National Institute on Drug Abuse

RESEARCH

MONOGRAPH SERIES

Drug

Discrimination:

Applications to

Drug Abuse

Research

U.S. Department of Health and Human Services • Public Health Service • National Institutes of Health

116
Drug Discrimination:

Applications to Drug

Abuse Research

Editors:

Richard A. Glennon, Ph.D.

Medical College of Virginia

Virginia Commonwealth University

Torbjörn U.C. Järbe, Ph.D.

Department of Psychology

University of Uppsala

Jerry Frankenheim, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

Research Monograph 116

1991

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, MD 20857
ACKNOWLEDGMENT

This monograph is based on the papers from the “International Drug

Discrimination Symposium” held from June 25 to June 27, 1990, in

Noordwijkerhout, The Netherlands.

COPYRIGHT STATUS

The National Institute on Drug Abuse has obtained permission from the

copyright holders to reproduce certain previously published material as noted in

the text. Further reproduction of this copyrighted material is permitted only as

part of a reprinting of the entire publication or chapter. For any other use, the

copyright holder’s permission is required. All other material in this volume

except quoted passages from copyrighted sources is in the public domain and

may be used or reproduced without permission from the Institute or the authors.

Citation of the source is appreciated.

Opinions expressed in this volume are those of the authors and do not

necessarily reflect the opinions or official policy of the National Institute on Drug

Abuse or any other part of the US. Department of Health and Human Services.

The U.S. Government does not endorse or favor any specific commercial

product or company. Trade, proprietary, or company names appearing in this

publication are used only because they are considered essential in the context

of the studies reported herein.

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

selectively included in the coverage of American Statistics Index, Biosciences

information Service, Chemical Abstracts, Current Contents, Psychological

Abstracts, and Psychopharmacology Abstracts.

DHHS publication number (ADM)92-1878

Printed 1991

ii
Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Richard A. Glennon

1

A Historical Perspective on Drug Discrimination . . . . . . . . . . . .

Donald A. Overton

5

Discriminative Stimulus Properties of Hallucinogens and

Related Designer Drugs . . . . . . . . . . . . . . . . . . . . . . . . 25

Richard A. Glennon

Discriminative Stimulus Properties of Amphetamine,

Cathinone, and Related Agents . . . . . . . . . . . . . . . . . . . . 45

A.J. Goudie

Discriminative Stimulus Effects of Cocaine . . . . . . . . . . . . . .

William L. Woolverton

61

Discriminative Stimulus Functions of

Cannabinoids/Cannabimimetics . . . . . . . . . . . . . . . . . . . .75

Torbjörn U.C. Järbe and Diane A. Mathis

Discriminative Stimulus Properties of Nicotine:

Mechanisms of Transduction . . . . . . . . . . . . . . . . . . . . . . 101

John A. Rosecrans and Heidi F.

Villanueva

Discriminative Stimulus Properties of Benzodiazepines

and Several New Anxiolytics . . . . . . . . . . . . . . . . . . . . . . 117

Richard Young

iii
Distinctive Discriminative Effects of Ethanol . . . . . . . . . . . . . . 131

Herbert Barry III

Training Dose: Influences in Opioid Drug Discrimination . . . . . . . 145

Sandra D. Comer, Charles P. France,

and James H. Woods

Discriminative Stimulus Properties of Phencyclidine and

Other NMDA Antagonists . . . . . . . . . . . . . . . . . . . . . . . 163

Robert L. Balster

Discriminative Stimulus Effects of Psychomotor

Stimulants and Benzodiazepines in Humans . . . . . . . . . . . . . 181

Chris-Ellyn Johanson

Tolerance to Drugs Acting as Discriminative Stimuli . . . . . . . . . 197

Alice M. Young

Tolerance: Role of Conditioning Processes . . . . . . . . . . . . . 213

Shepard Siegel

Responding to Drug-Related Stimuli in Humans as a

Function of Drug-Use History . . . . . . . . . . . . . . . . . . . . 231

Ronald N. Ehrman, Steven J. Robbins,

Anna Rose Childress, A. Thomas

McLellan. and Charles P. O’Brien

State Dependency as a Mechanism of Central Nervous

System Drug Action . . . . . . . . . . . . . . . . . . . . . . . . . . 245

Francis C. Colpaert

State-Dependent Learning With Social Drugs . . . . . . . . . . . . . 267

Geoff Lowe

Discriminative Stimulus Effects of Drug Mixtures in Rats . . . . . 277

I.P. Stolerman, E.A. Mariathasan, and

H. S. Garcha

iv
Application of Drug Discrimination With Drugs of Abuse

To Develop New Therapeutic Agents . . . . . . . . . . . . . . . . . 307

Theo Frans Meert

Intracranial Stimulation as Reinforcer for Neuropeptide

Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

Muriel Gewiss, Christian Heidbreder, and

Philippe De Witte

Drug Discrimination Used To Study Drug Withdrawal . . . . . . . . . 337

M. W. Emmett-Oglesby and G.A. Rowan

Schedule-Induced Self-Injection of Drugs . . . . . . . . . . . . . . . 359

George Singer

Use of Drug Discrimination in Drug Abuse Research . . . . . . . . . 369

James B. Appel, Lisa E. Baker, Rita L.

Barrett, Julie Broadbent, Elizabeth M.

Michael, Elizabeth E. Riddle, and Bette J.

Van Groll

Symposium Critique . . . . . . . . . . . . . . . . . . . . . . . . . . 399

L.S. Harris

v
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ISBN 0-16-036035-8
Introduction

Richard A. Glennon

The drug discrimination (DD) paradigm is essentially a drug detection

procedure whereby animals are trained to recognize or discriminate the

stimulus effects of a given dose of a particular training drug from those of (1) a

different dose of the same training drug, (2) a different training drug, or, more

commonly, (3) saline/vehicle (i.e., a nondrug condition). The most commonly

employed apparatus for conducting drug discrimination studies is a two-lever

operant chamber; however, several other types of test procedures, including

three-lever chambers, are also being used. Several species of animals are quite

popular, primarily rat, pigeon, and monkey; the use of other species, including

human, is now becoming more common.

The DD paradigm, by itself, cannot be used to completely characterize a novel

agent. This is true of any pharmacological procedure. However, the DD

paradigm can be used to investigate a wide variety of pharmacological aspects

relating to the stimulus properties of a drug. These aspects include, for

example, time of onset and duration of action, mechanism of action, similarity of

effect to other agents, structure-activity relationships, activity of metabolites,

and identification and development of potential antagonists. Recently, the

paradigm has been used to investigate the processes of tolerance and

withdrawal.

There have already been several international drug discrimination symposia.

For the most part, these symposia were concerned more with the technique

itself than with the application of the technique for the investigation of a

particular problem. Prior emphasis has been on, for example, the use of drug

discrimination in central nervous system pharmacology and on the transduction

mechanisms involved in the stimulus effects of drugs. Because many of the

drugs used in DD studies have a potential for abuse or, indeed, are already

noted for their abuse, we felt the time was right to address the application of DD

in drug abuse research.

1
This monograph presents selected papers from the 1990 International Drug

Discrimination Symposium held in Noordwijkerhout, The Netherlands, June

25-27,1990. The symposium was cosponsored by the European Behavioral

Pharmacology Society and the Society for the Stimulus Properties of Drugs.

The theme for the 1990 symposium was “Drug Discrimination: Applications in

Drug Abuse Research.” The meeting consisted of a number of invited

presentations on drug abuse research; it was also open to a general poster

session. Abstracts for most of the presentations were published as a

supplement to Psychopharmacology volume 101,1990. Unlike previous

symposia, this meeting brought together members of the international

academic, industrial, government, and drug enforcement communities to

discuss the relevance and application of the paradigm to a single health-related

issue: drug abuse. Presentations ranged from the basic science, such as

mechanisms of action and structure-activity relationships, to DD studies

involving human subjects, to the role of DD in the legal control of abused

substances. Another topic of interest was the use of abused substances as

training drugs for developing new therapeutic agents.

The consensus of the symposium participants was that the DD paradigm is an

important, useful, and very versatile tool for investigating drugs of abuse.

However, the committee invited several speakers to address this issue in a

formal sense. A separate session devoted to this topic was entitled “Invited

Perspectives.” The committee selected a member of the U.S. Drug

Enforcement Administration (Dr. F. Sapienza) to address the issue from a law

enforcement perspective. Two additional speakers were also requested to

critique the DD paradigm and/or the symposium per se. To obtain a balanced

viewpoint, one speaker (Dr. J. Appel) was selected because of his contributions

to the field of DD research and because he could address the issues as an

informed insider. The other speaker (Dr. L. Harris) has had extensive

experience in the field of drug abuse research and is familiar with the technique

of DD but does not actively conduct such studies in his own laboratory; his

comments may be taken as those of an unbiased outsider. Dr. Harris was given

the difficult task of critiquing the entire symposium.

The Scientific Organizing Committee for the symposium was composed of Dr.

Richard A. Glennon (United States, chair), Dr. Toby Järbe (Sweden, cochair),

Dr. John A. Rosecrans (United States), Dr. Ian P. Stolerman (United Kingdom),

and Dr. Alice M. Young (United States). The Scientific Organizing Committee

was assisted by a local organizing committee without whose help the

symposium would have been impossible. The local committee consisted of Drs.

2
C.L. Broekkamp, A.R. Cools, M.R. Kruk (chair), J.H.C.M. Lammers, B.M.

Spruyt, and A.M. Van der Poel. The Scientific Organizing Committee

acknowledges the efforts of the local committee in helping make this meeting a

success.

The Scientific Organizing Committee was awarded a conference grant from the

National Institute on Drug Abuse. These funds secured the success of the

symposium and provided awards for several young investigators on a

competitive basis. The award winners include Drs. SD. Comer, J.P. Druhan,

C.P. France, S. Gleeson, L.H. Gold, G.A. Rowan, S. Smurthwaite, H.F.

Villanueva, and E.I. Walker.

The Scientific Organizing Committee also gratefully acknowledges the financial

support of the following contributors:

Abbott Laboratories (United States)

Astra Research Center (Sweden)

Bayer AG (West Germany)

Boehringer lngeilheim Nederland B.V.

Bristol-Myers Company (United States)

Burroughs Wellcome Company (United States)

Duphar Nederland B.V.

E.I. du Pont de Nemours and Company (United States)

European Behavioral Pharmacology Society

Groupe de Recherche de Servier (France)

Hoechst-Roussel Pharmaceuticals Inc. (United States)

ICI Pharmaceuticals Groups (United States)

Lilly Research Laboratories (United States)

Merck Sharp & Dohme Research Laboratories (United States)

Ministry of Welfare, Health, and Cultural Affairs (Netherlands)

National Institute on Drug Abuse (United States)

Research Biochemicals, Inc. (United States)

Schering AG (Switzerland)

Schering-Plough Research (United States)

G.D. Searle Company (United States)

Society for the Stimulus Properties of Drugs

The Upjohn Company (United States)

Dr. S. van Zwanenbergstichting (Netherlands)

3

A Historical Perspective on Drug

Discrimination

Donald A. Overton

INTRODUCTION

Drug-induced state-dependent learning (SDL), as well as the ability of

physiological states to control retrieval of memories, has been known at least

since 1830. Until 1950, however, understanding of this area was based

primarily on clinical descriptions of fugue states, somnambulism, dream recall,

and cases of multiple personality. Since 1950, a series of experimental

demonstrations of the properties of SDL and drug discriminations (DDs), along

with progressive modifications of the DD procedure-each change in itself

relatively inconsequential-has led to the development of the DD paradigm that

is being employed now. Its properties make it an extremely useful tool for

preclinical investigation of a variety of pharmacological and psychological

questions. These technical and conceptual developments have allowed

widespread acceptance of the DD paradigm as a method for conducting

preclinical research. This paper reviews the 19th- and 20th-century history of

concepts, experiments, and clinical observations that led to the current status of

knowledge about drug-induced SDL and DDs.

SDL IN THE 19TH CENTURY

Throughout the 19th century there was widespread interest in hypnosis, fugue

states, somnambulism, multiple personality, and other forms of amnesia

(Ellenberger 1970). Various explanations for these clinical phenomena were put

forward, including (after 1830) the idea that the physiological state of the

organism determined, at each instant in time, which memories were accessible

to consciousness. Combe (1830, pp. 520-522) wrote as follows.

The patient was a girl of 16 [who had episodic somnambulistic

attacks]. . . . The circumstances [events] which occurred during the

5
paroxysm were completely forgotten by her when the paroxysm was

over, but were perfectly remembered during subsequent paroxysms.

Dr. Abel informed me of an Irish porter to a warehouse, who forgot,

when sober, what he had done when drunk; but being [again] drunk,

again recollected the transactions of his former state of intoxication.

On one occasion, being drunk, he had lost a parcel of some value,

and in his sober moments could give no account of it. Next time he

was intoxicated, he recollected that he had left the parcel at a certain

house, and there being no address on it, it had remained there

safely, and was got on his calling for it.

The only conclusion which seems to arise . . . is that before memory

can exist, the organs [have] to be affected in the same manner, or to

be in a state analogous to that in which they were, when the

impression was first received.

These facts cannot be accounted for in a satisfactory way; but by

communicating a knowledge of their existence, attention will be

drawn to them, and future observations and reflection may ultimately

throw light upon the subject.

Combe’s report was a direct extension of the zeitgeist of the time. It had been

known for a century that memories for the hypnotic state could not be retrieved

in the normal waking state, although they clearly persisted in unconscious form

(Chastenet de Puységur 1809). The similarities between hypnosis and

somnambulism were sufficient so that hypnosis was often called artificial

somnambulism (Ellenberger 1970). Somnambulistic patients were also known

to have state-dependent recall sometimes, as indicated in the first sentences of

the preceding quotation. However, the inclusion of a drug state (alcohol) as a

determiner of memory retrieval was probably a novel contribution by Combe,

because the case described above was quoted during the next half century as

evidence for drug-induced SDL and no other previous (or subsequent) evidence

was ever cited.

For many years after Combe’s “Irish porter” case was reported, the idea that

alcohol could produce SDL was carried forward through the medical literature

(Elliotson 1840, p. 646; Macnish 1834, p. 78; Macnish 1835, p. 30; Winslow

1860, p. 338). In 1868, Wilkie Collins incorporated SDL produced by drugs into

the plot of his novel The Moonstone. The novel was published in serialized form

6
and widely read, thus making the concept of SDL even more available to the

public.

INTEGRATION OF SDL INTO THEORIES OF LEARNING AND RECALL

The period 1880-l910 saw a major change in the status of SDL, in that it was

integrated into comprehensive theories of memory retrieval and personality.

Ribot proposed that the control of memory retrieval by bodily state was

specifically mediated by the mechanism of “organic sensations,” which he

enumerated, and which were essentially equivalent to stimuli later denoted by

the term “interoceptive stimuli” (Ribot 1891, pp. 23-30). Ribot was also

somewhat more explicit than previous writers in asserting that the stimulus

effects of the normal no-drug state were equally important as those of abnormal

states, and that the memory retrieval in the no-drug (N) state could occur only

as long as N cues were present. Hence he made an explicit assertion that

N-state cues were as salient as drug (D)-state cues, and that equally large

impairments in memory retrieval would be produced by D N and by N D

state changes (Ribot 1882, pp. 108-l15). This was the first mechanistic theory

for SDL, and it was a stimulus theory.

Semon explicitly integrated ethanol-induced SDL into his comprehensive model

for memory formation and retrieval (Semon [1904] 1921, pp. 144-145; [1909]

1923, p. 180); see discussions of Semon (Schacter et al. 1978; Schacter 1982,

p. 185). Coriat (1906) conducted experiments to show that memories for

periods of alcoholic blackout could sometimes be retrieved if special techniques

were employed, and he mentioned the Irish porter case as showing that such

memories sometimes became available if the subject again became intoxicated.

In 1914, Prince published a comprehensive description of dissociation and

unconscious processes, which included the following statement.

We may. . . lay it down as a general law that during any dissociated

state, no matter how extensive or how intense the amnesia,

[memories of] all the experiences that can be recalled in any other

state, whether the normal one or another dissociated state, are

conserved and, theoretically at least, can be made to manifest

themselves. And, likewise and to the same extent, during the normal

state [memories of] the experiences which belong to a dissociated

state are still conserved, notwithstanding the existing amnesia for

those experiences. (Prince 1914, p. 78)

7
It should be noted, however, that Prince did not explicitly refer to SDL in a

fashion comparable to Ribot, Semon, or Cornbe; instead, he mentioned

reintoxication only as a method of assisting retrieval for periods of alcoholic

blackout without proposing state equivalence as the mechanism that was

responsible for this retrieval (p. 81). Prince’s book was reprinted at least as late

as 1929. Semon’s books were translated into English in the 1920s, and some of

Ribot’s books were reprinted as late as 1910, thereby making the SDL concept

available at that time.

DISAPPEARANCE OF THE CONCEPT OF SDL

Although drug-induced SDL had been accepted as a property of the brain for

three-quarters of a century, I have not been able to trace the concept past the first

quarter of the 20th century. Although I am not very well informed about intellectual

trends during this period, I will venture a few comments about this development.

SDL had been an integral part of the exaggerated interest in the unconscious

that developed during the 19th century. For example, Whyte (1960, p. 168)

states that popular acceptance of the idea of unconscious determination of

behavior progressed from being “credible” in 1700 to being “topical” by 1800 to

being “fashionable” by 1875 (as reflected by the presence of SDL in The

Moonstone). Ellenberger (1970) characterizes the 19th century as the era of the

“first dynamic psychiatry,” which was primarily concerned with phenomena of

somnambulism, catalepsy, multiple personality, hysterical symptoms, and

hypnotism (p. 111); he also reports that this school of thought was actively

rejected starting in 1880 (p. 171); the usually accepted date for its demise was

1900 (p. 174).

During the transition phase before the “new dynamic psychiatry” was firmly

established by Freud and others, some workers still accepted the existence of

SDL, as indicated by mention of SDL in the writings of Ribot and Semon.

However, the phenomenon was not considered as important as it had been 50

years earlier, as indicated by its very brief mention in books published after

1900. Incidentally, it is not certain that Semon or Prince had even read the

original case report by Combe, because they cited Ribot as the source of the

Irish porter case and Ribot in turn described the Irish porter case as “well

known,” but did not explicitly cite Combe (Ribot 1882, p. 115).

Later, psychologists were influenced by Freud’s idea that most dissociation

occurred because of avoidance of one sort or another, that is, because of

8
strong emotional reactions leading to repression. This idea apparently led them

to reject the earlier tendency of viewing all dissociative phenomena as reflecting

the operation of physiological processes. Janet, for example, characterizes the

attempt to explain somnambulism (which term he used to refer to multiple

personality and hysterical symptoms), hypnotic suggestibility, and automatic

writing (other techniques previously accepted as routes to the unconscious) in

terms of physical brain function as “pure childishness” and “useless reveries”

(Janet 1907, p. 63). Possibly, as new ideas about the origins and contents of

the unconscious were developed, SDL just fell through the cracks and was lost.

SDL clearly was not a dynamic process, and it was not a frequently observed

physiological process. Indeed, no second case of drug-induced SDL had ever

been reported, and so the whole existence of the phenomenon rested on the

single case report by Combe.

Subsequently, experimental psychology apparently became increasingly

disinterested in dissociation of all types. Hilgard (1977, p. 10) has commented on

other factors that possibly underlay the rapid loss of interest in dissociation that

apparently occurred in the newly developing behavioral psychology in the 1930s; in

his view, interest waned more by social consensus than because of any new data

that explicitly reduced the significance of unconscious processes. For whatever

reasons—although knowledge of dissociation, somnambulism, fugues, and

multiple personality persisted—this writer has not found instances after Prince

(1914) in which state-dependent retrieval was mentioned in the literature.

PRECURSORS OF REDISCOVERY

Goodwin (1972) pointed out that Charlie Chaplin’s 1931 movie “City Lights”

depicts events remarkably resembling ethanol-induced SDL (McDonald et al.

1965), although alternative explanations for the events portrayed in the film are

possible. If the film depicts SDL, then where did Chaplin get the idea? Chaplin’s

autobiography does not clarify the issue (Chaplin 1964, p. 325); indeed, his

wording suggests that the film was not intended to portray SDL. Unfortunately,

Chaplin’s autobiographical statements are reported to be highly unreliable

(Geduld 1985) which leaves us with substantial doubts about what the film was

intended to portray. All that can be concluded is that “City Lights” may possibly

have reflected a knowledge of SDL on Chaplin’s part.

In 1937, Girden and Culler reported an experimental demonstration of

drug-induced dissociation between the drug state produced by raw curare and

the no-drug condition. Initially they studied the conditioned leg flexion response

9
in dogs. Later they expanded their work to include dissociation in other

response modalities. Girden’s experiments had serious methodological

difficulties resulting from his use of a drug that paralyzed the experimental

subjects. Although Girden tested a number of dogs, only a few were tested by

means of paradigms that would allow evaluation of whether SDL had occurred

or not, and Gardner’s replication effort was largely unsuccessful (Gardner 1961;

Gardner and McCullough 1962). Nonetheless, Gitden’s work was apparently

accepted by the scientific community and appeared in textbooks of

physiological psychology for 20 years after it was reported (e.g., Morgan and

Stellar 1950, p. 450). Girden did not cite the 19th-century literature on SDL.

Although few of the individuals who studied the stimulus properties of drugs

during the following 30 years acknowledged that their work was influenced by

Girden’s findings, his studies mark the starting point of modern experimental

work on SDL and DDs.

STATUS AS OF 1950

By 1950, the following ideas had been published: (1) Current physiological state

determines which memories are retrievable at any instant in time. (2) Both the

no-drug state and abnormal states have equally important influence on memory

retrieval. (3) Control of recall by bodily states may or may not be mediated by

the mechanism of interoceptive sensations. (4) Environmental contextual cues

also determine recall in a fashion analogous to the control of recall by

interoceptive stimuli. (5) Drugs may induce dissociation by functionally

decorticating the animal (Girden and Culler 1937).

The data underlying these assertions included many clinical observations of

hypnosis, somnambulism, fugue, dissociation, and multiple personality

collected during the 19th century, along with one reported case of SDL in a

delivery man who got drunk on the job around 1830, and some muscle spindle

twitches in Girden’s dogs.

Developments that directly underlie current DD methods started in 1951, and

the ensuing 25 years saw a progressive increase in the amount of experimental

attention devoted to stimulus properties of drugs. These studies can be

organized according to various themes. Some studies dealt with theoretical

models for SDL. Some measured whether significant amounts of SDL were

produced by clinically used doses of tranquilizers. Many were intended to

investigate neurochemical issues. The remainder of this paper will selectively

10
review this history, focusing on developments that contributed to the

development of the DD method as the practically applicable investigational tool

it is now.

THE FIRST DRUG DISCRIMINATION STUDY

Conger (1951) reported the first DD study. He was trying to study the effects of

alcohol on approach and avoidance behavior and realized that the effects he

was observing could be caused either by the intrinsic effects of ethanol or by

stimulus generalization deficits resulting from a change in drug state between

training and testing, that is, by SDL. In his words:

The avoidance was established under the condition of sober; one

group was then tested under the . . . condition of sober, and the

other group under the different condition of inebriation. Thus it is

logically possible that the decrease in the avoidance response might

be due solely to a change in the animal’s condition (regardless of the

direction of the change) rather than to any specific effect of alcohol

[because] it seems likely that a change from sobriety to inebriation

(or vice versa) produces a change in the animal’s stimulus situation.

(Conger 1951, p. 15)

Conger then made an unprecedented contribution by pointing out that if ethanol

did have stimulus effects, then the existence of these effects could be

demonstrated by using a discrimination training procedure. In an approach and

avoidance task, Conger’s rats learned to approach when drunk and avoid when

sober, or vice versa, thus becoming the first animals in history to learn a DD in

a laboratory setting. It is interesting that Conger’s study never answered the

question that led him to perform it. By showing that ethanol could exert

discriminative control, the rats indicated that stimulus generalization effects

might occur and that the intrinsic effects of ethanol might be confounded with

SDL in the experiments that Conger had conducted. However, his results never

answered the questions of whether or to what degree such confounding actually

had occurred. In Conger’s report, no prior literature was cited as indicating that

drugs could act as stimuli.

THE 2x2 EXPERIMENTAL DESIGN

In the same year, Auld (1951) published the first study I know of that used a 2x2

experimental design to evaluate drug stimulus effects. His experiment tested

11
the effect of tetraethylammonium (TEA) on escape and avoidance performance.

No SDL effects were found, which is reasonable because TEA primarily

produces effects outside the brain. Why was Auld concerned enough about

stimulus effects to use a 2x2 design?

In subsequent years, Miller repeatedly proposed the 2x2 experimental design

as a method for determining the relative strength of SDL effects and other drug

effects (Grossman and Miller 1961; Miller 1957; Miller and Barry 1960). He

actively promoted use of the 2x2 paradigm, arguing that it was unwise to

attempt to determine the intrinsic effects of drugs without including the extra

experimental groups that would demonstrate stimulus generalization

decrements if they were present. For reference, the following table describes

the structure and properties of that design.

Table 1. The 2 by 2 experimental design

Group Training

Session

State

Test Effects Present

Session in Test Session

State Data

1 N

2 N

3 D

4 D

N None

D Retrieval deficit +

performance deficit

+ SDL

N Memorization

deficit + SDL

D Memorization

deficit + retrieval

deficit +

performance deficit

The quantitative size of the SDL effect is computed from test session

performance by groups 1 + 4 - 2 - 3. Any effects of drug during training trials

on memory consolidation is computed from test performance of groups 1 + 2

- 3 - 4. Depressant drug effects on performance during test trials cannot be

distinguished from drug-induced impairments of memory retrieval, and the

combined size of these two effects is computed from test performance in

groups 1 + 3 - 2 - 4. The design assumes that all effects are linearly additive

and that SDL is symmetrical with equally large decrements after D N and

N D state changes. If any effects other than the postulated ones are present,

then the computed effect sizes will be incorrect.

12
STUDIES ON SDL

Conger had demonstrated discriminative control only after a moderately

prolonged series of training trials. However, a number of other investigators

soon tested whether the stimulus effects of drugs might be strong enough to

produce the generalization decrements postulated by Conger; they conducted

2x2 studies, many of which yielded evidence for weak SDL effects (Barry et al.

1962; Belleville 1964; Heistad 1957; Heistad and Torres 1958; Murphy and

Miller 1955; Otis 1964; Shmavonian 1956). Not all SDL studies from this era

resulted from the hypothesis that drugs produced stimulus effects; other models

for brain function, later termed “neurological” models by Bliss (1974)) also

predicted SDL, and several SDL studies were conducted by investigators

seeking to test these models (Overton 1964; Sachs 1966). In addition,

Holmgren (1964a, 1964b) reported SDL without, apparently, having any

theoretical predilection about where the phenomenon came from. Among these

studies, Shmavonian obtained the first results actually indicating the occurrence

of stimulus generalization decrements.

SOURCES OF REBIRTH OF INTEREST IN SDL

It appears that investigators in the 1950s and 1960s had no direct awareness of

the 19th-century literature on SDL and that their studies derived from other

sources. We should note that Guthrie’s stimulus elements theory was well

known in 1950 (Guthrie [1935] 1960). It was significantly similar to the theories

previously developed by Ribot and Semon, which had been devised in the first

place to explain SDL, among other phenomena. Hence, the possibility of SDL

was certainly not contrary to theories of learning extant in 1950, and it is hardly

surprising that investigators, even if they did not accept Guthrie’s model, at

least wanted to test whether their results might reflect its operation.

To clarify this issue, this writer recently interviewed (by telephone) several

investigators of that era. Girden reports that he had no knowledge of the earlier

published reports of SDL. Auld reports that he used the 2x2 design because

Miller encouraged him to do so, but without any particular expectation on his

part of finding drug stimulus effects. Conger attributes the stimulus change

explanation for his results as arising from the interest in drive stimuli that was

extant at the time and that made it appear reasonable to him that drugs also

could produce analogous stimulus effects. Barry’s thesis research was on the

effects of changes in the level of hunger on performance in a straight alley, and

he subsequently used a similar design when testing the effects of drugs on

13
performance in the same task. Auld, Conger, and Barry were students of Neal

Miller, and their publications report their research with him. Miller, then, was

centrally important in causing the experiments that rediscovered DDs and SDL

and he reports that he had no knowledge of the 19th-century literature on

drug-induced SDL. Barry reports that Miller encouraged him to use a 2x2

design to test for state change effects but discouraged him from performing DD

studies. Heistad reports that he was substantially influenced by Guthrie and

entertained the idea that a substantial portion of the effects of drugs on

behavior might result from stimulus generalization decrements. Shmavonian

reports that he used the 2x2 design, not as a tool to see stimulus effects, but

because it would allow detection of carryover effects. Otis had conducted an

interesting Ph.D. thesis on the possibility that drive stimuli might act as

conditioned stimuli and might, if paired with punishment during infancy, elicit

anxiety as a conditioned response later in life (Otis 1956). He later viewed

drugs as a convenient method to induce comparable changes in internal state

and for this reason used them in his 1964 study.

All the preceding reports are based on personal communications in 1990 and,

obviously, do not reflect the entire range of recollections by which these

investigators were influenced at the time they did their work. Nonetheless, the

investigators I contacted were unanimous in denying any direct knowledge of

the 19th-century work, and it seems reasonable to conclude that the interest in

SDL that reappeared in the 1950s and 1960s did not result from direct

knowledge of the 19th-century literature. Instead, the concept was apparently

reinvented on the basis of ideas prevalent at the time. Some of these ideas, in

turn, can be traced back to the 19th century, when they were first invented to

explain SDL and other dissociative phenomena.

The difficulty in evaluating the influence of prior ideas and findings is well

illustrated by the experience of this writer. My own “rediscovery” of SDL

occurred because of a neurological model (Overton 1964) that occurred to me

during a lecture while I was a graduate student. It seemed to me a novel

prediction of SDL based on ideas derived from the behavior of electronic

feedback systems, which I had recently studied as a student of engineering.

The idea led me to perform experiments that demonstrated SDL produced by

pentobarbital (Overton 1964). However, those experiments occurred about 12

months after I had read about (and forgotten) Girden’s work. I also probably had

read and forgotten Hebb’s 1949 (p. 201) paragraph on state-dependent cell

assemblies. So was my theory really novel, or was it a reevocation, in altered

form, of ideas that I had previously read? Subjectively, I was surprised when I

14
later learned of the work of Girden, Hebb, Conger, and Auld. Nonetheless,

many precedents for my ideas existed in the literature, and I had read some of

them.

PROLIFERATION OF THEORIES FOR SDL

Combe’s initial description of SDL did not propose a mechanism by which SDL

occurred; it was simply a statement of the fact that SDL did occur. As far as we

know, Ribot provided the first mechanistic explanation for SDL by postulating

that the physiological state of the body was reflected in “organic” sensations

and that recurrence of these sensations was a prerequisite for memory

retrieval. Girden proposed a different model based on the assumption that

drugs could functionally decorticate the animal (Girden and Culler 1937).

Conger, Auld, Miller, Otis, and others accepted a stimulus generalization model

for SDL (similar to Ribot’s model). Hebb proposed that cell assemblies should

be state specific; this was not a sensory model. Indeed, once the scientific

world was convinced again that SDL really existed, a plethora of mechanisms

for it were proposed; toward the end of this period I published a review paper

that summarized 22 different models that had been suggested as responsible

for SDL (Overton 1978).

These various competing theories have for the most part never been explicitly

tested. However, by the process of voting, the scientific community has come to

support a stimulus theory predominantly. Probably this support has come about

largely because the stimulus theory has proved so flexible in accommodating to

the very diverse and complicated phenomenology that DD experiments have

increasingly elucidated.

REDISCOVERY OF HISTORY

It appears that not one of the investigators who contributed to the experimental

development of the SDL and DD paradigms between 1937 and 1980 knew

explicitly of the 19th-century interest in SDL. Several writers related DDs and

SDL to multiple personality and other forms of dissociation, but not one single

citation of the early work was made. Finally, this amnesia about our history was

lifted by Siegel (1982) after someone attending one of his lectures pointed out

that the plot of The Moonstone implied knowledge of (1) contextual control of

retrieval, (2) SDL, and (3) one-trial tolerance. In The Moonstone, Collins quoted

the Irish porter case verbatim and mentioned Combe’s name; this allowed

15
Siegel to find part of the 19th-century literature on SDL and publish a

description of it (Siegel 1982,1983).

SYMMETRICAL TASKS

The first improvement in DD methodology after Conger’s study was that of

using a symmetrical task (Overton 1961). In such a task, stimulus effects of

drugs are reflected by response selection instead of by response failure, and

hence the rate-depressing effects of drugs are not confounded with stimulus

effects to anywhere near the same degree as in a single-response go/no-go

task (in which the rate of occurrence of a response is used as an index of

retention). The two-response T-maze task was selected to accomplish this

purpose after pilot experiments in a straight alley go/no-go maze yielded results

that were difficult to interpret because drug effects on rate and SDL effects were

confounded in that task.

RELATIONSHIP BETWEEN SDL AND DDS

Were the stimulus effects that produced discriminative control in Conger’s

experiment actually the same drug effects that produced SDL decrements in

2x2 experiments? The most convincing evidence for an affirmative answer to

this question comes from my own experiments. In a shock escape T-maze task,

I was able to demonstrate both SDL amnesias caused by changes in state and

D versus N DDs established by repeated training trials. With very high doses of

certain drugs, only two sessions were required to learn one response in the D

state and an opposite response in the N state, thus demonstrating SDL. With

tower doses of the same drugs, 30 or 40 training sessions were required to

establish discriminative control. When intermediate doses were tested, the

amount of training required to establish D versus N DDs turned out to be

inversely proportional to dosage. Hence, whatever action of the drugs was

producing SDL and DDs, it was apparently the same action (Overton 1974).

FURTHER IMPROVEMENTS IN THE DD PARADIGM

By 1970, it was possible to list about 20 different behavioral paradigms that had

been employed in DD studies by one investigator or another (Overton 1971)

and a few of these are identifiable as milestones in the development of the

paradigm that is most commonly employed at present. We mentioned

previously that introduction of a two-choice task was a major improvement over

tasks in which only a single response was measured. The next major step was

16
the use of operant tasks (Harris and Balster 1966). Even the earliest results

obtained in operant tasks indicated that they were sensitive to doses

considerably lower than could be detected in the T-maze task, and when the

symmetrical two-lever operant DD task was used, the operant paradigms

provided both high sensitivity and a relatively uncontaminated indication of

discriminable drug effects (Kubena and Barry 1969; Morrison and Stephenson

1969). One additional important development did not occur until 1975, when

Colpaert et al. (1975, 1976) introduced the use of a fixed ratio (FR-10)-

versus-extinction schedule of reinforcement. This schedule produced much

higher accuracy of lever selection than previously had been observed in

operant tasks, and it was soon adopted by the majority of DD investigators.

