concentrations of DADL,

noradrenaline and carbachol on the PGEl-stimulated cAMP formation in controls, morphine and DADL-pretreated preparations. For

further information see legend to figure 1.

induced changes in opioid receptor density become apparent at

concentrations which are 10- to 100-fold higher than those required for desensitization.

In summary, these results draw attention to the functional integrity of the opioid binding protein as the point where the

opioid-induced adaptive processes occur. It is concluded, therefore, that the binding sites in these cells are still capable of

binding opioid ligands, but have lost their ability to function

properly in mediating agonistic effects to the subsequent N-units.

The concept that chronic opioid treatment of NG 108-15 hybrid

cells results in an uncoupling of the binding sites from Ni-units

has gained considerable support from our experiments employing a

recently available toxin from Bordetella pertussis (pertussis toxin,

islet activating protein, IAP). IAP has been shown to catalyze the

ADP-ribosylation of a specific, neuronal membrane protein with the

molecular weight of 41,000 daltons and this coincides with an

elimination of all inhibitory hormone effects upon adenylate

cyclase (Katada and Ui 1981; Hazeki and Ui 1981; Aktories et al.

1983; Hildebrandt et al. 1983). Thus, by analogy with excitatory

hormone effects being abolished by cholera toxin, pertussis toxin

140
FIGURE 3. Effect of pertussis toxin pretreatment upon the inhibition of PGE1-stimulated CAMP accumulation by DADL. For further

explanation, refer to legend of figure 1.

impairs the signal transmission at inhibitory hormone receptors

by permanently affecting the function of Ni (Katada and Ui 1982;

Hildebrandt et al. 1983).

Using methods similar to chronic opioid treatment, NG 108-15

hybrid cells were grown in the presence of increasing concentrations of pertussis toxin and tested for the ability of DADL to

inhibit PGE-stimulated cAMP formation (figure 3). As was observed

with chronic DADL treatment (figure 1), pertussis toxin dosedependently reduced the maximal inhibitory effect of DADL. With

1 ng x ml-l of toxin all opioid effects were completely abolished.

Concerning the inhibitory effects of adrenergic and muscarinic

agonists on cAMP accumulation, the results are identical with

those of DADL (data not sham). The mechanism underlying the pertussis toxin effects may be related to the particular modulations

which are exerted in the binding of opioid agonists, but not of

antagonists, in neuroblastoma x glioma hybrid cells (Costa et al.

1983).

When comparing the kinetics of pertussis-toxin induced desensitization (figure 3) with those produced by chronic opioid treatment

(figure 11, the similarity is striking. In both cases the curves

are superimposable: the maximal opioid effects are changed without

141
alterations of half-maximal effective concentrations. This indicates that both treatments are non competitive with respect to

the ligand inhibitory effect. This may suggest that although the

phenomena are induced in both cases at different sites and they

differ in selectivity- both treatments have a similar consequence:

regulatory proteins (Ni) have an impaired ability to interact with

the receptor binding sites and therefore fail to receive hormone

signals.

CONCLUSIONS

The results presented here may advance our understanding of adaptive processes occurring as a consequence of chronic opioid action.

On theoretical grounds, a unitary mechanism underlying both tolerance and dependence has been postulated for many years and there

are several studies in support of such a concept. Indeed a significant role for adenylate cyclase in the expression of opiate tolerance and dependence appeared indicated (Sharma et al. 1975). In recent years, however, an increasing number of reports have investigated the relationship of the development of opioid tolerance and

dependence in vitro: in several cases data from these studies revealed a pronounced dissociation of the two phenomena (Schulz et

al. 1980; Schulz et al. 1982; Wüster et al. 1982; Andrade et al.

1983). Accordingly, the present data on NG 108-15 hybrid cells

further support the notion of a development of opioid tolerance

(desensitization) separate from the development of dependence.

This hypothesis is strengthened considerably by the cross-tolerance

studies, which reveal an unaltered sensitivity of the adenylate

cyclase pool of opioid-pretreated cells towards non-opioid inhibitory compounds. An analogous investigation, employing the identical

cell clone, has reported similar observations on carbachol-pretreated preparations (Green and Clark 1982). Thus, the earlier

concept which assumed a counterregulatory hypertrophy of adenylate

cyclase (Sharma et al. 1975) is in need of reevaluation. Evidence

now indicates that chronic opioid receptor activation results from

an inability of the binding proteins to properly interact with the

subsequent N-units, thus failing to transmit its signal to the

catalytic units of adenylate cyclase.

In contrast to these in vitro observations, chronic opioid administration into an intact animal or humans clearly induces the development of both tolerance and dependence. Extrapolating the observations derived from model preparations such as the NG 108-15 hybrid

cells, it may be speculated that drug tolerance and dependence are

initiated by different biochemical mechanisms and are localized at

separate neuronal levels. Opioid tolerance in vivo may, indeed, be

produced by an uncoupling of the opioid binding sites, analogous

to the events in in vitro preparations. Drug dependence, on the

other hand, is displayed in intact nervous circuits, and may be,

for example, caused by a compensatory hypertrophy of any intra- or

extracellularly localized mechanism which is involved in signal

transmission within an opioid-affected nervous pathway. A prerequisite for the initiation of such a hypertrophy, however, is a

continuing opioid inhibitory effect. Consquently, adaptation to142
wards opioids within the complexity of an intact nervous system may

be characterized by the simultaneous uncoupling of particular

opioid receptor populations, resulting in the phenomenon of drug

tolerance, whereas others remain coupled. Their persisting inhibitory effect generates a hypersensitivity within the inhibited

pathway, an event which gives rise to withdrawal symptoms upon

cessation of the opioid inhibitory effect.

ACKNOWLEDGEMENTS

We are grateful to Dr. B. Hamprecht, Würzburg, for the neuroblastoma x glioma hybrid clone; to Dr. H. Glossman, Gießen, for

the CAMP antibody; and to Drs. K. Aktories, K.H. Jacobs, and G.

Schultz, Heidelberg, for the generous supply of pertussis toxin.

The assistance of Ms. U. Bäuerle in cell culturing and S. John

and U. Reuss in conducting experiments is very much acknowledged.

Supported by the Deutsche Forschungsgemeinschaft, Bonn.

REFERENCES

Aktories, K.; Schultz, G.; and Jakobs, K.H. Islet activating protein prevents nicotinic acid-induced GTPase stimulation and GTP

but not GTP's-induced adenylate cyclase inhibition in rat

adipocytes. Febs Lett, 156:88-92, 1983.

Andrade, R.; van der Maelen, C.P.; and Aghajanian, G.K. Morphine

tolerance and dependence in the locus coeruleus: Single cell

studies in brain-slices. Europ J Pharmacol, 91:161-169, 1983.

Bläsig, J.; Meyer, G.; Höllt, V.; Hengstenberg, J.; Dum, J; and

Herz, A. Non-competitive nature of the antagonistic mechanisms

responsible for tolerance development to opiate induced

analgesia. Neuropharmacology, 18:473-481, 1979.

Collier, H.O.J. Cellular site of opiate dependence. Nature 283:

625-629, 1980.

Chang, K.-J.; Miller, R.J.; and Cuatrecasas, P. Interaction of

enkephalin with opiate receptors in intact cultured cells.

Mol Pharmacol 14:961-970, 1978.

Chang, K.-J.: Eckel, R.W.; and Blanchard, S.G. Opioid peptides

induce reduction of enkephalin receptors in cultured neuroblastoma cells. Nature 296:446-448, 1982.

Costa, T.; Aktories, K.; Schultz, G.; and Wüster, M. Pertussis

toxin decreases opiate receptor binding and adenylate cyclase

inhibition in a neuroblastoma x glioma hybrid cell line.

Life Sci 33:Suppl. 1, 219-222, 1983.

Gillan, M.G.C.; Kosterlitz, H.W.; Robson, L.E.; and Waterfield,

A.A. The inhibitory effects of presynaptic alpha-adrenoceptor

agonists on contractions of guinea-pig ileum and mouse vas

deferens in the morphine-dependent and withdrawn states produced

in vitro. Br J Pharmac 66:601-608, 1979.

Goldstein, A., and Goldstein, D.B. Enzyme expansion theory of

drug tolerance and physical dependence. Res Publ Assoc Res Nerv

Ment Dis 46:265-267, 1968.

Goldstein, A. Drug tolerance and physical dependence. In:

Principles of Drug Action, A. Goldstein, L. Aronow and S.M.

Kalman, eds. New York: Harper and Row, 1974. pp. 569-621.

143
Green, D.A., and Clark, R.B. Specific muscarinic-cholinergic desensitization in the neuroblastoma-glioma hybrid NG 108-15.

J Neurochem, 39:1125-1131, 1982.

Hamprecht, B. Structural, electrophysiological, biochemical and

pharmacological properties of neuroblastoma-glioma cell hybrids

in cell culture. Int Rev Cyt 49:99-170, 1977.

Hazeki, O., and Ui, M. Modification by islet-activating protein of

receptor-mediated regulation of cyclic AMP accumulation in isolated rat heart cells. J Biol Chem 256:2856-2862, 1981.

Hildebrandt, J.D.; Sekura, R.D.; Codina, J.; Iyengar, R.; Manclark,

C.R.; and Birnbaumer, L. Stimulation and inhibition of adenylyl

cyclases mediated by distinct regulatory proteins. Nature 302:

706-709, 1983.

Katada, T., and Ui, M. Islet activating protein. J Biol Chem 256:

8310-8317, 1981.

Katada, T., and Ui, M. ADP ribosylation of the specific membrane

protein of C6 cells by islet-activating protein associated with

modification of adenyiate cyclase activity. J Biol Chem 257:

7210-7216, 1982.

Law, P.Y.; Hom, D.S.; and Loh, H.H. Loss of opiate receptor

activity in neuroblastoma x glioma NG 108-15 hybrid cells after

chronic opiate treatment: A Multiple-step process. Mol Pharmacol

22:1-4, 1982.

Martin, W.R. A homeostatic and redundancy theory of tolerance to

and dependence on narcotic analgesics. Res Publ Assoc Res Nerv

Ment Dis 46:206-223, 1968.

McLawhon, R.W.; West, Jr., R.E.; Miller, R.J.; and Dawson, G.

Distinct high-affinity binding sites for benzomorphan drugs and

enkephalin in a neuroblastoma - brain hybrid cell line. Proc

Natl Acad Sci 78:4309-4313, 1981.

Rodbell, M. The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 284:17-22, 1980.

Sabol, S.L., and Nirenberg, M. Regulation of adenylate cyclase of

neuroblastoma x glioma hybrid cells by adrenergic receptors.

J Biol Chem. 254:1913-1920, 1979.

Schulz, R.; Seidl, E.M.; Wüster, M.; and Herz, A. Opioid dependence

and cross-dependence in the isolated guinea-pig ileum. Europ

J Pharmacol 84:33-40, 1982.

Schulz, R.; Wüster, M.; Krenss, H.; and Herz, A. Selective development of tolerance without dependence in multiple opiate receptors

of mouse vas deferens. Nature 285:242-243, 1980.

Sharma, S.K.; Klee, W.A.; and Nirenberg, M. Dual regulation of

adenylate cyclase accounts for narwtic dependence and tolerance.

Proc Natl Acad Sci 72:3092-3096, 1975.

Shuster, L. Repression and depression of enzyme synthesis as

a possible explanation of some aspects of drug action. Nature

189:314-315, 1961.

Traber, J.; Fischer, K.; Buchen, C.; and Hamprecht, B. Muscarinic

response to acetylcholine in neuroblastoma x glioma hybrid cells.

Nature 255:558-560, 1975a.

Traber, J.; Gullis, R.; and Hamprecht, B. Influence of opiates

on the levels of adenosine 3':5'-cyclic monophosphate in neuroblastoma x glioma hybrid cells. Life Sci 16:1863-1868, 1975b.

144
Mister, M.; Schulz, R.; and Herz, A. The development of opiate

tolerance may dissociate from dependence. Life Sci 31:1695-

1698, 1982.

Wüster, M.; Costa, T.; and Gramsch, Ch. Uncoupling of receptors

is essential for opiate desensitization (tolerance) in neuroblastoma x glioma hybrid cells NG 108-15. Life Sci 33:Suppl 1,

341-344, 1983.

Dr. Michael Wüster

Dr. Tommasco Costa

Department of Neuropharmacology, Max-Planck-Institut für Psychiatrie, Am Klopferspitz 18 A, 8033 Planegg-Martinsried, FRG.

145
Opiate Receptor Upregulation and

Functional Supersensitivity

R. Suzanne Zukin, Ann Tempel, and Eliot L. Gardner

Opiate narcotic analgesics are well known to produce tolerance

and dependence in vivo and desensitization in vitro. The observation of these phenomena originally raised the question as to

whether opiate receptor populations undergo up- or downregulation in response to long-term administration of opiate

drugs. Drug- and lesion-induced receptor up-regulation were

well documented in other neurotransmitter systems. Snyder and

coworkers (Burt et al. 1977; Creese et al. 1977) investigated

[

3

H]halaperidol binding. They found that chronic treatment with

the influence of denervation and functional dopamine blockade on

the neuroleptic drugs haloperidol or fluphenazine elicits a 25%

increase in striatal dopamine receptors. In addition, they

showed that 6-hydroxydopamine-induced lesion of the nigrostriatal tract results in enhanced apomorphine-induced rotational

behavior which is reflected in increased numbers of [

3

H]haloperidol binding sites in rat striatum.

Although the evidence for behavioral and biochemical supersensitivity in the CNS was greatest in catecholaminergic pathways, the clinical problems presented by opiate tolerance and

dependence made the endogenous opioid system an obvious target

for research into molecular mechanisms of neural plasticity.

Lesion-induced receptor up-regulation has been found in the

enkephalinergic system. Our laboratory studied the influence of

6-hydroxydopamine lesions of the substantia nigra on opiate

receptors of specific brain regions (Gardner et al. 1980). In

the case of lateral nigral lesions, enkephalin receptor nunber

was found to be significantly increased in the striatum and

amygdala on the lesioned side relative to that of the same

regions in the intact hemisphere.

Opiate receptor supersensitivity has also been induced by

chronic blockade of receptors with specific opiate antagonists.

In preliminary studies, Tang and Collins (1978) found that longterm treatment with naloxone resulted in enhanced morphineinduced analgesia. Because naloxone was rapidly metabolized, it

146
was necessary to prepare rats with indwelling venous cannulas.

Lahti and Collins (1978) determined that the enhanced analgesia

correlated with an increased number of [

3

H]naloxone binding

sites. Almost simultaneously, Schulz et al. (1979) reported

that guinea pigs exposed to naloxone for 1-2 weeks by implantation with naloxone pellets showed increased sensitivity to the

inhibitory properties of opiates in the isolated ileum preparation. Chronic naloxone treatment also resulted in increased

[

3

H]etorphine binding in the ileum and brainstem of guinea pigs.

These studies were preliminary reports and as such did not

control for 1) possible changes induced in endogenous opioid

peptide levels that might give rise to apparent changes in

receptor densities and 2) the effects of acute antagonist administration.

In 1982 our laboratory published the first detailed study of

opiate receptor up-regulation (Zukin et al. 1982). A coordinated up-regulation of Mu and Delta opiate receptors was observed following chronic, but not acute, exposure to naltrexone.

No change in the density or other properties was observed in the

case of the Kappa or Sigma receptors (Tempel et al. 1982).

Chronic, but not acute administration of antagonist was shown to

elicit the up-regulation phenomenon. Thorough washing and a

37°C preincubation of the brain were shown to be crucial for

obtaining accurate results. These procedures facilitate the

dissociation of endogenous ligand and of residual naltrexone.

FIGURE 1. Scatchard analysis of 3H-etorphine binding to whole

brain homogenates from chronic naltrexone and control-treated

rats. Reprinted with permission from [Life Sciences, 31, A.

Tempel, R.S. Zukin, and E.L. Gardner, Supersensitivity of brain

opiate receptor subtypes after chronic naltrexone treatment]

copyright (1982), Pergamon Press, Ltd.

147
This chapter presents a review of 1) the molecular events

associated with opiate receptor up-regulation, 2) the nature of

opiate functional supersensitivity, 3) the neuroanatomical

patterns of receptor up-regulation as visualized by in vitro

autoradiography and 4) the neurochemical correlates of this

receptor phenomenon. These studies document both quantitatively

and visually an important example of neuronal plasticity. An

understanding of the molecular mechanisms underlying this upregulation phenomenon will hopefully shed light on the

mechanisms subserving opiate tolerance and dependence.

DEMONSTRATION OF OPIATE RECEPTOR UP-REGULATION BY IN VITRO

BINDING STUDIES

Naltrexone, a long-acting opiate antagonist, has been used in

clinical studies of heroin withdrawal. Our laboratory investigated the molecular correlates of chronic naltrexone administration (Zukin et al. 1982; Tempel et al. 1982). In vitro binding

studies of [3H]etorphine binding to whole rat brain homogenates

were carried out. We observed a 95% increase in opiate receptor

number with no change in receptor affinity in brain tissue from

naltrexone-treated rats relative to that from control (untreated) animals (figure 1). Half-maximal increase in receptor

number occurred at 4 days. Receptor number increased until 8

days, after which it leveled off (figure 2a). Withdrawal from

chronic naltrexone treatment resulted in a decrease from elevated receptor levels to nearly control receptor levels in a period

of about 6 days, as revealed by [

3

H]etorphine binding (figure

2b).

In order to determine whether the state of coupling of the

receptors undergoes a change, the sensitivity of opiate binding

to inhibition by guanyl nucleotides was examined. The newly

synthesized or unmasked receptors were shown to be more sensitive to guanyl nucleotide modulation. That finding suggested

that up-regulation is accompanied by an increased coupling of

the receptors to a guanyl nucleotide binding protein (Zukin

et al. 1982).

FUNCTIONAL SUPERSENSITIVITY

The observation of a pronounced up-regulation of opiate receptor

sites raised the question as to whether long-term treatment with

naltrexone also produced a behavioral supersensitivity to

morphine. Chronic naltrexone treatment was seen to result in

enhanced morphine-induced analgesia as determined by the flinchjump paradigm for measuring pain threshold (figure 3). Morphine

produced an increased pain threshold in a dose-dependent manner

as measured by this paradigm. After chronic naltrexone treatment, a leftward shift in the dose-response curve for morphine

was observed. This finding suggested that a behavioral supersensitivity develops following long-term administration of naltrexone. Following removal of the naltrexone pellets, morphine

supersensitivity decreased monotonically to pre-naltrexone

148
FIGURE 2. Time-course of the a) increase in brain opiate receptors following implantation of naltrexone

mini-pumps and b) decrease in brain opiate receptor number following withdrawal from chronic naltrexone

treatment.
FIGURE 3. Morphine-induced analgesia as a function of morphine dose in animals treated chronically with

naltrexone (8 days). Thresholds were determined a) 24 hours or b) 6 days after removal of the naltrxone.

Male Sprague-Dawley rats were used in the flinch-jump paradigm. Reprinted with permission fran [Journal of

Pharm. Exp. Ther., in press, A. Tempel, E.L. Gardner, and R.S. Zukin] copyright (1984), The American Society

for Pharmacology and Experimental Therapeutics.
treatment levels within 6 days. These behavioral data suggested

a functional significance for the naltrexone-induced opiate

receptor up-regulation, as the time course is well correlated

with that for the disappearance of opiate receptor up-regulation

following withdrawal from chronic naltrexone. These results

serve to confirm those of Tang and Collins (1978) and are consistent with the functional supersensitivity observed in the

dopaminergic system (Burt et al. 1977; Creese et al. 1977).

VISUALIZATION OF OPIATE RECEPTOR UP-REGULATION BY LIGHT

MICROSCOPY AUTORADIOGRAPHY

Preliminary studies, in which the effects of chronic naltrexone

blockade on opiate receptors were examined in dissected brain

regions, revealed a heterogeneous distribution of receptor

density changes. In order to visualize neuroanatanical patterns

of brain opiate receptor up-regulation, light microscopy

autoradiography was carried out on thaw-mounted frozen brain

sections from chronic naltrexone-treated and control rats

(Tempel et al. 1984). Figure 4 shows autoradiograms of coronal

sections through the telencephalic region. In the striatum

discrete patches of densely-labeled Mu opiate receptors were

observed, surrounded by areas of diffusely organized receptors.

Dense labeling was also observed along the subcallosal streak.

This topographical organization of receptors is similar to that

previously reported for the striatum (Atweh and Kuhar 1977). In

the case of brain exposed to chronic naltrexone (figure 4b),

receptor density in the striatal patches was markedly increased

relative to that of control brain (figure 4a), whereas the

diffusely labeled receptors of surrounding areas remained

essentially unchanged. Additional structures shown in these

autoradiograms which exhibited pronounced receptor density

changes were the dopamine-rich nucleus accumbens and lateral

septal nuclei (figure 4). In the neocortex dense labeling by

[

3

H]dihydromorphine (DHM) along molecular layers was observed as

had been previously reported (figure 4a) (Goodman and Snyder

1982). Whereas layers I and III showed significant increases in

receptor density after chronic naltrexone treatment (figure 4b) ,

layers II, IV, V, and VI remained unchanged.

Nuclei of the thalamus exhibited high densities of [3H]dihydromorphine (DHM) binding (figure 5). Long-term naltrexone administration (figure 5b) resulted in an increase in binding to the

posterior thalamic nucleus (PO), but produced no significant

change in binding to the lateral or medial ventroposterior

thalamic nuclei (VPL or VPM) relative to control brains (figure

5a). In the region of the hippocampus (figure 5) very dense

labeling of Mu receptors was seen to overlie the molecular

layers; diffuse labeling was observed in the surrounding areas.

Autoradiography (figure 5b) revealed antagonist-induced

increases in opiate receptor density closely opposed to the

pyramidal cells within the molecular layers of the hippocampus

(e.g. mossy fiber tract and CA3 area) but not in the dentate

gyrus; surrounding areas were not affected. This pattern is

151
FIGURE 4. Autoradiographs of [3

H] DHM binding in the telencephalic region of brains fran (a) a control

animal and (b) from a chronic naltrexone-treated animal. Note the specific regions of high grain density

relative to control brain in the lateral septum and nucleus accumbens. Opiate receptor patches of the

striatum showed significant increases in optical density while surrounding areas of the striatal patches did

not show changes in density. Abbreviations are: N. ACC = nucleus accumbens; LS = lateral septum; CP =

caudate putamen; NC = neocortex.
FIGURE 5. Autoradiographs of [

3

H] DHM binding tobrain sections from (a) control and (b) chronic

naltrexone-treated animals. Large increases in density were observed in the posterior thalamic nuclei of

naltrexone-treated brain (b) relative to control brain (a). Note the high grain density in the molecular

layers of the hippocampus of naltrexone-treated brain (b) relative to control brain (a). Abbreviations:

LP = lateral posterior thalamic nucleus; PO = posterior thalamic nucleus; VPL and VPM = lateral and medial

ventroposterior thalamic nucleus; NC = neocortex; HP = hippocampus.
similar to that observed for the striatum, in that only densely

organized Mu receptors were observed to undergo up-regulation.

Table 1 summarizes the quantification of opiate receptor density

changes following chronic naltrexone treatment throughout the

brain. Largest increases were observed in Layer I of the neocortex, the nucleus accumbens, the amygdala, the ventral tegmental area, the ventromedial hypothalamus, and the substantia

nigra compacta. Moderate increases were observed in Layer III

of the neocortex, the striatum, the lateral septum, the

posterior thalamic nucleus and the superior colliculus. No

significant changes in density were found in areas surrounding

the striatal patches, the medial septum, the ventral thalamic

nuclei and the substantia nigra reticulata. Opiate receptor

binding to the rat brainstem at the level of the locus coeruleus

revealed dense labeling in the parabrachial nuclei and the locus

coeruleus as previously reported (Herkenham and Pert 1982;

1980). Chronic naltrexone treatment produced significant

increases in density in the parabrachial nuclei (+39%), but no

significant change in the locus coeruleus, which contains almost

exclusively norepinephrine cell bodies (table 1). These results

document in a visual manner neuroanatomical patterns of opiate

receptor up-regulation.

NEUROCHEMICAL CORRELATES OF OPIATE RECEPTOR UP-REGULATION

In order to examine correlate patterns of peptide regulation,

methionine-enkephalin content in brain regions of naltrexonetreated and control animals was measured by radioimnunoassay.

The distribution of methionine-enkephalin throughout the brain

following long-term exposure to naltrexone is summarized in

table 2. Two dopamine-rich structures, the striatum and nucleus

accumbens, Showed significant increases in methionine-enkephalin

content after chronic naltrexone treatment (+94% and +40%, respectively; t-test, p<.05). Moderate increases were detected in

the periaqueductal gray and hypothalamic areas. Neccortex did

not show a significant change in methionine-enkephalin levels

after chronic naltrexone treatment.

Changes in the levels of other opioid peptides have also been

reported. Ragavan et al. (1983) found dramatic decreases in ßendorphin levels in rat brain following long-term administration

(36 days) of naltrexone (4 mg/kg). Preliminary findings showed

greatest decreases in the hypothalamus and amygdala with no

significant change in the periaqueductal gray.

Thus, we have shown that specific brain regions vary markedly

with respect to peptide and receptor density changes. Two

possible mechanisms to account for this finding are 1) that the

drug does not have uniform effects throughout the brain or 2)

that the receptors or endogenous peptides themselves may be

associated with specific functional pathways. In support of the

latter possibility, several functionally defined pathways were

found to show particularly large increases in opiate receptor

154
TABLE 1. Optical Density Measurements of Opiate Receptors in

Chronic Naltrexone vs. Control Treated Rat Brain

Brain sections were labeled in vitro with 5 nM [

3

H]DHM, and

exposed to tritium-sensitive film for 12 weeks. Optical density

readings were taken from at least three sections from each

structure per rat and averaged. Averages across rats are

expressed as average ± deviation.

Region Control Brain Naltrexone %

Treated Brain Change

(n=2) (n=2)

Neocortex

Layer I

Layer II

Subcallosal streak

Striatun

Patches

Other areas

Nucleus Accumbens

Septun

Lateral

Medial

Amygdala

Habenular Nuclei

Lateral

Medial

Thalamus

Post. Thal. Nuc.

Dorsal Thal. Nuc.

Ventral Thal. Nuc.

Hippocampus

Molecular layer

Other areas

Hypothalamus

Ventromedial

Central Gray

Superficial Gray Layer

of Superior Colliculus

Substantia Nigra

Compacta

Reticulata

Ventral Tegmental Area

Locus Coeruleus

Parabrachial Nucleus

.11 ± .04

.17 ± .03

.36 ± .06

.38 ± .ll

.22 ± .03

.29 ± .06

.16 ± .03

.28 ± .01

.23 ± .01

.18 ± .01

.28 ± .02

.33 ± .02

.46 ± .04

.14 ± .01

.34 ± .03

.19 ± .03

.12 ± .02

.20 ± .01

.27 ± .01

.10 ± .02

.21 ± .02

.12 ± .01

.24 ± .03

.39 ± .03

.22 ± .02

.27 ± .01

.31 ± .04

.55 ± .02 +45%

.26 ± .04 +18%

.55 ± .02 +90%

.29 ± .01

.26 ± .01

.51 ± .03

.16 ± .01

.53 ± .01

.57 ± .03

.61 ± .02

.13 ± .02

.51 ± .02

.23 ± .02

.45 ± .02

.42 ± .04

.50 ± .02

.30 ± .04

.21 ± .03

.45 ± .02

.21 ± 0.0

.54 ± .04

+loo%

+59%

--- (-14%)

+81%

--- ( -7%)

+122%

--- (-11%)

+89%

+73%

+33%

--- ( -7%)

+50%

+21%

+275%

+110%

+85%

+200%

+275%

--- (-13%)

+39%

155
density after chronic naltrexone treatment. One example is the

limbic system, consisting of the nucleus accumbens, the lateral

septal nucleus, various nuclei of the hypothalamus, and the

amygdala. These areas are known to contain dopamine as a neurotransmitter. There is, to date, considerable evidence supporting the notion of dopaminergic-enkephalinergic anatanical and

functional interactions (Gardner et al. 1980; Zukin and Zukin

1981; Goodman et al. 1980; Biggio et al. 1978). Several

laboratories have reported that opiates and opioid peptides

inhibit dopamine release from rat striatal slices in vitro

(Pollard et al. 1977; Schwartz et al. 1978; Celsen and

Kaschinsky 1974) a finding which suggests a possible modulatory

effect on dopamine-mediated behaviors. A second important

system which exhibits pronounced up-regulation is the

periaqueductal gray matter, thought to play a role in

nociception and analgesia (Loh et al. 1976; Sabramanian et al.

1977). A third system includes several of the major structures

which receive input fran afferent sensory pathways. These areas

include the superficial layers of the superior colliculus, the

molecular layers of the hippocampus, layers I and III of

neocortex and the parabrachial nuclei. All of these structures

are particularly rich in opiate receptors relative to other

areas (Atweh and Kuhar 1977). These findings are congruent with

suggestions that opiates may function as neuromodulators in the

CNS. When the system is challenged, as by chronic opiate

antagonist treatment, specific enkephalinergic pathways (i.e.,

limbic, pain, sensory pathways) respond by synthesizing or

unmasking new receptors. It is also possible that enkephalin

levels increase first and that the receptor changes are of a

compensatory nature.

Other examples of drug- and lesion-induced opiate receptor upregulation have also been reported. Recently, Simantov and Amir

(1983) showed that lesions of the hypothalamic arcuate nucleus

of the mouse, produced by neonatal application of monosodium

glutamate (MSG) (Hong et al. 1981; Krieger et al. 1979; and

Romagnano et al. 1982), increased the binding of [

3

H]

dihydromorphine to membranes prepared from the midbrain. These

MSG-treated mice also exhibited an enhanced response to morphine

and naltrexone regarding thermal pain sensitivity. The some

lesion in neonatal rats produced an increase only in the number

of Delta receptors in the thalamus (as measured by [

3

H]DADLE),

with no changes in mu receptors (Young et al. 1982). The

apparent discrepancy in these two studies may be due to an

interspecies difference. Bardo and coworkers found that rat

pups (ages 1-21 days) treated chronically with naltrexone

exhibited an increased number of opiate binding sites, but did

not demonstrate a functional supersensitivity to morphine (Bardo

et al. 1982). These studies underline the importance of

monitoring neurochemical correlates of chronic drug or food

additive administration.

In contrast to the case of opiate receptor up-regulation, opiate

receptor down-regulation has been difficult to document.

156
Several groups have reported that chronic administration of

narcotic agonists in vivo does not produce any change in either

receptor number or affinity (Holaday et al. 1982; Hitzemann et

al. 1974; Klee and Streaty 1974; Pert et al. 1973; Simon and

Hiller 1978; Perry et al. 1982; Bardo et al. 1982). More

recently, Holaday and coworkers reported 17-20% increase in

opiate receptor density following chronic morphine treatment.

Using a different approach, Davis and markers (1979) showed

that the development of tolerance to morphine is accompanied by

a reduction of opiate binding as measured by [3H]naloxone, [3H]

etorphine and [

3

H] morphine binding in the brain-stem slice

preparation. Homogenization was carried out after ligand

incubation in this preparation, a procedure which may be important for maintaining membrane-receptor integrity. More

recently, down-regulation has been observed in neurotumor cell

lines following long-term exposure to enkephalin (Chang et al.

1982: Hazum et al. 1981: Blanchard et al. 1983) but not to

alkaioid agonists (Chang et al. 1982). Possible interpretations

of the latter findings are 1) opioid peptides and opioid

narcotic agonists bird differently to the same receptor or 2)

only a subpopulation of opioid receptors can be so modulated.

These findings are discussed in more detail in another chapter

in this monograph by Law et al.

The question arises as to the molecular mechanisms underlying

opiate receptor up-regulation and functional supersensitivity.

One possible hypothesis is that active opiate receptors in the

absence of drug treatment exist in a dynamic equilibriun with a

nearly equal concentration of inactive, "silent" receptors.

Long-term exposure of the system to opiate antagonist leads to

the activation or unmasking of the "silent" receptors, giving

rise to an apparent up-regulation. Long-term exposure of the

system to opiate agonists or opioid peptides might lead to

coupling of the receptors to cyclase, followed by internalization. As active receptors disappear, these would be replaced by

the conversion of "silent" receptors to active ones. In the

case in which activation paralleled internalization, no apparent

change in receptor density would be observed. Only in the case

in which internalization exceeded reactivation (i.e. opioid

peptides) would apparent down-regulation occur. It is possible

to test aspects of this hypothesis by binding studies involving

an in vitro system. Explant cultures of spinal cord with

attached dorsal root ganglia provide a valuable in vitro system

for studies of such mechanisms. Those experiments are discussed

in another chapter in this monograph by Crain.

The overall significance of these studies lies in their ability

to shed light on the mechanisms subserving the phenomena of

opiate supersensitivity and tolerance and neuronal plasticity in

general. There are potential clinical ramifications for naltrexone-induced supersensitivity. For example, naltrexone is in

use as a treatment for opiate dependence (Resnick et al. 1974;

O'Brien et al. 1978). After cessation of naltrexone therapy in

humans, a resulting supersensitivity could be reflected in

157
TABLE 2. Regional Distribution of Met-Enkephalin After

Long-Term Exposure to Naltrexone

Methionine-enkephalin content was measured by radioimmunoassay.

Values are reported as means of 4 determinations from 4 individual

sets (1 naltrexone brain, 1 control brain) of rats.

Naltrexone-Treated Control %

(ng/mg Protein) (ng/mg Protein) Change

(n=4) (n=4)

Neocortex 10.0 ± 0.6 11.8 ± 1.5 -15%

Hypothalamus 9.5 ± 0.1 7.3 ± 0.8 +30%

Periaqueductal Gray 9.6 ± 0.3 7.6 ± 0.7 +26%

Nucleus Accumbens 11.6 ± 0.5 8.3 ± 0.8 *+40%

Striatum 18.4 ± 2.1 9.5 ± 1.0 *+94%

*Asterisks indicate statistically significant differences (t-test,

p < .05).

increased susceptibility to a dosage of heroin which would have

been well tolerated initially, and thus lead to an unintentional

overdose. A potential use of naltrexone-induced supersensitivity might be to enhance the analgesic effects of opiates prior

to surgery. The studies of the effects of long-term agonist

treatment on opiate receptors are important to our understanding

of the molecular basis of opiate tolerance and of receptor downregulation in general. Together these studies should contribute

significantly to our understanding of how the brain works in

regard to the regulation of enkephalinergic transmission and

receptors for the opioid peptides. Ultimately, studies with the

purified opiate receptor subtypes should enable a precise determination of the molecular mechanisms underlying brain receptor

regulation.

REFERENCES

Atweh, S.F., and Kuhar, M.J. Autoradiographic localization of

opiate receptors in rat brain. Brain Res, 129:1-12, 1977.

Bardo, M.T., Bhatnagar, R.K., and Gebhart, G.F. Differential

effects of chronic morphine and naloxone on opiate

receptors, monoamines and morphine-induced behaviors in

preweanling rats. Dev Brain Res, 4:139-147, 1982.

Biggio, G., Casu, M., Corda, M.G., Dibello, C., and Gessa,

G.L. Stimulation of dopamine synthesis in caudate nucleus

by intrastriatal enkephalins and antagonism by naloxone.

Science, 200:552-554, 1978.

158
Blanchard, S.G., Chang, K.-J, and Cuatrecasas, P. Characterizaticn of the association of tritiated enkephalin with

neuroblastana cells under conditions optimal for receptor

down regulation. J Biol Chem, 258: 1092-1097, 1983.

Burt, D., Creese, I., and Snyder, S. Antischizophrenic drugs:

chronic treatment elevates dopamine receptor binding in

brain. Science, 196:326-327, 1977.

Celsen, B., and Kaschinsky, K. Effects of morphine on kinetics

of 1 4C-dopamine in rat striatal slices. NaunynSchmiedeberg's Arch Pharmacol, 284:159-165, 1974.

Chang, K.-J., Eckel, R.W., and Blanchard, S.G. Opioid peptides

induce reduction of enkephalin receptors in cultured

neuroblastoma cells. Nature (London), 296:446-448, 1982.

Creese, I., Burt, D., and Snyder, S. Dopamine receptor binding

enhancement accanpanies lesion-induced behavioral supersensitivity. Science, 197:596-598, 1977.

Davis, N.E., Akera, T., and Brody, T.M. Reduction of opiate

binding to brainstem slices associated with the development

of tolerance to morphine in rats. J Pharm Exper Ther, 211

(1): 112-119, 1979.

Gardner, E.L., Zukin, R.S., and Makman, M. Modulation of opiate

receptor binding in striatun and amygdala by selective

mesencephalic lesions. Brain Research, 194:232-239, 1980.

Goodman, R.R., and Snyder, S.H. Opiate receptors localized by

autoradiography to deep layers of cerebral cortex: Relation

to sedative effects. Proc Natl Acad Sci USA, 79:5703-5707,

1982.

Goodman, R.R., Snyder, S.H., Kuhar, M.J., and Young III, W.S.

Differentiation of delta and mu opiate receptor localizations by light microscopic autoradiography. Proc Natl Acad

Sci USA, 77: 6239-6243, 1980.

Hauzm, E., Chang, K.-J., and Cuatrecasas, P. Receptor

redistribution induced by hormones and neurotransmitters:

Possible relationship to biological functions.

Neuropeptides, 1:217-230, 1981.

Herkenham, M., and Pert, C.B. A general autoradiographic method

which preserves tissue quality. J Neuroscience, 2:1129-

1149, 1982.

Herkenham, M., and Pert, C.B. In vitro autoradiography of

opiate receptors in rat brain suggests loci of "opiatergic"

pathways. Proc Natl Acad Sci USA, 77:5532-5536, 1980.

Hitzemann, R., Hitzemann, B., and Loh, H. Binding of [

3

H]

naloxone in the mouse brain: Effect of ions and tolerance

development. Life Sci, 14: 2393-2404, 1974.

Holaday, J.W., Hitzemann, R.J., Curell, J., Tortella, F.C., and

Belenky, G.L. Repeated electroconvulsive shock or chronic

morphine treatment increases the number of [

3

H] D-Ala2, DLeu5

-enkephalin binding sites in rat brain membranes. Life

Sci, 31: 2359-2362, 1982.

Hong, J.-S., Lowe, C., Squibb, R.E., and Lamartiniere, C.A.

Monosodium glutamate exposure in the neonate alters

hypothalamic and pituitary neuropeptide levels in the

adult. Regulat Pep, 2: 347-352, 1981.

159
Klee, W.A., and Streaty, R.A. Narcotic receptor sites in

morphine-dependent rats. Nature, 248:61-63, 1974.

Krieger, D.T., Liotta, A.S., Nicholsen, G., and Kizer, J.S.

Brain ACTH and endorphin reduced in rats with monosodium

glutamate-induced arcuate nuclear lesions. Nature

(London), 278:562-563, 1979.

Lahti, R., and Collins, R. Chronic naloxone results in

prolonged increases in opiate binding sites in brain. Eur

J Pharmacol, 51:185-186, 1978.

Loh, H.H., Brase, D.A., Sampath-Khanna, S., Mar, J.B., and Way,

E.L. ß-Endorphin in vitro inhibition of striatal dopamine

release. Nature (London), 264:567-568, 1976.

O'Brien, C., Greenstein, R., Ternes, J., and Woody, G. Treatment approaches: Opiate antagonists. Ann NY Acad Sci,

311:232-242, 1978.

Perry, D.C., Rosenbaum, J.S., and Sadee, W. In vitro binding of

[

3

H] etorphine in morphine-dependent rats. Life Sci,

31:1405-1408, 1982.

Pert, C.B., Pasternak, G.W., and Snyder, S.H. Opiate agonists

and antagonists discriminated by receptor binding in

brain. Science, 182: 1359-1361, 1973.

Pollard, H., Llorens-Cortes, C., and Schwartz, J.C. Enkephalin

receptors in dopaminergic neurons in rat striatun. Nature

(London), 268:745-747, 1977.

Ragavan, V.V., Wardlaw, S.L., Kreek, M.J., and Frantz, A.G.

Effect of chronic naltrexone and methadone administration

on brain ß-endorphin in the rat. Neuroendocrinoloqy,

37:266-268, 1983.

Resnick, R., Volavka, J., Freedman, A.M., and Thomas, M.

Studies of EN-1639A (naltrexone): A new narcotic

antagonist. Am J Psychiatry, 131:646, 1974.

Romagnano, M.A., Chapel, T.L., Pilcher, W.H., and Joseph, S.A.

The distribution of enkephalin in the mediobasal

hypothalamus of the mouse brain: Effects of neonatal

administration of MSG. Brain Research, 236:497-504, 1982.

Sabramanian, N., Mitznegg, P., Sprugel, W., Domschke, W.,

Domschke, S., Wunsch, E., and Demling, L. Influence of

enkephalin on K

+

-evoked efflux of putative

neurotransmitters in rat brain. Naunyn-Schmiedeberg's Arch

Pharmacol, 299:163-165, 1977.

Schulz, R., Wuster, M., and Herz, A. Supersensitivity to

opioids following chronic blockade of endorphin activity by

naloxone. Naunyn Schmiedeberg's Arch Pharmacol, 306:93-96,

1979.

Schwartz, J.J., Pollard, H., Llorens, C., Malfroy, B., Gross,

C., Pradelles, P.L., and Dray, F. In: Costa, E. and

Trabucci, M., eds., The Endorphins. N.Y.: 1978. Raven

Press, pp. 245-264.

Simantov, R., and Amir, S. Regulation of opiate receptors in

mouse brain: Arcuate nuclear lesion induces receptor upregulation and supersensitivity to opiates. Brain Res.,

262:168-171, 1983.

Simon, E.J., and Hiller, J.M. In vitro studies on opiate

receptors and their ligands. Fed Proc, 37:141-146, 1978.

160
Tang, A., and Collins, R. Enhanced analgesic effects of

morphine after chronic administration of naloxone in the

rat. Europ J Pharmacol, 47: 473-474, 1978.

Tempel, A., Zukin, R.S., and Gardner, E.L. Supersensitivity of

brain opiate receptor subtypes after chronic naltrexone

treatment. Life Sci, 31: 1401-1404, 1982.

Young, E., Olney, J., and Akil, H. Increase in delta, but not

mu receptors in MSG-treated rats. Life Sci, 31(12&13):

1343-1346, 1982.

Zukin, R.S., Sugarman, J.R., Fitz-Syage, M.L., Gardner, E.L.,

Zukin, S.R., and Gintzler, A.R. Naltrexone-induced opiate

receptor supersensitivity. Brain Research, 245:285-292,

1982.

Zukin, R.S., and Zukin, S.R. MiniReview: Multiple opiate

receptors: Emerging concepts. Life Sci, 29:2681-2690,

1981.

ACKNOWLEDGEMENT

This research was supported by National Science Foundation Grant

BNS 83-08634, National Institute on Drug Abuse Grants DA 00069

and DA 01843 (awarded to R.S. Zukin), by National Institute of

Child Health and Human Development Training Grant HD 07154, and

by a generous private donation in support of neurobiological

research fran Mr. H.I. Corkin.

AUTHORS

R. Suzanne Zukin, Ph.D.: Ann Tempel, Ph.D.; and Eliot L.

Gardner, Ph.D.

Departments of Biochemistry (R.S.Z. and A.T.), Neuroscience

(R.S.Z. and E.L.G.) and Psychiatry (E.L.G.)

Albert Einstein College of Medicine

1300 Morris Park Avenue

Bronx, New York 10461

161
The Role of Dynorphin in Narcotic

Tolerance Mechanisms

Nancy M. Lee

A central unsolved problem in the study of opiate drugs is the

mechanism of the development of tolerance and physical dependence. Although there are many ways to prevent these phenomena

from occurring or to alter the rate at which they develop, there

is no known way of reversing tolerant and dependent states once

they have been established, except through the slow and painful

process of withdrawal. A promising new alternative approach to

this problem, however, has recently been offered by the discovery

of a variety of endogenous opioid peptides in the brain. Many of

these peptides are not analgesic, yet they seem to play a modulatory role; that is, they may affect the analgesic potencies of

other substances. This conclusion is supported by studies of

both the enkephalins and dynorphin, which have been shown under

certain conditions to enhance or inhibit the analgesic effect of

morphine and of the endogenous peptide endorphin (Vaught and

Takemori 1979; Friedman et al. 1981). These observations suggest that pain regulation may be a reflection not of a single

opiate, but of the interaction of several.

In the development of tolerance and dependence to opiates, dynorphin seems to be particularly important because it has been shown

to alter their analgesic potency differently in chronicallytreated and naive animals. While dynorphin inhibits morphine

analgesia in the latter, it potentiates it in tolerant animals.

Thus both analgesia and tolerance may result from disruption of a

balance or equilibrium normally existing among the three established endogenous opioid systems, represented, for example, by

dynorphin, B-endorphin and enkephalin. In this article, we will

discuss the evidence for this idea in some detail, as well as its

implications.

PHARMACOLOGICAL PROPERTIES OF DYNORPHIN

Dynorphin is a 17 amino acid peptide first isolated from pituitary glands by Goldstein and his colleagues (Goldstein et al.

1979). In in vitro bioassays such as the guinea pig ileum and

the mouse vas deferens, dynorphin and one of its fragments,

162
dynorphin-(1-13), proved to have greater potency than most other

opiates. It also displaced tritiated opiate ligands, including

dihydromorphine (DHM), D-ala2-D-leu5-enkephalin (DADLE) and naloxone from brain tissue in in vitro binding assays.

In the ileum, dynorphin exhibits properties similar to the prototypic k-agonist, ethylketocyclazocine (EKC). This conclusion was

established on the basis of naloxone sensitivity and on cross-tolerance studies between dynorphin and EKC in the guinea pig ileum

myenteric plexus preparation (Huidobro-Toro et al. 1981). In

further support, other investigators (Oka et al. 1981; Rezvani et

al. 1983) demonstrated that in the rabbit vas deferens, which appears to be a specific bioassay system for k-like opiates, dynorphin, but not B-endorphin nor the enkephalins, mimics the actions

of EKC. Finally, Goldstein and his group reported that dynorphin

also behaves like a k-ligand in the protection of brain opiate

receptors from alkylation (Chavkin et al. 1982). That is, if

opiate receptors are preincubated with dynorphin and the alkylating agent B-chlornaltrexamine, the EKC binding sites are protected from alkylation but those of or ligands, are not protected. Thus, all these in vitro studies provide strong evidence

for the notion that dynorphin is a k-ligand.

Additional studies in our laboratory in the intact animal, however, revealed that dynorphin does not behave in the classic

manner of prototypic k agonists. For example, dynorphin displayed

no analgesic activity in mice (Friedman et al. 1981). While this

lack of effect was originally ascribed to rapid degradation of

dynorphin in the brain, we were able to show that dynorphin

exhibited other pharmacological effects, indicating it should have

remained intact long enough to produce analgesia. Of particular

interest, dynorphin was found to reduce the analgesic response to

both morphine and B-endorphin. but not to the synthetic enkephalins, D-ala2

-D-leu5-enkephalin and FK-33824 (Sandoz).

For striking contrast, a subsequent study in our laboratory

revealed that dynorphin had the opposite effect in tolerant animals; it potentiated the analgesic effect of both morphine and

B-endorphin (Tulunay et al. 1981). In the same study, dynorphin

also suppressed abrupt morphine withdrawal signs. Dynorphin has

also been demonstrated to be effective in preventing withdrawal

signs in both opiate-dependent monkeys and humans. In monkeys,

this peptide suppressed withdrawal signs in a dose-dependent

manner, being effective at doses as low as 0.06 mg/kg (Aceto et al.

1982). In humans, 0.06 mg/kg of dynorphin(1-13) suppressed

withdrawal signs and symptoms of heroin addicts. Only 3 out of 12

subjects reported mild side effects such as warmness or dizziness

(Wen and Ho 1982). Similar results have been obtained with

dynorphin in the morphine-tolerant guinea pig ileum; the effect

of morphine was restored and the naloxone-induced contraction of

the tolerant-dependent ilea was suppressed (Rezvani and Way

1983).

More recently, we have investigated the effects of chronic infusion of dynorphin on tolerance and dependence development. Infu163
sion of doses as high as 3.1 nmole dynorphin/hr for 72 hrs by

itself resulted in no development of dependence, as measured by

naloxone-induced escape jumping. Dynorphin also modulates the

action of morphine on body temperature and respiration rate.

Unlike morphine, which produces pronounced hypothermia 30-60

minutes after administration, dynorphin has a slight hyperthermic

effect. When combined with morphine, however, it potentiates the

latter's hypothermic action. Dynorphin-(1-13) alone has no effect

on the respiratory rate; however, it enhances the inhibitory

effect of morphine on this parameter in naive mice, while antagonizing it in morphine-tolerant animals (Woo et al. 1983), just the

opposite of its modulating effect with respect to analgesia.

Another peptide closely related to dynorphin, neo-endorphin, was

shown to have similar modulatory effects in naive mice, but not

in tolerant animals.

The effects of dynorphin, in summary, can be conveniently understood in terms of a "set point" model. For example, in considering

analgesia, when dynorphin is administered with morphine, dynorphin tends to maintain the animals' sensitivity to the latter at

a more or less fixed level. In the naive animal, it raises the

ED50 for morphine from about 5 to 15-20 mg/kg, while in the tolerant animal, it lowers the ED50 from about 60 mg/kg to the range of

10-15 mg/kg. Thus, in the presence of dynorphin, both naive and

tolerant animals display approximately equal sensitivity to morphine; in the absence of dynorphin, this sensitivity is less than

that exhibited by naive animals, but more than that observed in

tolerant animals. In essence, the effects of tolerance have been

partially nullified, with the animals' response to morphine largely restored.

In the case of hypothermia and respiratory depression, a similar

model operates, but with opposite consequences. However, the

effect of dynorphin in tolerant animals is again to counteract the

acute responses to morphine; thus, dynorphin apparently acts to

maintain these physiological parameters at a level close to that

found in untreated animals. The overall effect of dynorphin in

the morphine-tolerant animal is, in every respect, a stabilizing

one on morphine action, attenuating the withdrawal signs, potentiating analgesia and reducing its side effects.

DYNORPHIN BINDING SITES

An important approach to understanding dynorphin's role in the

brain would be to determine the nature of its receptors and their

relationship to receptors for other opioid ligands. This, of

course, would enable us to decide whether or not it is actually

a k-agonist in this system and, if not, decide what is its relationship to other endogenous opioids. Tritiated dynorphin is

not yet commercially available, but we have studied the interaction of the unlabelled peptide with the binding of other tritiated opioids in vitro. Somewhat surprisingly, we have found

evidence of dynorphin interaction with each of the three distinct

types of binding sites now believed to exist in brain and

164
k (Garzon et al. 1982). These interactions are complex, with

concentration-displacement curves suggesting that dynorphin does

not interact with any of these sites in a simple competitive manner, but seemingly non-competitively (Garzon et al. 1984). These

results thus contrast with those carried out in the guinea pig

ileum, where dynorphin seems to behave as a k agonist (HuidobroToro et al. 1981), and suggest that dynorphin may not be a typical

k ligand like EKC.

The most important and unexpected result of these studies, however, is that a long-lasting inhibition of binding by dynorphin

was revealed. When mice were pretreated icv with 1 - 20μg of this

peptide, binding of 3H-DHM or 3H-DADLE to tissue fractions prepared from the brains of these animals was significantly reduced.

The maximum reduction was observed 30 min to 3 hrs after injection,

with recovery not taking place until 6-12 hrs after injection.

The Bmax but not the affinity, was reduced for both ligands; the

high affinity site was most affected in each case. A similar inhibition resulted from addition of dynorphin to tissue in vitro, although in this case complete recovery occurred within 90 minutes.

These results are very provocative, since earlier studies have

shown that dynorphin is rapidly metabolized in brain. We actually confirmed this fact in these studies, showing by means of the

mouse vas deferens assay that over 95% of the peptide had disappeared from the incubation media within 20-45 minutes. It is

unlikely that dynorphin's inhibitory effect is achieved through

the small amount of dynorphin not metabolized, since this would

make the peptide several orders of magnitude more potent than any

other opioid known. It is also unlikely that a metabolite of dynorphin is responsible for the effect, since fragments of less

than 11 amino acids had no effect on opiate binding. We have

thus tentatively concluded that dynorphin remains bound to its

receptor for a long period of time to exert protracted effects.

We believe it may be the endogenous inhibitor of opiate binding

that several investigators have described (Simantov et al. 1976).

THE ROLE OF DYNORPHIN IN TOLERANCE

The data discussed above indicate not only that dynorphin can

modulate the activity of other opioids, but that the nature of

this modulation depends on the state of the animal. In naive

animals, dynorphin inhibits the analgesic action of morphine and

B-endorphin, yet it potentiates it in animals chronically morphinized. Similar effects are observed with respect to other

opiate agonists, including meperidine, methadone, D-ala2-D-leu5-

enkephalin, FK33824, and ethylketocyclazocine (manuscript in preparation). At the same time, dynorphin potentiates the "side

effects" of these agonists, such as respiratory depression, in

naive animals, but inhibits them in chronically morphinized

animals.

Since dynorphin, as well as B-endorphin, is endogenous, these

findings suggest a fresh way of viewing analgesia and its regulation. The analgesic state is not simply the outcome of the

165
interaction of a single type of ligand with its specific receptors, but rather results from a complex interaction among several

ligands: B-endorphin, dynorphin and perhaps leu- and met-enkephalin as well. It is reasonable to speculate that in the normal

brain these substances exist in a state of dynamic balance; the

effects of morphine, both acute and chronic, are then seen as

upsetting this balance. This would explain why dynorphin, though

not itself analgesic, can suppress withdrawal signs and symptoms;

its administration restores an altered balance between endogenous

dynorphin and other peptides during tolerance. This operational

hypothesis can be subjected to experimental testing.

How exactly is this balance manifested? The most attractive, yet

unproven, possibility is that dynorphin and the other opiates

actually interact with a common receptor possessing multiple

binding sites. We have previously cited considerable evidence

indicating that morphine, B-endorphin and the enkephalins bind to

different sites on a common receptor complex (Lee and Smith

1980). One interpretation of the data presented in this paper is

that dynorphin also interacts with this receptor; alternatively,

it may interact with its own site, affecting B-endorphin binding

allosterically.

If this is true, of course, these peptides should coexist in certain areas of the brain; they definitely show marked differences

in their brain distribution (Weber et al. 1982). However, there

may be certain areas where the distribution and/or projections of

dynorphin and B-endorphin, at least, do coincide, e.g., the PAG

and hypothalamus; it is significant that pain transmission is

known to be mediated through both these areas. Alternatively,

these peptides may be in different, but interacting, regions of

the brain. Mapping studies should eventually settle this question.

In summary, dynorphin in the brain seems to regulate several

activities affected by opiates that may be a key factor in tolerance development. The latter results not from a perturbation

of one particular opiate in the brain, but from an imbalance among

several. A dramatic change in sensitivity to external opiates

may thus occur without any changes in receptors. Pharmacological,

electrophysiological and biochemical studies of the interactions

of these peptides should reveal more about these imbalances and

the way they underlie analgesia and tolerance.

REFERENCES

Aceto, M.D., Dewey, W.L., Chang, J.K., and Lee, N.M. Dynorphin-1-

13: effects in nontolerant and morphine-dependent rhesus monkeys. Europ J Pharmacol, 83:139-142, 1982.

Chavkin, C., James, I.F., and Goldstein, A. Dynorphin is a specific endogenous ligand of the k opiate receptor. Science, 215:

13-415. 1982.

Friedman; H.J., Jen, M.F., Chang, J.K., Lee, N.M. and Loh, H.H.

Dynorphin: a possible modulatory peptide on morphine or B-endorphin analgesia in mouse. Europ J Pharmacol, 69: 351-360, 1981.

166
Garzon, J.G., Jen, M.F., Sanchez-Blazquez, P., and Lee, N.M. Dynorphin-1-13: a long-lasting inhibition of opiate receptor binding in vitro. Life Sci, 31:1789-1792, 1982.

Garzon, J., Sanchez-Blazquez, P., Gerhart, J., Loh, H.H. and Lee,

N.M. Dynorphin-1-13: interaction with other ligand bindings in

vitro. Brain Res., in press, 1984.

Goldstein,A., Tachibana, S, Lowney, L.I., Hunkapiller, M., and

Hood, L. Dynorphin-1-13, an extraordinary potent opioid peptide. Proc Natl Acad Sci USA, 76:6666-6670, 1979.

Huidobro-Toro, J.P., Yoshimura, K., Lee, N.M., Loh, H.H., and Way,

E.L. Dynorphin interaction at the k-opiate site. Europ J

Pharmacol, 72:265-266, 1981.

Lee, N.M., and Smith, A.P. A protein-lipid model of the opiate

receptor. Life Sci, 26:1459-1464, 1980.

Oka, T., Negishi, K., Suda, M., Matsumiya, T., Inazu, T., and

Ueki, M. Rabbit vas deferens: a specific bioassay for opioid kreceptor agonists. Europ J Pharmacol, 73:235-236, 1981.

Rezvani, A., Höllt, V., and Way, E.L. k receptor activities

of the three opioid peptide families. Life Sci, 33:271-274,

1983.

Rezvani, A., and Way, E.L. Dynorphin-1-13 restores the potency

of morphine on the tolerant guinea pig ileum. Europ J Pharmacol, in press, 1984.

Simantov, R., Snowman, A.M., and Snyder, S.H. Temperature and

ionic influences on opiate receptor binding. Mol Pharmacol,

12:977-986, 1976.

Tulunay, F.C., Jen, M.F., Chang, J.K., Loh, H.H., and Lee, N.M.

Possible regulatory role of dynorphin on morphine and B-endorphin induced analgesia. J Pharmacol Exp Ther, 219:296-298, 1981.

Vaught, J.L., and Takemori, A.E. Differential effects of leucine

and methionine enkephalin on morphine-induced analgesia, acute

tolerance and dependence. J Pharmacol Exp Ther, 208:86-90, 1979.

Weber, E., Roth, K.A., and Barchas, J.D. Immunohistochemical distribution of -neo-endorphin/dynorphin neuronal systems in rat

brain. Evidence for colocalization, Natl Acad Sci USA, 79:3062-

3066, 1982.

Wen, H.L., and Ho, W.K.K. Suppression of withdrawal symptoms by

dynorphin in heroin addicts. Europ J Pharmacol, 83:134-142,

1982.

Woo, S.K., Tulunay, F.C., Loh, H.H., and Lee, N.M. Effect of dynorphin-1-13 and related peptides on respiratory rate and morphine-induced respiratory rate depression. Europ J Pharmacol,

96:117-122, 1983.

ACKNOWLEDGEMENT

Supported by Grants DA-02643 and KO2-DA-00020 from the National

Institute on Drug Abuse.

AUTHOR

Nancy M. Lee, Ph.D.

Department of Pharmacology

& Langley Porter Psychiatric Institute

University of California

San Francisco, CA 94143

167
Post-Translational Processing of

Neuropeptide Precursors

Richard E. Mains and Betty A. Eipper

Neuropeptides, and in fact a very large number of small peptides,

are synthesized as portions of larger precursor proteins which are

themselves inactive; this review will frequently use the ACTH/endorphin system as an example (figure 1; Mains et al. 1983).

FIGURE 1

Rapid DNA sequencing techniques have been instrumental in the

recent dramatic explosion of information about the structure of

such precursors, but there is still a great deal to be learned

about the enzymatic processing steps that must occur between the

synthesis of precursors and synthesis of their bioactive peptide

products. As it is now possible to proceed rapidly from the

amino acid sequence of a purified peptide to the nucleotide sequence and hence the entire amlno acid sequence of its precursor,

we are now in the position of knowing the sequences of a Iarge

number of precursors but not which product peptides are actually

made or even which product peptides are physiologically important.

Based on the limited number of examples that have been adequately

studied, It is clear that discrete cleavages in these precursors

result In the creation of a whole family of peptide products; alI

of the progeny peptides are secreted along with the particular

bioactive peptide that was originally thought to be the focus of

interest. Thus it is important to determine the structures of all

168
of the product peptides (i.e. secretory products). One important

example of this rule was the old clinical observation that

peptldes related to ß-Iipotropin are secreted along with ACTH

under a variety of circumstances. This was a key observation In

the discovery that the molecule already known to be the precursor

to ACTH (pro-ACTH) also contained the structure of ß-Iipotropin

and thus the potent opiate peptide ß-endorphin (hence, the name

pro-ACTH/endorphin) (Eipper and Mains 1980).

Although a set of general principles for peptide hormone processing has been developed over the past years, they really only

define a set of likely products; the actual product peptides

produced must be determined for each tissue where those peptides

occur. For example, in the pro-ACTH/endorphin system a pair of

basic amlno acids (Arg-Lys) precedes the sequence in the

precursor (figure 2); instead of producing peptides with

Residue number 48 49 50 51 52 53

16K fragment -Pro-Arg-Lys-Tyr-Val-Metas predicted

by removal of Tyr-Val-Metpairs of basic residues

ActuaI product Lys-Tyr-VaI-MetFIGURE 2. Sequences of 16K Fragment and Progeny

at their amino terminus, bovine and rat intermediate pituitary

cells produce peptides with at their amino terminus

(Esch et al. 1981). The shows far greater binding

affinity for adrenal cortical cells (the presumed target in vivo)

than (Pedersen and Brownie 1983).