MULTIPLICITY OF DRUG CUES

In 1960 it had never been demonstrated whether more than a single drug cue

existed, and investigators entertained the possibility that rats might be

discriminating “normal” versus “abnormal” irrespective of what drug was used

as the training compound. Hence, Overton (1966) felt obliged to conduct a

series of studies designed to demonstrate that atropine and pentobarbital

produced two qualitatively different states (or stimulus effects). Stewart (1962)

also produced data indicating that at least two different drug states existed.

By the end of the 1960s however, a remarkable development had occurred and

Overton (1971) was able to report 10 different types of drugs that were

discriminable from no drug and from one another. This report led to the

generalization that each different type (or class) of drugs would produce a

different discriminable effect (the “one cue per pharmacological class” idea) and

to the expectation that this pattern might continue to be found as additional

types of drugs were tested in the DD paradigm.

Analogous studies conducted in operant DD tasks yielded an additional

important result. In those tasks, after D-versus-N training with a particular drug,

animals would select the no-drug lever when tested with any novel drug. Hence

the no-drug lever was the default response selected by trained animals under

all drug conditions except the drug condition used for training (and drug

conditions closely related to the training condition). It should be noted that this

pattern of results differed from the one obtained 10 years earlier by Overton. In

his T-maze task, rats made 30-70 percent D choices under most novel drugs

and only a few test conditions led to consistent selection of the no-drug arm of

17
the maze. The operant task produced a different pattern of results, however, for

reasons that have never been adequately explained.

ONE MORE THEORY

In 1975, Colpaert et al. introduced an apparently minor restatement of the

stimulus theory, which in fact caused a fundamental revision in the predictions

of that theory. Colpaert argued that rats discriminated presence versus absence

of the training drug’s cues during D-versus-N DD training. Around the same

time, Frey and Winter (1977) made the same proposal even more pointedly,

referring to it as a “third cue” model. It followed as a prediction that the animals

would consistently select the no-drug lever during tests with a novel training

drug that produced cues different from the particular cues they had been trained

to detect.

This model differed radically from the one that had been accepted by most SDL

investigators who expected responding to be equally contingent on D cues and

N cues. It was not congruent with some data obtained by this writer during tests

for substitution in the T-maze DD task. However, most investigators were now

using the operant task, and Colpaert’s model did match the pattern of results

obtained in that task; probably for that reason, the “presence-versus-absence”

model has achieved wide acceptance. A variant of this model is also compatible

with the often observed “asymmetrical SDL” result in which loss of the response

occurs after D N but not after N D state changes (Overton 1968,1988).

SUMMARY OF PROGRESS SINCE 1975

The DD method most frequently employed after 1975 used a composite

paradigm incrementally constructed during the preceding 25 years by means of

the process that I have just described. By 1975, all the component pieces were

in place to allow the subsequent widespread use of DDs for investigation of

drug effects, causes of drug abuse, and other issues. The major components

included (1) simple two-response tasks in which lever selection was primarily

determined by stimulus effects—i.e., tasks with high specificity in that

discriminable effects could be distinguished moderately well from depressant or

other drug actions; (2) a paradigm that could be used with almost any type of

centrally acting drug; (3) specificity of recognition of different stimulus effects,

so that the stimulus effects of one drug could be distinguished from those of

most others; (4) a rational principle for predicting what stimulus effects might be

expected from a previously untested drug (the “one cue per class” model); and,

18
perhaps most important, (5) a simple and easy-to-understand theory (presence

versus absence), which made the results of the paradigm appear simple,

plausible, and interpretable. All that was now required was time for the news to

get around and for members of the pharmacological community to gradually

acquire confidence in the method. Stolerman has prepared a comprehensive

bibliography listing more than 1,000 DD studies and showing that the number of

DD publications per year has increased steadily since 1970 (slightly before the

method reached its current stage of development). The majority of DD studies

have been published since 1980 and have used the currently popular two-lever

FR-10-versus-extinction DD training paradigm.

THE FUTURE OF DRUG-INDUCED DISSOCIATION AND DISCRIMINATIVE

CONTROL

The study of dissociation, SDL, and DDs is now at an exciting juncture,

having split into several distinct subfields. DDs are used to investigate the

neurochemical effects of drugs, as illustrated by several other papers in this

volume, and to investigate the stimulus effects of drugs that are presumed to

underlie drug abuse (Overton 1987). Although DDs are believed to occur

because of the “stimulus effects of drugs,” very few studies intended to identify

these stimuli or to elucidate the properties of stimulus control by drug stimuli are

being reported (Overton 1988). Drug-induced SDL has not been extensively

investigated since it was concluded in about 1980 that the doses of

psychoactive drugs normally used for outpatient treatment do not produce the

phenomenon to an impressive degree (Eich 1980; but see Lowe 1982).

However, SDL produced by emotional states is under current investigation as a

possible etiological factor in depression and other mental illnesses (Blaney

1986). Recent years have seen a dramatic resurgence of interest in the multiple

personality syndrome, and the clinical syndrome has been redefined to include

“super multiples” reported to have as many as 50 distinct personalities, each

partially dissociated from the others. At the same time, something approaching

40 distinctively different drug stimuli have now been identified in the DD

literature. These various studies are being carried out by investigators in

several different fields, and a continuing challenge exists to identify findings in

one field that may have ramifications in another. We can optimistically hope

(with Schacter 1982, p. 263) that a “social amnesia” such as the one that

prevented our 19th-century predecessors from more directly influencing work

conducted between 1937 and 1980 will not dissociate too completely the

various fields in which work is currently being conducted on the stimulus

properties of drugs and on other forms of dissociation.

19
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ACKNOWLEDGMENTS

Supported in part by research grants AA-08174, DA-02403, DA-04725, and

MH-25136.

23
AUTHOR

Donald A. Overton

Departments of Psychiatry and Psychology

Temple University

Philadelphia, PA 19122

24
Discriminative Stimulus Properties of

Hallucinogens and Related Designer Drugs

Richard A. Glennon

CLASSICAL HALLUCINOGENS

Hallucinogenic agents are of several categories (see table 1). The present

review considers only those agents referred to as classical hallucinogens and

certain structurally related designer drugs. Most of the nonclassical

hallucinogens are treated separately elsewhere. The stimulus properties of the

classical hallucinogens have been previously reviewed (Glennon et al. 1982,

1983a).

Nature of the Stimulus

Several examples of hallucinogens have been widely used as training drugs

in drug discrimination (DD) studies. These include the simple tryptamine

5-methoxy-N, N-dimethyltryptamine (5-OMe DMT), the ergoline (+)lysergic

acid diethylamide (LSD), the phenethylamine mescaline, and the

phenylisopropylamine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane

(DOM) (Glennon et al. 1983a). Stimulus generalization occurs between these

four agents regardless of which is used as the training drug. This is part of the

justification for treating the classical hallucinogens as a group. On the other

hand, stimulus generalization does not typically occur between the classical

hallucinogens and the nonclassical hallucinogens, regardless of which is used

as the training drug. Indeed, the classical hallucinogens appear to share a

common mechanism of action, whereas (1) their mechanism of action seems to

be different from that of the nonclassical hallucinogens, and (2) each type of

nonclassical hallucinogen probably possesses its own distinct mechanism of

action. Also the DD procedure of using animals trained to discriminate a

classical hallucinogen from saline does not represent an animal model of

hallucinogenic activity (Glennon 1991). Stimulus effects of the classical

hallucinogens involve, at least in part, a mechanism of serotonin (5-HT); thus,

25
TABLE 1. Classification of hallucinogenic agents

Classical Hallucinogens

lndolealkylamine hallucinogens

Simple tryptamine (e.g., DMT, 5-OMe DMT, psilocin)

a-Methyltryptamines (e.g., a-MeT, 5-OMe a-MeT)

 -Carbolines (e.g., harmine, harmaline, 6-OMe harmalan)

Ergolines (e.g., (+)LSD, other lysergic acid analogs)

Phenalkylamines

Phenethylamines (e.g., mescaline)

Phenylisopropylamines (e.g., DOM, DOB, certain DMAs and TMAs)

Other Hallucinogens/Psychotomimetics

Cannabinoids

Phencyclidine (PCP)-related agents

Certain opiates

Certain cholinergic agents

Miscellaneous

stimulus generalization may occur between a classical hallucinogen stimulus

and stimuli produced by, for example, nonhallucinogenic agents capable of

acting as direct or indirect 5-HT agonists. For example, an LSD stimulus and/or

DOM stimulus generalizes with the 5-HT-releasing agent fenfluramine and the

nonselective 5-HT agonist quipazine. Both the LSD stimulus and the DOM

stimulus generalize to the nonselective 5-HT/dopamine (DA) agonist lisuride.

And yet, animals can be trained to discriminate between LSD and lisuride in a

three-lever paradigm (Callahan and Appel 1990). Although the DOM stimulus

(1 mg/kg) generalizes to 0.6 mg/kg of lisuride, administration of 0.01 mg/kg of

lisuride in combination with the training dose of DOM results in attenuation by

50 percent of the DOM stimulus (see figure 1; higher doses of lisuride in

combination with DOM result in disruption of behavior). It appears, then, that

lisuride may be a partial agonist, and Colpaert et al. (1982) have indeed shown

that at sufficiently high doses a variety of 5-HT “antagonists” can in fact mimic

the LSD stimulus, suggesting that they too may be acting as partial agonists.

26
FIGURE 1. Effect of TFMPP and lisuride administered in combination with

DOM in rats trained to discriminate 1 mg/kg of DOM from saline.

Doses of TFMPP and lisuride greater than 0.4 and 0.01 mg/kg,

respectively, in combination with DOM result in disruption of

behavior.

5-OMe DMT and LSD may produce their stimulus effects via a multiple (and

perhaps dose-dependent) serotonergic mechanism; DOM seems to produce a

more selective stimulus, and R(-)DOB may be even more selective than DOM

(Glennon 1988). In addition to any serotonergic involvement, recent work by

Meert et al. (1990) suggests that the stimulus effects of LSD involve a

catecholaminergic mechanism.

Mechanism of Action

Early studies with LSD, 5-OMe DMT, mescaline, and DOM concluded that

these agents act via a 5-HT agonist mechanism. For example, certain 5-HT

agonists mimicked the stimulus effects of hallucinogens, whereas known 5-HT

antagonists were capable of antagonizing these effects. Curiously, however, the

5-HT antagonist cinanserin was a relatively weak LSD antagonist and was

27
much more effective in attenuating the stimulus effects of mescaline (Glennon

et al. 1983a). initial arguments related these differences to the effectiveness of

the hallucinogens as discriminative stimuli. Furthermore, 5-OMe DMT and LSD

seemed to produce somewhat different stimulus properties depending on their

training dose (Glennon 1988). In addition, although hallucinogen stimuli

generalized with some 5-HT agonists, generalization did not occur with certain

other 5-HT agonists. These and other studies raised the possibility of some

subtle differences between the stimulus effects produced by indolealkylamine

hallucinogens and phenalkylamine hallucinogens.

Puzzling as it was at the time, we now know that there are multiple populations

of central 5-HT receptors (e.g., 5-HT1, 5-HT2,5-HT3). In 1983, Glennon et al.

(1983b) demonstrated that the stimulus effects of DOM, and DOM-stimulus

generalization to examples of other categories of classical hallucinogens such

as LSD, 5-OMe DMT, and mescaline, were potently antagonized by the 5-HT2

antagonist ketanserin. Colpaert et al. (1982) had reported 1 year earlier that

pirenperone was a specific LSD antagonist; pirenperone is now recognized as a

5-HT2 antagonist. Other 5-HT2 antagonists also potently inhibit the DOM

stimulus. These findings led to the hypothesis that hallucinogens produce their

stimulus effects via a 5-HT2 agonist mechanism (Glennon et al. 19836). Later,

with the use of LSD-trained rats, the LSD stimulus was also potently

antagonized by various 5-HT2

 antagonists (for a review see Cunningham and

Appel 1988).

On the basis that the DOM stimulus was a “cleaner” cue than that produced by

indolealkylamine hallucinogens, detailed mechanistic and structure-activity

relationship (SAR) studies were performed using DOM as the training drug.

Subsequent studies revealed the following:

DOM and DOM-related agents such as DOB and DOI are 5-HT2 agonists

(or at least partial agonists).

lndolealkylamine hallucinogens are nonselective 5-HT2 agonists (i.e.,

although they can act as 5-HT2 agonists, they bind at various

subpopulations of 5-HT1 sites with high affinity and also act as 5-HT1

agonists).

Hallucinogen-induced stimuli can be attenuated by a wide variety of 5-HT2

antagonists.

DOM-stimulus generalization does not occur with serotonergic agents that

are selective for other populations of 5-HT receptors (e.g., the 5-HT1A

agonist 8-OH DPAT).

28
For a wide variety of hallucinogens, DOM-stimulus generalization potency

is significantly correlated with their affinity for central 5-HT2

 receptors (for a

review see Glennon et al. 1984).

Stimulus effects produced by DOM are distinct from those produced by the

5-HT1A agonist 8-OH DPAT (Glennon 1988); in fact, in DOM-trained rats, low

doses of 8-OH DPAT (e.g., 0.1 mg/kg) produce less than 10 percent

DOM-appropriate responding. However, because we have recently

demonstrated that 5-HT1, agonists can modulate certain behavioral effects of

5-HT2 agonists (Glennon et al. 1990), we were interested in determining the

effect of 8-OH DPAT on the DOM stimulus. As shown in figure 2, 50 μg/kg

produces a shift to the left of the dose-response curve for DOM in DOM-trained

animals (ED50 = 0.45 vs. 0.19 mg/kg). Apparently 8-OH DPAT can augment the

stimulus effects of DOM. This is currently under further investigation.

FIGURE 2. Effect of 50 μg/kg of 8-OH DPAT on the dose-response curve of

DOM in DOM-trained rats. 8-OH DPAT was administered 5 min

prior to DOM and DOM was adminisfered 15 min prior to testing.

Inset shows the effect of different doses of 8-OH DPAT in

combination with the ED50 dose of DOM.

29
Hartig (1989) has demonstrated a significant homology between 5-HT1C and

5-HT2 receptors. Indeed, although DOM-like agents are quite selective for

5-HT2 receptors, they also bind (with a tenfold to fiftyfold lower affinity) at

5-HT1C receptors (Titeler et al. 1988). This raises the possibility that the

stimulus effects of certain classical hallucinogens may involve a 5-HT1C

component. To date, 5-HT1C-selective agents are unavailable to test this

hypothesis. 1-(3-trifluoromethylphenyl)piperazine (TFMPP) is a 5-HT1B/5-HT1C

agonist; stimulus generalization does not occur between DOM and TFMPP

regardless of which is used as the training drug. TFMPP also binds at 5-HT2

sites, and there is evidence that it may be a 5-HT2 antagonist. In stimulus

antagonism studies using rats trained to discriminate 1 mg/kg of DOM from

saline, 0.3 and 0.4 mg/kg of TFMPP attenuate the DOM stimulus by about 50

percent; higher doses (0.45 to 0.7 mg/kg) of TFMPP administered in

combination with DOM produce disruption of behavior. Most 5-HT2 antagonists

such as ketanserin are also 5-HT1Cantagonists, and their affinities for 5-HT1C

receptors and 5-HT2 receptors are rather similar. The one exception is the DA

antagonist spiperone; this agent is a more effective 5-HT2 antagonist than a

5-HT1C antagonist. Tests of stimulus antagonism were conducted with

spiperone using DOM-trained rats (figure 3); doses of up to 0.1 mg/kg were

without effect and higher doses produced disruption of behavior. Thus, at this

time it is not possible to rule out 5-HT1C involvement in the stimulus effects of

DOM. It might be noted that because of its DA antagonist actions, spiperone

was able to attenuate the stimulus effects of S(+)amphetamine in amphetamine

(AMPH)-trained animals.

Because DOM is a phenylisopropylamine, it has been speculated for years that

DOM might produce stimulus effects similar to those of the structurally related

phenylisopropylamine stimulant AMPH. In fact, it was once thought that DOM,

like AMPH, might be acting via a dopaminergic mechanism. However, it has

been demonstrated that DOM and AMPH produce distinct stimulus effects

regardless of which is used as the training drug. Nevertheless, certain other

DOM analogs to which the DOM-stimulus generalizes (i.e., 2,4,5-trimethoxy

analog [TMA], 3,4,5-TMA, 2,5-dimethoxy analog [DMA]) produce as much as 46

percent AMPH-appropriate responding in S(+)AMPH-trained animals (Glennon

et al. 1985). In fact, these agents may produce some central stimulant effects in

humans (Shulgin 1978). As a consequence, we sought to determine if these

agents would produce AMPH-like effects in AMPH-trained rats when the 5-HT2

response was blocked. Tests of stimulus generalization were conducted in

S(+)AMPH-trained rats that had been pretreated 45 min earlier with a dose of

ketanserin (0.5 mg/kg), which completely attenuates the effect of DOM in

30
FIGURE 3. Tests of stimulus antagonism with spiperone in rats trained to

discriminate either 1 mg/kg of DOM or S(+)AMPH from saline.

DOM-trained rats. Under these conditions, DOM failed to elicit more than 33

percent AMPH-like responding at doses of up to 2.7 mg/kg (figure 4); higher

doses resulted in disruption of behavior. Likewise, 2,4,5-TMA and 3,4,5-TMA

failed to produce more than 25 percent AMPH-appropriate responding (figure

5). Doses of 2,4,5-TMA greater than 15 mg/kg produced disruption of behavior.

3,4,5-TMA was evaluated at doses of up to 40 mg/kg; although at the higher

doses the animals’ response rates were reduced by about 60 percent,

responding was essentially saline-like.

Similar results were obtained with 2,5-DMA (figure 4); doses of up to 40 mg/kg

failed to engender more than 45 percent AMPH-appropriate responding.

N-Monomethylation of AMPH-like agents usually enhances their

amphetamine-like properties; consequently, the N-monomethyl analog of

3,4,5-TMA was evaluated (figure 5); here too, doses of up to 40 mg/kg resulted

31
FIGURE 4. Tests of (+)amphetamine generalization to (+)amphetamine (A),

(+)methamphetamine (M), DOM, and 2,5-DMA in rats pretreated

with 0.5 mg/kg of the 5-HT2 antagonist ketanserin.

in AMPH-appropriate responding that did not exceed 11 percent. Although

ketanserin binds at D2 DA receptors and may behave as a DA antagonist at

high doses, the dose of ketanserin used in the present study had no effect on

the AMPH stimulus; that is, 0.5 mg/kg of ketanserin did not attenuate the effect

of S(+)AMPH in AMPH-trained animals, nor did this dose of ketanserin interfere

with AMPH-stimulus generalization to 0.8 mg/kg of S(+)methAMPH (figure 4).

The results suggest that these agents do not produce AMPH-like stimulus

effects at the doses evaluated and further support lack of significant

dopaminergic involvement in their mechanism of action.

Pharmacokinetic lnvestigations

The DD paradigm can be used to investigate the pharmacokinetic and

biodispositional properties of hallucinogenic agents. Unfortunately, relatively

little has been reported in this regard.

32
FIGURE 5. Tests of (+)awhetamine stimulus generalization to 2,4,5-TMA,

3,4,5-TMA, and N-methyl 3,4.5-TMA in rats pretreated with 0.5

mg/kg of ketanserin. Rats were trained to discriminate 1 mg/kg of

(+)amphetamine from saline.

Locus of Action. The specific locus of action mediating the stimulus effects

of hallucinogenic agents is unknown. However, it seems likely that

hallucinogens produce their stimulus effects via a central mechanism. Several

lines of reasoning support this notion, For example, xylamidine (a 5-HT2

antagonist that does not readily penetrate the blood-brain barrier) is ineffective

in attenuating the stimulus effects of the classical hallucinogens. Also, although

5-OH DMT and 5-OMe DMT share similar 5-HT1/5-HT2 binding profiles, the

5-OMe DMT stimulus only partially generalizes to 5-OH DMT; the latter agent is

of low lipophilicii and is known to poorly penetrate the blood-brain barrier.

Another agent that should not penetrate the blood-brain barrier is the

quaternary analog of DOB (i.e., QDOB). In animals trained to discriminate

R(-)DOB from saline, stimulus generalization does not occur with QDOB.

However, it has been demonstrated that QDOB does not bind at 5-HT2 sites.

Minnema et al. (1980) conducted DD studies in which animals were implanted

33
with indwelling cannula; in this manner, hallucinogens could be administered

directly into the brain. Although this technique has not been widely used to

investigate such agents, it could prove valuable for investigating (1) agents that

do not readily penetrate the blood-brain barrier and (2) specific locations in the

brain that might be responsible for mediating the stimulus effects of

hallucinogens.

Temporal Properties. The time of onset and the duration of action of the

stimulus effects of various hallucinogenic agents have been examined. Of

course, these vary from agent to agent; the interested reader is referred to the

primary literature for details of such investigations.

Action of Metabolites. Although relatively little work has been done, there is

no evidence that the stimulus effects of classical hallucinogens are due

primarily to their metabolites. Phenylisopropylamines undergo

parahydroxylation in vivo, and it has been speculated that 4-OH 2,5-DMA might

be a metabolite of 2,5-DMA. The 4-COOH derivative of 2,5-DMA is a metabolite

of DOM. Neither of these agents produces DOM-like stimulus effects in rats,

nor do they bind at 5-HT2 receptors. lodinated compounds can undergo a rapid

deiodination in vivo; thus, the possibility exists that DOI, the iodo counterpart of

DOM, may be metabolized to 2,5-DMA. In DD studies, DOI is significantly more

potent than 2,5-DMA and the potencies of both compounds are consistent with

their affinities for 5-HT2 receptors. It appears unlikely that the stimulus effects of

DOI are due to its deiodinated derivative 2,5-DMA. DD may be a useful

procedure for examining the activity of metaboliies or potential metabolites,

particularly if this approach is coupled with cannulation studies (as mentioned

above) in order to avoid potential problems associated with penetration of the

blood-brain barrier.

Structure-Activity Relationship (SAR)

That the SAR of LSD has been neglected is probably a direct consequence of

the unavailability of LSD analogs. On the other hand, extensive SAR studies

have been conducted with 5-OMe DMT and DOM as training drugs; indeed, the

SAR of no other training drug has been as widely investigated as that of DOM.

The 2,5-dimethoxy substitution pattern of DOM is important. In general, little

can be done to increase the potency of DOM-like agents; the only structural

modifications that result in more potent DOM-like agents are replacement of the

4-methyl group of DOM with an ethyl (DOET) or n-propyl (DOPR) group, or with

certain halogens such as bromo (DOB) and iodo (DOI). The R(-)isomers of

34
DOM-like agents are several times more potent than their S(+)enantiomers,

whereas the reverse is true for derivatives of a-methyltryptamine. In neither

series does stereochemistry play a major role. The SARs of these agents have

been reviewed in detail (Glennon 1989a) and are summarized in figure 6.

Readdressing the structural similarity between the DOM-like agents and the

phenylisopropylamine stimulant AMPH, a separate and distinct SAR has been

formulated for AMPH-like stimulus effects; these are summarized in figure 7.

FIGURE 6. Summary of selected structure-activity relationships important for

DOM-like stimulus effects.

Relationship With Human Hallucinogenic Activity

When DOM-trained animals are used, there is a significant correlation between

the stimulus generalization potencies of variius classical hallucinogens and

their human hallucinogenic potencies. In fact, this was the first example of a

correlation between discrimination-derived data and any human measure of

activity. Because of the relationship between generalization potency and 5-HT2

35
FIGURE 7. Summary of selected structure-activity relationships important for

AMPH-like stimulus effects.

receptor affinity, there should also be a correlation between receptor affinity and

human hallucinogenic potency; such a correlation has been reported (Glennon

et al. 1984). Initially, the correlation was demonstrated using rat cortex as the

source of 5-HT2 receptors; this study has now been replicated using human

cortex as the source of tissue for the binding studies (Sadzot et al. 1989). Thus,

DD studies were directly responsible for aiding our understanding of the

mechanism of action of human hallucinogenic activity of the classical

hallucinogens.

Role in Drug Development

At first, one might think that DD studies using animals trained to discriminate a

classical hallucinogen from saline serve only the investigation of other

hallucinogenic agents. This is not the case; hallucinogen-trained animals can

be employed in several different applications.

36
Investigation of Basic Neurochemical Mechanisms. DOM-related agents

are fairly selective 5-HT2 agonists. indeed, it was speculated that these agents

were the first examples of 5-HT2-selective agonists on the basis of DD studies.

SAR for DOM stimulus generalization was later found to parallel SARs for the

binding of these agents at 5-HT2 sites. DOB and DOI are commonly used now

as 5-HT2 agonists, and [3H]DOB and [125I]DOI are commercially available for

radioligand binding and autoradiographic studies. DD studies using animals

trained to discriminate DOM, DOB, or DOI from saline may serve, then, as a

functional behavioral model of central 5-HT2

 receptor activation.

Indicator of Abuse Potential. Hallucinogen-trained animals can be used by

the pharmaceutical industry to evaluate the abuse potential of new therapeutic

entities. Stimulus generalization to a new agent suggests that the new agent be

further evaluated in other tests to determine whether it has any abuse liability.

To date, there are no examples of classical hallucinogens that are not

recognized by animals trained to discriminate DOM from saline. As mentioned

above, however, DD cannot be considered a model of human hallucinogenic

activity.

New Drug Development. There is evidence that 5-HT2

 antagonists possess

neuroleptic, antianxiety, and antidepressant properties. Thus, animals trained to

discriminate a 5-HT2 agonist could be useful tools for identifying novel 5-HT2

antagonists. Indeed, examples of this approach have already appeared in the

literature (e.g., Meert 1989; Meert and Awouters 1990).

DESIGNER DRUGS

Hallucinogen-related designer drugs were a topic of several recent symposia,

and the discriminative stimulus properties of these agents have been reviewed

(Glennon 1989a, 1989b, 1990; Nichols and Oberlender 1989). Separate and

distinct SARs have been formulated for the stimulus properties of

phenalkylamine hallucinogens and phenalkylamine stimulants (figures 6 and 7).

While in the process of formulating the SAR of phenylisopropylamines, we

became interested in 3,4-methylenedioxyamphetamine (3,4-MDA, MDA, “Love

Drug”). Because the methylenedioxy group was not in our SAR data base, and

because MDA had been popular during the 1960s as a mild hallucinogenic

agent with central stimulant properties, we investigated this agent and its

isomers in DOM- and S(+)AMPH-trained animals. Consistent with its street

reputation, MDA was recognized by both groups of animals. Its R(-)-isomer is

primarily responsible for DOM-like effects, whereas its S(+)-isomer is primarily

37
AMPH-like. Likewise, Nichols et al. (1989) have reported that racemic and

R(-)MDA, but not S(+)MDA, result in stimulus generalization in LSD-trained

rats. To date, MDA is the only phenalkylamine demonstrated to produce both

types of effects. Subsequently, animals were trained to discriminate MDA from

saline (table 2); confirming the foregoing observations, the MDA stimulus

generalizes both to DOM-like and AMPH-like agents.

TABLE 2. Drug discrimination studies involving methylenedioxy derivatives

of amphetamine as training drug in male rats

Drug and Study Route Schedule PSll Dose (mg/kg)

MDA

Glennon et al. 1982

Glennon and Young 1984

R(-)MDA

Appel et al. 1990

S(+)MDA

Appel et al. 1990

MDMA

Glennon et al. 1986

Schechter 1987

Oberlender and Nichols 1988

Glennon and Misenheimer 1989

MDE

Boja and Schechter 1987

(+)MBDB

Nichols and Oberlender 1989

IP

IP

IP

IP

IP

IP

IP

IP

IP

VI-15

VI-1 5

FR-10

FR-10

VI-15

FR-10

FR-50

VI-15

FR-10

15 1.50

15 1.50

15 1.50

15 1.50

15 1 .00

20 1.50

30 1.75

15 1.50

20 2.00

1.75

NOTE: PSll = Presession injection interval (min); MDA = 1-(3.4-Mefhylenedioxyphenyl)-2

-aminopropane; MDMA = N-Methyl MDA or N-Methyl-1-(3,4-methylenedioxyphenyl)-2

-aminopropane; MDE = N-Ethyl MDA or N-ethyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane;

MBDB = N-methyl-1-(3,4-methylenedioxyphenyl-2-aminobutane; VI-15 = variable interval

15-second schedule of reinforcement; FR-10 = fixed ratio (10) schedule of reinforcement.

38
Using DOM- and S(+)AMPH-trained animals, we examined several other

methylenedioxy phenalkylamines including 2,3-MDA, MMDA

(2-methoxy-4,5-methylenedioxy amphetamine), and MDMA (the N-monomethyl

derivative of MDA). During the early to mid-1980s MDMA became a rather

popular street drug (“Adam,” “Ecstasy”); once it became a scheduled (Schedule

I) substance, a series of MDMA-related designer drugs appeared on the

clandestine market. Two of the more popular agents include the ethyl homolog

of MDMA (MDE, “Eve”) and the N-hydroxy analog of MDA (N-OH MDA). We

had earlier demonstrated that MDMA differs from MDA in that it produces

AMPH-like, but not DOM-like, effects. MDA and MDMA also produce

AMPH-like stimulus effects in pigeons (Evans and Johanson 1986) and

monkeys (Kamien et al. 1986) trained to discriminate S(+)AMPH from saline,

and MDMA produces similar effects in rats trained to discriminate the

phenylisopropylamine stimulant cathinone from saline (Schechter 1987).

Stimulus generalization occurs between AMPH and cocaine regardless of which

is used as training drug, and Broadbent et al. (1989) showed that MDA and

MDMA produce cocaine-like effects (though not necessarily complete stimulus

generalization, depending on the training dose of cocaine) in rats trained to

discriminate cocaine from saline. The S(+)MDA stimulus generalizes to cocaine

but only partially generalizes to (+)AMPH (Appel et al. 1990). Oberlender and

Nichols (1988) failed to observe AMPH stimulus generalization to MDMA or to

either of its isomers; on the basis that the number of animals selecting the

drug-appropriate lever never exceeded 25 percent, it was claimed that MDMA

lacks AMPH-like character. However, they showed that the MDMA stimulus

generalizes to S(+)AMPH, and we have shown that the MDMA stimulus partially

generalizes to S(+)AMPH and S(+)methAMPH (Glennon 1990). Although some

of the reported inconsistencies may be attributable to differences in technique,

there is ample evidence from discrimination studies and others that MDMA is

capable of producing some AMPH-like effects. On the other hand, it has never

been claimed that MDMA, AMPH, or cocaine produce identical effects. (For

example, see Goudie in this volume for a comparison of the stimulus effects of

AMPH and cocaine.) On the contrary, all instances of AMPH-stimulus

generalization to MDMA have been accompanied by a significant decrease in

animals’ response rates. Taken together, these results suggest that, although

AMPH and MDMA may share some similarities, there also appear to be some

differences in their stimulus effects.

MDE and N-OH MDA reportedly produce effects in humans that are similar to

those of MDMA. Neither of these agents produces stimulus generalization in

AMPH-trained or in DOM-trained animals. However, both agents produce

39
MDMA-like effects in MDMA-trained rats (Glennon and Misenheimer 1989).

These results support the overall contentions of Oberlender and Nichols (1988)

that methylenedioxy derivatives of AMPH may possess properties that are

distinct from those considered simply AMPH-like or hallucinogen- (e.g.,

DOM-)like. In contrast to their conclusions, however, it seems very likely that

MDA and MDMA possess some AMPH-like qualities. MDE and N-OH MDA, on

the other hand, appear to lack significant AMPH-like and DOM-like character.

Relatively little has been reported regarding the SAR of MDMA analogs. The

a-ethyl homolog of MDMA (i.e., MBDB) is another MDMA-like agent that lacks

AMPH-like and LSD-like stimulus properties; several conformationally restricted

analogs have also been evaluated (for a review see Nichols and Oberlender

1989). Recently, we examined a new agent that has been confiscated from

several clandestine laboratories: PMMA. PMMA is a structural relative of

MDMA that possesses a 4-methoxy group in place of the 3,4-methylenedioxy

bridge. This agent, like MDE and N-OH MDA, fails to produce AMPH-like or

DOM-like stimulus effects, but it does produce MDMA-like effects (Glennon

1990). On a molar basis, PMMA (ED50 = 0.2 mg/kg) is about 3.5 times more

potent than MDMA (ED50 = 0.76 mg/kg). Consequently it appears that the

methylenedioxy group is not essential for MDMA-like activity and that an

entirely new SAR may need to be investigated.

SUMMARY

Animals trained to discriminate classical hallucinogens from saline have been

used in the past decade to examine other hallucinogenic agents. Time course

(onset, duration of action) and locus of action have been studied, SARs have

been formulated, and mechanism of action has been investigated in detail. On

the basis of DD studies in animals, it was proposed that hallucinogenic agents

may produce their actions in humans via a 5-HT2 agonist mechanism and that

certain phenalkylamine hallucinogens such as DOM and DOB might constitute

the first known examples of 5-HT2 agonists. This led to the development of

[3]HDOB and [125I]DOI for use in radioligand binding and autoradiographic

studies and to the use of hallucinogen-trained animals as a functional

behavioral model of 5-HT2 receptor activation. Animals trained to classical

hallucinogens are more recently being used to evaluate novel designer drugs. It

can be seen, then, that this paradigm, using hallucinogenic agents as training

drugs, has proven to be quite useful for the investigation of hallucinogens and

nonhallucinogens alike.

40
Note added in proof: We have recently found that both the DOM stimulus and

the MDMA stimulus can be attenuated by pretreatment of the animals with very

small doses of the 5-HT3 antagonist zacopride. Zacopride (0.001 mg/kg) in

combination with the training doses of DOM and MDMA (1 and 1.5 mg/kg,

respectively) reduces drug-appropriate responding from >90 percent to <25

percent in both groups of animals. These findings offer entirely new insight into

the mechanism of action of hallucinogenic agents and designer drugs as

discriminative stimuli; they suggest that 5-HT3 antagonists are capable of

modulating the effects of these agents and that they may be of value for

antagonizing the effects of these agents in a clinical setting.

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AUTHOR

Richard A. Glennon, Ph.D.

Professor of Medicinal Chemistry

Department of Medicinal Chemistry

School of Pharmacy

Medical College of Virginia

Virginia Commonwealth University

Richmond, Virginia 23298-0581

44
Discriminative Stimulus Properties of

Amphetamine, Cathinone, and Related Agents

A. J. Goudie

INTRODUCTION

The drug discrimination (DD) research reviewed here concentrates on

amphetamine (AMPH) and cathinone (CATH). The review excludes research on

cocaine, although it is known that AMPH, CATH, and cocaine have similar cue

properties. The justification for excluding cocaine comes from evidence that the

cue properties of AMPH and cocaine are not identical (Stolerman and D’Mello

1981). It is actually possible to train rats to discriminate between AMPH and

cocaine (Goudie and Reid 1988). In these studies rats were trained, over a

substantial number of sessions (about 140), to discriminate a constant dose of

AMPH (1 mg/kg) from varying doses of cocaine (5-12.5 mg/kg). In tests with

saline in trained animals, at the lowest training dose of cocaine (5 mg/kg)

animals selected the cocaine-associated lever. These animals therefore

interpreted saline as more like cocaine at 5 mg/kg than like AMPH at 1 mg/kg,

implying that this discrimination was the equivalent of a quantitative low dose

(cocaine) versus a high dose (AMPH) discrimination. However, at higher

cocaine training doses, saline tests produced “random” (50 percent)

responding. These data accord with predictions from Järbe and Swedberg’s

(1982) model of DD learning for a qualitative discrimination. Although it is not

known how such qualitative discriminations are learned, that it is possible to

train them suggests that conclusions about "stimulant" cues (Nielsen and

Scheel-Kruger 1988) should be considered with some caution if different

stimulants are used as training stimuli.

45
Nielsen and Scheel-Kruger (1988) and Barrett and Appel (1989) suggested that

AMPH and cocaine cues may be differentially sensitive to manipulations of

catecholamine systems, providing a further reason for considering these cues

as being different; parametric analyses directed at this question are clearly

needed, however. Woolverton and Cervo (1986) reported that, in AMPH-trained

rats, 6-OHDA lesions shifted the AMPH generalization curve to the right but had

no effect on cocaine’s ability to substitute for AMPH, again suggesting that the

cue properties of cocaine and AMPH may differ in some way. Schechter and

Boja (1988a) also suggested that the cue properties of cocaine may be more

complex than those of AMPH, involving significant local anesthetic actions (see

Zacany and Woolverton 1989). McElroy and O’Donnell (1989) reported that

although AMPH substituted for the cue induced by the beta-adrenergic agonist

clenbuterol, cocaine did not, again suggesting that these drugs may have subtly

different cue properties. However, because AMPH and CATH are closely

related structurally—CATH is the beta-keto side chain analog of AMPH (Young

and Glennon 1986; Goudie et al. 1986)-and because the pharmacology of

CATH resembles that of AMPH in many assays (Nencini and Ahmed 1989), it

seems reasonable to assume that CATH and AMPH possess very similar cue

properties and, therefore, to review research on these agents together.

Most of this review concentrates on studies with AMPH, because this drug has

been studied more extensively. However, to our knowledge, no studies have

shown important differences between CATH and AMPH in their cue properties,

although Schechter (1989) suggested that the CATH cue may have a more

rapid onset and a shorter duration of action than the AMPH cue. This issue

needs to be studied more systematically.

In this review we have chosen to exclude human studies of the AMPH cue. This

decision was taken to simplify the data to be reviewed. The validity of human

studies will ultimately have to be evaluated against the data base reviewed here.

Amphetamine has been extensively studied in DD assays, and previous

reviews have summarized basic findings (Nielsen and Scheel-Kruger 1988;

Young and Glennon 1986). This review concentrates on more recent work in

this area. However, before reviewing this literature it is relevant to reiterate the

main conclusions about the AMPH and CATH cues derived from earlier work.

The AMPH and CATH cues show the following properties.

1. They are dose dependent, being centrally mediated at “high” doses, and

possibly peripherally mediated at “low” doses (Colpaert et al. 1976; Goudie

46
et al. 1986). The extent to which drugs generalize to the cues may be

critically determined by the training dose (Stolerman and D’Mello 1981).

2. Catecholamine (specifically dopamine [DA]) systems are thought to be

involved in mediating the AMPH and CATH cues, both agents being

indirect DA agonists. lntracerebral AMPH injections in the nucleus

accumbens generalize to the cue produced by systemic AMPH. This

generalization is blocked by intra-accumbens sulpiride. However, AMPH

injected into the striatum does not produce such generalization (Nielsen

and Scheel-Kruger 1986) suggesting that the we is mediated by

mesolimbic DA systems (see Wood and Emmett-Oglesby [1989] for similar

results with cocaine). The nucleus accumbens may therefore play a critical

role in the AMPH cue. In support of this hypothesis, it has been shown that

typical and atypical neuroleptics block the AMPH cue (Nielsen and Jepsen

1985). Because atypical neuroleptics are believed to act at mesolimbii

rather than striatal sites, these data support the hypothesis that the AMPH

cue is mediated by the mesolimbii DA system. It has consequently been

proposed that the AMPH cue can be used to screen for neuroleptics,

including atypical agents devoid of extrapyramidal side effects (Nielsen

and Jepsen 1985). Although much evidence indicates that the AMPH and

CATH cues are in part DA mediated, roles for other neurotransmitters in

these cues have not been excluded (see “The Involvement of

Noradrenergic (NA) Systems in the Amphetamine Cue,” in this paper).

Furthermore, suggestions that endogenous phenylethylamine mediates the

cue properties of stimulants (Colpaert et al. 1980; Goudie 1982) have not

been followed up in recent years.

3. The cue properties of AMPH are not related to its action in stimulating

behavior, because the cueing actions of drugs that generalize to AMPH do

not correlate with their stimulant actions (Nielsen and Scheel-Kruger 1988).

This is perhaps not surprising if the AMPH cue is mediated by mesolimbic

DA, because motoric effects of stimulants are thought to be mediated

predominantly at striatal sites.

4. The AMPH cue shows stereoselectivity, d-AMPH being 3-5 times more

potent than I-AMPH. Because d- and I-AMPH are equipotent in peripheral

actions but d-AMPH is more potent in its central actions, these data accord

with the idea that, at doses typically studied, the AMPH cue is centrally

mediated. The CATH cue is also stereoselective (Schechter 1986) and

centrally mediated (Goudie et al. 1986).

47
5. A wide range of phenylethylamine and nonphenylethylamine drugs of

abuse substitute for AMPH and CATH, although other abused drugs (LSD,

morphine, ethanol) do not substitute. Such data led to the suggestion that

the AMPH cue may be used to assess novel drugs for abuse potential of

the AMPH type (Porsoft et al. 1984).

6. The structure-activity relationships for the AMPH and CATH cues have

been reviewed in detail by Young and Glennon (1986) and Glennon and

Young (1987). The interested reader is directed to these excellent reviews

for information on this topic.

The foregoing material summaries established knowledge about the AMPH

and CATH cues. We now turn to more recent research, which has built on this

basic information.

INVOLVEMENT OF DA SYSTEMS

General Studies

Recent studies provide further evidence that DA is involved in mediating the

AMPH and CATH cues. Schechter (1986) reported that in rats haloperidol

caused a parallel shift to the right in the CATH generalization curve, although

he noted that this shift was seen only with large haloperidol doses. He

tentatively suggested that non-DA systems also mediate the CATH cue. Goudie

et al. (1986) reported that haloperidol produced only 50 percent antagonism of

the CATH cue (and of AMPH substitution) in studies with the highest dose of

haloperidol that could be tested. It is not known whether more robust

antagonism of the CATH we with DA antagonists can be obtained under other

conditions or whether DA is less important in mediating the CATH cue than is

the AMPH cue (Schechter 1986).

In studies with AMPH, Schechter and Boja (1988a) examined the effect of CGS

10746B, which inhibits DA release but does not affect postsynaptic receptors.

The AMPH cue was antagonized by CGS 10746B, as was the ability of CATH

to generalize to AMPH. Schechter (1990) later reported that CGS 10746B

blocked CATH discrimination in animals trained on CATH. In addition, it blocked

acute tolerance to CATH (induced by CATH pretreatment), suggesting that DA

is involved in both the CATH cue and the tolerance that can develop for ft.

48
Nencini and Woolverton (1988) examined the effects of haloperidol and the

calcium channel blocker nimodipine on the AMPH cue. Nimodipine antagonized

the cue, although not to the extent haloperidol did. lt was suggested that the

antagonism seen with nimodipine was due to the inductbn of a third state

(unlike AMPH or saline) rather than to true antagonism. Such an interpretation

raises the question of how to differentiate specific from nonspecific antagonism

in DD studies. Nencini and Woolverton (1988) proposed that a specific

antagonist should (1) shift the generalization curve to the right, (2) possibly

antagonize effects of the drug tested on response rate, and (3) induce complete

antagonism at some dose. They noted that by these stringent criteria even

haloperidol only approximates being a specific antagonist of the AMPH cue

(see Goudie et al. 1986 for similar comments about the CATH cue).

Dworkin and Bimle (1989) showed that 6-OHDA lesions of the nucleus

accumbens cause a shift to the right in the AMPH generalization curve. These

data suggest that DA accumbens systems mediate the AMPH cue, in

agreement with earlier findings.

The foregoing studies further implicate mesolimbic DA in mediating the AMPH

and CATH cues. However, it is clear that (1) the criteria for assessment of

pharmacological antagonism in DD studies need to be clarified and (2) the

parametric conditions for obtaining maximal antagonism of the AMPH and

CATH cues are not established; it is possible that antagonism studies may be

influenced by pharmacokinetic factors related to agonist-antagonist

interinjection intervals (Callahan et al. 1991).

Studies With Partial DA Agonists

Further evidence for DA involvement in the AMPH cue comes from studies with

partial D2 agonists with high affinity but low efficacy for DA receptors. Partial D2

agonists are interesting drugs because they have inhibitory partial agonist

actions on autoreceptors but buffering actions on postsynaptic receptors

(Coward et al. 1989,1990). Typically, such agents act postsynaptically as DA

antagonists because of their low efficacy and high affinity. However, because of

their partial agonist actions at postsynaptic receptors, they maintain sufficient

DA activation to prevent the extrapyramidal side effects that normally result

from full postsynaptic DA receptor blockade (Coward et al. 1989,1990). Exner

et al. (1989) studied the effects of such partial agonists on the AMPH cue.

Complete blockade was seen with the prototypical partial agonist terguride

(transhydrolisuride). However, only incomplete blockade, with no increase in

49
antagonism over a very wide (eightfold to sixteenfold) dose range, was seen

with the partial agonists preclamol [-3PP] and SDZ 208-911. Exner et al. (1969)

concluded that the failure of SDZ 208-911 and preclamol to antagonize the

AMPH cue fully may have been due to postsynaptic agonist actions of these

agents, preclamol having been reported to substitute for a selective D2 agonist

cue (Appel et al. 1986). (Presumably, terguride does not possess such agonist

actions to the same extent, for it blocked the AMPH cue fully and acted like a

classical D2 antagonist.) Because some partial D2 agonists inhibited, but did not

fully block, the AMPH cue, and because they do not induce catalepsy (Coward

et al. 1989), Exner et al. (1989) suggested that these agents may be useful in

treating psychotic states without inducing extrapyramidal side effects. These

data obviously further implicate DA in mediating the AMPH cue. They also

develop earlier ideas about the potential use of the AMPH cue to screen for

neuroleptics (Nielsen and Jepsen 1985).

In limited studies with partial D1 agonists SKF 38393 and SKF 75760, Nielsen

et al. (1989) reported that these agents had very little effect in antagonizing the

AMPH cue. Because partial D2, but not DI, agonists inhibit the AMPH cue,

these preliminary data suggest that D2

 rather than D1 receptors may play the

primary role in the AMPH cue.

Involvement of D1 and D2 Receptors In the AMPH Cue

Recent years have seen much research into the roles of D1 and D2 receptors in

mediating the AMPH cue. These studies have generated a fairly consistent

pattern of data, although the interpretation of the data remains controversial.

Typically, D1 and D2 antagonists block the AMPH cue. However, although D1

agonists do not substitute for AMPH, D2 agonists do. The relevant empirical

data are summarized briefly as follows.

Studles With D1 Agonists. Most of these studies have been conducted with

SKF 38393, which has been found universally not to generalize to AMPH in rats

(Arnt 1988; Callahan et al. 1990; Furmidge et al. 1989a; Nielsen et al. 1989;

Smith et al. 1989) and monkeys (Kamien and Woolverton 1969). Nielsen and

Scheel-Kruger (1988) noted that the failure of SKF 38393 to generalize could

be due to its partial agonist actions . However, the full D1 agonist SKF 81297

also does not generalize fully to AMPH (Furmidge et al. 1989a; Nielsen et al.

1989) thus D1 agonists clearly do not substitute for AMPH.

50
Studies With D1

 Antagonists. Virtually all these studies have been conducted

with SCH 23390, which antagonizes the AMPH cue in rats (Arnt 1988; Callahan

et al. 1990; Exner et al. 1989; Nielsen and Jepsen 1985; Nielsen et al. 1989;

Smith et al. 1989) and monkeys (Kamien and Woolverton 1989). The D1

antagonist SKF 83566 also blocks the AMPH we (Furmidge et al. 1989a). There is

consequently reliable evidence that D1 antagonists block the AMPH cue.

Studies With D2 Agonists. In the vast majority of studies reported, various D2

agonists substitute for AMPH. In rats quinpirole has repeatedly been found to

substitute (Arnt 1988; Callahan et al. 1990; Furmidge et al. 1989a; Nielsen et al.

1969; Smith et al. 1989) as have D2 agonists such as RU 24213 (Furmidge et

al. 1989b), (–)-NPA, and pergolide (Arnt 1988; Nielsen et al. 1989). At low

doses quinpirole antagonizes the AMPH cue (Furmidge et al. 1989c), an effect

thought to be mediated by actions on D2 autoreceptors because in the same

study higher doses showed the typical generalization seen with quinpirole. In

pigeons, pirebedil also generalizes (Evans and Johanson 1987). However,

Kamien and Woolverton (1989) reported that in monkeys pirebedil,

bromocriptine, and propylbutyidopamine did not substitute for AMPH. The

reason or reasons for the discrepant monkey data are not clear.

Studies With D2 Antagonists. Various D2

 antagonists (including haloperidol,

raclopride, YM 09151-2, spiroperidol, sulpiride, and clebopride) block the AMPH

cue in rats (Amt 1988; Callahan et al. 1990; Furmidge et al. 1989a; Nielsen and

Jepsen 1985; Nielsen and Scheel-Kruger 1986; Nielsen et al. 1989). Studies in

pigeons (Järbe 1982) have also reported antagonism with haloperidol.

However, Kamien and Woolverton (1989) reported that pimozide antagonized

the AMPH cue in only one of three monkeys and raciopride antagonized the

cue only in one of two. Again, the cause or causes of the discrepant monkey

data are unclear.

If one ignores the monkey data, these studies suggest that D2 receptors play a

primary role in the AMPH cue (Arnt 1986; Callahan et al. 1990; Nielsen et al.

1989) and that D1 stimulation is a necessary but insufficient condition for

generalization to AMPH to occur. This pattern of data accords with a common

hypothesis about the nature of some D1-D2 receptor interactions. D1 receptor

activation may serve an “enabling” role, allowing some effects of D2 stimulation

to be observed (Clark and White 1967; Waddington 1989). Thus D1 antagonists

may block the AMPH cue because of inhibition ("disenabling”) of tonic D1

activation by endogenous DA. In support of this hypothesis, Smith et al. (1989)

reported that in AMPH-trained rats, generalization of a low dose of AMPH was

51
potentiated by SKF 38393. These data can be interpreted as demonstrating that

D1 stimulation enhances the effect of D1 activation caused by the DA-releasing

action of AMPH.

However, although the “enabling” hypothesis accounts for much of the available

data from studies with AMPH, it does not accommodate ail published findings.

For example, in studies with D1 and D2 agonist combinations, SKF 38393 did

not significantly potentiate the degree of AMPH generalization induced by

quinpirole (Smith et al. 1989). These data do not readily fit in with the enabling

hypothesis, because D1 activation did not potentiate effects of D2 stimulation.

Smith et al. (1989) also examined the ability of quinpiroie to substitute for

AMPH after treatment with SCH 23390. Substitution by quinpirole was not

blocked by SCH 23390, although it was by haloperidol, suggesting that, as far

as quinpiroie substitution for AMPH is concerned, no D1-D2 receptor

interactions occurred. In a similar study, Nielsen et al. (1989) examined the

ability of SCH 23390 to block substitution for AMPH by the D2 agonist

pergolide. SCH 23390 failed to block such substitution, whereas the D2

antagonist raclopride was effective. At the same doses, SCH 23390 blocked

pergolide-induced motor stimulation. Nielsen et al. (1989) suggested that

effects of D1 and D2 receptors are coupled in their actions on activity; however,

in the AMPH DD assay, no receptor coupling occurred. This conclusion was

supported by other studies in which combinations of D1 and D2 agonists were

investigated. SKF 38393 and SKF 81297 did not potentiate the ability of

pergolide and quinpirole, respectively, to substitute for AMPH, again suggesting

the absence of D1-D2 receptor coupling.

The precise role of D1 receptors in the AMPH we is obviously difficult to

specify. Many findings are compatible with the enabling hypothesis, although

other data do not support it. Furthermore, there is evidence from studies with

selective agonists that it is possible to train distinct D1 and D2 agonist cues so

that, after training with a D1 agonist, the cue is blocked only by D1 antagonists,

not by D1 antagonists. Conversely, after training with a D2 agonist, the we is

blocked only by D2 antagonists, not by D1 antagonists (Appel et al. 1988; Arnt

1988; Kamien et al. 1987). Such data suggest that in the DD assay there may

be little D1-D2 receptor coupling when specific agonists are used as training

stimuli (Clark and White 1987). Studies reporting that SCH 23390 does not

block the cue induced by the D2 agonists pirebedil (Kamien et al. 1987),

(-)-NPA (Amt 1988) and apomorphine (Schechter and Greer 1987; Woolverton

et al. 1987; but see Tang and Code 1989) are difficult to reconcile with a simple

form of the enabling hypothesis. lt is also perhaps surprising on the basis of the

52
enabling hypothesis that AMPH does not substitute fully for the cue induced by

D2 agonists such as quinpirole (Appei et al. 1988) and (-)-NPA (Arnt 1988). it

might be expected that AMPH would substitute for a D2 agonist, either because

of endogenous DA tone or because of stimulation of D1 receptors resulting from

AMPH-induced DA release. It is consequently possible that to observe D1-D2

receptor interactions in the DD procedure, it may be necessary to train and (of

critical importance) also to test animals with an indirect nonselective DA agonist

such as AMPH rather than with direct agonists such as apomorphine or with

specific D1 or D2 agonists (Waddington 1989). However, it is clearly necessary

to investigate this issue further before definitive conclusions can be reached.

Furthermore, it seems important to attempt to clarify why the training and test

drugs may be critical in determining the extent of DA receptor interactions

observed in DD studies.

INVOLVEMENT OF NORADRENERGIC (NA) SYSTEMS IN THE

AMPHETAMINE CUE

Snoddy and Tessel (l983) reported that mice discriminating low doses of AMPH

generalized to the selective NA uptake inhibitor nisoxetine. These data are

surprising in the light of earlier studies showing that NA receptor blockers such as

phentolamine and propranolol typically do not block the AMPH we (Young and

Glennon 1986). However, Snoddy and Tessel (l985) replicated their earlier results.

In addition, they showed that the AMPH we was blocked by the antagonist

prazosin and potentiated by the antagonist yohimbine. The potentiating actions

of yohimbine were attributed to blockade of presynaptic autoreceptors, which

were thought to be activated by AMPH-induced NA release, which reduced net NA

activation after AMPH treatment. Most significant, Snoddy and Tessel (1985)

reported that pimozide dii not affect the AMPH we at doses that decreased

AMPH-induced stimulation. Thus, these data suggest that the AMPH we may be

mediated by receptors and not by DA receptors.

A number of studies have followed up these reports. Evans and Johanson

(1987) found that nisoxetine generalized to AMPH in pigeons. Similarly, Kamien

and Woolverton (1989) found that nisoxetine generalized to AMPH in monkeys.

However, in these monkeys the a-NA antagonists phentolamine and prazosin

did not consistently block the AMPH cue. Arnt (1988) also reported that

prazosin does not antagonize the AMPH cue in rats. Schechter and Boja

(1988b) and Nielsen and Scheel-Kruger (1988) reported that nisoxetine does

not generalize to AMPH in rats, although both authors commented that

generalization with nisoxetine in mice was seen only with doses larger than

53
those studied in rats. Sanger (1988) reported that the antagonists yohimbine

and idazoxan, which may act at NA autoreceptors and thus potentiate NA

functioning, did not generalize to AMPH, suggesting no role for NA in the AMPH

cue in rats.

These data obviously show substantial inconsistencies, for which there are a

number of potential explanations. First, there may be species differences in the

involvement of NA in the AMPH cue (Schechter and Boja 1988b; Snoddy and

Tessel 1985). Second, the effects of nisoxetine may be mediated by DA

systems. Although nisoxetine is a relatively selective NA uptake inhibitor, it may

modify DA systems to some extent (Kamien and Woolverton 1969). However,

this explanation is difficult to reconcile with the finding (Nielsen and

Scheei-Kruger 1988) that the selective DA uptake inhibitor GBR 12909

produces only partial generalization to AMPH in rats. Third, the training dose of

AMPH may be critical, because Snoddy and Tessel (1983) found that mice

trained to discriminate the high dose of 3.2 mg/kg of AMPH did not generalize

to nisoxetine. The high-dose AMPH cue may be DA mediated and the low dose

NA mediated. Finally, Snoddy and Tessel (l985) suggested that studies with

nonselective a-NA antagonists such as phentolamine may be confusing

because and antagonists have opposite effects on the AMPH cue. Thus,

if nonselective antagonists are used, the actions on and receptors may

cancel each other out and no antagonism will be seen.

The data reviewed clearly suggest some role for NA in the AMPH cue.

However, the inconsistencies in the literature will probably be resolved only by

parametric studies involving a number of training doses of AMPH in various

species. It also seems desirable that such studies be accompanied by

neurochemical analyses of the effects of nisoxetine on NA and DA levels

(Schechter and Boja 1988b).

STUDIES ON FENFLURAMINE GENERALIZATION TO THE AMPHETAMINE

CUE

Evans and Johanson (1987) reported that pigeons discriminating AMPH

generalized to a range of anorectics and stimulants. These data are not

surprising, because they are in accord with earlier studies. However, the

nonstimulant serotonergic anorectic fenfluramine substituted fully for AMPH in

two pigeons, partially in a third, and not at all in a fourth. Evaluation of these

data is complicated by the relatively small number of pigeons studied and thus

depends on the significance attributed to data from subjects showing individual

54
differences in DD studies. However, if attention is paid to patterns of data

generated by individual subjects, these data are surprising because studies in

various species have shown that fenfluramine does not typically substitute for

AMPH (Young and Glennon 1986). Evans and Johanson (1987) suggested that

the observed generalization was mediated by anorectic properties shared by

fenfluramine and AMPH. However, De La Garza and Johanson (1987) reported

that although AMPH generalized to a range of anorectics in monkeys,

fenfluramine did not. On the basis of these data De La Garza and Johanson

(1987) concluded, in contrast to the pigeon studies, that the AMPH cue was not

related to the drug’s anorexic action.

The findings with fenfluramine in pigeons are potentially important because it

has often been suggested that generalization to AMPH may indicate that a drug

possesses abuse potential of the AMPH type (De La Garza and Johanson

1987; Evans and Johanson 1989; Goudie et al. 1988; Grant and Woolverton

1989; Porsolt et al. 1984). However, there is minimal evidence that fenfluramine

is either a stimulant or a drug of abuse. The generalization to AMPH seen in

pigeons therefore represents a potentially important example of a “false

positive” in terms of screening for drug abuse potential.

As a followup of their earlier work, Evans et al. (1990) developed a three-choice

discrimination in pigeons among AMPH, fenfluramine, and saline. They

demonstrated that an assay could be developed that differentiates the cue

properties of fenfluramine and AMPH in pigeons. In tests with fenfluramine,

subjects always responded on the fenfluramine lever; even at low doses they

did not generalize to AMPH. Conversely, in tests with AMPH, subjects

responded on the AMPH lever; they did not generalize to fenfluramine. Thus it

was possible to develop a DD assay that consisted of an exclusive

discrimination between AMPH and fenfluramine, in contrast to the earlier

two-choice results in pigeons (Evans and Johanson 1987). This is probably not

surprising, in that a three-choice discrimination would be expected to force

animals to attend to the differences between the cue properties of the two drugs

in much the same way animals can be trained to discriminate AMPH from

cocaine, even though they typically generalize between these agents (Goudie

and Reid 1988). Therefore, although it was possible to dissociate the cue

properties of AMPH and fenfluramine in pigeons, the original findings should be

considered false positives in terms of screening for abuse potential.

The tendency for some anorectics (mazindol and phendimetrazine) to

generalize to AMPH does not always predict whether the agent in question is

55
self-administered (De La Garza and Johanson 1987). The generalization to

AMPH seen by some authors with nisoxetine also seems to represent an

example of a false positive in terms of screening for abuse potential, because

this agent is not self-administered (Kamien and Woolverton 1989). Thus the

finding that a drug generalizes to AMPH does not necessarily mean that it will

be prone to abuse of the AMPH type. There is a clear need for refinements of

methodology, similar to those recently developed by Evans et al. (1990), for

abuse potential prediction. However, it should also be noted that, besides

generating a few false positive’s in screening for abuse potential, studies of the

AMPH cue have also generated very many true positive findings; the false

positive findings should be kept in perspective.

SUMMARY

Although the AMPH and CATH cues have been studied extensively,

inconsistent findings remain that need to be investigated. It is not known to

what extent, if at all, AMPH and CATH cues differ and how they differ from the

cocaine cue. The extent to which Dl and D2 receptor systems are coupled in

AMPH DD assays requires further investigation. The inconsistent findings in

substitution studies with nisoxetine need to be addressed in parametric studies

in various species. Finally, the possibility that the AMPH cue may generate

false positives in screening for drug abuse potential needs to be evaluated.

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ACKNOWLEDGMENTS

K.A. Cunningham, M. Exner, and D. Clark assisted in the preparation of this

paper by allowing access to unpublished data. I am grateful for their help. M.W.

Emmett-Oglesby provided insightful comments on a draft version of this paper.

AUTHOR

Dr. Andrew James Goudie, B.A. (Oxon), M.Sc., Ph.D.

Psychology Department, Eleanor Rathbone Building

University of Liverpool

P.O. Box 147

Liverpool L69 3BX UK

60
Discriminative Stimulus Effects of Cocaine

William L. Woolverton

These reviews address four aspects of the pharmacology of the discriminative

stimulus (DS) effects of various compounds: (1) assessment of novel

compounds, (2) investigations of mechanism of action, (3) structure-activity

relationships, and (4) problems that remain to be solved. This paper deals with

cocaine. In fact, the first three aspects can be considered part of the same

issue (i.e., mechanism of action). I hope to identify problems remaining to be

solved in each of these areas. At this time there are approximately 30 papers in

the literature that involve investigation of various aspects of the DS effects of

cocaine. Each of them has contributed to this review.

ASSESSMENT OF NOVEL COMPOUNDS

A large number of compounds have been evaluated for their ability to substitute

for cocaine as a DS. To summarize these data, I have somewhat arbitrarily

divided the compounds into three groups: a group found to substitute for

cocaine (81-100 percent drug lever responding), a group found to partially

substitute for cocaine (21-80 percent drug lever responding), and a group that

failed to substitute for cocaine (0-20 percent drug lever responding). Within a

category, the compounds are grouped according to predominant

pharmacological mechanism of action or into a group of miscellaneous

compounds. The data points for these figures are the maximum percentage of

cocaine-appropriate responses that have been found for each compound in any

study. This value cuts across species and procedures and therefore suffers any

limitations that such a summary may impose. In addition, the value equally

weights drugs for which several studies are available and drugs that have been

evaluated in only a single study. Finally, where study results differed for a

particular compound, the maximum value may not accurately represent the

consensus for that compound. In fact, consensus was rare; I will point out

where study findings differed. With those caveats in mind, the summaries are a

reasonable representation of the extant literature.

61
Among the compounds found to substitute for cocaine are a variety of

psychomotor stimulants and anoretics (see figure 1). Compounds such as

methamphetamine, amphetamine, methylphenidate, and diethylpropion have

consistently been found to substitute for cocaine as a DS. Gauvin et al. (1989)

reported that a caffeine-ephedrinephenylpropanolamine combination

substituted for cocaine in rats. A second group of compounds that are generally

known as selective dopamine (DA) reuptake blockers (nomifensine, bupropion,

GBR 12909) have been found to substitute fully for cocaine as a DS. A number

of cocaine analogs (e.g., WIN 35062-2) engendered 100-percent drug

responding at some dose in cocaine-trained animals. Colpaert et al. (1980)

evaluated the ability of monamine oxidase (MAO) inhibitors to substitute for

cocaine. In fact, several of the compounds substituted for cocaine. The effect

was correlated with their potency as MAC-B inhibitors, leading Colpaert et al.

(1980) to postulate that endogenous 8-phenethylamine was involved in the DS

effects of cocaine. This interesting effect has not, apparently, been pursued and

is an issue that remains to be solved. The direct DA agonists quinpirole and

apomorphine, primarily D2 agonists, have been reported to fully substitute for

cocaine. This is one point where summary data may misrepresent the literature:

the more general finding has been that DA agonists partially substituted for

cocaine. Phencyciidine (PCP), dexetimide, and procaine are grouped together,

not because of pharmacological similarity, but because they do not fii

particularly well into any of the other pharmacological categories or into our

notions of what should substitute for cocaine. Colpaert et al. (1979) reported

that PCP and the anticholinergic dexetimide substituted for cocaine and

postulated that the indirect DA actions of PCP accounted for the substitution.

Järbe (1984) reported that procaine substituted for cocaine, an effect that was

also found in some rhesus monkeys (de la Gatza and Johanson 1983).

Moreover, Woolverton and Balster (1982) found that cocaine substituted for

procaine as a DS. The behavioral commonalities between some local

anesthetics and cocaine clearly warrant further investigation, as do the

substitutions of PCP and dexetimide for cocaine.

Figure 2 summarizes drugs that have been found to substitute partially for

cocaine and includes agents from a number of different pharmacological

classes. The major point to be made from these data is that the DS effect of

cocaine is pharmacologically selective. The monoamine uptake inhibitors that

partially substituted for cocaine (pargyline, nialamide) have less MAO-B activity

that those that substituted for cocaine (figure 1). Nicotine, caffeine, and

strychnine are stimulants that, in contrast to the traditional psychomotor

stimulants shown in figure 1, only partially substituted for cocaine. Benztropine

62
FIGURE 1. Drugs found to substitute for cocaine

and amantadine are indirect DA agonists that only partially substituted for

cocaine, apparently because they have actions in addition to their indirect DA

agonist actions (e.g., the antichlolinergic actions of benztropine). it seems that

failure of a compound to substitute for cocaine can be due to lack of an action

63
FIGURE 2. Drugs found to partially substitute for cocaine

found in cocaine (indirect DA actions) or presence of an action lacking in

cocaine (e.g., anticholinergic effects). SKF 33393, piribedil, and bromocriptine

present a more typical picture of the direct DA agonists than noted in figure 1

(i.e., partial substitution for cocaine). Hallucinogens and norepinephrine (NE)

agonists have been found to substitute partially for cocaine. Partial substitution

with the local anesthetic lidocaine probably indicates that the local anesthetic

effect of procaine is not responsible for its substitution for cocaine. Cocaine

analogs that do not cross the blood-brain barrier do not engender

cocaine-appropriate responding, consistent with the belief that the DS effects of

cocaine involve an action in the brain Indirect serotin (5-HT) agonists only

partially substituted for cocaine, indicating that 5-HT actions of cocaine do not

play a primary role in this behavioral effect.

64
Compounds that failed to substitute for cocaine (figure 3) include MAO-A

inhibitors, delta-9-tetrahydrocannabinol, metabolites of cocaine, and one of the

isomers of cocaine. In addition, sedatives, hypnotics, anxiolytics, and

anticholinergics do not substitute for cocaine. The main point to be made from

these data is that the DS effect of cocaine, like that of other drugs, is

pharmacologically selective.

STRUCTURE-ACTIVITY RELATIONSHIPS

Although some work has been done with the DS effects of cocaine analogues,

the available data are limited. Figure 4 shows the structures of the cocaine

analogs that have been evaluated in cocaine-trained animals and their order of

potency as discriminative stimuli, again compiled across studies. The

compounds include modifications at three points of the molecule: the ester

linkage between the tropane and the phenyl ring, the phenyl ring itself, and the

amine group. The most potent compound, WIN 35428, does not have the ester

FIGURE 3. Drugs that failed to substitute for cocaine

65
linkage between the tropane and the phenyl ring and has a fluorine substituent

on the ring. Although Woolverton and Balster (1982) postulated that the ester

linkage was an important structural compound of the focal anesthetics with

cocaine-like effects, this compound demonstrates that ester linkage is not

essential for activity and that its presence can reduce potency. The second

most behaviorally potent compound is norcocaine, a compound in which the

methyl group has been removed from the terminal amine. Adding an N-allyl

group to the amine of CFT to produce CFNT, or to norcocaine to produce

N-allyl norcocaine, decreases potency relative to the methylated parent

compounds. Metabolites of cocaine (figure 5) do not engender

cocaine-appropriate responding.

MECHANISM OF ACTION

Table 1 is a comparison of relative potencies of various compounds at the

[

3 H]cocaine binding site in monkey brain and as a cocaine-like DS in rats,

pigeons, and rhesus monkeys. In rats, the potency order is identical except that

norcocaine is somewhat more potent as a DS than would be predicted based

on the binding data. In pigeons the potency order is similar, again with the

potency of norcocaine as a DS slightly greater than predicted. In monkeys the

potency relationships across preparations are also similar except that the

potency of cocaine as a DS is enhanced relative to the other compounds. In

short, although the potency orders are not identical across preparations, they are

remarkably similar considering the many ways in which pharmacokinetics could

alter the in vivo effects of cocaine. Madras et al. (1989) examined the relative

potency of blocking DA, NE, and 5-HT uptake with potency at the cocaine binding

site. The correlation between cocaine binding potency and DA uptake blockade

was quite high (r = 0.93) whereas the correlations with NE and 5-HT uptake

blockade were low (r = 0.52 and 0.38, respectively). Therefore, the available data

implicate cocaine binding sites and blockade of DA uptake as important

components of the mechanism of action of cocaine as a DS. It should be noted that

potency comparisons based on four or five compounds may be unreliable and that

additional analysis of this sort would make this a more valid conclusion.