Neuropeptides and peptide hormones display quite an array of

Important co- and post-translational modifications. Since the

precursors are bigger than their product peptides, proteolytlc

processing is clearly a very important modification during biosynthesis. For simplicity, these proteolytic modifications can be

grouped into three categories: removal of the signal peptide from

the precursor during or immediately after synthesis of the

precursor and its deposition into the lumen of the rough

endoplasmic reticulum; endoproteolytic steps which break the

precursor into the peptide regions which approximately comprise

the final product peptides; and exoproteolytic events which trim

the carboxyl and/or amino terminal ends of the peptides into the

forms found in the mature product. It is not yet clear how

distinct the endo- and exoproteolytic steps will be for any

peptlde family. It is not even clear whether a few proteases will

function in the biosynthesis of many different peptides or whether

a large number of proteases with narrow specificity will be

169
involved. It is clear that expression of the proinsulin gene in

monkey kidney cells results in secretion of proinsulin into the

medium without production of insulin and C-peptide (Laub et al.

1983); thus the biosynthetic processing proteases of peptide

hormone biosynthesis are not normally expressed in all cells.

In order to discern the number of proteolytic steps that were

actuaIIy used in the cell to produce a product peptide from its

precursor, one must identify intermediates in the biosynthetic

pathway. However, many other co- and post-translational covalent

modifications are easily detectable in the smaller products, and

In several instances these alterations are very important in

determining the bioactivity of the peptide products. This list

includes glycosylation (both asparagine-linked and serine/threonine-linked), N-acetylation, phosphorylation, sulfation, and

amidation. Neuropeptides are thought to function as

neurotransmitters or neuromodulators; synthesis of precursor and

the subsequent co- and post-translational modifications represent

the biosynthetic pathway for these molecules (Snyder 1984). In

order to understand the functioning and malfunctioning of these

peptidergic systems, we have to understand the physiological and

pharmacological factors that can regulate each step. Studying

these steps can be thought of as analogous to studying the role

and regulation of tyrosine hydroxylase, aromatic amino acid

decarboxylase, dopamlne ß-hydroxylase and phenylethanolamine Nmethyltransferase in catecholamine biosynthesis.

Another important point about bioactive peptides is that any given

peptide and its siblings can occur in more than one tissue in

major amounts. In addition, the final peptide products found in

the different tissues can differ structurally in very important

ways. For example figure 3), ACTH(1-39) is the peptide which

directs the adrenaI cortex to synthesize and reIease

glucocorticoids into the blood; ACTH(1-39) is a major product pepFIGURE 3. Biosynthetic Pathways

170
tide of pro-ACTH/endorphin in the anterior pituitary. Alphamelanotropin MSH; -N-acetyl-ACTH(1-13)NH2] stimulates skin

darkening in Iower vertebrates and may also have behavioral

act ions in higher vertebrates; MSH is a major product peptide

from pro-ACTH/endorphin in the intermediate pituitary, and

molecules similar to MSH also occur in the brain. Since it is

now clear that in several species there is only one proACTH/endorphin gene, the differences in the product peptides in

the different tissues must be due to differences in enzymatic

processing of the primary gene product.

FOCUS ON ENZYMES

This review will discuss two enzymatic activities which are

apparently important in the post-translational processing of proACTH/endorphin. Both enzymes are involved in late biosynthetic

events which determine the final structure and bioactivity of the

product peptides, after initial processing steps are done. Each

of the enzymatic activities is primarily localized in secretory

granules.

The first activity to be discussed will be the secretory granuleassociated N-acetyltransferase activity found in rat and beef

intermediate pituitary but not in rat anterior pituitary. Current

data argue that the same enzyme puts N-acetyl groups onto both

ACTH(1-13)NH2 [to produce or -meIanotropin] and ßendorphin(1-31) [to produce N-acetyl-ß-endorphin(1-31)] (figure

3, previous page).

The second enzymatic activity to be examined will be a peptidylglycine amidation activity. Many neuropeptides terminate in a

carboxyl-terminal amide and data accumulated to date suggest

that amidated peptides are derived from precursor peptides that

have an additional glycyl residue at the carboxyl terminus. This

enzymatic activity cleaves within the original COOH-terminal

glycyl residue to liberate glyoxylate and leave a product peptide

bearing a COOH-terminal amide group. Some similarities of this

enzyme activity to other known enzymes, most notably dopamlne ßhydroxylase, will be discussed.

N-ACETYLTRANSFERSE

The enzymatic activity utilizing acetyl coenzyme A (AcCoA) to

mediate the N-acetylation of ACTH(1-13)NH2 and ß-endorphin(1-31)

has been examined by Woodford and Dixon (1979), Glembotski (1982b)

and O'Donohue and coworkers (O'Donohue and Dorsa 1982). The

thorough subcellular fractionation work of Glembotski (1981;

1982a,b) made clear two important points. First, only about 10-

30% of the acetylating activity in intermediate pituitary was

found in a secretory granule fraction with the rest of the

activity associated with other cellular organelles or cytoplasm.

The secretory granule-associated activity is the only acetylating

activity of physiological relevance to the N-acetylatlon of

MSH, since previous work (Glembotski 1982a) demonstrated that the

171
-N-acetylation of ACTH(1-13)NH2 and ß-endorphin(1-31) in intact

cells occurs exclusively in secretory granules. In addition, it

was expected that other acetyltransferases would be detected in

the cell, mediating the acetylation of histones and the Nacetylatlon of many nascent polypeptide chains (Simpson 1978;

Belikoff et al. 1980; Traugh and Sharp 1977; Pestana and Pitot

1975). These latter acetyltransferase activities would not be

expected to be tissue speclflc.

Second, a secretory granule-associated N-acetyltransferase

activity was only found in the intermediate pituitary and not in

the anterior pituitary (Glembotski 1982b). Thus the observed

tlssue specificity of N-acetylatlon could be explained by tissue

specific expression of an enzyme. Alternate explanations for the

observed tissue specificity of N-acetylation would be that

anterior pituitary granules possessed the appropriate

acetyltransferase enzyme, but did not express this enzymatic

activity in vivo. One way to achieve this result would have been

for the anterior pituitary cells to fall to provide the secretory

granule lumen with AcCoA; however, given adequate AcCoA in a test

tube, anterior pituitary secretory granule extracts still do not

have demonstrable N-acetyltransferase activity. Thus, the

current data argue that the tissue specificity of N-acetylation

is achieved by the inclusion (or not) of N-acetyltransferase

enzyme in secretory granules. Mixing experiments will be

necessary, however, to show that anterior pituitary secretory

granules do not contain inhibitors of N-acetylation.

The enzymatic activity studied by Glembotski (1982b) is probably

the physiologilcally relevant acetyltransferase activity for

several reasons. The peptide substrates acetylated by the enzyme

activity in a test tube--ACTH(1-13)NH2, ACTH(1-39), ß-endorphin(1-

31), ß-endorphin(1-27)--are also N-acetylated in intermediate

pituitary secretory granules, while several other possible

substrates--CLIP [ACTH(18-39); corticotropin-like intermediate

lobe peptide], histones, 16K fragment (the NH2-terminal

glycoprotein region of the pro-ACTH/endorphin precursor; figure

1)--are not utilized (table 1, next page). The 3H-labeled peptide

products created by incubation of ACTH(1-13)NH2 or ß-endorphin(1-

31) with secretory granule extracts are correctly N-acetylated,

rather than N-acetylated on the groups of lysine residues as

expected for a histone acetyltransferase (Simpson 1978). Finally,

the amount of enzyme activity detected is reasonable for the rate

and extent of N-acetylation of peptides that actually occurs in

intermediate pituitary secretory granules; it takes roughly

twelve hours to N-acetylate the peptides during biosynthesis in

the intact tissue, and the secretory granule enzyme activity would

also take about a half day to N-acetylate the relevant peptides

In the granules.

GIembotski (1983) has also shown that the N-acetylatlon seen in

intact intermediate pituitary cells is effectively irreversible.

in pulse-chase experiments using [

35S]methionine and [3

H]acetate

double labeling, there was no decrease in the acetate:methionine

172
TABLE 1. N-Acetyl Transferase

ratio in MSH, diacetyl MSH or ß-endorphin-sized peptides during

incubations for up to 48 hours in nonradioactive medium. Both the

N-acetyI modifications of MSH and acetylated ß-endorphin and

the O-acetylation of diacetyl MSH were thus found to be quite

stable, at least while the peptides were still inside the

secretory granules. These double label experiments also suggest

that the same pool of 3H-acetyl CoA is used to N-acetylate

ACTH(1-13)NH2 and ß-endorphin and to O-acetylate

3

H/35S ratios for MSH, N-O-dlacetyl- MSH and acetylated ß-enMSH, since the

dorphins were found to occur in a 1:2:1 relationship.

A full understanding of the enzymatic mechanism of the -Nacetyltransferase from intermediate pituitary should be very

interesting. Current data of Glembotski (1982b; unpublished)

strongly suggest that the same enzyme N-acetylates both ACTHand ß-endorphin-related peptides. As expected, the N-acetylation

of ß-endorphin(1-31) is inhibited by product peptides such as

acetyl-ß-endorphin(1-31); interestingly, also inhibits the

acetylation of ß-endorphin(1-31). Likewise, N-acetylated

endorphins inhibit the acetylation of ACTH(1-13)NH2.

Substantially shortened peptides, such as ACTH(1-8) and ßendorphin(1-16) [also known as endorphin], are not detectably

N-acetylated under the same reaction conditions used for ACTH(1-

13)NH2 and ß-endorphin(1-31). Importantly, ACTH(1-8) and ßendorphin(1-16) each inhibit the N-acetylation of the longer

ACTH- and endorphin-related peptides. The very similar patterns

of inhibition of N-acetyltransferase activity by acetylated

product peptides and by shortened nonacetylated peptides are most

economically explained by the existence of one enzyme performing

the N-acetylatlon of both ACTH- and ß-endorphin-related

peptides. The requirements of the binding and catalytic sites of

this enzyme will be very interesting and probably quite complex.

173
When rat intermediate and anterior pituitary cells are placed in

tissue culture, they are deprived of many of the influences they

receive in the animal. Anterior pituitary cells no longer receive

occasional bursts of corticotropin releasing factor in the

hypothalamic portal blood system, and the varying levels of

feedback from circulating glucocorticoids are also lost in tissue

culture. Intermediate pituitary cells lose the several kinds of

direct innervation they receive in vivo. It might be expected

that the loss of some of these inputs, which are believed to

control secretory rates in vivo, would result in the loss of some

of the tissue-specific differences seen in the intact animal.

However, the anterior pituitary corticotropes do not detectably

begin to N-acetylate ACTH or ß-endorphin in culture, nor do

intermediate pituitary cells cease to N-acetylate ß-endorphin or

ACTH. This result is in striking contrast to the rapid loss of

amidation seen when intermediate pituitary cells are placed in

culture (see below).

PEPTIDYL-GLYCINE AMIDATION ACTIVITY

Dozens of bioactive peptides have COOH-terminal amide groups

which are essential for the expression of full biological potency

(reviewed in Malns et al. 1983; Elpper et al. 1983a,b). Using the

small synthetic peptide substrate originally designed by Bradbury

et al. (1982), we have found peptidyl-glycine amidating activity

in secretory granules from anterior, intermediate, and neural

lobes of the rat and beef pituitary as well as from AtT-20 mouse

corticotropic tumor cells and rat hypothalamus (Eipper et al.

1983b,c; Glembotski et al. 1984; Malns et al. 1984; Emeson 1984)

(figure 4). In this assay the conversion of D-Tyr-Val-Gly into DTyr-VaI-NH2 is monitored; substrate and product are easily

separated by ion exchange chromatography and the D-Tyr residue

provides a site for radioiodination as well as protecting against

proteolytic degradation.

[

125I]-D-Tyr-Val-Gly + goodies

(passes through)

[

125I]D-Tyr-Val-NH2 + glyoxylate

(binds; elutes)

Resin: SP-Sephadex

Bind/Wash: 10 mM NaPI, pH 5.0

Elute: 0.5 M NaCl, 10 mM NaPI, pH 5.0

FIGURE 4. Amidation Assay

174
For all these cases, optimal enzyme activity requires the presence

of copper ions, reduced ascorbic acid, and molecular oxygen.

Enzyme activity is: 1) greatly decreased by removal of copper ions

wlth micromolar amounts of diethyldithiocarbamate, and rejuvenated

by replacement of copper (no other metal ion tested will

substitute for copper); 2) stimulated by addition of ascorbate

better than by addition of any other redox cofactors tested, and

largely abolished by pretreatment with ascorbate oxidase; and 3)

substantially diminished by replacement of much of the dissolved

oxygen in the reaction tube with nitrogen or argon. The enzymatic

activity is operationally soluble, in that simple repeated

freezing and thawing releases the enzyme from secretory granules

(summarized in table 2).

TABLE 2. Properities of Amidation Activity

1. Similar enzymatic activity in different tissues:

rat anterior, intermediate, neural pituitary

bovine anterior, intermediate, neural pituitary

rat hypothalamus

rat serum

mouse corticotropic tumor cells

human plasma and cerebrospinal fluid

2. Secretory granule associated

3. Requires molecular oxygen

4. Stimulated by copper

5. Enhanced by ascorbate

6. Inhibited by endogenous inhibitors

7. Soluble

8. Secreted along wlth peptides

9. Broad specificity for many peptides wlth a carboxyl terminal

glycine residue

In striking contrast to the N-acetyltransferase, peptidylglycine amidation activity is found in a wide variety of tissues

and in particular is found in all three lobes of the rat and beef

pituitary. In most cases, the amidating activity cannot be

detected directly in crude tissue extracts made in hypotonic

buffers, due to the presence of high amounts of endogenous

inhibitory activity. The effect of the endogenous inhibitory

activity is greatly diminished when the amidation activity is

assayed using a secretory granule fraction, although some

inhibitory activity is still detectable even in secretory granule

preparations. Endogenous inhibitory activity is largely overcome

by addition of appropriate amounts of copper ions.

Its requirement for copper, ascorbate, and oxygen, plus the

175
presence of endogenous inhibitors which can be neutralized with

addition of optimal levels of copper, make the peptidyl-glycine

amidating activity very similar to the well-known enzyme, dopamine

ß-hydroxylase (Rush and Geffen 1980).

The peptidyl-glycine amidating activity studied by our group and

by Bradbury and Smyth (1983) exhibits a very different pattern of

specificity for peptide substrates from that seen for the -Nacetyltransferase (above). The amidating activity will produce

a peptide ending in -X-NH2 from a substrate with the structure -XGly; although there are quantitative differences, neutral amino

acids such as Phe, Val, and Gly function effectively as the

residue (X) to be amidated. Based on ability to inhibit

amidation of D-Tyr-Val-Gly, peptides where X is Trp can also

interact with the enzyme. In addition, the peptide to be amidated

need not be very long (unlike the case for N-acetylation); the

synthetic tripeptide D-Tyr-Val-Gly gives Michaelis constants

indistinguishable from the Kl values for longer and presumed

natural substrates such as N-acetyl-ACTH(1-14) and MSH.

The original work of Bradbury et al. (1982) showed that porcine

pituitary granules contained an enzymatic activity which utilized

D-Tyr-Val-Gly as peptide substrate and produced D-Tyr-Val-NH2 and

glyoxylate (HCOCOOH) as products. The reaction scheme suggested

by Bradbury et al. (1982) is:

D-Tyr-Val-NH-CH2-COOH + oxidized cofactor + H20

D-Tyr-Val-NH2 + HCOCOOH + reduced cofactor

This scheme does not incorporate the subsequent findings of the

need for copper, reduced ascorbate and oxygen, although copper

could presumably function as the cofactor. One argument against

this scheme is the observation that oxidized cofactors (NADP, NAD,

FAD, FMN, ascorbate oxidase-treated ascorbic acid) do not

stimulate the reaction when relatively clean secretory granules

are used as the enzyme source; In addition, the requirement for

molecular oxygen would not be explained (Eipper et al. 1983b,c;

Glembotski et al. 1984; Mains et al. 1984; Emeson 1984).

A second possible reactlon scheme that takes into account the

dependence of the reactlon on molecular oxygen resembles that of

the amine oxidases:

D-Tyr-Val-NH-CH2-COOH + O2 + H20

D-Tyr-Val-NH2 + HCOCOOH + H202

Since several of the amine oxidases characterized as diamine

oxidases utilize copper to perform their reactions, the above

scheme deserves serious consideration (Gorkin 1983; Kapeller-Adler

1970). It does utilize molecular oxygen, but does not explain why

reduced cofactors such as ascorbate (and, less well, biopterin)

stimulate the reaction. Addition of a range of monoamine oxidase

inhibitors (pargyline, tranylcypromine, iproniazid phosphate, D176
amphetamine) did not inhibit the amidation activity when the

inhibitors were used at concentrations of 1-2 mM. Addition of

semicarbazide, generally considered to be a relatively good

diamine oxidase inhibitor, had an inhibitory effect only at high

levels (slight inhibition at 2.5 mM; no inhibition at 0.5 mM).

Addition of millimolar levels of inhibitors such as cyanide and

azide does inhibit the enzyme but lower levels are ineffective.

The reaction scheme presently considered most plausible is that of

a monooxygenase:

D-Tyr-Val-NH-CH2-COOH + 2(ascorbate) + O2

D-Tyr-Val-NH2 + 2(semidehydroascorbate) + HCOCOOH + H20

A variant of the above scheme would involve only one ascorbate

molecule being fully oxidized to dehydroascorbate. This scheme

draws on our data and that of Bradbury et al. (1982) plus the

current data on the very similar reaction performed by dopamine ßhydroxylase (Skotland and Ljones 1980; Diliberto and Allen 1981),

in which semidehydroascorbate (ascorbate free radical) is now

believed to be the immediate reaction product (instead of

dehydroascorbate). SeveraI important aspects of this model must

be confirmed experimentally before it is accepted, however.

Peptide substrate-dependent ascorbate consumption will need to be

measured and compared to the simultaneous production of amidated

product peptides. Quantitation of glyoxylate production will be

crucial, as welI as the demonstration that the predicted amount of

oxygen is consumed without production of H2O2. Final confirmation

that this is a monooxygenase reaction would require use of 18O2

and detection of 18O in the glyoxylate by mass spectrometry

(Kaufman et al. 1962).

The fact that the peptidyl-glycine amidating activity is readily

released from secretory granules by simple freezing and thawing

suggested that the activity might be secreted along with peptide

hormones. When AtT-20 mouse corticotropic tumor cells are

stimulated to release ACTH- and ß-endorphin-related peptides,

amidating activity is also secreted. Both peptides and amidating

activity are released coordinately in response to severaI

secretagogues which can increase the secretory rate up to 20-fold

for brief periods: corticotropin releasing factor, isoproterenol,

BaCl2, and cycIic AMP (table 3, next page). The stimulated

release of peptides and of amidating activity is blocked by

addition of CoCl2 to the medium, consistent with previous

demonstrations that an inward flux of calcium ions is required for

the release of the soluble contents of the secretory granules by

exocytosis (Mains and Eipper 1981, Surprenant 1982, Adler et al.

1983). In each case cited above, the ratio of amidating

activity (measured at 0.5 μM D-Tyr-Val-Gly substrate) to hormone

in the stimulated secretion is the same as that ratio in the

secretory granules, again suggesting that release occurs by

exocytosis of all of the soluble contents of granules (table 3).

These co-release data also further strengthen the claim that the

177
TABLE 3. Ratio of Amidation Activity to ACTH/ ß-endorphin Content

Enzyme/Hormone

AtT-20 Cells 0.15

Granules 0.15

Medium 0.15

Bovine Intermediate Pituitary Granules 0.02

Rat Anterior Pituitary Granules 14

Rat or human plasma 6000

Units: Enzyme pmol/ml/h

Hormone pmol/ml (nM)

amidating activity being studied is localized in secretory

granules rather than in a minor contaminating organelle with

similar sedimentation properties to secretory granules.

Since corticotropic tumor cells release amidating activity along

with the peptides derived from pro-ACTH/endorphin, it was

considered possible that amidating activity could be detected in

plasma and/or cerebrospinal fluid (CSF). Indeed, when human

plasma and CSF are assayed, amidating activity is easily

demonstrated; the plasma and CSF activities are dependent on

copper ions, ascorbate, and molecular oxygen (Wand et al. 1984).

The correct product (D-Tyr-Val-NH2) is produced, as demonstrated

by high performance liquid chromatography, and the plasma and CSF

activities have several other properties consistent with their

being similar to the pituitary activity.

However, there is far more amidating activity in human and rat

plasma than can reasonably be expected to have come from anterior

pituitary corticotropes (table 3). This has led to an examination

of amidation activity in other tissues in the rat, with the

interesting finding that the submaxillary glands contain

sufficient amidating activity to be major candidates to have

secreted a significant amount of the amidating activity found in

plasma. The hypothalamus and cerebral cortex certainly contain

enough amidating activity to provide the enzyme activity found

in CSF. The cerebellum has very low levels of amidating

activity (using crude extracts at a wide variety of copper

levels), consistent wlth the very low levels of bioactive peptides

usually reported for the cerebellum.

Cellular Studies of Peptide Amidation

Rat intermediate pituitary cells maintain or increase their

content of pro-ACTH/endorphin-derived peptides for at least 2

weeks in tissue culture (Eipper et al. 1983a). The cultured

intermediate pituitary celIs continue to carry out a large number

of co- and post-translational modifications in culture; some of

178
the modifications are of the tissue-specific type [e.g., Nacetylation and cleavage of ACTH(1-39) into amino and carboxyI

terminal pieces] and some are not (e.g., phosphorylation and

glycosylation of the COOH-terminal region of ACTH and many of the

proteolytic cleavages). However, rat intermediate pituitary cells

in tissue culture specifically lose the ability to amidate MSH;

the loss of peptide amidation occurs with a half-life of about

15-18 hours (Eipper et al. 1983a; Glembotski et al. 1983).

Instead of producing various acetylated forms of ACTH(1-13)NH2,

the cells produce the corresponding set of non-, mono-, and

diacetylated ACTH(1-14). The COOH-terminal sequence of ACTH(1-14)

is -Lys11-Pro12-Val13-Gly14, while MSH terminates -Lys11-

Pro12-Val13-NH2; normally, <2% of the MSH-related peptides in rat

or beef intermediate pituitary terminate in -Val-Gly rather than

in -VaI-NH2. Given the above discussion of the intricate

properties of the amidation enzyme, it was important to examine

whether any of these properties of the amidation enzyme could be

associated with the loss of the ability of the rat intermediate

pituitary cells to amidate peptides in tissue culture.

The tissue culture media in our previous work, as in nearly all

work with primary cells and cell Iines in the Iiterature,

contained no added ascorbic acid and very low levels of copper

ions ( .0055 μM). Although a large proportion of the copper in <

serum is bound to other molecules and is unavailable to tissues

(total serum copper is about 16 μM; Riordan 1983; Sarkar 1981;

Danks 1980), it seemed reasonable to explore the effect of

additional copper on amidation by the cells in culture. Serum

levels of ascorbate are about 50 μM and it is all in the reduced

form (Hornig 1975; Basu and Schorah 1982; Kabrt et al. 1981).

Ascorbate is synthesized primarily in the Iiver of mammals such as

rats that can synthesize their own ascorbate, and it seemed likely

that exogenous ascorbate would be needed by the pituitary cells.

The simple inclusion of 2 or 20 μM CuSO4 in the serum-free tissue

culture medium had no effect on the rapid loss of the ability to

amldate peptides (Eipper et al. 1983d). When ascorbic acid was

included in the culture medium along with copper, a significant

recovery of the ability to amldate newly synthesized peptides

was always seen (table 4, next page). Experiments varying the

time of addition of ascorbate indicated that ascorbic acid added

only for the incubation with radiolabeled amino acid, (during

which time the ability to amidate newly synthesized peptides was

being tested) was just as effective as ascorbic acid added for the

entire time in culture (even when added fresh every few hours). A

rough dose-response curve for the response of amidation to the

dose of ascorbate was also seen, with the buIk of the recovery

from the loss of amidation occurring between 0 and 200 μM

ascorbate, but with significant increases in amidation seen up

to 1.0 mM ascorbic acid. The full ability to amidate peptides

was never regained in these experiments, although the fraction of

the newly synthesized MSH-related molecules which were correctly

amidated increased up to four-fold in the presence of 2 μM CuSO4

and 1 mM ascorbate.

179
TABLE 4. Reversal of Loss of Amidation in Tissue

Culture by Addition of Ascorbic Acid

Percent

Amidated

Experiment 1 : control 12

0.2 mM ascorbate 37

0.5 mM ascorbate 41

1 .0 mM ascorbate 45

Experiment 2: control 15

0.05 mM ascorbate 30

2.0 mM ascorbate 42

Rat intermediate pituitary cells were incubated for a day in

complete serum-free medium plus 2.0 μM CuSO4 and the indicated

concentration of ascorbate. At the end of the pre-incubation,

cells were incubated in the same medium plus [3H]tryptophan for 3

hours; RP-HPLC analyses of the tryptic peptides of the labeled

MSH-sized molecules separated peptides derived from ACTH(1-

13)NH2- and ACTH(1-14)-related molecules.

Possible Consequences of Inappropriate Copper and Ascorbate Levels

in Vivo

Ascorbate is made in the liver of most mammals including rats, but

primates and guinea pigs must rely on the diet for their

ascorbate. The pituitary has a higher concentration of ascorbic

acid than any other tissue in the body (Hornig 1975; Kabrt et al.

1981), although ascorbate is also very high in the central nervous

system, most notably in the hypothalamus (MiIby et al. 1981;

Schreiber et al. 1979). Copper is an essential trace element in

the diet, and modern human diets are frequently low or only

marginally adequate in copper (Danks 1980; Sarkar 1981; Riordan

1983). Two human genetic disorders, Wilson's and Menkes'

diseases, involve altered copper metabolism; the mottled series of

mutant mice are considered to be a good model for Menkes' disease

(Riordan 1983; Danks 1980; Hunt 1980). Copper levels can be low

In patients undergoing disulfiram (Antabuse) therapy for chronic

alcoholism, or in people on high zinc or high ascorbate diets

(Danks 1980; Riordan 1983; Schreiber et al. 1979).

Experimental animals (rats, guinea pigs, mice, mottled mutant

mice) have been examined for the effects of altered ascorbate

and/or copper metabolism on dopamlne ß-hydroxylase levels and on

catecholamine production, and interesting but somewhat conflicting

observations have appeared in the literature. Since the

amidating activity has many striking similarities to dopamine ßhydroxylase, consideration of the catecholamine Iiterature is

particularly relevant to the study of amidation of peptides.

Since dopamine ß-hydroxylase is not the rate-limiting step in vivo

for norepinephrine biosynthesis, a fairly major drop in the

180
expression of dopamine ß-hydroxyIase activity in intact tissue

would be needed in order to produce a decrease in norepinephrine

levels (Rush and Geffen 1980).

Copper deficiency due to dietary insufficiency or to a genetic

defect preventing an adequate supply of copper from reaching the

adrenaIs and the nervous system results in a marked decrease in

the norepinephrine content of the adrenals and the brain (Hunt

1980; Sourkes 1980; Danks 1980). A clear prediction from this

result is that copper deficiency (for dietary, genetic, or drug

therapeutic reasons) might also result in a marked decrease in the

levels of many crucial bioactive amidated peptides. The

response of dopamine ß-hydroxylase levels to copper deficiency,

when the dopamine ß-hydroxylase is tested in tissue extracts, is

less clear; some workers find enzyme levels to be decreased

(Holtzman 1976; Danks 1980; Sourkes 1980), while others report an

increase in enzyme activity (Hunt 1980; Prohaska and Smith 1982).

Some of the differences in results for the effect of copper

deficiency on dopamine ß-hydroxylase levels may reflect whether

assay conditions were optimized for copper levels (Hunt 1980). At

least for brief periods of time, purified dopamine ß-hydroxylase

is fully stable with all of its copper removed by chelation, and

activity is fully restored by the return of copper to the enzyme

(Skotland et al. 1980).

Ascorbate deficiency in guinea pigs results in a drop in brain

norepinephrine levels within 2 to 3 weeks in most (Hoehn and

Kanfer 1980; Saner et al. 1975; Deana et al. 1975) but not all

studies (Green et al. 1979). An elevated ascorbate diet in the

rat does not alter cerebral norepinephrine levels (Behrens and

Madere 1980). Since the rate of ascorbate reduction of enzymebound copper on dopamine ß-hydroxylase is not the rate-limiting

step of dopamine ß-hydroxylation (Skotland et al. 1980), it is

actually quite striking that a drop in norepinephrine levels is

detected so rapidly in most studies. The clear implication from

these studies is that ascorbate deficiency in vivo will manifest

itself as a drop in the levels of key amidated peptides; already

there are good data from the tissue culture studies that ascorbic

acid supplies directly affect the cellular expression of peptide

amidation activity (table 4, previous page).

CONCLUSIONS

The study of the co- and post-translational biosynthetic

processing events leading to the production of bioactive peptides

is a fast-growing field wlth many potential ramifications in

studies of human health and disease. The interaction of various

drugs of abuse wlth bioactive peptides, both at their plasma

membrane receptors and during biosynthesis, is only beginning to

be examined.

181
REFERENCES

Adler, M., Wong, B.S., Sabol, S.L., Busis, N., Jackson, M.B., and

Weight, F.F. Proc Natl Acad Sci (USA), 80:2086-2090, 1983.

Basu, T.K., and Schorah, C.J. Vitamin C In Health and Disease.

Westport, CT: AVI Publishing Co., 1982

Behrens, W.A., and Madere, R. J Nutr, 110:720-724, 1980.

Belikoff, E., Wong, L.J., and Alberts, B.M. J Biol Chem,

255:11448-11453, 1980.

Bradbury, A.F., and Smyth, D.G. Biochem Biophys Res Commun,

112:372-377, 1983.

Bradbury, A.F., Finnie, M.D.A., and Smyth, D.G. Nature (Lond.),

298:686-688, 1982.

Danks, D.M. CIBA Found Symp, 79:209-221, 1980.

Deana, R., Bharaj, B.S., VerJee, Z.H., and Galzigna, L. Int J

Vitam Nutr Res, 45:175-182, 1975.

Diliberto, E.J., and Allen, P.L. J Biol Chem, 256:3385-3393,

1981.

Eipper, B.A., Glembotski, C.C., and Mains, R.E. J Biol Chem

258:7292-7298, 1983a.

Eipper, B.A., Glembotski, C.C. and Mains, R.E. Peptides, 4:

921-928, 1983c.

Eipper, B.A., and Mains, R.E. Endocrine Rev, 1:1-27, 1980.

Eipper, B.A., Mains, R.E., and Glembotski, C.C. Proc Natl Acad

Sci (USA), 80:5144-5148, 1983b.

Eipper, B.A., Mains, R.E., and Glembotski, C.C. Ferring Symp, In

press, 1983d.

Emeson, R.B.J. Neurosci, 4: in press, 1984.

Esch, F.S., Shibasaki, T., Bohlen, P., Wehrenberg, W.B., and Ling,

N.C. In: Rich, D.H., and Gross, E., eds. Peptides. Rockford,

IL: Pierce Chemical Co., 1981.

Glembotski, C.C., Eipper, B.A., and Mains, R.E. J Biol Chem,

259:6385-6392, 1984.

Glembotski, C.C., Eipper, B.A., and Mains, R.E. J Biol Chem,

258:7299-7304, 1983.

Glembotski, C.C. J Biol Chem, 257:10493-10500, 1982a.

Glembotski, C.C. J Biol Chem, 257:10501-10509, 1982b.

Glembotski, C.C. J Biol Chem, 256:7433-7439, 1981.

Gorkin, V.Z. Amine Oxidases in Clinical Research, New York,

Pergamon Press, 1983.

Green, M.D., Bell, W., Kraut, C., and Omaye, S. Experientia,

35:515, 1979.

Hoehn, S.K., and Kanfer, J.N. J Nutr, 110:2085-2094, 1980.

Holtzman, N.A. Fed Proc, 35:2276-2280, 1976.

Hornig, D. Ann NY Acad Sci, 258:103-117, 1975.

Hunt, D.M. CIBA Found Symp, 79:247-260, 1980.

Kabrt, J., Pribyl, T., Ocenaskova, J., Dobrovolna, E., and Klein, O.

Cas Lek Cesk, 120:211-214, 1981.

Kapeller-Adler, R. Amine oxidases and Methods for Their Study.

New York: Wiley, 1970.

Kaufman, S., Bridgers, W.F., Eisenberg, F., and Friedman, S.

Biochem Biophys Res Commun, 9:497-502, 1962.

Laub, O., Rall, L., Bell, G.I., and Rutler, W.J. J Biol Chem,

258:6037-6042, 1983.

Mains, R.E., Eipper, B.A., Glembotski, C.C., and Dores, R.M.

182
Trends Neuroscience, 6:229-235, 1983,

Mains, R.E., and Eipper, B.A. J Cell Biol, 89:21-28, 1981.

Mains, R.E., Glembotski, C.C., and Eipper, B.A. Endocrinology,

114:1522-1530, 1984.

Milby, K.H., Mefford, I.N., Chey, W., and Adams, R.N. Brain Res

Bull, 7:237-242, 1981.

O’Donohue, T.L., and Dorsa, D.M. Peptides, 3:353-395, 1982.

Pedersen, R.C., and Brownie, A.C. Endocrinology, 112: 1279-1287,

1983.

Pestana, A., and Pitot, H.C. Biochemistry 14:1397-1403, 1975.

Prohaska, J.R., and Smith, T.L. J Nutr, 112:1706-1717, 1982.

Riordan, J.R., In: Sarkar, B., ed., Biological Aspects of Metals

and Metal-Related Diseases. New York, Raven Press, 1983.

pp. 159-170.

Rush, R.A., and Geffen, L.B. CRC Crit Rev Clin Lab Sci, 12:241-

217, 1980.

Saner, A., Welser, H., Hornig, D., DaPrada, M. and Pletscher, A.

J Pharm Pharmacol, 27:896-902, 1975.

Sarkar, B. Metal Ions in Biological Systems, 12:233-281, 1981.

Schreiber, V., Pribyl, T., and Jahodova, J. Endocrinologia

Experimentalis, 13:131-138, 1979.

Simpson, R.T., Cell, 13:691-699, 1978.

Skotland, T., and Ljones, T. Biochem Biophys Acta, 630:30-35,

1980.

Skotland, T., Petersson, L., Backstrom, D., Ljones, T., Flat-mark, T.

and Ehrenberg, A. Eur J Biochem, 103:5-11, 1980.

Snyder, S.H. Science, 224:22-31, 1984.

Sourkes, T.L. CIBA Found Symp, 79:143-154, 1980.

Surprenant, A.M. J Cell Biol, 95:559-566, 1982.

Traugh, J., and Sharp, S.B. J Biol Chem, 252:3738-3744, 1977.

Wand, G., Baylin, S., Ney, R. and Eipper, E. 7th International

Congress of Endocrinology, Quebec, Canada, Abstract, 1984.

Woodford, T.A., and Dixon, J.E. J Biol Chem, 254:4993-4999, 1979.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants

AM-32948 and AM-39249 and by the McKnight Foundation and the

Upjohn Company.

AUTHORS

Richard E. Mains, Ph.D.

Dept. of Neuroscience

The Johns Hopkins University School of Medicine

725 N. Wolfe Street

Baltimore, MD 21205

Betty A. Eipper, Ph.D.

Dept. of Neuroscience

The Johns Hopkins University School of Medicine

725 N. Wolfe Street

Baltimore, MD 21205

183
Opiate-Mediated Control of Luteinizing Hormone in the Male:

Role in the Development of

Narcotic Tolerance and Physical

Dependence

Theodore J. Cicero

In this review, the possible involvement of opiate-induced

perturbations in endocrine status in the development of narcotic

tolerance and physical dependence will be discussed. Although the

opiates affect a wide range of neuroendocrine axes, the discussion

will be limited to their effects on the endocrine secretions of

the hypothalamic-pituitary-gonadal axis in the male rat, since

this system has been by far the most extensively examined. The

review has been divided into three sections: the impact of opiates

on the function of the hypothalamic-pituitary-gonadal axis; the

plasticity and physiological significance of endogenous opioidmediated control of LHRH; and, finally, the preliminary data

implicating opiate-induced alterations in hormonal status in the

development of tolerance to and physical dependence on the

narcotics.

DIRECT EFFECTS OF THE OPIATES ON THE HYPOTHALAMIC-PITUITARYGONADAL AXIS

It has been clearly established that serum testosterone levels are

markedly depressed following acute or chronic narcotic

administration in the male of every species (e.g., Cicero 1980;

Cicero et al. 1976, 1977, 1979; Cicero and Badger 1977; Mendelson

et al. 1979; Mirin et al. 1976). There are four mechanisms that

could account for this: first, narcotics could enhance the

metabolism of testosterone by the liver; second, they could block

the biosynthesis of the steroid at the level of the testes; third,

they could suppress the synthesis and/or release of LH from the

pituitary and, thereby, inhibit LH-dependent testicular steroidogenesis; or, finally, they could inhibit the release of LHRH from

the hypothalamus.

Liver or other target organs. There are no reports in the

literature that either acute or chronic narcotic administration

enhances the catabolism of testosterone. Furthermore, it has been

shown (Cicero et al. 1976, 1977a) that narcotics do not affect the

fate of testosterone in its target organs.

184
Testes. It has been conclusively demonstrated that narcotics do

not depress serum testosterone levels by a direct effect on the

testes. The relevant information can be summarized as follows:

first, narcotics are totally ineffective in lowering serum

testosterone levels when narcotic-induced reductions in LH are

prevented by administration of LH (Cicero et al. 1976; Cicero et

al. 1977 a,b); second, narcotics do not interfere with the basal

or stimulated production of testosterone in the testes (Cicero et

al. 1977a); and, finally, it has been shown (Cicero et al.

1977a,b; Cicero 1980) that the narcotics do not interfere with the

uptake or binding of LH in the testes.

Hypothalamic-pituitary-LH axis. A large number of studies have

demonstrated that narcotics exert their primary effect on the

hypothalamic-pituitary-gonadal axis by depressing the elaboration

of LH by the hypothalamic-pituitary-axis, which leads to a

secondary depression in LH-dependent testicular steroidogenesis

(Cicero et al. 1977a, 1977b; Bruni et al. 1979; Blank et al. 1979;

Kalra 1982; Mendelson et al. 1979; Mirin et al. 1976; Ieiri et al.

1980).

The locus of action of the narcotics within the hypothalamicpituitary-LH axis appears to be at the hypothalamus. The evidence

supporting this conclusion is as follows. First, morphine blocks

the postcastration-induced increase in serum LH in the male rat,

and this effect can be completely overcome by the administration

of LHRH (Cicero et al. 1977b). Second, it has been shown that

even very large doses of morphine do not reduce the elevations in

serum LH produced by LHRH in the rat (Cicero 1980; Cicero et al.

1977b). Third, it has been demonstrated that the narcotics do not

inhibit the basal or LHRH-stimulated synthesis and secretion of LH

from anterior pituitaries incubated in vitro (Cicero et al.

1977b). Finally, it has been shown that both exogenous and

endogenous opiates inhibit the release of LHRH from the

hypothalamus under in vitro conditions (Rotsztejn et al. 1978a,b).

PARTICIPATION OF ENDOGENOUS OPIOIDS IN THE FUNCTION OF THE

HYPOTHALAMIC-PITUITARY-LH AXIS

The findings described above indicate quite clearly that opiate

alkaloid drugs are potent inhibitors of activity within the

hypothalamic-pituitary-gonadal axis in the male and that they

exert their effects exclusively by inhibiting the release of LHRH

from the hypothalamus. As will be more fully discussed below, it

is clear that these drugs affect the release of LHRH via specific

opiate receptors in the hypothalamus which are as yet

unidentified. Since it is quite obvious that these receptors

would not exist simply to bind synthetic opioid compounds, it has

been hypothesized that endogenous opioids exist which normally

play a role in the physiological regulation of LHRH release.

This hypothesis has been impressively supported by the following

data: first, several endogenously occurring opioid peptides

significantly inhibit the secretion of serum LH (Blank et al.

185
1979; Bruni et al. 1977; Meites et al. 1979); second, within

minutes after the subcutaneous injection of naloxone precipitous

increases in serum LH levels occur in the male and female of every

species (e.g., Blank et al. 1979; Mendelson et al. 1979; Mirin et

al. 1976; Cicero et al. 1979; 1980b; Ieiri et al. 1980; Sylvester

et al. 1980; Meites et al. 1979; Wilkes and Yen 1981); third,

under in vitro conditions it has been shown that opiate agonists

and antagonists increase and decrease, respectively, the efflux of

LHRH from the hypothalamus (Rotsztejn et al. 1978a.b; Wilkes and

Yen 1981); and, finally, the negative feedback control of the

hypothalamic-pituitary-LH axis exerted by testosterone (Cicero et

al. 1979) and estrogens (Ieiri et al. 1980; Sylvester et al. 1980;

Van Vugt et al. 1982) in the male and female, respectively, can be

blocked by the administration of naloxone.

The foregoing results have led to the hypothesis that opioidcontaining neuronal elements represent an intermediate link

between the effects of steroids on the hypothalamus and their

ultimate inhibition of the release of LHRH (Cicero 1980; Meites et

al.1979; Van Vugt et al. 1982). If this hypothesis is correct,

then it would follow that endogenous opioids, or narcotic drugs

with an affinity for the relevant opiate receptors, would mimic

the effects of gonadal steroids on the hypothalamic-pituitary-LH

axis. To assess this possibility, we have examined whether

morphine, as a prototypic opioid agonist, had androgen-like

effects in the castrated male rat (Cicero et al. 1980a). We found

that morphine was just as effective as testosterone in preventing

the initial castration-induced increase in serum LH levels and the

reduction in the hypothalamic content of LHRH in the male rat that

occurs 7 to 10 days following orchiectomy (Cicero et al. 198Oa).

Although it is now widely accepted that endogenous opioid systems

are in some way involved in regulating LHRH release from the

hypothalamus, many questions remain unanswered: (a) Which of the

many endogenously occurring opioids is the principal one

involved? (b) Which opiate receptor subtype mediates the effects

of endogenous and exogenous opiates on the hypothalamic-pituitaryLH axis? (c) Are these systems integral and physiologically

relevant components of this axis?

PHYSIOLOGICAL SIGNIFICANCE AND PLASTICITY OF ENDOGENOUS OPIOIDMEDIATED CONTROL OF LHRH.

As discussed above, a major problem encountered in assessing the

characteristics of opioid-mediated control of LHRH is the absence

of any information regarding the identity of the endogenous opioid

involved. This has precluded many crucial experiments, such as

increasing or decreasing the activity of the peptide to assess the

precise nature of the interaction between the relevant opioid

systems and LHRH release mechanisms-a traditional strategy used to

assess the role of neurotransmitters/neuro-modulators. As an

indirect means of examining this issue, we have developed an

different strategy in our laboratory over the last 2 years (Cicero

et al. 1983a,b,c). Specifically, we have used exogenous and

endogenous opioid agonists and antagonists, with presumed affinity

186
FIGURE 1. The effects of morphine, ketocyclazocine and SKF-10,047

on serum LH levels in normal rats (N=12 to 18). T.J. Cicero, D.P.

Owens, P.F. Schmoeker, E.R. Meyer. Opiate-Induced Enhancement of

Naloxone's Effects on Serum Luteinizing Hormone Levels in the Male

Rat: Specificity for mu Agonists. The Journal of Pharmacology and

Experimental Therapeutics, 226:770-775, 1983. ©1983, Williams

and Wilkins. Reprinted by permission.

for specific opiate receptor subtypes in brain, to selectively

enhance or block opiate receptors and then assessed the response

to subsequently administered opiate agonists and antagonists. Our

rationale was that, since endogenous opioids must interact with a

receptor to exert their effects, an alteration in the

responsiveness of these receptors would have the same effect as

enhancing or depressing the actions of the peptide, accomplished

by more traditional means (e.g., destruction of the pathways).

Thus, our strategy been two-fold: first, to determine whether one

or more receptors appear to mediate the effects of endogenous and

exogenous opioids on the release of LHRH; and, second, to

determine whether these receptors are influenced by the prior

administration of opioid agonists and antagonists.

187
FIGURE 2. Percent inhibition of serum LH levels produced by a

wide range of opiates. Each dose-response curve is the best-fit

line and represents at least 5 doses of each drug with an N of 18

to 40. The slopes of the lines were virtually identical except

for ketocyclazocine (p<.Ol) and cyclazocine (p<.O5) which were

significantly more steep than the others. Data for SKF have not

been included since it had no effect on serum LH levels. T.J.

Cicero, D.P. Owens, P.F. Schmoeker, E.R. Meyer. Opiate-Induced

Enhancement of Naloxone's Effects on Serum Luteinizing Hormone

Levels in the Male Rat: Specificity for mu Agonists. The Journal

of Pharmacology and Experimental Therapeutics, 226:770-775,

1983. ©1983, Williams and Wilkins. Reprinted by permission.

Effects of mu, kappa and sigma opiate agonists on serum LH

levels. The time-course for the effects of morphine.

ketocyclazocine and SKF on serum LH levels are shown in figure

1. As can be seen, following an injection of 5 mg/kg morphine

oketocyclazocine, serum LH levels declined sharply in a timedependent fashion, reaching peak levels of depression 1 to 3 hours

after the injection; thereafter, LH levels gradually returned to

control levels. Six to 8 hours after the injection, serum LH

levels "rebounded" significantly above control levels and then

returned to normal values 8 to 24 hours later. In contrast to the

effects of morphine and ketocyclazocine, 15 mg/kg SKF had no

188
TABLE 1

ED50s and potencies of various opiate agonists

in depressing serum LH levels

Agonist ED50s (95% confidence limits

Morphine 0.41 (0.37-0.43)

Potency

(morphine=1

Etorphine 0.0012 (0.0009-0-0.0016) 341.6

Ketocyclazoeine 0.21 (0.19-0.24) 1.95

Cyclazocine 0.27 (0.23-0.30) 1.52

Levorphanol 0.29 (0.25-0.36) 1.41

Methadone 1.30 (1.19-1.38) 0.32

Codeine 6.0 (5.81-6.20) 0.07

T.J. Cicero, D.P. Owens, P.F. Schmoeker, E.R. Meyer, OpiateInduced Enhancement of Naloxone's Effects on Serum Luteinizing

Hormone Levels in the Male Rat: Specificity for mu Agonists. The

Journal of Pharmacology and Experimental Therapeutics, 226:770-

775 1983 © 1983, Williams and Wilkins. Reprinted by .

permission.

TABLE 2

Naloxone antagonism of opiate-induced

inhibitions of serum LH levels

Agonist Ke (nM)

Morphine 1.41(±0.23) p A 27.86 (±0.07)

Levorphanol 1.44 (±0.05) 7.87 (±0.02)

Methadone 1.49 (±0.06) 7.82 (±0.01)

Ketocyclazocine 11.99 (±1.13) 6.92 (±0.05)

Cyclazocine 13.27 (±0.89) 6.87 (±0.50)

pA2 and Ke values

(Tallarida et al 1981).

were determined as described elsewhere

T.J. Cicero, D.P. Owens, P.F. Schmoeker, Meyer, E.R., OpiateInduced Enhancement of Naloxone's Effects on Serum Luteinizing

Hormone Levels in the Male Rat: Specificity for mu Agonists. The

Journal of Pharmacology and Experimental Therapeutics, 226:770-

775 1983 © 1983, Williams and Wilkens. Reprinted by .

permission.

significant effect on serum LH at any post-injection time interval

examined .

On the basis of the preceding studies, a 2-hour time interval was

selected to construct LH dose-response curves for a wide range of

opiate agonists. Figure 2 depicts the best-fit lines, plotted on

log-probit paper; all opiates tested depressed serum LH levels in

a dose-dependent fashion, with the exception of SKF which had no

189
FIGURE 3. Serum LH levels in animals injected with morphine (10

mg/kg) or saline and then injected with saline or naloxone (0.5

mg/kg) at the intervals shown. Values for saline + saline and

saline + naloxone groups have been pooled. T.J. Cicero, D.P.

Owens, P.F. Schmoeker, Meyer, E.R. Morphine-Induced Supersensitivity to Naloxone's Effects on Luteinizing hormone Secretion in

the Male Rat. The Journal of Pharmacology and Experimental

Therapeutics, 225:35-41, 1983. ©1983, William and Wilkins.

Reprinted by permission.

appreciable effect at any dose examined. The ED50s and the

potencies of these compounds, relative to morphine, are shown in

table 1.

Naloxone blockade of opiate-induced depressions in serum LH

levels. The efficacy of naloxone in competitively inhibiting the

LH-depressing effects of several representative opiates is shown

in table 2. As can be seen, naloxone effectively reversed the

effects of both putative mu and kappa agonists, but was nearly 10

times more potent in antagonizing the effects of mu agonists than

it was for kappa compounds. In addition, it is also apparent that

two distinct binding sites appear to mediate the effects of these

agonists.

190
FIGURE 4. The effects of naloxone (0.025 mg/kg) or saline on

serum LH levels in rats (N=20) implanted with morphine or placebo

pellets 48 hours earlier. *Significantly (p<.Ol) lower than

placebo-saline groups; **Significantly (p<.OOl) higher than all

other groups. T.J. Cicero, D.P. Owens, P.F. Schmoeker, Meyer,

E.R. Morphine-Induced Supersensitivity to Naloxone's Effects on

Luteinizing Hormone Secretion in the Male Rat. The Journal of

Pharmacology and Experimental Therapeutics, 225:35-41, 1983.

©1983, Williams and Wilkins. Reprinted by permission.

Influence of acute morphine administration on naloxone-induced

Increases in serum LH levels. The effects of a single morphine or

saline injection on the increase in serum LH levels produced by

naloxone at various intervals after the initial injection are

shown in figure 3. As can be seen, in animals receiving saline or

naloxone as the second injection, serum LH levels were substantially depressed in morphine-pretreated rats when compared to

saline-pretreated animals from 2 to 4 hours after the initial

injections. Thereafter, LH levels returned to normal levels in

control values 6 to 24 hours after the injection. With respect to

the effects of naloxone on serum LH levels in morphine and saline

pretreated rats, 2-3 hours after the morphine injection, naloxoneinduced increases in serum LH were suppressed, when compared to

saline-injected controls. By 4 hours post-morphine injection,

191
TABLE 3

Naloxone dose-response curves in controls and in

animals injected acutely with morphine or

implanted with morphine pellets.

Saline - Placebo

ED50(95% confidence limits)

0.23 (0.18-0.25)

Acute Morphine 0.124 (0.116-0.131)

Morphine Pellet 0.0053 (0.0048-0.0056)

normal LH responses to naloxone returned. However, 5-24 hours

after morphine, the increases in serum LH levels produced by

naloxone were markedly exaggerated in morphine pretreated animals

when compared to controls, with the peak degree of enhanced

sensitivity occurring at 6 hours.

Naloxone-induced increases in serum LH levels in morphine pellet

implanted animals. The effects of saline or naloxone on serum LH

levels in male rats implanted with a single morphine or placebo

pellet 48 hrs earlier are shown in figure 4. Serum LH levels were

significantly depressed in morphine-implanted animals when

compared to placebo controls. Moreover, at this dose of naloxone

(0.025 mg/kg) there was no statistically significant stimulation

of serum LH levels in placebo-implanted male rats. In marked

contrast to these results, naloxone was extremely effective in

elevating serum LH levels in morphine-implanted animals, producing

increases nearly 8 times greater than those found in controls.

Magnitude of the shift in the naloxone dose response curve after

acute and chronic morphine administration. The naloxone doseresponse curves generated in rats pretreated with saline/placebo,

a morphine injection or one morphine pellet implantation are shown

in figure 5. Both acute and chronic morphine administration

markedly shifted the naloxone dose-response curve significantly to

the left when compared to controls. However, this shift was much

more striking in chronically treated animals when compared to

controls or those animals receiving acute morphine pretreatment.

the ED50s in the respective groups are shown in table 3.

Opiate-induced enhancement of naloxone's effects on serum LH

levels. To assess whether the opiate-induced enhancement of

naloxene's effects was specific to morphine or represented a more

general property of the opiates, we assessed whether the wide

range of opiates utilized in the preceding studies (see figure 2)

produced an enhanced response to naloxone. The results of these

experiments are shown in figure 6. As can be seen, serum LH

levels in opiate-injected animals, receiving saline as the second

injection 6 hours later, were somewhat elevated with respect to

saline-pretreated animals (shaded area in this figure). When

naloxone was given as the second injection, a markedly enhanced

response was observed in those animals pretreated with mu

agonists, relative to saline-pretreated controls; the order of

potency was etorphine >levorphanol >morphine >methadone >codeine.

192
FIGURE 5. Naloxone dose-response curves in animals (N=17 to 57)

injected with saline or morphine (10 mg/kg) 6 hours earlier or

implanted with morphine or placebo pellets 48 hr earlier. T.J.

Cicero, D.P. Owens, P.F. Schmoeker, Meyer, E.R. Morphine-induced

Supersensitivity to Naloxone's Effects on Luteinizing Hormone

Secretion in the Male Rat. The Journal of Pharmacology and

Experimental Therapeutics, 225:35-41, 1983. © 1983 Williams and

Wilkins. Reprinted by permission.

In contrast to these results neither ketocyclazocine, cyclazocine

nor SKF produced any facilitation of naloxone's effects.

Effects of FK 33-824 on naloxone-induced increases in serum LH

levels. To determine whether FK 33-824 as a metabolically stable

analog of the endogenously occurring methionine enkephalin (Roemer

et al. 1977), produced an enhanced sensitivity to naloxone's

effects on serum LH levels, rats were injected with saline or FK

33-824 at a dose (0.05 mg/kg) selected to produce modest decreases

in serum LH levels (Cicero et al. 1983c). At intervals, thereafter, the animals in each group received a second injection of

either saline or naloxone (0.25 mg/kg). The results of this study

are shown in figure 7. Serum LH levels were modestly reduced in

FK 33-824-saline injected animals, when compared to controls at

intervals up to 2 hours after the initial injection: thereafter

the two groups were indistinguishable. When naloxone was given as

193
FIGURE 6. Serum LH levels in animals (N=20-28) injected with

saline or opiate followed 6 hours later by saline (see shaded area

within bar) or 0.25 mg/kg naloxone (open bars). The doses of each

opiate were selected to provide an initial 60-80% inhibition of

serum LH 2 hours after its adminitration, and were as follows:

etorphine .005 mg/kg, levorphanol 0.5 mg/kg, morphine 2.5 mg/kg,

methadone 2.5 mg/kg, codeine 7.5 mg/kg, cyclazocine 0.5 mg/kg,

ketocyclazocine 0.5 mg/kg, and SKF 5 mg/kg. *Significantly

(P<.01) greater than saline-naloxone injected animals;

†significantly (p<.05) greater than saline-saline injected

animals. T.J. Cicero, D.P. Owens, P.F. Schmoeker, E.R. Meyer.

Opiate-Induced Enhancement of Naloxone's Effects on Serum

luteinizing Hormone Levels in the Male Rat: Specificity for mu

Agonist. The Journal of Pharmacology and Experimental

Therapeutics, 226:770-775, 1983. ©1983, Williams and Wilkins.

the second injection to saline-pretreated rats, serum LH levels

were significantly elevated above values obtained in saline-saline

treated animals, but the increase was quite small at the very low

dose of the narcotic antagonist employed. In the case of FK 33-

824, the inhibitory effects of the peptide on serum LH levels were

initially (<2 hours) overcome by naloxone. However, at 4 to 6

194
FIGURE 7. Serum LH levels in animals (N=10 to 15) injected with

saline or FK 33-824 (0.05 mg/kg) followed by saline or naloxone

(0.25 mg/kg) at the intervals shown. Values for the saline-saline

and saline-naloxone groups were pooled (shown as continuous solid

or dashed lines). *Significantly (p<.01) higher than respective

control; †Significantly (p<.O5) lower than control. T.J. Cicero,

D.P. Owens, K.S. Newman, P.F. Schmoeker, Meyer, E.R. Exogenous

and Endogenous Opioid-induced Enhancements of Naloxone's Effects

on Serum Luteinizing Hormone Levels in the Male Rat. European

Journal of Pharmacology, in press. © 1984, Elsevier Biomedical Press.

hours after the FK 33-824 injection, naloxone was substantially

more effective in increasing serum LH levels than it was in

saline-pretreated animals. By 8 hours after the injection of FK

33-824 essentially normal responses to naloxone had returned.

Naloxone-precipitated withdrawal behavior following acute and

chronic morphine pretreatment. Naloxone-precipitated withdrawal

sians in rats implanted with morphine or placebo pellets 48 hours

earlier are shown in figure 8. Naloxone (0.5 mg/kg) produced a

vigorous withdrawal response in morphine-implanted rats. No other

group of animals, notably morphine-implanted rats injected with

0.005 mg/kg naloxone, displayed any opiate withdrawal behavior.

195
FIGURE 8. Naloxone-precipitated incidence of wet-dog shakes

(number/hour), body weight loss (gm/hour) and diarrhea (for rating

scale see Cicero and Meyer, 1973) in animals implanted with

morphine or placebo pellets 48 hours earlier. T.J. Cicero, D.P.

Owens, P.F. Schmoeker. E.R. Meyer. Opiate-Induced Enhancement of

Naloxone's Effects on Serum Luteinizing Hormone Levels in the Male

Rat: Specificity for mu Agonists. The Journal of Pharmacology and

Experimental Therapeutics, 226:770-775, 1983. © 1983 Williams

and Wilkins. Reprinted by permission.

Mechanisms involved in opiate-induced enhancement of naloxone's

effects on serum LH levels. In a series of studies we have

explored a number of mechanisms which could be involved in the

development of opiate-induced enhancement of naloxone's effects.

The experiments (Cicero et al. 1983a,b,c) can be summarized as

follows: first, morphine pretreatment did not influence the uptake

of naloxone into brain; second, changes in the sensitivity of the

pituitary to LHRH do not seem to be involved in this phenomenon,

since LHRH dose-response curves were identical in saline- and

morphine-pretreated animals; and, finally, we have been unable to

demonstrate any gross changes in the affinity or density of mu,

delta or kappa binding sites in whole brain or the hypothalamus of

morphine-pretreated rats when compared to controls.

196
DISCUSSION

The results of these studies demonstrate that either acute or

chronic morphine pretreatment markedly sensitizes male rats to the

subsequent effects of naloxone on serum LH levels. This

phenomenon appears to represent a true example of "supersensitivity", since the following criteria were met: a parallel shift

to the left in the dose-response curve, and an enhanced effect of

naloxone on serum LH levels at the same brain concentration of the

antagonist. Although both acute and chronic morphine

administration produced an enhanced sensitivity to naloxone,

relative to controls, there were substantial differences between

the two treatment regimens. For example, although acute and

chronic morphine administration produced an exaggerated LH

response to naloxone of approximately the same magnitude, the peak

effect was elicited by much lower doses of naloxone in morphineimplanted rats than it was in morphine-injected animals. In

addition, at comparable doses, naloxone-induced increases in serum

LH levels were up to 20 times greater in chronically morphinized

animals than they were in those rats receiving a single injection

of morphine. Finally, although a single injection of 10 mg/kg

morphine significantly lowered the dose of naloxone producing the

half-maximal increase in serum LH levels, relative to controls,

the naloxone ED50 morphine-pelleted animals was several orders of

magnitude less than that found in controls or in acutely

morphinized animals.

Our studies also revealed that an enhancement of naloxone's

effects on serum LH levels is not a common feature of all drugs

which depress activity within the hypothalamic-pituitary axis

since neither non-opiate (ethanol and Nembutal) nor certain opiate

compounds (cyclazocine and ketocyclazocine) produced this effect,

despite a profound initial depression in serum LH levels (Cicero

et al. 1983a,b,c). Moreover, we found that this phenomenon is not

a general feature of all opiates, but rather is restricted to only

those agents with an established affinity for mu binding sites in

the brain. We were unable to demonstrate a facilitation of

naloxone's effects by the administration of ketocyclazocine,

cyclazocine or SKF, representative kappa and sigma agonists

(figure 6).

Our observations indicate that at least two receptors appear to be

involved in suppressing LH release. In this regard, it is somewhat

puzzling that an enhancement of naloxone's effects develops only

as a function of mu receptor occupancy, particularly since

ketocyclazocine and cylazocine were more potent in depressing LH

levels than the mu agonists assessed, with the exception of

etorphine. However, it should be noted that functionally important

changes in the activity of the hypothalamic-pituitary-LH axis,

such as those produced by castration and/or excess steroid

administration, have previously been shown to attenuate the

effects of mu agonists and antagonists on LH secretion (Cicero et

al. 1982; Owens and Cicero 1981) and to alter the density (Bmax)

of mu binding sites in whole rat brain (Hahn and Fishman 1979).

197
If one makes the assumption that functionally important changes in

the activity of the hypothalamic-pituitary axis would be reflected

in changes in all components of the axis, including endogenous

opioid-containing neuronal systems, then the selective alterations

in mu receptor function observed in these and other studies could

indicate that they play a significant role in regulating the

release of LHRH.

Our data also indicate that a metabolically stable analog of

methionine enkephalin (FK 33-824) produced an enhanced response to

naloxone analogous to, and perhaps greater than, that generated by

opiate alkaloid agonists. These data indicate that an enhancement

of naloxone's effects on serum LH levels is not a unique event

associated with the administration of drugs, but can be generated

by endogenously occurring compounds. Moreover, since it has been

suggested in a number of recent reports (Cox and Baizman 1982;

Naber et al. 1981; Reid et al. 1982) that endogenous opioids, like

most neurotransmitters/neuro-modulators, are released in a

pulsatile manner, it seems probable that the changes in the

sensitivity of those opiate receptors involved in LHRH release

observed under our experimental conditions may occur under in vivo

conditions.