There is substantially less information concerning antagonism of the DS effects

of cocaine. The available data are summarized as the maximum reduction in

drug lever responding with the training dose of cocaine, again cutting across

studies (figure 6). We have found that D1 dopamine antagonists consistently

reduce the effects of the training dose of cocaine from 100 percent to 0 percent

(Kleven et al. 1990; Vanover et al. 1989). SCH 23390, SCH 39166, and A

66
FIGURE 4. Structures of compounds found to substitute for cocaine

67
FIGURE 5. Structures of compounds that failed to substitute for cocaine

66359 are all D1 antagonists and are from different chemical classes. It should

be noted, however, that Barrett and Appel (1989) evaluated SCH 23390 as an

antagonist of the DS effect of cocaine in rats and, although there was evidence

of blockade, it was not as clear-cut as in the monkey. A substantial number of

DA antagonists with primarily D2 activity have been evaluated as antagonists of

the DS effects of cocaine. The most common finding has been that these

compounds partially block the DS effects of cocaine to between 20 percent and

80 percent cocaine-appropriate responding at the training dose. The potency

order for binding at haloperidol sites is identical to that for reducing the effects

of the training dose of cocaine to 50 percent cocaine-appropriate responding

(table 2) suggesting some involvement of D2 receptors in the DS effects of

cocaine. Again, it should be noted that there is very little evidence on which to

base this conclusion (see also the chapter by Goudie). NE and 5-HT

antagonists do not alter the DS effects of cocaine.

In most experiments, antagonists have been combined only with the training

dose of cocaine, a strategy that does not allow detailed comparisons between

68
TABLE 1. Relative potency of several compounds at cocaine binding site

and as cocaine-like discriminative stimuli (DS)

Cocaine binding sitea:

Cocaine DS in ratb:

Nomifensine > methylphenidate > cocaine >

norcocaine

Nomifensine > methylphenidate > cocaine =

norcocaine

Cocaine binding sitea: WIN 35428 > 35065-2 > cocaine > norcocaine

Cocaine DS in pigeonc: WIN 35428 > norcocaine > WIN 35065-2 > cocaine

Cocaine binding sitea: Mazindol > nomifensine > GBR 12909 > cocaine >

bupropion

Cocaine DS in monkeyd: Cocaine >2 mazindol > nomifensine > GBR 12909 >

bupropion

aRelative potency displacing [3H]cocaine binding in monkey caudate-putamen (Madras et al. 1989). bRelative potency substituting for cocaine as a DS in rats (Bedford et al. 1981; Colpaert et al. 1979;

Ho et al. 1976; Järbe 1984; McKenna et al. 1979).

cRelative potency substituting for cocaine as a DS in pigeon (Järbe 1981).

dRelative potency substituting for cocaine as a DS in rhesus monkeys (Kleven et al. 1990).

antagonists. On the few occasions in which the entire dose-response function

for cocaine has been determined in the presence of a D2 antagonist, the effect

appears similar to noncompetitive antagonism (e.g., see Ho and Silverman

1978). Besides partial antagonism of the effects of cocaine, this effect could be

the result of one or more of a number of drug effects, such as loss of stimulus

control of behavior in general, that have nothing to do with the blockade of

cocaine. This effect should be contrasted with the effects of D1 antagonists in

monkeys (Kleven et al. 1990; Vanover et al. 1989). Cocaine dose-response

functions have been consistently shifted parallel to the right in combination with

D1 antagonists. In one case with SCH 39166, that shift was as large as

sixteenfold (monkey 8409; figure 7). There also was a mutual antagonism

between drugs in terms of the effect on response rate. Species differences may

contribute to these apparent differences between D1 and D2 antagonists in their

interaction with cocaine. In fact, we have found a small (approximately twofold)

shift to the right in the cocaine dose-response function in monkeys pretreated

with haloperidol (Kleven et al. 1990).

69
FIGURE 6. Effects of antagonists

Although these data clearly suggest that D1 and D2 receptors play a significant

role in the DS effects of cocaine, it remains to be established what that role may

be. For D1 receptors, an intriguing possibility is that the permissive role that has

been postulated for D1 receptors in the expression of DA-mediated behaviors

holds for the DS effects of cocaine. That is, blockade of the indirect DA agonist

effects of cocaine at D1 receptors may function as an on-off switch for the

effects of cocaine. In fact, we have found that several other behavioral effects of

cocaine (e.g., stereotypy and effects on food intake) can be blocked by D1

antagonists (Rapoza and Woolverton 1988; Rapoza et al. 1990). Another

possibility is that the cocaine DS is a complex stimulus that includes a D1

component and that, when that component is blocked, the remaining stimulus is

sufficiently unlike cocaine (but not neutral) to engender saline lever responding.

70
TABLE 2. Relative potency of several compounds binding at the haloperidol

binding site or reducing cocaine-appropriate responding to 50

percent at the training dose

Haloperidol binding sitea

: Spiperone > haloperidol > pimozide >

chlorpromazine (CPZ)

Cocaine DS in ratb: Spiperone > haloperidol > pimozide > CPZ

a

lnhibition of [

3 H]haloperidol binding in calf striatal membranes (Creese et al. 1976).

bRelative potency reducing the effect of training dose of cocaine to 50 percent (Colpaert et al. 1976,

1978; Järbe 1978).

FIGURE 7. SCH 39166 + cocaine

71
Finally, it will be of interest to know whether D1

 antagonists can block the

subjective effects of cocaine in humans.

Two additional points should be made with regard to problems remaining to be

solved, one pharmacological and one behavioral. Our efforts to this point with

cocaine have concentrated on DA function in the nucleus accumbens (e.g., see

Wood and Emmett-Oglesby 1989). The nucleus accumbens is not an isolated

unit in the brain; it has connections to other brain regions, connections involving

the action of several other neurotransmitters. By investigating the role of DA

and DA receptors, I think we have discovered something important about the

neuronal actions of cocaine, particularly its DS effects. However, the role of

other brain structures and neurotransmitters remains to be established. Finally,

I would like to reiterate a point made previously by Schuster. There have been

no investigations of the role of behavioral-environmental variables in the DS

effects of cocaine. This is clearly a gap that needs to be filled.

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72
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ACKNOWLEDGMENT

Preparation of this manuscript was supported by NIDA grant DA-00250.

AUTHOR

William L. Woolverton, Ph.D.

The University of Chicago

Departments of Pharmacological and Physiological Sciences and Psychiatry

947 East 58th Street

Chicago, IL 60637

74
Discriminative Stimulus Functions of

Cannabinoids/Cannabimimetics

Torbjörn U.C. Järbe and Diane A. Mathis

INTRODUCTION

The psychoactive effect of cannabis is unique. This cannabimimetic effect is

primarily responsible for cannabis abuse and for the unwanted side effects of

cannabis therapy. To isolate the chemical components that induce this

psychoactive property, experimental methods for detecting and quantifying

cannabimimetic activity in animals have been developed. Most of these

methods use measures of spontaneous behaviors such as locomotor activity,

ataxia, sleep time, and posture. However, one method specifically measures

detection of a cannabimimetic effect. That method is drug discrimination

learning (DDL). In DDL procedures, the effects of cannabinoids are used as

discriminative stimuli (DS) for a choice between response alternatives. These

studies are important for identifying commonalities in mechanisms between

cannabinoids and cannabimimetics, investigating factors that contribute to

cannabis dependence, and assessing the therapeutic potential of newly

synthesized cannabinoids (for previous reviews see Balster and Ford 1978;

Järbe et al. 19896; Krimmer and Barry 1977; Weissman 1978). As outlined

below, these compounds are easily discriminated and their effects are

pharmacologically specific.*

*A possible exception to pharmacological specificity of the tetrahydrocannabinol (THC) cue is the

observation that THC-discriminating rats at feast partly generalize the THC-appropriate response to

the stimulus effects of the benzodiazepine diazepem (Browne and Weissman 1981; Mokler et al.

1986). However, for THC-diicriminating pigeons and gerbils, very little generalization of the THC

response to benzodiazepines, such as diazepam and Ro 11-3128, has been found (Järbe and

Hiltunen 1988; Järbe et al. 1988a). Nevertheless, it has been reported that THC shows some

affinity for benzodiazepine receptor sites (Sethi et al. 1986; Sung and Jacubovic 1987) thus

indicating a possible neurochemical mechanism for THC response generalization to

benzodiazepine administration.

75
Two central concepts in DDL are discrimination and generalization.

Discrimination refers to the ability of an organism to distinguish between at least

two stimulus values that differ either qualitatively or quantitatively. The training

methods that are used to establish a drug discrimination have been

substantially reviewed elsewhere (Colpaert 1987; Järbe 1987; Järbe and

Swedberg 1982; Overton 1987; Overton et al. 1986). The most commonly used

procedure uses an operant task and a fixed ratio schedule of reinforcement

(e.g., Colpaert 1987; Lal and Emmett-Oglesby 1983; Mathis and

Emmett-Oglesby 1990; Spealman 1985; Woolverton et al. 1987). Once

discrimination of a cannabinoid has been established, generalization tests are

conducted to examine cannabimimetic effects.

Generalization reflects the inverse of discrimination, that is, it is the ability of an

organism to perceive other stimulus values as similar to the training stimulus

(e.g. Järbe 1986,1989). Thus, generalization of a cannabinoid discrimination to

the stimulus or stimulus complex produced by other drugs is used to identify

similarities or differences between the cannabimimetic effects of the training

drug and of the test compound.

Detailed descriptions of the generalization test procedures used in our

laboratory may be found in Järbe et al. (1981). Although discrimination of a

cannabis stimulus is readily attained, the standard testing procedures that are

used in DDL are untenable for cannabinoid research. For example, if the

potency and time course of a test compound are unknown, the standard DDL

procedure requires that separate tests be conducted at various times after

injections of each drug dose. Thus, many tests must be conducted at several

time intervals and with several drug doses. This procedure then requires large

amounts of the test compound to complete these preliminary tests. For

cannabinoids, large amounts of test compound are rarely available.

A procedure developed in our laboratory not only reduces the amount of

compound needed to assess the potency and duration of a test compound’s

effect but also decreases the time needed to conduct these tests. This

procedure uses a repeated test method in which both onset and duration of

effect can be assessed after a single injection of the drug (Hiltunen and Järbe

1986b; Järbe et al. 1981, 1986). Comparisons of the data obtained using this

procedure and the conventional procedure have shown that repeated testing is

reliable for assessing not only cannabinoids and cannabimimetics (Hiltunen and

Järbe 1986b; Järbe et al. 1981), but also other compounds (Haug and

Götestam 1982; Holtzman 1979; Woods et al. 1981). This method has been

76
particularly useful for assessing the structure-activity relationship (SAR) of

newly synthesized compounds with potential cannabimimetic effects.

The initial investigations of cannabinoids were limited to naturally occurring

compounds, such as marijuana and hashish smoke or derived tinctures.

However, once the active component or components of cannabinoids were

identified and the chemical structures elucidated (Mechoulam and Gaoni 1965)

synthetic compounds were available for DDL research. Thus, an

interdisciplinary approach using organic chemistry and choice behavior of

laboratory animals was developed to determine the chemical requirements for

inducing cannabis intoxication. One outcome of these experiments

demonstrated that the cannabimimetic discriminative stimulus (DS) (Weissman

1981) and the analgesic, antiemetic, and anticonvulsant activity of cannabinoids

are dissociated (Mechoulam and Feigenbaum 1987).

Investigations of the DS properties of cannabinoids have focused on the

tetrahydrocannabinols, including the naturally occurring compounds and their

metabolites, and synthetic derivatives. As discussed below, cannabis

intoxication is primarily induced by, but not limited to, the direct action of

delta-9-THC.

DELTA-9-THC AS A DISCRIMINATIVE STIMULUS

For humans, the subjective, psychoactive effects of hashish and marijuana

result primarily from the activity of THC. The most psychoactive of these

compounds are delta-9-THC and delta-8-THC. However, delta-8-THC is found

only in limited quantities in some plant materials (Bhargava 1978; Mechoulam

and Edery 1973) and is less potent than the delta-9-THC isomer (Järbe and

Henriksson 1974; Järbe et al. 1976,1977). The effect induced by delta-9-THC

in animals is apparently directly comparable to the effect of hashish smoke.

For example, rats (Järbe and Henriksson 1974; Järbe et al. 1976) and gerbils

(Järbe et al. 1975) perceive the effects induced by hashish inhalation as similar

to the effects induced by IP administered delta-9-THC. When these animals

were trained to discriminate delta-9-THC and were then exposed to hashish

smoke, they performed the THC-appropriate response. Similarly, when rats

were trained to discriminate between active smoke inhalation (hashish smoke)

and placebo (chervil smoke), they performed the hashish-appropriate response

after IP injections of THC. This effect was stronger for the delta-9-THC isomer

than for the delta-8-THC isomer (Järbe and Henriksson 1974). In addition, other

77
effects of delta-9-THC (e.g., vocalization and depression of water intake) are

similar to the effects of hashish smoke exposure (Henriksson and Järbe 1971;

Johansson et al. 1975). Thus, most DDL investigations of cannabinoid activity

have focused on training animals to discriminate the delta-9-THC stimulus.

When animals are trained to detect the presence or absence of a DS for THC,

speed of acquisition is a function of training dose and species of subject.

Because pigeons tend to be more sensitive than rats to the stimulus effects of

THC, many investigations of THC use pigeons as subjects. Acquisition of this

discrimination is fairly rapid and requires approximately 20-25 training trials

with each stimulus condition once initial shaping of the operant response has

been attained. Tests for stimulus generalization of the THC we result in a

characteristic dose-effect relationship. Generalization from the delta-9-THC

training stimulus to different doses of the training drug is illustrated in figure 1.

From the figure it can be seen that the animals emit progressively fewer drug

responses when tested with lowered amounts of the training drug. In addition,

similar to generalization tests for exteroceptive stimulus intensity (DeWitte

1978; Heinemann and Chase 1975; Mackintosh 1974), for doses higher than

the training dose there is no change in the curvature of the gradients (see also

Browne and Weissman 1981). The data in this figure, obtained from pigeons

trained with 1 mg/kg and rats trained with 3 mg/kg, demonstrate the good

stimulus control acquired with the delta-9-THC stimulus. The correlation

coefficients (r) and ED50 values for the generalization gradients were estimated

by logarithmic regression analysis-for pigeons, r = 0.935 (ED50 = 0.43 mg/kg);

for rats, r = 0.929 (ED50 = 1.17 mg/kg).

Similar to the ED5 0s obtained with other drugs used as discriminative stimuli,

the magnitude of the ED50 for delta-9-THC is affected by the training dose. This

effect of training dose within one species is illustrated in figure 2. Pigeons were

trained to discriminate between delta-9-THC (0.56 or 1 mg/kg) and vehicle, then

tested for generalization to different doses of the training drug. The obtained

ED50 value for the high-dose training condition was approximately three times

higher than for the low-dose condition. In addition, the magnitude of an ED50 for

delta-9-THC is affected by the route of and time since drug administration.

For example, when rats are trained to discriminate between IP injected

delta-9-THC (2 mg/kg) and no drug, tests conducted with IV administration of

lower doses of THC elicit more THC- appropriate responding than similar tests

using IP administration. Conversely, tests conducted with PO administration of

low THC doses elicit less THC-appropriate responding (Barry and Krimmer

78
FIGURE 1. Dose generalization gradients of delta-9-THC for pigeons and rats

trained to discriminate between delta-9-THC and vehicle. Pigeons

were trained with 1 mg/kg, injected IM 90 min prior to session

onset. Rats were trained with 3 mg/kg, injected IP 30 min prior to

sesstion onset. N = 8 at all points. (Adapted from Järbe and

McMillan 1980.)

1976). The time course for the generalization of the delta-9-THC DS is

illustrated in figure 3. Pigeons were trained to discriminate between the

presence and absence of 0.56 mg/kg of delta-9-THC, 90 min after IM

administration of THC or vehicle. By means of a repeated test procedure, a

generalization curve for doses of THC was determined at 30, 90, 270, and 540

min after drug administration. The peak of generalization to delta-9-THC

generally occurred at 90 min after THC administration. These data are also

similar to data obtained using a single test procedure (Hiltunen and Järbe

1986b).

79
FIGURE 2. Dose generalization gradients of delta-9-THC for pigeons trained

to discriminate between delta-9-THC (0.56 mg/kg, N = 7; 1 mg/kg,

N = 8) and vehicle. Training and test sessions were conducted

90 min after IM injection. Test sessions used a repeated test

procedure (see Järbe et al. 1981; Hiltunen and Järbe 1986b).

Data represent the average of at least two determinations for

each animal for the 0.56 mg/kg training condition and the average

of one determination for the 1.0 mg/kg training condition.

The time of peak generalization of THC does not vary as a function of training

dose. Pigeons were trained to discriminate delta-9-THC (0.28 mg/kg or 0.56

mg/kg) and vehicle, then tested for generalization to the drug (full or half

training dose) at 1.5 and 4.5 hr after administration (figure 4). The time course

of generalization did not differ, regardless of the original THC training dose.

Similar effects have been found with rats (Järbe et al. 1976) and gerbils (Järbe

et al. 1975).

80
FIGURE 3. Time course for dose generalization of delta-9-THC for pigeons

trained to discriminate between the presence and absence of

0.58 mg/kg of delta-9-THC. Training sessions were conducted 90

min after IM injections. Test sessions used a repeated test

procedure (see Järbe et al. 1981; Hiltunen and Järbe 1986b), and

data represent the average of at least two determinations. Data

for the 90-min test interval are the same as those shown for the

lower training dose in figure 2. N = 7 at all points.

In addition, retention of the THC discrimination is robust and tolerance to the

stimulus effects of delta-9-THC has not been reported, given the commonly

used DDL training and testing procedures. However, tolerance to high or

noncontingent doses of this compound does affect performance. For example,

when animals are trained to discriminate delta-9-THC from no drug and then

administered high doses of this drug, tests conducted 24 to 48 hr afterward

result in an attenuation of the delta-9-THC we (Semjonow and Binder 1985).

81
FIGURE 4. Time course for dose generalization of delta-9-THC for pigeons

trained to discriminate the presence or absence of low or high

doses of delta-9-THC. Subjects were first trained to discriminate

0.28 mg/kg (low), then tested with 0.28 mg/kg training dose (TD)

and 0.14 mgIkg (1/2 TD). Then subjects were retrained with

0.56 mg/kg and retested with 0.56 mg/kg (TD) and 0.28 mg/kg

(1/2 TD). Training sessions were conducted 90 min after IM

injections. Test sessions were conducted using a repeated test

procedure (Järbe et al. 1981; Hiltunen and Järbe 1986b), and

data represent the average of one determination for each animal.

N = 5 at all points.

Some attenuation of the THC stimulus after chronic, noncontingent

administration of THC has also been observed (Hirschhorn and Rosecrans

1974; Järbe and Henriksson 1973). Nevertheless, because tolerance to the DS

effects of THC under DDL procedures that do not administer noncontingent or

high doses of delta-9-THC has not been reported, it is unlikely that tolerance to

82
discrimination of the THC stimulus disrupts performance during generalization

testing. In addition, because the parameters controlling the magnitude of

generalization to this stimulus are well established, the DDL of delta-9-THC has

provided a useful model for detecting the cannabimimetic activity of other

naturally occurring cannabinoids.

CANNABIMIMETIC ACTIVITY OF CANNABIDIOL (CBD) AND CANNABINOL

(CBN)

A variety of other substances with potential psychoactive properties have been

detected in cannabis preparations, including CBD and CBN (Kettenes-Van den

Bosch et al. 1980; for an overview of these cannabinoids, see Sofia 1978).

Although the cannabinoid activity of CBD may be dissociated from its

cannabimimetic activity, CBN may share at least some of the cannabimimetic

effects of THC.

Investigations with CBD have shown that this compound has other cannabinoid

properties, such as effects on sleeping time (Monti 1977), anticonvulsive activity

(Consroe et al. 1981) and operant rate-depressant properties (Hiltunen et al.

1989). However, generalization of CBD to the subjective, DS effects of THC has

consistently not been found (e.g., see Hiltunen and Järbe 19866). Thus,

although this compound apparently does contribute to other effects of cannabis

preparations, it lacks cannabimimetic properties. In addition, the CBDs

monomethyl ether and cannabichromene are not cannabimimetic (Järbe et al.

1986; Järbe, unpublished data 1982).

On the other hand, the cannabimimetic activity of CBN is more certain. For

studies with animals, most authors report that the CBN stimulus is generalized

from the THC discriminative stimulus (Bueno et al. 1976; Järbe and Hiltunen

1987; Järbe et al. 1977; Weissman, 1978). However, some authors report that

CBN induces THC-like effects for humans (Perez-Reyes et al. 1973); others

report that it does not (e.g., Hollister and Gillespie 1975). Thus, these

investigations of the THC-like effects of CBD and CBN indicate that only CBN

has cannabimimetic activity. However, because cannabis preparations contain

all three cannabinoids (i.e., THC, CBN, and CBD), these compounds may have

different effects when administered in combination. For example, administration

of THC has been reported to induce anxiety in humans, but coadministration of

CBD with THC reduces that anxiety (Zuardi et al. 1982).

83
The results obtained from studies that assessed the interaction of the stimulus

effects of delta-9-THC, CBN, and CBD are summarized in tables 1 and 2.

These data indicate that THC does not appear to be solely responsible for the

cannabimimetic effects of cannabis preparations. The main interest of studies

on cannabinoid combinations has focused on THC administered with one of the

other compounds. The results of these studies indicate that the stimulus effects

of THC are generally enhanced or prolonged by coadministration of either CBD

or CBN. For example, when THC was given in combination with CBD, the DS

effects of THC were prolonged (Hiltunen and Järbe 1986b. However, this

interaction effect may differ among species; for example, for pigeons tested with

CBD and THC, no prolongation of THC-appropriate responding was obtained

(Hiltunen and Järbe 1986b). On the other hand, for studies in which THC was

coadministered with CBN to rats, discrimination of the THC stimulus was

enhanced but not prolonged (Järbe and Hiltunen 1967). Again, species

differences are apparent with these compounds; for pigeons, CBN only slightly

increased discrimination of the THC stimulus (Järbe and Hiltunen 1967).

Relatively fewer studies have focused on the combined effects of CBN and

CBD (Hiltunen and Järbe 1986a; Hiltunen et al. 1988,1989). The data obtained

so far indicates that CBD may reduce the stimulus effects of CBN. Some

studies have suggested that when animals are trained to discriminate THC, this

discrimination is generalized to the CBN stimulus (Bueno et al. 1976; Järbe and

Hiltunen, 1987; Järbe et al. 1977; Weissman 1978), but not to the CBD stimulus

(Henriksson et al. 1975; Hiltunen and Järbe, 1986a, 1986b; Järbe et al. 1977).

When THC-discriminating rats were administered CBN and CBD in

combination, this generalization of THC to CBN was attenuated (Hiltunen and

Järbe 1986a). Thus, CBD may antagonize or modulate the THC-like,

psychoactive effect of CBN. However, cannabis intoxication is not limited to the

direct action of these compounds. As discussed below, cannabimimetic effects

may also result partly from some metabolites of THC.

CANNABINOID METABOLITES

The metabolism of THC is complex and varies among species (Mechoulam et

al. 1976). Metabolites that have been isolated and examined with THC-trained

animals include the 11-hydroxy metabolites (11-OH-THCs). Collectively, the

data indicate that 11-OH-THCs are major, psychotropic metabolites exerting

THC-like activity of biological significance. Investigations with animals trained to

discriminate between THC and the no-drug state indicate that the 11-OH THCs

are similar to the delta-9-THC stimulus. The cannabimimetic activity of

84
TABLE 1. Generalization a

 of delta-9-THC (3 mg/kg) to delta-9-THC

administered in combination with cannabinol (CBN) or cannabidiol

(CBD)

Drug Combination (mg/kg) Time Post Administration (hr)

0.5 1.5 4.5 6.5

Delta-9-THC 0.0 CBD 0.0 0 0 0 0

30.0 0 0 2 0

0.3 0.0 27 8 0 0

30.0 0 22 0 0

1.0 0.0 66 41 6 0

30.0 63 83 69 46

Delta-9-THC 0.0 CBN 0.0 0 0 0 0

3.0 22 9 0 0

0.1 0.0 1 0 0 0

3.0 32 25 0 0

0.3 0.0 28 8 0 0

3.0 63 53 5 0

1 .0 0.0 67 41 9 1

3.0 89 73 10 0

aPercentage of responding to the THC-appropriate position for 8-12 rats at each injection test

interval.

SOURCES: Hiltunen and Järbe 1986b; Järbe and Hiltunen 1987.

11-OH-THCs (11-OH-delta-9-THC and 11-OH-delta-8-THC) has been

investigated using rats (Ford et al. 1984; Järbe and McMillan 1980; Weissman

1978), and pigeons (Järbe and McMillan 1980). Both of these species

generalized from the delta-9-THC stimulus to the test compounds in a

dose-related manner. However, 11-OH-delta-9-THC was more potent than

11-OH-delta-8-THC. In turn, 11-OH-delta-8-THC was at least as potent as

delta-9-THC.

85
TABLE 2. Generalization of delta-9-THC (3 mg/kg) to combinations of

cannabinol (CBN) and cannabidiol (CBD)

Drug Combination (mg/kg) Mean Time Post Administrationa (hr)

1.0 5.5

CBN 0.0 CBD 0.0 0.0 0.0

10.0 0.0 0.0

30.0 0.0 0.0

10.0 0.0 31.0 22.0

10.0 25.0 11.0

30.0 24.0 5.0

17.0 0.0 75.0 51.0

10.0 39.0 30.0

30.0 30.0 12.0

aAverage across two lime blocks (0.5 and 1.5 hr; 4.5 and 6.5 hr).

SOURCE: Hiltunen and Järbe 1986a

Other THC metabolites that have been studied for their cannabimimetic effect are

8-alpha-OH-delta-9-THC, 8-beta-OH-delta-9-THC, 8-alpha, 11 di-OH-delta-9-THC,

and 8-beta, 11 di-OH- delta-9-THC (Ford et at. 1984; Järbe and McMillan 1980).

Only the 8-beta, 11 di-OH-delta-9-THC produced complete generalization for the

delta-9-THC stimulus by pigeons. The metabolite 8-beta-OH-delta-9-THC did

produce partial generalization for the MC stimulus; however, this effect may

reflect species differences and may be limited to pigeons (Järbe and McMillan

1980). Other investigations using rats and this compound did not find

generalization for the THC stimulus (Ford et al. 1984). However, because lower

doses of the test compound were used for the tests with rats, and pigeons tend

to be more sensitive than rats to the the THC cue, further tests using higher doses

of this compound with rats must be conducted before a conclusion about

species differences can be drawn. Thus, apart from the 11-OH-THCs, the

remaining metabolites may possess MC-like action, but fairly high doses are

needed to produce a cannabimimetic effect (see ED50 values, estimated

according to a logarithmic regression analysis, presented in table 3). Thus,

these data suggest that the contribution of these metabolites to the

psychoactive properties of cannabis in natural settings is very minor. However,

86
TABLE 3. ED50 Values for generalization of drug-appropriate responding to

the training stimulus and THC metabolites by animals trained to

discriminate delta-9-THC

THC

Delta-9

11-OH-delta-9

11-OH-delta-8

Species N

RatC8

Pigeond6

Rat 7-8

Pigeon 8

Rat 8

Pigeon 8

The a

0.5

1.0

1.5

0.5

1.5

0.5

1.5

ED50 (r)b

1.15 (0.93)

1.07 (0.98)

0.43 (0.94)

0.65 (0.88)

0.10 (0.99)

1.12 (0.97)

0.38 (0.97)

8beta,11-di-OHdelta-9Pigeon 4-5 1.5 19.00 (0.99)

a Hr from injection until testing.

b

The ED50 (mg/kg) determinations assessed from the correlation coefficient (r ) by logarithmic

regression analysis of gradients for percentage of drug-appropriate (percent RDP) gradients.

c Rats were trained to discriminate 3 mg/kg administered IP 30 min prior to session onset.

d Pigeons were trained to discriminate 1 mg/kg administered IM 90 min prior to session onset.

SOURCE: Järbe and McMillan 1980.

examinations of the chemical structure of cannabinoids have helped to

elucidate the compounds contributing to cannabis intoxication.

SYNTHETIC CANNABINOIDS AND CANNABIMIMETICS

The SAR between chemical structure and drug action reveals the structural

requirements for the stereoselectivity or stereospecificity of a given drug effect

or action. Stereoselectivity is evidenced when a potency difference for a given

effect is obtained between isomers, whereas stereospecificity refers to a

complete absence of a drug effect for a particular isomer. A major purpose of

the SAR investigations with synthetic cannabinoids and cannabimimetics is to

eliminate the enantiomer that induces the undesirable, psychoactive properties

of these compounds (stereospecificiiy for a THC-like effect) and retain the

structures that induce medically useful effects. These studies have shown that

the THC-like properties of these compounds are highly dependent on particular

87
structural configurations. (See table 4 for compounds tested in our laboratory;

chemical structures are indicated in the original reports cited below.)

The relationship between the structure of cannabinoids and their stimulus

effects has been studied using delta-9-(11)-THC and (3”S)-3”-OH-delta-9-THC.

The results of these studies indicate that the stimulus effects of

delta-9-(11)-THC are less potent than those of delta-9-THC (Semjonow and

Binder 1985; see also Franke et al. 1985). However, the effect of this

compound on the development of rapid tolerance to the stimulus effects of

delta-9-THC is similar to the effect of the parent compound. For animals that

are pretreated with high doses of either delta-9-THC or delta-9-(11)-THC, then

tested for discrimination of the THC cue 24 to 48 hr after these high-dose

administrations, discrimination of the THC stimulus is attenuated. Thus, though

delta-9-(11)-THC may be less potent than the parent compound, it apparently

does have some similar psychoactive effects.

On the other hand, (3"S)-3”-OH-delta-9-THC may be more active than

delta-9-THC and the R epimer of this compound, and it may be an important

stereoselective determinant of the THC cue. For example, for rats trained to

discriminate delta-9-THC, (3"S)-3"-OH-delta-9-THC was approximately 5 times

more potent than the training compound and approximately 7 times more potent

than the R epimer (Martin et al. 1984). This difference in the effect of epimers

demonstrates the stereoselectivity of cannabimimetic effects. Selectivity of the

relationship between R and S epimers and cannabimimetic activity is further

demonstrated by studies with delta-10-THC structures.

Recent synthesis of stereoisomers for delta-10, 10a-THC and delta-10a,6a-THC

(Srebnik et al. 1984) has permitted further investigations of the relationship of R

and S epimers on cannabimimetic activity. Investigations of the SAR of

delta-10a,6a-THC have focused on the 9S and 9R enantiomers. In general,

these studies indicate that, for this structure, although the 9S enantiomer is less

potent than delta-9-THC, the 9R enantiomer may not be active. The

psychoactive properties of the delta-10a,6a enantiomers have been tested in

humans (Hollister et al. 1987) and pigeons (Järbe et al. 1988b). For humans,

the 9S enantiomer had psychoactive properties that were qualitatively similar to

the effects of delta-9-THC, but the effects of this enantiomer were quantitatively

less potent (i.e., 1:3-6). For pigeons, discrimination of the delta-9-THC stimulus

was generalized to this enantiomer. When the psychoactive properties of the

9R enantiomer were tested with humans, they were found to be not active

(Hollister et al. 1987). However, for the tests with humans, this compound could

88
TABLE 4. Generalization a

 of synthetic conqmmdsb to THC-appropriate

responding

Compound Species N Dose rangec

 ED50 (r)

d

Delta-8-THC

(+)-Delta-8-THC

(-)-Delta-8-THC-DMH

(-)-11-OH-delta-8-THC-DMH

(+)-11-OH-delta-8-THC-DMH

Benzofuran, 7

Benzofuran, 8

Delta-10-THC

(9S,6aR)-delta-10,10a-THC

(9R,6aR)-delta-10,10a-THC

(9S)-delta-10a,6a-THC

(9R)-delta-10a,6a-THC

Hexahydrocannabinols

(–)-11-OH-HHC(equatorial)

(-)-11-OH-HHC(axial)

Rate

4 5.6-10 none

Rat 14-16 0.1-0.56 0.21

Pigeon16-7 0.0175-0.175 0.05

Rat 11 0.003-0.03 0.01

Pigeon 5-7 0.0001-0.0056 0.002

Rat 4-9 3-10 none

Pigeon 4 3-10 none

Rat 9 up to 10 none

Pigeon 5 1-5.6 3.57

Rat 9 0.3-3.0 0.72

Pigeon 5 0.1-0.3 0.17

(none)

(0.94)

(0.95)

(0.99)

(0.99)

(none)

(none)

(none)

(0.78)

(0.98)

(0.99)

Pigeon

Pigeon

Pigeon

Pigeon

2-6

3-4

3-4

4

1-17.5 none (none)

3-17.5 4.78 (0.92)

1-10 6.68 (0.81)

3-30 11.90 0.98)

Rat 14 0.1-1 0.24 (0.95)

Pigeon 7 0.01-0.1 0.02 (0.87)

Rat 9 1-3 1.58 (0.98)

Pigeon 7 1 3 1.72 (0.97)

aPercentage responding to the drug-appropriate position (percent RDP) at postadministration time

interval at which compound disclosed highest potency.

bChemical structures are found in original reports cited in text.

c

In mg/kg.

dED50 (mg/kg) determinations assessed from correlation coefficient (r) by logarithmic regression

analysis of gradients.

e

ats were trained to discriminate 3 mg/kg administered IP 30 min prior to session onset.

fPigeons were trained to discriminate 0.56 mg/kg administered IM 90 min prior to session onset.

89
not be tested in high doses. In contrast, for pigeons, this 9R enantiomer did

generalize for the THC stimulus, but it was 2 to 3 times less potent. In addition,

these animal tests used the acetate form of this compound rather than the

parent phenol that was tested in humans. Thus, further tests with this

enantiomer must be conducted to determine its efficacy as a cannabimimetic.

Examinations of the structural activity of delta-10,10a-THC has focused on the

9R, 6aR and 9S, 6aR epimers in their acetate form. The 9R, 6aR epimer

disclosed THC-like activity (ED50= 4.78 mg/kg), whereas the 9S, 6aR epimer

did not, even at the highest dose tested (17.5 mg/kg) with pigeons (Järbe et al.

1988b). Thus, these SAR investigations of delta-10-THC compounds suggest a

stereoselectivity for induction of cannabimimetic action. The effect of S and R

epimers of delta-10, 10a-THC on cannabimimetic activity parallels findings

obtained with the epimers of 11-OH-hexahydrocannabinol (see below). Other

SAR studies of THC effects have focused on the (+)- and (-)-enantiomers,

particularly with regard to delta-8-THC compounds.