Based upon our results, it seems certain that the development of

an opiate-induced enhancement of naloxone's effects on serum LH

levels is due primarily to fundamental alterations in the

hypothalamus, rather than the pituitary. The nature of the

changes occurring in the hypothalamus are not clear, but we have

shown that the uptake, metabolism and regional distribution of

naloxone are not affected by prior opiate administration (Cicero

et al. 1983a) and, hence, it is unlikely that there is an increase

in the availability of the antagonist at its active binding

sites. Rather, the data are consistent with the interpretation

that the nature of the ligand-receptor interaction has been

altered in some way. However, we have been unable to demonstrate

any change in the Kd or Bmax of [

3

H]-naltrexone binding to its

receptors in whole brain or hypothalamus, nor have we been able to

discern differences in the relative proportion of mu and delta

binding sites following morphine administration (Cicero et al.

1983a,b,c). It would be premature, however, to dismiss the

possibility that such changes may occur under in vivo conditions

since it is possible that morphine pretreatment produces subtle

changes in opiate receptors in the intact membrane in highly

discrete areas of brain which could be obscured by the in vitro

analysis of receptor properties using whole brain or gross

regional analysis.

The factors leading to the development of supersensitivity to

naloxone's effects on serum LH levels are not understood, but on

the basis of our studies, the following conclusions can be

drawn. First, only those opiates with an affinity for mu binding

sites in brain produce an enhancement of naloxone's effects on

serum LH levels; opiates which bind selectively to kappa or sigma

binding sites are devoid of such activity (Cicero et al. 1983c)

198
Second, it appears that occupancy of mu receptors alone is

insufficient to promote the development of supersensitivity to

naloxone's effects, but that an initial depression in serum LH

levels, followed by a rebound to supranormal levels is an absolute

requirement (Cicero et al. 1983c). However, it should be noted

that a depression of LH per se cannot be the sole determining

factor, since many opiate and non-opiate compounds which depress

LH do not lead to the development of supersensitivity (Cicero et

al. 1983c).

The present results add to the growing evidence supporting the

hypothesis that endogenous opioid-containing neuronal systems play

an integral role in regulating LHRH release in the male and

female. Our results not only reinforce these conclusions but

indicate that these systems are markedly sensitive to transient

phases of depression and excitation.

As a potentially important offshoot of the work described in this

paper, it appears that naloxone-induced increases in serum LH

levels can serve as an extremely useful index of opiate

dependence. A sensitive and reliable index of the extent and

severity of opiate dependence should satisfy at least three

criteria: first, that naloxone produces increases in serum LH

levels in morphine pretreated rats which are greater than those

found in controls; second, that the naloxone dose-response curve,

and corresponding ED50 should shift in parallel to the left; and,

third, that the enhanced sensitivity to naloxone-induced increases

in serum LH levels should correlate with the development of other

behavioral and physiological indices of opiate withdrawal. From

the data presented above, it seems clear that the naloxoneprecipitated increase in serum LH levels in the morphine-exposed

male rat satisfies each of these criteria. Although acute

naloxone-precipitated withdrawal has been reported by a number of

investigators following morphine administration (Aceto et al.

1977; Cox et al. 1968; Kosersky et al. 1974; Meyer and Sparber

1977; Smits 1975; Wiley and Downs 19791, the increase in the

incidence of withdrawal signs has been generally unremarkablemaking quantifiable assessments difficult at best. In contrast,

we found in the present studies that a single morphine injection

produced an extremely exaggerated LH response to naloxone which

was readily quantifiable and highly reproducible. In addition, we

found that with chronic morphine exposure, naloxone-induced

increases in serum LH paralleled the development of other signs of

withdrawal and that the shift in the sensitivity to naloxone was

many times greater than that observed for these other signs.

CONSEQUENCES OF OPIATE-INDUCED ALTERATIONS IN HORMONES SECRETED BY

THE HYPOTHALAMIC-PITUITARY-GONADAL AXIS

Because of the pervasive influence of hormones in the biochemistry

and function of every organ in the body, it seems reasonable to

postulate that acute or persistent drug-induced alterations in any

of these hormones could play a significant role in the effects of

these drugs, particularly in those adaptive changes occurring

199
FIGURE 9. The effects of a single morphine or placebo pellet

implant on serum LH levels in male rats (N=10) that had been

castrated from 0 to 27 days earlier. * Significantly (p<.OOl)

lower when compared to placebo-implanted animals. T.J. Cicero,

E.R. Meyer, P.F. Schmoeker. Development of Tolerance to the

Effects of Morphine on Luteinizing Hormone Secretion as a Function

of Castration in the Male Rat. The Journal of Pharmacology and

Experimental Therapeutics, 223:784-789, 1982. © 1982 , Williams

and Wilkins. Reprinted by permission.

during the development of tolerance and physical dependence. We

have recently accumulated a good deal of evidence which supports

this hypothesis.

Functional tolerance. In earlier work (Cicero et al. 1980a). we

found that tolerance appeared to develop to the effects of

morphine on LH secretion as a function of castration. For

example, in male rats castrated for 3 days, morphine was a potent

suppressor of serum LH levels, but was virtually without effect in

animals that had been castrated for 2 weeks. In these studies,

however, morphine was administered by pellet implantation so that

it was not possible to ascertain the magnitude of the shift in

sensitivity to morphine. Because our earlier observations could

have considerable significance with respect to the plasticity of

200
FIGURE 10. The percent inhibition of serum LH levels produced by

various doses of morphine (mg/kg) 30 min after its administration

to male rats (N=8 to 10) castrated 3 or 31 days earlier. T.J.

Cicero, E.R. Meyer, Schmoeker, P.F. Development of Tolerance to

the Effects of Morphine on Luteinizins Hormone Secretion as a

Function of Castration in the Male- Rat. The Journal of

Pharmacology and Experimental Therapeutics, 223:784-789, 1982.

1982, Williams and Wilkins. Reprinted by permission.

the endogenous opioid system controlling the hypothalamicpituitary axis and the possible involvement of hormones in the

development of tolerance to and physical dependence on the

opiates, the difference in the sensitivity of castrated male rats

to an opiate agonist (morphine), antagonist (naloxone) and

testosterone has been further characterized in several recent

studies.

Differences in the response of castrated male rats to morphine.

To examine the time course for the change in the response to

morphine in the castrated animal, groups of rats were implanted

with a single morphine or placebo pellet at intervals after

castration and were killed 24 hr later. The results are shown in

figure 9. When morphine pellets were implanted at the same time

or 1 day after castration, the post-castration-induced increases

201
FIGURE 11. The percent inhibition of serum LH levels produced by

testosterone in rats (N=8) castrated 3 or 31 days earlier. T.J.

Cicero, E.R. Meyer, Schmoeker, P.F. Development of Tolerance to

the Effects of Morphine on Luteinizing Hormone Secretion as a

Function of Castration in the Male Rat. The Journal of

Pharmacology and Experimental Therapeutics, 223:784-789, 1982. ©

1982 Williams and Wilkins. Reprinted by permission.

in serum LH levels were nondetectable in morphine-treated rats.

morphine was administered 2 to 3 days after castration, it was

slightly less potent, but, even so, markedly (>80%) depressed

serum LH levels. Thereafter, morphine became progressively less

effective in reducing serum LH levels such that if it was

administered at 13 or 27 days after castration, it did not

significantly reduce serum LH levels.

Magnitude of the shift in sensitivity to morphine in castrates.

The foregoing studies revealed the time course of the change in

the sensitivity of LH secretion to morphine, but gave no

indication of the magnitude of the effect because only pellets

were employed. To determine the extent to which castrated rats

were resistant to the LH-reducing properties of morphine, acute

dose-response curves were determined in rats castrated 3 or 31

days earlier. The results are shown in figure 10. Morphine

202
produced a 50% inhibition in serum LH levels at a dose of

approximately 1.5 mg/kg in animals castrated 3 days earlier, which

is comparable to that previously found in normal rats or those

castrated for similar periods (Cicero et al. 1979, 1980a,b). In

contrast to these results, long-term castration (31 days) produced

a parallel shift to the right in the morphine dose-response curve

and the ED50 was markedly reduced.

Magnitude of the shift in sensitivity to testosterone in

castrates. The dose-response curves for testosterone-induced

decreases in serum LH levels in animals castrated 3 or 31 davs

earlier are shown in figure 11. Testosterone more effectively

suppressed LH levels in animals castrated 3 days earlier than it

did in 31-day castrates.

Uptake of morphine into the brains of long-term castrated

animals. To rule out the possibility that the diminished response

to morphine observed in long-term castrates was due to an enhanced

metabolism of morphine and/or an altered uptake of the drug into

the brain, the amount of morphine found in the brains of 31-day

castrates was compared with that found in sham-operated animals 30

min after its injection. There was no significant difference in

the amount of morphine present in brain 30 min after its

administration in 31-day castrates relative to sham-operated

controls (data not shown).

Effects of castration on the antinociceptive activity of

morphine. In an effort to determine whether the apparent

tolerance to the effects of morphine on LH secretion was

generalizable to all effects of morphine on the central nervous

system, the antinociceptive activity of morphine was assessed in

31-day castrates and sham-operated controls. Castrated rats did

not differ significantly from sham-operated controls.

DISCUSSION

The results of these studies indicate that castration renders male

rats less responsive to the effects of morphine on LH secretion.

The decrease in sensitivity to morphine appears to satisfy the two

pharmacological criteria for tolerance: a parallel shift in the

dose-response curve to the right, and a reduced effect of the drug

at the same brain concentration. Our results further suggest that

the tolerance observed in the present studies is not generalizable

to other effects of morphine on the brain because the doses of

morphine required to produce inhibitions of serum LH levels in

long-term castrates by 60% or more (50-75 mg/kg) produced a

significant number of deaths (30-40%) which is comparable to that

observed in normal animals (Cicero and Meyer 1973). Moreover, we

also found that the antinociceptive activity of morphine was not

reduced in long-term castrated animals when compared with shamoperated controls. Thus, our results indicate that the

development of tolerance to the effects of morphine in the

castrated male rat appears to be restricted to LH secretion. The

castration-induced development of tolerance to the effects of

203
morphine on LH secretion was accompanied by a change in the

responsivity of the hypothalamic-pituitary axis to testosterone;

however, it should be noted that the shifts in the morphine and

testosterone dose-response curves were not equivalent in our

studies since the ED5O for morphine increased nearly 10-fold in

long-term castrates, relative to short-term castrates, whereas

only a 4- to 5-fold increase was found in the case of

testosterone. Furthermore, in terms of the absolute decrease in

serum LH levels, at specific doses of testosterone and morphine,

long-term castrates appeared to be slightly more resistant to

testosterone than they did to morphine. Nevertheless, it seems

clear that long-term castration produces decreases in the

sensitivity of the hypothalamic-pituitary axis to both morphine

and testosterone. These data, while correlational in nature,

support the previously suggested hypothesis that opioid-containing

neuronal elements tonically inhibit LHRH release and may represent

a bridge between the actions of steroids on the hypothalamus and

their ultimate inhibition of LHRH release.

The mechanisms underlying the development of tolerance to the

effects of morphine on LH secretion in castrated male rats are not

clear. Two prominent possibilities exist, however; first,

castration could produce testosterone-reversible changes in the

affinity (Kd) or density (Bmax) of the relevant opiate receptors

involved in LHRH/LH release; and, second, castration could elevate

the levels of endogenous opioids in certain areas of brain. With

respect to the first of these possibilities, Hahn and Fishman

(1979) found marked increases in the Bmax of [

3

H]-naltrexone

binding sites in whole brains of male rats castrated for 3 or more

weeks; testosterone readily overcame these effects. Although

these data support the hypothesis that castration-induced changes

in opioid receptors could be involved in the development of

tolerance to the effects of morphine on LH secretion that we have

observed in the present studies, we (Cicero et al. 1983d) and

several other groups (Bhanot and Wilkinson 1983; Diez and Roberts

1982: Wilkinson et al. 1981) have been unable to replicate their

observations. It is not clear what accounts for this difference

in results, but on balance it appears that castration does not

influence opiate receptor populations in brain.

The possibility that castration may produce testosteronereversible elevations in endogenous opioid levels in critical

regions of brain has not been systematically examined, but it has

been reported that a number of endocrine manipulations significantly alter opioid levels in brain, pituitary and a number of

target organs (Mueller 1980; DuPont et al. 1982; Hong et al.

1982). If castration does in fact increase opioid levels in the

hypothalamus or other regions of brain, this mechanism could be

significantly involved in the effects we have observed.

Specifically, prolonged elevations in endogenous opioids could

lead to a desensitization of opioid receptors such that the

effects of subsequently administered exogenous or endogenous

opioids are attenuated, much as is seen in the opiate-dependent

animal. If this occurs, then one would expect, as we found, that

204
the morphine dose-response curve with respect to increases in LH

would be shifted in parallel to the right. This explanation is a

parsimoniuous one and explains all of our data quite well, but it

should be reiterated that castration-induced changes in endogenous

opioid levels, synthesis and/or release in rat brain have yet to

be demonstrated.

The preceding results could have considerable significance with

respect to the plasticity of the endogenous opioid system

regulating the secretion of LH. In previous work, we have shown

that tolerance develops to the effects of naloxone on the

secretion of LH after acute or chronic administration (Owens and

Cicero 1981) and that morphine tolerance markedly sensitizes male

rats to the effects of naloxone. These observations, coupled with

the present results, suggest that there is a great deal of

plasticity in the opioid-regulated control of LHRH release. It

seems probable that these changes could have considerable

physiological significance and further studies directed at this

issue appear warranted.

In addition, our results suggest that the hormonal status of the

animal greatly influences the development of tolerance and

possibly physical dependence to the opiates. Since these

compounds exert potent effects on most neuroendocrine systems, it

seems probable that many of these changes may be intimately

involved in both the acute and chronic effects of this class of

compounds. Moreover, because most psychoactive compounds

influence neuroendocrine status, it may well be that the crosstolerance and interactions between these compounds may share a

common biochemical mechanism (i.e. perturbations in endocrine

status).

REFERENCES

Aceto, M.D., Flora, R.E. and Harris, L.S. The effects of naloxone

and nalorphine during the development of morphine dependence

in rhesus monkeys. Pharmacology, 15:1-19, 1977.

Bhanot, R., and Wilkinson, M. Opiate binding to brain slices and

antagonists of hypothalamic [3H]naloxone binding sites. Int J

Develop Neurosci, 1983, in press.

Blank, M.S., Panerai, A.E., and Friesen, H.G., Opioid peptides

modulate luteinizing hormone secretion during sexual

maturation. Science, 203:1129-1131, 1979.

Bruni, F.F, Van Vugt, D., Marshall, S., and Meites, J. Effects of

naloxone, morphine and methionine- enkephalin on serum

prolactin, luteinizing hormone, follicle-stimulating hormone,

thyroid-stimulating hormone, and growth hormone. Life Sci,

21:461, 1977.

Cicero, T.J., and Meyer, E.R., Morphine pellet implantation in

rats: Quantitative assessment of tolerance and dependence. J

Pharmacol Exp Ther, 184:404-408, 1973.

205
Cicero, T.J., Meyer, E.R., Bell, R.D., and Koch, G.A., Effects of

morphine and methadone on serum testosterone and luteinizing

hormone levels and on the secondary sex organs of the male

rat. Endocrinology, 98:367-372, 1976.

Cicero, T.J. and Badger, T.M., A comparative analysis of the

effects of narcotics: alcohol and the barbiturates on the

hypothalamic-pituitary-gonadal axis. Adv Exp Med Biol,

858:95-116, 1977. (WE HAVE NO REPRINTS)

Cicero, T.J., Bell, R.D., Meyer, E.R. and Schweitzer, J.,

Narcotics and the hypothalamic-pituitary-gonadal axis: Acute

effects on luteinizing hormone, testosterone and androgendependent systems. J Pharmacol Exp Ther, 201:76-83, 1977a.

Cicero, T. J., Badger, T.M., Wilcox, C.E., Bell, R.D., and Meyer,

E.R.. Morphine decreases luteinizina hormone bv an action on

the -hypothalamic-pituitary axis. J Pharmacol Exp Ther,

203:548-555, 1977b.

Cicero, T.J., Schainker, B.A., and Meyer, E.R. Endogenous opioids

participate in the regulation of the hypothalamic-pituitaryluteinizing hormone axis and testosterone's negative feedback

control of luteinizing hormone. Endocrinology, 104:1286-1291,

1979.

Cicero, T.J., Effects of exogenous and endogenous opiates on the

hypothalamic-pituitary-gonadal axis in the male. Fed Proc,

39:2551-2554. 1980.

Cicero, T.J., Meyer, E.R., Gabriel, S.M., Bell, R.D., and Wilcox,

C.E.. Morphine exerts testosterone-like effects in the

hypothalamus of the castrated male rat. Brain Res, 202:151-

164. 1980a.

Cicero, T.J., Wilcox, C.E., Bell, R.D., and Meyer, E.R.,

Naloxone-induced increases in serum luteinizing hormone in the

male: Mechanisms of action. J Pharmacol Exp Ther, 212:573-

578, 1980b.

Cicero, T.J., Meyer, E.R., and Schmoeker. P.F., Development of

tolerance to the effects of morphine on luteinizing hormone

secretion as a function of castration in the male rat. J

Pharmacol Exp Ther, 223:784-789, 1982.

Cicero, T.J., Owens, D.P., Schmoeker, P.F., and Meyer, E.R.,

Morphine-induced supersensitivity to naloxone's effects on

luteinizing hormone secretion in the male rat. J Pharmacol

Exp Ther, 225:35-41, 1983a.

Cicero, T.J., Owens, D.P., Schmoeker, P.F. and Meyer E.R.,

Opiate-induced enhancement of naloxone's effects on serum

luteinizing hormone levels in the male rat: Specificity for mu

agonists. J Pharmacol Exp Ther, 226:770-775, ; 1983b.

Cicero, T.J., Owens, D.P., Newman, K.S., Schmoeker, P.F. and

Meyer, E.R., Exogenous and endogenous opioid-induced

enhancements of naloxone's effects on serum luteinizing

hormone levels in the male rat. Eur J Pharmacol, in press,

1983c.

Cicero, T.J., Newman, K.S., and Meyer, E.R. Testosterone does not

influence opiate binding sites in the whole rat brain. Life

Sci, 33:1231-1239. 1983d.

Cox, B.M., Ginsberg, M., and Osmond, O.H. Acute tolerance to

narcotic analgesic drugs in rats. Br J Pharmacol Chemother.

33:245-256, 1968.

206
Cox, B.M. and Baizman. E.R. Physiological functions of

endorphins. In: Malick, J.B. and Bell, R.M.S. ed. Endorphins:

Chemistry, Physiology, Pharmacology and Clinical Relevance,

Marcel Dekker, Inc., 1982. p. 141.

Diez, J.A. and Roberts, D.L. Evidence contradicting the notion

that gonadal hormones regulate brain opiate receptors.

Biochem Biophys Res Commun, 108:1313-1319, 1982.

DuPont, A Merand, Y., Rouleau, D., Cusan, L., Lemay, A., Vaudry,

H ., Jegan, S., Lepin. J., and Barden, N. In: Shah, N.S. and

Donald, A.G. eds. Endorphins and Opiate Agonists in Psychiatry

Research: Clinical Implications, New York: Plenum Press,

1982. p. 99.

Hahn, E.J. and Fishman, J., Changes in rat brain opiate receptor

content upon castration and testosterone replacement. Biochem

Biophys Res Commun, 90:819-823, 1979.

Hong, J-S, Yoshikawa, K., Huvson, P.M., and Uphouse, L.L.

Regulation of pituitary and brain enkephalin systems by

estrogen. Life Sci, 31:2181-2184, 1982.

Ieiri, T., Chen, H.T., Campbell, G.A., and Meites, J. Effects of

naloxone and morphine on the proestrous surge of prolactin and

gonadotropins in the rat. Endocrinology, 106:1568-1570, 1980.

Kalra, S.P. In K. W. McKerns, and V. Pantic eds. HormonallyActive Brain Peptides: Structure and Function, New York:

Plenum Press, 1982 p. 141

Kosersky, D.S., Harris, R.A., and Harris, L.S. Naloxoneprecipitated jumping activity in mice following the acute

administration of morphine. Eur J Pharmacol, 26:122-124,

1974.

Meites, J., Bruni, J.F., and Van Vugt, D. In Collu, R.,

Ducharmie, O.R., and Rochefort, G.G. eds. Central Nervous

System Effects of Hypothalamic Hormones and Other Peptides,

New York:Raven Press: 1979 pp.261-268.

Mendelson, J.H., Ellingboe, J., Kuehnle, J.C., and Mello, N.K.

Effects of naltrexone on mood and neuroendocrine function in

normal adult males. Psychoneuroendocrinology 3:231-236, 1979.

Meyer, D.R., and Sparber, S.B. Evidence of possible opiate

dependence during the behavioral depressant action of a single

dose of morphine. Life Sci, 21:1087-1094, 1977.

Mirin, S.M., Mendelson, J.H.. Ellingboe, J., and Meyer, R.E.

Acute effects of heroin and naltrexone on testosterone and

gonadotropin secretion: a pilot study.

Psychoneuroendocrinology 1:359-369, 1976.

Mueller, G.P. Attentuated pituitary beta-endorphin release in

estrogen-treated rats. Proc Soc Exp Biol Med, 165:75, 1980.

Naber. D., Cohen, R.N., Pickar, D., Kalin. N.H., Davis. G., Pert.

C.B.; and Bunney, W.E. Episodic secretion of opioid activity

in human plasma and monkey CSF: evidence for diurnal rhythm.

Life Sci, 28:931-935, 1981.

Owens, D.P. and Cicero, T.J. Development of acute tolerance to

the effects of naloxone on the hypothalamic-pituitaryluteinizing hormone axis in the male rat. J Pharmacol Exp

Ther, 216:135-141, 1981.

Reid, L.D., Konecka, A.M., Przewlocki, R., M

A. Endogenous opioids, circadian rhythm,

eating and drinking. Life Sci, 31:1829-

illan, M.J., and Herz.

nutrient deprivation,

1832, 1982.

207
Roemer, D., Buescher, H.H., Hill, R.C., Pless, J., Bauer, W.,

Cardincaiux, F., Closse, A., Hauser, D., and Hughenin, R. A

synthetic enkephalin analogue with prolonged parenteral and

oral analgesic activity. Nature, 268:547, 1977.

Rotsztejn, W., Drouva, S.V., Patton, E., and Kordon, C. Effect of

morphine on the basal and the dopamine-induced release of LHRH from mediobasal hypothalamic fragments in vitro. Eur J

Pharmacol, 50:285-289, 1978a.

Rotsztejn, W.H., Drouva. S.V., Patton, E., and Kordon, C. Metenkephalin inhibits in vitro dopamine-induced LH-RH release

from mediobasal hypothalamus of male rats. Nature, (Lond),

274:281-283, 1978b.

Smits, S.E. Quantitation of physical dependence in mice by

naloxone-precipitated jumping after a single dose of

morphine. Res Comm Chem Path Pharmacol, 10:651-661, 1975.

Sylvester, P.W., Chen, H.T., and Meites, J. Effects of morphine

and naloxone on phasic release of luteinizing hormone and

follicle stimulating hormone. Proc Soc Exp Biol Med, 164:207,

1980.

Tallarida, R.J., Cowen. A., and Adler, M.W. pA2 and receptor

differentiation: A statistical analysis of competitive

antagonism. Life Sci, 28:2355-2360, 1981.

Van Vugt, D.A., Sylvester, P.W., Aylsworth, C.F., and Meites, J.

Counteraction of gonadal steroid inhibition of luteinizing

hormone release by naloxone. Endocrinology 34:274-280. 1982.

Wiley, J.N., and Downs, D.A. Naloxone-precipitated jumping in

mice pretreated with acute injections of opioids. Life Sci,

25:797-802, 1979.

Wilkes, M.M., and Yen. S.S.C. Augmentation by naloxone of efflux

of LRF from superfused medial basal hypothalamus. Life Sci,

28:2355-2360, 1981.

Wilkinson, M., Herdon, H., and Wilson, C.A. Gonadal steroid

modification of adrenergic and opiate receptor binding in the

central nervous system. In Fuxe, K., Gustafsson, J.A., and

Wetterby, L. eds. Steroid Regulation of the Brain, Pergamon

Press: New York, 1981, pp. 253-263.

ACKNOWLEDGEMENTS

The author's research reported in this review was supported in

part by grants DA-00259 from the National Institute on Drug Abuse

and AA-03539 from the National Institute on Alcohol Abuse and

Alcoholism. The author also is a recipient of Research Scientist

award DA-00095 from the National Institute on Drug Abuse.

AUTHOR

Theodore J. Cicero, Ph.D.

Washington University School of Medicine

Department of Psychiatry

4940 Audubon Avenue

St. Louis, Missouri 63110

208
Modulation of Opioid Physical

Dependence During the Hibernation Cycle

Alexander L. Beckman, Carmen Llados-Eckman,

Steven K. Salzman, and Toni L. Stanton

INTRODUCTION

One early cornerstone of the conventional wisdom

regarding opioid physical dependence is that it is an

inevitable consequence of the prolonged administration

of opioid agonists in the mammalian CNS. Although there

may be some species exceptions (see Dewey, this volume)

to this concept, each of the model systems that have

been most extensively studied commonly displays a

uniform profile of dependence liability to a given

opioid compound throughout its natural range of

function. However, experiments in the mammalian

hibernator suggest that, rather than being an invariant

feature of the response of the nervous system to the

presence of opioids, physical dependence is a CNS

state-dependent phenomenon whose presence is qualitatively and quantitatively determined by a requisite set

of variable characteristics of the CNS. Within one

domain of activity (the euthermic, or nonhibernating,

state in which the functional characteristics of the

CNS are like those of other more commonly studied

mammalian systems), prolonged morphine administration

produces robust signs of physical dependence.

Moreover, seasonal changes in the functional characteristics of the euthermic CNS, as inferred by changes in

naturally occurring behavioral and physiological

variables, are associated with changes in the display

of morphine physical dependence. In contrast, a shift

in the operating mode of the CNS to the state that

produces and maintains hibernation, which is globally

characterized by a radical alteration of CNS activity

and, consequently, of the physiological and behavioral

Systems it controls, is associated with the failure of

prolonged morphine administration to produce physical

dependence.

This observation is interesting for reasons that go

beyond the sheer novelty of identifying a mammalian

system that displays a naturally occurring state209
dependent resistance to the development of morphine

physical dependence. We may now pose questions regarding the cellular responses that mediate the modulating

actions of opioids on physiological and behavioral

neural control systems, and how they compare to the

opioid-induced changes in neuronal function that

produce physical dependence and tolerance in those

systems.

This paper will establish a perspective for studying

opioid actions in the euthermic/hibernating model. We

will first review the characteristics of morphine physical dependence in hibernators (Citellus lateralis, the

golden-mantled ground squirrel) and then examine some

of the salient characteristics of the CNS during the

state of hibernation. Finally, we will consider the

potential of CNS mechanisms associated with the hibernating state to modulate morphine physical dependence.

These will be discussed in relation to data obtained

from species that do not hibernate that are concerned

with morphine-induced changes in CNS neurotransmitter

dynamics and on neurotransmitter/neuromodulator-induced

reduction of morphine physical dependence and tolerance.

CHARACTERISTICS OF MORPHINE PHYSICAL DEPENDENCE DURING

THE NONHIBERNATING AND THE HIBERNATING STATES

We have described physical dependence on morphine in

the euthermic ground squirrel according to the commonly

used technique of administering morphine sulfate subcutaneously via two 75-mg pellets inserted in the

interscapular region. In parallel with the development

of physical dependence during the 72-hour morphine

exposure period, we observed a biphasic effect on

behavioral activity (Beckman et al. 1981, 1982a, 1982b)

in which the initial phase was excitatory. The increased locomotor activity usually resulted in the

destruction of the normally well-maintained cotton

nest, which the animals did not rebuild for the duration of the morphine exposure. Following the excitatory phase, the animals became quiet, generally remaining

in a supine position. Those animals whose nests were

not flattened during the period of increased activity

generally remained outside their nest for the balance

of the behaviorally depressed phase of the development

of physical dependence on morphine. Seventy-two hours

after inserting the pellets, abstinence was precipitated by a subcutaneous injection of naloxone hydrochloride (1 mg/kg). In this manner, we established that the

euthermic ground squirrel develops strong physical

dependence on morphine, as evidenced by a pronounced

display of a substantial number of autonomio and behavioral responses. These included most of the signs that

have been observed in many other species, plus some

signs that are novel to the ground squirrel and which

210
resemble behaviors in the natural repertoire of this

animal. This constellation of signs, recorded in the

30-min period following naloxone injection, included

exploratory behavior, nesting behavior, grooming, vocalization, body and/or head shakes, mouth gaping

(giving the appearance of yawning), chewing, milky

dacryorrhea, digging, dyspnea, eyetwitch, flattened

posture, ptosis, and a forward-extended, fanned tail.

Seasonal Variations in Morphine Physical Dependence

Seasonal variations in the display of the abstinence

syndrome were apparent in the majority of the 14 abstinence signs. Figure 1 presents an example of this

variation for the 6 signs that were quantified. It is

apparent that among these 6 signs, those most intensely

displayed in a given animal were vocalizations and

shakes. Statistical analysis of the differences in the

mean number of occurrences of each sign showed that

significant differences were present in the seasonal

display of nesting, vocalizations, shakes, and mouth

gaping (Beckman et al. 1982a). Nesting responses were

significantly greater in the fall than in the summer.

Vocalizations were significantly reduced in the fall

compared with the winter and spring. Shakes were significantly elevated in the fall compared to spring and

summer. In the fall, the occurrence of mouth gaping

was significantly greater than in any other season.

Exploratory behavior was more prominently displayed in

the summer and fall than in the winter and spring,

although these differences did not reach statistical

significance.

Another way of measuring physical dependence, that of

determining the percentage of the morphine-exposed

population exhibiting particular abstinence signs, also

demonstrated seasonal differences. This measure showed

that in 4 of the 6 quantified signs (exploring, nesting, vocalizations, and mouth gaping), differences of

22-35% were apparent in the percentage of animals

displaying the signs across the four seasons. For example, exploratory behavior was displayed by the fewest

animals during the winter, with an increasing percentage of the population displaying the sign through the

spring and the fall. Nesting and mouth gaping were

most commonly displayed in the fall, with a steadily

decreasing incidence through winter, spring and summer.

An increasing percentage of the population displayed

vocalizations in winter and spring compared to the

relatively small percentage observed in summer and

fall. Five of the 8 signs that were tabulated as being

present or absent in each animal following naloxone

administration (as opposed to being quantified on a per

occurrence basis) showed progressive seasonal differences. For example, chewing occurred in the highest

percentage of the population in the fall and declined

211
progressively in winter, spring, and summer. The

incidence of milky dacryorrhea was lowest in the winter

and rose rapidly thereafter in the spring, reaching a

peak in the summer before declining in the fall.

The occurrence of dyspnea in the population rose

steadily from winter through fall. The display of

eyetwitch was lowest in the spring and increased

progressively in summer and fall before declining in

FIGURE 1. Seasonal changes in the number of occurrences

of quantified naloxone-precipitated abstinence signs in

morphine-dependent animals. Values are mean ± SEM.

(Reprinted with permission, from Beckman et al. 1982a,

Copyright 1982, Pergamon Press, Ltd.)

212
winter. The sign of a forward, fanned tail was least

common in the summer and increased rapidly in the fall

to reach a peak occurrence in the spring. In contrast

to these progressive changes, abrupt changes occurred

in two signs, ptosis and flat posture. Ptosis alternated between a high incidence in the summer and fall

and low values in winter and spring. A substantially

higher percentage of the population displayed flat

posture in the fall compared to the other three

seasons. No seasonal differences in the population

display of digging were apparent.

The seasonal differences in the display of various

abstinence signs must be a reflection of regional

differences in the development of morphine physical

dependence in the CNS. That is, although it is clear

that physical dependence is manifest throughout the

year, subtle changes in the action of morphine upon

particular behavioral or autonomic control systems

occur as a consequence of natural changes in the

nervous system. These changes, in effect, alter the

potency of morphine with respect to the production of

physical dependence.

Although there is currently no information available

comparing functional properties of the CNS throughout

the year in euthermic C. lateralis, reports of seasonal

changes in physiological and behavioral responses of

this species clearly point to underlying changes in CNS

mechanisms. These include differences in sleep cycles

(Walker et al. 1977), food consumption and body weight

(Pengelley and Fisher 1963; Mrosovsky 1975),

reproductive behavior (Heller and Poulson 1970) and, of

course, hibernation (Mrosovsky 1978). We have

suggested (Beckman et al. 1982a) that such changes

might conceivably involve the level of excitability or

activity of neural networks that controls these

variables. That is, slowly increasing levels of

excitability in particular networks would be expected

to eventually reach the threshold for the presentation

of seasonally observed responses. This concept is in

agreement with other observations (Muchlinski et al.

1983) in which the regional brain concentration of

neuropeptides (bombesin, neurotensin, somatostatin, and

cholecystokinin varied during the period of the year in

which C. lateralis undergoes a rapid increase in body

weight.

Thus, we might interpret our results to suggest that

the development of morphine physical dependence is

contingent upon morphine acting on neuronal populations

that exhibit a requisite level of intrinsic activity.

Differences in the level of endogenous activity within

these networks would therefore modulate the intensity

of physical dependence, as shown by differences in the

intensity of individual abstinence signs. This concept

213
is supported by reports that discrete regions in the

CNS display unique patterns of neural responses to

morphine (Dafny et al. 1979) and that the influence of

morphine on striatal dopamine (DA) release varies

according to the frequency of nigrostriatal spike

activity (Moleman and Bruinvels 1979). Other evidence

of a relationship between neuronal activity and

response to morphine may be found in an autoradiographic study of 2-deoxyglucose (2-DG) uptake in rats

(Wooten et al. 1982). Brain regions generally

displaying higher baseline levels of 2-DG

incorporation responded to chronic administration of

morphine with a significant reduction of 2-DG uptake

whereas those with lower baseline values showed

significant increases (Wooten et al. 1982).

The observation of seasonal differences in the

abstinence syndrome raises an important question

regarding the process underlying the development of

physical dependence: are the associated variables

primarily those of changes in the binding

characteristics of opioids, or is the variability

contained in the sequence of events that the agonistreceptor complex initiates? Our hypothesis concerning

the relationship between morphine dependence and the

basal level of activity in target neural networks

contains an assumption that the basic dynamics of

morphine's cellular action do not change on a

seasonal basis in C. lateralis. However, data from an

invertebrate species, a marine mollusc, suggest that

seasonal variation in the binding of morphine may also

be a contributing factor (Stefano et al. 1980). The

seasonal differences in enkephalin binding that were

observed in the marine mollusc were associated with

differences in the ability of d-ala,met-enkephalinamide

(DALA) and met-enkephalin to alter dopamine parameters

in the pedal ganglia of this animal. The possibility

that a similar mechanism operates in the ground

squirrel will be considered in a subsequent section.

The profound difference in the hibernating ground

squirrel's responses to chronic administration of

morphine underscores the question as to where the

sequence of events leading to the development of

physical dependence may be broken. Our data (Figure 2)

show that physical dependence on morphine fails to

develop during deep hibernation (Beckman et al. 1981).

These animals received morphine either through

subcutaneous implantation of two 75-mg morphine sulfate

pellets or directly into the brain via an icv cannula

at the same dose parameters as those which produced a

robust naloxone-precipitated abstinence syndrome in

euthermic animals. Although the hibernating animals

only received morphine following entrance into deep

hibernation, they were tested for an abstinence

syndrome during the euthermic period immediately

214
FIGURE 2. Quantified signs of abstinence precipitated

by naloxone during (A) the nonhibernating (euthermic)

state and (B) the hibernating state in ground squirrels

implanted with morphine pellets. Brackets denote SEM.

(From Beckman et al. 1981. copyright 1981 by AAAS).

following arousal. Whereas no abstinence syndrome was

apparent, some of the animals did respond to morphine

with a display of stereotyped behavior which began

while the animals were starting to arouse (i.e., prior

to naloxone administration) and continued into the

Post-arousal euthermic state. It is thus clear that,

although morphine does not produce physical dependence

in the hibernating state (at least at the dose

215
parameters that we tested), it does evoke a strong

response in the CNS.

PROPERTIES OF THE CNS DURING THE STATE OF HIBERNATION

Whereas the euthermic ground squirrel presents itself

as a model of subtle modulation of opioid physical

dependence, the hibernating ground squirrel is a model

of the full extent of that modulation, in which the

phenomenon disappears with a change in the state of the

brain, Entrance into hibernation begins during sleep

and is characterized by a striking reduction of body

temperature and metabolic rate that reaches maximum

depression during deep hibernation. It is so characteristic a feature that we commonly identify the departure of the euthermic state by the fall in these two

variables. Thus, whereas internal temperature during

euthermia is regulated at approximately 38°C, during

deep hibernation in natural conditions, internal temperature decreases to nearly 0°C (Wang 1978).

Current evidence clearly indicates that the entrance

into hibernation is controlled by the CNS and suggests

that this control is manifest by an increase in central

inhibitory level. For example, heart and respiratory

rates, which are depressed during hibernation, begin to

decline prior to any significant decrease in body

temperature during euthermia (Lyman 1958; Strumwasser

1959a). Likewise the amplitude of the EEG has been

observed to begin declining before any significant fall

in body temperature occurred (Wunnenberg et al. 1978).

As the entrance into hibernation progresses, the

amplitude of the EEG continues to decrease (Strumwasser

1959a; Shtark 1970; Walker et al. 1977), and changes in

the frequency distribution of the EEG occur with the

decline in body temperature (South et al. 1969; Shtark

1970). Observations taken from multiple EEG recording

sites within the same animal suggest that suppression

of EEG activity is first evidenced in the cerebral

cortex, followed by the midbrain reticular formation,

thalamus and, lastly, in areas of the limbic system

(notably in the hippocampus and hypothalamus)

(Shtark 1970).

Studies of the CNS regulator of body temperature

provide additional evidence for a controlled entry into

hibernation. During entrance into hibernation, the

threshold hypothalamic temperature for activating an

increase in metabolic rate was demonstrable at all

times, but at increasingly lower levels as time

progressed (Heller et al. 1977; Florant et al. 1978).

Electroencephalographic studies indicate that once

animals achieve the resting state of hibernation, the

level of electrical activity is similar to that of the

entry phase. That is, the cortex remains electrically

216
depressed except for varying periods of increased EEG

activity (South et al. 1969; Shtark 1970; Walker et al.

1977) and, similarly, the midbrain reticular formation

displays long periods of silence that are punctuated by

short bursts of spiking activity (Strumwasser 1959b;

South et al. 1969; Shtark 1970). The limbic system

appears to diverge from this picture of general

electrical depression in that limbic structures

(notably the hippocampus, septum, and hypothalamus)

have been reported to display nearly continuous EEG

activity throughout the hibernation bout (Strumwasser

1959b; South et al. 1969; Shtark 1970). This characteristic of limbic structures, contrasted with the

relative electrical silence of cortical and midbrain

areas, has prompted suggestions (South et al. 1972;

Beckman and Stanton 1976) that the proposed increase in

central inhibitory level accompanying the transition

from the euthermic state to the hibernating mode

(Luecke and South 1972; Hammel et al. 1973) is controlled by neurons within the limbic system.

More recent analysis of regional CNS metabolic activity

using the 14C-2-DG autoradiographic method in ground

squirrels (Kilduff et al. 1982) confirms the earlier

descriptions of a general decline in activity throughout the brain during hibernation. Of particular

interest is the fact that not all structures undergo a

similar reduction in activity. Those that undergo the

smallest reduction in relative 2-deoxyglucose uptake,

in comparison to euthermia, include the medial preoptic

area, ventral hippocampus, locus coeruleus, suprachiasmatic nucleus, and the lateral septal nucleus.

Presumably, these structures retain functional

significance during deep hibernation. Likewise, in

addition to the locus coeruleus and the suprachiasmatic

nucleus, we might expect structures such as the dorsal

tegmental nucleus, inferior co1liculus, superior

colliculus, and the habenular nucleus to perform

important control functions during deep hibernation

because this group of structures exhibits the greatest

relative 2-deoxyglucose uptake. It is worth noting

that these structures are components of the endogenous

opioid system, as shown by the presence of opoid

receptors (Hiller et al. 1973; Kuhar et al. 1973; Pert

et al. 1975; Simon and Hiller 1978; Pearson et al.

1980; Duka et al. 1981) or endogenous opioid compounds

(Watson et al. 1977; Matsukura et al. 1978; Gramsch

et al. 1979; Dupont et al. 1980).

Evidence for the maintenance of control functions

during hibernation has been provided by studies on the

CNS thermoregulatory system which show that it retains

functional activity and can defend the hibernating

animal against decreases in internal temperature below

a threshold level (Hammel et al. 1968; Heller and

Colliver 1974). Similarly, work in our laboratory has

217
demonstrated the activation of a mechanism that is

capable of inhibiting disturbance-induced arousal from

hibernation (Beckman et al. 1976b).

Studies of the responsiveness of the hibernating brain

to the microinjection of putative neurotransmitters

such as norepinephrine (NE), 5-hydroxytryptamine (5-

HT), acetylcholine (ACh), and the neuropeptide thyrotropin-releasing hormone (TRH), have demonstrated that

these compounds continue to exert a potent effect on

the brain during this state (Beckman and Satinoff 1972;

Beckman et al. 1976a; Stanton et al. 1980). Microinjection of low concentrations of these compounds into

the hypothalamus, hippocampus, or midbrain reticular

formation can activate small thermogenic responses or

the trigger process for arousal from hibernation.

Additional studies in our laboratory, using ACh microinjections into the midbrain reticular formation or the

preoptic/anterior area of the hypothalamus, have

demonstrated that the hibernating brain undergoes a

decrease in excitability coincident with the entrance

into hibernation. CNS responsiveness then begins a

recovery towards euthermic levels of excitability as

time in the hibernation bout elapses (Beckman and

Stanton 1976; Stanton and Beckman 1977). Interestingly, the profile of the increase in responsiveness to

ACh is different in the midbrain reticular formation as

compared with the preoptic/anterior area of the

hypothalamus. In the former structure, the increase in

responsiveness is gradual and progressive, whereas in

the latter structure it occurs more abruptly at the

half-way point of the bout. This increased

responsiveness is probably a reflection of the higher

level of EEG (Strumwasser 1959b; South et al. 1969;

Shtark 1970) and metabolic (Kilduff et al. 1982)

activity observed during hibernation in the hypothalamus.

It is therefore clear, both from the maintenance of

neural activity and from the physiological responses

that actively maintain the state of hibernation against

disruption, that the hibernating brain is a functionally intact system.

CHARACTERISTICS OF THE HIBERNATING CNS THAT MAY MEDIATE

THE MODULATION OF PHYSICAL DEPENDENCE ON MORPHINE

From the foregoing description of the functional

characteristics of the hibernating brain, it is

apparent that a number of major differences from the

euthermic brain could contribute to the blockade of

morphine physical dependence. We have outlined

(Beckman et al. 1981) four possibilities which merit

early consideration in light of recent findings in

nonhibernating species: a) restrictions in the

intracerebral distribution of morphine following icv

218
administration in the hibernating brain, b) differences

in the turnover (synthesis, release, reuptake and/or

degradation) of neurotransmitters or neuromodulators

across the two states, c) differences in the characteristics of opioid receptors (changes in regional receptor density or ligand binding affinity), and d) low

temperature-induced differences in the development of

physical dependence that are associated with the

conditions of deep hibernation.

Intracerebral Distribution of Morphine During

Hibernation

It is conceivable that some of the characteristics of

the state of hibernation, such as the widespread

reduction in the level of function of physiological

systems or possible changes in tissue characteristics

(Beckman and Stanton 1982) could reduce the efflux of

icv-administered compounds into the brain and/or

restrict patterns of intracerebral distribution

compared to the euthermic state. In consideration of

this, we have begun to examine autoradiographically the

intracerebral distribution of tritiated morphine

following icv administration during hibernation. In

these experiments, tritiated morphine was infused

continuously into the lateral ventricle of hibernating

ground squirrels for periods of 4 to 30 hours.

Qualitative results obtained by visual inspection of

the autoradiographs revealed that the radiolabeled

compound did leave the ventricular space and diffuse

widely into the brain. The zones of increased optical

density were clearly evident in the cortex, lateral

ventricles, third ventricle, caudate nucleus, dorsal

hippocampus, amygdala, bed nucleus of the stria

terminalis, septum, medial preoptic area, thalamic

structures, the aqueduct of Sylvius, and the periaqueductal gray. These regions were among those

identified in previous studies as being sites of

importance to the development of physical dependence

and the expression of naloxone-precipitated abstinence,

as shown by regional 2-DG uptake in dependent and

withdrawing animals (Wooten et al. 1982) and by the

ability to modify the display of physical dependence

following focal lesions or drug administration (Kerr

and Pozuelo 1971; Herz et al. 1972; Wei et al. 1972;

Wikler et al. 1972; Wei et al. 1973).

We are beginning to analyze these autoradiographic data

using computer-assisted densitometry in order to

improve the qualitative resolution and to quantify the

magnitude of regional optical density. We are, in

addition, in the preliminary stages of analyzing the

regional content of brain areas displaying increased

autoradiographic density to determine whether the

radioactive label is associated with morphine or its

metabolites. Thus, while yet qualitative, our data do

219
not indicate any decrement in the intracerebral

distribution of morphine in the hibernating brain

following continuous icv infusion.

Changes in Neurotransmitter Turnover During Hibernation

The second feature of the hibernating CNS that may

contribute to the state-dependent block of physical

dependence concerns the changes that occur in neurotransmitter level or turnover during deep hibernation.

The notion that these changes, described below, may be

of significance to the development of physical

dependence is derived from the literature on the

effects of morphine administration on neurotransmitter

level or turnover, as well as the effect of changes in

the brain level or activity of putative neurotransmitters on physical dependence and tolerance. The

catecholamines (NE and DA), 5-HT, and ACh have received

the most attention.

The earlier literature describing morphine's effects on

the brain levels of these compounds is contradictory in

that morphine has been reported to increase (Gunne

1959; Akera and Brody 1968) and decrease (Vogt 1954;

Gunne 1959; Maynert and Klingman 1962; Gunne 1963; Lee

and Fennessy 1970) NE levels, to increase DA levels

(Clouet and Ratner 1970) and to decrease (Lee and

Fennessy 1970) or have no effect (Maynert et al. 1962;

Sloan et al. 1962) on 5-HT levels in the brain.

Similarly, morphine may increase the level of ACh

(Large and Milton 1970; Domino and Wilson 1973) or,

depending upon the dose administered, produce biphasic

effects on ACh level (Vasco and Domino 1978). Other

reports have described effects of morphine administration on the turnover of these compounds. In view of

the data on morphine's effects on neurotransmitter

content, it is not surprising that morphine administration

has been reported to increase or decrease the turnover

of NE (Costa et al. 1974; Gomes et al. 1976; Arbilla

and Langer 1978; Bensemana and Gascon 1978), DA (Loh et

al. 1972; Costa et al. 1974; Bensemana and Gascon 1978;

Moleman and Bruinvels 1979), 5-HT (Bensemana and Gascon

1978; Snelgar and Vogt 1980; Vasko and Vogt 1982), and

ACh (Vasco and Domino 1978). The heterogeniety of these

effects is most probably due to methodological

differences (species, dose, sites of administration).

In any case, the major point established by these

studies is that chronic morphine administration affects

the dynamics of major neurotransmitter systems in the

brain. It seems reasonable to assume that such changes

would be reflected in a functionally significant change

in the matrix of inputs received by neural networks

within the CNS, thereby producing an altered output

that underlies the development of physical dependence.

This interpretation is supported by ample evidence for

morphine-induced supersensitivity in monoamine220
sensitive systems, particularly with respect to DA

(Schulz and Herz 1977; Kromer and Steigemann 1982;

Ritzmann et al. 1982; Rae and DeMorhes 1983).

The foregoing evidence established that chronic

morphine administration produces changes in the turnover of several neurotransmitter systems in the CNS and

therefore supports the concept that physical dependence

is, at least in part, a manifestation of these changes.

If this is indeed the case, then experimental

manipulation of neurotransmitter level or turnover

should result in the modulation of physical dependence.

The results of such experiments, while not conclusive,

do provide some support for this notion. They show

that decreases in 5-HT levels resulted in decreases

(Way et al. 1968; Ho et al. 1972) or no change

(Schwartz and Eidelberg 1970; Cheney and Goldstein

1971) in the intensity of the morphine abstinence syndrome and in diminished (Way et al. 1968; Ho et al.

1972) or unaltered (Cheney and Goldstein 1971)

development of tolerance to morphine. Decreases in the

level of catecholamines have been reported to have no

effect on the development of tolerance (Bhargava et al.

1973) and to increase (Friedler et al. 1972) or

decrease (Schwartz and Eidel berg 1970) the intensity of

the morphine abstinence syndrome.

Given the matrix of intense responses that are elicited

during the antagonist-precipitated withdrawal from

morphine, it seems appropriate to characterize

morphine-induced changes in neurotransmitter dynamics

as a disruption of normal CNS function. Viewed from

this perspective, the hibernating CNS is a particularly

interesting model because the dynamics of several

neurochemical systems are markedly changed from that

observed in euthermia. Low levels of brain protein

renewal during hibernation, and dramatic increases

during arousal, have been measured in ground squirrels

(Belik and Kracho 1961). In hibernating golden

hamsters, brain cholinesterase activity is depressed,

perhaps reflecting the decrease in electrical activity

of the cortex (which is high in cholinesterase concentration in the euthermic state) (Robinson and Bradley

1963). In contrast, glutamic acid decarboxylase activity, which is associated with the formation of GABA,

increased in the brain of hibernating golden hamsters

(Robinson and Bradley 1963). Measures of the turnover

of NE and 5-HT during euthermia and hibernation generally indicate that the activity of 5-HT systems is

accelerated prior to entrance and during deep hibernation whereas that of NE-releasing systems is depressed prior to entrance and remains low during deep

hibernation (Wang 1982).

As we have pointed out, evidence obtained from nonhibernating species clearly suggests that the

221
development of morphine physical dependence may stem

from the effects of alterations in neurotransmitter

dynamics. The hibernating CNS also displays changes in

neurotransmitter dynamics which may contribute to the

failure of morphine to produce physical dependence

during hibernation. For example, we note that although

the transition from euthermia to hibernation is accompanied by changes in distribution and quantity of activity within the brain as well as changes in neurochemical parameters, brain function does not appear to

be disrupted or functionally impaired. It is likely

that the preservation of this functional integrity is

dependent upon such changes which are themselves manifestations of the hibernator’s adaptive specializations

that buffer the CNS against the effects of otherwise

disruptive conditions.

With this in mind, we studied the turnover of DA in the

caudate nucleus of drug-naive ground squirrels during

euthermia and hibernation, as a prelude to examining

the effect of chronic morphine administration on DA

turnover in these two states. As we noted earlier,

morphine-induced changes in DA turnover, and supersensitivity of DA systems following chronic opioid

administration, suggest that sustained perturbation of

normal dopaminergic activity is an important component

of opioid physical dependence. In our experiments, we

stereotaxically implanted four ground squirrels with a

unilateral push-pull cannula in the caudate nucleus and

another unilateral (same side) cannula in the lateral

ventricle (for eventual chronic infusion of morphine).

Resting euthermic animals were tested in a plexiglass

chamber (ambient temperature, Ta: 20-22°C) over a 2-3

hr period in which successive 30-min caudate perfusates

(perfusion rate: 20.6 μl/min) samples were collected.

Animals were allowed free movement by means of a swivel

coupling in the perfusion system. Hibernating animals

were tested in their cotton nests (Ta: 5°C) for a

similar period. Perfusion samples were analyzed for DA

(free and conjugate forms) and its metabolites (3,4-

dihydroxyphenylacetic acid [DOPAC], 3-methoxy-4-

hydroxyphenylacetic acid [homovanillic acid, HVA], and

3-methoxy-4-hydroxyphenethanol [MOPET] by high performance liquid chromatography with electrochemical

detection (HPLC/EC). Conjugates of DA were cleaved by

acid hydrolysis prior to HPLC/EC analysis.

Our results (summarized in schematic form in Figure 3)

show clear differences between the euthermic and

hibernating brain in the performance of the caudate DA

system. Our data from the euthermic state are based on

a total of 26 samples collected from 4 animals. Free

DA was not detectable in 21 of the samples. It was

present in 5 of the samples at a mean level of 3.9

pmol/30 min. HVA was detected consistently at high

levels (12.1 pmole/30 min.) in all 26 samples. DOPAC

222
EUTHERMIA

RELEASE

RE-UPTAKE,

DEGRADATION, SYNTHESIS

HIBERNATION

DIMINISHED RE-UPTAKE AND SYNTHESIS

DIMINISHED RELEASE ALTERED DEGRADATION, CONJUGATION

FIGURE 3. Caudate nucleus DA turnover during euthermia

and hibernation in drug-naive ground squirrels. Upper

half summarizes events of release, reuptake, degradation, and synthesis during euthermia as suggested by

measurements of DA and its metabolites. Lower half

summarizes these events during hibernation; note the

shift in metabolism to the enzyme pathway utilizing

alcohol dehydrogenase and the increased formation of

conjugated DA (DA-SO4). HVA was not detected in half

of the samples collected during the early portion of

the bout and in none of the samples obtained during the

late portion of the bout.

223
was not detectable in 12 of the samples, but was

present in 14 at a level of 0.9 pmol/30 min. Conjugated DA was not detected in 14 samples, but was

present in 12 of the perfusates at a level of 1.9

pmol/30 min.

In contrast, results from the hibernating state (N=13

samples, 4 animals) showed that free DA was present in

all samples at 35.0 pmol/30 min, but that HVA was not

present in 10 samples. In 3 samples, HVA was detected

at a level of 5.0 pmol/30 min. DOPAC was not detected

in any of the samples. We did, however, detect MOPET

in each sample, in quantities of 5.6 pmol/30 min.

Conjugated DA was present in 7 of 13 samples at a level

of 13.5 pmol/30 min.

Other interesting features of the hibernating DA system

became apparent when these data were grouped according

to the period of the hibernating bout in which they

were collected. Early in the bout, levels of DA (in 7

out of 7 samples) averaged 22.2 pmol/30 min, whereas

late-bout levels increased to 45.9 pmol/30 min (in 6 of

6 samples). The early-bout level of HVA in 3 samples

was 5.0 pmol/30 min and was not detectable in 3

samples; late in the bout, HVA was not detectable in

any of the 7 samples collected. MOPET was present

during the early portion of the bout at a level of 4.7

pmol/30 min (6/6 samples) and during the late portion

of the bout, it increased by 37% to 6.5 pmol/30 min

(7/7 samples). Conjugated DA was not detectable in any

of the 6 early-bout samples, but was consistently found

in all 7 of the late-bout samples (13.5 pmol/30 min).

Thus, the data show that caudate perfusates from

euthermic animals displayed consistent, high levels of

HVA and very little (usually no) DA. This probably

indicates a relatively constant and high turnover rate.

It appears that the released DA was rapidly and

efficiently converted to HVA through the normal route

of DA metabolism, as the perfusates contained small

amounts of DA and always contained HVA.

Caudate perfusates from the hibernating animals

appeared quite different. The level of DA was approximately 10-fold higher than in euthermic animals and

occurred in each perfusate sample (as opposed to euthermia wherein DA appeared in few samples). Furthermore, there was very little HVA present. DA metabolism

continued to occur, however, albeit by a completely

different route. Rather than being metabolized through

the normal route to HVA, DA was converted preferentially to MOPET (where levels in each sample were about

one-half the molar quantity of the euthermic levels of

HVA), indicating a shift from aldehyde dehydrogenase to

alcohol dehydrogenase. Thus, these data probably indicate that DA was still being released by caudate

224
neurons (although we cannot say how the rate of release

compared to that during euthermia), but was not converted as efficiently to a metabolite. Because DA

appeared to be accumulating in the extracellular space,

it is likely that overall turnover was less. However,

DA turnover appeared to accelerate late in the bout,

but remained qualitatively similar to that in the early

portion of the hibernation bout.

Our data also suggest that DA conjugates may play an

important role in the conservation of DA during

hibernation. That is, conjugated DA appears to serve

as a reservoir for the large amount of unmetabolized DA

that accumulates during hibernation.

The results of these experiments suggest that the

hibernating ground squirrel, in order to efficiently

utilize the DA that is released from slowly firing

caudate neurons, alters the metabolism of DA such that

the synaptic concentration of DA is high enough to

maintain a requisite level of tone in the DA pathway.

This alteration appears to involve, at least in Part, a

shift in metabolism from HVA to MOPET and the use of

conjugated DA (which is resistant to oxidative and

methylating enzymes) as a storehouse for free DA. The

resulting increase in the potential availability of

free DA would serve as a buffer against the effects of

opioid-induced decreases in DA release, thereby

increasing the resistance of the dopaminergic system to

opioid-induced dopamine supersensitivity. Assuming

that DA supersensitivity is a component in the matrix

of CNS changes that constitute opioid physical

dependence, this adaptive change in DA metabolism

could, in part, explain the failure of physical

dependence to develop in the hibernating CNS. In order

to test this hypothesis, we are beginning experiments

to determine the effect of morphine administration

during hibernation and euthermia on these parameters Of

DA metabolism.

There is another aspect to the concept that the

development of morphine physical dependence involves

alterations in the activity of CNS neurotransmitter

systems. We have already noted that the experimental

manipulation of central monoamine dynamics can

influence physical dependence. Evidence has been

presented in recent years that extends the concept of

opioid-neurotransmitter interactions in physical

dependence to another class of neuroactive compounds,

the neuropeptides. Although there is some evidence to

the contrary (Olson et al. 1982), the results of

several studies on melanotropin-release inhibiting

factor (MIF) and TRH favor an inhibitory influence of

these compounds on opioid tolerance and physical

dependence. For example, MIF has been reported to

inhibit tolerance to the analgesic and cataleptic

225
effect of beta-endorphin (Bhargava 1981a) and to block

beta-endorphin-induced increases in DA responsiveness

(Bhargava 1981d). Other studies found that MIF can

block morphine-induced increases in DA-responsiveness

(Bhargava 1980a) and reduce tolerance to morphineinduced analgesia (Bhargava 1980a; Bhargava 1982),

locomotor depression (Bhargava 1982), hypothermia

(Bhargava 1981c), and catalepsy (Bhargava 1981c).

Similarly, an analogue of MIF(cyclo[Leu-Gly]) has been

reported to inhibit tolerance to morphine-induced

analgesia (Ritzmann et al. 1982; Bhargava 1980a, 1981a,

1982) as well as some signs of morphine physical

dependence, morphine-induced DA supersensitivity, and

increases in the ligand affinity of striatal DA

receptors (Ritzmann et al. 1982). TRH has been shown

to block tolerance to the analgesic effects of morphine

(Bhargava 1981b) and to exert an inhibitory effect on

the development of morphine physical dependence

(Bhargava 1980b). TRH and other peptide compounds have

also been shown to antagonize opioid-induced responses.

For example, TRH inhibited morphine-induced locomotor

depression and decreases in body temperature (Bhargava

et al. 1982), somatostatin inhibited beta-endorphininduced adrenal medullary release of epinephrine (Van

Loon et al. 1981), and cholesystokinin inhibited betaendorphin-induced catalepsy in rats (Olson et al.

1982).

These findings are potentially relevant to the hibernating model system’s reduced morphine dependence liability because changes in the brain levels of these and

other neuroactive peptide compounds occur with hibernation. Thus, the level of TRH is lower in the brain

of hibernating ground squirrels than in euthermic animals in all areas studied (cortex, septum, hippocampus,

preoptic area, hypothalamus, thalamus, midbrain, and

medulla) (Stanton et al. 1982). Curiously, TRH levels

in the pineal gland increased dramatically during the

late portion of the bout (Stanton et al. 1982). The

levels of somatostatin, cholecystokinin, bombesin, and

neurotensin were measured in five brain regions of

hibernating and summer euthermic ground squirrels,

with some interesting differences being displayed

(Muchlinski et al. 1983). Somatostatin levels generally

increased in hibernation in the thalamus and decreased

in cortex, cerebellum, medulla, and hypothalamus,

compared with average values measured during the summer

season. Hibernation levels of cholecystokinin increased, relative to average summer values, in the

cortex and cerebellum, tended to decrease in medulla

and hypothalamus and showed no change in the thalamus.

Hibernating levels of bombesin were lower than average

Summer euthermic levels in the hypothalamus, thalamus,

and medulla but showed no changes in the cortex or

cerebellum. Neurotensin levels were unchanged in the

cortex during hibernation, but decreased in hypothal226
amus, thalamus, and medulla compared to summer animals,

In another study, the content of met-enkephalin, leuenkephalin, and ACTH was measured in whole-brain extracts obtained from hibernating and winter euthermic

ground squirrels. The level of both met- and leuenkephalin was increased, and that of ACTH decreased,

in hibernating brain (Kramarova et al. 1983).