The stereoselectivity of delta-8-THC may be a function of the (-)- and

(+)-enantiomers. For example, (+)-delta+THC is considerably less potent (Järbe

et al. 1981) than (-)-delta&THC (Järbe and Henriksson 1974; Järbe et al. 1976) in

eliciting the delta-9-THC discriminative response. In addition, tests with

THC-trained rats and pigeons (Järbe et al. 1981, 1989a; Järbe and Mechoulam,

unpublished data 1983) have shown that the dimethyheptyl (DMH) homolog of

delta-8-THC, i.e., delta-8-THC-DMH in its levorotation [(-)delta-8-THC-DMH] is a

potent cannabimimetic with a slow onset and long duration.

In order to examine a possible dissociation between the effects of delta-8-THC

enantiomers, monohydroxylated dimethyl heptyl homologs of a major

metabolite of delta-8-THC (11-OH-delta-8-THC) have been studied [i.e.,

(-)-11-OH-delta-8-THC-DMH and (+)-11-OH-delta-8-THC-DMH]. These studies

also indicated that the (-)-enantiomer may elicit the cannabimimetic effects of

delta-8-THC. The compound (-)-11-OH-delta-8-THC-DMH was more potent

than naturally occurring delta-9-THC by approximately 87 times in rats

(ED50 = 0.01 mg/kg) and 73 times in pigeons (ED50 = 0.002 mg/kg) (Järbe et al.

1989a); Mechoulam et al. 1988). However, the (+)-enantiomer (+)-delta-8-THC

was much less potent than the (-)-enantiomer in generalization for the THC

cue. It was inactive at doses higher than the ED50 value by approximately 1,000

times for rats and 4,500 times for pigeons. Furthermore, similar effects of these

enantiomers on cannabinoid activity have been obtained for performance on the

ring test with mice and the rotarod neurotoxicity test with rats (Mechoulam and

90
Feigenbaum 1987; Mechoulam et al. 1988). Recently obtained data (Järbe and

Mechoulam unpublished data 1990) also indicate that this stereoisomeric

differentiation is important for cannabimimetic activity by naturally occurring

compounds. Stereospecificity of the (+)-isomer was obtained with pigeons

trained to discriminate between delta-9-THC (0.56 mg/kg) and vehicle. Tests

with the (+)- and (-)-isomers of the major psychoactive metabolite

11 -OH- delta-8-THC indicated that the (+)-isomer did not induce THC-like

responding after administration of doses up to 10 mg/kg. Studies with

hexahydrocannabinols (HHCs) have further demonstrated the structural

requirements for cannabimimetic effects, including the importance of the

position of the methyl group on cannabimimetic activity.

Tests with HHCs indicate that conversion of THC to 11-OH-THC is not a

necessary prerequisite to achieve a THC effect (Browne and Weissman 1981;

Ford et al. 1984; Weissman 1978) and that HHCs may be highly stereoselective

and stereospecific. For example, examinations of the relative potencies of the

HHCs levonantradol and dextronantradol have demonstrated that levonantradol

was considerably more potent than dextronantradol for eliciting the

THC-appropriate response. In addition, the dextro form did not substitute for

delta-9-THC at the dose of 3.2 mg/kg, whereas the levo form disclosed an ED50

value of 0.02 mg/kg, making it one of the more potent cannabimimetics

examined so far with drug discrimination of delta-9-THC (Browne and

Weissman 1981). Furthermore, two epimers of 11-OH-HHC elicited

cannabimimetic activity in both rats and pigeons (Järbe et al. 1986). The

difference in the potency of these isomers was determined by the position of the

methyl group in relation to the plane of the cyclohexane ring. Generalization to

the THC stimulus was more potent for the isomer in which the methyl group is

equatorial (i.e., lies approximately in the plane of the cyclohexane ring) than for

the isomer in which the methyl group was axial (i.e., protrudes from the plane of

the cycbhexane ring). However, species differences in response to these

compounds make elucidation of their SAR less clear. For example, the

difference in the relationship between the structure of isomers and elicited

cannabimimetic activity is considerably less for rats than for pigeons (for

chemical structures, see Järbe et al. 1986).

Although most of the studies on the SAR of cannabinoids support a contention

that a dihydrobenzopyran structure is a necessary component for

cannabimimetic activity (Mechoulam and Edery 1973; Mechoulam and

Feigenbaum 1987), some studies suggest that this structure is not a central

requirement for THC- like activity. For example, an examination of a derivative

91
of 3-phenylcyclohexanol (CP 47497), which lacks this structure, indicates that

this compound has potent cannabimimetic activity (Weissman et al. 1982). In

addition, other tests conducted with rats and pigeons trained to discriminate

delta-9-THC indicated that there was profound generalization to a DMH

homolog of CP 47497 (Järbe and Mechoulam unpublished data 1990). Other

compounds that lack this structure but have cannabimimetic properties are

benzofuran cannabinoids. These compounds have a benzofuran, rather than

the delta-9-THC benzopyran, moiety and induce marked cannabimimetic

activity for both rats and pigeons (Mechoulam et al. 1990). In addition,

levonantradol, a hexahydrocannabinol with potent THC-like activity, also

deviates from this structural requirement.

It is not yet known why compounds that lack a dihydrobenzopyran structure

have cannabimimetic properties. One suggestion might be that generalization of

the THC response to the effects of these compounds may result from analgesic

actions. CP 47497, levonantradol, and delta-9-THC all have analgesic

properties. Thus, if analgesia is part of the stimulus effects of THC, then

generalization to these compounds may reflect generalization of THC

analgesia. However, the analgesic properties of THC have been shown to be

dissociated from its DS effects. Other compounds having a standard

cannabinoid configuration and strong analgesic properties (e.g., the dextro form

of THC such as (+)-11-OH-delta-8-THC-DMH) do not have cannabimimetic

properties (Järbe et al. 1989a; Mechoulam et al. 1988). Therefore, the

possibility that the cannabimimetic properties of compounds that lack a

dihydrobenzopyran structure reflect their analgesic action seems unlikely, and

firm confirmation of the chemical structure needed to induce THC intoxication

remains to be elucidated.

CONCLUSION

Determining the necessary chemical components for inducing the DS

properties of delta-9-THC is important for understanding the factors that

contribute not only to cannabis abuse but also to the unwanted side effects of

cannabis therapy. By means of DDL procedures to examine naturally occurring

cannabinoids, metabolites, and synthetic compounds, the cannabimimetic and

medicinal properties of cannabinoids (e.g., analgesia, antiemesis, and

anticonvulsive) have been dissociated, and some requirements for inducing

THC intoxication have been delineated.

92
Studies using the DDL procedure with naturally occurring compounds have

shown that cannabis intoxication is induced primarily, but not solely, by the

direct action of delta-9-THC. The stimulus effects of THC may be modulated by

coadministration of other cannabinoids, such as CBD or CBN. In fact, CBN may

have direct cannabimimetic properties that are attenuated by coadministration

of CBD. In addition, cannabimimetic activity can be induced by some THC

metabolites, specifically the 11-OH- and 8-beta, 11 di-OH-delta-9-THC

metabolites. However, in order to determine the chemical requirements for

inducing cannabis intoxication, studies of the SAR of cannabinoids using

organic chemistry and DDL are necessary.

Examinations of cannabinoid SAR have shown that the structures determining

THC intoxication vary among compounds and are highly stereoselective,

perhaps even stereospecific. Some of the relevant structural features that have

been identified are epimerii and enantiomeric preferences (e.g., R and S

epimers, (+) and (-) enantiomers), and the position of the C-11 substituent on

the cyclohexane ring. For example, the (3"S")-3”-OH-delta-9-THC is more potent

than the R epimer. For delta-10,10a-THC, the 9R,6aR epimer disclosed

THC-like activity, whereas the 9S,6aR epimer did not. in addition, the 9s

enantiomer for delta-10a,6a-THC is more potent than the 9R enantiomer.

Stereoselectivity of THC intoxication is also demonstrated with delta-8-THC

enantiomers and HHC isomers. For example, generalization of delta-8-THC for

the THC cue may be induced by the (-)-enantiomer but not by the

(+)-enantiomer. In addition, SAR studies of HHCs have demonstrated that

cannabimimetic activity also shows isomeric selectivity. For the compounds

examined in both rats and pigeons, the equatorial isomer is more potent than

the axial isomer (this SAR is also described above for delta-10,10a-THC).

Furthermore, some SAR studies suggest that a dihydrobenzopyran structure is

not a necessary requirement for cannabimimetic activity. A derivative of

3-phenylcyclohexanol that lacks this structure (CP 47497), an HHC (levonantradol),

and a benzofuran cannabinoid have potent cannabimimetic activii.

Thus, the complexity of the SAR of cannabinoids and cannabimimetics is

apparent. However, the utility of DDL and synthesis of new compounds as

research tools for understanding this SAR are also apparent. It is only with

further use of these research tools that a fin understanding of cannabimimetic

activity may be gained.

93
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ACKNOWLEDGMENTS

This work was supported by research grants from the Swedish Medical

Research Council, the Bank of Sweden Tercentenary Foundation, the Mangnus

Bergvall Foundation, and the Swedish Social Welfare Board.

AUTHORS

Torbjörn U.C. Järbe

Diane A. Mathis

University of Uppsala

Department of Psychology

Box 1854

S-751 48 Uppsala

Sweden

99

Discriminative Stimulus Properties of

Nicotine: Mechanisms of Transduction

John A. Rosecrans and Heidi F. Villanueva

INTRODUCTION

Cholinergic drugs, for the most part, are orphans among the drugs studied by

behavioral pharmacologists. Until recent evidence was found that Alzheimer’s

disease might entail a cholinergic deficit, few researchers paid attention to this

class of pharmacologic agents. Even the knowledge that more humans

maintain nicotine (or cotinine) levels more of the time than any other drug

(except perhaps caffeine) has engendered little interest in this area of research.

In spite of this general lack of interest in cholinergic pharmacology, however,

these drugs do produce many interesting effects that may eventually provide

some answers about several disease states involving memory deficits,

Parkinson’s disease, and even drug dependencies. In addition, these agents

might offer some unique approaches to treatment if studied more intensely.

The major goal of this review, however, is to summarize recent experiments

concerning how nicotine acts at its respective receptor with a focus of learning

more about how this interaction can elicit nicotine’s discriminative stimulus (DS)

effect. Because of the many recent reviews on nicotine’s DS effects (Rosecrans

1989; Stolerman 1987; Stolerman and Reavil 1989), this paper spotlights the

current state of the art and attempts to fill in the gaps where appropriate.

ARRANGEMENTS OF CHOLINERGIC NEURONS AND ACETYLCHOLINE

RECEPTORS (AChRs)

Our appreciation of the physiological importance of both the autonomic and

central nervous systems has relied heavily on evaluating the in viva effects of

cholinergic drugs such as nicotine. Because drug discrimination (DD)

techniques have been used for studying these drugs, it has become

increasingly apparent that much is yet to be learned about the function of

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central nicotinic-cholinergic receptors (n-AChRs) and muscarinic-cholinergic

receptors (m-AChRs). Research in molecular biology has advanced far in the

knowledge of how these drugs affect specific AChRs. We may be at a point in

our own science where we can draw on this information to evaluate the cellular

transduction mechanisms that permit these drugs to exert DS control of

behavior.

The most important contribution made by cholinergic in vivo and in vitro

neuroscience has been the knowledge that m-AChRs and n-AChRs have

different brain region distributions and are not monolithic in relation to receptor

type. Even though acetylcholine (ACh) appears to be the major endogenous

ligand (or neurotransmitter) at both the m-AChR and the n-AChR, its function

and mechanism of neuronal transduction appear uniquely different at both

receptor sites (figure 1). The n-AChR appears linked to a cation channel that

serves as its major transductional signal, whereas the m-AChR appears to

employ a more traditional second messenger, inositol triphosphate (IP3) to carry

out its function.

From the perspective of evaluating the DS properties of drugs affecting these

receptors, we find little overlap. Arecoline, for example, appears to act at only

the muscarinic site and is antagonized by atropine but not by mecamylamine,

whereas nicotine uniquely acts at the nicotinic site and is antagonized by

mecamylamine but not by atropine (Rosecrans 1989; Stolerman 1987). In

addition, only those drugs that act at the muscarinic site appear to generalize to

increases in brain ACh-via the administration of the acetylcholinesterase

(AChE) inhibitor physostigmine (Meltzer and Rosecrans 1988)-prompting us

to rethink how nicotine might be interacting with its cholinergic receptor (figure

1).

The neuroanatomical arrangement of the cholinergic system has also been

described in great detail. Cholinergic pathways appear to consist of neurons

affected by a variety of cholinergic drugs, but for the most pan postsynaptic (or

presynaptic) receptors of these cholinergic systems behave as though they

were all muscarinic (Jung et al. 1987; 1988a). Thus, although these

arrangements have been delineated and there seems to be some separation

between n-AChRs and m-AChRs (from DD experiments; Rosecrans and

Meltzer 1981), there is little evidence to intimate which neuronal pathways

innervate n-AChRs or m-AChRs. Furthermore, there is little evidence to support

the contention that n-AChRs are in fact innervated by specific cholinergic

neurons (figure 2).

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ACETYLCHOLINE ACETYLCHOLINE

Nicotinic Muscarinc

FIGURE 1. A schematic comparison of nicotinic and muscarinic cholinergic

neurons. The n-AChR is viewed as linked to a cation channel;

whereas the m-AChR mediates its effects via a second

messenger, IP3 or CAMP. (Redrawn from Shephard 1988, with

permission.)

Thus the question: Is the n-AChR localized at only presynaptic sites of both

cholinergic and noncholinergic neuronal systems? This important question has

been posed by several investigators. The evidence that many n-AChRs are

located on presynaptic dopamine (DA)-containing neurons is rather convincing

at this time (Wonnocott et al. 1989). In addition, lwamoto (1989) has also

provided a model in which the n-AChR appears to play a pivotal role at select

presynaptic cholinergic neurons. In this model nicotine is viewed as interacting

with an n-AChR, eliciting the release of ACh onto an m-AChR (figure 3). This

model is engaging and reminiscent of the peripheral autonomic nervous

system, the ganglia being represented by a presynaptic n-AChR. Thus, many

questions remain concerning the arrangement and interrelationships between

these two receptor classes.

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FIGURE 2. A schematic presentation of select ascending cholinergic

pathways. Cell body groups correspond to the Ch subdivisions.

Abbreviations: ac=anterior commissure; amg=amygdala;

cbl=cerebellum; ci=inferior colliculus; cp=caudata putamen;

cs=superior collliculus; gp=slobus pallidus; h=hippocampus;

ltn=laterodorsal tegmental nucleus; ms=medial septal nucleus;

nb=nucleus basalis; n=neurocortex; nhl=nucleus of the horizontal

limb complex; nvl=nucleus of the vertical limb complex;

ob=olfactoty bulb; th=thalamus; ppn=pedunculopontine tegmental

nucleus. (From Wainer et al. 1984.)

SPECIFICITY AND SELECTIVlTY OF THE NICOTINE-ELICITED DS

The ability of nicotine to exert DS control of behavior is time and dose related,

and it appears to correlate well with blood and brain levels of nicotine (and

analogs) even though no major brain area site appears to concentrate this drug

(Rosecrans 1989; Stolerman 1987). The nicotine DS appears to be elicited at

central cholinergic receptors specific to nicotine and located in at least two brain

area sites, the hippocampus and reticular formation. In addition, these studies

indicate that slight changes in the molecular structure of nicotine can greatly

reduce its effectiveness as a DS (figure 4).

Comparing data obtained in squirrel monkeys (-)-nicotine metabolites,

(-)-cotinine, and (-)-nornicotine generalized with the nicotine (0.2 or 0.4

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FIGURE 3. A model of the cholinergic neuron showing the relationship

between n-AChRs and m-AChRs. A model of postulated

mechanisms and sites of action on nicotine-induced

antinociceprion at a cholinergic nerve terminal in the mesoponrine

tegmentum. (From lwamoto 1989, with permission.)

μmole/kg IV) DS but were 29-2,000 times less potent (Takada et al. 1988). In

addition, two compounds, (+)-nicotine and 3-pyridylmethylpyrrolidineoptical-positional isomers of nicotine (figure 4)-appear to have a similar

pharmacology but are one-tenth as potent (Rosecrans 1989; Stolerman and

Reavil 1989). These compounds, besides generalizing with nicotine, can also

be antagonized by mecamylamine, indicating they also share a common

molecular site of action with (-)-nicotine. Thus, the spectrum of nicotinic activity

across a variety of chemical structures, from the vantage point of its DS

properties, is quite limited.

Stolerman and coworkers also remind us that dose and schedule of

reinforcement are important variables to be considered in nicotine

generalization studies. In one such investigation, Stolerman et al. (1984)

demonstrated that the nicotine analogs anabasine and cytisine were recognized

105
Nicotine 3- pyridylmethylpyrrolidine

(3 PMP)

Nornicot ine Cotinine

FIGURE 4. Chemical structures of nicotine analogs or metabolites studied.

more readily as nicotine if the training dose was reduced to 0.1 mg/kg of

nicotine versus saline. More recently, Stolerman (1989) further showed that

nicotine-trained rats would generalize to (+)-amphetamine (predominant DA

receptor action) when the training dose was reduced from 0.4 to 0.1 mg/kg in

rats trained under either a tandem or a fixed ratio schedule. Previous work

indicated that nicotine would generalize only partially to (+)-amphetamine in rats

trained at 0.4 mg/kg under a VI-15 second schedule of reinforcement (Chance

et al. 1977). Besides demonstrating the reliability of evaluating nicotine’s effects

across schedules of reinforcement, this study also demonstrated the need to

consider training dose when evaluating other compounds that have the

potential to act at similar sites of action. Thus, this study appears to

demonstrate that nicotine, while having a predominant effect at some n-AChRs,

may have overlapping effects at DA receptors (Rosecrans 1988) as well. This

potential DA interaction has been difficult to study in DS experiments, possibly

because the cholinergic versus dopaminergic activity may dominate at higher

nicotine training doses. The approach used clearly demonstrates the need to

106
evaluate drugs at several training doses before judging the singularity of

mechanism of drug action.

MECHANISM OF ACTION OF THE NICOTINE DS

A major assumption of research involving nicotine’s mechanism or mechanisms

of action is that it is capable of eliciting DS control of behavior by activating

specific ACh-sensitive cholinergic receptors. That is, nicotine was envisioned to

act as though ACh were released at some cholinergic receptor, a site also

antagonized by mecamylamine (figures 1 and 3). To test this hypothesis, a

series of experiments was designed to determine whether the nicotine-elicited

DS could be mimicked by elevating brain ACh by inhibiting AChE. Attempts to

accomplish this goal, however, were negative in outcome (Meltzer and

Rosecrans 1988). This approach, on the other hand, has been quite useful in

demonstrating that the DS effects of the m-AChR receptor agonist arecoline are

similar in action to ACh at this site.

Rosecrans and coworkers have attempted a variety of approaches to

circumvent our failures, including training a group of rats to discriminate

subcutaneously (SC) administered physostigmine (0.25 mg/kg) and

(-)-hyoscyamine (2 mg/kg SC) as a cocktail (Rosecrans 1989). The rationale

was that this drug combination would be pharmacologically equivalent to

nicotine. Although theoretically sound, the drug combination did not generalize

to nicotine, nor were these investigators able to antagonize the DS elicited with

mecamylamine. Similar findings have been observed when physostigmine

alone was used as a DS (Jung et al. 1988b; Tang and Franklyn 1988). Most

m-cholinergic agonists generalized with the physostigmine-elicited DS.

Mecamylamine was also incapable of antagonizing the physostigmine-induced

DS, even though both scopolamine and atropine were potent antagonists of this

stimulus. This result, in addition to the physostigmine DS being unable to

generalize to nicotine, again suggests that the DS is primarily muscarinic.

These studies, therefore, suggest that in contrast to our initial cholinergic model

(figure 1), nicotine may not be acting at a specific ACh-sensitive n-AChR. On

the other hand, it may be that the pharmacological effects measured by our

approaches, which are antagonized by mecamylamine, do not involve an

n-AChR. As inferred from the research of DiChiara and lmperato (1988)

nicotine could be exerting stimulus control of behavior by acting via a

mesolimbii dopaminergic pathway common to several other drugs of

dependence such as morphine and (+)-amphetamine. These workers, using in

107
vivo microdialysis techniques, demonstrated that nicotine was able to release

brain DA from these areas at readily discriminable doses.

Even though nicotine could ultimately be shown to elicit DS control of behavior

via a noncholinergic mechanism (such as acting at presynaptic DA n-AChRs),

there is no reason to believe that all of nicotine’s effects are the result of a

noncholinergic neuronal interaction. Mecamylamine (a drug classified as

anticholinergic), for example, appears unable to antagonize chronic

nicotine-induced n-AChR up-regulation, suggesting that this antagonist is acting

at a different site that is cholinergic (Schwartz and Kellar 1985). This finding is

also supported by earlier work demonstrating mecamylamine’s inability to

compete with either ACh or nicotine at n-ACh binding sites (Schwartz et al.

1982). More importantly, mecamylamine appears to be unable to attenuate the

development of tolerance to nicotine but yet readily antagonizes nicotine’s

acute behavioral disruptive effects in the same paradigm or in the nicotine DS

(Rosecrans et al. 1989). Thus, these findings strongly suggest that nicotine is

acting at two different receptors, one mecamylamine sensitive and ACh

insensitive, and a second n-AChR that is ACh sensitive.

DESENSITIZATION OF THE n-AChR

The preceding discussion leads us to conclude that nicotine may be acting at

either of two receptors, but we might also review what we know about the

mecamylamine-nicotine interaction. As Stolerman (1987) pointed out, this drug

interaction appears to be noncompetitive and may be acting presynaptically or

postsynaptically at some cation channel. This concept has much support and

also points out that nicotine and mecamylamine do not have to act at the similar

sites of action to explain the data thus far collected. Furthermore, such a

hypothesis gives much flexibility to the alternative concept that nicotine is acting

at an ACh-sensitive receptor; perhaps our approaches to the problem and to

mecamylamine have driven us to our current models.

We are at a stage in our quest of nicotine’s molecular mechanisms to begin

asking how nicotine can alter the receptors to which it binds. The research of

several investigators (Marks et al. 1983; Nordberg et al. 1985; Schwartz and

Kellar 1985; Wonnocott 1987; Wonnocott et al. 1989) has provided us with

much evidence that nicotine may be acting partially by inducing a

desensitization of the n-AChRs, especially when administered chronically. This

possibility has been manifested by the ability of nicotine to induce an

up-regulation of nicotinic receptors following repeated dosing. Furthermore, it

108
has also been established that one of the major differences between cholinergic

receptors is the ability of ACh to desensitize the nicotinic receptor. Ochoa et al.

(1989) in an impressive review, describe research that clearly demonstrates

how ACh may be acting at the n-AChR receptor (figure 5). The major concept

put forward is that ACh, after being released at the synapse, interacts with the

n-AChR to form an activated state that promotes cation movement (via a

channel) across the postsynaptic membrane. As receptor affinity for ACh

increases, the n-AChR moves toward a deactivated (desensitized) state that

closes the same cation channel. As these investigators pointed out, this

mechanism may be essential to terminating the synaptic transduction effects of

ACh and may be an essential mechanism to several pathological states

involving learning and memory deficits as well as myasthenia gravis.

In all, a picture emerges that may assist our understanding of how nicotine is

eliciting its effects at the n-AChR and, consequently, how it may act to exert

stimulus control of behavior. The basic theoretical model presented views

nicotine as an agonist that can induce a secondary desensitization (in in vivo

pharmacological terms, tachyphylaxis or acute tolerance). In this conceptual

model, nicotine is envisioned as mimicking ACh directly or indirectly (inducing

ACh release presynaptically) at a cholinergic receptor, thus opening a channel

and allowing some cation to enter the postsynaptic or presynaptic cell-possibly

Ca++ (the transductional signal is the entrance of a specific cation). Depending

on dose and time parameters, and possibly on the excitability level of the

n-AChR, the nicotine levels (or increase in ACh) achieved at a specific

hippocampal site or reticular formation site or both will consequently provide the

neuronal mechanism (opening of the cation channel) that is important to

exerting stimulus control. It is further postulated that, following the initial agonist

effect, a rapid desensitization of the receptor will occur (close the channel to

further cation entry), reducing the strength of the nicotine-elicited DS (figure 5).

To test this hypothesis, we initiated a series of experiments from which

preliminary evidence suggested that nicotine was capable of inducing rapid in

vivo desensitization-that is, tolerance (figure 6). Rats were initially trained to

discriminate nicotine (0.4 mg/kg, SC) from saline when a VI-20 second

schedule of reinforcement was used; responding was reinforced during test

sessions. Following initial training, individual rats were (1) administered 0.8

mg/kg of nicotine (SC) at time zero in their home cage; (2) administered a

second training dose of nicotine 15-180 min after the first nicotine challenge

dose; and (3) tested for ability to discriminate nicotine during a 2-min test

session (5 min after training dose) in the behavioral environment associated

109
FIGURE 5. Mechanisms of n-AChR desensitizations. Represents an idealized

nicotinic cholinergic synaptic junction containing acetylcholine

(black dots) contained within synaptic vesicles (in circles) and

postsynaptic membrane. (From Ochoa et al. 1989, with

permission.) A-Receptor exists in an equilibrium condition of a

mixture of two forms: resting (R) and desensitized (D) states.

B-An action potential facilitates the release of ACh. The result is

a sudden increase in ACh concentration at the synaptic cleft and

occupies one of the two receptor states, R and D. This induces a

conformational change, which leads to the activated state (A), in

which the channel opens allowing cation movements essential for

the development of a postsynaptic action potential. As soon as

ACh occupies its sites, the affinity of the receptors toward ACh

increases and the D state is promoted, resulting in the termination

of the action of ACh.

110
TIME AFTER 1ST NICOTINE DOSE (Minutes)

FIGURE 6. Evidence of in vivo desensitization in rats trained to discriminate

nicotine (0.4 mg/kg SC) from saline. Data represent

nicotine-correct responding tested at various time periods

following an initial challenge of nicotine (0.80 mg/kg SC). Rats

were tested for two minutes and responses on both levers were

rewarded.

111
with learning the nicotine DS. The results of this study indicated that 11 of 15

rats exhibited tachyphylaxis (desensitization) to nicotine-elicited DS;

tachyphylaxis, when observed, occurred at different intervals between nicotine

challenge doses (figure 6).

These results, although preliminary, are encouraging in that they may shed

some light on how nicotine exerts DS control of behavior. Of special interest is

that these data may help explain why researchers (Jung et al. 1988b; Meltzer

and Rosecrans 1988; Rosecrans 1988; Stolerman 1987) have been unable to

mimic nicotine’s effects by increasing brain ACh through AChE inhibition,

especially if our contention is correct that nicotine is exerting its effects via an

interaction at the n-AChR. This is especially puzzling because m-cholinergic

agonists readily generalize to increases in brain ACh. An explanation of these

observations is that n-AChRs were desensitized by physostigmine, which would

produce an “antagonistic state” and prevent any generalizations in rats under

nicotine-induced stimulus control. Additional studies are obviously needed to

determine, whether desensitization is a mechanism common to all rats or a

special case involving the ability of some n-AChRs to respond to nicotine in this

manner. Thus, the experimental approach has not been at fault in

demonstrating the relationship between nicotine and its n-AChR; perhaps the

fault has been with the differential sensitivity of n-AChR to ACh or nicotine or

both.

SUMMARY AND CONCLUSIONS

Our thinking about how nicotine might be inducing DS control of behavior has

changed drastically in the past 25 years. Our first inclination was that nicotine

was mimicking ACh at a variety of specific and select n-AChRs. Then several

nicotine researchers suggested that nicotine might be acting via specific and

select noncholinergic receptors. At present, we seem to have returned to the

view that nicotine may have pronounced effects at the n-AChR (figure 1), at

least in some specific cases such as in the development of tolerance and in rats

trained to discriminate nicotine (figure 6).

The endogenous ligand has not changed, but the mechanism of how it affects

presynaptic and postsynaptic receptors appears to have been rediscovered.

The concept of rapid ACh-induced desensitization at the n-AChR is not new

and appears basic to cholinergic neuronal function (figure 5). The

desensitization concept has been revitalized by several investigators who also

consider this mechanism important to how nicotine might act in protecting DA

112
neurons from neurotoxicity of chemicals such as 6-OHDA or MPTP (Janson et

al. 1988). The overall concept suggests that n-AChR desensitization at

presynaptic DA sites may reduce neuronal accessibility to select exogenous

neurotoxins and thus attenuate neuronal destruction. This hypothesis has also

been partially validated in relation to the cholinergic neuron by providing

preliminary evidence that nicotine was able to reduce cholinergic neuron

destruction (measured by brain ACh levels) via the intraventricular

administration of the neurotoxin AF64A, an ACh nitrogen mustard (Villanueva et

al. 1990). Thus, nicotine or compounds acting like nicotine could possibly be

beneficial to patients exhibiting the select neurological problems observed in

Parkinson’s and Alzheimer’s diseases.

The process of desensitization might also be useful to understanding why

humans choose to smoke tobacco products. Perhaps the ability of nicotine to

induce such neuronal effects at a specific n-AChR makes it reinforcing (or

aversive) to behavior. Such a mechanism of action might explain why nicotine

appears to both increase and decrease arousal levels in animals (or humans)

exhibiting differential basal level of excitability (Hendry and Rosecrans 1982), or

why some people never become dependent on nicotine.

How nicotine alters the n-AChR may also be beneficial to our understanding of

the subtle nature of cholinergic neuronal function in learning, memory, and

other behavioral states. The abiliiy of ACh (or nicotine) to induce an activation

or attenuation of some cholinergic or noncholinergic neuron or both may be

important to these brain processes. lt addition, there are important

interrelationships between cholinergic and noncholinergic pathways that may

assist in understanding how cholinergic receptors control behavior. Robinson

(1983, 1984) and Robinson et al. (1979), for example, have shown that both

dopaminergic- and serotonergic-projecting neurons play an important role in

controlling ACh turnover in oholinergio-rich brain areas (hippocampus and

frontal cortex) that may affect the level of m- and n-AChR excitability.

Conversely, we should also consider how cholinergic presynaptic receptors

located on DA- and serotonin-containing neurons are able to control a variety of

behavioral states. The importance of central ACh-containing neurons to

behavior (cognitive and affective), therefore, may be as important as these

neurons are to the autonomic nervous system (Pomerleau and Rosecrans

1989).

113
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ACKNOWLEDGMENTS

This research was supported by the following grants: Commonwealth of Virginia

Center on Drug Abuse Research, Virginia Commonwealth University; National

Institute on Drug Abuse grants DA-04002 and DA-07027; and the German

Smoking and Health Council, Bonn

AUTHORS

John A. Rosecrans, Ph.D.

Professor of Pharmacology and Toxicology

Department of Pharmacology and Toxicology

Heidi F. Villanueva, Ph.D.

Department of Pharmacology and Toxicology

Medical College of Virginia

Virginia Commonwealth University

Box 613, MCV Station

Richmond, VA 23298-0001

116
Discriminative Stimulus Properties of

Benzodiazepines and Several New

Anxiolytics*

Richard Young

INTRODUCTION

The clinical efficacy of the benzodiazepines in treating anxiety is well

documented. Although they are relatively safe drugs, they possess a number of

unwanted side effects, such as sedation. In recent years compounds have

emerged as purported “anxioselective” agents. This quest was given

considerable impetus by two factors: the discovery of benzodiazepine receptors

in the brain, and the emergence of the serotonergic anxiolytic busiprone and

related compounds, which appear to constitute a novel mechanistic class of

anxiolytic agents.

The drug discrimination (DD) paradigm is a sensitive procedure for evaluating

the interoceptive stimulus effects produced by a drug. For example, using a

typical two-lever operant procedure, animals can be trained to press one lever

following administration of an anxiolytic drug and the other lever after

administration of vehicle. Once the animals have been trained, tests of stimulus

generalization (substitution) can be conducted. The occurrence of stimulus

generalization suggests that a challenge drug is capable of producing

behavioral (stimulus) effects similar to those produced by a particular training

drug.

This chapter is organized into three main parts: (1) a review of DD studies

concerning benzodiazepine stimulus effects, including studies of the activity of

benzodiazepine metabolites and stereoisomers, antagonism tests, and

correlations of DD data with benzodiazepine binding data and human

*This chapter was not presented at the International Drug Discrimination Symposium held on

June 25-27, 1990

117
therapeutic potency; (2) the stimulus effects of newer compounds that are

chemically unrelated to benzodiazepines but most likely interact with

benzodiazepine receptors (i.e., benzodiazepine receptor mediated agents); and

(3) the stimulus effects of pyrimidinylpiperazines, such as buspirone,

ipsapirone, and gepirone, which seem to produce their effects through a

serotonergic mechanism. Table 1 lists some of these different classes of drugs

that have been used as training stimuli.

BENZODIAZEPINES

Several benzodiazepine derivatives have been used as discriminative stimuli in

animals. Early studies (table 1) sought to determine whether the stimuli

produced by these drugs differed from those produced by members of other

classes of agents active in the central nervous system; these studies have been

previously reviewed (see Colpaert et al. 1976) and are not discussed here.

More recent studies with benzodiazepines as discriminative stimuli have been

directed, for the most part, toward two goals: (1) characterizing the

benzodiazepine stimulus and (2) determining a possible commonality of effect

with newer types of purported anxiotytic agents. In general, DD studies have

shown that stimulus control established with one psychoactive benzodiazepine

generalizes to other benzodiazepines. These studies have also shown that

there are several important positions on the benzodiazepine molecule where

the presence of substituents can have a significant effect on activity or potency.

Although the structure-activity relationship (SAR) for the benzodiazepines was

relatively well established many years ago (for a review see Sternbach 1973), it

should be noted that the SAR generated from DD studies parallels known SAR

generalities for the benzodiazepines (Young and Glennon 1967).

Metabolites

The activity and potency of metabolites have been shown to be important

considerations in evaluating the stimulus properties of benzodiazepines. For

example, major metabolites of diazepam include desmethyldiazepam,

temazepam, and oxazepam. In the rat, significant quantities of the hydroxylated

metabolites 4'-hydroxydiazepam and 4'-hydroxydesmethyldiazepam are also

produced. Young et al. (1966) examined the activity and potency of these

metabolites in rats trained to discriminate diazepam from vehicle.