Whereas it is hazardous to infer particular changes in

the overall turnover (thus, in substance release and

alteration of neuronal activity) from data on changes

in brain neurotransmitter or neuromodulator levels, it

appears safe to assume that these results reflect

changes, albeit unspecified, in the functional activity

of these neuroactive compounds. This assumption

appears to be particularly valid in the case of the

increased levels of enkephalin. Recent work in our

laboratory (Llados-Eckman and Beckman 1983) suggests

that these increased levels reflect an increase in the

functional activity of the endogenous opioid system

during hibernation. We demonstrated that opioid

blockade produced by continuous icv infusion of

naloxone during hibernation produced a dose-dependent

decrease in bout duration. These and other results

(Margules et al. 1979; Kromer 1980) suggest that

disruption of the baseline activity of endogenous

opioid peptide systems during hibernation destabilizes

the state, perhaps by interfering with a fundamental

component of its controlling mechanism (Oeltgen et al.

1982).

As noted above, the proposed changes in functional

activity of neuropeptides such as TRH, somatostatin,

and cholesystokinin during hibernation could contribute

to the blockade of the development of morphine physical

dependence during this state. Another contributing

factor, alluded to earlier, may involve an increase in

the level of GABA in the hibernating brain (Robinson

and Bradley 1963); inhibition of the degradation of

GABA has been reported to attenuate physical dependence

(Contreras et al. 1979).

When we proposed (Beckman et al. 1981) that changes in

opioid binding might be a potential factor in

explaining the resistance of the hibernating state to

the development of physical dependence, our rationale

was quite straightforward: if morphine binding

decreased, either through increased competition with

naturally available endogenous ligands or through a

frank decrease in opioid binding sites, the potency of

morphine (and, hence, physical dependence) would

diminish. There is very recent evidence (Kramarova et

al. 1983), as reviewed above, that enkephalin levels do

increase during hibernation. Whether endorphin levels

(or, more to the point, endorphin release) also increase during this state is still unknown.

227
Changes in the Characteristics of Opioid Binding

In considering the role that changes in opioid receptor

parameters may play in the blockade of the development

of physical dependence during hibernation, we have

begun to study with Dr. James K. Wamsley regional brain

opioid binding in hibernating and euthermic ground

squirrels, using in vitro receptor autoradiography in

conjunction with computer-assisted densitometry. In

our initial studies, we have sought to avoid

preferential labeling of specific opioid receptor

subtypes and therefore have used a high concentration

(4 nM) of tritiated dihydromorphine as the agonist

ligand. The preliminary results show that, indeed,

overall opioid binding does decrease in the hibernating

brain, particularly in the dorsal aspects of the

cerebral cortex, in the septum, hippocampus, thalamus,

and in the amygdala. The decrease in intracerebral

binding during hibernation is not, however, uniform.

For example, binding in the rostra1 hypothalamus and

the habenula remain comparable to that during the

euthermic state. In addition, the overall decrease in

binding cannot simply be ascribed to the low body

temperatures associated with the hibernating state

because the trend toward decreased binding is evident

in euthermic winter animals, as compared to that observed in the brains of animals during the fall.

It is tempting to speculate that the decline in opioid

binding in the winter euthermic and hibernating ground

squirrel brain is due to a down-regulation of opioid

receptors produced by the elevation of regional brain

concentrations of endogenous opioids. Although it

seems clear that the opposite phenomenon, upregulation, occurs following chronic treatment with

opioid antagonists such as naloxone or naltrexone

(Lahti and Collins 1978; Bardo et al. 1982; Zukin

et al. 1982), it remains uncertain whether chronic

administration of opioid agonists reliably induces

receptor down-regulation (Davis et al. 1975; Davis

et al. 1979; Tsang and Ng 1980; Sivam et al. 1981).

Contribution of Low Body Temperature to Reduced

Morphine Dependence Liablility During Hibernation

Although the evidence we have presented thus far

suggests that hibernation-related adaptive changes in

neurotransmitter or neuromodulator parameters (e.g.,

changes in DA metabolism or in opioid receptor binding)

may provide the main source of stabilization against

the development of morphine physical dependence, the

potential role of low body temperature yet needs to be

defined in the ground squirrel. Certainly, evidence

obtained in nonhibernating species has identified a

temperature-dependent component in the development of

dependence and tolerance. Thus, lowered temperature

228
exerted a short-term (less than 4 hours) inhibition of

dependence on normorphine in the isolated guinea pig

ileum (Collier et al. 1981), and also blocked the

development of tolerance to morphine depression of

sensory evoked responses in mouse spinal cord-dorsal

root ganglion explants (Crain et al. 1979).

In initial experiments, we have begun to compare the

naloxone-precipitated abstinence syndrome in euthermic

animals that have received continuous icv infusion of

morphine (55 μg/μl/hr) with that displayed by postarousal euthermic animals that had just completed

receiving the same dosage of morphine, while

hibernating at 20°C. Our initial results indicate that

animals hibernating at this elevated temperature (usual

hibernation temperature, 5°C) display a feeble

abstinence syndrome that includes a few body shakes and

vocalizations, and the occasional presence of milky

dacryorrhea, chewing, dyspnea, eyetwitch, and forward,

fanned tail during the 30-minute period following

naloxone administration. One animal also displayed

stereotypy, as observed previously in animals that

received morphine during deep hibernation (Beckman et

al. 1981). These data are too preliminary to adequately

describe the effect of low temperature on the

hibernation-induced block of physical dependence. They

do however, indicate that low body temperature may

contribute to this phenomenon. Continuing work will

determine its extent.

CONCLUSION

These studies on the ground squirrel have progressed to

the point where it is clear that the hibernating model

can make an important contribution to our understanding

of the neural correlates of opioid physical dependence.

Its unique advantage is that it permits a direct

comparison of the performance of designated CNS systems

following chronic opioid administration in states of

high or low dependence liability. We can, therefore,

identify changes in performance that do, or do not,

correlate with the development of physical dependence

and thereby describe the important determinants of

morphine dependence. It is not inconceivable that the

critical elements of the natural stabilizing mechanisms

utilized by the hibernating CNS could be adapted for

the therapeutic prevention of opioid physical

dependence.

229
REFERENCES

Akera, T., and Brody, T.M. The addiction cycle to

narcotics in the rat and its relation to

catecholamines. Biochem Pharmacol, 17:675-688,

1968.

Arbilla, S., and Langer, S.Z. Morphine and ß-endorphin

inhibit release of noradrenalin from cerebral cortex

but not dopamine from rat striatum. Nature,

271:559-561, 1978.

Bardo, M.T.; Bhatnagar, R.K.; and Gebhart, G.F. Differential effects of chronic morphine and naloxone on

opiate receptors, monoamines, and morphine-induced

behaviors in preweanling rats. Develop Brain Res, 4:

139-147, 1982.

Beckman, A.L.; Llados-Eckman, C.; Stanton, T.L.; and

Adler, M.W. Physical dependence on morphine fails

to develop during the hibernating state. Science,

212:1527-1529, 1981.

Beckman, A.L.; Llados-Eckman, C.; Stanton, T.L.; and

Adler, M.W. Seasonal variation of morphine physical

dependence. Life Sci, 30:147-153, 1982a.

Beckman, A.L.; Llados-Eckman, C.; Stanton, T.L.; and

Adler, M.W. Differential effect of environmental

temperature on morphine physical dependence and

abstinence. Life Sci, 30: 1013-1020, 1982b.

Beckman, A.L., and Satinoff, E. Arousal from hibernation by intrahypothalamic injections of biogenic

amines in ground squirrels. Am J Physiol, 222: 875-

879, 1972.

Beckman, A.L.; Satinoff, E.; and Stanton, T.L.

Characterization of midbrain component of the trigger

for arousal from hibernation. Am J Physiol, 230:

368-375, 1976a.

Beckman, A.L., and Stanton, T.L. Changes in CNS

responsiveness during hibernation. Am J Physiol,

231:810-816, 1976.

Beckman, A.L., and Stanton, T.L. Properties of the CNS

during the state of hibernation. In: Beckman, A.L.,

ed. The Neural Basis of Behavior. New York:

Spectrum, 1982. pp. 19-45.

Beckman, A.L.; Stanton, T.L.; and Satinoff, E.

Inhibition of the trigger process for arousal from

hibernation. Am J Physiol, 230:1018-1025, 1976b.

Belik, B., and Kracho, L.S. Protein metabolism in the

brain of hibernating animals. Ukrain Biochim Zhrl

(Kiev), 33:684-692, 1961.

Bensemana, D., and Gascon, A.L. Relationship between

analgesia and turnover of brain biogenic amines. Can

J Physiol Pharmacol, 56:721-730, 1978.

Bhargava, H.N. Cyclo (leucylglycine) inhibits the

development of morphine induced analgesic tolerance

and dopamine receptor supersensitivity in rats. Life

Sci, 27:117-123, 1980a.

230
Bhargava, H.N. The effects of thyrotropin-releasing

hormone on the central nervous system responses to

chronic morphine administration. Psychopharmacology,

68:185-189, 1980b.

Bhargava, H.N. Inhibition of tolerance to the pharmacological effects of human ß-endorphin by prolylleucyl-glycinamide and cyclo (leucylglycine) in the

rat. J Pharmacol Exp Ther, 218:404-408, 1981a.

Bhargava, H.N. Dissociation of tolerance to the

analgesic and hypothermic effects of morphine by

using thyrotropin releasing hormone. Life Sci, 29:

1015-1020, 1981b.

Bhargava, H. N. The effects of peptides on tolerance to

the cataleptic and hypothermic effects of morphine in

the rat. Neuropharmacology, 20:385-390, 1981c.

Bhargava, H.N. Enhanced response to apomorphine in

rats treated with multiple injections of human beta

endorphin and its blockade by pro-leu-gly-NH and

cyclo (leu-gly). Life Sci, 29:1945-1949, 1981d.

Bhargava, H.N. Hypothalamic peptide hormone, prolylleucyl-glycinamide and analog, inhibit tolerance to

the analgesic and locomotor depressant but not to the

locomotor stimulant effects of morphine in the mouse.

Neuropharmacology, 21:227-233, 1982.

Bhargava, H.N.; Afifi, A.; and Way, E.L. Effect of

chemical sympathectomy on morphine antinociception

and tolerance development in the rat. Biochem Pharm,

22: 2769-2772, 1973.

Bhargava, H.N.; Matwyshyn, G.A.; Currie, B.L.; and

Goebel, R.J. Structure activity relationship studies

with hypothalamic peptide hormones II. Effects of

thryotropin releasing hormone analogs on morphineinduced responses in mice. Life Sci, 30:711-718,

1982.

Cheney, D.L., and Goldstein, A. The effect of pchlorophenylalanine on opiate-induced running,

analgesia, tolerance, and physical dependence. J

Pharmacol Exp Ther, 177:309-315, 1971.

Clouet, D.H., and Ratner, M. Catecholamine biosynthesis in brains of rats treated with morphine.

Science, 168:854-856, 1970.

Collier, H.O.J.; Cuthbert, N.J.; and Francis, D.L.

Effect of temperature on the induction of opiate

dependence in guinea-pig isolated ileum. Br J

Pharmacol, 73:300P-301P, 1981.

Contreras, E.; Tamayo, L.; and Quijoda, L. Effects of

irreversible inhibition of GABA transaminase upon

some morphine effects. Neuropharmacology, 18:309-

313, 1979.

Costa, E.; Carenzi, A.; Guidotti, A.; and Revuelta, A.

Narcotic analgesics and the regulation of neuronal

catecholamine stores. In: Usdin, E., and Snyder, S.,

eds. Frontiers in Catecholmaine Research 1973.

London: Pergamon Press, 1974. pp. 1003-1010.

231
Crain, S.M.; Crain, B.; Finnigan, T.; and Simon, E.J.

Development of tolerance to opiates and opioid

peptides in organotypic cultures of mouse spinal

cord. Life Sci, 25:1797-1802, 1979.

Dafny, N.; Brown, M.; Burks, T.F.; and Rigor, B.M.

Patterns of unit responses to incremental doses of

morphine in central gray, reticular formation, medial

thalamus, caudate nucleus, hypothalamus, septum, and

hippocampus in unanesthetized rats. Neuropharmacol,

18:489-495, 1979.

Davis, M.E.; Akera, T.; and Brody, T.M. Saturable

binding of morphine to rat brain-stem slices and the

effect of chronic morphine treatment. Res Commun

Chem Path Pharmacol, 12:409-418, 1975.

Davis, M.E.; Akera, T.; and Brody, T.M. Reduction of

opiate binding to brainstem slices associated with

the development of tolerance to morphine in rats. J

Pharmacol Exp Ther, 211:112-119, 1979.

Domino, E.F., and Wilson, A.E. Enhanced utilization of

brain acetylcholine during morphine withdrawal in the

rat. Nature, 243:285-286, 1973.

Duka, T.; Wuster, M.; Schubert, P.; Stoiber, A.; and

Herz, A. Selective localization of different types

of opiate receptors in hippocampus as revealed by in

vivo autoradiography. Brain Res, 205:181-186, 1981.

Dupont, A.; Lepine, J.; Langelier, P.; Merand, Y.;

Rouleau, D.; Vaudry, H.; Gros, C.; and Barden, N.

Differential distribution of ß-endorphin and

enkephalins in rat and bovine brain. Reg Peptides,

1:43-52, 1980.

Florant, G.L.; Turner, B.M.; and Heller, H.C.

Temperature regulation during wakefulness, sleep, and

hibernation in marmots. Am J Physiol, 235:R82-R88,

1978.

Friedler, G.; Bhargava, H.; Quock, R.; and Way, E.L.

The effect of 6-hydroxydopamine on morphine tolerance

and physical dependence. J Pharm Exp Ther, 183:49-

55, 1972.

Gomes, C.; Svensson, T.H.; and Trolin, G. Effects of

morphine on central catecholamine turnover, blood

pressure, and heart rate in the rat, NauynSchmiedeberg's Arch Pharmacol, 294:141-147, 1976.

Gramsch, C.; Hollt, V.; Mehraein, P.; Pasi, A.; and

Herz, A. Regional distribution of methionineenkephalin- and beta-endorphin-like immunoreactivity

in human brain and pituitary. Brain Res, 171:261-

270, 1979.

Gunne, L. Noradrenaline and adrenaline in the rat

brain during acute and chronic morphine

administration and withdrawal. Nature, 184:1950-

1951, 1959.

Gunne, L. Catecholamines and 5-hydroxytryptamine in

morphine tolerance and withdrawal. Acta Physiol

Scand, 58 (Suppl 204):1-91, 1963.

232
Hammel, H.T.; Dawson, T.J.; Abrams, R.M.; and Andersen,

H.T. Total calorimetric measurements on Citellus

lateralis in hibernation. Physiol Zool, 41:341-347,

1968.

Hammel, H.T.; Heller, H.C.; and Sharp, F.R. Probing

the rostral brainstem of anesthetized, unanesthetized, and exercising dogs and of hibernating and

euthermic ground squirrels. FedProc, 32:1588-1597,

1973.

Heller, H.C., and Colliver, G.W. CNS regulation of

body temperature during hibernation. Am J Physiol,

227:583-589, 1974.

Heller, H.C.; Colliver, G.W.; and Beard, J.

Thermoregulation during entrance into hibernation.

Pflugers Arch, 369:55-59, 1977.

Heller, H.C., and Poulson, T.L. Circannian rhythms-II.

Endogenous and exogenous factors controlling

reproduction and hibernation in chipmunks (Eutamius)

and ground squirrels (Spermophilus). Comp Biochem

Physiol, 33:357-383, 1970.

Herz, A.; Teschemacher, H.J.; Albus, K.; and

Zieglgansberger, S. Morphine abstinence syndrome in

rabbits precipitated by injection of morphine

antagonists into the ventricular system and

restricted parts of it. Psychopharmacologia, 26:

219-235, 1972.

Hiller, J.M.; Pearson, J., and Simon, E.J.

Distribution of stereospecific binding of the potent

narcotic analgesic etorphine in the human brain:

Predominance in the limbic system. Res Commun Chem

Pathol Pharmacol 6:1052-1062, 1973.

Ho, I.K.; Lee, S.E.; Stolman, S.; Loh, H.H.; and Way,

E.L. Influence of p-chlorophenylalanine on morphine

tolerance and physical dependence and regional brain

serotonin turnover studies in morphine tolerantdependent mice. J Pharmacol Exp Ther, 182:155-165,

1972.

Kerr, F.W.L., and Pozuelo, J. Suppression of physical

dependence and induction of hypersensitivity to

morphine by stereotaxic hypothalamic lesions in

addicted rats. Mayo Clin Proc, 46:653-665, 1971.

Kilduff, T.S.; Sharp, F.R.; and Heller H.C. [

14C]2-

deoxyglucose uptake in ground squirrel brain during

hibernation. J Neuroscience, 2:143-157, 1982.

Kramarova, L.I.; Kolaeva, S.H.; Yukhananov, R.Y.; and

Rozhanets, V.V. Content of DSIP, enkephalins, and

ACTH in some tissues of active and hibernating ground

squirrels (Citellus suslicus). Comp Biochem Physiol,

74c:31-33, 1983.

Kromer, W. Naltrexone influence on hibernation.

Experientia, 36:581-582, 1980.

Kromer, W., and Steigemann, N. Opiate tolerance/dependence in the isolated guinea pig ileum is associated

with an increased sensitivity to acetylcholine.

Pharmacology, 25:294-296, 1982.

233
Kuhar, M.J.; Pert, C.B., and Snyder, S.H. Regional

distribution of opiate receptor binding in monkey and

human brain. Nature, 245:447-450, 1973.

Lahti, R., and Collins, A. Chronic naloxone results in

prolonged increases in opiate binding sites in brain.

Eur J Pharmacol, 51:185-186, 1978.

Large, W.A., and Milton, A.S. The effect of acute and

chronic morphine administration on brain acetylcholine levels in the rat. Br J Pharmac, 38:451P452P, 1970.

Lee, J.R., and Fennessy, M.R. The relationship between

morphine analgesia and the level of biogenic amines

in the mouse brain. Eur J Pharmacol, 12:65-70, 1970.

Leucke, R.H., and South, F.E. A possible model for

thermoregulation during deep hibernation. In: South,

F. E., Hannon, J.P., Willis, J.R., Pengelley, E.T.,

and Alpert, N.R., eds. Hibernation and Hypothermia.

Perspectives and Challenges. Amsterdam: Elsevier,

1972. pp. 577-604.

Llados-Eckman, C., and Beckman, A.L. Reduction of

hibernation bout duration by ICV infusion of naloxone

in Citellus lateralis. Soc Neurosci Abstr, 9:796,

1983.

Loh, H.H.; Brase, D.A.; Sampath-Khanna, S.; Mar, J.B.;

and Way, E.L. ß-endorphin in vitro inhibition of

striatal dopamine release. Nature, 264:567-568,

1972.

Lyman, C.P. Oxygen consumption, body temperature and

heart rate of woodchucks entering hibernation. Am J

Physiol, 194:83-91, 1958.

Margules, D.L.; Goldman, B.; and Finck, A. Hibernation: An opioid-dependent state? Brain Res Bull, 4:

721-724, 1979.

Matsukura, S.; Yoshimi, H.; Sueoka, S.; Kataoka, K.,

Ono, T.; and Ohgushi, N. The regional distribution of

immunoreactive ß-endorphin in the monkey brain. Brain

Res, 159:228-233, 1978.

Maynert, E.W., and Klingman, G.I. Tolerance to

morphine. I. Effects on catecholamines in the brain

and adrenal glands. J Pharmacol Exp Ther, 135:285-

295, 1962.

Maynert, E.W.; Klingman, G.I.; and Kaji, G.K.

Tolerance to morphine. II. Lack of effects on brain

5-hydroxytryptamine and gamma-aminobutyric acid. J

Pharmacol Exp Ther, 135:296-299, 1962.

Moleman, P., and Bruinvels, J. Morphine-induced

striatal dopamine efflux depends on the activity of

nigrostriatal neurones. Nature, 281:686-687, 1979.

Mrosovsky, N. The amplitude and period of circannual

cycles of body weight in golden-mantled ground

squirrels with medial hypothalamic lesions. Brain

Res, 99:97-116, 1975.

Mrosovsky, N. Circannual cycles in hibernators. In:

Wang, L.C.H., and Hudson, J.W., eds. Strategies in

Cold: Natural Torpidity and Thermogenesis. New York:

Academic Press, 1978. pp. 21-65.

234
Muchlinski, A.E.; Ho, F.J.; Chew, P.; and Yamada, T.

The concentrations of four neuropeptides in various

brain regions of summer active and hibernating

185-189, 1983.

Spermophilus lateralis. Comp Biochem Physiol, 74:

Oeltgen, P.R.; Walsh, J.W.; Hamann, S.R.; Randall,

D.C.; Spurrier, W.A.; and Myers, R.D. Hibernation

"trigger": Opioid-like inhibitory action on brain

function in the monkey. Pharmac Biochem Behav, 17:

1271-1274, 1982.

Olson, G.A.; Olson R.A.; Kastin, A.J.; and Coy, D.H.

Endogenous opiates: 1981. Peptides, 3:1039-1072,

1982.

Pearson, J.; Brandeis, L.; Simon, E.; and Hiller, J.

Radioautography of binding of tritiated diprenorphine

to opiate receptors in the rat. Life Sci, 26:1047-

1052, 1980.

Pengelley, E.T., and Fisher, K.C. The effect of

temperature and photoperiod on the yearly hibernating

behavior of captive golden-mantled ground squirrels

(Citellus lateralis tescorum). Canad J Zool, 41:

1103-1120, 1963.

Pert, C.B.; Kuhar, M.J.; and Snyder, S.H. Autoradiographic localization of the opiate receptor in rat

brain. Life Sci, 16:1849-1854, 1975.

Rae, G.A., and De Morhes, S. Supersensitivity to

noradrenaline in vas deferens from morphine-dependent

mice is confirmed. Eur J Pharmacol, 86:347-352,

352, 1983.

Ritzmann, R.F., Lee, J.M., and Fields, J.Z. Peptide

inhibition of morphine-induced dopaminergic supersensitivity. Life Sci, 31:2287-2290, 1982.

Robinson, J.D., and Bradley, M. Cholinesterase and

glutamic acid decarboxylase levels in the brain of

the hibernating hamster. Nature, 197:389-390, 1963.

Schulz, R., and Herz, A. Naloxone precipitated

withdrawal reveals sensitization to neurotransmitters

in morphine tolerant/dependent rats. NauynSchiedeberg's Arch Pharmacol, 299:95-99, 1977.

Schwartz, A.S., and Eidelberg, E. Role of biogenic

amines in morphine dependence in the rat. Life Sci,

9:613-624, 1970.

Shtark, M.B. The Brain of Hibernating Animals. NASA

Technical Translation TTF-619, Washington, 1972.

Translation of Mozg Zimnespyashchekt. Novosibirsk,

Nauka Press, Siberian Branch, 1970.

Simon, E.J., and Hiller, J.M. The opiate receptors.

Ann Rev Pharmacol Toxicol, 18:371-394, 1978.

Sivam, S.P.; Nabeshima, T.; and Ho, I.K. Effects of

morphine administration on opiate receptor binding in

mice. Fed Proc, 40:289, 1981.

Sloan, J.W.; Brooks, J.W.; Eisenman, A.J.; and Martin,

W.R. Comparison of the effects of single doses of

morphine and thebaine on body temperature, activity,

brain and heart levels of catecholamines and

serotonin. Psychopharmacologia, 3:291-301, 1962.

235
Snelgar, R.S., and Vogt, M. Mapping, in the rat

central nervous system, of morphine-induced changes

in the turnover of 5-hydroxytryptamine. J Physiol,

314:395-410, 1980.

South, F.E.; Breazile, J.E.; Dellmann, H.D.; and

Epperly, A. D. Sleep, hibernation and hypothermia in

the yellow-bellied marmot (M. flaviventris). In:

Mussachia, X.J., and Saunders, J.F., eds. Depressed

Metabolism. New York: El sevier, 1969. pp. 277-312.

South, F.E.; Heath, J.E.; Luecke, R.H.; Mihailovic,

L.T.; Myers, R.D.; Panuska, J.A.; Williams, B.A.;

Hartner, W.C.; and Jacobs, H.K. Status and role of

the central nervous system and thermoregulation

during hibernation and hypothermia. In: South, F.E.,

Hannon, J.P., Willis, J. R., Pengelley, E.T., and

Alpert, N.R., eds. Hibernation and Hypothermia,

Perspectives and Challenges. Amsterdam: Elsevier,

1972. pp. 629-633.

Stanton, T.L., and Beckman, A.L. Thermal changes

produced by intrahypothalamic injections of acetylcholine during hibernation and euthermia in Citellus

lateralis. Comp Biochem Physiol, 58A:143-150, 1977.

Stanton, T.L.; Winokur, A.; and Beckman, A.L. Reversal

of natural CNS depression by TRH action in the

hippocampus. Brain Res, 181:470-475, 1980.

Stanton, T.L.; Winokur, A.; and Beckman, A.L. Seasonal

variation in TRH content of different brain regions

and the pineal in the mammalian hibernator, Citellus

lateralis. Reg Peptides, 3:135-144, 1982.

Stefano, G.B.; Kream, R.M.; Zukin, R.S.; and Catapane,

E.J. Seasonal variation of stereospecific enkephal in

binding and pharmacological activity in marine

molluscs nervous tissue. In: Rozan, K.S., ed.

Advances in Physiological Science Vol. 22, Neurotransmitters in Invertebrates. London: Pergamon

Press, 1980. pp. 453-458.

Strumwasser, F. Thermoregulatory, brain and behavioral

mechanisms during entrance into hibernation in the

squirrel, Citellus beecheyi. Am J Physiol, 196:15-

22, 1959a.

Strumwasser, F. Regulatory mechanisms, brain activity,

and behavior during deep hibernation in the squirrel,

Citellus beecheyi. Am J Physiol, 196:23-30, 1959b.

Tsang, D., and Ng, S.C. Effect of antenatal exposure to

opiates on the development of opiate receptors in rat

brain. Brain Res, 188:199-206, 1980.

Van Loon, G.R.; Appel, N.M.; and Ho, D. ß-endorphininduced increases in plasma epinephrine, norepinephrine and dopamine in rats: Inhibition of adrenal

medullary response by intracerebral somatostatin.

Brain Res, 212:207-214, 1981.

Vasko, M.R., and Domino, E.F. Tolerance development to

the biphasic effects of morphine on locomotor

activity and brain acetylcholine in the rat. J

Pharmacol Exp Ther, 207:848-858, 1978.

236
Vasko, M.R., and Vogt, M. Analgesia, development of

tolerance, and 5-hydroxytryptamine turnover in the

rat after cerebral and systemic administration of

morphine. Neuroscience, 7:1215-1225, 1982.

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.

Walker, J.M.; Glotzbach, S.F.; Berger, R.J.; and

Heller, H.C. Sleep and hibernation in ground

squirrels (Citellus spp.): Electrophysiological

observations. Am J Physiol, 233:R213-R221, 1977.

Wang, L.C.H. Energetic and field aspects of mammalian

torpor: The Richardson’s ground squirrel. In: Wang,

L.C.H., and Hudson, J.W., eds. Strategies in Cold:

Natural Torpidity and Thermogenesis. New York:

Academic Press, 1978. pp. 109-145.

Wang, L.C.H. Hibernation and the endocrines. In:

Lyman, C.P., Willis, J.S., Malan, A., and Wang,

L.C.H., eds. Hibernation and Torpor in Mammals and

Birds. New York: Academic Press, 1982. pp. 206-236.

Watson, S.J.; Akil, H.; Sullivan, S.; and Barchas, J.D.

Immunocytochemical localization of methionine enkephal in: Preliminary observations. Life Sci, 21:733-

730, 1977.

Way, E.L.; Loh, H.H.; and Shen, F. Morphine tolerance,

physical dependence, and synthesis of brain 5-

hydroxytryptamine. Science, 162: 1290-1292, 1968.

Wei, E.; Loh, H.H.; and Way, E.L. Neuroanatomical

correlates of morphine dependence. Science, 177:

616-617, 1972.

Wei, E.; Loh, H.H.; and Way, E.L. Brain sites of

precipitated abstinence in morphine dependent rats. J

Pharmacol Exp Ther, 185:108-115, 1973.

Wikler, A.; Norrell, H.; and Miller, D. Limbic system

and opioid addiction in the rat. Exptl Neurol, 34:

543-557, 1972.

Wooten, G.F.; DiStefano, P.; and Collins, R.C. Regional

cerebral glucose utilization during morphine withdrawal in the rat. Proc Natl Acad Sci, 79:3360-3364,

1982.

Wunnenberg, W.; Merker, G.; and Speulda, E. Thermosensitivity of preoptic neurons and hypothalamic

integrative function in hibernators and non-hibernators. In: Wang, L.C.H., and Hudson, J.W., eds.

Strategies in Cold: Natural Torpidity and Thermogenesis. New York: Academic Press, 1978. pp. 267-

297.

Zukin, R.S.; Sugarman, J.R.; Fitz-Syage, M.L.; Gardner,

E.L.: Zukin. S.R.: and Gintzler, A.R. Naltrexoneinduced opiate receptor supersensitivity. Brain Res,

245:285-2921 1982.

237
ACKNOWLEDGEMENTS

The work reported here was supported by National

Institute on Drug Abuse grant DA-02254 and the Alfred

I. duPont Institute.

AUTHORS

Alexander L. Beckman, Ph.D.

Carmen Llados-Eckman, B.S.

Steven K. Salzman, Ph.D.

Toni L. Stanton, Ph.D.

Alfred I. duPont Institute, P.O. Box 269, Wilmington,

DE 19899

238
Neurophysiological Investigations

of Opiate Tolerance and Dependence in the Central Nervous

System

Steven Jon Henriksen

Most pharmacological studies investigating opiate- and opioidinduced tolerance, dependence or addiction liability have utilized

In vitro bioassay systems or whole animal preparations. These

studies, although critical for the elucidation of rank order

potency series and the characterization of the variety of opiate

receptor subtypes, have not led to the understanding of the

cellular processes underlying opiate tolerance and dependence.

More recently, studies using hybrid cell lines (see previous papers

in this volume) and the investigation of opiate receptor up-or-down

regulation (Zukin et al., this volume) have suggested important

receptor Iigand and second messenger processes that are candidates

for participation in cellular events leading to the phenotype of

the opiate-tolerant organism. Neurophysiological approaches have

also been employed in the study of opiate and oploid neuropharmacology. These studies, using a variety of electrophysiological

approaches at the cellular level, have begun to address those

bioelectric events associated with acute and chronic drug exposure.

Electrophysiological Techniques: An Inherent Advantage For The

Investigation Of Tolerance And Dependence:

(1) They allow the opportunity for real-time analysis of the cellular processes occurring during acute, chronic, and drug

withdrawal conditions; (2) they provide a unique wide dynamic range

of measured events exemplified by encephalographic and field

potential analysis as well as analysis of intracellular events

associated with synaptic and nonsynaptic processes; and (3)

electrophysiological measurements can be made in several different

in vitro and in vivo animal preparations of which each provides

particular technical advantages for answering specific questions

regarding mechanisms of opiate tolerance and dependence.

In this essay, I will review the evidence of the neurophysiological

correlates of acute and chronic opiate (and oploid) exposure, and

will discuss how these data generate hypotheses regarding the

cellular mechanisms underlying these phenomena. This review is in

no way meant to be exhaustive; rather, it has been focused solely

239
on the central nervous system, and then only on particular examples

from investigators using specific neurophysiological tools to study

the mechanisms of tolerance.

Four considerations are important when discussing central neuronal

responses to acute or chronic opiate treatment: (1) What is the

nature of event being measured, i.e., is it evoked or spontaneous?

(2) What is the time course of the alteration in the event

recorded, and what is the stability of this effect?; (3) Can

dependence be ascertained by precipitation with naloxone? and (4)

Are the changes at the cellular level following chronic opiate

exposure due to the same mechanisms that produce the acute effects

of these drugs?

Encephalographic Analysis

The electroencephalograph to record spontaneously generated

cortical bioelectric events has long been used as a tool to assess

both acute and chronic effects of opiates (Khazan 1975). Systemic

or intraventricular administration of acute doses of morphine, In

numerous experimental animal species, results in a widespread

cortical desynchrony and 4-10 Hz burst activity associated with

behavioral stupor (Khazan et al. 1967; Khazan and Colasanti 1971;

Nakamura and Winters 1973; Tortella et al. 1978). In addition,

changes in cortical and subcortical electrical activity in response

to repeated morphine administration has been investigated and

electroencephalographic tolerance to opiates has been observed

(Khazan et al. 1967; Teitelbaum 1976).

Multiple intracerebral injections of morphine into specific subcortical brain loci can result In electrographic tolerance at those

injected sites (Teitelbaum et al. 1976). In addition, specific

brain lesions have been shown to reverse electrographically

assessed morphine tolerance (Teltelbaum et al. 1979).

In addition to those studies evaluating the action of opiates on

the spontaneous electroencephaIogram, other investigations have

focused on the effects of opiates on evoked potentials in naive,

tolerant and withdrawn animals (Straw and MitchelI 1964; Domino

1968; Gildenberg et al. 1976). These studies have demonstrated

that particular components of stimulation-evoked potentials, and

not others, are dramatically altered by acute opiate administration. These effects diminish on continued administration and

therefore exhibit tolerance.

Studies such as these have demonstrated that the electroencephalographic technique is a valuable tool to monitor those cellular

electrical events that parallel the development of behavioral

tolerance to opiates. This approach has also been used to demonstrate more localized cerebral electrical changes associated with

the opiate dependence cycle.

The same encephalographic techniques used to assess the acute and

chronic effects of opiates on brain electrical activity have been

applied to the study of the endogenous opioid peptides.

240
ß-endorphin, methionine and leucine enkephalin, as well as several

enkephalin derivatives, have been shown to have nonconvulsive

epileptogenic properties when administered intraventricularly to

rats (Henriksen et al. 1978; Urca et al. 1977; Neal and Keane 1978;

Tortella et al. 1978). This epileptiform activity seems to have

its origin in limbic structures (an area rich in opiate receptors)

as determined by localized recording and 2-deoxyglucose techniques

(figure 1). Another major opioid peptide, Dynorphin A (1-17), is

not epileptogenlc in rats, suggesting specific opiate receptor

specificity for the generation of this activity (Henriksen et al.

1983).

LATERAL

SEPTUM

VENTRAL

CA3

FIGURE 1. 2-DG. EEG traces and 14C-2 deoxyglucose (2DG) autoradiographic illustrations of the response to intraventricularly

administered ß-endorphin (B-E, 3 nM) in a representative rat.

A) Electrographic tracing of ictal seizure episode elicited by B-E.

B) 14C-2DG autoradiographs of representative sections of control

rat brain and a brain of a rat exhibiting a seizure following B-E.

(Henriksen et al. 1983. © Alan R. Liss, Inc. 1983).

241
Visual inspection of the autoradiographs reveals a marked uptake of

DG, bilaterally, in the dorsal and ventral hippocampus, posterior

and ventral subiculum, dentate gyrus, lateral septum, and the

posterior part of the medial nucleus of the amygdala (amygdalahippocampal area). There was also a marked decrease of uptake

bilaterally in the medial geniculate body.

The electrographic changes seen in the EEG following repeated

ventricular or in situ administration of opioid peptides result in

the development of tolerance to the effect of the peptide simiIar

to opiates (Elazar et al. 1979). Figure 2 illustrates the development of tolerance to the epileptogenic action of a daily injection

of ß-endorphin (3 nM i.c.v.) in a rat.

These encephalographic studies provide important information

regardlng localized electrographic patterns associated wlth acute

and chronic opioid/opiate administration. Therefore they suggest

specific brain areas, and even nuclei, that are highly sensitive to

the effects of these drugs. On the other hand, this experimental

approach is unable to distinguish between a direct pharmacological

effect and an indirect, secondary, effect of these drugs. Because

of this technical limitation other neuropharmacological approaches

have been employed to investigate more directly the action of

narcotics and opioid-peptides on single neurons.

Extracellular Analysis

1. Acute effects of opiates and opioid peptides

Prior to the knowledge of the endogenous opioid peptides or

specific opiate receptors, most central pharmacological effects of

systemically administered opiate alkaloids were attributed to their

action on known transmitter systems (Clouet 1971; Way 1972; Way and

Shen 1971; Clouet and lwatsubo 1975). However, in their extensive

review, Clouet and lwatsubo (1975) could not attribute a causative

role to any single transmitter system for the development of

tolerance to opiates.

In an attempt to characterize more precisely the possible interaction of opiates with other neurotransmitters, the neuropharmacological technique of microiontophoresis combined with single cell

recording has been employed. This approach has the important

advantage of allowing the direct assessment of drug (or putative

transmitter) efficacy on a single recorded neuron thereby alleviating the complication of indirect effects of systemically administered drugs. In addition, the effects of iontophoretically applied

opiates (or, as we are now aware, opioid peptides) on cellular

responses to other neurotransmitters can be evaluated. Early

studies using this approach demonstrated compIex neuronal act ions

of iontophoretically applied opiates. In the brainstem of the rat,

iontophoretically applied morphine produced both excitatory (43%)

as well as inhibitory (22%) actions on the spontaneous discharge of

individual neurons (Bradley and Dray 1974). In addition, iontophoretically applied morphine was able to block, in many cases, the

242
CHANGES IN BETA-ENDORPHlN INDUCED ICTAL EPISODES FOLLOWING

REPEATED INJECTIONS

FIGURE 2. Endorphin Tolerance. Amygdala EEG traces taken from a

rat injected once daily wIth ß-endorphin (3 nM) intraventricularly.

IPSILAT: refers to same side of the brain as the injection;

CONTRA: refers to the opposite side from the injection. Note the

slow but marked tolerance that develops to the ictal epileptiform

events as recorded from the amygdala. WDS: wet-dog-shakes; NM=no

movement.

normal excitatory response of these neurons to iontophoretically

applied acetylcholine, noradrenaline and 5-hydroxytryptamine. On

the other hand, the excitatory effect of glutamate on these neurons

could not be blocked by morphine (Bradley and Dray 1974). In an

attempt to assess the specificity of these morphine effects, it was

243
found that only the depressions in brain stem unit activity were

capable of being antagonized by naloxone, suggesting that the

direct excitatory effects of morphine observed in a majority of the

neurons tested was a nonspecific effect (Bramwell and Bradley

1974). In another important observation, these investigators

rarely observed the phenomenon of tachyphylaxis (i.e., desensitization) to repeated iontophoretic applications of opiate and never

observed such an effect to the inhibitory (i.e., specific) action

of morphine.

Numerous other investigators have studied the action of opiates in

other brain areas using the microiontophoretic technique. In

general, these studies show that the specific (i.e., naloxonereversible) effect of electrophoretically applied opiates is to

depress the spontaneous activity of recorded neurons (Bradley and

Dray 1974; Henry and Neuman 1975; Zieglgansberger and BayerI 1975;

Zieglgansberger et al. 1975; Satoh et al. 1976; Fry et al. 1980;

but see Davies 1976). Figure 3 illustrates a representative

example of the effect of normorphine, iontophoretically applied

onto rat amygdala neurons.

Soon after the isolation, characterization, and synthesis of the

first endogenous opioid peptide, neuropharmacologists began to

evaluate this new, growlng list of peptides for central opiate-Iike

activity. Of paramount importance in these investigations has been

the localization of oploid receptors through autoradiographic

analysis (Goodman et al. 1980; Duka et al. 1981; Goodman and Snyder

1982) as well as the distribution of the endogenous opioid peptides

thorough the use of immunohistochemical techniques (Bloom et al.

1977, 1978; Watson et al. 1977 and others).

In this way, the cellular physiologists have been able to direct

their studies to those areas in the mammalian brain appropriate for

each opiate receptor subtype being investigated as well as for the

specific oploid peptide locally dlstributed. Early surveys of the

response of neurons to iontophoretically applied opioid peptides

suggested great similarities to what had previously been observed

with opiate alkaloids (Nicoll et al. 1977; Siggins et al. 1978;

Zleglgansberger et al. 1975; French and Siggins 1980). In most

brain areas studied, the great majority of single neurons demonstrate naloxone-reversible inhibition of spontaneous discharge

following electrophoresis of the enkephalins (either leucine or

methionine) or ß-endorphin (figure 4). A notable exception to this

general inhibitory effect is observed in the hippocmapal formation

where mostly excitatory effects are observed (NicolI et al. 1980;

and Zieglgansberger et al. 1979). These results supported earIier

encephalographic data suggesting excitatory, epileptogenic, actions

of intraventricularly administered opioid peptides on the hippocampal formation (Henriksen et al. 1978). Because of the unique

excitatory actions of most opioid peptides on hippocampal neurons,

a great number of investigators have focused their attention on

this structure in an attempt to explain this paradox. Simply

stated, it now appears that these opioid-induced excitatory effects

244
FIGURE 3. Amygdala. Inhibitory effects of morphine (MS; panel A)

on a single neuron in anterior central amygdala, and of methionineenkephal in (ME) and ß-endorphin (ß; panel B) on a neuron in

anterior basolateral amygdala. Apparent differences here in sensitivity of these neurons to the opiates or peptides were not consistently observed in other neurons. Note: the magnitude of response

to MS in panel A is correlated with the iontophoretic current

(French and Slggins 1980, ©

Biomedical Press).

are most likely the result of

the peptides on adjacent

Zieglgansberger et al. 1979).

1980 by Elsevier North Holland -

an indirect disinhibitory action of

inhibitory interneurons (see

These physiological data are supported by anatomical evidence suggesting the presence of opioid-containing neurons in close

proximity to putative inhibitory interneurons (Gall et al. 1981).

2. Extracellular effects of opiates and oploids in tolerant

preparatlon

Microlontophoretic and micropneumatic techniques have been used to

assess the cellular response to applied opioid peptides and opiates

in animals previously made tolerant to opiate alkaloids. In most

brain areas studied in tolerant rats, extracellular application of

opiates and opioids results in a diminished responsiveness to their

action compared to their effect in naive animals (Satoh et al.

1974, 1975 1976; Zieglgansberger et al. 1976; Zieglgansberger and

Fry 1976; Fry et al. 1979; Aghajanian 1978). In addition, cellular

245
FIGURE 4. Excitatory action of methionine-enkephalin (ME) and

acetylcholine (ACh) in the hippocampus, and the blockade of the ME

act ion by iontophoretic naIoxone. Thin bar indicates ME application and thicker bar, ACh application. Same neuron throughout

record. Note that firing rate and ACh response is not much

affected at 2-3 minutes of naloxone application, when ME responses

are totally blocked, but less selective naloxone effects emerge

later, when the ACh response is reduced by about 30% (French and

Siggins 1980, © 1980 by Elsevier North Holland - Biomedical Press).

dependence to morphine has been demonstrated by studies indicating

a higher incidence of excitatory responses to microiontophoretically administered naloxone has been observed for neurons recorded

in the medial thalamus (Frederickson et al. 1975), frontal cortex

(Satoh et al. 1976), locus coeruleus (Aghajanian 1978) and striatum

(Fry et al. 1980) of morphine-tolerant rats.

These studies offer strong evidence that both tolerance, as well as

dependence, to opiates and opioid peptides can be observed at the

single neuron level in the vertebrate CNS.

Recent studies have attempted to assess opioid tolerance and dependence using a CNS in vitro preparation, the hippocampal, or locus

coeruleus slice. This preparation offers several technical advantages over the in vivo preparation for the study of opiate tolerance and dependence. Of foremost importance is the abiIity to

accurately control drug concentration, which allows true pharmacological studies to be done with agonist and antagonist drugs.

As was previously discussed, opiates and opioids produce excitatory

effects in the hippocampal formation. In rat hippocampal slices,

opiates and opioid peptides potentiate the synaptic activation of

246
pyramidal neurons as revealed by a shift to the left in the inputoutput curves of monosynaptically evoked CA1 field potentials

(Robinson and Deadwyler 180; Corrigall et al. 1981; French and

Zieglgansberger 1982; Valentino and Dingledine 1982; Robinson et

al. 1982). Figure 5 illustrates the effect of normorphine and

methionine-enkephalin on hippocampal slice CA1 field potentials in

a slice obtained from a naive rat.

FIGURE 5. Effect of normorphine (NM, 1 μM) on the population spike

evoked in hippocampal pyramidal cells (CA1) by stimulation of

Schaffer collaterals. A. Spikes at several time points (3,4,6

minutes) after onset of opioid perfusion. Washout taken at 20

minutes after termination of drug application. Note the emergence

of a secondary wave (6 minutes). Stimulus intenslty was 40 V,

duration 0.1 ms. Calibration: 1 mV, 5 ms, B. Effect of

methionine-enkephalin (ME, 1 μM) at various time points (5, 25

minutes) and with different stimulus intensities (23,30,40 V).

Note the large secondary and tertiary wave forms at the 25-minute

time point. The responses to ME + NX were taken 5 minutes after

the onset of perfusion of ME (1 μM) + naloxone (NX, 1 μM).

Calibration: 1 mV, 5 ms (same recording and stimulation sites as

In A) (French and Zieglgansberger 1982, © 1982 by Springer-Verlag).

Hippocampal slices obtained from rats made tolerant to morphine

exhibit tolerance to morphine but not to opioids when challenged in

vitro (French and Zieglgansberger 1982). These results suggest

that, while both opiate agonists increase neuronal excitability of

hippocampal pyramidal celIs, this effect is likely to be mediated

via different opiate receptor populations (figure 6).

247
FIGURE 6. Effect of methionine-enkephalin (ME) and lack of effect

of a high concentration of normorphine (NM) when applied to slices

obtained from chronically morphinized rats:

control; ME, 5 μM; NM, 5 μM;~ washout

of Me response (French and Zieglgansberger 1982, © 1982 by

Springer-Verlag).

In a recent study utilizing the locus coeruleus slice preparation

(Andrade et al. 1983), tolerance was observed to the normal

decrease in spontaneous firing of locus coeruleus neurons produced

with acute administration of morphine. However, in contrast to

what has been reported in in vivo studies (Aghajanian 1978),

naloxone administered to the tolerant slice did not precipitate

supranormal firing rates of locus coeruleus neurons. These results

strongIy suggest that, at least for noradrenergic locus coeruleus

neurons in vitro, tolerance and dependence can be dissociated. It

also suggests that dependence (withdrawal activation) in locus

coeruleus neurons is mediated by factors present in vivo but not in

vitro (i.e., extrinsic excitatory inputs to this area).

248
FIGURE 7. Acute desensitization to phoretically applied

methionine-enkephalin. A: methionine-enkephal in (ME, 20 nA)

reduces spontaneous and glutamate-induced (G, 10 nA/20 s) discharge

activity. Despite ongoing application of ME, the firing rate

increased again and showed a clear rebound after termination of

drug application. The inhibitory action of GABA (5 nA) before (not

shown), during and after ME application was not altered significantly. Applications are indicated by bars. Doses are indexed.

B: ordinates in B and C give activity during pulses (10 s) of

glutamate (10 nA). The applications were spaced 60 s apart.

Microiontophoretic administration of D-Ala2-D-Leu5-enkephalin (D,

50 nA) causes inhibition which is not changed despite the desensitization to morphine (M, 100 nA). C: the inhibitory effect of

morphine (M, 40 nA) was lost after the neuron was rendered insensitive to ME (65 nA). After termination of drug administration the

neuron showed a marked rebound in its firing rate and reached

control levels and sensitivity to morphine after about 15 minutes

(Williams and Zieglgansberger 1981, © 1981 by Elsevier North

Holland - Biomedical Press).

3. Acute desensitization to opiates/opioids

Cellular studies of opiate/opioid tolerance and dependence have the

unique capability of accurately monitoring the developmental time

course of subsensitivity to opiate agonists. Some investigations

have suggested very short-term tolerance, or tachyphylaxis, to

opiates can occur (Fry et al. 1980; Gahwiler 1981; Williams and

Zieglgansberger 1981. Figure 7 illustrates an example of

249
FIGURE 8. Dynorphin A(1-17), 30 nA, induces excitatory response in

a CA3 hippocampal neuron recorded from anesthesized rat. Because

space does not allow, this rate-meter represents only the last four

of 50 dynorphin A 30-second pulses. No acute desensitization was

observed.

desensitization of a frontal cortical neuron to a continuous

iontophoretic puIse of methionine-enkephalin. In addition, in this

same study, an apparent one-way cross-desensitization was observed

for morphine and D-Ala-D-Leu-enkephalin (figure 70).

Other studies in other brain areas faiIed to demonstrate acute

tolerance to either opiates or opioids peptides. Figure 8 illustrates the effect of iontophoretically applied dynorphin on a rat

hippocampal neuron and the lack of tachyphylaxis.

In addition, studies of opiate/opioid effect on hippocampal neurons

in the slice preparation have failed to show any short-term tolerance to either agent (Valentino and Dingledine 1982; Robinson and

Deadwyler 1980). In fact, even after 4-hours of superfusion with

morphine (20 M), no diminution in the augmentation of the evoked

population spike was observed.

It is possible that some of the earlier investigations demonstrating tachyphylaxis to opiates may have been confounded by

technical problems associated with long-term ejection artifacts

observed when using iontophoretic pipettes. The issue of acute

tolerance to opiates and opioid peptides, therefore, remains

unresolved.

Intracellular Analysis

Following the lead of in vivo pharmacological studies of the action

of opiates and opioid peptides on single neurons in brain, several

investigators recently have begun to investigate the intracellular

processes associated with acute and chronic drug exposure.

Recordings have been made from neurons penetrated in the hippocampal and locus coeruleus slice preparation (See Siggins and

Zieglgansberger 1981; Masukawa and Prince 1982; Robinson and

Deadwyler 1981; Nicoll et al. 1980; Dingledine 1980; Haas and RyalI

1980; Pepper and Henderson 1980; WiIIiams et al. 1982). In the

locus coeruleus slice preparation, methionine5-enkephalin, D-Ala2-

D-Leu5-enkephalin (DADLE) and morphine produce a naloxone-sensitive

increase in potassium conductance resulting in hyperpolarization of

250
FIGURE 9. Enkephal inhibits cell firing. The figure shows an

intracellular recording from a spontaneously firing locus coeruleus

neuron. DADLE was applied by a brief (50 ms) pulse of pressure (70

kN M

-2, 10 p.s.i.) to a pipette positioned just above the brain

slice at the time indicated by the arrows. The cell hyperpolarized

and the action potential discharge was inhibited. The pen recorder

did not reproduce the full action potential amplitude, which was

70-100 mV among different neurons. These recordings were made from

a completely submerged slice of pons (thickness 300 M) continuously

superfused with a solution of the following composition (nM): NaCL

11, which was equilibrated a 37°C with 95 O2, 5% CO2 (Williams et

126, KCl 2.5, CaCI2 2.4, NaH2PO4 1.2, MgCI2 1.3, NaHCO3 25, glucose

al. 1982, © 1982 by MacMillan Journals Ltd.).

the cell and cessation of spontaneous firing (Williams et al. 1982;

North and Williams 1983; see figure 9). Note the lack of acute

desensitization to this response.

in contrast, pyramidal cells penetrated in the hippocampal slice

preparation show little or no direct transmembrane effects of

either morphine or several opioid peptides. On the other hand

these agents do alter synaptic responses to afferent stimulation,

which is consistent with a modulatory action, of opioids in this

structure (Masukawa and Prince 1982; Siggins and Zieglgansberger

1981). As yet, there exists no consensus as to the exact

mechanisms by which opioids alter these hippocampal synaptic potentials but most studies suggest a reduction of recurrent and feed

forward inhibitory post-synaptic potentials (IPSPs) implying a

disinhibitory mechanism. This interpretation is also suggested by

extracellular studies previously discussed.

One recent study has investigated the changes in transmembrane

properties and sensitivity to opiates following prolonged exposure

to morphine (Williams and North 1983). In this interesting study,

intracellular recordings were made from locus coeruleus neurons in

251
the slice preparation after three treatment paradigms: (1)

recordings made from slices cut from naive rats and superfused with

morphine for 8-12 hours, (2) recordings made from slices taken from

rats previously implanted with morphine pellets and superfused with

morphine; and (3) recordings made from slices taken from pelleted

rats but superfused in a morphine-free solution. In "naive" locus

coeruleus slices, normorphine (1-3 M) produced the usual hyperpolarization of these neurons; however, the hyperpolarization

lasted for as long as the superfusion continued (up to 12 hours).

That is, no tolerance was observed to the hyperpolarizing effect of

normorphine in this time period. Neurons recorded from pelleted

rats showed only mild tolerance to the hyperpolarizing action of

superfused normorphine (3-10-fold less potent). In studies taken

from pelleted rats, naloxone caused a depolarization of recorded

neurons and the return to baseline of the spontaneous firing rate.

These studies indicate that no acute tolerance to normorphine is

seen during 8-12 hours of perfusion in the locus coeruleus slice.

In addition, only minimal tolerance is observed in the slices taken

from peIIeted rats. This is to be compared to strong behavioraI

and cellular tolerance seen in vivo with the same treatment

schedule. Therefore, at least for neurons in this preparation,

much of those observed signs of tolerance and dependence seen in

vivo must derive from a larger neural net influencing secondarily

the excitability of locus coeruleus neurons, Studies such as these

intracellular investigations will be very important to follow for

neurons from other brain areas where acute tolerance has been

observed.

SUMMARY

In this essay I have presented several examples of cellular neurophysiological approaches used to investigate opiate tolerance and

dependence. In many ways, these central nervous system studies

parallel earlier investigations carried out in in vitro preparations and celI culture systems reviewed by others in this volume.

The examples presented here have demonstrated that opiate/opioid

tolerance and dependence are observed at alI levels of electrophysiological analysis. Moreover, recent studies utiIizing the

locus coeruleus slice preparation have shown the feaslbiIity of

separating the processes underlying opiate tolerance and those of

dependence (Andrade et al. 1983). Several important issues remain

unresolved: (1) Can acute tolerance to opiates/opioids be demonstrated in alI preparations (and in alI brain areas studied) or is

this phenomenon, in part, due to technical shortcomings of iontophoresis? (2) What are the characteristics of the subpopulations

of opioid receptor agonists and their differential ability to

alter, in naive or tolerant preparations, synaptic and nonsynaptic

processes in neurons? and (3) What are the intracellular events

associated with dependence on other brain areas known to contain

neurons that are post-synaptic to endogenous opioid peptides? In

addition, what is the relationship, at the synaptic level, of

opioid-induced synaptic activity and the cascade of intracellular

events coupIed to "second messenger" processes? Are these Intracellular processes ubiquitous for all opioid agonist subtypes or

252
are regional as well as intracellular coupling mechanisms

heterogeneous?

Many of these questions currently remain unanswered. However,

taking our lead from investigators using molecular approaches to

study receptor function (see Sigworth 1982; Walker et al. 1982;

Taylor et al. 1983; Dunn et al. 1983 and others vis-a-vis

muscarinic ACh receptor), "opiold" physiologists are likely to play

a major role in elucidating and characterizing endogenous

ligand/receptor interactions in the coming years.

REFERENCES

Aghajanian, G. Tolerance of locus coeruleus neurones to morphine

and suppression of withdrawal response by clonidine. Nature,

276: 186-187, 1978.

Andrade, R., Vandermaelen, C. and Aghajanian, G. Morphine

tolerance and dependence in the locus coeruIeus: single cell

studies in brain slices. European J Pharmacol, 91:161-169,

1983.

Bloom, F., Battenberg, E., Rossier, J., Ling, N., LeppaIuoto, J.,

Vargo, T. and Guillemin, R. Endorphins are located in the

intermediate and anterior lobes of the pituitary gland not in

the neurophyphysis. Life Sci, 265:49-51, 1977.

Bloom, F.E., Battenberg, E., Rossier, J., Ling, N. and Guillemin,

R. Neurons containing beta-endorphin in rat brain exist

separately from those containing enkephalin: immunocytochemical studies. Proc Natl Acad Sci USA, 75:1591-1595, 1978.

Bradley, P.B. and Dray, A. Morphine and neurotransmitter

substances: microiontophoretic study in the rat brain stem.

Br J Pharm, 50:47-55, 1974.

Bramwel I, G.J. and Bradley, P.B. Actions and interactions of

narcotic agonists and antagonists on brain stem neurons.

Brain Res, 73:167-170, 1974.

CIouet, D.H. The alteration of brain metabolism by narcotic

analgesic drugs. In: Lajtha, A., ed. Handbook of Neurochemistry. Vol. 6 New York: Plenum Press, 1971. pp.479-

508.

Clouet, D.H. and Iwatsbo, K. Mechanisms of tolerance to and

dependence on narcotic anaIgesic drug. Ann Rev Pharm, 15:49-

70, 1975.

Corrigall, W., Linseman, M.A., Lucato, R. and ElIiott, M.

Differential tolerance to the effects of morphine on evoked

activity in the hippocampal slice. Life Sci, 28:1613-1620,

1981.

253
Davies, J. Effects of morphine and naloxone on renshaw cells and

spinal interneurons in morphine dependent and non-dependent

rats. Brain Res, 113:311-326, 1976.

Dingledine, R. Enkephalins excitatory action on hippocampal

neurons cannot be explained by attenuation of recurrent

inhibition. Neurosci Abstr, 6:612, 1980.

Domino, E.F. Effects of narcotic analgesics on sensory input,

activating system and motor output. Res Pull Ass Nerv Ment

Pis, 46:150-155, 1968.

Duka, T., Wuster, M., Schubert, P., Stoiber, R. and Herz, A.

Selective localization of different types of opiate receptors

in hippocampus as revealed by in vitro autoradiography. Brain

Res, 205:181-186, 1981.

Dunn, S., Conti-Tronconi, B. and Raftery, M. Separate sites of low

and high affinity for agonists on torpedo californice

acetylcholine receptor. Biochemistry, 22:2512-2518, 1983.

Elazar, Z., Motles, E., Ely, Y. and Simantov, R. Acute tolerance

to the excitatory effect of enkephalin microinjections into

hippocampus. Life Sci, 24:541-548, 1979.

Frederickson, R.C.D., Norris, F.H. and Hewes, C.R. Effects of

naloxone and acetylcholine on medial thalamic and cortical

units in naive and morphine dependent rats. Life Sci, 17:81-

82, 1975.

French, E. and Siggins, G. An iontophoretic survey of opioid

peptide actions in the rat limbic system: In search of opiate

epileptogenia mechanisms. Reg Pep, 1:127-146, 1980.

French, E. and Zieglgansberger, W. The excitatory response of in

vitro hippocampal pyramidal cells to normorphine and

methionine-enkephalin may be mediated by different receptor

populations. Exp Brain Res, 48:238-244, 1982.

Fry, J.P., Zieglgansberger, W. and Herz, A. Specific versus

nonspecific actions of opioids on hippocampal neurones in the

rat brain. Brain Res, 163:295-305, 1979.

Fry, J.P. Herz, A. and Zieglgansberger, W. A demonstration of

naloxone-precipated opiate withdrawal on single neurons in the

morphine-tolerant/dependent rat brain. Brit J Pharmac,

68:585-592, 1980.

Fry, J.P., Zieglgansberger, W. and Herz, A. Tachyphylaxis to

enkephalin-tolerance and dependence at the single neuron

level? In: Ryall and Kelley, eds. Iontophoresis and

Transmitter Mechanisms in the Mammalian Central Nervous

System, Elsevier, 1978. pp. 323-325.

254
Fry, J.P., Zieglgansberger, W. and Herz, A. Development of acute

opioid tolerance and dependence in rat striatal neurons.

Naunyn Schmiedebergs Arch Pharmacol, 313:145-149, 1980.

Gahwiler, B.H. Development of acute tolerance during exposure of

hippocampal explants to an opioid peptides. Brain Res,

217:196-200, 1981.

Gall, C., Brecha, N., Chaung, K.J. and Karten, H.I. Localization

of enkephalin-like immunoreactivity to identified axonal and

neuronal popuIations of the rat hippocampus. J Comp Neurol,

198:335-350, 1981.

Gildenberg, P., Murthy, K.S., Adler, M. and Frost, E.A. The effect

of morphine on evoked potentials in naive, tolerant and

withdrawn rats. in: Opiates and Endogenous Opioid Peptides.

Amsterdam: Elsevier Biomedical Press, 1976, pp. 247-256.

Goodman, R.R., Snyder, S., Kuhar, M. and Young, W.S. III.

Differentiation of delta and mu opiate receptor localizations

by light microscopic autoradiography. Proc Natl Acad Sci USA,

77:6239-6243, 1980.

Goodman, R. and Snyder, S. k opiate receptors localized by

autoradiography to deep layers of the cerebral cortex:

relation to sedative effects. Proc Natl Acad Sci USA,

79:5703-5707, 1982.

Haas, H. and Ryall, R. Is excitation by enkephalins of hippocampal

neurons in the rat due to presynaptic of activation or to

dlslnhibition? J Physiol, 308:315-330, 1980.

Henriksen, S.J., Bloom, F.E., McCoy, F., Ling; N. and Guillemin, R.

Beta-endorphin induces nonconvulsive limbic seizures. Proc

Natl Acad Sci USA, 75:5221-5225, 1978.

Henriksen, S., Chouvet, G., McGinty, J. and Bloom, F.

Neuropeptides and the epilepsies: Current Perspectives. In:

Epilepsy: An Update on Research and therapy. Nistico, D.,

Perry, & Meinardi , eds. Progress in Clinical and Biological Research, V. 24. New York: Alan R. Liss, 1983. pp. 105-120.

Henry, J.L. and Neuman, R.S. Morphine depression of dorsal horn

neurons in the cat. Proc Canad Fed Biol Soc, 17:158, 1975.