Generalization was found to occur with temazepam and oxazepam, which were

nearly equipotent with diazepam, and also with desmethyldiazepam, which was

about half as potent as diazepam. The two hydroxylated metabolites

116
TABLE 1. Benzodiazepines and newer types of purported anxiolytics that

have been used as discriminative stimuli in animals

Training Drug Reference

Benzodiazepines

Chlordiazepoxide

Clorazepate

Diazepam

Flurazepam

Lorazepam

Midazolam

Oxazepam

Pyrazoloquinolines

CGS 9696

lmidazopyridines

Zolpidem

Pyrimidinylpiperazines

Buspirone

lpsapirone

Colpaert et al. 1976

Dantzer and Perio 1962

Haug and Gotestam 1962

Overton 1976

Ator and Griffiths 1966

Garcha et al. 1985

Hendry et al. 1983

Bennett 1985

Sanger and Zivkovic 1986

Hendry et al. 1983

Spencer and Traber 1987

4'-hydroxydiazepam and 4'-hydroxydesmethyldiazepam were inactive. These

results indicate that benzodiazepine metabolites may contribute to the

discriminative stimulus (DS) effects of a benzodiazepine.

Stereoisomers

Optical isomers of biologically active compounds often display differences in

potency and sometimes can also display differences in effect. Unfortunately,

very few pairs of benzodiazepine optical isomers have been studied in DD

tests. The effects of (±)-, R(-)-, and S(+)-3-methytflunitrazepam have been

examined in animals trained to discriminate diazepam from vehicle (Shannon

and Herling 1983b; Young et al. 1984). Generalization of the diazepam stimulus

was found to occur to (±)- and S(+)-3-methyiflunitrazepam, with the S(+)-isomer

being twice as potent as the racemate. The administration of

119
R(-)-3-methylflunitrazepam did not produce significant drug lever responding,

even at 8-10 times the dose of S(+)-Smethylflunitrazepam that produced

diazepam generalization. However, Hiltunen and Järbe (1986) have shown that

animals trained to a S(+)-3-methylflunitrazepam stimulus generalize to

R(-)-3-methylflunitrazepam, with the S(+)-isomer being 10 times more potent

than the R(-)-isomer. S(+)-3-methylflunitrazepam is also recognized by animals

trained to discriminate chlordiazepoxide from saline (Hiltunen and Järbe 1986).

More recently, Järbe et al. (1986) trained animals to discriminate both diazepam

and the S(+)-isomer of meclonazepam from vehicle. The administration of the

R(-)-isomer of meclonazepam did not produce significant drug lever responding

in either group of trained animals. Taken together, these limited data suggest

benzodiazepine stereoselectivity, with the S(+)-isomers of

3-methylflunitrazepam and meclonazepam being more potent than their

corresponding R(-)-isomers.

Antagonism Studies

Evidence that the DS effects produced by benzodiazepines are mediated by

benzodiazepine receptors is derived from the finding that these effects can be

blocked by the administration of benzodiazepine antagonists such as flumazenil

(Ro 15-1788) and CGS 6216 (Herling and Shannon 1982; Young and Dewey

1982). Flumazenil may also possess agonist activity, because stimulus

generalization occurs in clorazepate- or chlordiazepoxide-trained animals

(Dantzer and Perio 1962; De Vry and Slangen 1966). In fact, flumazenil itself

has been used as a training drug (Bennett et al. 1985). Generalization of the

flumazenil stimulus was found to occur to CGS 8216 and diazepam. In that

same study, animals were also trained to discriminate CGS 8216 from saline.

Generalization did not occur to flumazenil, indicating an asymmetrical

cross-generalization between the two training drugs. CGS 8216 stimulus

generalization did occur to the purported anxiogenic agent pentylenetetrazol.

The authors concluded that the stimuli produced by the two “antagonists” are

qualitatively different, in that flumazenil has partial agonist

(benzodiazepine-like) activity and CGS 8216 has inverse agonist (i.e.,

anxiogenic) activity.

Correlation Studies

A significant correlation (r= .78) exists between potencies of benzodiazepines

in a DD task and their affinities for benzodiazepine binding in rat cerebral cortex

(Shannon and Herling 1983b). More recently, Young and Glennon (1987)

120
compared discrimination-derived ED50 values for a series of benzodiazepines

with data on displacing aft inities (Ki values) for 3H-diazepam brain binding in

humans, and with human therapeutic potency. A significant correlation (r = .88)

was found between benzodiazepine binding affinities and ED50 values derived

from the discrimination assay. A significant correlation (r= .92) was also found

between DD ED50 values and human therapeutic potencies (figure 1). Thus, a

relationship might exist between benzodiazepine-induced stimulus effects in

animals and benzodiazepine-induced subjective effects in humans.

BENZODIAZEPINE RECEPTOR-MEDIATED COMPOUNDS

The triazolopyridazine CL 218,672 (3-methyl-6-[3-trifluromethylphenyl]-1,2,4-

triazolo [4,3-b] pyridazine) displays high affinity for benzodiazepine receptors.

Indeed, it was the first nonbenzodiazepine to selectively displace brain-specific

benzodiazepine binding at a potency level comparable to the benzodiazepines

(Lippa et al. 1979). Further studies on the binding properties of CL 218,672

suggest that it has a high affinity for a distinct subpopulation of receptors in the

cerebellum, termed BZ1, and a lower affinity for a subpopulation of receptors in

the hippocampus, termed BZ2 (Klepner et al. 1979). Behaviorally, CL 218,872

possesses the anxiolytic-anticonvulsant properties typical of a psychoactive

benzodiazepine, but if appears to lack the sedative-hypnotic and musclerelaxant effects of benzodiazepines (Klepner et al. 1979; Lippa et al. 1979).

Taken together, these data led to the proposal that the BZ1 receptor might

mediate anxiolytic activity, whereas the BZ2 receptor might mediate the side

effects (i.e., sedation, ataxia) associated with the benzodiazepines. However,

based on historical brain structure and function relationships, that proposal

seems paradoxical. That is, ataxic-sedative effects would be thought to be more

closely associated with the activity of the cerebellum (BZ1), whereas anxiolytic

activity might be more intimately involved with the function of the hippocampus

(BZ2). Consistent with the latter proposal is the activity of zolpidem (see below).

The pyrazoloquinoline CGS 9896 (2-(4-chlorophenyl)-2,5-dihydo-3H pyrazolo

[4,3-c] quinolin-3-one), the pyrolopyrazine zopiclone, and the imidazopyridine

zolpidem are also potent inhibitors of benzodiazepine binding to rat brain

receptors (Julou et al. 1985; Sanger et al. 1987; Yokoyama et al. 1982). CGS

9696 and zopiclone show no selectivity between BZ1 and BZ2 receptors,

whereas zolpidem displays a preference for benzodiazepine receptors in the

cerebellum (BZ1) over those in the hippocampus (BZ2). All three wmpounds

produce the anxiolytic-anticonvulsant effects associated with benzodiazepines

(e.g., Bernard et al. 1985; Julou et al. 1985; Sanger et al. 1987). In addition,

121
FIGURE 1. Correlation between human therapeutic potency and

discrimination-derived ED50 values using diazepam as training

drug (r = .92). Compounds are bromazepam (BROM),

chlordiazepoxide (CDP), clonazepam (CLON),

desmethydiazepam (DSMT), diazepam (DZP), flunitrazepam

(FLUN), flurazepam (FLUR), lorazepam (LZP), nitrazepam (NIT),

and temazepam (TEM). (Data from Young and Glennon 1987.)

CGS 9896 may be a mixed agonist-antagonist, because it blocks

diazepam-induced rotorod impairment in animals (Bernard et al. 1985). CGS

9896 is purported to be an anxioselective agent, whereas zopiclone and

zolpidem are reported to be sedative-selective agents (Bernard et al. 1985;

Julou et al. 1985; Sanger et al. 1987).

In DD studies, animals have been trained to discriminate either

chlordiazepoxide, diazepam, lorazepam, midazolam, CGS 9896, or zolpidem

122
from vehicle (table 2). In tests of stimulus generalization, all of those training

drugs generalized to chlordiazepoxide or CL 218,872 (e.g., Ator and Griffiths

1986; Bennett 1985; Leidenheimer and Schechter 1988; McElroy and Feldman

1982; Sanger and Zivkovic 1986; Spealman 1965; Young and Dewey 1982;

Young et al. 1967). The stimuli also generaliied to zopiclone or diazepam (e.g.,

Ator and Griffiths 1986; Bennett 1985; Colpaeft et al. 1976; Sanger and

Zivkovic 1986; Sanger et al. 1965; Spealman 1985; Young et al. 1987),

although zopiclone has not been tested in CGS 9896-trained animals and

diazepam has not been tested in zolpidem-trained animals. To date, there has

not been any detailed report of zopiclone or CL 218,872 as a training drug.

The activities of CGS 9896 and benzodiazepines in DD studies have not always

produced consistent results. For example, stimulus generalization occurs

between CGS 9896 and chlordiazepoxide when either drug is used as the

training stimulus (e.g., Leidenheimer and Schechter 1988; Sanger et al. 1985).

In comparison, lorazepam-trained animals reportedly do not generalize to CGS

9896 (Ator and Griffiths 1986), whereas diazepam-trained animals reportedly do

not (Shannon and Herling 1983a) and do (Young et al. 1987) generalize to CGS

9896. Finally, diazepam is recognized by animals trained to discriminate CGS

9896 from vehicle (Bennett 1985). These apparent discrepancies may perhaps

be accounted for by procedural differences in the experiments (Young et al.

1987) or by the purported mixed agonist-antagonist properties of CGS 9696

(Bernard et al. 1985; Sanger and Zivkovic 1987), or both.

TABLE 2. Results of generalization studies in animals trained to discriminate

chlordiazepoxide, diazepam, lorazepam, midazolam, CGS 9896,

and zolpidem from vehicle

123
The sedative-selective profile of zolpidem has been emphasized in DD

experiments by Sanger and Zivkovic (1986, 1987). For example, in

zolpidem-trained animals, stimulus substitution occurs to chlordiazepoxide, but

in chlordiazepoxide-trained animals, zolpidem produces only partial (i.e., 70-75

percent chlordiazepoxide-appropriate responding) generalization with significant

decreases in response rates (Sanger and Zivkovic 1986). The authors suggest

that zolpidem has chlordiazepoxide-like stimulus properties only at doses that

are highly sedative. A zolpidem stimulus does not generalize to CGS 9896.

However, CGS 9896 does antagonize a zolpidem cue (Sanger and Zivkovic

1987). Because zolpidem shows selectivity for BZ1 receptors and CGS 9896

diaplsys high affinity for both BZ1 and BZ2 receptors, an intriguing possibility is

that CGS 9896 may be an agonist at BZ2 receptors (mediating anxiolytic

action -and thus cross-generalization may occur between it and

chlordizepoxide-and an antagonist at BZ1 receptors (mediating

ataxic-sedative activities) and thus may antagonize the sedative stimulus of

zolpidem.

PYRIMIDINYLPIPERAZINES

Prototypical of this novel series of second-generation anxiolytics (SGAs) are

busiprone, gepirone, and ipsapirone. Buspirone, the most extensively studied

member of this series, is a clinically effective antianxiety agent (e.g., Riblet et

al. 1982) All three of these drugs form a common metabolite, 1-(2-pyrimidinyl)

piperazine (1-PP).

In animal studies these drugs, unlike benzodiazepines, lack anticonvulsant,

sedative, and muscle-relaxant effects (Eison et al. 1986; Riblet et al. 1982).

They also appear to lack abuse and physical dependence properties (e.g.,

Riblet et al. 1982). Behaviorally, the SGAs can (but do not always) exhibit

anxiolytic activity in animals, but such effects are not attenuated by

benzodiazepine receptor antagonists (Traber et al. 1984; Young and Glennon

1988). Neurochemical studies have demonstrated that these agents do not bind

to benzodiazepine receptors. They do, however, interact with serotonin 5-HT1A

receptors (Eison et al. 1986; Glaser and Traber 1983).

DD studies have been used to distinguish SGAs from benzodiazepines. For

example, animals have been trained to discriminate either buspirone,

ipsapirone, or a benzodiazepine from vehicle (table 3). In tests of stimulus

generalization, the SGAs and 1-PP are not recognized by animals trained to

discriminate a benzodiazepine from vehicle, and benzodiazepines are not

124
TABLE 3. Results of generalization studies in animals trained to discriminate

buspirone, isaprone, 8-OH DPAT, diazepam, lorazepam,

midazolam, and oxazepam from vehicle

Training Drug

Test Agent Buspirone lpsapirona 8-OH DPAT Diazepam Lorazepam Midazolam Oxazepam

Buspirone Gen Gen Gen NGen NGen NGen NGen

lpsapirone - Gen Gen NGen - - -

Gepirone Gen Gen Gen NGen - - -

1-PP - - NGen NGen - - -

8-OH DPAT Gen Gen Gen NGen - - -

Diazepam - NGen NGen Gen Gen Gen -

Midazolam NGen - - Gen Gen Gen -

NOTE: See text for references; Gen = Generalization; NGan = No Generalization; - = Not Tested

recognized by animals trained to discriminate an SGA from vehicle (Hendry et

al. 1983; Mansbach and Barrett 1987; Pierson et al. 1987; Spealman 1985;

Spencer and Traber 1987; Young et al. 1987). Finally, animals trained to

discriminate buspirone or ipsapirone from vehicle generalize to gepirone,

buspirone, and ipsapirone (Cunningham 1989; Pierson et al. 1987; Spencer

and Traber 1987; Young and Glennon 1988). Taken together, these data

indicate that the SGAs do not share stimulus properties with the

benzodiazepines.

To further differentiate SGAs and benzodiazepines, DD studies have

emphasized their different mechanisms of action. For example, stimulus

generalization occurs between an SGA and the 5-HT1A agonist 8-OH DPAT

when either drug is used as the training stimulus (Mansbach and Barrett 1987;

Spencer and Traber 1987; Young and Glennon 1988). In contrast, stimulus

generalization does not occur between diazepam and 8-OH DPAT when either

agent is used as the training stimulus. An 8-OH DPAT stimulus also fails to

generalize to 1-PP (Young and Glennon 1988). These results indicate that the

SGAs (but not 1-PP) produce stimulus effects similar to those of the 5-HT1A

agent 8-OH DPAT. Lastly, a benzodiazepine stimulus but not an 8-OH DPAT

stimulus, can be blocked by Ro 15-1788 (flumazenil), whereas an 8-OH

DPAT-stimulus can be antagonized by the purported 5-HT1A antagonist

(-)pindolol (Tricklebank et al. 1987; Young and Glennon 1988). Thus, the

125
stimulus effects of SGAs appear to be independent of the effects of the

common metabolite 1-PP and, unlike those of benzodiazepines, appear to be

mediated by a 5-HT1A mechanism.

CONCLUSION

Although benzodiazepines still constitute one of the most widely prescribed

classes of drugs in the world, a number of new drugs have been investigated

for their anxiolytic potential. Some are related to the benzodiazepines by their

interaction at benzodiazepine receptors, while others interact at 5-HT1A

systems.

The ability of various benzodiazepines to serve as discriminative stimuli has

been well documented. Benzodiazepine stimuli have been demonstrated to

generalize to other benzodiazepines (including certain metabolites), and a

significant correlation exists between their relative potency and both their

affinity for benzodiazepine receptors in the brain and their human anxiolytic

potency. Moreover, the benzodiazepine stimulus is stereoselective and can be

attenuated by benzodiazepine receptor antagonists. Taken together, these data

provide evidence that benzodiazepines exert their stimulus effects through an

interaction with benzodiazepine receptors.

Newer compounds have been developed that are chemically unrelated to the

benzodiazepines but exert their effects by interacting with benzodiazepine

receptors, and they appear to be anxioselective (CL 218,872, CGS 9896). An

interesting byproduct of this research is that some of these compounds may be

sedative selective (zopiclone, zolpidem). In DD studies, CL 218,872 and

zopiclone appear to produce stimulus effects that are typical of the

benzodiazepines, although neither drug itself has been used as a training

stimulus. In comparison, CGS 9896 and zolpidem appear to produce stimulus

effects that might indicate an anxioselective profile for the former and a

sedative-selective profile for the latter.

Finally, although both the benzodiazepines and the SGAs, such as busiprone

and ipsapirone, can be used to establish stimulus control, their stimulus

properties are clearly dissimilar. By extrapolation, this suggests that the SGAs

probably produce qualitatively different subjective effects in human patients, as

compared to those produced by benzodiazepines. The significant interaction of

the SGAs with 5-HT1A systems qualify these drugs as a novel mechanistic

class of antianxiety agents.

126
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AUTHOR

Richard Young, Ph.D.

Department of Medicinal Chemistry

Box 540

Medical College of Virginia/Virginia Commonwealth University

Richmond, VA 23298-0540

130
Distinctive Discriminative Effects of Ethanol

Herbert Barry III

Experiments on discriminative effects of ethanol in laboratory animals have

contributed important information on the comparison of ethanol with other

drugs. This paper summarizes and interprets that research and also proposes a

model to explain differential discriminative effects of ethanol from the

discriminative effects of barbiturates and benzodiazepines. Stolerman et al.

(1989), a bibliography of publications on discriminative drug effects through

1988, is a useful resource that includes ethanol and alcohol in an excellent

index of drugs and topics.

COMPARISONS WITH BARBITURATES AND BENZODIAZEPINES

After animals have been trained to discriminate a drug effect from the control

condition, they can be tested with other drugs to determine whether these novel

drugs resemble the training drug condition or the control (i.e., nondrug)

condition. In studies with animals trained to discriminate ethanol from the

nondrug condition, the other drugs that have been tested most often are

barbiturates and benzodiazepines. These studies consistently show that the

drug response is induced when sufficiently high doses of barbiturates or

benzodiazepines are tested. In animals trained to discriminate a barbiturate or

benzodiazepine from the nondrug condition, however, the drug response is not

consistently induced during tests with ethanol.

An early report by Overton (1966) reported similar discriminative stimulus

effects of several sedative or hypnotic drugs, including ethanol, chloral hydrate,

pentobarbital, phenobarbital, and chlordiazepoxide. The author pointed out that

in rats trained to discriminate the benzodiazepine chlordiazepoxide from

nondrug, ethanol did not dependably induce the drug choice. Subsequently,

Barry (1974) and Barry and Krimmer (1977, 1978) summarized several studies

showing generalization of the ethanol response to barbiturates and

benzodiazepines but not consistent generalization of the barbiturate or

benzodiazepine response to ethanol.

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These differential effects were tested systematically and demonstrated clearly

by De Vry and Slangen (1986). Discrimination of three drugs (ethanol,

pentobarbital, and chlordiazepoxide) from the nondrug vehicle was trained by

using six groups of rats, including separate groups trained with lower and higher

doses of each of the three drugs. In tests with other drugs, the response trained

with ethanol generalized to the other two drugs. The response trained with

pentobarbital generalized to chlordiazepoxide but not to ethanol. The response

trained with chlordiazepoxide did not generalize to ethanol, and a much higher

dose of pentobarbital was needed to induce the chlordiazepoxide response

than to induce the drug response in rats trained with pentobarbital or ethanol.

These findings indicate asymmetrical generalization. The ethanol response

generalizes to barbiturates and benzodiazepines, but neither the barbiturate nor

the benzodiazepine response generalizes equivalently to ethanol. The ethanol

effect, therefore, has the greatest generality, whereas the benzodiazepine effect

has the greatest specificity. Pentobarbital is intermediate in these attributes of

generality and specificity.

MODEL FOR ASYMMETRICAL GENERALIZATION

Barry and Krimmer (1979) suggested that the strength or distinctiveness of the

drug stimulus depends partly on the dose and partly on the characteristics of

the drug. Two drugs at a dose with equivalent potency might differ in

distinctiveness of difference from the nondrug condition. This conception

implies that the difference from the nondrug condition is more distinctive for

pentobarbital than for ethanol and more distinctive for chlordiazepoxide or

diazepam than for pentobarbital.

A model for the differences among these three types of drugs attributes the

most diverse effects to ethanol and the most specific effects to

benzodiazepines; barbiturates are intermediate. These differential drug effects

are consistent with known pharmacological actions of these drugs.

Ethanol is a small molecule with pervasive effects on the central nervous

system (CNS). Impairment of neurotransmission is seen in the anesthetic effect

at doses only moderately higher than the doses required for behavioral effects.

The mechanism for the ethanol effect appears to be primarily an increase in

fluidity of the membranes of the cell. This uniform increase in membrane fluidity

occurs at many sites in the CNS and hence induces a wide variety of responses

because of the diversity of functions in the CNS. The effects on

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neurotransmission are secondary to these generalized effects on the

functioning of the cells rather than due primarily to binding of the ethanol

molecule to a particular neurotransmitter receptor.

The principal behavioral effects of benzodiazepines appear to result from the

drug binding to a specific endogenous receptor. Drugs that block the receptor

therefore antagonize the effects of benzodiazepines, indicating a specific rather

than a diverse effect on the CNS. Benzodiazepines impair neurotransmission

and cause anesthesia only at doses that are far above the range used for

clinical treatment and behavioral experiments.

The specificity of the barbiturate effect is intermediate. In common with

benzodiazepines, an important effect of barbiturates is binding to the

endogenous benzodiazepine receptor complex. Like ethanol, barbiturates

increase membrane fluidity. Anesthesia is a prominent effect of barbiturates, as

of ethanol, at doses only moderately higher than the doses required to induce

behavioral changes.

An analogy for this difference among the three types of drugs is the

distinctiveness of an auditory stimulus. The nondrug condition corresponds to a

generalized noise, low in volume and constituting a mixture of all the 12 pitches

(7 natural tones, 5 sharps). Waterfalls and electric fans produce this type of

generalized noise. The ethanol stimulus corresponds to five tones with adjacent

pitches, such as F, F sharp, G, G sharp, and A. The magnitude of the dose is

determined by the loudness of the group of five tones. Pentobarbital

corresponds to two tones with adjacent pitches, such as F and F sharp.

Chloridazepoxide or diazepam corresponds to a single tone, such as F. These

sounds can be reproduced with an organ, pitch pipe, or piano.

According to this model, any of the three types of drugs can easily be

discriminated from the nondrug condition if the dose is high enough. Differences

between the drugs are indicated by tests that substitute one of the other two

drugs for the training drug. Generalized noise is more similar to the five

adjacent pitches (ethanol) than to the single tone (benzodiazepine). In animals

trained to discriminate a group of five pitches (ethanol) from generalized noise,

two pitches or a single tone is more similar to the group of five pitches than to

the generalized noise. In animals trained to discriminate the single tone

(benzodiazepine) from generalized noise, the two pitches and especially the

group of five pitches might be perceived as more similar to the generalized

noise than to the single tone. In animals trained to discriminate two pitches

133
(barbiturate) from generalized noise, the single tone is more similar to the two

pitches, but the group of five pitches might be perceived as more similar to the

generalized noise.

This model of differences between these three types of sedative drugs indicates

a progression of increasing specificity from the nondrug condition to ethanol to

barbiturates to benzodiazepines. This progression is consistent with the results

of experiments in which animals are trained to discriminate one of these types

of drugs from the nondrug condition and then tested with the other types of

drugs. Other experiments provide further information on the validity and

usefulness of this model.

ETHANOL COMPARED WITH OTHER DRUGS

Some drugs in addition to barbiturates and benzodiazepines have been

reported to induce the ethanol response. Overton (1966) found that the ethanol

response was chosen in tests with volatile anesthetic agents, such as chloral

hydrate and ether, and also with meprobamate. Rees et al. (1987) found that

the ethanol response trained in mice was chosen in tests with toluene,

halothane, and 1,1,1-trichloroethane. Signs and Schechter (1988) found that

the ethanol response trained in rats was chosen in tests with a serotonin

agonist, 1-(3-trifluoromethylphenyl) piperazine.

The ethanol response has been reported to be chosen to a partial degree in

tests with 3-carboxysalsolinol, which is generated during ethanol metabolism

(Chipkin et al. 1979; Schechter 1980) and in tests with acetaldehyde, another

metabolite of ethanol (York 1981). A partial ethanol response has also been

reported in tests with the opioid antagonist naloxone (Altshuler et al. 1981).

These partial similarities suggest that the discriminative ethanol effect has

diverse attributes.

Several drugs have shown evidence of sharing discriminative effects of ethanol

because the ethanol effect was augmented when ethanol was administered

together with the other drug. This interaction with the effect of ethanol was

found for apomorphine (Schechter 1985), chlordiazepoxide (Schechter and

Lovano 1985), and nicotine (Signs and Schechter 1966); it may be useful as a

sensitive measure of the existence of common attributes of the discriminative

effect. In animals trained to discriminate ethanol from the nondrug condition,

tests with apomorphine or nicotine would probably induce the nondrug

response rather than the drug response.

134
Most types of drugs do not seem to show any similarity to ethanol. These

include opioid analgesics, neuroleptics, antimuscarinics,

delta-9-tetrahydrocannabinol, hallucinogens, and stimulants. Animals trained to

discriminate ethanol from the nondrug condition predominantly choose the

nondrug response in tests with these drugs (Barry 1974). Also, animals trained

to discriminate any of these drugs from the nondrug condition predominantly

choose the nondrug response in tests with ethanol. The discriminative ethanol

effect therefore is differentiated from most other pharmacological conditions.

ANTAGONISTS OF ETHANOL

Specificity of a drug effect is indicated if this effect can be antagonized when

the drug is combined with a single other drug. An example is the antagonism of

morphine by naloxone. After animals have been trained to discriminate

morphine from the nondrug condition, a sufficiently high dose of naloxone

together with the training dose of morphine shifts the choice from the morphine

to the nondrug response.

Experiments on rats have failed to identify a single consistent antagonist of the

discriminative ethanol response. The ethanol response in rats has been

antagonized by bemegride (Krimmer 1974) by amphetamine (Schechter 1974)

and by p-chlorophenylalanine, which depletes brain serotonin (Schechter 1973).

The ethanol response was not antagonized by the benzodiazepine inverse

agonist Ro 15-4513 in rats (Hiltunen and Järbe 1988) or in mice (Middaugh et

al. 1989). Contrary to these findings, however, Rees and Balster (1988)

reported that the discriminative ethanol response in mice was antagonized by

Ro 15-4513.

In contrast to the failure to find a consistent, specific antagonist of the

discriminative ethanol effect in rats, barbiturates and benzodiazepines have

specific antagonists. The stimulant drug bemegride antagonizes the

discriminative barbiturate response in rats (Barry and Krimmer 1979; Krimmer

1974; Overton 1966). The benzodiazepine inverse agonist Ro 15-4513

antagonizes the discriminative benzodiazepine response but not ethanol

response in rats (Hiltunen and Järbe 1989a, 1989b).

When rats are trained to discriminate Ro 15-4513 from the nondrug control

condition, the Ro 15-4513 response is induced by the stimulant drug

pentylenetetrazol. This response to pentylenetetrazol is antagonized by

135
diazepam or pentobarbital but not by ethanol (Harris et al. 1987; Hiltunen and

Järbe 1989b; Järbe and Hiltunen 1988).

DISCRIMINATION BETWEEN TWO DRUGS

If animals are trained to discriminate a drug from the nondrug condition, tests

with other drugs provide only indirect comparisons between the stimulus

attributes of the two drugs. A more direct comparison is to train the animals to

discriminate between the two drugs. Successful training of this discrimination

demonstrates that the drugs have differential effects.

Krimmer and Barry (1973) reported on successful training of rats to discriminate

ethanol (1,000 mg/kg) from pentobarbital(10 mg/kg); both drugs were injected

in a volume of 10 mL/kg. Tests with higher doses of either drug reliably induced

the response for that drug. Tests with lower doses of pentobarbiial, and tests

with saline, shifted some animals to the ethanol response.

Overton (1977) reported that a group of rats learned to discriminate ethanol

from pentobarbital and another group learned to discriminate ethanol from

phenobarbital. A third group failed to discriminate pentobarbital from

phenobarbital. These findings give evidence for close similarity of the

discriminative effects of the two barbiturates.

Barry et al. (1981) trained rats to discriminate either of two ethanol doses from

either of two chlordiazepoxide doses. This procedure required the animals to

discriminate the qualitative differences between the two drugs rather than a

possible quantitative difference between a stronger effect of a single dose of

one drug and a weaker effect of a single dose of the other drug.

Other studies have tested effects of ethanol in rats trained to discriminate

pentobamital from chlordiazepoxide. Barry and Krimmer (1979) summarized

tests with conflicting results on whether tests with ethanol induced the

chlordiazepoxide or the pentobarbital response. In a subsequent experiment

(Barry et al. 1982) rats were trained to discriminate pentobarbiial from

chlordiazepoxide with two doses of both drugs in order to establish

discrimination between the qualitatively different effects of the two drugs. After

this training, tests with ethanol induced the pentobarbital response rather than

the chlordiazepoxide response.

136
Another type of discrimination between two drugs is to train discrimination

between drugs that have contrasting effects, such as a depressant and a

stimulant. Gauvin et al. (1989) trained rats to discriminate chlordiazepoxide

from pentylenetetrazol. After this training, ethanol was found to resemble the

chlordiazepoxide rather than the pentylenetetrazol condition. The similarity of

ethanol to the benzodiazepine as a depressant drug, therefore, is more obvious

when the benzodiazepine is discriminated from a stimulant than from the

nondrug condition.

A further test by Gauvin et al. (1989) gave evidence that the withdrawal illness

after a high ethanol dose resembles a stimulant drug, pentylenetelrazol, more

than a depressant drug, chlordiazepoxide. A saline injection normally induced

nearly equal choice of the diazepam and pentylenetetrazol responses. At 12 hr

after a high ethanol dose of 3 or 4 g/kg, the saline injection induced a

preponderance of pentylenetetrazol choices. This finding is consistent with a

report by Lal et al. (1988) that the ethanol withdrawal illness resembles the

effect of pentylenetetrazol. Rats trained to discriminate pentylenetetrazol from

the saline control injection chose the saline response in tests after ethanol.

Ethanol withdrawal illness was subsequently demonstrated at 24 hr folIowing

4 days of gastric intubation of a high ethanol dose. During this ethanol

withdrawal illness, the saline injection induced a preponderant choice of the

pentylenetetrazol response.

DIFFERENT ETHANOL DOSES AND TIME INTERVALS

Studies of discrimination between ethanol and the nondrug condition have used

a wide variety of doses. The highest were 2.4 g/kg intraperitoneally in rats

(Overton 1966) and 3 g/kg orally in pigeons (Järbe and McMillan 1983). These

doses have incapacitating effects in some animals. The lowest doses were

0.2 g/kg (Schechter 1980) and 0.3 g/kg (York 1978) intraperitoneally in rats and

0.1 g/kg intravenously in rats (Ando 1975). These doses have no observable:

behavioral or physiological disruptive effects.

A low dose and a high dose of a drug differ quantitatively, but they might also

produce qualitatively different stimulus conditions. Ethanol appears to have

behaviorally stimulant effects at low doses and depressant effects at high

doses. Accordingly, the drug stimulus might be a different type at different

doses (York 1978). A finding by Barry et al. (1981) might indicate qualitatively

different effects of different doses. Rats failed to learn a discriminative response,

when one response was reinforced after injection with either of two doses of

137
ethanol (1.0 or 1.5 g/kg) and the alternative response was reinforced in the

saline control condition. Another group of rats learned to discriminate either of

the same two ethanol doses from either of two chlordiazepoxide doses. The two

ethanol doses thus were more differentiated from each other and from

chlordiazepoxide than from the saline control condition.

Tests for generalization to other drugs indicate greater specificity with high

training doses, resulting in less generalization of the drug response to other

drugs. For example, York (1978) trained three groups of rats to discriminate one

of three ethanol doses (0.33, 0.66, 0.99 g/kg) from saline. Barbital induced the

ethanol choice in the animals trained with 0.33 g/kg but not in those trained with

0.99 g/kg. This finding suggests that the high dose has a more specific effect,

generalizing to a lesser degree to other drugs. Different doses of ethanol

therefore are not merely different magnitudes of the same stimulus.

Different time intervals after administration also might induce different stimuli.

Shippenberg and Altshuler (1985) trained a discrimination of 1.0 g/kg ethanol

from the nondrug condition at an interval of 6 min for one group of rats and 30

min for another group. Both groups showed only a partial drug response when

tested with ethanol after the alternative interval. A sedative rather than

excitatory effect of ethanol at the longer interval was inferred from the finding

that naloxone partially antagonized the ethanol response trained at the interval

of 6 but not 30 min. Schechter (1989) trained a discrimination of a lower ethanol

dose (0.6 g/kg) from the nondrug condition in two groups of rats at the same

intervals of 6 and 30 min. The drug response learned at 30 min was induced at

6 min, but the drug response learned at 6 min was only partially induced at 30

min. This difference suggests that the effect of 0.6 g/kg ethanol is a more

specific stimulus at the shorter time interval, thus generalizing only partially to

the more diverse stimulus of ethanol at the longer interval. The more diverse

ethanol stimulus at the longer interval may include actions of metabolites, such

as acetaldehyde, and the beginnings of the withdrawal symptoms in addition to

the same effects as at the shorter interval.

EXPERIMENTAL PROCEDURES FOR ETHANOL DISCRIMINATION

The discriminative ethanol response has been trained with a wide variety of

procedures. The ethanol stimulus thus differs from the nondrug condition and is

trainable and measurable under many different conditions.

138
The most prevalent procedure is to train the animals to select equivalent but

different responses, such as levers on opposite sides of a food cup. The

incentive for the discrimination learning is usually a pellet of food in a hungry

rat. The lever pressing is usually on a fixed ratio schedule of 10 or more

responses to obtain a pellet of food.

The earliest studies used the locomotor response of running in a straight alley

(Conger 1951) or choosing opposite directions in a T-shaped maze (Barry et al.

1965; Overton 1966). The operant lever-pressing response was introduced by

Barry (1968) and Kubena and Barry (1969a, 1969b) as a technique for training

and testing discriminative effects of ethanol and other drugs, The discrimination

was learned with lower drug doses with this procedure, perhaps because

distracting stimuli were minimized by the less active physical response of

pressing a lever in a chamber or by the use of food reward instead of escape

from painful shock as the incentive for the discriminative response.

A different type of choice is between responding and refraining from responding

instead of between two equivalent, alternative responses. Some studies have

associated the ethanol or nondrug condition with delivery of a food pellet and

the alternative condition with no food (Winter 1975; York 1978), with a painful

shock (Kubena and Barry 1969b), or with both food and shock (Conger 1951).

Animals learn to respond or to refrain from responding, depending on which

consequence is associated with their ethanol or nondrug condition. Conger

(1951) also demonstrated more rapid learning of the discrimination by rats that

received food after ethanol and food plus shock after the nondrug treatment

than by another group of rats that received food after the nondrug treatment

and shock after ethanol. This finding is in accordance with a tendency for

ethanol to diminish avoidance of an aversive event.