Khazan, N. Implications and significance of EEG and sleep-awake

activity in the study of experimental drug dependence on

morphine. In: Ehrenpreis, S. and Neidle, A., eds.

Methods in Narcotic Research. New York: Marcel Dekker, Inc.,

1975. pp. 173-215.

Khazan, N., Weeks, J.R. and Schroeder, L.D. Electroencephalographic electromyographic and behavioral correlates during a

cycle of self-maintained morphine addiction in the rat. J

Pharmacol Exp Ther, 155:521-531, 1967.

255
Khazan, N. and Colasanti, B. EEG correlates of morphine challenge

in post-addict rats. Psychopharmacologia, 22:56-63, 1971.

Masukawa, L. and Prince, D. Enkephalin inhibition of inhibitory

input to CA1 and CA3 pyramidal neurons in the hippocampus.

Brain Res, 249:271-280; 1982.

Nakamura, J. and Winters, W.D. Attenuation of the morphine EEG

continuum following a repeat dose within 16 days: delayed

tolerance in the rat. Neuropharm, 12:607-617, 1973.

Neal, H. and Keane, P.E. The effects of local microinjections of

opiates and enkephalins into the forebrain on the electrocorticogram of the rat. Electroenceph Clin Neurophysiol,

45:655-665, 1978.

Nicoll, R., Siggins, G., Ling, N., Bloom, F. and Guillemin, R.

Neuronal actions of endorphins and enkephalins among brain

region: A comparation microiontophoretic study. Proc Natl

Acad Sci USA, 74:2584-2588, 1977.

Nicoll, R., Alger, B. and Jahr, C. Enkephalin blocks inhibitory

pathways in the vertebrate CNS. Nature, 287:22-25, 1980.

North, R. and Williams, J.T. Opiate activation of potassium

conductance inhibitory calcium action potentials in rat locus

coeruleus neurons. Br J Pharmac, 80:225-228, 1983.

Pepper, C.M. and Henderson, G. Opiates and opioid peptides

hyperpolarize locus coeruleus neurons in vitro. Science,

209:394-596, 1980.

Peri, R. and Meinardix, H., eds. An update on Research

and Therapy. New York: Alan Liss, Inc., 1983. pp. 105-120.

Robinson, J., Dunlap, C.E. III and Deadwyler, S. Differences in

opiate-induced synaptic excitability of hippocampal slices

prepared from tolerant rats. Exper Neurol, 77:590-598, 1982.

Robinson, J.H. and Deadwyler, S. Morphine excitation: effects on

field potentials recorded in the in vitro hippocampal slice.

Neuropharmacology, 19:507-514, 1980.

Robinson, J.H. and Deadwyler, S.A. Intracellular correlates of

morphine excitation in the hippocampal slice preparation.

Brain Res, 224:375-383, 1981.

Satoh, M., Zieglgansberger, W., Fries, W. and Herz, A. Opiate

agonist-antagonist interaction at cortical neurones of naive

and tolerant/dependent rats. Brain Res, 82:378-382, 1974.

256
Satoh, M., Zieglgansberger, W. and Herz, A. Interaction between

morphine and putative excitatory neurotransmitters in cortical

neurones in naive and tolerant rats. Life Sci, 17:75-80,

1975.

Satoh, M., Zieglgansberger, W. and Herz, A. Actions of opiates

upon single unit activity in the cortex of naive and tolerant

rats. Brain Res, 115:99-110, 1976.

Siggins, G.R., Zieglgansberger, W., French, E., Ling, N. and Bloom,

F. Opiate and opioid peptides may excite hippocampal (HPC)

neurons (HPN) by inhibiting adjacent inhibitory interneurons.

Neurosci Abstr, 4:141, 1978.

Siggins, G.R. and Zieglgansberger, W. Morphine and opioid peptides

reduce inhibitory synaptic potentials in hippocampal pyramidal

cells in vitro without alteration of membrane potential. Proc

Natl Acad Sci USA, 78:5235-5339, 1981.

Sigworth, F. Fluctuation in the current through open ACh-receptor

channeIs. Biophys J, 37:309a, 1982.

Straw, R.N. and MitchelI, C.L. The effects of morphine,

pentobarbital, pentazocine and nalorphine on bioelectric

potentials evoked in the brain stem of the rat by electrical

stimulation of the tooth pulp. J Pharmacol Exp Ther, 146:7-

15, 1964.

Taylor, P., Brown, R. and Johnson, D. The linkage between ligand

occupation and response of the nicotinic acetylcholine

receptor. In: Current Topics in Membranes and Transport.

Vol. 18. New York: Academic Press, 1983. pp. 407-443.

Teitelbaum, H., Catravas, G. and McFarland, W.L. Reversal of

morphine tolerance after medial and thalamic lesions in the

rat. Science, 185:449-451, 1979.

Teitelbaum, H., Blosser, J. and Catravas, G. Bilateral electroencephalographic response and unilateral tolerance to

unilateral intracerebral morphine injections. Nature,

260: 158-159, 1976.

Tortella, F.C., Moreton, J.E. and Khazan, N. Electroencephalographic and behavioral effects of D-Ala2

-methionine-enkephaIinamide and morphine in the rat. J Pharm Exp Therap,

206:636-643, 1978.

Urca, G., Frenk, H., Liebeskind, J.C. and Taylor, A.N. Morphine

and enkephal In: analgesia and epileptic properties. Science,

197: 83-96, 1977.

Valentino, R. and Dingledine, R. Pharmacological characterization

of opioid effects in the rat hippocampal slice. J Pharmacol

Exp Therap, 223:502-509, 1982.

257
Walker, J., Takeyasu, K. and McNamee, M. Activation and inactivation kinetics of torpedo californica acetylcholine receptor in

reconstituted membranes. Biochemistry, 21:5384-5389, 1982.

Watson, S.J., AkiI, H., Sullivan, J. and Barchas, J.D. Immunocytochemical localization of methionine enkephalin: preliminary

observation. Life Sci, 21:733-738, 1977.

Way, E.L. Role of serotonin in morphine effects. Fed Proc,

31:113-120, 1972.

Way, E.L. and Shen, F.H. Catecholamine and 5-hydroxytryptamine.

In: Clouet, D., ed. Narcotic Drugs Biochemical Pharmacology.

New York: Plenum Press, 1971, pp. 229-253.

Williams, J.T. and Zieglgansberger, W. Neurons in the frontal

cortex of the rat carry multiple opiate receptors. Brain Res,

226:304-308, 1981.

Williams, J.T., Egan, T.M. and North, D. Enkephalin opens

potassium channel on mammalian central neurons. Nature,

229:74-77, 1982.

Williams, J. and North, R.A. Tolerance to opiates in locus

coeruleus actions? Abst lnternational Narcotic Res Conf, 49

1983.

Zieglgansberger, W. and Bayer I, J. Interference of microelectrophoretically applied substances with the depolarizing action

of glutamate and acetylcholine. Pflugers Arc ges Physiol, 355

(Suppl):R170, 1975.

Zieglgansberger, W., Satoh, M. and Bayer I, J. Actions of microelectrophoretically applied opiates on cortical and spinal

neurons. Naunyn-Schmiedebergs Arch exp Path Pharmacol,

287:(Suppl)R16, 1975.

Zieglgansberger, W. and Fry, J.P. Actions of enkephalin on

cortical and striatal neurons of naive and morphine

tolerant/dependent rats. In: Kosterlitz, H.W., ed. Opiate

and Endogenous Opioid Peptides. Amsterdam: Elsevier/North

Holland Biomed. Press, 1976. pp. 213-238.

Zieglgansberger, W., Fry, J.P., Herz, A., Moroder, L. and Wunsch,

E. Enkephalin-Induced inhibition of cortical neurons and the

lack of this effect in morphine tolerant/dependent rats.

Brain Res, 115:160-164, 1976.

Zieglgansberger, W., French, E., Siggins, G. and Bloom, F. Opioid

peptides may excite hippocampal pyramidal neurons by

inhibiting adjacent inhibitory interneurons. Science,

205:415-417, 1979.

258
AUTHOR

Steven J. Henriksen, Ph.D.

Scripps Clinic and Research Foundation

Department of Basic and Clinical Research

Division of Preclinical Neuroscience and Endocrinology

10666 North Torrey Pines Road

La Jolla, California 92037

259
Spinal Cord Tissue Culture Models

for Analyses of Opioid Analgesia,

Tolerance and Plasticity

Stanley M. Crain

I. MODELS OF OPIOID FUNCTIONS USING FRESHLY ISOLATED TISSUES

OR CELL CULTURES

In vitro preparations of freshly isolated guinea pig ileum and

mouse vas deferens have provided simple model systems for bioassays of opioid agonists and antagonists (Kosterlitz and

Waterfield 1975; Kosterlitz and Hughes 1977). The validity of

these models rests on demonstrations of close correlation between

the agonist potencies of opioid agents as measured by their

ability to depress electrically induced contractions and: 1) their

analgesic potencies in man (Kosterlitz et al. 1973); 2) their

relative opiate receptor binding potencies in rat brain homogenates as well as in guinea pig ileum (Creese and Snyder 1975; Simon

1976). Although peripheral nerve muscle preparations may be limited in their usefulness for analyses of the specific opioid systems

within the CNS (e.g. Van Neuten et al. 1976; Ward and Takemori

1976a,b), they have indeed provided valuable insights into some of

the cellular mechanisms involved in opioid tolerance and dependence. Studies of isolated guinea pig ileum during chronic exposure to opioids for periods up to 24 hours (e.g. Hammond et al.

1976; Collier et al. 1980, 1981a,b; North and Karras 1978a,b;

North and Zieglgansberger 1978; North and Vitek 1979; Johnson and

North 1980; see reviews by Duggan 1980; North 1979; Collier 1980)1

have demonstrated that some of the basic phenomena characteristic

of opioid tolerance and dependence-withdrawal in the CNS can be

elicited in isolated neurons. (See, however, discussion in Sect.

III-A on differences in the opioid tolerance properties observed

in freshly isolated preparation of myenteric neurons innervating

peripheral muscle vs. dorsal root ganglion neurons co-cultured

with spinal cord.) The data obtained by North and co-workers

from myenteric ganglia suggest that significant components of

opioid tolerance and dependence may be mediated by plastic changes

in the bioelectric excitability and propagative properties of individual neurons (see, however, results with brainstem slices,

below)--whereas Collier has emphasized the possible role of “an

hypertrophy of the cyclic AMP system in these neurons in response

to inhibition by opiate of a neuronal adenylate cyclase” (see

below).

260
Freshly isolated slices or strips of neonatal spinal cord with

attached dorsal root ganglia (DRGs) (Otsuka and Konishi 1974)

have provided useful preparations for electrophysiologic analyses

of the acute effects of opioids on primary afferent networks of

the CNS (Suzue and Jessell 1980; Hentall and Fields 1983). Furthermore, intracellular recordings of neurons in slices from adult rat

spinal cord have recently demonstrated that morphine and D-alaD-leu enkephalin (DADLE) can directly hyperpolarize dorsal-horn

inter-neurons by opening membrane K

+

 channels (Yoshimura and North,

1983), in addition to previous evidence from cell cultures that

opioids may depress the release of transmitter from nociceptive

DRG fibers (see below and Sect. II, III). However, studies of

the long- term effects during chronic exposure to opioids require

maintenance of these isolated neural tissues under more stable

conditions such as those provided by tissue culture techniques

(see Sect. III). For example, no signs of tolerance to the hyperpolarizing action of opiates on locus ceruleus neurons were detected when freshly isolated slices of normal rat brainstem were exposed to normorphine for 8-12 hours (Williams and North 1983),

whereas slices prepared from morphine-tolerant rats were 3- to

10-fold less sensitive to opiates as determined by similar electrophysiologic tests. Since the degree of tolerance to the hyperpolarizing action of opiates on locus ceruleus neurons was, nevertheless, small compared to the tolerance assessed in these rats by

behavioral tests, the authors suggested that “tolerance (and dependence) observed in experiments which examine the behavior of

complex networks of neurons results from or is amplified by an

adaptive process which occurs in transmission between cells”

(Williams and North 1983). 2

Cultures of dissociated fetal rodent spinal cord neurons and DRG

cells (Ransom et al. 1977) have also been fruitfully used to

analyze physiologic mechanisms of action of opioids. With intracellular recording techniques, MacDonald and Nelson (1978)

demonstrated that etorphine depressed excitatory postsynaptic

potentials (EPSPs) elicited in spinal cord cells by activation of

co-cultured DRG cells. Statistical analysis of the quantal parameters for the DRG-evoked EPSPs in cord neurons revealed that

etorphine reduced EPSP quantal content at this synapse without

altering quantal size, thereby indicating a presynaptic depressant action. The data suggested that etorphine may have a

direct depressant effect on transmitter release at the DRG-cord

synapse, but partial blockade of spike propagation into the

presynaptic terminals could not be ruled out. Analyses by Mudge

et al. (1979) in cultures of dissociated DRG neurons provided

more direct evidence that opioids depress transmitter release,

possibly by decreasing the influx of Ca++ across the membrane of

th DRG presynaptic terminal (see Sect. III-C).

Although no studies of the effects of chronic opioid exposure of

dissociated DRG or cord neurons have been reported, cultures of

dissociated mouse neuroblastoma x glioma hybrid cells, which show

261
high stereospecific opiate binding levels (Klee and Nirenberg

(1974), develop significant alterations in cyclic nucleotide and

adenylate cyclase levels during chronic exposure to opiates.

Whereas acute exposure of these cells to opioids results in inhibition of adenylate cyclase and a decreased cyclic AMP level

after chronic exposure to morphine, basal levels of adenylate cyclase

increase so that higher concentrations of opiates are required to

produce decrements in cyclic AMP (Sharma et al. 1975a). These

workers suggested a provocative model for tolerance and dependence,

based on their demonstrations of sequential early inhibition and

compensatory increase in adenylate cyclase activity and cyclic AMP

levels during chronic opiate exposure (Klee et al. 1975; Sharma

et al. 1975b; Lampert et al. 1976; see also Traber et al. 1975,

and reviews by Hamprecht 1977 and Collier 1980).3 No correlative

electrophysiological data have been reported on alterations in

dissociated neuroblastoma x glioma cells during chronic exposure

to opioids. However, electrophysiological analyses of myenteric

neurons in the guinea pig ileum (Karras and North 1979), dorsalhorn neurons in cat spinal cord (Duggan and Griersmith 1979), and

dorsalhorn networks in mouse cord-ganglion explants in culture

(see Sect. II; Crain, unpublished observations) have not detected

any signs of altered sensitivity to opiates during acute exposure

to cyclic nucleotides or phosphodiesterase inhibitors (see reviews

by Duggan 1980 and Collier 1980). These physiological data do

not, however, preclude possible alterations in cyclic AMP functions

in neurons during development of tolerance and dependence following

chronic exposure to opiates (see Sect. III-B).

Although these cultures of tumor-derived cells do not develop

sufficient organization to permit analyses of mechanisms involved

in the specific physiologic effects of opioids on synaptic networks of the CNS, recent biochemical studies of these cells by

Sharma and Khanna (1982) have, nevertheless, provided interesting

clues to a possible role of vitamin C in the development of opioid

tolerance. They reported that introduction of ascorbate (1-5mM)

attenuated the delayed etorphine-induced compensatory increase

in cyclic AMP levels (tested at 4 hrs of incubation) without

effecting the short-term (15 min) inhibitory response of these

neuroblastana cells to the drug. Prompted by the data obtained

from this in vitro model system, co-administration of high levels

of vitamin C (-1mg/kg) with morphine in mice was shown to “suppress

the development of tolerance and physical dependence on the drug

without significantly affecting its analgesic properties” (Khanna

and Sharma 1983).

In addition to the enhancement of adenylate cyclase activity and

cyclic AMP levels in these neuroblastoma cells during chronic exposure to opioids, Dawson et al. (1979) have shown that a stereospecific dose-dependent, naloxone-reversible inhibition of the

biosynthesis of membrane glycosphingolipid and glycoprotein also

occurred within 24 hours. It is not yet clear whether these

opiate-induced cell-surface changes are a consequence of cyclic

nucleotide changes or vice versa.

262
II. SPINAL CORD TISSUE CULTURE MODELS OF OPIOID ANALGESIA

Cultures of organized explants of fetal mouse spinal cord crosssections with attached sensory dorsal-root ganglia (DRGs) (Crain

and Peterson 1974; Crain 1976) have been particularly useful for

analyses of cellular mechanisms underlying opioid analgesia and

tolerance (Crain et al. 1977, 1978, 1979, 1982b). Single focal

DRG stimuli evoke prominent negative slow-wave responses restricted to dorsal regions of spinal cord explants (Fig. 1A,D), arising

abruptly after latencies of 2-3 msec, with amplitudes up to 2 mv,

and often lasting more than 500 msec (Crain and Peterson 1974).

These potentials resemble primary afferent depolarization (PAD)

and secondary sensory-evoked synaptic network responses in dorsal

cord in situ (e.g. Eccles, 1964). Simultaneous recordings in

ventral cord regions generally show small positive or polyphasic

slow-wave potentials and spike barrages, after latencies of 5-10

msec (Fig. 1).

DRG-evoked ‘PAD’ responses in dorsal cord are generally maintained or augmented in the presence of 1 mM -aminobutyric acid

(GABA), whereas most other synaptic network discharges in the cord

explants are rapidly depressed (Crain and Peterson, 1974). In

contrast to the enhancing effects of GABA on sensory-evoked

dorsal-horn network responses in cord-DRG explants, these responses are selectively depressed by acute exposure to low concentrations of morphine and other opiates (Crain et al. 1977). Introduction of 0.1-1 μM morphine into the culture leads to marked and

sustained depression of major components of the DRG-evoked, PADlike, slow-wave responses in dorsal cord within a few min, whereas ventral cord discharges are either unaltered or enhanced (Fig.

1B). A series of opioid peptides with a wide range of analgesic

potencies produced similar selective depressant effects on the

sensory-evoked dorsal-horn network discharges of cord-DRG explants

(Table 1) (Crain et al. 1978), at concentrations remarkably proportionate to their potency in the intact animal. After return

to control medium, restoration of opioid-depressed dorsal cord

responses gradually occurs during periods of 30-60 min (Fig. 1C).

Exposure to opiate antagonists, e.g. naloxone (10-100 nM) or

diprenorphine (1-10 nM), prevents development of the characteristic depression of sensory-evoked dorsal cord responses by these

opiates and opioid peptides (Fig. 1D) (Crain et al. 1977, 1978).

whereas they are ineffective against the acute effects of nonopioid-receptor-mediated depressant agents, e.g. high-Mg++

,

xylocaine, serotonin (see Sect. III-B).

The depressant effects of opiates and opioid peptides on dorsal

cord explant responses were often markedly reduced or prevented

when the Ca++ level was raised to 5mM, especially in the

presence of 0.lmM 4-aminopyridine (4-AP) (Fig. 2) (Crain et al.

1982a). Receptor assays showed that 0.1mM 4-AP ± 5mM Ca++ had no

effect on stereospecific opiate binding, indicating that the

antagonist actions of these agents in our cultures do not occur at

the level of the opiate receptor. 4-AP has been shown to

markedly potentiate synaptic transmitter release by depressing

263
FIGURE 1

Selective opiate depression of sensory-evoked dorsal-horn network

responses in cord-DRG explant (4 weeks in vitro). A: Short-latency

negative slow-wave potential in dorsal cord (d) and small longerlatency, positive slow-wave response in ventral cord (v) are evoked

by a single large DRG stimulus. B: 5 min after introduction of

morphine (1μM), major components of the dorsal cord responses

have disappeared. In contrast, the ventral cord response is enhanced. C: 10 min after return to BSS, the dorsal cord response

to DRG stimuli remains small, and the ventral cord discharge is

still large. Dl: 3 min after introduction of naloxone (3μM) the

dorsal cord slow-wave response is restored and enhanced; shortlatency components are seen at faster sweep (D2). E: 20 min after

return to BSS, 1μM morphine partly depresses DRG-evoked dorsal

cord response (more complete depression occurred after increase to

10μM morphine). Note: Upward deflection indicates negativity at

recording electrode; onset of stimulus is indicated by first

sharp pulse or break in baseline of each sweep. All records in

Figs. l-4 show maximal response evoked by large stimuli with standard placements of recording microelectrodes within the cord and

stimulating electrodes within the DRG tissue. BSS is balanced salt

solution consisting of (in mmol/l distilled water) of NaCl, 137;

KCl, 5.5; KH2PO4, 0.4; CaCl2, 1.3; MgCl2, 0.5; MgSO4, 0.4; Na2HP04,

0.3; NaHCO3, 4.2; and glucose, 5.5 (From Crain et al. 1977.

©1977, Elsevier Biomedical Press.)

264
TABLE 1

Comparison of depressant potency in vitro and analgesic

potency in situ for a series of opioid peptides

Compound

Effective depressant

concentration (μM)

Molar potency ratios

in vitroa

Sensory network Analgesic potency

depressant potency in rodents

in vitro in situb

Tyr-gly-gly-phe-met 10 1

Tyr-gly-gly-phe-leu 10

0.1-1.0

1

Tyr-D-ala-gly-phe-met 10-100 100;700

Tyr-D-ala-gly-phe-D-leu 0.02 500 1000

D-ala-tyr-gly-gly-phe-met 1 0 1

[D-Met2,Pro5]-enkephalinamide

1

[D-ala2-MePhe4,Met-(0)5-ol] -

0.005-0.01 1000-2000 2000

enkephalin (Sandoz FK 33-824) 0.002-0.005 2000-5000 30,000

ß-Endorphin 0.1 100 200-1000

Endorphin 1.0 10 < 1

Morphine 0.3 30 30

a

Molar concentration required to produce marked and sustained depression (>50%) of sensory-evoked

dorsal-horn network responses in explants of mouse spinal cord with attached dorsal root ganglia.

411 of these opiate depressant effects were prevented by naloxone (0.01-0.1μM) or diprenorphine

(0.001-0.01 μM).

b

Determined by tail-flick test after intracerebral. intraventricular. or spinal subarachnoid injections

(see references to in situ studies in Crain et al. 1978).

(From Crain et al. 1978. © 1978. Elsevier Biomedical Press.)
FIGURE 2

Antagonism by 4-aminopyridine (4-AP) of the depressant effects

of morphine on sensory-evoked dorsal-horn network responses in

cord-DRG explants. (Al): Characteristic short-latency negative

slow-waves are evoked in two sites in dorsal cord by DRG stimulus.

(A2): Dorsal cord responses are blocked after 10 min in 1μM

morphine. (A3): Cord responses gradually recover after addition

of 0.1 mM 4-AP along with 1μM morphine and can still be elicted

one hour later (A4). (B): Dorsal horn responses in another

explant (B1) are remarkably enhanced in 0.1 mM 4-AP + 5 mM Ca++

(B2). (B3): Cord responses are not significantly attenuated

after 10 min exposure to 10μM morphine + 0.1 mM 4-AP + 4 mM Ca++,

nor after 10 min in 100μM morphine + same 4-AP/Ca++ levels (B4).

(From Crain et al. 1982a. © 1982, Pergamon Press Inc.)

266
membrane K

+

 conductance during depolarization of nerve

terminals, thereby prolonging the presynaptic spike and enhancing Ca++ influx (e.g. Illes and Thesleff 1978). Furthermore,

4-AP antagonized the depression of synaptic transmission in

spinal cord produced by high Mg++ or EGTA (Galindo and Rudomin

1978) and the depressant effects of adenosine on cerebral neurons

(Perkins and Stone 1980; Stone 1981a). The effects of 4-AP and

high Ca++ on cord-DRG cultures are in good agreement with studies

in the mouse vas deferens where similar concentrations of these

agents were shown to reverse the inhibitory action of morphine

on neuromuscular transmission (Illes et al. 1980; Stone 1981a).

Although these effects of 4-AP and high Ca++ on cord-DRG cultures

do not clearly distinguish between presynaptic or postsynaptic

mechanisms of opiate depressant action on afferent cord networks,

a primary action of opioids at presynaptic DRG terminals in the

dorsal horn appears to be more probable (see below) but does not

preclude direct inhibitory effects on cord interneurons (Yoshimura

and North 1983; Murase et al. 1982). Either mode of opioid action

could probably be overcome by 4-AR/high Ca++-enhanced transmitter

release, as in studies of botulinum- or curare-blocked motor

endplates (Kim et al. 1980). Nevertheless, our in vitro results

demonstrate that the depressant effects of opiates on afferent

spinal cord networks involve relatively restricted physiological

actions that can be readily antagonized by Ca++, even in the

presence of extremely high opioid concentrations (Crain et al.

1982a). The effects of increased Ca++ concentration on the

opioid-sensitivity of the sensory-evoked responses in cord-DRG

explants are in agreement with the suppression of opiate analgesic

effects in situ by intracerebroventricular injections of calcium

(Kakunaga et al. 1966; Harris et al. 1975) and the reversal of

morphine analgesia in rats by injections of 4-AP (Tung and Brandom

1981). They are also consonant with evidence that the depressant

effects of opioids may be due to interference with Ca++ conductance

in presynaptic terminals, thereby impeding transmitter release

(see Sect. III C; also Dunlap and Fischbach 1978; Macdonald and

Nelson 1978).

The development of opiate-sensitive DRG-evoked responses restricted to dorsal-horn regions of our cord explants is in good agreement with microelectrophoretic injection of morphine into the substantia gelatinosa in cat spinal cord, resulting in selective

depression of cell responses to noxious, but not tactile, stimuli

(Duggan et al. 1977). Furthermore, physiologic analyses of these

opioid networks in vitro and in situ correlate well with binding

assays showing that high levels of stereospecific opiate receptors

develop in the dorsal-horn regions of the adult monkey spinal

cord (Lamotte et al. 1976) and in our fetal mouse cord-DRG

explants (Hiller et al. 1978b). In addition, we have demonstrated

that even higher levels of receptors develop in the neuritic

outgrowth of isolated DRG cultures (Hiller et al. 1978a). The

latter assays provided the first direct evidence that opiate

receptors are located on DRG nerve fibers which are destined to

establish presynaptic afferent connections with the spinal cord

(sec also Lamotte et al. 1976; Fields et al. 1980).

267
III. CORD TISSUE CULTURE MODELS OF OPIOID TOLERANCE AND

PLASTICITY

A. Development of Tolerance in Cord-DRG Explants During

Chronic Exposure to Opiates and Opioid Peptides

Most in vitro analyses of tolerance to opiates have utilized

peripheral neurons or neuroblastoma cells (as noted in Sect. I).

Our electrophysiologic experiments during chronic exposure of

cord-DRG explants to opiates and opioid peptides (Crain et al.

1979) have provided the first demonstration that isolated mammalian

CNS tissues can, indeed, develop marked tolerance after several

days of chronic exposure in vitro. Earlier studies of tolerance

in chronic spinal dogs by analyses of spontaneous and reflex activities during chronic morphine exposure and hyperactivity of

these reflexes after withdrawal of morphine (Wikler and Frank 1948;

Wikler 1953; Wikler and Carter 1953) suggest that this cord-DRG

explant model is reasonable and likely to provide significant

insights into some of the complex problems of opiate addiction.

Following exposure of cord-DRG explants to low levels of morphine

(1 μM) for 2 or more days (at 35°C), the initial opiate-depressant

effects on sensory-evoked dorsal-horn network responses disappeared

and characteristic dorsal cord responses could again be evoked by

DRG stimuli in the presence of morphine-- even after large increases in concentration (up to 100-fold: Fig. 3B2,3) (Crain et

al. 1979). These manifestations of tolerance to opiates could

still be observed for 2 days after transfer to drug-free culture

medium, and characteristic sensitivity to opiates returned during

the subsequent few days. The stereospecificity of these opiatetolerance effects was demonstrated by the fact that chronic exposure of cord-DRG explants to 1 μM levorphanol produced similar

tolerance to opiates as occurred after 1 uM morphine, whereas

chronic exposure to 1 μM dextrorphan was ineffective.

Tolerance also developed after chronic exposure of cord-DRG

explants to low concentrations (10 nM) of the synthetic enkephalin

analog, {D-ala2, MePhe4, Met - (0) 5-o1}-enkephalin (Sandoz FK 33-824),

consonant with the tolerance that occurs in rodents following daily

injections of this potent, long-lasting analgesic (Roemer et al.

1977). Acute application of this opioid peptide to our explants

depressed DRG-evoked dorsal cord responses at levels as low as

1 nM (Table 1) (Crain et al. 1978). Furthermore, tolerance developed after chronic exposure of cord-DRC explants to opioid

peptides (0.1-1 μM) that bind predominantly to delta-type

receptors, e.g. D-ala-D-leu enkephalin (DADLE) (Wuster et al.

1979; Kosterlitz et al. 1980) as well as to mu-type receptors,

e.g., tyr-D-ala-gly-N-Me-Phe-Gly-ol (DAGOL) (Handa et al. 1981).

Morphine-tolerant cultures showed cross-tolerance to FK 33-824

met-enkephalin, DADLE, and ß-endorphin when tested at concentrations up to 10-fold higher than required to produce acute depressant effects on naive cord-DRG explants. On the other hand,

cultures rendered tolerant to FK33-824 (at 0.01 μM) showed un268
FIGURE 3

Development of tolerance to acute opiate-depressant effects

on sensory-evoked dorsal-horn network response in cord-DRG explants (3 weeks in vitro). A1

: Short- latency negative slow-wave is

evoked in dorsal cord (d) by DRG stimulus. A2: Within 10 min in

0.1 μM morphine, dorsal cord response is attenuated, whereas

ventral cord discharge (v) shows little change. A3: In 0.3 μM

morphine, dorsal cord slow-wave response is almost abolished.

(No recovery occurred during exposure to 0.3-1 μM morphine for

several hrs.) B1

: Large dorsal-cord network response is evoked in

another cord-DRG explant after chronic exposure to 1 μM morphine

for 2 days at 35°C (of. A2,3). B2: Dorsal cord response remains

large even after increasing morphine concentration to 10 μM.

(Ventral cord discharges were often enhanced at high opiate

levels.) B3

: Even after increase to 100 μM morphine dorsal cord

response shows no further attenuation (10 min test; partial depression developed after longer exposure at 100 μM level). C1

:

Only small dorsal-horn responses can be evoked in another explant

(2 sites) after exposure to 1 μM morphine for 4 days at 20°C,

resembling attenuation after acute exposure to morphine (cf. A2).

C2: 1 μM naloxone restores characteristic dorsal cord response

within a few min; some recording sites as in C1. Sharp rising

phases of these DRG-evoked dorsal cord responds are shown at

faster sweep (C3). (From Crain et al, 1979. © 1979, Pergamon

Press Inc.)

269
usually strong cross-tolerance to morphine; dorsal cord responses

could still be evoked even after acute exposure to high levels of

morphine (about 100 μM), or 1,000-fold higher than the depressant

level required to depress cord responses in a naive explant (i.e. ,

cross-tolerance ratio = 1,000).

In contrast to the high degree of selective tolerance to specific

types of opiate receptors (delta vs. mu) that has been observed

in mouse vas deferens (Schulz et al. 1980)5

, preliminary tests on

cord-DRG explants chronically exposed to either DADLE or DAGOL (at

0.01-1 μM) have often shown evidence of at least sane degree of

cross tolerance to agonists binding to other receptor subtypes

including the kappa agonists, dynorphin and ethylketocyclazocine

(Chang et al. 1981; Chavkin et al. 1982; Kosterlitz et al. 1981)

However, cross-tolerance ratios were often much lower than in tests

made with agonists binding to similar opiate receptor subtypes.

The variability observed in these cross-tolerance tests may be due

in part to marked heterogeneities in the distribution of opiate

receptor subtypes that develop in the DRG neurons (Werz and Macdonald, 1982a,b, 1983), as well as the cord neurons, explanted from

various levels of the neuraxis into these cultures. In guinea

pig ileum tissue, where cross-tolerance was observed between

delta and mu agonists, Schulz et al. (1981) interpreted these data

as support for “the notion that these drugs act via a common

receptor, possibly a mu-receptor type” (see alternative views in

sect. III-R).

The tolerant state did not develop if morphine was prevented from

binding to the opiate receptors by concomitant chronic exposure

to low levels of naloxone (01μM). Within an hour after removal

of naloxone from the culture medium, morphine produced characteristic depressant effects on dorsal cord responses. Furthermore,

when the incubation temperature was lowered to < 25°C, no evidence

of tolerance was detected after sustained exposure to 1 μM

morphine (as long as 7 days). Dorsal cord synaptic network responses remained depressed in these cord-DRG cultures during chronic

incubation in 1 μM morphine at 20°C (Fig. 3C1); the opiate

sensitivity tests were carried out after returning the cultures to

35°C. However, characteristic sensory-evoked cord responses were

rapidly restored after introduction of 0.1-1 μM naloxone (Fig. 3

C2,3), demonstrating that the depression of dorsal cord responses

in these cultures was opiate-mediated and not merely due to

metabolic deficits resulting from incubation at suboptimal temperature. Control 2-week-old cultures incubated in regular media at

20°C for 1 week showed no significant electrophysiologic deficits.

The data indicate that a temperature-dependent metabolic change

occurs in the neurons of these mouse cord-DRG explants after 2

days of chronic exposure to morphine at 35% leading to a sustained decrease in sensitivity to opiate-depressant effects (Crain

et al. 1979).4 These results are compatible with evidence that

tolerance to opiates in rats is blocked by inhibitors of protein

and RNA synthesis (Cox and Osman 1970); more critical tests are

required to characterize the specific molecular alterations that

may underlie the development of tolerance in neurons.

270
The temperature-dependence of the opiate-tolerance mechanism in

our mouse spinal cord-DRG explants is in contrast to the opiate

tolerance which develops in myenteric neurons of freshly isolated

guinea pig ileum within 1 day after incubation in 1 μM morphine

at 4°C (Hammond et al. 1976) and at 21-24°C (North and Karras 1978

a,b) . These differences in temperature requirements may be due

to differences in processes involved in the development of tolerance in central vs. peripheral neurons and they may provide clues

to some of the molecular mechanisms underlying the development

of tolerance in CNS neurons. It should also be noted that the

tolerance observed following the 1-day morphine exposure of freshly isolated guinea pig ileum preparations disappeared within a

few hours after transfer to drug-free medium (Hammond et al. 1976),

whereas our cord-DRG explants continued to show tolerance effects

for several days after return to regular culture medium. The

marked tolerance to opiates that develops in cord-DRG explants

may also be enhanced due to the relatively immature stage of these

fetal mouse tissues during the period of chronic exposure in vitro

as observed in neonatal mice at 1-2 weeks of age (Nicak and Kohut

1978).

As noted above, in the presence of 4-AP and high Ca++ acute introduction of morphine to cord-DRG cultures at concentrations as high

as 100-1,000 times the level required to depress afferent cord

responses in regular medium is ineffective (Fig. 2B), resembling

the tolerance observed in these explants several days after chronic

exposure to opiates (Fig, 3B). These results are consonant with

evidence suggesting that neurons may become tolerant during chronic

exposure to opioids by developing higher levels of intra-cellular

calcium in their presynaptic terminals (e.g. Harris et al. 1977;

Yamamoto et al. 1978; Huidobro-Toro et al. 1981)--as occurs during

acute 4-AP/high Ca++ exposure of naive explants--thereby overcoming

opioid inhibitory effects produced at presynaptic, and perhaps also

at postsynaptic, sites (Crain et al. 1982a).

It is generally considered to be unlikely that tolerance to

morphine is caused by down-regulation of opiate receptors since

most analyses in vivo have not detected alterations in the number

nor the affinity of these receptors after chronic treatment with

morphine (e.g. Pert and Snyder 1976; Chang et al. 1981; Bardo

et al. 1982). Nevertheless, Davis et al. (1975, 1979) reported

a significant decrease in opiate binding after development of

analgesic tolerance in rats when the assays were made on thin

slices of brainstem tissue rather than on brain homogenates, and

Kirby(1983) observed decreased opiate binding in the spinal cord

of young rats that had been chronically exposed to morphine during

the last half of gestation. Furthermore, chronic incubation of

aggregating brain cells cultured with etorphine appeared to induce

a down-regulation of mu and kappa opiate receptors (Lenoir et al.

1983), in extension of previous evidence demonstrating downregulation of delta receptors following 4-hour DADLE exposures of

cultured neuroblastoma-glioma hybrid cells (Chang et al. 1982;

Simantov et al. 1982) and freshly isolated rat hippocampal slices

(Chang et al. 1983). In view of the ambiguities in the data on

271
possible receptor down-regulation following chronic exposure

of CNS tissues to opiate agonists, cord-DRG explants may provide

a useful system to determine if opiate receptors do, in fact,

undergo significant alterations during the development of tolerance, just as we have demonstrated up-regulation during chronic

exposure of these explants to opiate antagonists (see Sect. III-D).

Marked increases in the spike-firing rate of opiate-tolerant

myenteric neurons occurred in the guinea pig ileum preparation

after agonist removal, or after introduction of an antagonist

(North and Karras 1978 a,b). This was interpreted as evidence

of opiate-dependence in the ileum preparation (see also Hammond

et al. 1976). Although we have not detected such increased

spike-firing in our CNS explants, we have observed that the sensoryevoked dorsal-horn network responses in opiate-tolerant cultures

were generally much larger in amplitude (e.g. Fig. 3B), and could

often be recorded over larger areas of the dorsal cord, than the

responses evoked in control cultures. The generation of unusually

large amplitude dorsal cord responses during chronic exposure to

opiates may, indeed, be an expression of dependence following

development of tolerance to the opioid level available to the

neurons in these cultures. Furthermore, in tolerant cord-DRG

explants that did not show unusually large amplitude sensoryevoked dorsal cord responses, acute exposure to naloxone often

produced marked increases (up to 50%) in the amplitude of these

responses. {Naloxone also enhanced dorsal cord responses in some

of our naive cord-DRG explants (Crain et al. 1977), but the

effects in these untreated cultures were generally smaller and

more variable}.

B. Cross-Tolerance to 5-Hydroxytryptamine in Morphine-Tolerant

Cord-DRG Explants

Explants that had become tolerant to morphine after chronic exposure for >2 days often developed a significant degree of crosstolerance to the depressant effect of serotonin (5-HT) on dorsalhorn network responses (Crain et al. 1982b), as well as to other

opioid peptides (Crain et al. 1979). In some morphine-tolerant

explants (Fig. 4Bl) 3- to 10-fold increases in the usual 5-HT concentration were required in order to produce depression of the dorsal cord responses comparable to that in naive explants (Fig.4B2

,3

versus 4A2,3 and Fig. 5). In contrast, morphine-tolerant explants did not show cross-tolerance to other types of agents that

depressed cord responses, e.g. high Mg++, xylocaine, alcohol.

Moreover, acute exposure of morphine-tolerant explants to

naloxone (1 μM) further attenuated the effects of 5-HT so that the

minimum depressant levels of 5-HT were often increased up to 30-

fold (Fig. 4B5,6 and Fig. 5).

Increasing the extracellular Ca++ concentration from the usual

1 mM to 5 mM level or introduction of 4-AP markedly antagonized

the depressant effects of 5-HT on DRG-evoked cord responses, so

that 5-HT concentrations comparable to those used on morphinetolerant explants were required to depress naive explants. Furthermore, exposure of cord-DRG explants to 4-AP in the presence of

272
FIGURE 4

Depressant effects of 5-HT on sensory-evoked dorsal-horn network

responses in fetal mouse spinal cord-DRG explants (A) and

development of cross-tolerance to 5-HT after chronic exposure to

morphine (B). (Al): Short-latency negative slow-waves are evoked in

two regions of dorsal cord by DRG stimulus. (A2): Cord responses

are marked depressed after 10 min in 25 nM 5-HT (in other

cultures levels as low as 3 nM were effective). (A3): After

several min in 250 nM 5-HT, responses are almost completely blocked. (A4

): Cord responses recover in BSS (+ 1 μM naloxone).

(A5.6): Similar 5-HT-induced depression of cord responses occurs

in presence of 1 μM naloxone (cf. A5,6 and A2,3. (5-HT antagonists

methysergide and cyproheptadiene were ineffective as naloxone

up to 10 μM.) (B1): After 3-day exposure to 1 μM morphine,

tolerance develops and dorsal-horn responses can be evoked in the

presence of morphine. (B2): Relatively little attenuation of the

cord responses occurs in this opiate-tolerant explant after 10 min

in 25 nM 5-HT (+ morphine) (cf. A 2). (B3): A significant cord response can still be evoked even after 5 min in 250 nM 5-HT (cf. A3

).

(B4): Cord response recovers within 5 min after adding 1 μM naloxone

to bathinq fluid. (B5): No depression of cord response occurs

after 10 min in 25 nM 5-HT in the presence of naloxone (cf. A2,B2)

and a response can still be elicited even after 10 min in 250 nM

5-HT (cf. A3,B3). (From Crain et al. 1982b. © 1982, Pergamon

Press Inc.)

273
FIGURE 5

Shift in 5-HT dose-response curve after chronic exposure of cordDRG explants to morphine. Naive explant in BSS; morphinetolerant explant in BSS (after 3 days in 1 μM morphine);

shortly after exposure of morphine-tolerant explant to naloxone

(1 μM). (*Based on response data in Fig. 4 and similar tests

in >20 cultures) (From Crain et al. 1982b. © 1982, Pergamon

Press Inc.)

5 mM Ca++ was even more effective in preventing the usual

depressant effects of 5-HT -- as well as morphine (Fig. 2)-- on

cord responses. In the presence of 4-AP/high Ca++, large dorsal

cord responses could still be evoked after introduction of concentrations of 5-HT about 10- to 100-fold higher than required to

produce comparable depression in control medium (Crain et al.

(1982b) .

Our in vitro results are consonant with evidence that 5-HT administration near the cell bodies of dorsal horn neurons and in the

substantia gelatinosa of the cat reduced the excitation of these

neurons by noxious stimuli (Headley et al. 1978; Belcher et al.

1978) and depressed spinal segmental nociceptive reflexes (Bell

and Matsumiya 1981). Furthermore, depression of opiate-sensitive

dorsal-horn network responses by bath perfusion with 5-HT provides

an in vitro model that correlates well with analgesia produced in

the rat by intrathecal administration of 5-HT (Yaksh and Wilson

1979). The absence of naloxone-antagonism of 5-HT depressant

effects on DRG-evoked cord responses in naive explants indicates

that the 5-HT effects are not mediated by opioid receptors in

these cultures, in agreement with studies of 5-HT-induced antinociception and analgesia in situ (Headley et al. 1978; Bell and

Matsumiya 1981; Yaksh and Wilson 1979). Similarly, the absence

of methysergide- and cyproheptadine-antagonism of the depressant

274
effects of 5-HT on DRG-evoked cord responses suggests that these

effects may not be mediated by 5-HT receptors. Alternatively,

another type of 5-HT receptor may be involved (e.g. Peroutka

et al. 1981). Further studies are required to clarify the

mode of action of 5-HT in producing these potent depressant

effects on dorsal-horn responses and the remarkable similarities in the effects of 5-HT and opiates on the responses of

both naive and opiate-tolerant explants. In cultures of

dissociated chick DRG neurons where 5-HT and opiates attenuate

the duration of Ca++-mediated soma action potentials, the effects

of 5-HT also failed to be antagonized by methysergide (Dunlap and

Fischbach 1978). These data suggest that 5-HT may block neuronal

components of dorsal-horn networks at similar regions to those

that are depressed by opiates, e.g. presynaptic DRG nerve terminals

where abundant opiate receptors are located (see above; and related studies of 5-HT effects in situ by Belcher et al. 1978;

Carstens et al. 1981). Furthermore the marked attenuation of the

depressant effects of both 5-HT and opioids on cord-DRG explants

by high Ca++ and 4-AP (Crain et al. 1982a,b) raises the possibility

that cross-tolerance to 5-HT and to other types of opioid peptides

in morphine-tolerant explants (see Sect. III-A) may result from

a more generalized physiological alteration in presynaptic components of dorsal-horn networks {e.g., enhanced Ca levels (Harris

et al. 1977; Yamamoto et al. 1978; Huidobro-Toro et al. 1981)} that

renders them tolerant to a broad spectrum of agents whose depressant actions are mediated by interference with Ca++-dependent

synaptic transmitter release (Crain 1984).

Finally, the marked increase in degree of cross-tolerance to 5-HT

after acute introduction of naloxone in morphine-tolerant cord-DRG

cultures may be an expression of opiate dependence, possibly mediated by the same mechanism underlying naloxone-enhancement of

the amplitude of sensory-evoked dorsal-horn responses observed in

opiate-tolerant explants (Crain et al. 1979). Both of these

naloxone-elicited alterations may reflect the onset of hyperexcitability properties in dorsal-horn networks associated with sudden

withdrawal from chronic exposure to opiates (see reviews by Duggan

1980; Wikler 1980; Johnson and North 1980).6

These interpretations have been strengthened by recent studies of

the effects of Mn++ on opioid-tolerant explants. In previous tests

with elevated Mg++concentrations, no clearcut evidence of crosstolerance to this Ca++-antagonist had been detected (as noted

above). Mn++ is much more potent than Mg++ in selectively competing with Ca++ for a common site on presynaptic membrane in

neuromuscular junctions (Meiri and Rahamimoff 1972; Balnave and

Gage 1973) and other synapses (e.g. Nichols and Nakajima 1975).

At concentrations of about 1mM, Mn++ produces negligible depressant

effects on postsynaptic membrane conductances or action potentials,

and it appears to act primarily in blocking excitation-secretion

coupling mechanisms in presynaptic terminals (Nichols and Nakajima

1975). In preliminary tests on cord-DRG cultures, introducticm

of 1mM Mn++ often produced much weaker depressant effects on

opioid-tolerant vs. naive explants, especially after acute addition

275
of naloxone, resembling the effects of 5-HT on these cultures

(see above) .

C. Alterations in Presynaptic DRG Functions in Cord-DRG

Explants Monitored by Intracellular Recordings from DRG Neurons

Analyses by Mudge et al. (1979) in cultures of dissociated chick

embryo DRG neurons demonstrated that D-ala 2 enkephalinamide

inhibited the K

+

-evoked (Ca++-dependent) release of substance

P from these DRG cells. Furthermore, enkephalin also decreased

the duration and magnitude of the Ca++ component of the spike

recorded in the DRG perikarya without affecting the resting

membrane potential or resting membrane conductance. Since Ca++

spikes in perikarya appear to be generated by specialized membranes

with high densities of Ca++ channels comparable to those present

in the less accessible presynaptic terminal membrane (Stinnakre

and Tauc 1973), the data suggest that opioids may depress synaptic

transmitter release by decreasing the voltage-dependent influx of

Ca++ across the active membrane of the DRG terminal as well as

the perikaryon.

Our extracellular bioelectric recordings of DRG-evoked dorsal-horn

responses in cord-DRG explants during chronic opioid exposure have

demonstrated that tolerance to the depressant effects of opioids

develops after 2-3 days in vitro. A mechanism that may possibly

underlie thus sustained decrease in responsiveness to opioids is

that some functional alteration occurs in the presynaptic DRG

terminals in the dorsal cord which attenuates the effectiveness

of the postulated opioid-induced depression of voltage-dependent

Ca++ influx in these terminals (see Section III-B). For example,

an increased density of Ca++ channels uncoupled to opiate receptors might develop in DRG terminals during chronic exposure

to opioids, or voltage-dependent Ca++ influx might become much

larger due to altered ionic concentration gradients or conductances across the membrane of DRG terminals (as may occur during

acute introduction of 4-AP and high Ca++ in naive cord-DRG

cultures; Fig. 2). We are attempting to test this hypothesis by

carrying out intracellular electrophysiologic recordings from DRG

neuron perikarya in cord-DRG explants during chronic exposure to

opiates to determine whether significant alterations in Ca++-

mediated components of DRG action potentials, or other evidence

of enhanced Ca++-mediated functions, can be detected in tolerant

explants. In order to establish baselines for evaluating

possible alterations in DRG functions that may develop in tolerant

explants, we have carried out intracellular recordings fran DRG

neuron perikarya in organotypic cord-DRG explants (grown for 3-5

weeks in regular culture media) during acute application of

opioids (Chalazonitis and Crain 1983). Intracellular recordings

were made with beveled micropipettes (40-80 M on DRG neuron

somas (20-50 μm diam) (Fig. 6A). Opiate agonists were tested on

DRG spikes evoked in a BSS containing 5 mM Ca++ and 5 mM Ba++ in

order to enhance inward current through Ca++ channels and block

delayed voltage-dependent K

+

 channels. A “hump" developed in the

repolarizing phase of the spike under these conditions (Fig. 6B)

which was blocked by 5-10 mM Co++, confirming Ca++ mediation of

276
FIGURE 6

Exposure to DADLE results in sustained attenuation of the duration

of the action potential of a DRG neuron in an explant of mouse

spinal cord with attacked DRGs (2 months in vitro). (A): Typical.

somatic action potential is evoked by intracellular depolarizing

current during initial perfusion of culture with BSS containing

5.4 mM Ca++. (B): After addition of 5mM Ba++ to the high-Ca++ BSS,

the duration of the action potential increased more than 3-fold;

note prominent 'hump' on repolarizing phase. (C1): By 2 min after

bath perfusion with 10μM DADLE (in high-Ca++, high-Ba++ BSS) the

duration of the action potential was sharply reduced and remained

short as long as the agonist was present (4-10 min tests). (C2):

After 5 min in DADLE, action potential duration was still attenuated (8 superimposed sweeps at 0.lHz). (D): After return to highCa++, Ba++ BSS, action potential became markedly prolonged again.

(E): Introduction of l0μM DADLE in the presence of 3μM naloxone

was ineffective in altering the action potential. Calibration

bars: 20mV and 5 msec. (Unpublished data from Chalazonitis and

Crain 1983 and in preparation).

277
this hump. Opiate agonists were bath-perfused (0.5 ml/min) for

test periods up to 10 min. In about 80% of the neurons in isolated

DRG explants, the duration of the Ca++ component of the action

potential was reduced by about 50% in the presence of DADLE at

10 μM (Fig. 6C; Table 2). These effects were reversed after

return to Ca++-Ba++ BSS and prevented by perfusion of naloxone

(3 μM) together with DADLE. This widespread distribution of

opioid-sensitive neuron perikarya in relatively mature, organotypic

DRG explants is similar to the large fraction of opioid-sensitive

cells (ca. 75%) observed in dissociated chick and mouse embryo

DRG cultures (Mudge et al. 1979; Werz and Macdonald 1982a). On

the other hand, no significant alterations in the action potentials

generated by adult mouse DRG neurons were detected during acute

application opioid to freshly isolated ganglia (Williams and

Zieglgansberger 1981; see also North 1979). The marked difference

in opioid sensitivity of DRGsin these in vitro vs. in situ studies

may reflect normal developmental changes that occur in at least

some types of DRG perikarya during establishment of synaptic

connections with cord neurons or, alternatively the in vitro

results may be due to abnormal opioid hypersensitivity resulting

from isolation of these embryonic cells and their maintenance in

a relatively immature state. In order to clarify this ambiguity,

we compared the opioid sensitivity of sensory neurons in

organotypic explants of DRGs attached to spinal cord (Fig. 1) vs.

isolated DRG explants. These tests showed that a significantly

smaller fraction of DRG neuron somas were sensitive to DADLE when

grown attached to spinal cord vs. isolated DRGs: 51% vs. 78%

(Table 2; Chalazonitis and Crain 1983). Furthermore, recordings

made 8-16 days after dorsal-root transection in 2-to 3-week-old

cord-DRG explants revealed that 84% of these decentralized DRG

neurons were opioid-sensitive (Table 2). Although the duration

of the Ca++ hump of the somatic DRG action potential was

significantly shorter in attached vs. isolated cultures, some of

the opiate-insensitive neurons still generated prominent Ca++

humps. This suggests that a decrease in functional opiate receptors rather than in Ca++ channels may underly the observed

decrease in opioid-sensitive DRG somas in DRG-cord explants. The

data obtained thus far are consistent with the hypothesis that

as the central terminals of fetal DRG neurites participate in the

formation of opioid synaptic networks within the dorsal-horn

region of DRG-cord explants the opioid sensitivity of the

perikaryal region of these DRG neurons may decrease (Chalazonitis

and Crain 1983).

During maturation opiate receptors on DRG neuron sanas may decrease in number or they may become non-functional, e.g. by uncoupling from Ca++ channels in the soma membrane, whereas at

embryonic stages, opiate receptors may be distributed all over the

surface of DRG neurons just as acetylcholine receptors are distributed all along embryonic un-innervated muscle fibers (Diamond

and Miledi 1962). Perhaps opiate receptors are translocated

during maturation to the terminal regions of DRG neurites as these

neurites establish synaptic connections in the spinal cord,

analogous to the clustering of ACh receptors during formation of

278
TABLE 2

Opioid-Sensitivity of DRG Neurons Grown

Either Attached to Cord or Alone

DRG neurons with opioid-sensitive Ca++-mediated

action potentialsa

Type of explant

Explants DRG neuron

tested somas tested

Opioid-sensitive

neuronsb

Isolated DRG

DRG attached to cord

DRG-cord, 8-16 days

after dorsal-root

transection in vitro

n n n Fraction

36 49 38 78%

58 67 34 51%*

17 19 16 84%

a

Tests made by bath-perfusion of 10μM D-ala-D-leu enkephalin (DADLE) during

intracellular recordings in BSS containing 5mM Ca++ and 5mM Ba++ (at 3-5 weeks

in vitro; sac text).

bNeurons showing marked reduction (about 50%) in duration of Ca++ component of

action potential during exposure to DADLE (see text).

*This fraction is significantly lower than that of the isolated DRG population

(x2 test: p < 0.005) and from the DRG population that had been decentralized

(p < 0.01).

(Unpublished data from Chalazonitis and Crain 1983 and in preparation.)

neuromuscular junctions. Alternatively, opiate receptors might

still be present on many DRG somas- -as evidented by autoradiographic

localization of 3H-etorphine on some types of adult DRG neurons

(Ninkovic et al. 1982)--but the distribution of Ca++ channels on

these somas may decrease during maturation (Matsuda et al. 1978;

Yoshida and Matsuda 1979; Spitzer 1979) or these channels may no

longer be regulated by linkage with opiate receptors. These

ambiguities may be clarified by more systematic intracellular recordings from DRG neurons at different stages of maturation in

cultures of DRG-cord explants and is isolated DRG explants, and by

correlative autoradiography of opiate receptor distributions on

these DRG neurons. Studies of these types of plasticity in opioid

networks during development of DRG-cord synapses or after decentralization of DRG neurons may also provide insights into mechanisms

underlying plastic alterations in opiate receptor distributions

that occur following chronic pharmacologic interventions (see

Sect. III-D).

We have demonstrated that about 50% of the DRG neuron perikarya in

mature cord-DRG explants still show marked sensitivity to opiates

(Table 2) under culture conditions where reliable tolerance develops

in dorsal cord afferent networks after chronic opioid exposure.

Intracellular recording from these DRG perikarya during chronic

exposure of cord-DRG explants to opiates may, therefore, lead to

significant clues to alterations in DRG functions that may play a

role in the development of opioid tolerance and dependence in the

279
dorsal horn of the spinal cord6

. Although such data are not yet

available, we have observed an interesting type of short-term

“desensitization” during intracellular recordings from about 30%

of the opioid-sensitive DRG neurons in both cord-DRG and isolated

DRG explants. In these neurons, after an initial reduction (-50%)

in the duration of the DRG action potential within 2-3 minutes after

introduction of DADLE, the characteristic Ca++-hump progressively

reappeared and the action potential regained its original width

during the following 5-6 minutes, even though the agonist was

still present (Chalazonitis and Crain 1983, and unpublished

observations). In the rest of the DRG neurons, the opioid-induced

decrease in the duration of the Ca++-hump was stably maintained

(during 4-10 min tests). It will be of interest to determine if

this desensitization is temperature-dependent and fails to occur

at 20°C, as in the “acute tolerance” studies of hippocampal explants

exposed to FK 33-824 (Gahwiler 1981) and in our long-term tolerance

test on cord-DRG explants (Crain et al. 1979). We have not

observed signs of desensitization in our previous extracellular

recordings of DRG-evoked dorsal-horn field potentials in cord-DRG

explants exposed to DADLE (or morphine) for periods up to several

hours, and the transitory depressant effects of DADLE on DRG neuron

perikarya may, therefore, be a type of tachyphyllaxis2 unrelated

to cellular processes mediating long-term opioid tolerance.

D. Opiate Receptor Upregulation During Chronic Exposure of CordDRG Explants To Opiate-Antagonists

Chronic administration of naloxone in rats resulted in enhanced

morphine-induced analgesia (Tang and Collins 1978) and an increased number of opiate binding sites in brain tissue (Lahti and

Collins 1978). Chronic naloxone treatment of guinea pigs also led

to increased opiate binding in the ileum and brainstem and enhanced sensitivity to opiate-depressant effects on the isolated

ileum preparation (Schulz et al. 1979). These preliminary reports

of enhanced opiate binding after chronic naloxone have been confirmed and extended by Zukin et al. (1982), using improved assay

techniques and controls, demonstrating that chronic naltrexone in

rats resulted in a nearly two-fold increase in brain opiate receptor number (the actual increase ranged widely in specific regions of the (CNS) (see Zukin et al., this volume).

Since high levels of stereospecific opiate receptors develop in

fetal mouse cord-DRG explants (Hiller et al. 1978 a,b; see

Sect. II), we used this system to examine the mechanism underlying

the regulation of opiate receptors in response to chronic

antagonists (Tempel et al. 1983). Cord-DRG explants were grown

for two weeks, and then exposed to naloxone (10μM) for an additional

week. Assays of cultures exposed to naloxone for 1 week revealed

a 50% increase in 3H-dihydromorphine binding (Tempel et al. 1983)

relative to that in either control cultures (Table 3) containing

regular nutrient media or 10μM (+)naloxone {the (+)enantiomer of

naloxone which does not bind to opiate receptors (Ijima et al. 1978)}.

In order to answer the question as to whether antagonist-induced

280
TABLE 3

Opiate Receptor Upregulation During Chronic Exposure

of Cord-DRG Cultures to Naloxone

Drug Exposure* No. of Opiate Receptor % Change

Agents(s) Period Assays** Binding § in Binding

(days) (fmoles bound Relative to

per mg protein) Control

Control medium 8 100

Naloxone (10μM) 0-1 3 100 0

" 2 2 125 +25%

"

5 2 150 +50%

"

7 4 151 +51%

(+)Naloxone (10μM) 7 1 88 -12%

Naloxone (10μM) +

cyclohexamide (1μM) 5 1 93 +50% § §

Cyclohexamide (1uM) 5 1 62

*Cultures were grown in regular medium for 2 weeks prior to drug

exposure

**For each assay, 7-9 cultures were pooled, homogenized and

washed free of residual naloxone

§ Aliquots of homogenates were incubated in 3H-dihydromorphine(5nM)±

levorphanol(10μM)

§§ %change in binding relative to cyclohexamide control; 1μM

cyclohexamide blocked >90% of protein synthesis in cord-DRG

cultures (measured by incorporation of 14C-leucine into total

protein)

(Unpublished data from Tempel, Crain, Simon and Zukin 1983, and

in preparation.)

281
receptor upregulation requires the synthesis of new receptors,

cultures were exposed to naloxone (10μM) together with

cycloheximide (1μM), a relatively selective inhibitor of protein

synthesis, for 5 days at 35°. These conditions blocked protein

synthesis by greater than 90% in cord-DRG explants, as measured

by incorporation of 14C-leucine into total protein, yet

upregulation of opiate receptors still occurred (Table 3). This

result suggests that upregulation may be due to an unmasking of

previously-existing receptors. No significant change in receptor

density was observed following 1 day of naloxone treatment; a

modest increase was observed after 2 days; and maximal upregulation,

after 5 days of chronic exposure (Table 3). The maximal increase

of 50% was quite comparable to the 52% increase observed in assays

of spinal cord after chronic naltrexone in situ (Tempel and

Zukin, in preparation; see also Zukin et al. 1982 and this volume).

These data demonstrate the feasibility of utilizing cord-DRG

explants as an in vitro model system for analyses of cellular

mechanisms underlying the plasticity of opioid networks during

chronic exposure to opioid antagonists, as well as agonists (see

Sect. III-A). Attempts are also being made to detect electrophysiologic evidence of opioid supersensitivity in correlation

with experimental upregulation of these receptors as observed

in situ by Tang and Collins (1978), Schulz et al. (1979), Zukin

et al. (this volume); see, however, Bardo et al. (1982) .

IV. CONCLUDING REMARKS

Electrophysiologic analyses of dissociated DRG and cord neurons

have provided valuable insights into some of the primary effects

of opioids on transmitter release and conductance changes at DRG

terminals and cord-DRG synapses (Sect. I and III-C). However, many

important aspects of sensory-evoked dorsal-horn network functions

mediating opioid analgesia, tolerance and dependence may be

difficult to study in randomly dispersed, monolayer arrays of DRG

and cord neurons in dissociated cultures. Cord-DRG explants, on

the other hand, provide a specific opioid-sensitive network of

CNS and DRG neurons which can maintain not only an organized set

of physiological properties, but also an organotypic multi-layered

geometrical arrangement of neuron perikarya and fiber tracts

clearly related to the original body axes of the explanted tissue

(Crain 1976; Crain et al. 1978; Crain 1980, 1984). Studies

with organotypic CNS explants can, therefore, bridge the gap between experiments in the whole animal -- which are often difficult

to analyze because of the large number of complex interacting

networks -- and experiments in cell cultures of relatively disorganized, but more accessible, arrays of dissociated neurons.

Our experiments with cord-DRG explants show the potentialities of

this model system for analyses of cellular mechanisms associated

with opioid tolerance (Sect. III) and they provide a foundation

for more systematic experiments with these tissue culture models

to gain further insights into physico-chemical factors that regulate development of tolerance and dependence during chronic

exposure to opioids.

282
Furthermore, developmental studies of the effects of opioids in

cultures of immature fetal CNS tissues may be useful in relation

to problems arising in the human fetus during maternal narcotic

addiction. Finally, analyses of factors leading to increased

opiate receptor levels and possible opioid supersensitivity in

cord-DRG explants after chronic exposure to naloxone at various

stages of maturation provide an opportunity to correlate studies

of opioid tolerance with the more general problem of plasticity

in opioid systems of the CNS.

FOOTNOTES

1

See reviews by Herz, Tucker, Klee, Loh and Wuster (this volume).

2

See review by Henriksen (this volume).

3

See reviews by Tucker, Klee, Loh and Wuster (this volume).

4

See review by Beckman (this volume).

5

See review bv Herz (this volume).

6Hyperexcitability in opioid-tolerant cord-DRG explants observed

after sudden withdrawal of opiates may, indeed, be mediated by

compensatory alterations in the cord, as well as DRG , neurons of

these complex dorsal-horn networks (see reviews by Herz and Wuster,

this volume). Similarly, emphasis in this review on presynaptic

DRG factors that may be involved in opioid analgesia and tolerance

does not, of course, preclude a significant role for postsynaptic

cord neurons (e.g. Williams and North 1983).

REFERENCES

Balnave, R.J., , and Gage, P.W. The inhibitory effect of manganese

on transmitter release at the neuromuscular junction of the toad.

Brit. J. Pharmacol, 47: 339-352, 1973.

Bardo, T., Bhatnagar, R.K., and Gebhart, G.F. Differential

effects of chronic morphine and naloxone on opiate receptors,

monoamines, and morphine-induced behaviors in preweanling rats.

Dev. Brain Res, 4: 139-147, 1982.

Belcher, G. Ryall, R.W., and Schaffner, R. The differential effects

of 5-hydroxytryptamine, noradrenaline and raphe stimulation on

nociceptive and non-nociceptive dorsal-horn interneurones in the

cat. Brain Res 151: 307-321, 1978.

Bell, J.A., and Matsumiya, T. Inhibitory effects of dorsal-horn and

excitant effects of ventral horn intraspinal micro-injections of

norepinephrine and serotonin in the cat. Life Sci, 29: 1507-1514,

1981.

Carstens, E., Klumpp, D., Randic, M., and Zimmerman, M. Effects of

iontophoretically applied 5-hydroxytryptamine on the excitability

of single primary afferent C- and A-fibers in the cat spinal cord.

Brain Res, 220: 151-158, 1981.

Chalazonitis, A., and Crain, S.M. Opioid-sensitivity of neurons in

organotypic cultures of fetal mouse dorsal root ganglion (DRG)

explants grown either attached to spinal cord or in isolation.

Soc. Neurosci Abstr, 9: 943, 1983.

283
Chang, K.J., Hazum, E., and Cuatrecasas, P. Novel opiate binding

sites selective for benzanorphan drugs. Proc. Nat. Acad. Sci.

USA, 78: 4141-4145, 1981.

Chang , K. J. , Eckel , R.W. , and Blanchard S.G. Opioid peptides

induce reduction of enkephalin receptors in cultured neuroblastoma

cells. Nature, 296: 446-448, 1982.

Chang, K.J. Cuatrecasas, P., Valentino, R.J., Bostock, E.,

King, M.E., and Dingledine, R. Down-regulation of opiate receptors

in the hippocampal slice after prolonged incubation with agonist

but not μ-agonists. INRC Symp. Abstr:L-30, 1983 (Life Sci, 33:333-336).

Chavkin, C., James, I.F., and Goldstein, A. Dynorphin is a specific

endogenous ligand of the K opioid receptor, Science, 215: 413-415,

1982.

Collier, H.O.J. Cellular site of opiate dependence. Nature, 283:

625-629, 1980.

Collier, H.O.J.) Cuthbert, N.J. and Francis, D.L. Tolerance,

dependence and quasi-dependence in the guinea-pig isolated ileum.

In: Way, E.L., ed. Endogenous and Exogenous Opiate Agonists

and Antagonists. Oxford: Pergamon Press, 1980. pp.509-512.

Collier, H.O.J., Cuthbert, N.J. and Francis, D.L. Clonidine

dependence in the guinea-pig isolated ileum. Brit. J. Pharmacol,

73: 443-453, 1981a.

Collier, H.O.J. Cuthbert, N.J., and Francis, D.L. Model of

opiate dependence in the guinea-pig isolated ileum. J. Brit.

Pharmacol, 73: 921-932, 1981b.

Cox, B.M., and Osman, O.H. Inhibition of the development of

tolerance to morphine in rats by drugs which inhibit ribonucleic

acid or protein synthesis. Brit. J. Pharmacol., 38: 157-170, 1970

Crain, S.M. Neurophysiologic Studies in Tissue Culture New York,

Raven Press, 1976.

Crain, S.M. Development of specific sensory-evoked synaptic networks

in organized CNS tissue cultures. In: Giacobini, E., Vemadakis, A.,

and Shahar, A. , ed. Tissue Culture in Neurobiology. New York:

Raven Press, 1980. pp. 169-185.

Crain, S.M. Development and plasticity of specific sensory synaptic

networks in fetal mouse spinal cord cultures. In: Tsukada, Y., ed.

Perspectives of Neuroscience: from Molecule to Mind. Tokyo: Univ.

of Tokyo Press, 1984 (in press)

Crain, S.M. and Peterson E.R. Enhanced afferent synaptic functions

in fetal mouse spinal cord-sensory ganglion explants following NGFinduced ganglion hypertrophy. Brain Res, 79: 145-152, 1974.

Crain, S.M., Crain, B., Peterson, E.R., and Simon, E.J. Selective

depression by opioid peptides of sensory-evoked dorsal-horn network responses in organized spinal cord cultures. Brain Res,

157: 196-201, 1978.

Crain, S.M. , Crain, B. , Finnigan, T., and Simon, E.J. Development

of tolerance to opiates and opioid peptides in organotypic cultures

of mouse spinal cord. Life Sci, 25: 1797-1802, 1979.

Crain, S.M., Peterson, E.R. Crain, B., and Simon, E.J. Selective

opiate depression of sensory-evoked synaptic networks in dorsal-horn

regions of spinal cord cultures. Brain Res, 133: 162-166,

1977.

284
Crain, S.M., Crain, B., Peterson, E.R., Hiller, J.M., and Simon, E.J.

Exposure to 4-aminopyridine prevents depressant effects of opiates

on sensory-evoked dorsal-horn network responses in spinal cord

cultures. Life Sci, 31: 235-240, 1982a.

Crain, S.M., Crain, B., and Peterson, E.R. Development of crosstolerance to 5-hydroxytryptamine in organotypic cultures of mouse

spinal cord-ganglia during chronic exposure to morphine. Life Sci,

31: 241-247. 1982b.

Creese, II, and Snyder, S.H. Receptor binding and pharmacological

activity of opiates in the guinea pig intestine. J. Pharmacol.

exp. Ther, 194: 205-219, 1975.

Davis, M.E., Akera, T., and Brady, T.M., Saturable binding of

morphine to rat brain-stem slices and the effect of chronic

morphine treatment. Res. Commun. Chem. Path. Pharmacol, 12:

409-418, 1975.

David, M.E., Akera, T., and Brody, T.M. Reduction of opiate binding

to brainstem slices associated with the development of tolerance

to morphine in rats. J. Pharmacol. exp. Ther, 211: 112-119, 1979.

Dawson, G., McLawhon, R., and Miller, R.J. Opiates and enkephalins

inhibit synthesis of gangliosides and membrane glycoproteins in

mouse neuroblastoma cell line N4TG1. Proc. Natl. Acad. Sci. USA,

76: 605-609, 1979.

Diamond, J., and Miledi , R. A study of foetal and new-born rat

muscle fibres. J. Physiol, (London) 162: 393-408, 1962.

Duggan, A.W. Single neurone studies related to opiate tolerance and

dependence. Proc. Austral. Physiol. and Pharmacol. Soc, 11: 15-19,

1980.

Duggan, A.W., and Griersmith, B.T. Methyl xanthines, adenosine 3’,

5’-cyclic monophosphate and the spinal transmission of nociceptive

information. Brit. J. Pharmacol, 67: 51-57, 1979.

Duggan, A.W., Hail, J.G., and Headley, P.M. Suppression of transmission of nociceptive impulses by morphine: Selective effects of

morphine administered in the region of the substantia gelatinosa.

Brit J Pharmacol. 61:65-76, 1977.

Dunlap, K., and Fischbach. G.D. Neurotransmitters decrease the

calcium component of sensory neurone action potentials. Nature

276:837-839. 1978..

Eccles, J.C. The Physiology of Synapses. Berlin, Springer-Verlag,

1964.

Fields, H.L., Emson, P.C., Leigh, B.K., Gilbert, R.F.T., and

Iversen, L.L. Multiple opiate receptor sites on primary afferent

fibres. Nature, 284: 351-353, 1980.

Gahwiler, B.H. Development of acute tolerance during exposure of

hippocampal explants to an opioid peptide. Brain Res; 217:

196-200, 1981.

Galindo. J., and Rudomin, P. Facilitation of synaptic activity in

the frog spinal cord produced by 4-aminopyridine. Neurosci.

Letters. 19: 299-304. 1978.

Hammond, M.D., Schneider, C., and Collier, H.O.J. Induction of

opiate tolerance in isolated guinea pig ileum and its modification

by drugs. In: Kosterlitz, H.W. ed. Opiates and Endogenous

Opioid Peptides. Amsterdam: Elsevier, 1976, pp.169-178.

pp. 169-178.

285
Hamprecht, B. Structural, electrophysiological, biochemical,

and pharmacological properties of neuroblastoma-glioma cell hybrids

in cell culture. In: Bourne, G.H., Danielli, J.F., and Jeon, K.W.,

eds. International Review of Cytology. New York: Academic

Press, 1977. pp. 99-170

Handa, B.K., Lane, A.C., Lord, J.A.H., Morgan, B.A., Rance, M.J.,

and Smith, C.F.C. Analogues of ßLPH 61-64 possessing selective

against activity at μ-opiate receptors. Eur. J. Pharmacol,

70: 531-540. 1981.

Harris, R.A. , Loh, H.H. , and Way, E.L. Effects of divalent cations,

cation chelators and an ionophore on morphine analgesia and

tolerance. J. Pharmacol. exp. Ther, 195: 488-498, 1975.

Harris, R.A., Yamamoto, H., Loh, H.H. and Way, E.L. Discrete changes

in brain calcium with morphine analgesia, tolerance-dependence,

and abstinence. Life Sci, 20: 501-506, 1977.

Headley, P.M., Duggan, A.W., and Griersmith, B.T. Selective reduction

by noradrenaline and 5-hydroxytryptamine of nociceptive responses

of cat dorsal-horn neurons. Brain Res, 145: 185-189, 1978.

Hentall, I.D., and Fields, H.L. Actions of opiates, substance P,

and serotonin on the excitability of primary afferent terminals

and observations on intemeuronal activity in the neonatal rat’s

dorsal horn in vitro. Neuroscience, 9: 521-528, 1983.

Hiller, J.M. , Simon, E.J. , Crain, S.M. , and Peterson, E.R. Opiate

receptors in cultures of fetal mouse dorsal root ganglia (DRG)

and spinal cord: Predominance in DRG neurites. Brain Res,

145: 396-400, 1978.

Hiller, J.M. Simon, E.J., Crain, S.M., and Peterson, E.R. Opiate

receptor distribution in organized cultures of fetal mouse spinal

cord and dorsal root ganglia. In: Van Ree, J.M. , and Terenius, L.,

eds. Characteristics and Function of Opioids. Amsterdam:

Elsevier/North Holland Press, 1978. pp. 477-478.

Huidobro-Toro, N.P., Hu, J., and Way, E.L. Calcium antagonism

of the inhibitory effect of normorphine on the ileum of the

morphine-tolerant and nontolerant guinea pig. J. Pharmacol.

exp. Ther, 218: 84-91, 1981.

Ijima, I., Minamikawa, J., Jacobson, A.E. Brassi, A., and

Rice, K.C. Studies in the (+)-morphinan series 5. Synthesis and

biological properties of (+)-Naloxone. J. Med. Chem, 21: 398-

400, 1978.

Illes, P., and Thesleff, S. 4-Aminopyridine and evoked transmitter

release from motor nerve endings. Brit. J. Pharmacol, 64:

623-629, 1978.

Illes, P., Zieglgansberger, W., and Herz, A. Calcium reverses

the inhibitory action of morphine on neuroeffector transmission

in the mouse vas deferens. Brain Res, 191: 511-522, 1980.

Johnson, S.M., and North, R.A. Membrane potential changes in

neurones undergoing withdrawal from opiates. Brain Res, 190:

559-563, 1980.

Kakunaga, T., Kaneto, H., and Hano, K. Pharmacologic studies on

analgesics. VII. Significance of the calcium ion in morphine

analgesia. J. Pharmacol. exp. Ther, 153: 134-141, 1966.

Karras, P.J., and North, R.A. Inhibition of neuronal firing by

opiates: evidence against the involvement of cyclic nucleotides.

Brit. J. Pharmacol, 65: 647-652, 1979.

286
Khanna, N.C., and Shanna, S.K. Megadoses of vitamin C prevent

the development of tolerance and physical dependence on morphine

in mice. Life Sci, 33:401-404, 1983.

Kim, Y.I, , Goldner, M.M. , and Sanders, D.B. Facilitatory

effects of 4-aminopyridine on normal neuromuscular transmission.

Muscle and Nerve, 3: 105-111, 1980.

Kirby, R.L. Changes in {

3H}naloxone binding in spinal cord of rats

treated prenatally with morphine. Neuropharmacol, 22: 303-307,

1983.

Klee, W.A. , and Nirenberg, M. A neuroblastoma x glioma hybrid cell

line with morphine receptors. Proc. Natl. Acad. Sci. USA, 71:

3474-3477, 1974.

Klee, W.A., Sharma, S.K. , and Nirenberg, M. Opiate receptors as

regulators of adenylate cyclase. Life Sci, 16: 1869-1874, 1975.

Kosterlitz, H.W. , and Hughes, J. Peptides with morphine-like

action in the brain. Brit. J. Psychiat, 130: 298-304, 1977.

Kosterlitz, H.W., and Waterfield A.A. In vitro models in the

study of structure-activity relationships of narcotic analgesics.

Ann. Rev. Pharmacol, 15: 29-47, 1975.

Kosterlitz, H.W., Waterfield, A.A. and Berthoud, V. Assessment

of the agonist and antagonist properties of narcotic analgesic

drugs by their actions on the morphine receptor in the guinea

pig ileum. In: Braude, M.C., Harris, L.S., May, E.L.,

Smith, J.P., and Villarreal, J.E., eds. Advances in Biochemical

Psychopharmacology, Vol. 8, Narcotic Antagonists. New York:

Raven Press, 1973. pp. 319-334.

Kosterlitz, H.W., Lord, J.A.H., Paterson, S. J. , and Waterfield

A.A. Effects of changes in the structure of enkephalins and of

narcotic analgesic drugs on their interactions with μ- and

receptors. Brit. J. Pharmacol, 68: 333-342, 1980.

Kosterlitz, H.W., Paterson, S.J., and Robson, L.E., Characterization

of the K-subtype of the opiate receptor in the guinea-pig brain.

Brit. J. Pharmacol, 73: 939-949, 1981.

Lahti, R., and Collins, R. Chronic naloxone results in prolonged

increases in opiate binding sites in brain, Eur. J. Pharmacol,

51: 185-186, 1978.

Lamotte, C., Pert, C.B., and Snyder, S.H. Opiate receptor binding

in primate spinal cord: distribution and changes after dorsal

root section. Brain Res, 112: 407-412, 1976.

Lampert, A. , Nirenberg, M., and Klee, W.A. Tolerance and

dependence evoked by an endogenous opiate peptide. Proc. natl

Acad. Sci. USA, 73: 3165-3167, 1976.

Lenoir, D., Barg, J., and Simantov, R. Down-regulation of opiate

receptors in serum-free cultures of aggregating fetal brain

cells. Life Sci, 33:337-340, 1983.

Macdonald, R.L., and Nelson, P.G. Specific opiate-induced depression of transmitter release fran dorsal root ganglion cells in

culture. Science, 199: 1449-1451, 1978.

Matsuda, Y., Yoshida, S., and Yonezawa, T. Tetrodotoxin sensitivity

and Ca component of action potentials of mouse dorsal root ganglion

cells cultured in vitro. Brain Res, 154: 69-82, 1978.

Meiri , U. , and Rahamimoff, R. Neuromuscular transmission: Inhibition

by manganese ions. Science, 176:308-309, 1972.

287
Mudge, A.W., Leeman, S.E., and Fischbach, G.D. Enkephalin

inhibits release of substance P from sensory neurons in culture

and decreases action potential duration. Proc. natl. Acad.

Sci. USA, 76: 526-530, 1979.

Murase, K., Nedeljkov, V., and Randic, M. The actions of

neuropeptides on dorsal-horn neurons in the rat spinal cord

slice preparation an intracellular study. Brain Res, 234: 170-

176, 1982.

Nicak, A., and Kohut, A. Development of tolerance to morphine

and pethidine in rats is dependence on age. Activitas Nervosa

Superior (Praha), 20: 231-235, 1978.

Nichols, R.A., and Nakajima, Y. Effects of manganese and cobalt

on the inhibitory synapse of the crustacean stretch receptor

neuron. Brain Res, 86: 493-498, 1975.

Ninkovic, M., Hunt, S.P., and Gleave, J.R. Localization of

opiate and histamine H1-receptors in the primate sensory

ganglia and spinal cord. Brain Res, 241: 197-206, 1982.

North, R.A. Opiates, opioid peptides and single neurones. Life

Sci, 24: 1527-1546, 1979.

North, R.A. , and Karras, P.J. Opiate tolerance and dependence

induced in vitro in single myenteric neurones. Nature, 272:

73-75, 1978a.

North, R.A., and Karras, P.J. Tolerance and dependence in vitro.

In: Van Ree, J.M., and Terenius, L., eds. Characteristics and

Function of Opioids. Amsterdam: Elsevier/North Holland Press,

1978. pp. 25-36b.

North, R.A., and Vitek, L. The effect of chronic morphine treatment on excitatory junction potentials in the mouse vas deferens.

Brit. J. Pharmacol, 68: 399-406, 1979.

North, R.A., and Zieglgansberger, W. Opiate withdrawal signs in

single myenteric neurones. Brain Res, 144: 208-211, 1978.

Otsuka, M., and Konishi, S. Electrophysiology of mammalian spinal

cord in vitro. Nature, 252: 733-735, 1974.

Perkins, M.N. and Stone, T.W. 4-aminopyridine blockade of neuronal

depressant responses to ATP. Brit. J. Pharmacol, 70: 525-428, 1980.

Peroutka, S.J. , Lebovitz, R.M. , and Snyder, S.H. Two distinct

central serotonin receptors with different physiological functions.

Science, 212: 827-829, 1981.

Pert, C., and Snyder, S. Opiate receptor binding-enhancement of

opiate administration in vivo. Biochem. Pharmacol, 25: 847-853,

1976.

Ransom, B.R., Neale, E., Henkart, M., Bullock, P.N., and Nelson, P.G.

Mouse spinal cord in cell culture. 1. Morphology and intrinsic

neuronal electrophysiologic properties. J. Neurophysiol, 40:

1132-1150, 1977.

Roemer, D., Buescher, H.H. , Hill, R.C. , Pless, J., Bauer, W.,

Cardinaux, F., Closse, A., Hauser, D., and Hugenin, R. A

synthetic enkephalin analogue with prolonged parenteral and oral

analgesic activity. Nature, 268: 547-549, 1977.

Schulz, R., Wuster, M., and Herz, A. Supersensitivity to opioids

following chronic blockade of endorphin activity by naloxone.

Naun. Schmied. Arch. Pharmacol, 306: 93-96, 1979.

288
Schulz, R., Wuster, M., Krenss, H., and Herz, A. Selective development of tolerance without dependence in multiple opiate receptors

of mouse vas deferens. Nature, 285: 242-243, 1980.

Schulz, R., Wuster, M., Rubini, P., and Herz, A. Functional opiate

receptors in the guinea-pig ileum: Their differentiation by

means of selective tolerance development. J. Pharmacol. exp. Ther,

219: 547-550, 1981.

Sharma, S.K. , and Khanna, N.C. Ascorbate suppresses the opiateinduced compensatory increase in cyclic AMP in neuroblastoma

x glioma hybrid cells. Biochem. J, 208: 43-46, 1982.

Sharma, S.K. , Nirenberg, M. , and Klee, W.A. Morphine receptors

as regulators of adenylate cyclase activity. Proc. natl. Acad.

Sci. USA, 72: 590-594, 1975a.

Sharma, S.K. , Klee, W.A. , and Nirenberg, M. Dual regulation of

adenylate cyclase accounts for narcotic dependence and tolerance.

Proc. nat. Acad. Sci. USA, 72: 3092-3096, 1975b.

Simantov, R., Baram, D. , Levy, R. , and Nadler, H. Enkephalin and

adrenergic receptors: Evidence for both common and differentiable regulatory pathways and down-regulation of the enkephalin

receptor. Life Sci, 31: 1323-1326, 1982.

Simon, E.J. The opiate receptors. Neurochemical Res, 1: 3-28,

1976.

Spitzer, N. C. Ion channels in development. Ann. Rev. Neurosci,

2: 363-397, 1979.

Stinnakre, J., and Tauc, L. Calcium influx in active aplysia

neurones detected by injected aequorin. Nature (New Biol.),

242: 113-115, 1973.

Stone, T.W. Physiological roles for adenosine and adenosine 5’-

triphosphate in the nervous system. Neuroscience, 6: 523-555,

1981a.

Stone, T.W. The effects of 4-aminopyridine on the isolated vas

deferens and its effects on the inhibitory properties of adenosine,

morphine, noradrenaline and aminobutyric acid. Brit. J.

Pharmacol, 73: 791-796, 1981b.

Suzue, T., and Jessell, T. Opiate analgesics and endorphins inhibit

rat dorsal root potential in vitro. Neurosci. Lett, 16: 161-166

1980.

Tang, A.H., and Collins, R.J. Enhanced analgesic effects of

morphine after chronic administration of naloxone in the rat.

Eur. J. Pharmacol, 47: 473-474, 1978.

Tempel, A., Crain, S.M., Simon, E.J., and Zukin, R.S. Opiate

receptor upregulation in explants of spinal cord-dorsal root

ganglia. Soc. Neurosci. Abstr, 9: 327, 1983.

Traber, J., Gullis, R. and Hamprecht, B. Influence of opiates on

the levels of adenosine 3’:5’-cyclic monophosphate in neuroblastana

x glioma hybrid cells. Life Sci, 16: 1863-1868, 1975.

Tung, A.S., and Brandom, B.W. 4-Aminopyridine reversal of

morphine analgesia. Soc. Neurosci. Abstr, 7: 798, 1981.

Van Neuten, J.M., Janssen, P.A.J., and Fontaine, J. Naloxone

reverses inhibitory effects of fatigue and of compounds not

related to narcotic analgesics in the guinea-pig ileum. Arch.

int. Pharmacodyn, 220: 349-350, 1976.

Ward, A., and Takemori, A.E. Studies on the narcotic receptor in

the guinea-pig ileum. J. Pharmacol. exp. Ther, 199: 117-123,

1976a.

289
Ward, A. , and Takemori, A. E. Effect of 6-hydroxydopamine and

5,6-dihydroxytryptamine on the response of the coaxially

stimulated guinea-pig ileum to morphine. J. Pharmacol. exp.

Ther, 199: 124-130, 1976b.

Werz, M.A. and Macdonald, R.L. Opioid peptides decrease calciumdependent action potential duration of mouse dorsal root ganglion

neurons in cell culture. Brain Res, 239: 315-321, 1982a.

Werz , M.A., and Macdonald, R.L. Heterogeneous sensitivity of

cultured dorsal root ganglion neurones to opioid peptides

selective μ-and opiate receptors. Nature, 299: 730-732, 1982b.

Werz, M.A., and Macdonald, R.L. Dynorphine specifically decreased

calcium-dependent action potential duration in cultured primary

sensory neurons. INRC Symp, Abstr: P-84, 1983.

Wikler, A. Opiate Addiction. Psychological and Neurophysiological Aspects in Relation to Clinical Problems.

Illinois: C.C. Thomas, 1953.

Wikler, A. Opioid Dependence. New York: Plenum Press, 1980.

Wikler, A., and Carter, R.L. Effects of single doses of Nallylnormorphine on hindlimb reflexes of chronic spinal dogs

during cycles of morphine addiction. J. Pharmacol. exp. Ther,

109: 92-101, 1953.

Wikler, A., and Frank, K. Hindlimb reflexes of chronic spinal dogs

during cycles of addiction to morphine and methadon. J. Pharmacol.

exp. Ther, 94: 382-400, 1948.

William, J.T. and North, R.A. Tolerance to opiates in locus

coeruleus neurones? INRC Symp, Abstr: P-49, 1983.

Williams, J., and Zieglgansberger, W. Mature spinal ganglion cells

are not sensitive to opiate receptor mediated action. Neurosci .

Lett, 21: 211-216, 1981.

Wuster, M.R., Schulz, R., and Herz, A. Specificity of opioids towards

the μ-, and E-opiate receptors. Neurosci. Lett, 15: 193, 1979.

Yaksh, T.L., and Wilson, P.R. Spinal serotonin terminal system

mediates antinociception. J. Pharmacol. exp. Ther, 208: 446-453,

1979.

Yamamoto, H., Harris, R.A. Loh, H.H., and Way, E.L. Effect of acute

and chronic morphine treatments on calcium localization and binding

in brain. J. Pharmacol. exp. Ther, 205: 255-264, 1978.

Yoshida, S., and Matsuda, Y. Studies on sensory neurons of the

mouse with intracellular-recording and horseradish peroxidaseinjection techniques. J. Neurophysiol, 42: 1134-1145, 1979.

Yoshimura, M., and North A.R. Substantia gelatinosa neurones

hyperpolarized in vitro by enkephalin. Nature, 305: 529-530,

1983.

Zukin, R.S. , Sugarman, J.R., Fitz-Syage, M.L. , Gardner, E.L.,

Zukin, S.R., and Gintzler, A.R. Naloxone-induced opiate supersensitivity. Brain Res, 245: 285-292, 1982.

290
ACKNOWLEDGEMENTS

This work was supported by a research grant from the National

Institute on Drug Abuse (DA-02031). Collaborative research with

Drs. Eric J. Simon and Jacob M. Hiller (New York University) was

supported by NIDA grant DA-00017 (to E.J.S.). Collaborative

research with Drs. R. Suzanne Zukin and Ann Tempel (at Einstein)

was supported by NIDA grants DA-01843 and DA-00069 (to R.S.Z.).

The cord-DRG cultures used in these studies were prepared by Edith

E. Peterson. Tissue culture facilities were kindly provided by

Dr. Murray B. Bornstein. Bea Crain provided skillful technical

assistance in carrying out many of the electrophysiologic

experiments.

AUTHOR

Stanley M. Crain, Ph.D.

Departments of Neuroscience and Physiology, and the

Rose F. Kennedy Center for Research on

Mental Retardation and Human Development

Albert Einstein College of Medicine

Bronx, New York 10461

291
NOTE ADDED IN PROOF

Following the discussions at the NIDA Symposium (Boston,

Nov. 1983) on Dr. Collier’s hypothesis regarding the possible

roles of adenylate cyclase (AC)/cyclic AMP in opioid tolerance

and dependence, attempts were initiated to utilize forskolin,

a selective activator of AC (Seamon and Daly), to facilitate

evaluation of the effects of increased AC/CAMP levels on the

sensitivity of cord-DRG explants to opiates (see above: Sect. I).

When naive explants were treated with forskolin (10-50μM) for

10-30 min prior to and during exposure to 0.3μM morphine, the

usual depressant effects on dorsal-horn responses often failed

to occur (10-30 min tests) (Crain et al, 1984). Dibutyryl

cAMP (10μM) or the more potent analog, dioctanoyl cAMP(0.1mM),

produced a similar degree of “tolerance” to opiates as 10μM

forskolin. With high levels of forskolin (50μM), even concentrations of morphine up to 10μM were far less effective in

depressing cord responses. These anti-opiate effects of exogenous cAMP and forskolin are probably both mediated by

increases in intracellular cAMP. The onset of opioid-“tolerance” in cAMP-or forskolin-treated cord-DRG explants provides

the first electrophysiologic support for the hypothesis that

neurons may develop tolerance/dependence during chronic opioid

exposure by a compensatory enhancement of their AC/CAMP system

following initial opioid-depression of AC activity (see review

by Collier, 1980). Previous evidence relied primarily on

behavioral tests (Ho et al. 1973; Collier et al. 1975) and

biochemical analyses in cell cultures (Sharma et al. 1975).

It will be of interest to determine if dorsal-horn tissues

of cord-DRG explants do, in fact, develop increased AC/cAMP

levels as they become tolerant during chronic exposure to

opiates.

ADDITIONAL REFERENCES

Crain, S.M., Crain, B. and Peterson, E.R. Cyclic AMP or

forskolin produces rapid “tolerance” to the depressant

effects of opiates on sensory-evoked dorsal-horn responses

in spinal cord-dorsal root ganglion (DRG) explants. Soc.

Neurosci Abstr 10 (in press).

Ho. I.K., Loh. H.H. and Way, E. L. Cyclic adenosine monophosphate antagonism of morphine analgesia. J Pharmacol

Exp Ther, 185:336-345, 1973.

Seamon, K.B. and Daly, J.W. Forskolin: a unique diterpene

activator of cyclic AMP-generating systems. J Cyclic

Nucleotide Res, 7:201-224, 1981.

292
Experimental Approaches Toward

Determining the Neuronal Circuits

Most Affected by Chronic Opiate

Administration

Robert Elde

INTRODUCTION

A wide variety of studies have explored the expression of

tolerance to opiates at behavioral, cellular and molecular levels.

However, very little is known concerning the anatomical identity

of neuronal systems that are most affected by chronic opiate

administration. Since the discovery of opiate receptors and the

characterization of opioid peptides, a great deal of information

has been gathered concerning the regional distribution of opiate

binding sites as well as the distribution of neuronal cell bodies,

fibers and terminals that contain enkephalins, endorphins and

dynorphins. It has been hypothesized that the activity of neurons

that bear opiate receptors is likely to be perturbed by chronic

opiate adminstration, perhaps by alterations in receptor number or

affinity (Klee et al., Law et al., Zukin et al, this volume). In

addition, it has been suggested that the activity of opioid neurons

themselves may be altered in the tolerant state, perhaps through

receptor-mediated autofeedback mechanisms. It is also quite

possible that the major effects of chronic opiate administration are

expressed by neurons that are regulated by the above classes of

neurons, but separated from them by one or more synaptic connections

(Wuster and Costa, this volume). Moreover, it has not been

established if chronic opiate administration leads to chronic

excitation or inhibition of the affected neuronal circuits.

PREVIOUS STUDIES

Little is known of the neuroanatomical substrates of opiate

tolerance because of limitations inherent to techniques used to

measure neuronal activity. Microelectrode physiological methods

provide precise records of neuronal activity of single cells or

small groups of cells over times that range from milliseconds to

minutes. Chronic or periodic recording from chronically implanted

electrodes may extend the period for recording of activity to

several days. However, the number of neurons that can be sampled by

these methods is very small. Considering the large number of brain

regions and the large number of neurons within each region, it is

clear that unit recording of neuronal activity is not an adequate

293
method for sampling the entire nervous system for the effects of

chronic opiate administration on neuronal activity.

Electroencephalographic methods overcome some of the limitations of

microelectrode recording in that large regions of brain can be

measured for net neuronal activity. However, the neuroanatomical

resolution of these methods is poor.

Introduction of the autoradiographic method for detecting regional

accumulation of radiolabeled 2-deoxyglucose provided the technical

means to survey the entire brain for changes in glucose utilization.

Regional deoxyglucose accumulation has been found to closely

parallel neuronal activity during the time of exposure to

deoxyglucose, the duration of exposure usually being an hour or less

(Sokoloff et al. 1977).

Several studies have been reported on the effects of opiate

administration on 2-deoxyglucose accumulation. Lightman and

colleagues (1982) found that acute morphine produced a naloxonereversible increase in glucose utilization in the posterior lobe of

the pituitary. Chronic administration of morphine also produced an

increase in glucose utilization in the posterior pituitary, although

a subsequent acute challenge with morphine produced a decrease in

glucose utilization. Wooten and colleagues (1982) surveyed

regional glucose utilization in the di- and telencephalon of

morphine-dependent rats and in rats in which withdrawal was

precipitated with naloxone. Optical densitometry of autoradiograms

from morphine-dependent animals revealed significant increases in

some regions, significant decreases in other regions and no change

in glucose utilization in other regions (table 1).

These data are of considerable interest, however it is important to

note that the relative activity of neurons as measured in these

studies may not genuinely reflect the sites most prominent in

expressing the tolerant state. In the first place, it is debatable

whether or not a genuine state of tolerance was achieved during the

periods of measurement, since the morphine injections were halted or

morphine pellets were removed some hours prior to infusion of

radiolabeled deoxyglucose. Thus, it is possible that some of the

changes in deoxyglucose accumulation could be due to early phases of

withdrawal from the tolerant state. Secondly, at least some of the

changes in glucose utilization may reflect the morphine-treated

animals' behavioral response to the handling that was necessary for

cannulae insertion and deoxyglucose administration.

It is important to stress that limitations inherent to the

deoxyglucose method produce difficulties for the design of

experiments that will differentiate changes in glucose utilization

that are directly attributable to a drug's action from those which

occur as secondary effects. General hypoxia secondary to opiateinduced respiratory depression represents another example of

possible counfounding effects that may be detected by the

deoxyglucose method. Wuster and colleagues (1981) attempted to

294
TABLE 1

Regions with Changes in Brain Glucose Utilization

after Chronic Morphine Treatment*

Increased

Medial preoptic area, lateral preoptic area, bed nucleus stria

terminalis, preoptic magnocellularis, parasubiculum, subiculum,

medial entorhinal cortex, lateral entorhinal cortex; CA-1 pyramidal

layer, CA-1 striatum radiatum, perforant path, dentate gyrus, CA-3

pyramidal layer, CA-Z.

Decreased

Frontal motor cortex, medial frontal cortex; striatum; thalamic

anterior ventral nucleus; medial habenula nucleus.

*Data from Wooten et al. (1982)

avoid the effects of opiate-induced respiratory depression by

studying glucose utilization in an in vitro whole brain perfusion

system. Whereas such an approach may provide information on the

sites of acute effects of opiates, it is unlikely to provide

information on the sites where the chronic effects of opiate

administration may be manifest.

Recent work on levels of enzymes involved in brain energy metabolism

suggests an alternate approach for studying the sites involved in

the expression of tolerance to opiates. Wong-Riley (1979) and

colleagues (Wong-Riley et al. 1978; Wong-Riley and Welt 1980) have

explored the intensity of histochemically-revealed cytochrone

oxidase activity as a function of neuronal activity. When auditory,

visual and somatosensory systems were denervated, cytochrome oxidase

staining intensity was shown to decrease within neurons as far as

several synapses distal to the site of sensory deprivation. Using

more quantitative assays, several other enzymes related to energy

metabolism were shown by Dietrich and colleagues (1981) to be

significantly depressed within specific targets of whisker afferent

pathways after cauterization of whiskers. Thus, there is striking

evidence that long-term inactivation of neuronal pathways leads to

decreases in energy-related enzyme levels.

Kageyama and Wong-Riley (1982) have further reported that the level

of cytochrome oxidase staining within hippocampal neurons correlates

positively with the known spontaneous activity of those neurons.

That is, spontaneously active neurons have higher levels of

cytochrome oxidase activity than those which are tonically silent.

Most importantly, Dietrich and colleagues (1982) have demonstrated

increases in levels of three energy metabolism-related enzymes in

cortical barrel fields that are the targets of intact whisker

afferents in animals in which other whisker afferents had been

removed. The authors suggest this is the first demonstration of an

increase of energy-related enzymes in brain in response to chronic,

295
experimental stimulation of a pathway. As they point out, however,

these changes are not without biological precedent, since similar

long-tern changes in levels of enzymes of energy metabolism are

observed in skeletal muscle as a function of exercise and disuse

(see Karpati and Engel 1968, Holloszy and Booth 1976).

Metabolic enzyme histochemistry provides a possible means to detect

changes in neuronal activity that are manifest and maintained over a

period of weeks. The technique is attractive for identification of

sites affected by chronic opiate administration, since transient

phenomena that may accompany handling and treatment of animals are

unlikely to be detected. In contrast to the deoxyglucose method,

enzyme histochemistry does not require intensive manipulation of

animals (such as restraint, intravenous cannulation and injection)

immediately prior to and during the time of the measurement.

Thus, it can be expected that neurons identified by enzyme

histochemistry as most altered by chronic exposure to opiates will

represent those neurons that are responsible for the physiological

and behavioral expression of tolerance.

PILOT STUDIES

An experiment was conducted to compare the intensity of cytochrome

oxidase staining in brains of animals chronically exposed to

morphine and in brains from control animals. Morphine was made

available to Swiss-Webster mice ad libitum in drinking water that

contained 10% sucrose (Khavari and Risner 1973). During the first

16 days, mice consumed an average of 69 mg morphine sulfate/kg/day.

During the last 9 days the concentration of morphine was doubled

resulting in mean consumption of 146 mg/kg/day. Control mice

received sucrose drinking water for 25 days. At several intervals

during the study, tolerance was assessed in cohorts by naloxoneprecipitated withdrawal. Brains of mice were fixed by vascular

perfusion with 4% formaldehyde. Frozen sections sampling the entire

neuraxis were processed for cytochrome oxidase activity according to

Wong-Riley and Welt (1980). Specificity controls for staining were

obtained by inclusion of sodium azide in the incubation media.

Staining patterns and density were examined and, in limited areas to

date, quantified by optical densitometry.

Cytochrone oxidase staining was heterogeneous within the brain.

Staining for this enzyme complex appeared to be specific since

inclusion of the electron transport inhibitor, sodium azide,

prevented staining. The intensity of staining in many regions did

not appear to differ between morphine-treated and control mouse

brains (figures 1 and 2). However, in certain discrete regions such

as the mammillary nuclei, a striking decrease in the staining

intensity was observed in morphine treated animals (74% of control,

p<0.05; figures 1 and 2). Decreases in staining in

296
FIGURES 1 and 2. Photomicrographs of coronal sections of rat

midbrain after processing for cytochrome oxidase histochemistry.

Many regions in vehicle control (figure 1) and chronic morphinetreated animals (figure 2) exhibit similar staining intensity.

However, the mammillary nuclear complex (arrows) of the morphinetreated animals was strikingly decreased in staining intensity.

morphine-treated mice were apparent in some other regions. No

increases in cytochrome oxidase staining were noted in morphinetreated mice.

This pilot study demonstrates the feasibility of using enzyme

histochemistry to determine sites where neuronal activity is most

altered as a consequence of chronic morphine administration.

Previous reports (Dietrich et al. 1981, 1982) have suggested that

levels of enzymes involved in energy metabolism change only over a

time course of several weeks. This phenomenon is exploited by

enzyme histochemistry and thereby avoids detection of transiently

occurring events. Thus histochemical detection of metabolic enzymes

has an advantage over the 2-deoxyglucose method in that it is less

susceptible to revealing changes in neuronal activity that are

actually a consequence of periodic handling of the animal or

responses to manipulation of the animal in the period immediately

prior to sacrifice.

However, cytochrome oxidase histochemistry is unlikely to

demonstrate all of the sites involved in tolerance to morphine. As

noted above, neither this pilot study nor previous studies in other

systems have noted increases in levels of cytochrome oxidase. The

findings of Dietrich and colleagues (1982) suggest that increases as

well as decreases of malate dehydrogenase or citrate synthase may be

histochemically demonstrable, Thus histochemical study of these

enzymes may be more appropriate to determine the variety of changes

in neuronal activity which occur as a consequence of long-term

perturbation by opiates.

SUMMARY AND FUTURE QUESTIONS

The investigation of neuroanatomical substrates, through which

opiate tolerance and dependence phenomena are manifest, now appears

to be feasible. Histochemical processing of brains to reveal certain

enzymes of energy metabolism is likely to identify neurons most

297
involved in the tolerant state. The following specific questions

can be addressed.

1. IS the net activity of neurons bearing opiate receptors altered

by chronic opiate adninistation? Are neurons bearing different

classes of opiate receptors differentially affected? Are neurons

that produce opioid peptides affected? Do opioid neurons in

discrete regions, or those producing particular opioid peptides,

differ in their activity during chronic exposure to morphine? Is

the net effect inhibition or excitation?

2. What neurotransmitters are produced by neurons most affected by

chronic exposure to morphine? Such inquiry may suggest the use of

new therapeutic means of countering the untoward effects of

tolerance to opiates.

3. Which neurons in the brain are principally involved in the

manifestation of withdrawal from chronic opiates? Which

neurotransmitters do they contain and which receptors do they bear?

Such data may suggest new therapeutic means of countering the

untoward effects of withdrawal from opiates.

REFERENCES

Dietrich, W.D., Durham, D., Lowry, O.H., and Woolsey, T.A.

Quantitative histochemical effects of whisker damage on single

identified cortical barrels in the adult mouse. J Neuroscience,

1:929-935, 1981.

Dietrich, W.D., Durham, D., Lowry, O.H., and Woolsey, T.A.

"Increased" sensory stimulation leads to changes in energyrelated enzymes in the brain. J Neuroscience, 2:1608-1613, 1982.

Holloszy, J.O. and Booth, F.W. Biochemical adaptations to

endurance exercise in muscle, Annu Rev Physiol, 38:273-291, 1976.

Kageyama, G.H., and Wong-Riley, M.T.T. Histochemical localization

of cytochrome oxidase in the hippocampus: Correlation with

specific neuronal types and afferent pathways. Neuroscience,

7~2337-2361, 1982.

Karpati, G., and Engel, W.K. Correlative histochemical study of

skeletal muscle after suprasegmental denervation, peripheral

nerve section and skeletal fixation. Neurology, (N.Y.) 18:681-

692, 1968.

Khavari, K.A., and Risner, M.E. Opiate dependence produced by

ad libitum drinking of morphine in water, saline, and sucrose

vehicles. Psychopharmacologia, 30:291-302, 1973.

Lightman, S., Hunt, S.P., and Iversen, L.L. Effects of opiates

and osmotic stimuli on rat neurohypophyseal metabolic activity

monitored with [3H]-2-deoxyglucose. Neuroendocrinology, 35:104-

110, 1982.

Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M.H., Patlak,

C.S., Pettigrew, K.D., Sakurada, O., and Shinohara, M. The

(

14C)-deoxyglucose method for the measurement of local cerebral

glucose utilization: Theory, procedure, and normal values in the

conscious and anaesthetized albino rat. J Neurochem, 28:897-916,

1977.

298
Wong-Riley, M.T.T. Changes in the visual system of monocularly

sutured or enucleated cats demonstrable with cytochrome oxidase

histochemistry. Brain Res, 171:11-28, 1979.

Wong-Riley, M.T.T., Merzenich, M.M., and Leake, P.A. Changes in

endogenous enzymatic reactivity to DAB induced by neuronal

inactivity. Brain Res, 141:185-192, 1978.

Wong-Riley, M.T.T. and Welt, C. Histochemical changes in

cytochrome oxidase of cortical barrels after vibrissal removal

in neonatal and adult mice. Proc Nat1 Acad Sci USA 77:2333-

2337. 1980.

Wooten, G.F., DiStefano, P., and Collins, R.C. Regional cerebral

glucose utilization during morphine withdrawal in the rat. Proc

Nat1 Acad Sci USA 79:3360-3364, 1982.

Wuster, M., Dirks, B., Krieglstein, J., and Herz, A. The effect

of the potent opiate agonist etorphine on local energy

metabolism in the isolated perfused rat brain. Neuropharmacology,

20:901-904, 1981.

ACKNOWLEDGMENTS

Studies in the authors' laboratory were supported by USPHS Grant

DA 02148 from the National Institute on Drug Abuse. Drs.

L. Lichtblau, V. Seybold, A. Takemori, and M. Wessendorf, at the

University of Minnesota, provided helpful ideas and advice.

AUTHOR

Robert Elde, Ph.D.

Associate Professor

Department of Anatomy

University of Minnesota Medical School

321 Church Street S.E.

Minneapolis, MN 55455

299
Effects of Opiates and

Neuropeptides on Immune

Functions

Robert E. Faith, N. P. Plotnikoff, and A. J. Murgo

INTRODUCTION

In recent years there has been an increase in interest in the

effects of drugs and pharmaceuticals on immune function. Indeed a

number of chemicals have been identified and developed because they

possess immunomodulatory properties. This includes both immunostimulants and immunosuppressants. In addition to drugs developed

specifically for their immunomodulatory properties, it has become

clear that a number of chemicals, including environmental pollutants, various anesthetics and analgesics, drugs of abuse, and

hormones, including neuropeptides, can influence immune functions.

In this chapter we will discuss the effects of the opiates and

neuropeptides on immune function.

OPIATE EFFECTS

While the opiates have been abused, or used as recreational drugs

for centuries, very little is known about the possible effects of

these chemicals on immune function, direct or indirect. Most of

what is known was recently reviewed by Sheagren and Tuazon (1977).

It is difficult to investigate the effects of opiates on immune

function in the addict. However, some knowledge has been gained

from studying these individuals. It is recognized clinically that

infectious disease is often a complication of drug abuse. It must

be kept in mind that a number of factors other than direct immunosuppression could lead to increased incidence of infections in

opiate addicts. These include nutritional factors, living conditions and contamination of injection paraphernalia. However,

Sheagren and Tuazon (1977) state, "Most authors do agree that the

incidence of infections is increased in most common forms of drug

abuse and that immune alterations probably do exist."

The immune system is composed of nonspecific as well as specific

components. Cellular elements, macrophages and neutrophils, and

serum factors, complement and interferon, participate nonspecifically in immunity. Several studies have shown these elements of

300
immunity to be affected by opiates. One study has shown that

neutrophil chemotaxis is suppressed by morphine (Stanley et al.

1976) while another has shown that phagocytic and bactericidal

functions of neutrophils from patients taking methadone and morphine are normal (Nickerson et al. 1970). Studies in mice have

shown that injections of morphine, dilaudid or methadone significantly reduce the ability of these animals to produce interferon in

response to induction by either poly I:C or bacterial endotoxin

(Hung et al. 1973). Finally, Wetli et al. (1974) have shown that

some heroin addicts have elevated complement levels, others have

depressed complement levels and the rest are not affected.

Recent studies have presented evidence that lymphocytes possess

receptors which bind opiates such as morphine (Wybran et al. 1979)

and naloxone (McDonough et al. 1980, Mehrishi and Mills 1983). The

presence of these receptors would indicate that the opiates may be

able to affect the immune system directly. Indeed there is some

evidence to suggest that this is the case. It has been reported

that opiate users often have hyperglobulinemia (Sheagren and Tuazon

1977). The IgM levels are most frequently increased, but increases

in IgG are also seen. These increases in immunoglobulin levels are

most likely a result of either repeated injections of antigenic

materials along with the abused drug or the presence of chronic

infections which affect many addicts. Opiates have been reported

to inhibit specific antibody production in experimental animals.

Mice given large doses of morphine or methadone had reduced antibody production when compared to nontreated controls (Lefkowitz and

Yang 1974; Gungor et al. 1980).

There is also some evidence that opiates may directly affect

T-lymphocytes. Brown et al. (1974) investigated the ability of

peripheral blood lymphocytes from heroin addicts to proliferate in

response to mitogenic stimulation. They found the responses of

cells from heroin addicts to stimulation with phytohemagglutinin

(PHA), Concanavalin A (Con A) or pokeweed mitogen (PWM) to be

significantly less than the responses of cells from normal volunteers. The proliferative responses of PBL's to mitogenic stimulation tended to return to normal when the study subjects were not

taking heroin. McDonough et al. (1980) have shown that opiate

addicts have a significant reduction in the number of total Tlymphocytes in circulation as measured by the ability of the

lymphocytes to form rosettes with sheep red blood cells (a marker

for T-lymphocytes). They also showed an increase in the number of

"null" lymphocytes, but there was no significant change in the B

lymphocytes or total white blood cell count in opiate addicts. The

T cell decrease and "null" cell increase were reversible by naloxone. Similarly, Wybran et al. (1979) have shown that in vitro

treatment of peripheral blood lymphocytes from normal human volunteers with morphine significantly reduces their ability to form

active rosettes with sheep red blood cells. This effect is completely reversed by naloxone.

One final set of studies must be mentioned when discussing the

effects of opiates on host defense mechanisms. These are studies

involving the effects of opiates on tumor growth and survival times

301
of mice implanted with a transplantable neuroblastoma cell line.

Zagon and McLaughlin (1981a, b, 1983) have shown the heroin,

naloxone or naltrexone inhibit tumor growth and prolong survival

times at relatively low doses, while high doses may enhance tumor

growth. These studies are somewhat difficult to interpret because

the neuroblastoma possesses receptors for the opiates, and the

drugs may be exerting their action on the tumor cells rather than

on host defense mechanisms. The opiates may exert their effects on

both the tumor cells and host defense mechanisms, or their action

on the tumor cells may not be a factor in host resistance to this

tumor. Studies with other tumor systems are required to more fully

elucidate the effects of the opiates on the ability of a host to

resist a tumor challenge.

NEUROPEPTIDE EFFECTS (ENKEPHALINS - ENDORPHINS)

Recently several studies have been performed to investigate the

effects of the opioid neuropeptides, endorphins and enkephalins on

immune function. Initially Wybran et al. (1979) produced data

indicating that human lymphocytes possess receptors for methionineenkephalin. At the same time Hazum et al. (1979) reported that

cultured human lymphocytes possess specific receptors for betaendorphin. Subsequently it has been shown that alpha-, beta- and

gamma-endorphin are capable of suppressing the in vitro antibodyproducing cell response of mouse splenic lymphocytes immunized in

vitro (Johnson et al. 1982). Suppression with beta- and gammaendorphin occurred at high concentrations. There have been two

studies reported in which the effects of beta-endorphin on the

proliferative response of lymphocytes to mitogen stimulation were

investigated. McCain et al. (1982) reported that beta-endorphin is

a potent suppressor of PHA-induced T-lymphocyte blastogenesis when

human peripheral blood lymphocytes are exposed early in the course

of mitogenic activation. This suppression was not blocked by

pretreatment with naloxone. In contrast, Gilman et al. (1982) have

reported that beta-endorphin significantly enhances the proliferative response of rat splenic lymphocytes to the T cell mitogens PHA

and Con A. Treatment with naloxone did not reverse the enhancing

effect of the beta-endorphin. The differences observed in these

two studies may be due to one or more of the following:

1) Different concentrations of beta-endorphin were utilized in the

two studies; 2) Rat lymphocytes may respond differently to betaendorphin than do human lymphocytes; or 3) Lymphocytes in spleen

contain somewhat different subpopulations than those found in

peripheral blood, which may result in a different effect observed

following treatment with beta-endorphin. Based on results obtained

with the enkephalins in our laboratory, the first possibility

listed may be the most likely. We have observed that varying

concentrations of enkephalins exert a biphasic effect on lymphocyte

function with some concentrations exerting stimulatory effects

while other concentrations are suppressive. The final reported

study on the effects of beta-endorphin on immune functions is that

of Mathews et al. (1983). This study reports that beta-endorphin

significantly enhances natural cytotoxicity exhibited by natural

killer (NK) cells in peripheral blood lymphocytes of normal human

volunteers. In these studies lymphocytes were isolated from the

302
peripheral blood of normal volunteers and incubated with betaendorphin. This treatment resulted in enhanced NK cell activity as

measured in a cytotoxicity assay. This enhancement appears to

result from an increase in the numbers of NK cells and increased

cytotoxic efficiency of the NK cells. This enhancement of NK

activity was reversible with naloxone treatment.

TABLE 1. Effect of enkephalins on thymus

and spleen weights in micea

Enkephalin dosage Thymus wt. Spleen wt.

(mg/kg) (mg±SE) (mg±SE)

Methionine-enkephalin

0 68.6±3.6 101.8±3.1

10 78.1±2.9 68.3±2.2

(p=O.O5)b (p<O.OO1)b

30 79.1±4.5

(p<0.1)b

69.8±l.7

(p<O.OO1)b

Leucine-enkephalin

0 56.1±O.9 115.2±6.1

10 49.3±2.8

(p<0.05)b

98.0±4.0

(p<0.05)b

30 63.O±1.7

(p<0.005)b

71.3±1.9

(p<O.OO1)b

a . Mice were injected daily for 1 week with varying dilutions of methionine-enkephalin or leucine-enkephalin.

b . Statistical difference from control; N.S. = no significant difference (p>0.1).

The effects of the enkephalins on immune functions have been

studied more extensively than beta-endorphin. It is becoming quite

clear that the enkephalins have the ability to modulate a number of

immune functions. Studies in our laboratory have shown that daily

injection of mice for 1 week with either methionine- or leucineenkephalin results in significant changes in lymphoid organs

(table 1). This treatment resulted in significant increases in

thymus size and significant decreases in spleen size. Further

303
studies are required to completely explain the mechanism(s) of

these lymphoid organ weight changes.

The finding that lymphocytes possess receptors for enkephalins

(Wybran et al. 1979) prompted us to investigate the potential

effects of the enkephalins on various immune functions. Wybran

et al. (1979) reported that lymphocytes not only possess receptors

for enkephalins but also were influenced by enkephalins. Their

studies showed that peripheral blood lymphocytes from normal human

volunteers were enhanced in their ability to form rosettes with

sheep red blood cells (SRBC) following treatment with methionineenkephalin. This enhancement could be inhibited by naloxone.

Studies in our laboratory confirmed these findings and were expanded to include a population of immunosuppressed individuals (Miller

et al. 1983). Peripheral blood lymphocytes from a group of lymphoma patients were used to investigate the effects of methionine- and

leucine-enkephalin on the ability of lymphocytes from immunosuppressed individuals to form rosettes with SRBCs. It was found that

both enkephalins significantly enhance the ability of these cells

to form active T cell rosettes (table 2). Methionine-enkephalin

was active over a wide range of concentrations (1O-2 to 10-14 mg/ml)

while leucine-enkephalin was only active at one low concentration

(10-14 mg/ml).

TABLE 2. Effect of methionine- and leucine-enkephalin

on the percentages of active T-cell rosettes formed

by peripheral blood lymphocytes from lymphoma patients

Enkephalin

concentration

(mg/ml)

Methionine- Leucineenkephalin enkephalin

10-2 27.6±5.3 32.0±4.1

10-6 46.6±4.2* 41.1±5.6

10-10 46.8±3.0* 40.3±5.7

10-14 48.5±5.6* 52.7±2.8*

*

= significant difference from untreated samples, p<0.05.

Clin. Immun. Immunopath. 26, 446-51, Copyright (1983).

Academic Press

In order to gain some insight into the possible effect of enkephalin treatment on host defense mechanisms the following study was

undertaken (Plotnikoff and Miller 1983). BDF1 mice were inoculated

intraperitoneally with either 102 or 104 L1210 tumor cells. Following tumor challenge the animals were injected daily with saline

vehicle, methionine-enkephalin or leucine-enkephalin. The animals

were observed daily following tumor challenge and deaths recorded

304
on the day of occurrence. Table 3 illustrates results obtained in

mice challenged with 104 tumor cells and treated with 30 mg/kg

daily of either enkephalin. Methionine-enkephalin provided significant protection from tumor challenge. Similar results were obtained in mice challenged with 102

 tumor cells and treated with

10 mg/kg daily leucine-enkephalin.

Day of

study

13

14b

15

16

17

18

19

20

21

TABLE 3. Effect of enkephalins on

survival of mice challenged

with L1210 tumor cells

Treatment groupa

Non-treated Methionine- Leucineenkephalin enkephalin

7/25c

5/30 3/30

10/25 7/30 5/30

16/25 8/30 12/30

20/25 12/30 17/30

21/25 15/30 22/30

21/25 18/30 22/30

21/25 l8/30 26/30

22/25 19/30 26/30

22/25 19/30* 26/30

a. BDF1 mice were inoculated with 104 L1210 tumor cells ip.

Following tumor challenge the animals were injected daily

with saline vehicle, 30 mg/kg methionine-enkephalin or

30 mg/kg leucine-enkephalin. Deaths were recorded on the

day of occurrence.

b . The approximate median time to death is underlined for

each group.

c. The data is reported as the number of dead over the total

* number of animals in the group.

Indicates a significant difference (p<0.05) between

treated and nontreated animals.

Int. J. Immunopharm., 5, 5, 437-441, 1983. Copyright (1983).

Pergamon Press Ltd.

The effect of enkephalins on in vitro lymphocyte proliferative

responses has been studied in our laboratory and in the laboratory

of Gilman et al. In our laboratory, using mouse splenic lymphocytes and varying dilutions of either methionine-enkephalin or

305
leucine-enkephalin, the response to varying dilutions of PHA was

investigated. Both enkephalins were found to significantly enhance

the blastogenic response of lymphocytes to PHA (Plotnikoff and

Miller 1983). Results obtained with PHA at a dilution of 1:1OO are

illustrated in table 4. Similar results were obtained with other

dilutions of PHA. Methionine-enkephalin tended to enhance the

response over a broader range of dilutions than did leucine-enkephalin, while the leucine-enkephalin was active at lower concentrations than the methionine-enkephalin. These results are in

contrast to those obtained by Gilman et al. (1982) who reported no

stimulation of the blastogenic response of rat splenic lymphocytes

to stimulation with Con A following treatment of the lymphocytes

with enkephalin.

TABLE 4. Effect of enkephalins

on lymphocyte blastogenesisa

Enkephalin Methionine- Leucineconcentration enkephalin enkephalin

(mg/ml)

0 1458±90b

1458±90

1 4393±664* 1114±310

10-1 5258±990* 1516±207

10-2 3144±500* 165O±180

10-4 2123±228* 1918±293

10- 6 2357±155* 2425±323*

10-8 1884±188* 2759±387*

10-10 1538±170 2065±360*

a . Murine splenic lymphocytes were treated in vitro

with various concentrations of either methionine- or

leucine-enkephalin and then assayed for lymphocyte

transformation in response to PHA stimulation.

b. Stimulation was measured by incorporation of 3

Hthymidine into acid insoluble material. The data is

* presented as mean cpm± SE.

Indicates significant enhancement of transformation

in enkephalin treated cells as compared to nontreated cells (p<0.05).

Int. J. Immunopharm. 5, 5, 437-441, 1983. Copyright

(1983). Pergamon Press Ltd.

Johnson et al. (1982) have studied the effect of the enkephalins on

the in vitro antibody response. Murine splenic lymphocytes were

305
cultured in the presence of sheep red blood cells and varying

dilutions of either methionine- or leucine-enkephalin. After 5

days of culture the number of specific antibody-forming cells was

enumerated in each culture. Both enkephalins were found to cause

suppression of the antibody response.

Recently studies have been undertaken in our laboratory to investigate the effects of the enkephalins on natural killer cell activity- Initially isolated peripheral blood lymphocytes from normal

human volunteers were utilized in these studies. These cells were

incubated for 1 hour with varying concentrations of either methionineor leucine-enkephalin. Following incubation the cells were washed

and then used in NK cell assays. Table 5 illustrates data obtained

in these studies. Both enkephalins have the ability to significantly enhance natural killer activity over a range of concentrations. Leucine-enkephalin appears to be more active in this

enhancement than is methionine-enkephalin. We are presently

performing similar studies with peripheral blood lymphocytes from a

variety of cancer patients. Our results to date indicated the

enkephalins are capable of enhancing NK activity in cancer patients

as well as normal volunteers.

TABLE 5. Effect of enkephalins on natural

killer cell activitya

Mean difference of enkephalintreated cells vs. nontreated cells

Enkephalin

concentrationb

(mg/ml)

Methionine- Leucineenkephalin enkephalin

10-4 1.41±3.19(NS)C10.00±4.75(p<0.05)

10-6 6.41±3.41(p<O.O5) 11.29±6.06(p<0.05)

10- 8 5.35±3.74(p<0.05) 12.44±5.99(p<O.O5)

10-10 6.56±4.53(p<0.05) 11.20±6.16(p<0.05)

10-14 2.55±3.19(NS) 8.72±6.26(p<O.O5)

a. NK assays were performed at an effector to target

cell ratio of 11:1.

b . Effector cells were incubated in the presence of the

indicated concentration of enkephalin for 1 hour,

washed 3 times and used in NK assays.

c. Significance of difference from control value. NS

indicates not significant.

CONCLUSIONS

While there are still many questions to be answered, it is now

clear that opiates and the natural opioid neuropeptides can influence immune function. We feel that part of the normal function of

337
the enkephalins and endorphins is to participate in immune regulatory pathways. Many more studies are required to fully define the

role that these neuropeptides play in immune regulations.