A differential response is not necessary for discriminative learning. Barry (1968)

demonstrated that rats learned the discriminative response when pressing a

lever that controlled the environmental condition (lighted or dark chamber).

Food was delivered in one illumination condition after ethanol injection and in

the alternative illumination condition after saline injection.

CHARACTERISTICS OF THE ANIMALS

Most of the experiments on discriminative ethanol effects have been in rats.

Other species tested include gerbils (Järbe 1977), pigeons (Järbe and McMillan

1983), mice (Rees and Balster 1988; Rees et al. 1987), and squirrel monkeys

139
(York and Bush 1982). Most of the findings do not appear to differ among

species.

York (1981) compared two lines of albino rats that had been bred for 33

generations for difference in consumption of a 10 percent ethanol solution in

tests with simultaneous availability of water and the ethanol solution. The

discrimination between ethanol and the nondrug condition was learned more

rapidly by the line of rats with low ethanol consumption. This difference gave

evidence that in this ethanol-nonpreferring line of rats, ethanol provided a more

salient cue that was probably aversive. This study also reported a greater

contribution of acetaldehyde to the ethanol stimulus in the ethanol-nonpreferring

line of rats.

Krimmer (1990) compared two lines of rats, differing in the duration of loss of

righting reflex after injection of a high ethanol dose. After training to discriminate

0.6 g/kg ethanol from the saline control condition, the initial tests with 0.15 and

0.3 g/kg induced higher percentages of ethanol choice by the line of rats with

longer duration of loss of righting reflex. This finding indicates that greater

susceptibility to the effect of a high ethanol dose on righting reflex was

associated with greater sensitivity to the discriminative effect of low ethanol

doses.

CONCLUSIONS

Pharmacological classifications view ethyl alcohol as a hypnotic sedative,

closely similar to general depressants such as barbiturates, ether, and chloral

hydrate and also related to antianxiety agents such as benzodiazepines.

Studies of discriminative effects of ethanol in laboratory animals agree with the

pharmacological classifications. Several studies indicate discriminative effects

of ethanol in rats at lower doses than the minimum sufficient to cause

observable changes in behavior.

Ethanol has distinctive discriminative effects, demonstrated by discriminability

of this drug from barbiturates or benzodiazepines. A model of the comparison

among these three types of drugs indicates that the discriminative effects are

most diverse for ethanol and most specific for benzodiazepines. Barbiturates

are intermediate, sharing characteristics of both ethanol and benzodiazepines.

140
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AUTHOR

Herbert Barry, Ill, Ph.D.

Department of Pharmacology and Physiology, School of Dental Medicine

University of Pittsburgh

Pittsburgh, PA 15261

144
Training Dose: Influences in Opioid Drug

Discrimination

Sandra D. Comer, Charles P. France, and James H. Woods

INTRODUCTION

Drug discrimination procedures have been used in a number of species and

under a variety of conditions to investigate the effects of drugs from many

different pharmacological classes. Typically animals are trained to respond on

one manipulandum after administration of a drug and to respond on another

manipulandum after administration of drug vehicle. After repeated

presentations, selective discriminative stimulus control is established: animals

reliably respond on the drug-associated lever following drug administration and

on the vehicle-associated lever following vehicle administration. Although drugs

from various pharmacological classes have been used to establish stimulus

control, the discriminative stimulus effects of opioids will be the topic of this

paper. Drugs are classified as opioid based on well-characterized sets of

pharmacological effects, on the susceptibility of these effects to antagonism by

opioid antagonists such as naloxone or naltrexone, and on the ability of these

compounds to induce tolerance and selectively exhibit cross-tolerance to other

opioid agonists.

One interesting feature that has been investigated using the opioid drug

discrimination paradigm is the effect of changes in training dose on the slopes

of generalization gradients, on profiles of substitution, and on antagonism by

opioid antagonists. A potentially useful way to interpret data collected in opioid

drug discrimination experiments is based on receptor theory involving the

concepts of pharmacological selectivity and efficacy. A behavioral context from

which to interpret results obtained when training dose is manipulated involves

the issue of discriminative stimulus control. The changes in slopes of

generalization gradients, profiles of substitution, and antagonism by opioid

antagonists that occur when training dose is manipulated will be the topic of this

paper. This analysis includes both behavioral and pharmacological perspectives.

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CHANGES IN TRAINING DOSE

Changes in Generalization Gradients

A number of investigators have shown that training dose is an important

variable in the drug discrimination paradigm (Colpaert et al. 1980a, 1980b;

Hottzman 1982; Koek and Woods 1989; Shannon and Holtzman 1979). For

example, in a study examining the effects of progressively tower training doses

in rats discriminating 0.04 mg/kg fentanyl from saline, Colpaert et al. (1980b )

showed that stimulus control could be maintained at a training dose as low as

0.0025 mg/kg. In this study, the slope of the fentanyl generalization gradient

decreased as the training dose was decreased (figure 1). Similarly, the slope of

morphine generalization gradients was shallower as training dose was reduced.

Although the ED50 for stimulus generalization also decreased with a reduction

in training dose, the concomitant decrease in slope may indicate that the

apparent change in potency might be due simply to a loss of stimulus control.

Different Patterns of Substitution

Another effect of varying training dose is to alter the pattern of stimulus

generalization to other drugs, both opioid and nonopioid (Colpaert et al. 1980a ;

Koek and Woods 1989; Shannon and Holtzman 1979). For some test

compounds, increasing the dose of the training compound decreases the

percentage of drug-appropriate responding produced by those test compounds,

while decreasing the training dose increases the percentage of

drug-appropriate responding. Shannon and Holtzman (1979), for example,

showed that nalbuphine and cyclazocine produced more drug-appropriate

responding in rats discriminating between saline and 1.75 mg/kg morphine than

in rats discriminating between saline and 5.6 mg/kg morphine, while profadol

and pentazocine completely substituted for the morphine discriminative

stimulus in rats discriminating either dose of morphine (figure 2). Similarly, Koek

and Woods (1989) found that pigeons discriminating morphine from saline

partially generalized cyclazocine, (-)-N-allylnormetazocine ((-)-NANM) and

ketamine, but not U50488 when the training dose was 5.6 mg/kg. However,

decreasing the training dose of morphine to 1.8 mg/kg resulted in a shift to the

left in the morphine generalization gradient and increased the percentage of

drug-appropriate responding after administration of cyclazocine but not (-)-

NANM or ketamine. Increasing the training dose of morphine to 18.0 mg/kg

resulted in a rightward shift in the morphine generalization gradient and a

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FIGURE 1. Log-probit plot comparing fentanyl dose-response functions in rats

trained to discriminate 0.04, 0.02, 0.01, or 0.005 mg/kg fentanyl

from saline (n = 8 per group). Each point represents the

percentage of rats making the first 10 responses on the drug

lever. (Replotted from Colpaert et al. 1980b.)

decrease in the percentage of drug-appropriate responding after administration

of cyclazocine or (-)-NANM, but not ketamine.

Using a different training drug, Colpaert et al. (1980b) examined the ability of

morphine, cyclazocine, apomorphine, and damphetamine to induce stimulus

generalization with the fentanyl discriminative stimulus in rats discriminating

either 0.04, 0.02, 0.01, or 0.005 mg/kg fentanyl from saline (figure 3). When the

fentanyl training dose was 0.04 mg/kg, neither apomorphine, d-amphetamine,

nor cyclazocine produced any fentanyl-appropriate responding. However,

cyclazocine produced an increasing amount of fentanyl-appropriate responding

as the fentanyl training dose was decreased from 0.02 (29 percent) to 0.01 (50

percent) to 0.005 (83 percent) mg/kg. Damphetamine also produced an

increasing amount of fentanyl-appropriate responding when the training dose

was progressively lowered, although the maximum percentage of

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FIGURE 2. Comparison of the dose-related effects of four analgesics with

agonist and antagonist properties in rats trained to discriminate

between saline and 1.75 or 5.6 mg/kg morphine. Each point is the

mean number of trials completed on the morphine-appropriate

choice lever in a 20-trial session; the remaining trials were

completed on the saline-appropriate lever. Means are based on

one observation in each of 4-5 rats. The mean number of trials

completed on the morphine-appropriate fever after an injection of

saline (Sal). lactic acid vehicle (Veh), or the morphine (M) training

dose are represented by the points above Sal, Veh, and M,

respectively. (From Shannon and Holtzman 1979.)

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FIGURE 3. Stimulus generalization of 0.31 mg/kg apomorphine (AP), 0.63

mg/kg d-amphetamine (AM), and 0.04 mg/kg cyclazocine (CY) in

rats trained to discriminate 0.04, 0.02, 0.01, or 0.005 mg/kg

fentanyl from saline (n = 8 per group). Each bar represents the

percentage of rats making the first 10 responses on the drug

lever. Where there is an apparent absence of data for the

conditions such as for all drugs at 0.04 mg/kg fentanyl Paining

dose, no rats selected the drug lever. (Replotted from Colpaert et

al. 1980b.)

fentanyl-appropriate responding with amphetamine was only 35 percent,

regardless of fentanyl dose. Apomotphine produced a maximum of only 17

percent fentanyl-appropriate responding at all training doses of fentanyl. In

contrast, morphine produced greater than 90 percent fentanyl-appropriate

responding at each of the fentanyl training doses, although the potency of

morphine decreased as training dose decreased. The results of these studies

thus show that varying the dose of the training drug can produce differences in

the pattern of stimulus generalization using both opioid and nonopioid test

drugs. While some drugs such as morphine completely substitute for the

fentanyl discriminative stimulus regardless of fentanyl training dose, other

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drugs, both opioid (e.g., cyclazocine) and nonopioid (e.g., d-amphetamine),

partially substitute for fentanyl only when the dose of training drug is small.

Agonist, Antagonist Effects

As described above, under some conditions, some opioids and nonopioids

produce only partial substitution. In an attempt to more fully characterize the

partial substitution that occurs with various test compounds, Koek and Woods

(1989) evaluated the effects of the opioid antagonist naltrexone on the

discriminative stimulus effects produced by morphine, cyclazocine, (-)-NANM,

U50488, and ketamine in pigeons discriminating between 5.6 mg/kg morphine

and saline. Morphine produced greater than 90 percent and U50488 produced

less than 10 percent drug-appropriate responding, while cyclazocine, (-)-

NANM, and ketamine produced intermediate levels of drug-appropriate

responding. Naltrexone completely antagonized the discriminative effects of

morphine, cyclazocine, and (-)-NANM but not ketamine, indicating that

morphine, cyclazocine, and (-)-NANM most likely produce their discriminative

stimulus effects through opioid mechanisms whereas, under these conditions,

ketamine probably produces its discriminative effects through a nonopioid

mechanism.

Drug Combinations

Another method of evaluating the effects produced by drugs that either partially

or fully substitute for a training drug is to examine the effects of these

compounds administered in combination. For example, (-)-NANM, cyclazocine,

and ketamine were evaluated in combination with morphine in pigeons

discriminating between saline and one of several doses of morphine (Koek and

Woods 1989). When the training dose of morphine was 1.8 mg/kg, cyclazocine

completely substituted for the morphine discriminative stimulus while (-)-NANM

and ketamine partially substituted for morphine; (-)-NANM, but not ketamine,

antagonized the ability of cyclazocine to substitute for the morphine

discriminative stimulus. When the training dose of morphine was increased to

18.0 mg/kg, the morphine-like discriminative stimulus effects of cyclazocine and

(-)-NANM decreased. Under these conditions where cyclazocine and (-)

-NANM failed to substitute for morphine, both compounds antagonized the

discriminative stimulus effects of morphine. In contrast, the percentage of

morphine-appropriate responding produced by ketamine was not changed by

increasing the training dose of morphine to 18.0 mg/kg; moreover, ketamine

failed to antagonize morphine at either training dose. Together with the results

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showing no antagonism by naltrexone of the apparent morphine-like

discriminative stimulus effects of ketamine, these results further support the

notion that the effects of ketamine under these conditions are nonopioid. The

potency of naltrexone in antagonizing the discriminative stimulus effects

produced by different agonists provides strong evidence that these agonists

produce their discriminative stimulus effects through a similar mechanism (e.g.,

Bertalmio and Woods 1987). However, changes in the percentage of

morphine-appropriate responding and the ability of some compounds to

antagonize morphine when training dose is varied can also be used to evaluate

whether or not drugs are producing discriminative stimulus effects through a

similar mechanism.

Colpaert and Janssen (1984) evaluated a larger range of drugs in rats trained to

discriminate 0.04 mg/kg fentanyl from saline and found that cyclazocine,

nalorphine, ketocyclazocine, and ±NANM partially substituted for the fentanyl

discriminative stimulus. These compounds also partially antagonized the

fentanyl discriminative stimulus, which is consistent with, but does not prove

that the discriminative stimulus effects of these compounds were similar to each

other. These investigators also found that analysis of data from individual

animals was important in evaluating the discriminative stimulus effects of

agonists because not all of the animals produced the same pattern of

responding: some rats responded completely on the fentanyl-associated lever

while others never responded on the fentanyl-associated lever. In animals in

which cyclazocine did not substitute for fentanyl, cyclazocine antagonized

fentanyl. In animals in which cyclazocine did substitute for fentanyl, doses of

cyclazocine with no apparent discriminative stimulus effects failed to antagonize

the discriminative stimulus effects of fentanyl. Finally, nalorphine and

ketocyclazocine substituted for fentanyl in the same animals in which

cyclazocine substituted for fentanyl. Thus, different patterns of responding

occur in groups of subjects when training dose is manipulated; different patterns

of responding also occur among individual subjects with training dose.

WHY?

In summary, changes in the dose of a training drug in a drug discrimination

study can produce very dramatic effects including the following: changes in the

potency of different agonists; changes in slopes of generalization gradients to

the training drug as well as to other drugs; different patterns of substitution for

drugs from the same and different pharmacological classes (e.g., opioids and

nonopioids); and changes in the patterns of discriminative stimulus effects when

151
drugs are studied in combination. These effects that occur as training dose is

manipulated could be a result of a variety of factors including changes in levels

of discriminative stimulus control, changes in the pharmacological selectivity of

a training drug, or differences in efficacy among drugs and efficacy

requirements across experimental conditions. One or more of these factors may

contribute to the results obtained as training dose is varied.

Discriminative Stimulus Control

The notion that changes in levels of discriminative stimulus control account for

some of the effects observed when training dose is varied is supported by

experiments (Colpaert et al. 1980a, 1980b) in which rats discriminating 0.04

mg/kg fentanyl from saline were trained to progressively smaller doses of

fentanyl. In these animals, the two measures of performance were the number

of sessions required to reach a predetermined criterion level of performance

and the accuracy in responding after criterion had been met. The number of

sessions to criterion increased as training dose was decreased, possibly

indicating that the stimulus produced by the training drug was more difficult to

differentiate from the nondrug condition as training dose was reduced. Two

types of errors occur in a simple two-choice (drug, vehicle) discrimination: (1)

responding on the drug lever in the absence of the training stimulus or (2)

responding on the saline lever in the presence of the training stimulus. Colpaert

et al. (1980a) found that the total number of errors increased both on drug

training days and on saline training days as the training dose was decreased;

however, the accuracy of responding during drug training sessions was better

than during saline training sessions as training dose was decreased, indicating

the possibility of a drug-lever bias and not simply a loss of stimulus control.

In describing effects that are produced by different agonists under conditions of

different training doses, loss of discriminative stimulus control, as evidenced by

errors in lever selection, could account for decreases in the slope of

generalization gradients as training dose is decreased. Furthermore, to the

extent that a drug-lever bias occurs with small training doses, animals will have

a higher probability of showing drug-appropriate responding in the absence of

the training stimulus (e.g., pharmacologically unrelated drugs). Thus, while

stimulus control might contribute to the changes in slopes of generalization

gradients and different patterns of substitution that result from manipulations of

training dose, it does not provide a satisfactory explanation for why the

compounds that produce less drug-appropriate responding antagonize the

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compounds that produce more drug-appropriate responding. Other factors must

also contribute to the effects that occur when training dose is altered.

Pharmacological Selectivity

Differences in the pharmacological selectivity of a training drug at different

doses also might contribute to the changes in potencies and slopes of the

generalization gradients and the patterns of substitution that occur as training

dose is altered. Kuhn et al. (1976) showed that in rats trained to discriminate

10.0 mg/kg pentazocine from saline, morphine completely substituted for the

pentazocine stimulus. Moreover, naloxone antagonized the discriminative

stimulus effects of morphine and of pentazocine, indicating that these two drugs

produced their discriminative stimulus effects through similar mechanisms.

White and Holtzman (1982) trained squirrel monkeys to discriminate 3.0 mg/kg

pentazocine from saline and showed that the opioid mu agonist levorphanol, as

well as the nonopioids phencyclidine and dextrorphan, substituted for the

pentazocine discriminative stimulus. The opioid kappa agonists ketocyclazocine

and ethylketocyclazocine and the mixed-action opioid nalbuphine produced less

than 50 percent pentazocine-appropriate responding. The nonopioids

apomorphine, d-amphetamine, secobarbital, and mescaline produced no

pentazocine-appropriate responding. In these animals, naltrexone antagonized

the discriminative effects of L-pentazocine and levorphanol, but not

dextrorphan, indicating that the discriminative stimulus effects of pentazocine

could have both an opioid and a nonopioid component.

This hypothesis could explain the different patterns of responding sometimes

observed when the training dose is varied; that is, at some training doses, drugs

like pentazocine might be selective for a particular receptor type and as the

training dose is increased pentazocine might also act at other receptors. Under

the condition in which the training dose is such that more than one receptor

type is activated, drugs that act at either receptor type may substitute, while at

lower training doses, drugs that activate only a single receptor type may

substitute. The notion that selectivity differences occur with different training

doses can also account for why naltrexone antagonizes the discriminative

effects of some, but not all, of the compounds that substitute for the

discriminative stimulus effects of pentazocine. At larger training doses, when

pentazocine might have discriminative stimulus effects that are nonopioid, some

nonopioid test drugs might substitute and these discriminative stimulus effects

would not be antagonized by naltrexone.

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Efficacy

Because for any agonist small doses presumably occupy fewer receptors than

large doses, different amounts of receptor stimulation probably occur when

different doses are administered. If receptor occupancy was the only

determinant of the magnitude of a response, all agonists would be equally

effective in producing a maximal response and simply increasing the dose of

agonist would be sufficient to produce that level of response. However, the

dose of drug administered is not the sole determinant of the effectiveness of

drugs in producing biological responses. Agonists also vary in a property,

independent of potency, that enables them to activate receptors and thereby

produce responses. Because different agonists vary in their abilities to produce

a response once the receptor is activated, they are said to have different

efficacies. For a given level of effect, high-efficacy agonists occupy fewer

receptors while low-efficacy agonists occupy more receptors. Conversely, at

equal levels of receptor occupancy, an agonist with higher efficacy will produce

a larger effect than an agonist with lower efficacy (e.g., Kenakin 1987).

Regarding results with drug discrimination procedures then, a third explanation

that could account for the changes in potency, slopes of the generalization

gradients, and patterns of substitution produced by varying the training dose of

a compound is that the efficacy requirements of the drug discrimination

paradigm change with training dose. A lower efficacy agonist will not substitute

for an agonist with higher efficacy when the training dose of that high-efficacy

agonist is large, but the low-efficacy agonist will substitute under small training

dose conditions. Many drugs substitute for a small training dose of high-efficacy

agonist, even drugs that need to occupy all of the receptors to produce this

small stimulus. However, when a large dose of a high-efficacy agonist is used

as the training stimulus, agonists with low efficacy do not substitute because

the stimulus cannot be produced, even though in principle all of the receptors

are occupied.

Under conditions in which a low-efficacy agonist fails to substitute for a

high-efficacy agonist, it is important to confirm that these drugs produce their

effects through similar receptor types. One method of confirming that agonists

with different efficacies are producing their effects through similar receptor

types is to antagonize the discriminative stimulus effects of both low- and

high-efficacy agonists with an antagonist such as naloxone, which has no

efficacy. If these drugs are producing their discriminative stimulus effects

through similar receptor types, the potency of naloxone in antagonizing the

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discriminative stimulus effects of these agonists should be the same.

Furthermore, according to receptor theory, low-efficacy agonists that fail to

substitute for the discriminative stimulus effects produced by a high-efficacy

agonist should antagonize the discriminative stimulus effects of agonists that do

substitute. Once it has been established that similar receptor types mediate the

discriminative stimulus effects of a set of drugs, a number of different methods

can be used to determine whether or not these drugs vary in efficacy.

Irreversible Antagonists. One way of evaluating efficacy differences among

agonists is to study drugs under conditions in which the number of available

receptors is systematically decreased. Irreversible antagonists are tools that

can be used to inactivate a portion of the receptor population (Furchgott and

Bursztyn 1967). Although, like irreversible antagonists, competitive antagonists

also produce rightward shifts in agonist dose-effect curves, they cannot be used

to differentiate agonists with varying efficacies. Since the agonist-competitive

antagonist interaction is reversible and governed by the law of mass action, the

maximum level of effect produced by an agonist is not altered. In contrast, once

bound, irreversible antagonists do not dissociate from receptors; therefore,

increasing amounts of irreversible antagonist result in decreasing numbers of

available receptors. When a proportion of the receptor population is inactivated

by a particular dose of an irreversible antagonist, dose-effect curves for

high-efficacy agonists may be shifted to the right without a decrease in the

maximum effect. Dose-effect curves for low-efficacy agonists, on the other

hand, will also be shifted to the right in the presence of the same dose of

irreversible antagonist, but a decrease in the maximum effect also occurs. A

larger dose of irreversible antagonist would be required to produce a decrease

in the maximum effect produced by a higher efficacy agonist.

Since low-efficacy agonists must occupy a larger number of receptors than

high-efficacy agonists to produce a given level of effect, inactivating a

proportion of receptors with an irreversible antagonist results in a decrease in

the maximum effect produced by low-efficacy agonists before a decrease

occurs in the maximum effect produced by a high-efficacy agonist. Thus,

administration of different doses of an irreversible antagonist can be used to

differentiate agonists with different efficacies. While several investigators have

indeed shown that irreversible antagonists can reduce the maximum effect

produced by certain agonists in rodent analgesia assays (Adams et al. 1990;

Porreca et al. 1982; Tallarida and Cowan 1982), very little is known about the

effects of the interaction between irreversible antagonists and agonists that vary

in efficacy in the drug discrimination paradigm.

155
Tolerance. Another pharmacological tool that can produce agonist

dose-effect curves that are qualitatively the same as those obtained with

irreversible antagonists is chronic treatment with doses of agonist sufficient to

induce tolerance. While chronic treatment with an agonist does not necessarily

produce a reduction in receptor number, this method appears to produce the

same result as treatment with an irreversible antagonist and therefore might be

a useful technique to evaluate and confirm efficacy differences among agonists.

One experiment demonstrating that agonist efficacies can be differentiated in

tolerant animals was conducted by Young et al. (1991). Rats discriminating 3.2

mg/kg morphine from saline were chronically treated with 10.0 mg/kg morphine

either once or twice daily for at least 2 weeks, during which time training was

suspended. After twice daily treatments with morphine, the dose-effect curves

for morphine, etorphine, methadone, and buprenorphine were shifted to the

right with no decrease in maximum levels of drug-appropriate responding, as

compared to dose-effect curves for these compounds prior to chronic treatment.

Furthermore, the morphine dose-effect curve was shifted further to the right

than etorphine, buprenorphine, or methadone, suggesting that morphine is less

efficacious than these compounds. While the dose-effect curve for nalbuphine

was also shifted to the right after chronic morphine treatment (10 mg/kg every

12 hr) as compared to pretreatment dose-effect curves, the maximum level of

drug-appropriate responding was also decreased (figure 4). In contrast, in

animals chronically treated with only a single dose of 10.0 mg/kg morphine per

day, the nalbuphine dose-effect curve was shifted to the right but the maximum

was not decreased. To the extent that etorphine, buprenorphine, methadone,

and nalbuphine are producing their discriminative stimulus effects through the

same receptor type, these data are consistent with the notion that nalbuphine

has lower efficacy than the other agonists tested because the dose-effect curve

for nalbuphine was shifted down and to the right rather than simply to the right

as were the other agonists. Because tolerance and treatment with an

irreversible antagonist appear to produce similar results, they are both useful as

tools to differentiate agonists with different efficacies.

Results from yet another study (France and Woods 1990) demonstrated that

efficacy differences could be detected among various agonists in animals

chronically treated with morphine. Pigeons received 10.0 mg/kg/day morphine

6 hr prior to sessions and discriminated among injections of 0.032 mg/kg

naltrexone, saline, and 10.0 mg/kg morphine. The discriminative stimulus

effects of a number of mu-receptor-selective agonists, including etonitazene,

nalbuphine, meperidine, butorphanol, and buprenorphine, were evaluated in

156
FIGURE 4. Comparison of dose-response functions for morphine-like

stimulus (MS) effects before and during repeated treatment with

morphine, arranged by potency before treatment, Open circles

represent pooled generalization tests conducted before repeated

treatment. Closed circles represent pooled generalization tests

conducted after 1 and 2 weeks of repeated treatment with twice

daily injections of 10.0 mg/kg morphine. Tolerance ratios and 95

percent confidence levels were calculated by parallel line assay.

Dose-response functions for etorphine, methadone, and morphine

were parallel across treatments (n = 5-7 per group). (from Young

et al. 1991.)

157
pigeons that had received either morphine (the dependent condition) or saline

(the abstinent or withdrawal condition). In the dependent condition, while

etonitazene completely substituted for morphine, buprenorphine only partially

substituted for the morphine discriminative stimulus. Butorphanol, meperidine,

and nalbuphine, on the other hand, partially substituted for naltrexone. In the

abstinent condition, pigeons responded primarily on the naftrexone-associated

key when saline was administered under test conditions. Under this condition,

increasing doses of etonitazene produced a switch in responding from the

naltrexone to the saline to the morphine key. In contrast, buprenorphine and

butorphanol produced a switch from the naltrexone to the saline key without a

further switch to the morphine key. Nalbuphine and meperidine produced only a

partial switch from responding on the naltrexone to the saline key.

Both the antagonism of morphine and naltrexone produced by buprenorphine,

butorphanol, and nalbuphine and the patterns of substitution that occur with

these drugs under these conditions are generally consistent with results

obtained using the standard two-key discrimination. The efficacy requirements

of switching responding from the naltrexone to the saline key in the abstinent

condition might be analogous to a low training dose condition, while a further

switch in responding from the saline to the morphine key is analogous to the

high training dose condition. Thus, only agonists with high efficacy (e.g.,

etonitazene) will produce a switch in responding from the naltrexone to the

saline to the morphine key, while tower efficacy agonists (e.g., buprenorphine)

will only produce a switch from the naltrexone to the saline key. One

inconsistency between the data collected in the two-key procedure (Young et

al. 1991) and the three-key procedure (France and Woods 1990) is that

buprenorphine is apparently more efficacious than morphine in the two-key

procedure and is less efficacious than morphine in the three-key procedure.

Further experimentation is required to resolve this issue.

Also consistent with predictions based on results using two-key procedures is

that drugs with low efficacy antagonize drugs with high efficacy. In the

dependent condition, doses of buprenorphine, butorphanol, and nalbuphine that

produced saline-appropriate responding antagonized the discriminative stimulus

effects of both morphine and naltrexone. While nalbuphine does not have

enough efficacy to mimic the discriminative stimulus effects of morphine,

nalbuphine antagonizes the effects of morphine, a higher efficacy agonist.

These results confirm then that efficacy differences do indeed exist among

agonists and may be detected using both the two-choice and the three-choice

drug discrimination paradigm.

158
CONCLUSION

While toss of discriminative stimulus control or changes in pharmacological

selectivity might play a role in the effects that are produced when training dose

is varied in opioid discrimination experiments, differences in efficacy and

changes in efficacy requirements are also probably important factors

contributing to the results obtained. The notion that efficacy differences exist

among agonists that activate the same receptors has been supported

experimentally in animals chronically treated with an agonist. The fact that

some opioids, such as nalbuphine, do not produce a full effect under conditions

in which the chronic treatment conditions induce a large amount of tolerance

suggests that these agonists have tower efficacies than agonists that do

produce a full effect under similar conditions. Thus, when the efficacy

requirement of the assay is changed, such as when the training dose is varied,

low-efficacy agonists show different patterns of substitution.

Other drugs, for example, morphine, etonitazene, or fentanyl, that presumably

have higher efficacies will produce a full effect even when the efficacy

requirement is high, such as when the training dose is increased. In addition,

the notion of efficacy differences might also explain why drugs such as

cyclazocine antagonize the discriminative stimulus effects of morphine when

the training dose of morphine is increased. While the efficacy requirements in

the high training dose condition are such that cyclazocine cannot produce a full

effect, cyclazocine still occupies the same receptors that morphine activates to

produce its discriminative stimulus effects; cyclazocine effectively acts as an

antagonist under these conditions. Thus, the notion of efficacy differences

among agonists appears to explain a number of results obtained when training

dose is varied in the drug discrimination paradigm. Other variables, however,

such as loss of discriminative stimulus control and changes in pharmacological

selectivity of agonists may also contribute to this profile of effects. Hence, both

a behavioral and pharmacological analysis of these outcomes will be necessary

to account for the varied results of changes in training dose in opioid

discrimination studies.

REFERENCES

Adams, J.U.; Paronis, C.A.; and Holtzman, S.G. Assessment of relative intrinsic

activity of mu-opioid analgesics in vivo by using beta-funattrexamine. J

Pharmacol Exp Ther 255:1027-1032,1990.

159
Bertalmio, A.J., and Woods, J.H. Differentiation between mu and kappa

receptor-mediated effects in opioid drug discrimination: Apparent pA2

analysis. J Pharmacol Exp Ther 243:591-597, 1987.

Colpaert, F.C., and Janssen, P.A. Agonist and antagonist effects of prototype

opiate drugs in rats discriminating fentanyl from saline: Characteristics of

partial generalization. J Pharmacol Exp Ther 230:193-199, 1984.

Colpaert, F.C.; Niemegeers, C.J.E.; and Janssen, P.A.J. Factors in regulating

drug cue sensitivtty: Limits of discriminability and the role of a progressively

decreasing training dose in fentanyl-saline discriminations. J Pharmacol Exp

Ther 212:474-480, 1980a.

Colpaert, F.C.; Niemegeers, C.J.E.; and Janssen, P.A.J. Factors regulating

drug cue sensitivity: The effect of training dose in fentanyl-saline

discrimination. Neuropharmacol 19:705-713, 1980b.

France, C.P., and Woods, J.H. Discriminative stimulus effects of opioid agonists

in morphine-dependent pigeons. J Pharmawl Exp Ther 254:626-632, 1990.

Furchgott, R.F., and Bursztyn, P. Comparison of dissociation constants and of

relative efficacies of selected agonists acting on parasympathetic receptors.

Ann NY Acad Sci USA 68:1742-1747, 1967.

Holtzman, S.G. Discriminative stimulus properties of opioids in the rat and

squirrel monkey. in: Drug discrimination: Applications in CNS Pharmacology,

Colpaert, F.C. and Slangen, J.L. (eds.). Elsevier: Amsterdam, pp.17-36,

1982.

Kenakin, T.P. Pharmacologic Analysis of Drug-Receptor Interaction. New York:

Raven Press, 1987.

Koek, W., and Woods, J.H. Partial generalization in pigeons trained to

discriminate morphine from saline: Applications of receptor theory. Drug

Develop Res 16:169-181, 1989.

Kuhn, D.M.; Greenberg, I.; and Appel, J.B. Stimulus properties of the narcotic

antagonist pentazocine: Similarity to morphine and antagonism by naloxone.

J Pharmacol Exp Ther 196:121-127, 1976.

Porreca, F.; Raffa, R.B.; Cowan, A.; and Tallarida, R.J. A comparison of the

receptor constants of morphine and ethylketocyclazocine for analgesia and

inhibition of gastrointestinal transit in the rat. Life Sci 31:1955-1961, 1982.

Shannon, H.E., and Hoftzman, S.G. Morphine training dose: A determinant of

stimulus generalization to narcotic antagonists in the rat. fsychopharm

61:239-244, 1979.

Tallarida, R.J., and Cowan, A. The affinity of morphine for its pharmacologic

receptor in vivo. J Pharmacol Exp Ther 222:198-201, 1982.

White, J.M., and Holtzman, S.G. Properties of pentazocine as a discriminative

stimulus in the squirrel monkey. J Pharmacol Exp Ther 223:396-401, 1982.

160
Young, A.M.; Kapitsopoulos, G.; and Makhay, M.M. Tolerance to morphine-like

stimulus effects of mu opioid agonists. J Pharmacol Exp Ther 257:795-805,

1991.

161

Discriminative Stimulus Properties of

Phencyclidine and Other NMDA Antagonists

Robert L. Balster, Ph.D.

INTRODUCTION

Although phencyclidine (PCP) was initially developed as an injectable

anesthetic, most of the scientific interest in it is based on its abuse. It should be

noted that PCP is a problem almost exclusively in North America, with little

evidence of abuse in other areas of the world. Worldwide interest in PCP

research has been sparked by the discovery that PCP and other PCP-like

drugs, such as ketamine and dizocilpine (MK-801), are antagonists of

glutamatergic neurotransmission (Lodge et al. 1987) and thus could offer some

protection against excitotoxic neural injury (Foster et al. 1987; Olney 1990). The

possibility that excitatory amino acid antagonists may have therapeutic potential

as neuroprotectants and/or anticonvulsants has led to concern about their

potential to produce PCP-like psychotomimetic side effects and, more generally,

to an interest in understanding more fully the neural basis for PCP abuse.

PCP produces an intoxication in humans characterized by altered perceptual

experiences and feelings of dissociation from the environment (Luby et al.

1962; Pollard et al. 1965). PCP abusers report a dreamy, carefree state with

impaired judgment and heightened enjoyment of various activities (Siegel

1978). Occasionally, PCP intoxication results in panic states and highly

disordered thinking that can be dangerous to both the user and to others

(McCarron 1986). The fact that this pattern of effects is not produced by other

common drugs of abuse suggests that PCP might produce a unique profile of

behavioral and pharmacological effects relevant to its abuse.

Considerable research has been carried out in animals using drug

discrimination procedures to further study the perception of intoxication with

PCP-like drugs. However, since the late 1950s no experimental laboratory

studies of PCP intoxication in humans have been carried out, presumably

163
because of risks to human subjects. Thus, drug discrimination studies with

PCP-like drugs have been the only source of experimental information on the

nature and underlying neural basis of PCP intoxication. This situation is

somewhat unique among drugs of abuse in that no reference data are available

on the subjective effects of PCP using standard assessments such as the

Addiction Research Center Inventory or the Profile of Mood States. Often, this

has led to considerable confusion concerning the nature of PCP-like effects.