Studies with lymphocytes from normal human volunteers as well as

cancer patients suggest that methionine-enkephalin is more active

than leucine-enkephalin in enhancing active T-cell rosettes (Miller

et al. 1983). In addition, the two enkephalins exhibit potency

differences in mouse splenic lymphocyte blastogenesis induced by

PHA. Leucine-enkephalin was active at low concentrations and

inactive at higher levels (Plotnikoff and Miller 1983).

These apparent differences in activity or potency of the enkephalins may indeed represent major differences in affinities for

cellular receptors. Thus, Vaught and Takemori (1979) reported that

leucine-enkephalin, but not methionine-enkephalin, potentiated

effects of morphine. Different subpopulations of receptors for

morphine, enkephalins and endorphins have now been established

(Bloom 1983). These various receptors are only partially antagonized by naloxone. The difference in receptors for morphine, which

has been reported to be immunosuppressive, and the enkephalins

supports the concept that natural enkephalins may have separate

and/or complementary functions in the immune system.

We are proposing that the enkephalins play a role in a central

nervous system-endocrine-immune interrelationship in which dysfunctions in the immune system may be related to chronic stress and be

accompanied by perturbations in enkephalin levels in the adrenals,

the primary source of enkephalins in peripheral circulation (Viveros

et al. 1980; Hanbauer et al. 1982; Dumont et al. 1983). Large

amounts of enkephalins, catecholamines and steroids are secreted

simultaneously from the adrenals during stress (Amin et al. 1980).

It is well known that the central nervous system and behavior can

influence immune function (Stein et al. 1976; Ader 1981; Brooks

et al. 1982; Renoux et al. 1983). Recently behavioral states have

been shown to influence cerebral spinal fluid levels of endorphins

(Pickar et al. 1981), and exercise has been shown to increase

plasma levels of endorphins (Carr et al. 1981; Farrell et al.

1982). Exercise has also been shown to enhance human NK activity

(Targan et al. 1981). Due to these facts and the effects of

enkephalins referred to above, we feel that the enkephalins,

influenced by a number of factors, may play a central role in

natural immunomodulation.

REFERENCES

Ader, R., ed. Psychoneuroimmunology. New York: Academic Press,

1981.

Amin, S., Brown, Z.W., and Amit, Z. The role of endorphins in

stress: Evidence and speculation. Neurosci Biobehav Rev 4:77-

86, 1980.

Bloom, F.E. The endorphins: A growing family of pharmacologically

pertinent peptides. Ann Rev Pharmacol Toxicol 23:151-170, 1983.

Brooks, W.H., Cross, R.J., Roszman, T.L., and Markesbery, W.R.

Neuroimmunomodulation: Neural anatomical basis for impairment

and facilitation. Ann Neurol 12(1):56-61, 1982.

308
Brown, S.M., Stimmel, B., Taub, R.N., Kochwa, S., and Rosenfield,

R.E. Immunologic dysfunction in heroin addicts. Arch Intern Med

134(6):1001-1006, 1974.

Carr, D.B., Bullen, B.A., Skrinar, G.S., Arnold, M.A., Rosenblatt,

M ., Beitins, I.Z., Martin, J.B., and McArthur, J.W. Physical

conditioning facilitates the exercise-induced secretion of

beta-endorphin and beta-lipotropin in women. N Engl J Med

305(10):560-563, 1981.

Dumont, M., Day, R., and Lemaire, S. Distinct distribution of

immunoreactive dynorphin and leucine enkephalin in various

populations of isolated adrenal cromaffin cells. Life Sci

32(3):287-294, 1983.

Farrell, P.A., Gates, W.K., Maksud, M.G., and Morgan, W.P. Increases in plasma ß-endorphin/ß-lipotropin immunoreactivity after

treadmill running in humans. J Appl Physiol 52(5):1245-1249,

1982.

Gilman, S.C., Schwartz, J.M., Milner, R.J., Bloom, F.E., and

Feldman, J.D. ß-endorphin enhances lymphocyte proliferative

responses. Proc Natl Acad Sci 79(13):4226-4230, 1982.

Gungor, M., Genc, E., Sagduyu, H., Eroglu, L., and Koyuncuoglu, H.

Effect of chronic administration of morphine on primary immune

response in mice. Experientia 36(11):1309-1310, 1980.

Hanbauer, I., Kelly, G.D., Saiani, L., and Yang, H.Y.T. [met5]-

enkephalin-like peptides of the adrenal medulla: Release by

nerve stimulation and functional implications. Peptides 3:

469-473, 1982.

Hazum, E., Chang, K.J., and Cuatrecasas, P. Specific nonopiate

receptors for beta-endorphin. Science 205(4410):1033-1035, 1979.

Hung, C.Y., Lefkowitz, S.S., and Geber, W.F. Interferon inhibition

by narcotic analgesics. Proc Soc exp Biol Med 142:106-111, 1973.

Johnson, H.M., Smith, E.M., Torres, B.A., and Blalock, J.E.

Regulation of the in vitro antibody response by neuroendocrine

hormones. Proc Natl Acad Sci 79(13):4171-4174, 1982.

Lefkowitz, S.S., and Yang, C. Effects of highly abused drugs on

antibody production (abstract). J Reticuloendothel Soc 16:25,

1974.

Mathews, P.M., Froelich, C.J., Sibbitt, W.L., Jr., and Bankhurst,

A.D. Enhancement of natural cytotoxicity by ß-endorphin. J

Immunol 130(4):1658-1662, 1983.

McCain, H.W., Lamster, I.B., Bozzone, J.M., and Gabic, J.T.

ß-endorphin modulates human immune activity via non-opiate

receptor mechanisms. Life Sci 31(15):1619-1624, 1982.

McDonough, R.J., Madden, J.J., Falek, A., Shafer, D.A., Pline, M.,

Gordon, D., Bokos, P., Kuehnle, J.C., and Mendelson, J. Alteration of T and null lymphocyte frequencies in the peripheral blood

of human opiate addicts: In vivo evidence for opiate receptor

sites on T lymphocytes. J Immunol 125(6):2539-2543, 1980.

Mehrishi, J.N., and Mills, T.H. Opiate receptors on lymphocytes

and platelets in man. Clin Immunol Immunopathol 27(2):240-249,

1983.

Miller, G.C., Murgo, A.J., and Plotnikoff, N.P. Enkephalins -

enhancement of active T-cell rosettes from lymphoma patients.

Clin Immunol Immunopathol 26(3):446-451, 1983.

309
Nickerson, D.S., Williams, R.C., Boxmeyer, M., and Quie, P.G.

Increased opsonic capacity of serum in chronic heroin addiction.

Ann Intern Med 72:671-677, 1970.

Pickar, D., Naber, D., Post, R.M., van Kammen, D.P., Bellenger, J.,

Kalin, N., and Bunney, W.E., Jr. Measurement of endorphins in

CFS. Relationship to psychiatric diagnosis. Mod Probl Pharmacopsychiatry 17:246-262, 1981.

Plotnikoff, N.P., and Miller, G.C. Enkephalins as immunomodulators.

Int J Immunopharm, 5, 5, 437-441, 1983.

Renoux, G., Biziere, K., Renoux, M., and Guillaumin, J.M. The

production of T-cell-inducing factors in mice is controlled by

the brain neocortex. Scand J Immunol 17:45-50, 1983.

Sheagren, J.N., and Tuazon, C.U. Immunological aspects. In:

Pradhan, S.N., Dutta, S.N., eds. Drug Abuse. Clinical and Basic

Aspects. St. Louis: C.V. Mosby Co., 1977. pp. 321-331.

Stanley, T.H., Hill, G.E., Portas, M.R., Hogan, N.A., and Hill,

H.R. Neutrophil chemotaxis during and after general anesthesia

and operation. Anest Anal 55(5):668-672, 1976.

Stein, M., Schiavi R.C., Camerino, M. Influence of brain and

behavior on the immune system. Science 191(4226):435-440, 1976.

Targan, S., Britvan, L., and Dorey, F. Activation of human NKCC by

moderate exercise: increased frequency of NK cells with enhanced

capability of effector-target lytic interactions. Clin Exp

Immunol 45(2):352-360, 1981.

Vaught, J.L., and Takemori, A.E. A further characterization of the

differential effects of leucine enkephalin, methionine enkephalin

and their analogs on morphine-induced analgesia. J Pharmacol Exp

Therapeut 211(2):280-283, 1979.

Viveros, O.H., Diliberto, E.J., Jr., Hazum, E., and Chang, K.J.

Enkephalins as possible adrenomedullary hormones: Storage,

secretion, and regulation of synthesis. In: Costa, E., and

Trabucchi, M., eds. Neural Peptides and Neuronal Communication.

New York, Raven Press, 1980. pp. 191-204.

Wetli, C.V., Noto, T.A., and Fernandez, C.A. Immunologic abnormalities in heroin addiction. South Med J 67:193-197, 1974.

Wybran, J., Appleboom, T., Famaey, J.P., and Govaerts, A. Suggestive evidence for receptors for morphine and methionine-enkephalin

on normal blood T lymphocytes. J Immunol 123(3): 1068-1070,

1979.

Zagon, I.S., and McLaughlin, P.J. Heroin prolongs survival time

and retards tumor growth in mice with neuroblastoma. Brain Res

Bull 7(1):25-32, 1981a.

Zagon, I.S., and McLaughlin, P.J. Naloxone prolongs the survival

time of mice treated with neuroblastoma. Life Sci 28(10):1095-

1102, 1981b.

Zagon, I.S. and McLaughlin, P.J. Naltrexone modulates tumor

response in mice with neuroblastoma. Science 221(4611):671-673,

1983.

310
AUTHORS

Robert E. Faith, D.V.M., Ph.D.

Director, Biomedical Research Center

Oral Roberts University

7777 South Lewis

Tulsa, Oklahoma 74171

Nicholas P. Plotnikoff, Ph.D.

Department of Pharmacology

School of Medicine

Oral Roberts University

7777 South Lewis

Tulsa, Oklahoma 74171

Anthony J. Murgo, M.D.

Section of Hematology/Oncology

Department of Medicine

University of West Virginia

Morgantown, West Virginia 26506

311
Changes in CNS Sensitivity to

Cannabinoids with Repeated

Treatment: Tolerance and

Auxoesthesia

R. Karler, L.D. Calder, and S.A. Turkanis

INTRODUCTION

Repeated or chronic treatment with drugs can result in pharmacodynamic changes, which classically manifest themselves in decreases

in drug sensitivity or tolerance. There are, however, reports

that, in some instances, repeated treatment results not in tolerance but in the quantitatively opposite effect, that is, an increase in drug sensitivity or auxoesthesia*. Although this seemingly paradoxical response to repeated treatment was first described over fifty years ago for cocaine (Tatum and Seevers 1929),

the phenomenon was not investigated until it was recently rediscovered (Post and Kopanda 1975). Now it is of particular significance to drug-abuse research because, in addition to cocaine

other drugs in repeated doses have been found to cause auxoesthesia; these include amphetamine (Segal and Mandell 1974), PCP

(Smith et al. 1981), alcohol (Pinel and Van Oot 1976) and morphine

(Shuster et al. 1975). This kind of change in pharmacodynamics

presents an entirely new dimension to our understanding of the

chronic effects of these drugs. With respect to the cannabinoids,

the reported effects of repeated treatment range from tolerance to

auxoesthesia (Pried 1977). Although what determines the direction

of the change in drug responsiveness has not been elucidated, it

*Any descriptive consideration of a change in an effect which is

quantitatively the opposite of tolerance suffers at the outset

from the lack of a word to refer to the phenomenon. "Reverse tolerance" has been used, but it is at once inadequate and misleading. Other words that are sometimes used include sensitization,

hypersensitivity and supersensitivity. None of these is appropriate because each already has a restricted meaning in pharmacology: Sensitization and hypersensitivity classically refer to allergic phenomena; while supersensitivity is the enhanced responsiveness of an effector system following denervation. In the absence of an appropriate existing word, we offer "auxoesthesia,"

which is both pristine and novel, as well as etymlogically apposite. We are indebted to Dr. B.Z. Karler for coining the term.

312
is clear that any interpretation of the available data is complicated by species differences, dosage and treatment differences,

and, possibly most important, differences in drug effects, which

include functional as well as behavioral effects. The data described below were derived from experiments designed to minimize

sane of these variables, specifically by using only one species

treated daily with a constant dose of drug. Under these conditions, several different effects on CNS excitability were measured

simultaneously in order to compare the quantitative character of

changes in drug responsiveness with repeated treatment.

REPEATED CANNABINOID TREATMENT AND CHANGES IN PHARMACODYNAMICS IN

CONVULSIVE THRESHOLD TESTS

In figures 1, 2 and 3 are data obtained from mice that were

treated daily for 22 days with an anticonvulsant dose 50

determined in a maximal electroshock test (Karler et al. 1974).

FIGURE 1. Repeated THC treatment: ESTs and brain- and plasma-drug

concentrations. In each test 60 mice given 100 mg/kg THC (i.p.),

another 60, vehicle; median effective currents or voltage determined at peak drug effect time. l = differences in EST between

drug and vehicle groups. --- = period of daily treatments.

--- = period of withdrawal. Brain and plasma values come from

6-8 mice (Karler et al. 1982). © 1982, Springer-Verlag.

The activities of the drugs were assessed in three different electroconvulsive threshold tests at weekly intervals. In this study,

the effects of delta-9-tetrahydrocannabinol (THC) were compared

with those of cannabidiol (CBD), another major constituent of

313
marijuana. Both drugs have previously been extensively investigated for their acute anticonvulsant properties (Karler et al.

1974; Turkanis et al. 1974). Phenytoin (PHT) was included as a

standard anticonvulsant drug in these tests. The results from the

THC study are shown in figure 1. As can be seen, THC acutely (day

1) raises both the 6-Hz (6-Hz-EST) and maximal electroshock

thresholds (MEST); but the identical dose decreases the threshold

for convulsions in the 60-Hz-electroshock threshold (60-Hz-EST)

test. With repeated treatment, however, tolerance developed to

all three effects. Similar results were obtained for another proconvulsant effect not shown in figure 1; that is, THC acutely can

lower the minimal seizure threshold to pentylenetetrazol (PTZ)

and, here again, tolerance develops to that effect. In summary,

in two seizure tests in which THC is anticonvulsant and in two

tests in which it is proconvulsant, tolerance develops. That the

tolerance is cellular in nature rather than metabolic is evidenced

by the brain concentrations of THC which remained unchanged at the

test times throughout the experiment.

FIGURE 2. Repeated CBD treatment (120 mg/kg): ESTs and brain- and

plasma-drug concentrations. On day 1 only 6-HZ-EST and MST vehicle-control values were significantly different from CBD-treated

groups. See figure 1 for details. © 1982, Springer-Verlag.

The data shown in figure 2 are the results of an experimental

design identical to that used to generate the data in figure 1,

except that CBD rather than THC was administered daily. There are

some striking differences in the results. First, acutely (on day

1), CBD has no apparent activity on the 60-Hz-EST, although, like

THC, it raises the thresholds on both the 6-Hz-EST and the MEST.

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In the 6-HZ-EST, however, daily treatment resulted in a progressive increase rather than the decrease in activity found in the

THC study. In contrast, tolerance developed on the MEST as it did

for THC. Although there was no CBD effect initially on the 60-HzEST, after one week, activity was evident, and thereafter appeared

to increase progressively. Again, as in the case of THC, the

changes in the activity of CBD were independent of any changes in

brain-drug concentration, which remained relatively constant at

the various test times.

The data from the CBD experiment clearly illustrate that the cannabinoids have the potential of producing auxoesthesia, as well as

tolerance, with repeated treatment. The development of tolerance

in one of the tests of CNS excitability demonstrates that under

identical conditions quantitatively opposite changes in drug sensitivity can occur simultaneously. These data emphasize the

necessity of identifying the specific effect when repeated treatment results in a pharmacodynamic change. A priori, there is no

reason to assume, for example, that, if tolerance develops to one

effect, tolerance will generalize to other effects (except in the

case of a metabolic type of tolerance); therefore, to state simply

that tolerance develops to a drug may be an inappropriate description of the consequences of repeated treatment on some effects.

The CBD results also raise a point in relation to the general

characteristics of effects to which increased vs. decreased responsiveness develops with repeated treatment. Tolerance has

generally been associated with depressant effects, such as the

depressant effects of the opiates, the barbiturates and alcohol.

A simplistic explanation of tolerance to a CNS depressant effect

is that the nervous system compensates for the effect, thereby

diminishing drug potency. The withdrawal hyperexcitation associated with CNS depressant drugs can be construed to support the

concept that these agents elicit compensatory excitatory responses. On the other hand, auxoesthesia has generally been observed with excitatory effects; for example, the production of

seizures by subconvulsant doses of convulsant drugs such as PTZ

(Mason and Cooper 1972), and the increase in motor stimulant

activity of morphine, cocaine and amphetamine (Shuster et al.

1975; Shuster et al. 1977; Segal and Mandell 1974). Because many

drugs, like the cannabinoids, produce both depressant and excitatory effects, the influence of repeated treatment on a specific

effect may depend upon whether the effect is excitatory or depressant. With respect to the cannabinoids, Fried (1977) surveyed the

literature and concluded that tolerance developed to THC's depressant effects and an increase in sensitivity developed to its excitatory effects. The threshold data described above for THC, however, do not fit this pattern, because tolerance developed to both

anticonvulsant and proconvulsant effects. Furthermore, the

hypothesis is not supported by the CBD data which illustrate that

increases in drug sensitivity can develop to anticonvulsant or

depressant effects. The notion that chronic treatment results in

apposite quantitative responses to depressant and excitatory effects does not appear to be valid, at least not for the

cannabinoids.

315
FIGURE 3. Repeated PHT treatment (7 mg/kg): ESTs and brain- and

plasma-drug concentrations. On day 1 only 6-HZ-EST and MEST vehicle-control values were significantly different from PHT-treated

groups. See figure 1 for details. © 1982, Springer-Verlag.

The data shown in figure 3 represent the results obtained for PHT

under conditions identical to those in the experiments of figures

1 and 2. The PHT results differ from those of THC and CBD in that

there is no change in the drug's activity in 6-HZ-EST. Like CBD,

there is an increase in activity in the 60-Hz-EST and, like THC

and CBD, tolerance developed in the MEST test. As in the THC and

CBD experiments, the PHT results were independent of any change in

brain-drug concentration.

The PHT data illustrate that all three possibilities resulting

from repeated treatment can occur simultaneously: that is, tolerance, no change in activity and auxoesthesia. Here again, the results emphasize the hazard of generalizing from one chronic effect

of a drug to other chronic effects. In addition, the enhanced

anticonvulsant efficacy of PHT in one test reinforces the CBD observation that an increase in drug activity can occur in response

to a depressant effect, or that such reactions are not necessarily

the domain of excitatory phenomena.

The PHT data also raise the question: How common is the development of auxoesthesia with chronic drug treatment? Although PHT

has been investigated extensively for about fifty years, the

failure of others to observe this phenomenon earlier is

316
noteworthy. This failure probably relates to the almost complete

absence of chronic studies with PHT, even though clinically the

drug is used chronically. Studies of the influence of chronic

treatment on the pharmacodynamics of many drugs are lacking, and

the PHT data suggest that enhanced drug responsiveness with repeated treatment maybe a more widespread phenomenon than hitherto

recognized.

INTERACTION OF THC ON KINDLING REACTIONS

We have approached the study of auxoesthesia with the use of kindling as a potential model for this type of reaction. The term

kindling was first introduced by Goddard et al. (1969), who observed that repeated electrical stimulation of the brain with a

subconvulsant stimulus could ultimately elicit convulsions. In

other words, the sensitivity of the nervous system to the stimulus

increased with repeated application of the stimulus. Subsequently,

chemical kindling to convulsions was demonstrated with the daily

administration of subconvulsant doses of such drugs as PTZ (Mason

and Cooper 1972). Conceivably, the development of increased responsivity to a drug or stimulus could be affected by drug interactions. Starting with this hypothesis, we tested the influence of

THC treatment on electrical and chemical kindling to convulsions.

0 2 4 6 8 1 0 12 14 1 6

DAYS

FIGURE 4. THC effects on electrical kindling to minimal convulsions. 3 Groups of 25 mice were given THC or vehicle (i.p.) every

other day for 13 days; at peak brain concentration (2 hr), corneal electroshock-kindling stimulus (2 sec, 3.5 mA, 60 Hz) was

given. After day 13, drug withdrawn; on day 16, electroshock only.

317
The results in figure 4 represent the interaction of THC with

electrical kindling. Here we see that high doses enhance the

development of convulsions, whereas low doses retard the phenomenon. The persistence of the drug-caused enhancement of kindling

after cannabinoid withdrawal suggests that the interaction is a

true effect on the kindling process as opposed to a readily reversible effect on the convulsive activity of the electrical stimulus. The complexity of the THC interaction with electrical stimulation is illustrated by the opposite low- and high-dose effects

of the drug on the development of seizures.

FIGURE 5. THC effects on PTZ kindling to minimal convulsions.

3 Groups of 25 mice were given THC or vehicle (i.p.) every 3rd

day for 16 days; at peak brain concentration (2 hr), PTZ kindling stimulus (40 mg/kg, s.c.) was given. After day 16, drug

withdrawn; on day 23, kindling stimulus only.

The ability of THC to enhance kindling to chemical stimuli is

shown in figures 5 and 6. Figure 5 shows that THC augments the

development of convulsions to PTZ and, like the results fran the

electrical experiment, the enhancement persisted after cannabinoid

withdrawal. Figure 6 depicts the influence of THC on kindling by

picrotoxin. In this instance, the interaction appears to be

fundamentally different from that observed in the electrical and

PTZ studies because, after cannabinoid withdrawal, there was no

persistence of the convulsions associated with the THC treatment.

The rapidly reversible nature of the interaction argues against a

true effect on kindling, which is known to be be a long-lived phenanenon (Racine 1978; Sangdee et al. 1982).

318
The diversity of results from the study of THC's interactions with

the various kindling stimuli illustrates that the drug-caused

increase in sensitivity to convulsions appears to be a relatively

selective, rather than a generalized, effect. This conclusion is

based on the failure of THC to promote kindling to picrotoxin.

FIGURE 6. THC effects on picrotoxin kindling to minimal convulsions. 3 Groups of 25 mice were given THC or vehicle (i.p.) once

daily, 5 days/week for 2 weeks; at peak brain concentration

(2 hr), picrotoxin-kindling stimulus (1 mg/kg, s.c.) was given.

After day 14, drug withdrawn; on day 16, kindling stimulus only.

Supporting evidence for the selectivity in the drug interaction

was observed in other experiments in which we demonstrated that

subconvulsant doses of PTZ do not affect the development of convulsions to an electrical stimulus (unpublished data). This

failure of PTZ was surprising because it is a convulsant drug,

which suggests that the mechanism of PTZ kindling is different

fran that of electrical kindling. This conclusion is further

strengthened by our observation that animals electrically kindled

to minimal convulsions do not show cross-sensitivity to PTZinduced minimal convulsions (unpublished data).

If kindling represents a valid model for the study of auxoesthetic

reactions in general, then the present data suggest that THC may

augment the development of the phenomenon to some drugs but not to

others. The ability of THC to enhance these reactions represents

a toxic potential of the drug that has not yet been investigated.

319
CONCLUSIONS

Classically, repeated exposure to drugs has been viewed pharmacodynamically in terms of only two alternatives, either tolerance or

no change in sensitivity. The data presented above, however,

serve to reinforce the evidence that suggests that the consequences of repeated drug exposure may also include auxoesthesia

or an increase in sensitivity. The frequency of the occurrence of

auxoesthesia in pharmacology is not known. To date many such reported phenomena have involved drugs of abuse, such as amphetamine

(Segal and Mandell 1974), cocaine (Shuster et al. 1977), morphine

(Shuster et al. 1975), PCP (Smith et al. 1981) and alcohol (Pinel

and Van Oot 1976). In general, the observed effect is an increase

in locomotor activity with repeated drug exposure; the extent to

which other effects are involved is also unknown. In addition,

the significance of auxoesthesia to the pharmacology of drugs of

abuse is not clear, but some ideas have been proposed (Post et al.

1976). Our ignorance, in part, reflects the paucity of information available on the phenomenon; nevertheless, because auxoesthesia has been described for a number of drugs of abuse, it should

be considered in our attempts to understand the pharmacology of

these substances.

There are many questions about auxoesthesia that should be addressed. To wit: What is the relationship of this response to

dose and to the frequency and duration of exposure? How

persistent and how generalized are these adaptations of the CNS?

Can concomitant drug exposure influence the development of auxoesthesia? Is there a relationship between the development of tolerance and auxoesthesia? These are but a few of the questions that

need to be answered in order to appreciate the significance of

this phenomenon.

The data here presented on the cannabinoids represent only a

beginninq of the description of the characteristics of auxoesthesia as related to these drugs. Specifically, within the context

of the limited number of drugs and effects observed, both tolerance and auxoesthesia can develop and these can exist simultaneously. The data indicate that to describe properly the influence

of chronic treatment on the pharmacodynamics of the cannabinoids,

both the specific drug and the effect must be identified. The

changes, for example, do not generalize either to all cannabinoids

or to all effects. Finally, THC's enhancement of kindling reactions raises the possibility that the use of marijuana may

adversely interact with some environmental or pathological or

pharmacological factors to result in the very long-lasting changes

in CNS excitability characteristic of kindling (Racine 1978;

Sangdee et al. 1982). To illustrate, the development of some

types of human epilepsy, especially the posttraumatic type, appear

to be kindling-like, which raises the possibility that marijuana

use in such individuals may increase their liability for epilepsy.

The THC potential to interact with other drugs is supported by the

observation that the cannabinoid enhances PTZ kindling; in addition, others have reported that THC can also augment the motorstimulant activity of amphetamine (Garriott et al. 1967) and of

320
morphine (Ayhan et al. 1979). Such drug interactions deserve

further investigation, especially among the drugs of abuse because

many of them have been reported to produce auxoesthesia and many

of them are used concomitantly.

REFERENCES

Ayhan, I.H., Kaymakcalan, S., and Tulunay, F.C. Interaction

between delta-9-tetrahydrocannabinol and morphine and the motor

activity of mice. Psychopharmacol, 63:169-172, 1979.

Fried, P.A. Behavioral and electroencephalographic correlates of

the chronic use of marijuana--A review. Behav Biol, 21:163-

196, 1977.

Garriott, J.C., King, L.J., Fomey, R.B., and Hughes, F.W. Effects of some tetrahydrocannabinols on hexobarbital sleeping

time and amphetamine induced hyperactivity in mice. Life Sci;

6:2119-2128, 1967.

Goddard, G.V., McIntyre, D.C., and Leech, C.K. A permanent change

in brain function resulting from daily electrical stimulation.

Exp Neurol, 25:295-330, 1969.

Karler, R., Cely, W., and Turkanis, S.A. Anticonvulsant properties of delta-9-tetrahydrocannabinol and other cannabinoids.

Life Sci, 15:931-947, 1974.

Karler, R., Borys, H.K., and Turkanis, S.A. Influence of 22-day

treatment on the anticonvulsant properties of cannabinoids.

Naunyn-Schmiedebergs Arch Pharmacol, 320:105-109, 1982.

Mason, C.R., and Cooper, R.M. A permanent change in convulsive

threshold in normal and brain-damaged rats with repeated small

doses of pentylenetetrazol. Epilepsia, 13:663-674, 1972.

Pinel, J.P.J., and Van Oot, P.H. Generality of the kindling phenomenon: Some clinical implications. In: Wada, J.A., ed.

Kindling. New York: Raven-Press, 1976. pp. 151-171

Post, R.M.. and Kopanda. R.T. Cocaine, kindling, and reverse tolerance. Lancet, 1:409-410, 1975.

Post, R.M., Kopanda, R.T., and Black, K.E. Progressive effects of

cocaine on behavior and central amine metabolism in rhesus

monkeys: Relationship to kindling and psychosis. Biol

Psychiatry, 11:403-419, 1976.

Racine, R. Kindling: The first decade. Neurosurgery, 3:234-252,

1978.

Sangdee, P., Turkanis, S.A., and Karler, R. Kindling-like effect

induced by repeated corneal electroshock in mice. Epilepsia,

23:471-479, 1982.

Segal, D.S., and Mandell, A.J. Long-term administration of d-amphetamine: Progressive augmentation of motor activity and

stereotype. Pharmacol Biochem Behav, 2:249-255, 1974.

Shuster, L., Webster, G.W., and Yu, G. Increased running response

to morphine in morphine-pretreated mice. J Pharmacol Exp Ther,

192:64-72, 1975.

Shuster, L., Yu, G., and Bates, A. Sensitization to cocaine

stimulation in mice. Psychopharmacology, 52:185-190, 1977.

321
Smith, R.C., Leelavathi, D.E., Hsu, L., Ho, B.T., Tansey, W.,

Taylor, D., and Biggs, C. Acute versus chronic administration

of phencyclidine: Effects on behavior and brain biochemistry.

In: Domino, E.F., ed. PCP (Phencyclidine) Historical and Current Perspective. Ann Arbor: NPP Books, 1981. pp. 243-291.

Tatum, A.L., and Seevers, M.H. Experimental cocaine addiction. J

Pharmacol Exp Ther, 36:401-410, 1929.

Turkanis, S.A., Cely, W., Olsen, D.M., and Karler, R. Anticonvulsant properties of cannabidiol. Res Commun Chem Pathol

Pharmacol, 8:231-246, 1974.

ACKNOWLEDGEMENT

This research was supported by research grant DA-00346 from the

National Institute on Drug Abuse. Dr. Monique C. Braude, NIDA

Division of Preclinical Research, served as Project Officer.

AUTHORS

Ralph Karler, Ph.D.

Larry D. Calder, B.S.

Stuart A. Turkanis, Ph.D.

Department of Pharmacology

University of Utah School of Medicine

Salt Lake City, UT 84132

322
Catecholamine-Independent

Behavioral and Neurochemical

Effects of Cocaine in Rats

Karen Gale

Classically, the mechanism of action of cocaine has been attributed to the augmentation of catecholamine neurotransmission.

Many of the acute and chronic effects of cocaine administration

on the CNS, and consequently on behavior, have been ascribed to

such an enhancement of norepinephrine- and/or dopamine-mediated

function. However, data has recently begun to accumulate indicating that cocaine may influence certain behaviors, both acutely and chronically, by mechanisms that do not involve stimulation of either dopaminergic or adrenergic receptors. The

present report will describe some actions of cocaine on behavior

and neurochemistry in rats that cannot be blocked or attenuated

by catecholaminergic receptor antagonists: (1) behavioral

sensitization produced by repeated administration of cocaine,

(2) changes in GABA-related neurochemical parameters in the

striatum of rats treated chronically with cocaine, and (3)

cocaine-induced conditioned taste aversions.

Behavioral Sensitization Following Chronic Cocaine Treatment

Repeated administration of cocaine in rats consistently has been

found to result in “sensitization” to the motor stimulant

act ions of this drug (Downs and Eddy 1932; Tatum and Seevers

1929; Post and Rose 1976; Stripling and Ellinwood 1976; Post et

al. 1981). Activation of dopaminergic transmission is an

essential feature of the stereotyped hyperactivity induced by

cocaine, since drugs that block dopamine receptors can prevent

the appearance of these behaviors in both naive as well as in

“sensitized” animals The experiments to be described here were .

designed to determine firstly, whether the blockade of dopamine

receptors could prevent the sensitization to cocaine, and

secondly, whether animals sensitized to cocaine would also show

sensitization to a directly-acting dopamine receptor agonist.

In our studies, cocaine was administered subcutaneously to 250

gram rats; each rat received 0.1 ml of a 400 mg/ml solution.

323
This route of administration results in a slow rate of absorption due to the vasoconstrictive action of cocaine. By 30 min

after injection, the cocaine-treated rats showed hyperactivity

and sympathomimetic activation; this increased in intensity to a

peak (stereotyped head movements and some gnawing) by 90 min.

The stereotypies were maintained for 8-10 hr after a single

injection. Injections were given daily for 10 days. After the

last injection, rats were tested for behavioral responses to

cocaine or apomorphine.

The chronic treatment groups were as follows:

(1) cocaine

(3) droperidol

(2) cocaine + droperidol (given simultaneously)

(4) saline

Droperidol was selected as the dopamine antagonist for this

study because its short half-life made it possible to match the

duration of action with the duration of the cocaine stimulation.

The treatment regimen involved giving droperidol (1 mg/kg i.p.)

twice a day, 3 hr apart. This resulted in noticeable catalepsy--even in those animals receiving cocaine concurrently; the

catalepsy lasted for approximately 3 hr after each droperidol

injection. In rats receiving cocaine and droperidol simultaneously, no stereotypy or hyperactivity of any kind was observed.

Three days following the last drug treatment, the rats were

tested for their behavioral responses to cocaine (20 mg/kg i.p.)

or apomorphine (.25 mg/kg s.c.). The scale used to rate the

stereotyped behavior is shown in the legend to table 1.

Table 1 shows the behavioral scores of the animals after the

challenge dose of cocaine, as a function of time following

inject ion. A marked difference between “controls” (rats that

received chronic injections of saline) and the “chronic cocaine”

group is evident: all rats in the “chronic cocaine” group

showed restricted stereotypies with gnawing during the first 30

min. Controls engaged in locomotor activity with some rearing

and sniffing. This difference reflects the phenomenon of

“sensitization” to the motor stimulant effects of the drug.

Although not shown in table 1, we found that animals showing

sensitization to cocaine also exhibited enhanced hyperactivity

in response to d-amphetamine.

The rats which had been receiving chronic droperidol + cocaine

simultaneously were similar to those receiving cocaine alone.

The behavior of these rats was clearly distinguishable from both

“control” and “chronic droperidol” groups.

Thus, despite the fact that concurrent treatment with droperidol

prevented the behavioral manifestations of cocaine administration, this treatment did not prevent the development of “sensitization” to a subsequent cocaine challenge. This indicates

that the acute psychomotor stimulant actions of cocaine and the

changes in drug sensitivity caused by its chronic administration

are separable phenomena.

324
TABLE 1

Stereotyped behavior induced by cocaine (20 mg/kg i.p.)

in rats treated chronically with cocaine and/or droperidol

Chronic Treatment Time After Cocaine (min)

10 2 0 3 0

control 2.0 2.0† 2.0

droperidol 2.0 3.0† 2.5

cocaine 4.6 5.5† 3.2

cocaine + droperidol 3.6 4.0 5.0†

Values represent behavioral ratings based on the

following scoring scale: 0 = little or no activity--

occasional grooming; 1 = intermittent rearing and

exploratory activity; 2 q continuous activity involving

locomotion, rearing and sniffing; 3 = repetitive

sniffing of cage walls or floors; 4 = repetitive head

movements and restricted sniffing of small areas of the

cage; 5 = continuous sniffing with discontinuous gnawing

or licking; 6 = gnawing with occasional sniffing;

7 = continuous gnawing; 8 q self-directed gnawing.

Each value represents the mean of 4 rats. The range of

scores for any given determination was not greater than

1 unit on either side of the mean; at peak effect, the

largest difference between any 2 rats was 1 scoring

unit. † Denotes peak effect.

The behavioral scores of the chronic treatment groups in

response to apomorphine are shown in table 2. The control rats

showed marked stereotypies in response to apomorphine. In contrast, the rats which had been chronically exposed to cocaine

showed only mild stereotyped responses which were of short duration. Rats in the chronic droperidol’ group were similar to

controls in their response to the apomorphine challenge, except

that many of them exhibited restricted gnawing behavior during

the peak effect. The groups of rats that had been chronically

treated with the combination of cocaine and droperidol were

similar to controls in their response to apomorphine.

Thus, it appears that exposure to chronic cocaine causes a

reduction in the subsequent responsiveness to the behavioral

stimulant actions of apomorphine. We have since replicated this

study using additional test doses of apomorphine (.17-.4 mg/kg)

and found that the dose-response relationship was shifted to the

right in animals which had received chronic cocaine. This

apparent tolerance to apomorphine was prevented by concurrent

droperidol treatment, suggesting that it may be mediated by

stimulation of dopamine receptors.

325
TABLE 2

Stereotyped behavior (scored as described in table 1)

induced by apomorphine (0.25 mg/kg s.c.) in rats treated

chronically with cocaine and/or droperidol.

Chronic Treatment Time After Apomorphine (min)

10 2 0 30

control 2.0 4.4† 1.2

droperidol 2.5 5.5† 1.8

cocaine 2.5† 2.0 0.0

cocaine + droperidol 3.8 4.0† 0.5

The range of scores within each group at each time point

was within 1 scoring unit. † Denotes peak effect.

The two aspects of this study, taken together, raise some interesting points. First, the same treatment--namely, chronic

cocaine--which caused “sensitization” to cocaine, caused “desensitization” or tolerance to apomorphine. Second, chronic treatment with a dopamine receptor antagonist which is also, incidentally, an alpha-adrenergic receptor antagonist, blocked the

development of tolerance to apomorphine but not the development

of sensitization to cocaine. This suggests that at least two

different phenomena are taking place in response to repeated,

prolonged exposure to cocaine: (1) chronic stimulation of dopamine transmission, leading to an eventual decrease in the sensitivity of the dopamine receptors (or changes in other neural

links in series with dopamine-receptive cells), and (2) alterations in parameters independent of dopamine and alpha-adrenergic

receptors, which augment the sensitivity of the system to

cocaine. In addition, it is noteworthy that the presence of the

chronic behavioral stimulation is not required for the development of sensitization to cocaine.

Changes in CABA Synthesis and GABA Receptors in Striatum Following Chronic Cocaine Treatment

Measurements of GABA-related neurochemical parameters were made

in the striatum of rats treated chronically with cocaine and/or

droperidol. The striatum was examined because of its known

involvement in the generation of dopamine-related stereotyped

behavior; GABAergic parameters were examined because of the

importance of GABAergic interneurons in striatum and the fact

that GABA synthesis in this region has been previously shown to

change in response to chronic interference with dopamine transmission (Gale and Casu 1981).

Table 3 shows the activity of glutamic acid decarboxylase (GAD),

the enzyme responsible for GABA synthesis, in rats that had been

chronically treated in the exact manner as described for the

behavioral studies. A significant increase in GAD activity in

326
striatum was seen in rats in the 'chronic cocaine' group; this

effect was still present in rats that had received concurrent

treatment with droperidol. The increase in GAD activity was

still observed 10 days after cessation of chronic treatment.

Studies are now in progress to determine the time course of

recovery. It should be noted that the behavioral ‘sensitization’ to cocaine that we have described is still present at 10

days after stopping chronic treatment.

TABLE 3

Glutamic acid decarboxylase activity (GAD) in rat

striatum after chronic cocaine administration

GAD Activity

Chronic Treatment (n) umol/mg prot/hr % Control

control (12) .35 ± .02

cocaine (10) .50 ± .03* 143%

cocaine + droperidol (8) .48 ± .03* 137%

droperidol (6) .31 ± .03

10 days after

chronic cocaine (8) .43 ± .04* 123%

*Significantly different from control p < .05

GAD activity was measured in tissue homogenates, in the

presence of saturating concentrations of substrate

(glutamate) and cofactor (pyridoxal phosphate), based on

the method of Sims and Pitts (1970). See text for

details of experimental design.

Table 4 shows the effect of the chronic cocaine treatment on the

rate of striatal GABA accumulation in vivo following irreversible inhibition of GABA degradation. This gives us an indication

of GABA turnover. The GABA-transaminase inhibitor, gamma-vinylGABA (GVG) was injected directly into the striatum and the

accumulation of GABA was measured at 3 hr. The net GABA accumulation in striata of rats that had been chronically exposed to

cocaine was significantly greater than that of controls. No

change in steady-state levels of GABA was found. This suggests

that chronic exposure to cocaine results in an enhanced turnover

rate of GABA in striatum, an observation consistent with our

finding of increased GAD activity in striatal homogenates.

The effect of chronic cocaine exposure on GABA binding in striatum is shown in table 5. It can be seen that a significant

decrease in GABA binding in striatum was associated with chronic

cocaine treatment. This effect was not prevented by concurrent

treatment with droperidol. As in the case of GAD, the change in

GABA binding was still present at 10 days following cessation of

cocaine treatment.

327
TABLE 4

Effect of chronic cocaine administration on accumulation

of GABA in viva in striatum.

Chronic

Treatment

GABA (nmol/mg prot)

3 hr Net

Basal After GVG Increase % Control

control 24.0 ± 0.4 77.2 ± 2.0 53

cocaine 24.5 ± 0.5 97.4 ± 3.8* 73

*Significantly different from control p < .05

138%

GABA accumulation was measured 3 hr after the microinjection of gamma-vinyl-GABA (GVG, 20 ug) into striatum.

Basal values are those obtained from tissue not treated

with GVG. Each value is the mean ± S.E. of 5 rats.

TABLE 5

Specific [

3

H]-GABA binding in rat striatum after chronic

cocaine administration

[

3

H]-GABA Binding

Chronic Treatment (n) fmol/mg prot % Control

control (12) 424 ± 30

cocaine (10) 301 ± 25* 71%

cocaine + droperidol (8)

(6)

288 ± 29* 68%

droperidol 373 ± 35

10 days after

chronic cocaine (8) 350 ± 32* 83%

*Significantly different from control p < .05

Binding was done in the presence of 80-120 ug membrane

protein, 30 nM [

3

H]GABA, at 0°C in a Na+-free medium;

binding in the presence of 10 mM nonradioactive GABA was

subtracted from total binding to calculate specific

binding. Membranes were prepared by freeze-thawing,

Triton-X 100 incubation (.01% at 37°C for 60 min) and

extensive washing.

To determine whether the changes in GABA binding and GAD activity represented a general response of GABAergic synapses to

prolonged cocaine treatment, cerebellum and cerebral cortex of

the same rats were examined for these parameters. No significant changes in GAD or GABA binding were found in neocortex or

cerebellum. This suggests that the changes in GABAergic parameters observed in the striatum of rats exposed chronically to

328
cocaine represent adjustments which may be specifically related

to the neural circuitry of this nucleus. In view of these

results, it is unlikely that cocaine exerts a direct effect on

GABAergic transmission.

Cocaine-Induced Conditioned Taste Aversion

We have also obtained evidence for acute actions of cocaine that

are insensitive to dopaminergic and/or adrenergic blockade using

a conditioned taste aversion paradigm. Since the reinforcing

properties of cocaine have been demonstrated to depend upon

central dopaminergic and possibly noradrenergic transmission

(Gill et al. 1978; Wilson and Schuster 1972), we were interested in determining whether aversive properties of cocaine

would be mediated by the same neurotransmitter systems. In

these experiments, rats were trained to drink water for one 20

min session each day. On the first day of drug exposure, the

rats received saccharin instead of water, and 20 min following

the drinking session drugs were administered.

Cocaine was administered subcutaneously as it had been for the

chronic studies previously described. In addition to cocaine,

separate groups of rats received one of the following dopamine

and/or adrenergic antagonists: haloperidol (1 mg/kg), chlorpromazine (20 mg/kg), pimozide (1 mg/kg), or propranolol

(5 mg/kg). An additional group that received both chlorpromazine and propranolol in addition to cocaine was also examined.

In this way, we were able to completely block all hyperactivity,

stereotypy and/or sympathomimetic stimulation due to cocaine for

the entire duration (approximately 8 hr) of the cocaine effect.

The antagonist drugs, under our experimental conditions, did not

by themselves cause conditioned taste aversion. Cocaine administration caused a marked taste aversion, which was evident on

the first post-drug saccharin exposure (saccharin test 1)

initiated 4 or 5 days after the first saccharin exposure. The

rats that had received cocaine following the pre-drug saccharin

exposure showed a 50-60% decrease in saccharin intake on the

first saccharin test (table 6). No change in saccharin consumption was seen in controls and in rats receiving the dopamine

and noradrenergic antagonists without cocaine. None of the

antagonists, or antagonist combinations, when administered

concurrently with cocaine, were able to prevent or attenuate the

taste aversion produced by exposure to cocaine. It should be

noted that the dose of cocaine used to produce the taste

aversions was equivalent to only twice the minimum dose necessary for causing a significant (but submaximal) decrease in

saccharin consumption. Thus, it should have been sensitive to

alteration by antagonists if the antagonists acted on a system

involved in mediating the aversion produced by cocaine.

329
TABLE 6

Cocaine-induced conditioned taste aversions: Effect of

dopaminergic and adrenergic antagonists

Saccharin Consumption: % Control

1† 2†

control

cocaine

chlorpromazine

propranolol

propranolol + cocaine

chlorpromazine + propranolol

chlorpromazine + propranolol +

cocaine

haloperidol

pimozide

pimozide + cocaine

100%

50%

105%

90%

53%

92%

45%

110%

90%

59%

100%

20%

98%

93%

21%

85%

10%

105%

95%

25%

† 1 = first (2 = second) saccharin test session.

Each group contained 4 or 5 rats, matched for initial

(pre-drug) saccharin consumption. Standard errors of

the mean saccharin consumption for each exposure were

less than 20% of the mean value. Control saccharin

consumption = 25 ml on test session 1 and 23 ml on test

session 2. All tests involved access to a single bottle

of saccharin only.

To determine whether the cocaine-induced taste aversions were

related to the local anesthetic action of cocaine, we tested a

group of animals with equivalent doses of lidocaine in combination with epinephrine (the epinephrine was included to simulate

the local vasoconstrictive action of the cocaine injections).

This treatment resulted in no measurable change in subsequent

saccharin consumption, indicating that local anesthetic actions

per se are not responsible for the taste aversions produced by

cocaine.

Thus, it appears that the conditioned taste aversion that

develops with cocaine is mediated by a system other than one

containing catecholamines. While blockade of catecholaminergic

transmission eliminated all behavioral signs of motor and autonomic stimulation induced by cocaine, the aversive characteristics of the exposure to cocaine were not altered. Since the

reinforcing properties of cocaine can be eliminated by blockade

of central dopaminergic transmission, it appears that the

reinforcing and aversive properties are mediated by distinctly

different neurotransmitter systems. We have yet to identify the

system responsible for the aversive characteristics of cocaine.

330
Discussion

We have seen several instances in which cocaine-induced effects,

either chronic or acute, are obtained despite blockade of

noradrenergic and dopaminergic transmission. Evidently, there

are one or more noncatecholaminergic neurotransmitter systems

with which cocaine can interact directly in order to induce

these effects. At present, we do not know whether the relevant

system or systems even involve known neurotransmitters; it is

possible that the neuromodulator mediating some of these actions

has yet to be described. In this context, the findings of

Sershen, Reith and Lajtha (1980) are of interest. These authors

have described a binding site for [

3

H]cocaine in brain tissue

which appears to be related to central stimulatory actions of

cocaine (as opposed to local anesthetic actions). The binding

of cocaine to this site was relatively insensitive to displacement by classical neurotransmitters such as dopamine, norepinephrine, acetylcholine, serotonin and GABA or receptor antagonists for these neurotransmitters (Reith et al. 1980a). These

same authors have found that a low molecular weight peptide

extracted from brain homogenates is capable of inhibiting the

saturable binding of cocaine to crude mouse brain membranes

(Reith et al. 1980b). Thus, it is possible that cocaine may

interact with receptors for a yet-to-be-identified neuroactive

peptide. It is interesting that while no loss of these cocaine

binding sites occurred following lesions of dopamine neurons, at

least a portion of these binding sites appeared to be associated

with terminals of serotonergic neurons (Reith et al. 1983).

Thus, even if cocaine may exert some of its actions via uncharacterized receptors, it is likely that one or more classical

transmitter substances may indirectly mediate these effects. It

will therefore be worthwhile examining the ability of agonists

and antagonists of serotonin and other noncatecholaminergic

neurotransmitters to prevent the development of cocaine-induced

sensitization, conditioned taste aversions, and/or changes in

striatal GABA utilization. In addition, by examining different

cocaine analogs for their ability to elicit catecholamineindependent actions, we may be able to determine: (1) whether

the different catecholamine-independent actions are correlated

with each other (in terms of relative potencies of cocaine

analogs), and (2) whether any of the cocaine-induced effects

that are catecholamine-independent are related to the cocaine

binding site described by Reith et al. (1980).

By pursuing this line of investigation, we may find that the

classical actions of cocaine as an uptake inhibitor for catecholamines represent only a small part of the mechanism by which

this drug can have an impact on central nervous system function,

in the context of both short-term and long-term exposure to the

drug.

331
References

Downs, A.W., and Eddy, N.B. The effect of repeated doses of

cocaine on the rat. J Pharmacol Exp Ther, 46:199-200, 1932.

Gale, K., and Casu, M. Dynamic utilization of GABA in substantia nigra: Regulation by dopamine and GABA in the striatum,

and its clinical and behavioral implications. Mol Cell

Biochem, 39:369-405, 1981.

Gill, C.A., Holz, W.C., Zirkle, C.L., and Hill, H. Pharmacological modification of cocaine and apomorphine self-administration in the squirrel monkey. Proc 10th Cong of Collegium Int

Neuropsychopharm, 2:1477-1484, 1978.

Post, R.M., Lockfeld, A., Squillace, K.M., and Contel, N.R.

Drug-environment interaction: Context dependency of cocaineinduced behavioral sensitization. Life Sci, 28:755-760,

1981.

Post, R.M., and Rose, H. Increasing effects of repetitive

cocaine administration in the rat. Nature, 260:731-732,

1976.

Reith, M.E.A., Sershen, H., Allen, D.L., and Lajtha, A. A

portion of [

3

H]cocaine binding in brain is associated with

serotonergic neurons. Molec Pharmacol, 23:600-606, 1983.

Reith, M.E.A., Sershen, H., and Lajtha, A. Saturable [

3

H]-

cocaine binding in central nervous system of mouse. Life Sci,

27:1055-1062, 1980.

Reith, M.E.A., Sershen, H., and Lajtha, A. Endogenous peptide(s) inhibiting [

3

H]cocaine binding in mouse brain.

Neurochem Res, 5:1291-1299, 1980.

Sershen, H., Reith, M.E.A., and Lajtha, A. The pharmacological

relevance of the cocaine binding site in mouse brain. Neuropharmacology, 19:1145-1148, 1980.

Sims, K.L., and Pitts, Jr., F.N. Brain glutamate decarboxylase:

Changes in the developing rat brain. J Neurochem, 17:1607-

1612, 1970.

Stripling, J.S., and Ellinwood, Jr., E.H. Sensitization to

cocaine following chronic administration in the rat. In

Ellinwood, E.H., and Kilbey, M.M., eds. Cocaine and Other

Stimulants. New York: Plenum Press, 1976 pp. 327-351.

Tatum, A.L., and Seevers, M.H. Experimental cocaine addiction.

J Pharmacol Exp Ther, 36:401-410, 1929.

Wilson, M.C., and Schuster, C.R. The effects of chlorpromazine

on psychomotor stimulant self-administration in the rhesus

monkey. Psychopharmacologia, 26:115-126, 1972.

Author

KAREN GALE, Ph.D.

Department of Pharmacology

Georgetown University

Schools of Medicine and Dentistry

3900 Reservoir Road, N.W.

Washington, DC 20007

332
Barbiturate Tolerance and

Physical Dependence: Contribution of Pharmacological Factors

Michiko Okamoto

INTRODUCTION

Sedative-hypnotics and anti-anxiety drugs comprise a large group of

chemically different compounds that are classified pharmacologically

as CNS depressants. These compounds manifest a spectrum of effects

ranging from calming and sedative activity through profound CNS

depression, manifested as anesthesia, coma and respiratory paralysis, and finally death. For the most part, the acute effects

exhibited by individual compounds are nonspecific, and any selectivity, in terms of the clinical indications for which an individual compound is used, is conferred by (1) the dose and schedule of

administration, (2) the slope of the dose-response curve along the

continuum of CNS depression, and (3) the pharmacokinetic profile of

the compound. Therefore, a clear understanding of these pharmacologic properties is essential to the appropriate selection of a

sedative-hypnotic or anti-anxiety drug for therapeutic use.

Since the introduction of sodium barbital into clinical medicine in

1903, considerable effort has been devoted to identifying and

understanding the risks associated with both acute and chronic

administration of sedative-hypnotics. Although all of these drugs

have similar acute effects, there is no question that abuse among

this class of drugs differs, and so do the risks. For example, the

large number of deaths each year from overdose of self-administered

barbiturates attests to the narrow margin of safety of these

drugs. On the other hand, the more widely available benzodiazepines rarely cause death from overdose unless they are used in

combination with other sedative-hypnotics.

Despite these differences, all sedative-hypnotics, including nonbarbiturates, can produce tolerance, psychological and physical

dependence, and a barbiturate-like withdrawal syndrome with the

risk of convulsions when they are taken in sufficient amounts.

Thus we chose barbiturates as the prototype sedative-hypnotic to

study the underlying pharmacodynamic and pharmacokinetic factors

which govern the production of tolerance and physical dependence.

333
Clinical studies indicate that the pattern of sedative-hypnotic use

that culminates in withdrawal hyperexcitation is a prolonged period

of continuous drug intoxication. This suggests that the functional

depression of the CNS is an important factor in the development of

physical dependence. Accordingly, in designing an animal model of

sedative-hypnotic dependence, techniques for measurements of drug

response (CNS depression), in addition to measurement of dose and

frequency of drug administration throughout chronic treatment,

enable the evaluation of this factor.

Quantitative measurement of response is also essential to the

characterization of the tolerance to sedative-hypnotics which

invariably develops during their chronic administration. According

to Kalant et al. (1971), this tolerance can be dispositional (i.e.,

primarily due to an increased rate of elimination) or functional

(i.e., decreased sensitivity of the CNS to depression) or both.

The dissection of "total" tolerance into these components and the

separate characterization of their development and mechanisms can

be achieved by concurrent measurements of daily drug effect (CNS

depression) and drug load (dose and blood or brain concentration)

during chronic administration.

RESULTS

Using these techniques and pentobarbital and barbital as prototype

barbiturates, we characterized the development of functional and

dispositional tolerance during chronic barbiturate treatment in our

animal model (Okamoto and Boisse 1981). Cats were treated with Na

pentobarbital twice daily or Na barbital once every 3 days for 5

weeks. Each dose was adjusted to produce a preset peak level of

CNS depression; in this case, a light surgical anesthesia (Boisse

and Okamoto 1978a). The kinetics of barbiturate elimination were

determined once a week to monitor the dispositional component of

tolerance (Boisse and Okamoto 1978b).

The average dose required to produce the peak effect of surgical

anesthesia increased during chronic treatment for both pentobarbital and barbital, showing the development of "total" tolerance

to both drugs (figure 1). At the beginning of treatment, the dose

of Na pentobarbital increased more rapidly than the dose of Na

barbital. Elimination kinetics showed that the half-life of

pentobarbital decreased rapidly during this initial rapid increase

in the dose of Na pentobarbital, a demonstration of the quick onset

of the development of dispositional tolerance to Na pentobarbital.

The decrease in the pentobarbital half-life was presumably due to

induction of the hepatic microsomal drug-metabolizing enzymes which

biotransform pentobarbital (Remer 1969). In contrast, the halflife of barbital did not change during the Na barbital treatment;

thus, there was no dispositional component of tolerance to

barbital, a drug which is known to be eliminated unchanged

primarily through the kidney.

The development of the functional component of tolerance was shown

by the steady increase in the blood concentration of pentobarbital

334
FIGURE 1. Chronic pentobarbital and barbital treatments and

characteristics of tolerance in each. Ordinates: (a) average CNS

depression ratings: peak after each morning dose residual

depression just prior to subsequent dose (b) average total

CNS depression based on areas under time-action curves summed at

3-day intervals; (c) average administered dose; (d) elimination

half-life; a decrease indicates dispositional tolerance; (e)

average blood concentration at the time of peak drug response; an

increase indicates functional tolerance. Abscissa: time during

chronic treatment (Okamoto and Boisse 1981. Copyright 1981,

Elsevier Publications).

or barbital which was required to achieve the preset level of CNS

depression. Cellular tolerance increased slowly and steadily as

the treatment duration increased (figure 1, Boisse and Okamoto,

1978b). Most interestingly, when animals were treated with these

"chronically equivalent" doses (Boisse and Okamoto 1978a) of

pentobarbital and barbital (that is, doses which produce the same

peak and residual CNS depression shown in figure 1, functional

tolerance developed in a parallel fashion to pentobarbital and

barbital.

335
FIGURE 2. Recovery from cellular (functional) tolerance and

dispositional tolerance after chronic Na pentobarbital treatment.

Ordinates: (A) pentobarbital concentration (ug/ml blood) that produced CNS depression ratings of 11 (surgical anesthesia), 5 (ataxia),

and 1 (loss of fine motor coordination). (B) pentobarbital elimination half-life from blood. Vertical bars indicate ± S.E. (N=6).

Abscissae: "C" stands for control, prior to chronic Na pentobarbital treatment, "WD" stands for day 1 of withdrawal or just after

the last dose of chronic Na pentobarbital, and numbers indicate days

after the last chronic Na pentobarbital dose (Okamoto et al.

unpublished observations).

The "reverse" of the development of functional and dispositional

tolerance, i.e., the return to normal after abrupt termination of

chronic drug administration, was also studied. To investigate

this, Na pentobarbital chronic administration was abruptly

terminated, and the pentobarbital elimination kinetics and CNS

sensitivity to a challenging dose of Na pentobarbital were evaluated

every 3 days until recovery was complete. The 3-day interval was

chosen because preliminary experiments had shown that animals

treated chronically with Na pentobarbital once every 2 days

did not develop functional or dispositional tolerance (Okamoto et

al. unpublished observations); thus, the testing procedure was not

expected to affect the processes being studied.

336
FIGURE 3. Peak withdrawal intensity rating after various durations

of chronic "high" or "low" dose barbiturate treatment.

Ordinate: peak withdrawal intensity rating, mean ± S.E.

Abscissa: duration of chronic treatment in weeks. Number of

observations per point: 10 to 67 for high dose barbiturate, 8 to

19 for low dose barbiturate (Okamoto et al. 1981. Copyright 1981,

American Society for Pharmacology and Experimental Therapeutics).

Recovery from the two types of tolerance occurs after the abrupt

termination of Na pentobarbital (figure 2). These results show that

the kinetics of recovery were almost the mirror image of the induction of tolerance. Dispositional tolerance was reversed within 2

weeks. In contrast, recovery from functional tolerance was slow

and not complete at the end of the 2-week observation period.

The relationship between chronic dosing levels and the degree of

functional tolerance and physical dependence that develops is also

an important issue to be investigated. Accordingly, we compared

the development of tolerance and dependence in groups of animals

treated chronically either with a "high" or "low" dose of

barbiturate (Okamoto et al. 1978). The "high" dose group received

Na pentobarbital twice daily sufficient to produce light surgical

anesthesia (this corresponds to a CNS Depression Rating of 10-11 on

our scale). The "low" dose group received Na barbital sufficient

to produce gross ataxia (this corresponds to a CNS Depression

Rating of 5-6). Both groups were treated for 1 to 5 weeks and

groups of animals were abruptly withdrawn after treatment for

various durations to evaluate the time-course of development of

dependence. The severity of physical dependence was measured by an

overall withdrawal intensity rating system developed in our

laboratory (Okamoto et al. 1981), and the development of functional

tolerance was measured as described above.

The "low" dose produced less physical dependence than the "high"

dose after equal durations of treatment (figure 3). However,

physical dependence increased in a parallel fashion in both

treatment groups and was still increasing in the "low" dose group

after 5 weeks. Indeed, when the "low" dose treatment was further

increased to 100 days, it produced approximately the same severity

337
FIGURE 4. Incidence of convulsions and total number of convulsions

per animal after various durations of chronic "low" dose barbital or

pentobarbital treatment. Abscissa: duration of chronic treatment in

weeks. Ordinates: (A) percent incidence of cats with grand mal type

convulsions during withdrawal. (B) average total number of grand

mal-type convulsions per animal during withdrawal. Vertical bars represent ± S.E. Number of observations per point: 10 to 15 animals

(Okamoto and Hinman 1984, Copyright 1984, Pergamon Press, Ltd.).

of physical dependence as had the 5 weeks of "high" dose treatment

(figure 4). These results indicate that physical dependence develops

more slowly with "low" dose barbiturate treatment, but that the same

level of dependence eventually develops if the treatment is continued

at doses sufficient to achieve a preset level of CNS depression

(Okamoto and Hinman 1984).

Another important factor to consider during chronic sedative

hypnotic treatment is the frequency of administration. This was

clearly demonstrated by Goldstein (1972) for the production of

ethanol physical dependence. The relationship between dosing

frequency and elimination determines the drug concentration at any

time. The maintenance of certain drug concentrations may be a major

factor in the development of functional tolerance and physical

dependence. Small individual differences in the half-life of drug

elimination affected the development of physical dependence to Na

pentobarbital when the drug was given on a fixed schedule (figure

5). A group of animals was treated with "high" dose Na pentobarbital twice a day for 5 weeks. The elimination half-life of

pentobarbital was determined for each animal on the last day of

treatment. The number of grand mal-type convulsions was tabulated

for each animal upon abrupt termimation of the drug as a measure of

withdrawal severity. The range of the half-life of pentobarbital

in these animals was 3-13 hours, and the longer the half-life, the

more severe was the physical dependence (Okamoto and Hinman 1983).

338
FIGURE 5. Relationship between pentobarbital half-life and total

number of convulsions displayed during pentobarbital withdrawal.

Abscissa: pentobarbital half-life in hours. Ordinate: total

number of convulsions displayed by each animal during the

withdrawal (Okamoto and Hinman, 1983. Copyright 1983, American

Society for Pharmacology and Experimental Therapeutics).

FIGURE 6. Relationship between the peak withdrawal intensity and

total number of "high" Na pentobarbital doses. Abscissa: total

number of Na pentobarbital doses. Ordinate: average peak withdrawal intensity. dosed every 12 hours; dosed every 24

hours, dosed every 48 hours (Okamoto et al. unpublished

preliminary observations).

339
Reducing the frequency of chronic barbiturate administration

reduces the risk of development of physical dependence. That is,

the longer the interval between doses, the less severe was the

withdrawal (figure 6). It is apparent that dosing once every 2

days produced a negligible level of physical dependence. In other

experiments, as the dosing interval exceeded 36 hours, the drug

concentration just prior to succeeding doses was below the limits

of detection ( <500 ng/ml blood; Okamoto et al., unpublished

communications). These findings underscore the importance of

adjusting dosing frequency so as not to maintain sufficient drug

levels within biological systems that would risk producing physical

dependence.

Other studies have demonstrated how critical the kinetics of

elimination of sedative-hypnotics are in understanding the

underlying state of physical dependence. Since physical dependence

can only be measured by the intensity of the withdrawal syndrome,

it is difficult to dissect the phenomena of physical dependence

from withdrawal so as to study either independently. To start

with, it has been accepted that long-acting barbiturates such as

barbital or phenobarbital are less liable to produce physical

dependence than the short-acting barbiturate drugs such as

pentobarbital or secobarbital (cf. Jaffe 1980). Also, in clinical

subjects and in animals, the onset of withdrawal was slower and its

intensity less severe in barbital dependency than in pentobarbital

dependency (Boisse and Okamoto 1978c). For example, the average

elimination half-lives of barbital and pentobarbital in these

animals were 30 and 5 hours, respectively. To determine whether

the slow rate of barbital elimination was responsible for the

slower onset of withdrawal, we experimentally "slowed" the

elimination of pentobarbital by readministering proportionally

smaller doses of pentobarbital intravenously at hourly intervals

according to a computer calculation based on first-order

elimination kinetics (Boisse and Okamoto 1978d).

The added drug effectively altered the apparent rate of

pentobarbital elimination from a half-life of 5 hours to 27 hours

(table 1). This procedure also slowed the onset of withdrawal and

dramatically reduced its severity, as indicated by the withdrawal

intensity rating and the incidence of grand mal convulsions (Boisse

and Okamoto 1978d). Additional evidence that the elimination of

the drug is critical for the expression of withdrawal signs was

obtained by experimentally increasing the rate of barbital elimination to mimic that of pentobarbital. The rate of barbital

elimination was increased by peritoneal dialysis after the last

dose of chronic barbital. This procedure decreased the apparent

half-life of barbital elimination from 30 hours to 7 hours (table 1).

As a result, the barbital withdrawal signs appeared much sooner and

were markedly intensified (Boisse and Okamoto 19784).

These results support Hollister's hypothesis (1980) that the

severity of the withdrawal reaction is related to the rate of

disappearance of sedative drugs from plasma. He predicted that the

sedative-hypnotics with half-lives in the range of 12-24 hours

(e.g., pentobarbital, secobarbital, and glutethimide) will produce

the most severe withdrawal syndrome in patients.

340
TABLE 1. Reconstructed from Table 3 in Boisse and Okamoto 1978d.

Copyright 1978, American Society for Pharmacology and Experimental

Therapeutics).

In contrast, drugs which have short half-lives have little

potential for producing tolerance and physical dependence because

their residual CNS concentrations at the time of successive doses

are small. There is insufficient drug to produce and maintain

functional tolerance and physical dependence. In the other

direction, drugs which have long half-lives are less likely to

result in severe withdrawal signs and symptoms despite the fact

that they produce severe physical dependence. That is, the CNS can

gradually readapt, when the drug in no longer administered, due to

the slow elimination of the drug from the CNS.

Although the behavioral aspects of sedative-hypnotic withdrawal

have been well documented, the pathophysiology of physical

dependence in terms of underlying neuronal mechanisms has not yet

been well elucidated. Barbiturate withdrawal in humans consists of

a variety of overt signs (Isbell et al. 1950); Fraser et al. 1958;

Wulff 1959) accompanied by characteristic EEG abnormalities

recorded through surface electrodes (Isbell et al., 1950; Wikler et

al. 1955; Wulff 1959). These abnormalities consist of high

amplitude slow waves, paroxysmal discharges, and spike-and-dome

activity. Interestingly, paroxysmal discharges were more prominent

during withdrawal from moderate doses of barbiturate than in

withdrawal from high doses (Wikler et al. 1955). Seizure activity

usually originated in subcortical rather than in cortical areas

(Essig 1962, 1967); Sharpless and Jaffee 1967; c.f. Kalant et al.,

1971). Although barbiturates and other sedative-hypnotics depress

all CNS activity as a function of dose, it is possible that chronic

use of these drugs affects various CNS loci differently in relation

to the chronic dose administered. Therefore further information

regarding the most sensitive site(s) and characteristics of

341
FIGURE 7. EEG recordings during moderate-intensity barbiturate

withdrawal, 40 hours after last dose. Recordings are from

hippocampus (HPC) with spike-and-wave activity, amygdala (AMY) with

paroxysmal discharge, motor cortex (MC), auditory cortex (AC),

visual cortex (VC), caudate (CAU), hypothalamus (HYP), mid-brain

reticular formation (MRF), and lateral geniculate (LGB) (Hinman and

Okamoto 1984. Copyright 1984, Academic-Press).

abnormal brain activity during withdrawal, especially in

subcortical loci, is essential to understanding the mechanisms of

barbiturate tolerance and physical dependence.

To pursue this information, we have recorded electroencephalograms from cortical and subcortical brain regions during

moderate-intensity barbiturate withdrawal (Hinman and Okamoto

1984). The EEG pattern showed 4-6 Hz rhythmic bursts in the hippocampus and visual cortex during withdrawal (figure 7). The bursts

in the hippocampus were a spike-and-wave type. In animals which

had spontaneous seizures, 4-6 Hz bursts occurred also in the motor

cortex; this motor cortex abnormality did not occur in animals

which did not have spontaneous generalized seizures. Prolonged

high frequency, high voltage discharges and spikes were also

recorded frequently from the amygdala and the LGB during withdrawal

(figure 8). Animals displayed bizarre behaviors suggestive of

hallucinations during these periods of prolonged discharges,

especially during prolonged bursts from the hippocampus and the

visual cortex (Hinman and Okamoto 1984).

In order to explore more fundamental neuronal mechanisms of the

development of tolerance and physical dependence and the neuronal

mechanisms responsible for the expression of CNS hyperexcitation

during withdrawal, electrophysiological studies of well-characterized spinal cord reflexes were performed in our physically

barbiturate-dependent animals. After 5 weeks of treatment with our

standard chronic "high" dose pentobarbital regimen (Rosenberg and

Okamoto 1974; Okamoto et al. 1975), animals were prepared for

spinal cord reflex function studies 48 hours after the last dose.

Several alterations in spinal cord reflex function during

342
FIGURE 8. EEG recording during a spontaneous partial seizure

during moderate-intensity barbiturate withdrawal. The animal

remained standing during the seizure. Recordings are from various

loci: motor cortex (MC), visual cortex (VC), hippocampus, right

side (RHPC), hippocampus, left side (LHPC), amygdala (AMY), nucleus

centralis centralis (CC), and lateral geniculate (LGB). (Hinman

and Okamoto 1984. Copyright 1984, Academic Press).

FIGURE 9. Recurrent inhibition of the reflex discharge (2N

response). Measured in 12 control and 14 withdrawing animals

48 hours after the last dose of chronic pentobarbital treatment. Vertical bars indicate ± S.E.; asterisks indicate P .05 by

t-test. Inset: A schematic drawing of electrode locations: GS

(gastrocnemius-soleus muscle nerve), St (stimulating electrode,

test impulse), VRL7 (ventral root L-7), Sc (stimulating

electrode on one-sixth to one-third of L-7 ventral root to deliver

conditioning impulse), R (recording electrode). (Rosenberg and

Okamoto 1978. Copyright 1978, American Society for Pharmacology

and Experimental Therapeutics).

343
FIGURE 10. Presynaptic inhibition of the 2N response. Measured in

14 control and 14 withdrawing animals 48 hours after the

last dose of chronic pentobarbital. Vertical bars indicate ± S.E.;

asterisks indicate P < .05 by t-test. Inset: Schematic drawing of

experimental electrode locations: GS (gastrocnemius-soleus muscle

nerve), St (stimulating electrode, test impulse), PSBT (posterior

biceps and semitendinosus muscle nerve), SC (stimulating

electrode to deliver conditioning impulse), (VRSl (ventral root

S-l), R (recording electrode) (Rosenberg and Okamoto 1978.

Copyright 1978, American Society for Pharmacology and Experimental

Therapeutics).

barbiturate withdrawal were demonstrated (Rosenberg and Okamoto

1976). These included (1) increased polysynaptic but not

monosynaptic (2N) reflex discharges; (2) decreased synaptic

refractoriness, allowing more efficient transmission of repetitive

impulses; (3) increased sensitivity to lower frequency tetanic

stimulation in the production of post-tetanic potentiation (PTP);

and (4) augmented spontaneous discharge. All of these results

indicate generalized augmentation of neuronal activity during

withdrawal.

Since the overall excitability of the CNS depends to a large extent

on a delicate balance between excitatory and inhibitory functions,

the hyper-excitability displayed in withdrawing animals may be a

consequence not only of increased activity in excitatory pathways

but also of decreased activity in inhibitory pathways. Further

electrophysiological studies of inhibitory function at the spinal

reflex level (Rosenberg and Okamoto 1978) indicate that recurrent

inhibition disappeared almost totally during barbiturate withdrawal

(figure 9). Presynaptic inhibition also decreased dramatically

(figure 10). Loss of inhibition during withdrawal may be causally

related to the enhancement of inhibition by the presence of the

344
barbiturate, as has been demonstrated in the hippocampus (Nicoll et

al. 1975), the olfactory bulb (Nicoll 1972), and possibly in the

(Hurlbrink and Boyd 1969). The neurochemical basis of this drug

enhancement of inhibition and reduced inhibition during withdrawal

is of great interest. Barbiturates appear to selectively

facilitate GABA-mediated responses (Ransom and Barker 1976; cf. Ho

1981) and to increase presynaptic inhibition in the spinal cord,

which is thought to be GABA mediated (Eccles et al. 1963; Schmidt

1963; Bell and Anderson 1972; Davidoff 1972). Accordingly it was

possible that our findings of decreased presynaptic inhibition

during withdrawal might be related to altered GABA levels or

kinetics, although this would not explain our findings of the loss

of recurrent inhibition, since GABA is not thought to be involved

in the recurrent inhibitory pathway. Glycine is the proposed

transmitter for both the Renshaw cell and also for neurons which

mediate Ia inhibition (Werman et al. 1968; Curtis 1969). However,

our findings do not specifically implicate glycinergic mechanisms

either. Although recurrent inhibition, involving the Renshaw cell,

was nearly abolished during withdrawal, direct inhibition involving

the Ia inhibitory interneuron was not changed.

The results of these studies support the hypothesis of a

nonspecific adaptation of the nervous system during chronic

barbiturate treatment, i.e., adaptation not specific to a

particular neurochemical system. A major part of this adaptation

is a decreased activity in certain inhibitory pathways. This view,

if correct, means that the adaptation is a response to chronic

depression of the nervous system rather than a specific response to

the particular drug. Further studies should be forthcoming to

elucidate the underlying mechanisms which contribute to a

development of cellular tolerance and physical dependence.

REFERENCES

Bell, J.A., and Anderson, E.G. The influence of

semicarbazide-induced depletion of aminobutyric acid on

presynaptic inhibition. Brain Res, 43:161-169, 1972.

Boisse, N.R., and Okamoto, M. Physical dependence to barbital

compared to pentobarbital. I. "Chronically equivalent" dosing

method. J Pharmacol Exp Ther, 204:497-506, 1978a.

Boisse, N.R., and Okamoto, M. Physical dependence to barbital

compared to pentobarbital. II. Tolerance characteristics. J

Pharmacol Exp Ther, 204:507-513, 1978b.

Boisse, N.R., and Okamoto, M. Physical dependence to barbital

compared to pentobarbital. III. Withdrawal characteristics. J

Pharmacol Exp Ther, 204:514-525, 1978c.

Boisse, N.R., and Okamoto, M. Physical dependence to barbital

compared to pentobarbital. IV. Influence of elimination

kinetics. J Pharmacol Exp Ther, 204:526-540, 1978d.

Curtis, D.R. The pharmacology of spinal postsynaptic inhibition.

Progr Brain Res, 31:171-189, 1969.

Davidoff, R.A. Gamma-aminobutyric acid antagonism and presynaptic

inhibition in the frog spinal cord. Science (Washington),

175:331-333, 1972.

345
Eccles, J.C., Schmidt, R., and Willis, W.D. Pharmacological

studies

on presynaptic inhibition. J Physiol (London), 168:500-530,

1963.

Essig, C.F. Convulsive and sham rage behaviors in decorticate dogs

during barbiturate withdrawal. Arch Neurol Psychiat (Clin),

7:471-475, 1962.

Fraser, H.F., Wikler, A., Essig, C.F., and Isbell, H. Degree of

physical dependence induced by secobarbital or pentobarbital.

JAMA, 166:126-129, 1958.

Goldstein, D.B. Relationship of alcohol dose to intensity of

withdrawal signs in mice. J Pharmacol Exp Ther, 180:203-215,

1972.

Hinman, D.J., and Okamoto, M. EEG patterns recorded from cortical

and subcortical areas during moderate-intensity barbiturate

withdrawal. Experimental Neurology, 83:555-567, 1984.

Ho, I.K. Mechanisms of action of barbiturates. Ann Rev Pharmacol

Toxicol, 21:83-111, 1981.

Hollister, L.E. Dependence on benzodiazepines. In: Szara, E.I.,

and Ludford, J.P., eds. Benzodiazepines: A Review of Research

Results. National Institute on Drug Abuse Research Monograph 33.

DHEW Pub. No. (ADM) 81-1052. Washington, D.C.: Supt. of DOCS.,

U.S. Govt. Print. Off. 1981. pp. 70-82.

Hurlbrink, E.E., and Boyd, E.S. Some effects of pentobarbital and

strychnine on transmission through the ventrobasal complex of the

cat thalamus. J Pharmacol Exp Ther, 170:181-189, 1969.

Isbell, H., Altschul, S., Kornetsky, C.H., Eiseman, A.J., Flanary,

H.G., and Fraser, H.F. Chronic barbiturate intoxication. An

experimental study. Arch Neurol Psychiat (Clin), 64:1-28, 1950.

Jaffe, J.H. Drug addiction and drug abuse. In: Goodman, L.S.,

and Gilman, A., eds. The Pharmacological Basis of

Therapeutics. New York: MacMillan, 1980. pp. 535-584.

Kalant, H., LeBlanc, A.E., and Gibbins, R.J. Tolerance to and

dependence on some non-opiate psychotropic drugs. Pharmacol Rev,

23:135-191, 1971.

Nicoll, R.A. The effects of anesthetics on synaptic excitation and

inhibition in the olfactory bulb. J Physiol (London),

223:803-814, 1972.

Nicoll, R.A. Eccles, J.C., Oshima, T.C., and Rubia, F.

Prolongation of hippocampal inhibitory postsynaptic potentials by

barbiturates. Nature (London), 258:625-627, 1975.

Okamoto, M., and Boisse, N.R. Sedative-hypnotic tolerance and

physical dependence. TIPS, 2:9-13, 1981.

Okamoto, M., and Hinman, D.J. Effects of individual variations in

drug elimination kinetics for production of pentobarbital

physical dependence. J Pharmacol Exp Ther, 226:52-56, 1983.

Okamoto, M., and Hinman, D.J. Physical dependence produced by long

duration, low dose chronic barbital treatment. Subst Alcohol

Actions/Misuse, 1984. (in press).

Okamoto, M., Boisse, N.R., Rosenberg, H.C., and Rosen, R.

Characteristics of functional tolerance during barbiturate

physical dependency production. J Pharmacol Exp Ther,

207:906-915, 1978.

346
Okamoto, M., Hinman, D.J., and Aaronson, L.M. Comparison of

ethanol and barbiturate physical dependence. J Pharmacol Exp

Ther, 218:701, 1981.

Okamoto, M., Rosenberg, H.C., and Boisse, N.R. Tolerance

characteristics produced during the maximally tolerable chronic

pentobarbital dosing in the cat. J Pharmacol Exp Ther,

192:555-569, 1975.

Ransom, B.R., and Barker, J.L. Pentobarbital selectively enhances

GABA-mediated post-synaptic inhibition in tissue cultured mouse

spinal neurons. Brain Res, 114:530-535, 1976.

Remmer, H. Tolerance to barbiturates by increased breakdown. In:

Steinberg, H., ed. Scientific Basis of Drug Dependence. New

York: Grune and Stratton, 1969. pp.111-128.

Rosenberg, H.C., and Okamoto, M. A method for producing maximal

pentobarbital dependence in cats: Dependence characteristics.

In: Singh, J.M., and Lal, H., eds. Drug Addiction: Neurobiology

and Influences on Behavior. Miami: Symposia Specialist, 1974.

Vol. 3, pp. 89-103.

Rosenberg, H.C., and Okamoto, M. Electrophysiology of barbiturate

withdrawal in the spinal cord. J Pharmacol Exp Ther,

199:189-197, 1976.

Rosenberg, H.C., and Okamoto, M. Loss of inhibition in the spinal

cord during barbiturate withdrawal. J Pharmacol Exp Ther,

205:563-568, 1978.

Schmidt, R.F. Pharmacological studies on the primary afferent

depolarization of the toad spinal cord. Pfugers Arch Gesmate

Physiol Menschen Tuse, 277:325-346, 1963.

Sharpless, S.K., and Jaffe, J.H. The electrical excitability of

isolated cortex during barbiturate withdrawal. J Pharmacol Exp

Ther, 151:321-329, 1967.

Werman, R., Davidoff, R.A., and Aprisan, M.H. Inhibitory action of

glycine on spinal neurons in the cat. J Neurophysiol, 31:81-95,

1968.

Wikler, A., Fraser, H.F., Isbell, H., and Pescore, F.T., EEGs

during cycles of addiction to barbiturates in man. EEG Clin

Neurophysiol, 7:1-13, 1955.

Wulff, M.H. The barbiturate withdrawal syndrome; a clinical and

electro-encephalographic study. EEG Clin Neurophysiol Suppl,

14:1-173, 1959.

ACKNOWLEDGEMENT

This study has been supported by research grant DA 00591 from the

National Institute on Drug Abuse. Drs. Norman R. Boisse, Donald J.

Hinman, and Howard C. Rosenberg contributed to various phases of

this study.

AUTHOR

Michiko Okamoto, Ph.D.

Department of Pharmacology

Cornell University Medical College

1300 York Avenue

New York, NY 10021

347
Behavioral and Biochemical

Studies in Rats After Chronic

Exposure to Nicotine

Leo G. Abood, Susan Grassi, Maria Costanzo, and

Jeffrey Junig

INTRODUCTION

Since nicotine is the major constituent in cigarette smoke responsible

for the reinforcing effect of smoking, studies on the behavioral and

pharmacologic tolerance of nicotine are of considerable interest to

the health consequences of smoking. As discussed by Russell (1979)

in a NIDA monograph, there are diverse pharmacologic, as well as

psychologic, factors involved in the assessment of smoking as a

dependence process; but attempts to develop appropriate animal

models towards this end have not been very successful. Although

tolerance is a requirement for a dependence producing drug,

tolerance occurs with a wide variety of psychotropic drugs not

producing dependence and can involve behavioral mechanisms as well

as alterations in drug disposition and pharmacodynamic

responsiveness (Jaffe 1980).

A review of the literature on the effect of chronic administration of

agonists or antagonists on various neurotransmitter receptors reveals

findings which are often conflicting and difficult to interpret.

Chronic administration of amphetamine (a dopamine agonist) has

been reported to decrease both agonist and antagonist binding (Muller

and Seeman 1979; Howlett and Nahorski 1979), while others failed to

observe any effect on striatal binding of 3 H-haloperidol (a

dopaminergic receptor antagonist).

It was recently reported that chronic treatment of rats with

diisopropylfluorophosphate (DFP) (a cholinesterase inhibitor)

decreased 3H-acetylcholine binding to cerebral cortex by 23%;

whereas chronic exposure to nicotine increased the amount of binding

by 25% (Schwartz and Kellar 1983). No change was observed on the

affinity of 3H-acetylcholine with either agent.

In another study involving continuous infusion of various doses of

nicotine to DBA female mice for 10 days, significant increases in 3Hnicotine binding, as well as 125I-bungarotoxin binding, occurred in

the midbrain and hippocampus with no change in the Kd values (Marks

et al. 1983). Although nicotine infusion had no effect on baseline

rotarod performance to acute injections of nicotine, the nicotineinduced bradycardia was diminished. They also observed a significant

tolerance to the acute effects of nicotine on rotarod performance

and body temperature.

348
Tolerance after Chronic Administration of Nicotine

In order to determine whether behavioral tolerance develops after

chronic administration of nicotine to rats, nicotine was chronically

administered either intraperitoneally of by infusion into the lateral

ventricles (table 1).

TABLE 1

Effect of Various Dosage Regimens and Routes of Administration on

Prostration Following Nicotine Administration into Fourth Ventricles

of Rats.

Dosage Schedule

Syndrome

Incidence of Prostration

5 nmoles nicotine twice daily

into fourth ventricles for 4 days

All rats responded day 1; 60%

responded day 2; 30% responded

day 4; 90% responded fully

after 3 days without nicotine.

Alzet minipump (50 nmoles/hr)

into lateral ventricle for 10 days

Normal response to 5 nmoles of

nicotine into fourth ventricle

on last day of infusion.

Acute injection of 12 μmole/kg of Most rats responded to 5nmoles

ip twice daily for 14 days. of nicotine into fourth

ventricles 12 hr after last

injection. Response was

slightly less than in controls.

When 5 nmoles of (-)-nicotine (neutralized with HCl and in a volume

of 1 μl) was administered twice daily for 4 consecutive days into the

fourth ventricle, all rats exhibited prostration on day 1, 60% on day

2, and only 30% on day 4. At least 90% of the rats responded to

5 nmoles of nicotine when administered into the fourth ventricle on

day 7 (3 days after the 4 daily doses of nicotine). After nicotine was

infused continuously into the lateral ventricle for 10 days at a rate of

50 nmoles/hr, the rats exhibited full prostration when 5 nmoles

nicotine was administered into the fourth ventricle on the last day of

infusion. In a third protocol, after rats were injected intraperitoneally with 12 μmoles/kg of nicotine twice daily for 14 days,

they exhibited prostration when given 5 nmoles of nicotine into the

fourth ventricle 12 hr after the last injection of nicotine.

A number of conclusions can be inferred from these studies. The

administration of nicotine into the fourth ventricle for 4 days appears

to produce some tolerance in the prostration syndrome; however, the

tolerance vanishes 3 days after the last dose. When nicotine is

349
infused continuously into the brain for 14 days, no tolerance develops

to the administration of nicotine directly into the fourth ventricle.

Finally, the chronic administration of nicotine systemically for 14

days resulted in only slight tolerance to nicotine given into the fourth

ventricle. It would appear that behavioral tolerance to nicotine, as

assessed by the prostration syndrome, is transient and diminishes with

chronic administration. The findings are in agreement with those of

Stollerman et al. (1974) that following chronic exposure to nicotine,

the development of tolerance initially increased and then decreased

as the dose of nicotine was increased.

Effect of Chronic Nicotine Treatment on 3H-Nicotine Binding in Rat

Brain

After administering 12 μmole/Kg of nicotine ip twice daily to a total

of 10 rats, the Kd and B max for (-)- 3H-nicotine binding were

determined 15 hr after the last injection on membrane preparations

of whole brain employing Scatchard analyses. A control group of rats

maintained under similar environmental conditions received saline. A

third group of 5 rats, which received a continuous infusion of nicotine

into the lateral ventricle from an Alzet minipump at the rate of

50 nmole/hr for a period of 10 days, was analyzed similarly 15 hr

after infusion ceased. The binding studies revealed no significant

differences between the control and nicotine-exposed groups in either

the high or low Kd or Bmax values (table 2).

TABLE 2

Effect of Chronic Nicotine Treatment on Bmax and Kd of 3H-(-)-

Nicotine Binding to Rat Brain Membranes

Conditions

B max K d

moles/mg ×101 4 x10 1 0

High Low High Low

Normal 0.5 ± 0.1 2.9 ± 0.4 1.0 ± 0.3 3.5 ± 0.2

Chronic (twice 0.4 ± 0.1 2.7 ± 0.2 0.8 ± 0.2 3.3 ± 0.3

daily)

Chronic (infusion) 0.4 ± 0.1 2.8 ± 0.3 1.1 ± 0.2 3.5 ± 0.3

Values are expressed as mean ± sd with n=3-4 rat brains. Data were

derived from Scatchard plots.

These findings, which fail to demonstrate any alteration in the

nicotine receptor in rat brain following chronic exposure to nicotine,

are consistent with the behavioral studies demonstrating only a slight

decrease in the sensitivity of the nicotine-induced prostration.

Although they would appear to be at variance with the study of Marks

et al. (1983) reporting up-regulation of nicotine binding, their studies

350
were performed on DBA mice which developed a significant behavioral and pharmacologic tolerance after nicotine infusion. It is also

conceivable that regional brain areas may reveal differences not

observable in whole rat brain.

Binding of Nicotine Analogues and Other Agents to Rat Hepatocytes

Using Either (-)- or (+)-3H-Nicotine

In the course of investigating the metabolism of nicotine by isolated

rat hepatocytes, it was observed that the hepatocytes exhibited a

specific affinity for nicotine and its analogues and that the affinity

appeared to be greater for the (+)- than the (-)-enantiomer (table 3).

TABLE 3

Binding of Nicotine Analogues and Other Agents to Rat Hepatocytes

Using Either (-)- or (+)-3H-Nicotine.

Agent

I C 5 0 M

(-)-3H-nicotine (+)-3H-nicotine

(-)-nicotine

(+)-nicotine

nornicotine

N'-nicotonium

(-)-cotinine

N'-ethyl nornicotine

N-methyl nicotine

N'-ethyl nornicotine

N'-propyl nornicotine

N'-butyl nornicotine

mecamylamine

haloperidol

chlorpromazine

imipramine

carbamylcholine

hexamethonium

1 x 10-6

3 x 10-7

3 x 10-5

1 x 10-3

5 x 10-4

3 x 10-6

1 x 10-3

5 x 10-6

8 x 10-6

2 x 10-5

2 x 10-5

2 x 10-4

3 x 10-4

1 x 10-4

1 x 10-3

1 x 10-3

5 x 10-7

3 x 10-8

1 x 10-5

1 x 10-3

3 x 10-4

1 x 10-6

1 x 10-3

2 x 10-6

2 x 10-6

8 x 10-6

1 x 10-5

1 x 10-4

1 x 10-4

8 x 10-5

1 x 10-3

1 x 10-3

At a final concentration of 1 x 10-9M (-)- or (+)-3H-nicotine, the IC50

values were found to be 1 x 10-6 and 3 x 10-7M in the presence of a

100-fold excess of the unlabeled (-)- and (+)-nicotine respectively;

351
whereas, with (+)-3H-nicotine the values were 5 x 10-7 and 3 x 10-8M

in the presence of a 100-fold excess of the unlabeled enantiomers,

With increasing chain length of alkyl substituents on the pyrrolidine N

of nicotine, there was a corresponding decrease in the affinity, using

either (-)- or (+)-3H-nicotine. As discussed elsewhere (Abood et al.,

1983), there was an excellent correlation between the binding affinity

of this homologous series of nicotine analogues and their ability to

produce prostration when administered ivc. Quaternisation of either

the pyrrolidine or pyridine N of nicotine greatly reduced the affinity

for hepatocytes; while nicotinic cholinergic agents such as

carbamylcholine and hexamethonium exhibited no affinity.

It can be concluded from these studies that specific nicotine binding

exists in isolated hepatocytes. The binding is reversible and

nonstereoselective; and since it is not saturable at concentrations of

(-)- or (+)-nicotine lower than 10-4M, the binding may be related to a

transport system involved in the metabolism-disposition of nicotine.

When the hepatocytes are even slightly damaged, they lose their

ability to bind or take up nicotine. It is noteworthy that the

relationship of binding affinity of the various nicotine analogues in

hepatocytes agrees with the relative psychotropic potency of the

analogues.

Effect of Chronic Nicotine Treatment on the Metabolism of (-)- and

(+)-3H-Nicotine by Isolated Hepatocytes

In an effort to determine whether chronic nicotine administration

(systemic administration for 14 days) altered its metabolism by liver,

hepatocytes were isolated from rat liver after collagenase perfusion

and modification of the method of Baur and Heldt (1971).

The hepatocytes were incubated for various times at 37°C in Hank’s

buffer containing 10- 9M (-)-3H-nicotine + 10- 5M unlabeled (-)-

nicotine + l0-3M nicotinamide at pH 7.5. The rate of disappearance

of the nicotine present was 2.5 x 10-6 mmoles/min/mg protein for the

control preparation compared to a value of 3.0 x l0-6 for the rats

chronically exposed to nicotine. Since the difference was not

statistically different, it would appear that chronic nicotine

treatment does not alter its metabolism by liver. The results are in

agreement with those of Marks et al. (1983) who failed to observe any

effect of chronic nicotine infusion on the rate of nicotine

disappearance from the blood of DBA mice in vivo.

Effect of Chronic Nicotine Administration on Lever Press Avoidance

of Aversive Shock

A number of groups have reported on the antinociceptive actions of

nicotine in rodents (Tripathi et al. 1982; Sahley and Bemtson 1979).

Dewey et al. (1970) have reported that the analgetic action of

nicotine on a molar basis was comparable to that of morphine. A

number of mechanisms have been proposed for the antinociceptive

action of nicotine including a nicotinic cholinergic one (Sahley and

352
Berntson 1979; Tripathi et al. 1982), an opioid one in mice but not

rats (Tripathi et al. 1982), and one involving a-adrenergic receptors

(Tripathi et al. 1982). It is apparent from such studies that the action

of nicotine on antinociception is a complex one. There also remains

the issue as to whether nicotine is analgetic or is simply elevating the

animal’s threshold to an aversive stimulus (Abood et al. 1981). In an

effort to further investigate this action of nicotine, another paradigm

was employed for measuring the response to an aversive stimulus.

In a previous study (Abood et al. 1981), it was reported that nicotine

administered intraventricularly to rats significantly elevated the

threshold to an aversive shock, employing a shock-avoidance

paradigm in which the animals were delivered 16 random shocks over

a 5-min period beginning with an initial near-subthreshold shock

(0.4 mA) and extending to a maximal shock of 1.4 mA. (Abood et al.

1981; D’Amato and Fazzaro 1966; for experimental details.) A

comparison of the effects of acute and chronic nicotine

administration on the frequency of lever pressing for shock avoidance

is presented in table 4. After rats had established a frequency

TABLE 4

Effect of Acute and Chronic Nicotine Administration on

Discriminative Lever-Pressing for Shock Avoidance

Mean Frequency % Decrease

Acute Study

Before nicotine 40.4 ± 2.6

After nicotine 19.9 ± 2.3 50

Naloxone + nicotine 22.0 ± 3.0 45

After Before nicotine 35.5 ± 3.0

chronic

nicotine After nicotine 25.5 ± 2.5 28

Results are presented as mean frequency of lever pressing before and

after saline or 15 nmoles (-)-nicotine ivc. Analysis of variance

employing a univariate and multivariate regression problems program

yields p <0.0001. Values are expressed as mean frequency of

responses with variance. Chronic rats received nicotine ip twice

daily for 14 days as described in text. A dose of naloxone of 4 mg/kg

ip was administered 20 min prior to nicotine ivc.

baseline of lever pressing, they were administered 15 nmoles of (-)-

nicotine (5 μl) ivc and again tested. In the group not receiving

nicotine chronically, a 50% decrease in the frequency of lever

353
pressing occurred after nicotine ivc; and, in the chronically treated

groups the decrease was 28%. After chronic administration of

nicotine, the mean frequency of response prior to ivc nicotine was

lowered while the frequency after nicotine ivc was elevated. When

naloxone was administered 20 min prior to nicotine ivc, there was no

effect on the frequency of response. It would appear from these

studies that following chronic nicotine administration, some

tolerance had developed to the ability of nicotine to elevate the

threshold to shock. Furthermore, the effect of nicotine on

aversiveness does not appear to be mediated by an opioid system.

CONCLUSION

A number of conclusions concerning the problem of tolerance to

nicotine may be derived from the present study. Although tolerance

develops in rats during the earlier stages of chronic administration,

behavioral tolerance to intraventricularly administered nicotine tends

to diminish with further exposure to nicotine. Some tolerance

appears to develop in the ability of nicotine to attenuate aversiveness

to electric shock; while the initial tolerance to nicotine-induced

prostration appears to disappear. There does not appear to be any

alteration in either the affinity or density of 3H-nicotine binding

sites in rat brain with chronic administration of nicotine, nor any

increase in its rate of metabolism by liver.

REFERENCES

Abood, L.G.; Reynolds, D.T.; Booth, H.; and Bidlack, J.M. Sites and

mechanisms for nicotine’s action in the brain. Neurosci Biobehav,

5:479-486. 1981.

Abood, L.G.; Grassi, S.; and Costanza, M. Binding of optically pure

(-)-3H-nicotine to rat brain membranes. FEBS Lett 157:147-149,

1983.

Baur, H., and Heldt, H.W. Use of Isolated Liver Cells and Kidney

Tubules in Metabolic Studies. North Holland, Amsterdam, 1971,

357 pp.

D’Amato, M.R., and Fazzaro, J. Discriminated lever-pressing avoidance learning as a function of type and intensity of shock. J Comp

Physiol Psychol, 61:313-315, 1966.

Dewey, W.L.; Harris, L.S.; Howes, J.F.; and Nuite, J.A. The effect of

various neurohumoral modulators on the activity of morphine and

the narcotic antagonists in tail-flick and phenylquinoline test. J

Pharmacol Exp Ther, 175:435-442, 1970.

Howlett, D.R., and Nahorski, S.R. Acute and chronic amphetamine

treatments modulate striatal dopamine receptor binding sites.

Brain Res, 161:173-178, 1979.

Jaffe, J.H. Drug addiction and drug abuse. In: Gilman, A.G.,

Goodman, L.S., and Gilman, A., eds. Goodman and Gilman’s The

Pharmacological Basis of Therapeutics. 6th edition. New York:

MacMillan, 1980, pp. 535-584.

Marks, M.J.; Burch, J.B.; and Collins, A.C. Effects of chronic nicotine infusion on tolerance development and nicotinic receptors. J

Pharmacol Exp Ther, 226:817-825, 1983.

354
Muller, P., and Seeman, P. Pre-synaptic subsensitivity as possible

basis for sensitization by long term mimetics. Europ J Pharmacol,

55:149-157, 1979.

Russell, M.A.H. Tobacco dependence: Is nicotine rewarding or

aversive? In: Krasnegor, N.A., ed. Cigarette Smoking as a

Dependence Process. National Institute on Drug Abuse Research

Monograph 23. DHEW Pub. No. (ADM) 79-800. Washington, D.C.:

Supt. of Docs., U.S. Govt. Print. Off., 1979. pp. 158-184.

Sahley, T.L., and Berntson, G.G. Antinociceptive effects of central

and systemic administration of nicotine in the rat.

Psychopharmacology 65:279-283, 1979.

Schwartz, R.D., and Kellar, K.J. Nicotinic cholinergic receptor of

binding sites in the brain: Regulation in vivo. Science 220:214-216,

1983.

Stollerman, J.P.; Bunker, P.; and Jarvik, M.E. Nicotine tolerance in

rats: Role of dose and dose interval. Psychopharmacologia 30:329-

342, 1974.

Tripathi, H.L.; Martin, B.R.; and Aceto, M.D. Nicotine-induced

antinociception in rats and mice. Correlation with nicotine levels.

J Pharmacol Exp Ther 221:91-96, 1982.

ACKNOWLEDGEMENTS

This research was supported by DA00464 from The National Institute

on Drug Abuse and a grant from the Council for Tobacco Research.

AUTHORS

Leo G. Abood, Ph.D.

Center for Brain Research-Box 605

University of Rochester Medical Center

601 Elmwood Avenue

Rochester, New York 14642

Susan Grassi

Center for Brain Research-Box 605

University of Rochester Medical Center

601 Elmwood Avenue

Rochester, New York 14642

Maria Costanzo

Center for Brain Research-Box 605

University of Rochester Medical Center

601 Elmwood Avenue

Rochester, New York 14642

Jeffrey Junig

Center for Brain Research-Box 605

University of Rochester Medical Center

601 Elmwood Avenue

Rochester, New York 14642

355
Psychobiological Foundations of

Behaviors Induced by

Amphetamines

Philip M. Groves and David S. Segal

The abuse of psychomotor stimulants such as amphetamine and

cocaine is a problem of major proportions in contemporary life.

It has been well documented that drug abuse results in serious

untoward medical and social consequences. Research on the effects

of amphetamine has attempted to establish models of the behavioral

consequences of stimulant administration in animal subjects and to

determine the biomedical sequelae of acute and long-term abuse of

amphetamine, especially as the stimulant drug affects the structure and function of the brain. It is now well known that longterm amphetamine administration can result in the development of

psychosis in humans. Further, when amphetamine is administered to

animal subjects it produces a characteristic pattern of behavioral

response which includes locomotor activation and the occurrence of

repetitive species-specific stereotyped behaviors. Extensive

research efforts have now established numerous sites of action of

stimulant drugs in the brain and have begun to relate the biochemical and neurophysiological effects of the drug to the characteristic behavioral profiles that have been described.

BEHAVIORAL CONSEQUENCES OF ACUTE AND CHRONIC AMPHETAMINE ADMINISTRATION

When administered acutely to animal subjects, amphetamine produces

a characteristic dose- and time-related pattern of behavioral

responding. The laboratory rat has now been studied extensively

as a model for understanding the biomedical and behavioral effects

of the drug. Some of the features of the amphetamine response

following acute administration of the drug and following multiple

daily injections are illustrated in figure 1 where it can be seen

that the acute effects of the drug as well as the changes that

occur in the behavioral response with chronic administration are

both dose- and time-dependent. Repeated administration of relatively low doses (0.5 - 1.5 mg/kg), which initially produce

enhanced locomotion as the predominant response, results in a gradual increase in locomotor activity with respect to both peak

effect and duration. With moderate doses of the drug (1.5 - 2.5

mg/kg), amphetamine-induced locomotion during the early period of

356
FIGURE 1. Alterations in response pattern with repeated daily

injections of a constant dose of d-amphetamine sulfate. Top: For

low doses, 5 to 10 days of administration results in a progressive

increase in locomotion as reflected in the magnitude and duration

of the response. Middle: For intermediate doses, the duration of

stereotypy episodes is increased, and by 3 to 5 days, a continuous

stereotypy phase (stippled shading) emerges. Poststereotypy

hyperactivity is also enhanced. Bottom: For the high dose range,

stereotypy (stippled shading) appears more rapidly and is intensified by the second injection, although the duration of the stereotypy phase is not correspondingly prolonged. Locomotion after the

stereotypy phase is progressively increased. Reprinted from David

S. Segal, Marc A. Schuckit, 1983, with permission. @Raven Press,

1983.

357
the response pattern is gradually replaced by progressively longer

episodes of focused stereotypies. Repeated injections of higher

doses (2.5 - 7.5 mg/kg), which acutely produce triphasic response

patterns consisting of an initial increase in locomotion followed

by a period of intense stereotyped behavior and a subsequent phase

of enhanced locomotion, result in a more rapid onset of the

storeotypy and a progressive augmentation in the later phase of

hyperactivity. Observation of the animal subjects confirms the

more rapid onset of stereotypy with a corresponding decrease in

locomotion.

Although repeated amphetamine administration augments various components of the acute response without apparent qualitative change,

the behavior that emerges does not simply reflect a shift to the

left in the amphetamine dose-response curve. Although the stereotypy appears more rapidly and is intensified at some doses, the

duration of the stereotypy phase is not correspondingly increased,

as it is with increasing doses administered acutely. Evidence

such as this has nor accumulated and shows a complex, dose- and

time-dependent alteration which can be characterized as a sequential pattern of behavioral change resulting from multiple amphetamine injections in an extensive array of animal species. Such

evidence is clearly important in the evaluation of potential

underlying mechanisms. Further, alterations in the behavioral

response to amphetamine have nor been detected in animal subjects

at least one month following a single acute injection of the drug,

suggesting an extremely powerful and potentially long-lasting

influence of amphetamine and rolatod psychomotor stimulant drugs

on the brain.

NEURONAL MECHANISMS OF ACTION OF AMPHETAMINE

Substantial evidence accumulated ovor the past 2 decades suggests

that a significant site of action of amphetamine in the brain can

be identified with the catecholamine cell groups of the brainstem.

By releasing catecholamines, amphetamine is believed to act

indirectly on various targets in the brain including both postsynaptic targets of catecholamine neuron projections as roll as

the presynaptic neurons themselves (Groves et al. 1975; Groves and

Rebec 1976; Groves and Tepper 1983; Segal and Schuckit 1983).

This has been demonstrated convincingly by recording the activity

of catecholamine neurons during the administration of amphetamine

to experimental animals. Among the early and best documented evidence illustrating the effect of amphetamine on catecholamine neurons was the demonstration that amphetamine administration led to

a marked inhibition of neuronal activity in two intensively studied cell groups of the brain, the dopaminergic pars compacta of

substantia nigra (Bunney et al. 1973) and the noradrenergic locus

coeruleus (Graham and Aghajanian 1971). This effect is illustrated for two representative neurons in figure 2. Intravenous

administration of amphetamine produces a decline in neuronal firing of dopaminergic neurons of tho substantia nigra while a similar and even more pronounced effect also occurs for neurons in the

noradrenergic locus coeruleus.

358
This profound influence of amphetamine on the activity of catecholamine neurons was originally believed to be due to changes in

long-axoned feedback loops from sites of catecholaminergic innervation but is now thought to include the release of catecholamines

locally within those cell groups. For example, amphetamine has

now been shown to release dopamine from dopaminergic neurons of

substantia nigra both in vitro (Paden et al. 1976) and in vivo

(Cheramy et al. 1978). Anatomical evidence suggests that both

catecholaminergic presynaptic axons and dendrites are found within

the catecholaminergic cell groups (Groves et al. 1979; Groves and

Wilson 1980; Grovos and Linder 19833) and would be available to

release these neurotransmitters in response to amphetamine.

FIGURE 2. Illustration of the difference in sensitivity to

intravenously administered amphetamine between noradrenergic neurons of the nucleus locus coeruleus and dopaminergic neurons of

the substantia nigra pars compacta. The solid line indicates the

spontaneous firing rate of a representative dopamine neuron; the

dashed line is the firing rate of a noradrenergic neuron. Sequential intravenous injections of 0.25 mg/kg d-amphetamine sulfate

were given (arrows) for a cumulative dose in each case of 0.75

mg/kg. Whereas the locus coeruleus cell shows greater than 80%

inhibition to 0.75 mg/kg, the substantia nigra neuron only exhibits a 25% reduction in firing rate to this doss. Reprinted from

Philip M. Groves, James M. Tepper, 1983, with permission. ©Raven

Press, 1983.

359
NEUROPHYSIOLOGICAL CONSEQUENCES OF PRESYNAPTIC RECEPTOR ACTIVATION

Since activation of catecholamine receptors on dopaminergic and

noradrenergic neurons causes an inhibition of neuronal firing by

these cells, it was of particular interest to determine if activation of presynaptic receptors presumed to exist on the terminals

of these neurons would affect excitability in the same way. Groves

and associates have now developed a method for measuring the excitability of catecholaminergic synaptic endings in the brain. This

method is illustrated in figure 3. Electrical stimulation of the

dopaminergic terminal field is affected while recording simultaneously from the region of the dopaminergic cell body. Antidromic

action potentials reaching the initial segment and/or cell body of

the dopaminergic neuron are recorded to determine whether the

stimulus has produced an action potential. By plotting the probability of antidromic activation of the neuron against a range of

stimulation currents, a excitability curve may be constructed that

may be used to define the excitability of the synaptic terminal

field. Now by administering various pharmacological or other

treatments, variations in terminal excitability can be seen and

appear to correlate with the release of catecholamines by the

catecholaminergic synaptic endings. Using this paradigm for

determining terminal excitability of individual dopaminergic and

other monoaminergic neurons in the brain, Groves and associates

have now been able to show that activation of these receptors,

like those in the region of the cell body and dendrites, also

seems to be accompanied by a decrease in terminal excitability, as

illustrated in figure 4. As shown there, the currents necessary

to activate dopaminergic cells antidromically by stimulation of

their terminal fields in the neostriatum are uniformly increased

by systematically administered amphetamine, and a similar effect

occurs when amphetamine is infused in low concentrations and

volumes directly into the vicinity of the terminals activated by

the electrical stimulus. As illustrated in figure 5, antidopaminergic drugs such as the receptor-blocking agent haloperidol produce increased terminal excitability and can reverse the agonistinduced decrease in terminal excitability, the effects being

represented by a uniform shift to the left in the excitability

curve.

Evidence such as this suggests that the extracellular accumulation

of dopamine that is produced by amphetamine causes activation of

presynaptic receptors and that these, in turn, result in a change

in polarization and/or conductance of the dopaminergic synaptic

ending. Such an effect could, for example, represent a hyperpolarization of the synaptic ending produced by presynaptic receptor

activation (Groves et al. 1981).

When similar experiments are carried out while monitoring the

excitability of noradrenergic terminals in frontal cortex, a similar influence is also seen. Catecholaminergic agonists for the

alpha-2-noradrenergic receptor lead to reduced terminal excitability when infused locally into the region of the terminal field,

including low concentrations and volumes of d-amphetamine

360
FIGURE 3. Schematic illustration of the experimental paradigm used

for measuring nigrostriatal dopaminergic terminal excitability. A

bipolar electrode is positioned within the neostriatum for

antidromic stimulation of the dopaminergic striatal afferents originating in the ipsilateral substantia nigra pars compacta. An

extracellular recording electrode is positioned within the ipsilateral pars compacta, and the frequency of antidromic response to

neostriatal stimulating currents of varying strengths is measured.

361
FIGURE 4. Top: Decrease in neostriatal dopamine terminal excitability following intravenous administration of d-amphetamine sulfate. Following an initial injection of 0.5 mg/kg d-amphetamine,

the plot of neostriatal stimulating currents versus the proportion

ofantidromic responses detected by an extracellular recording in

the ipsilateral substantia nigra pars compacta is shifted uniformly to the right, indicating a decrease in terminal excitability

at all current values. A second injection of 0.5 mg/kg damphetamine produces a further shift to the right in the excitability curve and an additional decrease in terminal excitability.

Bottom: Decrease in excitability of dopaminergic terminal in neostriatal induced by the local microinfusion of d-amphetamine directly into the stimulating site. Following an infusion of 0.31

μl of 1 μM d-amphetamine, the excitability curve is shifted to the

right, indicating a decrease in terminal excitability. A second

infusion of d-amphetamine (0.31 μl, 1 μM) 5 min later produces a

further decrease in terminal excitability. Reprinted from Philip

M. Groves, James H. Tepper, 1983, with permission. ©Raven Press,

1983.

362
(Nakamura et al. 1981). However, systemically administered amphetamine produces a more complex influence on terminal excitability.

Low doses of the drug that do not lead to a profound inhibition of

firing produce a decrease in terminal excitability, but this

effect is reversed when the drug is administered in quantity sufficient to produce a cessation of impulse flow in the neuron

(Nakamura et al. 1981). This has been interpreted to indicate

that the release of catecholamine by the noradrenergic neuron in

response to amphetmine is partly dependent upon impulse traffic.

Evidence such as this has now demonstrated persuasively that

amphetamine, by releasing catecholamines, affects the activity and

excitability of the catecholamine neuron and, by inference, the

various postsynaptic targets of these systems throughout the

brain. While there has been a continuing effort to ascertain the

mechanisms underlying the inhibition of neuronal activity in there

cell groups following amphetamine administration, there has only

recently been a concerted attempt to determine mechanisms of

action at these manifold sites where amphetamine produces alterations in the brain. A recent review of such evidence indicates

that amphetamine administration leads to marked alterations in

neuronal firing throughout the reticular formation of the brainstem, the caudate nucleus, putamen and globus pallidus and related

striatal cell groups, the hypothalamus, cerebellum, and the cerebral cortex (see recent review by Groves and Tepper 1983). From

evidence such as this, it becomes apparent that the influence of

the drug is extremely widespread, is both dose- and timedependent, and cannot be easily explained by a monotonic increase

in the release of catecholamines.

A recent theoretical framework for intrastriatal neuronal circuitry has emphasized the interconnections of the common spiny neurons of neostriatum and has proposed that these form a large,

lateral inhibitory network which has been termed the Spiny I cell

matrix (Groves 1983). When this network is affected by amphetamine, we can imagine that at low doses of the drug, the processing

of information through this lateral inhibitory matrix is improved

but as the dose is increased or as repeated doses are administered, the ability of the neostriatum to respond to the patterns

of activation that arrive from substantia nigra, midline thalamus,

and cerebral cortex in time and space becomes compromised, and

stereotyped behaviors and other emergent patterns of behavioral

responding become apparent. The analysis of the environment and

the interaction with it that normally require information processing by the neostriatum become impaired and only those most primitive and securely represented patterns of behavioral responding

emerge, signalling a progressive constriction of the behavioral

repertoire. The various components of the emergent behaviors may

reflect an augmentation or diminution of component behaviors that

appeared upon acute administration of the drug, and lateral inhibitory processes intrinsic to the neostriatum may be expected to

lead to the evolution of these different degrees of apparent

behavioral sensitization and tolerance. New approaches toward

understanding the manifold mechanisms of action of amphetamine and

363
FIGURE 5. Top: Increase in neostriatal dopaminergic terminal excitability induced by intravenous injection of haloperidol lactate

(Haldol). Following an initial injection of 0.025 mg/kg haloperidol the excitability curve is shifted nearly uniformly to the

left, indicating an increase in terminal excitability inferred

from the reduced neostriatal stimulating currents necessary to

elicit varying proportions of antidromic responses in the ipsilateral substantia nigra pars compacta. A second injection of 0.025

mg/kg haloperidol produces a further increase in terminal excitability. Bottom: Reversal of the increase in dopaminergic terminal

excitability caused by a local infusion of amphetamine by a subsequent local infusion of haloperidol. In this representative example, after a control level of excitability was determined, an

infusion of d-amphetamine (0.31 μl, 1 μM) was made into the neostriatal stimulating rite which resulted in a shift to the right

in the excitability curve (O--O), indicating a decrease in excitability. Approximately 5 min later, an infusion of haloperidol

(0.31 μl, 0.10 μM) was made into the same site via a second cannula. This infusion reversed the effects of the first infusion

and within 5 min the excitability curve was shifted dramatically

to the left (X--X), past the baseline, representing an overall

increase in terminal excitability. Reprinted from Philip M.

Groves, James R. Tepper, 1983. ©Raven Press, 1983.

364
the way in which its actions are altered by long-term administration of the drug have now begun (e.g., Gale, this volume). Continued effort along these various lines of research holds considerable promise for further understanding the ways in which this

and related drugs affect the brain and behavior.

REFERENCES

Bunney, B.S., Walters, J.R.. Roth, R.H. and Aghajanian, G.K.

Dopaminergic neurons: Effect of antipsychotic drugs and

amphetamine on single cell activity. J Pharmacol Exp Ther,

185: 560-571, 1973.

Cheramy, A., Nieoullon, A. and Glowinski, J. In vivo

changes in dopamine release in cat caudate nucleus and

substantia nigra induced by nigral application of various

drugs including GABAergic agonists and antagonists. In

Garattini, S., Pujol, J.F., and Samanin, R., eds. Interactions

Between Putative Neurotransmitters in the Brain. New York:

Raven Press, 1978. pp. 175-190.

Graham, A. and Aghajanian, G. Effects of amphetamine on single

cell activity in a catecholamine nucleus, the locus coeruleus.

Nature, 234: 100-102. 1971.

Groves, P.M. A theory of the functional organization of the

neostriatum and the neostriatal control of voluntary movement.

Brain Res Rev, 5:109-132, 1983.

Groves, P.M., Fenster, G.A., Tepper, J.M., Nakamura, S., and

Young, S.J. Changes in dopaminergic terminal excitability

induced by amphetamine and haloperidol. Brain Res,

221:425-431, 1981.

Groves, P.M., and Linder, J.C. Dendro-dendritic synapses in

substantia nigra: Descriptions based on analysis of serial

sections. Exp Brain Res, 49:209-217, 1983.

Groves, P.M., and Rebec, G.V. Biochemistry and behavior: Some

central actions of amphetamine and certain antipsychotic drugs.

Annu Rev Psychol, 27:91-127, 1976.

Groves, P.M., Staunton, D., Wilson, C.J., and Young, S.J. Sites

of action of amphetamine intrinsic to catecholaminergic nuclei:

Catecholaminergic presynaptic dendrites and axons. Prog

Neuropsychopharmacol, 3:315-335, 1979.

Groves, P.M., and Teppe, J.M. Neuronal mechanisms of action

of amphetamine. In: Creese, I.. ed. Stimulants: Neurochemical,

Behavioral, and Clinical Perspectives. New York: Raven Press,

1983. pp. 81-129.

Groves, P.M.. and Wilson, C.J. Monoaminergic presynaptic axons

and dendrites in rat locus coeruleus seen in reconstructions of

serial sections. J Comp Neurol, 193:853-862, 1980.

Groves, P.M., Wilson, C.J., Young, S.J., and Rebec, G.V. Selfinhibition by dopaminergic neurons. Science, 190:522-529,

1975.

Nakamura, S., Tepper, J.M., Young, S.J., and Groves, P.M.

Neurophysiological consequences of presynaptic receptor

activation: Changes in noradrenergic terminal excitability.

Brain Res, 226:155-170, 1981.

365
Paden, C., Wilson, C.J., and Groves, P.M. Amphetamine-induced release

of dopamine from the substantia nigra in vivo. Life Sci,

19:1499-1506, 1976.

Segal, D.S., and Schuckit, M.A. Animal models of stimulant-induced

psychosis. In: Creese, I., ed. Stimulants: Neurochemical,

Behavioral, and Clinical Perspectives. New York: Raven Press,

1983. pp. 131-167.

ACKNOWLEDGEMENTS

This work was supported in part by USPHS Grants DA-02854 and DA01568 from the National Institute on Drug Abuse; P.M.G. and D.S.S.

are the recipients of Research Scientist Awards DA-00079 from the

National Institute on Drug Abuse and MH-70183 from the National

Institute of Mental Health.

AUTHORS

Philip M. Groves, Ph.D., Professor of Psychiatry, Department of

Psychiatry, School of Medicine, University of California, San

Diego, La Jolla, California 92093

David S. Segal, Ph.D., Professor of Psychiatry, Department of

Psychiatry, School of Medicine, University of California, San

Diego, La Jolla, California 92093

366
Index

Abstinence Signs

alpha-N-Acetyltransferase

Acetylcholine

ACTH

Adenosine

Adenyl Cyclase

Adrenergic Neurons

Alcohol

alpha-Amidation

Amphetamine

Amygdala

Analgesia

Anticonvulsant Activity

Apomorphine

Ascorbic Acid

Autoradiography

Aversive Stimuli

Barbiturates

Benzodiazepines

Binding Sites

Bombesin

alpha-Bungarotoxin

Buprenorphine

Calcium

Calmodulin

Cannabidiol

Caudate Nucleus

Chemoreceptor

beta-Chlornaltrexamine

Chloroquine

Cholecystokinin

Cholera Toxin

Cholinergic Neurons

Cocaine

Convulsant Activity

Copper

Cortex, Cerebral

Cross-Dependence

Cross-Desensitization

Cross-Tolerance

Cyclazocine

Cyclic AMP

Cycloheximide

Cyclohexyladenosine

16, 19, 20, 210

171

84, 348

169

82, 83, 131

96, 109, 119, 137, 261

51, 121, 139

51, 87, 197

170, 175, 178

28, 177, 315, 348, 356

155, 242

58, 164

31

325

175, 180

148, 294

329, 352

333

98, 162, 169, 173

72, 164

213, 226

348

114

96, 104, 130, 267

96, 130

314

99, 151, 222

13

163

127

226

77, 90, 140

83, 121, 139, 221

58, 315, 323

313, 341

175

151, 155, 226

28. 72

139; 250

47, 70, 270

123, 188

89, 97, 110, 262

110, 124, 281

86

367
Cytochrome Oxidase 295

DADLE (d-ala, d-leu-enk) 71, 98, 127, 137, 155, 268

DAME (d-ala, met-enk) 64, 120

DAMEA (d-ala, met-enk-amide) 63, 111

2-Deoxyglucose 217, 241, 294

Desensitization 126, 133, 138, 146, 204, 244

Dihydromorphine 60, 63, 151

Diprenorphine 111, 121, 125

Dopamine 95, 146, 151, 222, 323, 358

Down Regulation 77, 100, 115, 127, 137, 146

Drug Disposition 31, 43, 335

Dynorphin 73, 125, 162, 241

EEG 218, 240, 342

Electroshock 313

beta-Endorphin 102, 125, 302

Enkephalin 64, 98

Ethylketocyclazocine 60, 71, 125, 163

Etorphine 31, 101, 115, 147

Evoked Potentials 240, 261

FK-33-824 193, 268

Funaltrexamine 73

GABA 104 323, 326

Ganglia 157: 260, 278

Genetics 50

Glutamic Acid Decarboxylase 221, 326

Glycosylation 170

Ground Squirrel 210

GTP or Guanyl Nucleotide 96, 111, 119. 148

Guinea Pig Ileum 72, 81, 136, 147, 163, 260

Heroin 28

Hibernation 209

Hippocampus 100, 151, 155, 228, 242, 348

Histochemical Studies 297

Human Subjects 18, 307

Hydromorphine 39

Hyoscine 84

Hyperpolarization 100

Hypophoria 15, 21

Hypothalamus 154, 180, 185, 226

Inbreeding 51

Ion Fluxes 96, 97, 100, 276

Intracellular Recordings 251

Ketocyclazocine 60, 63, 187

Kindling 317

LAAM (1-alpha-acetomethadol) 45

Leu-enkephalin 64, 227, 303

Levorphanol 123, 189, 268

Locus Coeruleus 136, 154, 250, 358

Luteinizinq-Hormone 185, 193

Mammillary Nuclei 297

alpha-Melanotropin 169

Melanotropin-release Inhibiting Factor 225

Meperidine 31

Met-enkephalin 83, 154, 227, 303

368
Methadone

Monkeys

Morphine

Mouse vas Deferens

Nalorphine

Naloxone

Naltrexone

Needs (States)

alpha-Neo-Endorphin

Neuropharmacology

Neuroanatomy

Neuroblastoma x Glioma Cells

Neuroleptic Drugs

Neurotensin

Neurotransmitters

Nicotine

Noradrenergic Neurons

Norepinephrine

Normorphine

Nucleus Accumbens

N-Units

Opioid Receptors

Opioids

Pain Threshold

Pentazocine

Pentylenetetrazol

Pertussis Toxin

Pharmacokinetic Factors

8-Phenyltheophylline

Phenytoin

Phospholipase C

Pimozide

Pituitary Gland

Postsynaptic Potentials

Post-translation Modification

Precursors to Opiopeptides

Propranolol

Protein Kinase

Prostaglandin

Protein Phosphorylation

Purinoceptor

Rabbit Vas Deferens

Receptor Density

Receptor Internalization

Receptor Occupancy

Saccharin

Secretory Granules

Sedative-Hypnotic Activity

Seizure Threshold

Sensitization

Serotonin, 5-hydroxytryptamine

SKF-10047

Single Cells

, 101, 110,

19, 101

39

16, 28, 44, 58

123, 164, 191

71, 136

39, 112

39, 60, 110, 119, 147, 186

112, 147, 154, 302

13, 21

73, 125

27

151

110, 119, 137

98, 146, 324, 329, 348, 364

213, 226

51, 97, 155, 220

30, 348

358

180

53, 76, 82

155

111, 113, 140

70, 115, 120, 146, 157,

228, 260

28, 154

28, 148

30

314

81, 89, 111, 140

333

82

314

130

329

170, 178, 180, 185, 294

251, 344

169

168

329

9 7

97, 121 138

96, 170

83

163

126, 147, 197, 293

127

33, 137

329

171

333, 341

32

104

218, 263, 272

125, 187

261

369
Single-Dose Tolerance

Sleep Cycle

Somatostatin

Spinal Cord

Stereotypy

Striatum

Substance P

Substantia Nigra

Sufentanil

Supersensitivity

Telencephalon

delta-9-Tetrahydrocannabinol

Testosterone

Tetrodotoxin

Thalamus

Thermic Effects

Thyroid Releasing Hormone

Tunicamvcin

Tyrosine Hydroxylase

Up-Regulation

Withdrawal Symptoms

Yohimbine

29

24

226

268

329, 356

95, 99, 151, 155, 326, 360

83

97, 15l, 155, 358

83

100, 137, 146, 156, 199,

226, 312

151

29, 313

184, 201

82

151, 155

31, 164, 210, 228

226

124

96, 170

100, 146, 280

18, 22, 40, 46, 195, 339

82

370
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