There is no need here to provide an extensive historical review of drug

discrimination studies with PCP-like drugs because this has been done recently

in other publications (Balster 1987; Balster and Willetts 1988). However, before

proceeding to a more thorough discussion of recent studies, it will be helpful to

summarize some of the main findings of earlier work.

PHARMACOLOGICAL SPECIFICITY OF PCP DISCRIMINATION

Substitution studies in subjects trained to discriminate PCP or another PCP-like

drug, such as ketamine, generally demonstrate considerable pharmacological

specificity for the PCP stimulus (Browne 1986; Poling et al. 1979; Shannon

1983). Opioids, sympathomimetic stimulants, anticholinergics, and other

classes of hallucinogens do not generalize from PCP. Perhaps the only

exceptions are central nervous system (CNS) depressants such as ethanol and

the barbiturates, in which cross-substitution with PCP appears to depend

somewhat on the test conditions. Barbiturates can often result in partial

substitution for PCP, although this is often accompanied by response-rate

suppression (McMillan 1982; McMillan and Wenger 1983; Overton 1975;

Willetts and Balster 1988b). Recent evidence (Mansbach and Balster 1991)

suggests that the level of generalization from PCP to pentobarbiial is affected

by training dose of PCP, with lower training doses associated with more

PCP-lever selection. This is consistent with the results of other studies showing

less pharmacological specificit at lower training doses of other drugs (Colpaert

et al. 1980; Shannon and Holtzman 1979).

Further evidence for cross-generalization among PCP and CNS depressants

comes from recent studies with ethanol discrimination. In pigeons trained to

discriminate ethanol from vehicle, both PCP and ketamine fully substituted for

the ethanol stimulus (Grant et al. 1991). In ethanol-trained mice, PCP and

ketamine also substituted for ethanol, but unlike the results in pigeons,

PCP-lever responding produced by ethanol was accompanied by response-rate

disruption (Grant et al. 1991). This emerging evidence for a degree of overlap in

164
the discriminative stimulus properties of PCP and classical CNS depressants

may reflect the ability of PCP-like drugs to produce other depressant behavioral

and pharmacological effects (Balster and Wessinger 1983).

Although the discriminative stimulus properties of PCP are not generally shared

by other drug classes, certain chemical analogs of PCP produce complete

cross-generalization with PCP or ketamine, and the structural requirements for

the production of PCP-like discriminative stimulus effects of

arylcyclohexylamines are quite well established (Balster and Willetts 1988;

McMillan et al. 1988; Overton et al. 1989).

A number of other drugs are capable of producing PCP-like discriminative

stimulus effects (figure 1). They do not bear an obvious structural relationship to

PCP and themselves comprise diverse chemical groups.

Studies with these classes of PCP-like drugs have made important

contributions to our understanding of the neural basis of PCP discrimination.

PCP and Sigma

Working independently, two laboratories used receptor binding technology with

3H-PCP to identify a specific binding site in the mammalian nervous system for

PCP-like drugs (Vincent et al. 1979; Zukin and Zukin 1979). The functional

significance of this site for the discriminative stimulus effects of PCP was

established by Zukin and Zukin (1979), who found a good structure-activity

correlation between binding affinity and potency for PCP-like discriminative

stimulus effects as reported by Shannon (1981). Of particular importance was

the finding that (±)-N-allylnormetazocine (NANM) both displaced radioligands at

the PCP site and substituted for PCP in drug discrimination studies (Brady et al.

1982; Holtzman 1982; Shannon 1981). This drug had been widely studied as

the putative sigma ( )-opioid agonist SKF-10,047 as defined by Martin and his

colleagues (1976) on the basis of a selective pharmacological profile in the dog.

This led to the identification of the PCP binding site as the elusive o-receptor

(Quirion et al. 1981; Zukin and Zukin 1983).

This association of PCP with a-receptors has now led to considerable

confusion, which some historical perspective will help to unravel. The problem

developed when biochemical pharmacologists using radioligand binding

techniques discovered that some -agonist benzomorphans, including

(+)-NANM, had potent actions on a central nervous system site that was clearly

165
FIGURE 1. Structures of drugs with PCP (phencyclidine)-like discriminative

stimulus effects.

166
not the PCP site (Su 1982; Tam 1983). Indeed, (+)-NANM had an even higher

affinity for this site than for the PCP receptor. Among the most potent ligands

for this site was haloperidol, a drug with little affinity for PCP receptors. Another

drug that became widely used in binding studies for this site was the dopamine

analog (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine[(+)-3-PPP] (Largent et al.

1984). Thus, this site could properly, if not redundantly, be referred to as the

haloperidol-sensitive, high-affinity (+)-NANM/(+)-3-PPP binding site. Various

combinations of these terms have been widely used. The result of the discovery

of this site is that we now have two sites, the PCP/ site and the high-affinity

site.

To address the nomenclature problem, a group convened at a PCP conference

in Ann Arbor, Michigan, to propose some consensus. The proposed solution

(Quirion et al. 1987) was to name the haloperldol-sensitive high-affinity site the

receptor and the historical PCP/ site the PCP receptor, a proposal that has

done little to resolve the confusion, particularly among new entrants to the field

who are not familiar with the PCP/ legacy. This proposal was doubly

unfortunate since this binding site had not as yet met criteria established to

label it a receptor, and there was little evidence that this site mediated the

profile of a-agonist effects produced by PCP, (+)-NANM, or other o-agonists.

Which site is responsible for mediating the discriminative stimulus properties of

PCP and o-agonist drugs? Most evidence currently points to the PCP receptor

as an important neural substrate for PCP discrimination and generalization to

arylcyclohexylamines and benzomorphan a-agonists (Balster and Willetts 1988;

Kozlowski et al. 1986). There is also considerable evidence that the

discriminative stimulus properties of (+)-NANM are mediated through its

interaction with the PCP site (Balster 1989; Steinfels et al. 1987b). On the other

hand, reports of haloperidol antagonism of (+)-NANM discrimination (Steinfels

et al. 1987a) and substitution by (+)3-PPP (Steinfels et al. 1986) are

inconsistent with PCP-receptor mediation.

Studies of the discriminative stimulus properties of more selective drugs for the

site have produced results that have done little to clarify the role of this site in

acute drug effects. In rats trained on a 1,3-di-ortho-tolyl-guanidine (DTG)

discrimination (Holtzman 1989), PCP, (+)-NANM, and (+)-pentazocine all

substituted for DTG, consistent with a role for the site in DTG discrimination.

On the other hand, substitution was also obtained with drugs having little or no

affinity for the site. Indeed, there were negative correlations between

o-binding affinity and potency for producing DTG-like effects. Steinfels et al.

167
(1988) trained a discrimination based on a relatively low dose of the potent

ligand (+)-pentazocine. In these animals, (+)-NANM substituted for

(+)-pentazocine, and it was more potent-the reverse of the affinity of these

compounds for sites. PCP only partially substituted for (+)-pentazocine.

In summary, it is clear that the PCP receptor is involved in the discriminative

stimulus properties of PCP and many -agonist benzomorphans. The role, if

any, of the high-affinity a-binding site in the discriminative stimulus effects of

drugs needs to be determined.

Dioxolanes

Drug discrimination studies with the 1,3-substituted dioxolanes such as

etoxadrol and dexoxadrol and their isomers have provided important

information about the steric requirements for producing PCP-like effects. PCP

itself lacks a chiral center, and steroisomers of other arylcyclohexylamines such

as ketamine provide evidence for only relatively small potency differences

(Brady and Balster 1982). On the other hand, 1,3-substituted dioxolanes show

pronounced stereospecificity. For example, dexoxadrol, but not levoxadrol,

substitutes fully for PCP and binds to the PCP receptor (Mendelsohn et al.

1984; Slifer and Balster 1984). Further assessment of the enantiomeric and

diastereomeric selectivity of dioxolanes for PCP-like effects has led to an

advanced understanding of the active conformations of PCP receptor agonists

and the development of molecular models of the PCP pharmacophore

(Jacobson et al. 1987; Thurkauf et al. 1988).

Dizoclipine

Dizocilpine (MK-801) has recently played a very important role in furthering our

understanding of the function of the PCP receptor in drug discrimination.

Dizocilpine is a potent and selective ligand for the PCP site (Wong et al. 1986).

It substitutes completely in PCP- or ketamine-trained animals with a potency

consistent with its 5- to 10-fold greater affinity than PCP for the PCP receptor

(Beardsley et al. 1990; Jackson and Sanger, 1988; Koek et al. 1988;

Tricklebank et al. 1987; Willetts and Balster 1988a). Furthermore, PCP-like

drugs substitute completely in dizocilpine-trained animals (Tricklebank et al.

1989). Thus, dizocilpine, because of its potency and selectivity for PCP

receptor activation, has become a widely used tool for probing the function of

this cellular site. Of particular importance is its identification as an excitatory

amino acid antagonist (Wong et al. 1986).

168
PHENCYCLIDINE AS AN N-METHYL-D-ASPARTATE ANTAGONIST

Certainly the most important recent development in the neurobehavioral

pharmacology of PCP was the discovery that PCP and ketamine were able to

selectively block the effects of activation of the N-methyl-D-aspartate (NMDA)

subtype of excitatory glutamate receptor (Lodge et al. 1987). It was also found

that all drugs which bound to the PCP receptor and had PCP-like discriminative

stimulus effects, including dizocilpine, were able to antagonize NMDA (see

Lodge and Johnson 1990 for review). Biochemical and electrophysiological

studies have led to the development of a model for the NMDA receptor

complex, which is represented in figure 2.

In this model, glutamate, or possibly other endogenous ligands, bind to the

NMDA-preferring site, resulting in the opening of a cationic channel. The

resultant influx of Ca++ produces excitatory postsynaptic potentials and, in

cases of excessive stimulation, excitotoxicity. Excessive release of glutamate

may occur with ischemic stroke or head injury and thus excitatory amino acid

antagonists may offer a means of providing neuroprotection. The NMDA

receptor may also be involved in epileptogenesis, anxiety, and/or learning and

memory, providing additional therapeutic possibilities for drugs that may modify

activity at this receptor complex.

The PCP site is thought to lie within the cationic channel. Binding of ligands to

the site may physically block the passage of ions, functionally antagonizing

NMDA receptor activation. PCP can thus be referred to as a noncompetitive

NMDA antagonist. PCP-like drugs are effective antagonists of NMDA-produced

convulsions (Church and Lodge 1990; Koek and Colpaert 1990) and other

NMDA-produced toxicity (Leander et al. 1988; McDonald et al. 1989). PCP and

dizocilpine also have been shown to provide protection against traumatic

(Hayes et al. 1988) and ischemic neural injury (Olney et al. 1989).

The emergence of NMDA antagonists as possible therapeutic agents has

sparked interest in whether all NMDA antagonists will produce PCP-like

intoxication and abuse liability. As reviewed above, there is good evidence that

binding to a PCP site is predictive of PCP-like discriminative stimulus effects,

and this is certainly true for the potent NMDA antagonist dizocilpine. This fact,

combined with the excellent correlation between the potency of PCP-site

ligands for antagonism of NMDA and their PCP-like discriminative stimulus

effects (Koek et al. 1990; Koek and Woods 1988; Martin and Lodge 1988),

provides support for the prediction that all noncompetitive NMDA antagonists

169
FIGURE 2. Model of the NMDA (N-methyl-D-aspartate)/PCP (phencyclidine)

receptor complex.

acting on the PCP site are likely to produce PCP-like subjective effects and

abuse liability. Results of human testing with dizocilpine and/or other of these

drugs will be important in providing validation of the drug discrimination model

of subjective effects.

Another class of NMDA antagonist has been developed that does not have

affinity for the PCP site. These drugs are competitive blockers of NMDA

receptor site activation (see figure 2) and include APH, CPP, CGS 19755, and

NPC 12626 (see Watkins et al. 1990 for review). Although competitive NMDA

antagonists can produce certain observable PCP-like effects at high doses

(Koek and Colpaert 1990; Woods 1989), evidence of differences in their

discriminative stimulus effects is beginning to emerge (Willetts et al. 1990).

Although studies in pigeons generally show cross-substitution between PCP

and competitive NMDA antagonists (Koek et al. 1986; Leander 1989), studies in

rats and rhesus monkeys reveal important differences. Competitive NMDA

antagonists produce at most only partial PCP-like or dizocilpine-like

discriminative stimulus effects, and drug-lever selection when it does occur is

usually associated with marked response-rate reductions (Ferkany et al. 1989;

170
Jackson and Sanger 1988; Koek and Colpaert 1990; Tricklebank et al. 1989;

Willetts and Balster 1988b). In ketamine-trained rhesus monkeys, CGS 19755

completely fails to substitute (France et al. 1989). In addition, in rats trained with

NPC 12626, PCP and dizocilpine produce only partial substitution accompanied

by response-rate decreasing effects (Willetts et al. 1989). Of particular

importance is the evidence that competitive NMDA antagonists can have

behavioral effects, as evidenced by response-rate reductions and antagonism

of NMDA, at doses lower than those that produce any evidence of PCP or

ketamine-like discriminative stimulus effects (France et al. 1989; Willetts and

Balster 1989).

These differences in the profiles of discriminative stimulus effects of

noncompetitive and competitive NMDA antagonists are summarized in figures 3

and 4. Figure 3 shows the results of tests with PCP as an antagonist of NMDA

discrimination and as a substitute for PCP and NPC 12626. It can be seen that

PCP-like effects can be produced at doses of PCP that do not antagonize

NMDA, and that NPC 12626-like effects, to the extent that they occur, are

produced only at high doses. This is in contrast to the results with CPP. a

typical competitive NMDA antagonist (figure 4). It can be seen that CPP is a

potent and effective antagonist of NMDA at doses considerably lower than

those that produce even partial PCP-like effects. Unlike PCP, CPP substitutes

fully for NPC 12626.

There are a number of possible implications of the differences in the

discriminative stimulus effects of competitive and noncompetitive PCP-like

NMDA antagonists. Of practical importance is the possibility that competitive

antagonists may not have PCP-like subjective effects or at least may have good

dosage separation between therapeutically useful actions and PCP-like effects.

Studies in humans will be needed to confirm this hypothesis, and the results will

help in the future interpretation of drug discrimination results in animals.

These results also show that antagonism of NMDA per se is not sufficient to

produce PCP-like discriminative stimulus effects. Does this mean that NMDA

antagonism does not play a role in PCP discrimination? This seems unlikely

because of the strong structure-activity relationship between NMDA antagonism

and PCP-like discriminative stimulus effects. I am not aware of any drug that

substitutes fully for PCP that does not also antagonize NMDA. One possibility is

that the functional consequences for the cell of competitive and noncompetitive

antagonism are different enough to result in different profiles of behavioral

effects. The analogy with the GABA/benzodiazepine receptor may be worth

171
FIGURE 3. Effects of various doses of PCP (phencyclidine) in three drug

discrimination procedures, antagonism of NMDA

(N-methyl-D-aspartate) discrimination (Willetts and Balster 1989),

substitution for PCP (Willetts and Balster 1988b), and substitution

for NPC 12626 (Willetts et al. 1989).

considering, in which direct GABA receptor agonists such as muscimol have a

somewhat different profile of discriminative stimulus and other behavioral

effects than do indirect acting GABA agonists such as the benzodiazepines and

barbiturates (Grech and Balster 1990; Sanger 1985). Differences can also be

shown between benzodiazepines and barbiturates (Ator and Griffiths, 1989).

It is also possible that NMDA and PCP receptors are not always functionally

coupled to each other (Rao et al. 1990) or perhaps subtypes of NMDA

172
FIGURE 4. The effects of various doses of CPP in three drug discrimination

procedures; antagonism of NMDA (N-methyl-D-aspartate)

discrimination (Willetts and Balster 1989), substitution for PCP

(phencyclidine) (Willetts and Balster 1988b), and substitution for

NPC 72626 (Willetts et al. 1989).

receptors exist with differing propensity for PCP-like channel blockade

(Monaghan et al. 1988). In any case, the results of drug discrimination studies

with NMDA antagonists have shown once again the power of this methodology

to dissect subtle differences in the behavioral effects of psychoactive drugs.

173
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ACKNOWLEDGMENTS

The preparation of this review and the data reported from the author’s

laboratory were supported by grant DA-01442 from NIDA. Joyce Willetts

assisted in the collection and interpretation of these data and in the preparation

of figures 3 and 4.

AUTHOR

Robert L. Balster, Ph.D.

Professor of Pharmacology and Toxicology

Medical College of Virginia

Virginia Commonwealth University

Richmond, VA 23298-0613

180
Discriminative Stimulus Effects of

Psychomotor Stimulants and

Benzodiazepines in Humans*

Chris-Ellyn Johanson

This chapter will review a series of studies on the discriminative stimulus (DS)

effects of psychomotor stimulants and benzodiazepines in humans. Although

there has been an enormous increase in studies of the DS effects of drugs in

animals over the last two decades (Stolerman and Shine 1985; Stolerman et al.

1989), relatively few studies have been conducted with humans. Human studies

are necessary for several reasons. First, as is always the case, our acceptance

of results obtained with other species is increased if under comparable conditions,

similar results are generated in humans. Second, drug discrimination is presumed

to be an animal model of subjective drug effects in humans (Schuster and

Johanson 1988). However, to validate this model, it is necessary to measure

DS and subjective effects simultaneously and that is possible only in studies

that use human subjects.

The present series of studies was designed to determine the similarity between

drug discrimination results obtained in animals and results obtained with

humans using drugs from two different pharmacological classes, amphetamine

and diazepam, as DS. In addition, the subjective effects of these drugs as well

as of test compounds were measured simultaneously in order to compare the

DS and subjective effects of drugs directly. Because almost no studies on the

DS effects of drugs had been conducted with humans when these studies were

initiated, it was also necessary to demonstrate the feasibility of conducting such

studies in humans despite not being able to expose subjects to large doses of

drugs over tong periods of time. Thus, it was necessary to develop a procedure

that used relatively low doses and minimized the duration of the training and

*The research reviewed was supported by grants from the National Institute on Drug Abuse

DA00250 and DA06030 and from Uniformed Services University of the Health Sciences. The

opinions expressed by the author are not necessarily those of the United States Government.

181
testing period. At the same time, it was considered essential to try to mimic as

closely as possible the procedural characteristics used in animal studies. When

a determination had been made that humans could learn an AMP or DZ

discrimination under these conditions, further evaluations were conducted to

verify that the discrimination was pharmacologically sensitive (dose

responsitivity) and specific.

METHODS

Subjects

Two series of studies were conducted using either 10 mg d-amphetamine

(AMP) or 10 mg diazepam (DZ) as the DS. Unless noted, the procedure used in

these two series of studies was identical. However, the AMP studies were

conducted at the University of Chicago and the results have been published

previously (Chait and Johanson 1988; Chait et al. 1984; 1985; 1986a; 1986b;

1989). The DZ studies were conducted at the Uniformed Services University of

the Health Sciences. Subjects from both series were recruited using advertisements posted in their respective university areas and published in local

newspapers. In addition, the DZ studies were listed by the normal volunteer

recruiting office of the National Institutes of Health.

Potential candidates underwent a physical and a psychiatric examination and

received a blood screen prior to participation. Candidates were not accepted if

they had a previous history of drug abuse or dependence other than tobacco

dependence, a history of psychiatric illness, or any significant present or past

medical or psychiatric problem that would place them at risk if they participated

in the experiment. Females were not allowed to participate if they were

pregnant as determined by a urine test or if they planned to become pregnant in

the near future. The protocol was reviewed and approved by the local

institutional human use committees and informed consent was obtained.

Subjects were paid for their participation.

General Procedure

Subjects were instructed that their job was to learn to discriminate between two

different drugs, drug A and drug B. They were told that they could receive either

stimulants, sedatives, or placebo, and that drugs A and B would differ. Subjects

were not informed that they would be learning to discriminate between an active

drug and placebo. Subjects reported to the laboratory three to four times each

182
week between 8:15 a.m. and 10:00 a.m. over a 6- to 10-week period. Upon

arrival, subjects filled out three subjective effects questionnaires as described

below. After completion, subjects received a capsule but were then free to leave

the laboratory. They took three additional sets of questionnaires with them to fill

out 1, 3, and 6 hr later.

Phase 1: Sampling/Training (Sessions 1-4). The purpose of phase 1 was to

familiarize subjects with the effects of drug A and drug B and to begin training

the discrimination. Subjects received drug A on the first and third sessions, and

drug B on the second and fourth sessions. The order of presentation of drugs A

and B on the third and fourth sessions was reversed for half the subjects in the

DZ experiments. On each of these four sessions, the letter code of the capsule

was revealed to the subject prior to ingestion. For half of the subjects in each

individual experiment, drug A was placebo and drug B was 10 mg

&hetamine (AMP) or 10 mg diazepam (DZ). The assignments were

reversed for the other subjects.

Phase 2: Training/Selection (Sessions 5-11). The purpose of phase 2 was to

provide additional training and then select subjects that reliably learned the

discrimination. In these seven sessions, subjects received drug A three times

and drug B four times or vice versa. They were administered in a mixed order

with the restriction that the same drug was not scheduled more than two

sessions in succession. The order was different for different subjects. On these

sessions, subjects were not told which drug they received when they ingested

the capsule. At 1, 3, and 6 hr after capsule ingestion, in addition to the

questionnaires described below, subjects filled out a form on which they

identified the drug they believed they had received using the A and B letter

codes, and indicated on a 100-mm visual analog scale (VAS) how certain they

were that their identification was correct. This VAS was labeled ‘Unsure (Just

Guessing)” at the left end and “Positive (Absolutely Sure)” on the right end.

Subjects were told that they were free to change their identification from hour to

hour, based on what they believed at the time. There were no consequences

attached to the 1- and 3-hr identifications. However, at the 6-hr period, subjects

telephoned the experimenter, identified themselves, and reported their 6-hr

letter code identification. If their response was correct, they were told so and

received a monetary bonus when they returned to the laboratory for the next

session. Those whose responses were incorrect were also informed and

received no money at the next session.

183
The discrimination was considered reliable if at least five of the seven

identifications were correct.’ Subjects were not informed of this criterion at the

beginning of the experiment. Those subjects that failed to reach this level of

performance were asked to terminate participation after session 11.

Phase 3: Testing (Sessions 12-end): Subjects that were reliable

discriminators as defined above entered phase 3 but were not told that a new

phase had started. The purpose of phase 3 was to determine whether subjects

would identiiy lower doses of the training drug (DZ or AMP) and other drugs as

placebo or the training drug (DZ or AMP). The test phase consisted of six test

sessions intermixed with additional training sessions. Test sessions were

identical to training sessions except that subjects were not informed when they

telephoned whether their response was correct-they were told only that it was

a test session and that they would receive money at the next session. Subjects

were not told the purpose of test sessions, nor did they know when test

sessions were scheduled until after they had reported their 6-hr drug

identification.

The additional training sessions were interspersed with test sessions in order to

determine whether subjects maintained the discrimination. These training

sessions were exactly like the training sessions during phase 2; that is, subjects

received either drug A or drug B, were told whether their response was correct

when they telephoned, and were reinforced appropriately. Subjects received

placebo and the training drug an equal number of times in mixed order on these

training sessions. The training sessions were interspersed among the test

sessions in an unsystematic fashion with the restriction that no more than two

test sessions or two training sessions occurred in succession. The order varied

across subjects.

Subjective Effects Questionnaires

Profile of Mood States (POMS). An experimental version of the 65-item POMS

(McNair et al. 1971) consisting of 72 adjectives commonly used to describe

momentary mood states was used. Subjects indicated how they felt at the

moment in relation to each of the 72 adjectives on a 5-point scale from “not at

all” (0) to “extremely” (4). There are eight clusters (scales) of items that have

been separated using factor analysis. The names of these scales (Anxiety,

Depression, Anger, Vigor, Fatigue, Confusion, Friendliness, and Elation)

*The criteria for the series of AMP studies was six of seven correct or five correct in a row. For the

DZ experiments the criterion was five of seven coned.

184
describe the adjectives in the cluster. Two additional (unvalidated) scales

(Arousal and Positive Mood) were derived from the other scales as follows:

Arousal=(Anxiety+Vigor)-(Fatigue+Confusion)

Positive Mood=Elation-Depression.

Addiction Research Center Inventory (ARCI). The ARCI is a hue-false

questionnaire with empirically derived scales that are sensitive to the effects of

a variety of classes of abused drugs. A short form of the inventory consisting of

five scales with a total of 49 items was used (Marlin et al. 1971). The five scales

were the Morphine-Benzedrine Group (MBG), a general measure of

drug-induced euphoria; the Amphetamine (A) and Benzedrine General (BG),

which measure amphetamine-like effects; the Pentobarbital-ChlorpromazineAlcohol Group (PCAG), a measure of sedation; and the LSD, a measure of

dysphoria and somatic symptoms.

Visual Analog Scales (VAS). This form has six horizontal 100-mm lines, each

labeled with an adjective. These adjectives are “stimulated,” “high,” “anxious,”

“sedated,” “down,” and “hungry.” The left ends of the lines were labeled “not at

all” and the right ends, “extremely.” Subjects were instructed to place a mark on

each line indicating how they felt at that moment.

RESULTS AND DISCUSSION

Amphetamine Discrimination

Across four studies (Chait and Johanson 1988; Chait et al. 1985; 1986a; 1986b;

1989), 53 of the 100 participants were considered reliable discriminators,

having identified the training compounds correctly five times in a row or on six

of the seven sessions during phase 2. In subjects that learned the

discrimination, the percent of correct identifications and the certainty of

identification increased from hour 1 to hour 6, and there were no differences

between AMP and placebo. Post hoc analyses revealed that all participants

experienced subjective effects typical of those for stimulant drugs, although the

discriminators had a tendency to be more sensitive (Chait et al. 1989).

The 53 participants who learned the discrimination entered the test phase.

During each of the four studies, different drugs were evaluated during the test

phase as follows:

185
Study 1 (N = 17): Placebo, AMP, DZ, and 2 and 5 mg

d-amphetamine.

Study 2 (N = 27): Placebo, AMP, 25 and 50 mg phenmetrazine, and

20 and 40 mg fenfluramine.

Study 3 (N = 20): Placebo, AMP, 25 and 75 mg

phenylpropanolamine (PPA), and 0.5 and 2 mg mazindol.

Study 4 (N = 36): Placebo, AMP, 100 and 300 mg caffeine, and 25

and 50 mg benzphetamine.

In study 1, seven participants learned the discrimination between placebo and

AMP. When tested with these training drugs, they responded correctly.

Participants consistently identified 2 mg amphetamine as placebo; at the 5 mg

dose, the capsule was identified as placebo 50% of the time across

participants. DZ was identified as placebo by five of the seven participants and

produced a profile of subjective effects typical for a benzodiazepine. In study 2,

14 participants learned the discrimination and identified phenmetrazine at both

doses as AMP. The low dose of fenfluramine was identified as placebo,

whereas the high dose produced intermediate levels of AMP-appropriate

responding, reflecting both within- and between-subject variability. In study 3,

12 participants learned the discrimination and identified the high doses of both

mazindol and PPA as drug. The lower doses were identified as placebo or the

identifications were variable. In study 4, 20 participants learned the

discrimination and did not reliably identify either caffeine or benzphetamine as

AMP.

The profile of subjective effects of drugs (i.e., fenfluramine and DZ) that did not

substitute for AMP differed from AMP. On the other hand, phenmetrazine and

the high dose of PPA that substituted for AMP produced subjective effects that

were similar to AMP. However, mazindol, despite the fact that the high dose

substituted for AMP as a DS, produced subjective effects that differed

somewhat from those of AMP. More specifically, mazindol’s subjective effects

were restricted to increases in anxiety, an effect also produced by AMP

(Schuster and Johanson 1988). Since these anxiety-increasing effects (as well

as the more positive effects such as increased scores on the Arousal scale)

were also seen with phenmetrazine, if is possible that they were the basis of the

discrimination.

186
In participants who learned the discrimination, the results with test drugs were

similar to those seen in animals. That is, phenmetrazine, mazindol, and

PPA-but not fenfluramine or benzphetamine-have been shown to substitute

for amphetamine as a DS in rhesus monkeys (de la Garza and Johanson

1987). Furthermore, it may be concluded that the discrimination was dose

related and was also pharmacologically specific because DZ was identified as

placebo.

DZ Discrimination

One study using DZ as the DS has been completed and a second study is

presently under way. In the first study, 16 of 18 subjects learned the

discrimination reliably, and 14 of them entered phase 3. The drugs that were

tested during phase 3 were 2 and 5 tng DZ, 1 and 2 mg brazepam, 50 mg

pentobarbital, and 10 mg AMP. Figure 1 shows the percent of subjects

identifying each of the test compounds as DZ across hours 1 to 6. As can be

seen, the lowest doses of diazepam and lorazepam were identified as placebo

FIGURE 1. The percent of subjects (N = 14) that identified each of the test

drugs as DZ across hours 1, 3, and 6. Only the identification at

hour 6 resulted in monetary bonus and subjects were aware that

they could change their drug identification at each hour.

Abbreviations: DZ=diazepam; LZ=lorazepam; PB=50 mg

pentobarbital; AMP= 10 mg d-amphetamine. The numbers next to

the drug name indicate dose in mg units.

187
by the majority of subjects, whereas the 5 mg dose of diazepam was identified

as DZ by 64 percent of the 14 subjects. The highest dose of lorazepam was

identified as DZ by the majority of subjects. Pentobarbital was similar to the

5 mg dose of diazepam in terms of hour 6 identifications whereas AMP was

identified as placebo. Identifications did not change across hours as was also

true for identifications of DZ and placebo during training sessions. This

indicates that the effects of DZ and drugs identified as DZ had a rapid onset of

action. Certainty ratings increased across hours but were already high at hour 1.

In the second ongoing study, eight subjects have completed phase 3. The drugs

that were tested during this phase included 25 and 50 mg ttipelennamine, 0.125

and 0.25 mg triazolam, and 5 and 10 mg buspirone. Neither dose of

tripelennamine was identified as DZ, nor was either of the lower doses of the

other two compounds. However, 0.25 mg triazolam was identified as DZ by

seven of the eight subjects. The subject that dii not identify this dose of

triazolam as DZ was tested with 0.5 mg triazolam and identified it as DZ. Finally,

the 10 mg dose of buspirone was identified as DZ by five of the eight subjects.

In general the results of these DZ discrimination studies are similar to those

found with animals. Diazepam and related benzodiazepines have been shown

to function as DS in a variety of species, including rats (Colpaert et al. 1976;

Haug and Gotestam 1982), baboons (Ator and Griffiths 1983), squirrel monkeys

(Spealman 1985), and pigeons (Evans and Johanson 1989; Garza et al. 1987;

Järbe and McMillan 1983). In animals trained to discriminate diazepam, other

benzodiazepines substitute as DS and the discrimination is dose dependent.

For instance, Shannon and Herling (1983) trained rats to discriminate between

1 or 3 mg/kg DZ and found that lorazepam in the same dose range substituted

completely for the DZ cue. Triazolam also substituted but was approximately 10

times more potent. Pentobatbital at doses 10 times higher also substituted for

the diazepam DS. Drugs from other pharmacological classes, including

stimulants and antihistamines have not substituted for benzodiazepines (Evans

1987; Evans and Johanson 1989; Spealman 1985). However, in animals

trained to discriminate benzodiazepines, buspirone has consistently failed to be

discriminated as drug-like (Evans and Johanson 1989).

GENERAL DISCUSSION

In summary, the results of the studies described above indicate that humans

can be trained to discriminate 10 mg d-amphetamine or 10 mg diazepam from

placebo using a procedure that (1) does not require laboratory space, (2) limits

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exposure to drugs by using low doses and a relatively short experimental

protocol, and (3) in many ways is comparable to procedures used in animal

studies. In the AMP series, it is interesting to note that only half of the

participants learned the discrimination. While this finding may indicate that

additional methodological studies would be desirable to improve training, the

results with the DZ experiment suggest that DZ and AMP differ in

discriminability. However, because only a small number of subjects have been

tested with DZ and because the differences may have been due only to dose of

the training drug (Chait et al. 1989), additional studies are needed to verify this

hypothesis. Nevertheless, the procedure appears feasible, the results indicate

that the discriminations are pharmacologically sensitive and specific, and the

similarities between the findings in humans and in other animals are promising.

One of the goals of the studies that have been described was to evaluate the

relationship between DS and subjective effects. Many strategies can be used to

determine the correlation between these two measures of drug effect. Because

the results of the AMP experiments have been extensively presented in

previous publications, these strategies will be illustrated for the most part using

results generated from the initial DZ discrimination study.

First, it must be determined whether drugs used as training stimuli produce

reliable time-related changes on measures of subjective effects relative to

placebo that are similar to those that have been previously reported. In the

present experiment, DZ produced effects on several scales of the POMS, ARCI,

and VAS that were similar to those previously reported for DZ. For instance, as

shown in figure 2, DZ significantly increased scores on the Confusion scale and

decreased scores on the Arousal scale of the POMS. Differences from placebo

were greatest at hour 1 and had largely disappeared by hour 6. DZ also

produced significant increases on Fatigue (POMS), PCAG (ARCI), Down

(VAS), and Sedated (VAS) and decreases on Vigor (POMS), A (ARCI), BG

(ARCI), MBG (ARCI), and Stimulated (VAS) relative to placebo while following

a generally similar time course to that seen in figure 2. This time course was

similar to that found in previous studies using acute administration of diazepam.

A second strategy that can be used to evaluate the degree of relationship

between subjective and DS effects is to compare subjective effects in subjects

that do and do not learn the discrimination. If subjects who do not learn the

discrimination also do not report changes in subjective state relative to placebo,

there is indirect evidence that these effects form a basis for the discrimination.

Because only a few subjects in the DZ experiment failed to learn the

189
FIGURE 2. Effects of 10 mg diazepam (DZ) compared to placebo on two

scales of the Profile of Mood States (POMS) in subjects (N = 16)

that reliably learned the discrimination.

discrimination, it is necessary to illustrate this strategy using results from the

AMP experiment. As shown in figure 3, AMP produced increases in ratings of

High on the VAS relative to placebo in subjects that learned the discrimination,

whereas the minimal effects in nondiscriminators on this scale were minimal.

That was true for other subjective effects as well and is presumptive evidence

of the importance of subjective effects in acquiring the discrimination (Chait et

al. 1989).

A third strategy is to compare the subjective effects of drugs that are identified

as the training drug and those identified as placebo in relationship to the

subjective effects of the two training compounds. For instance, as shown in

figure 4, most subjects in the DZ experiment identified 2 mg diazepam as

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FIGURE 3. Differences in the effects of amphetamine (squares) and placebo

(circles) on one of the scales of the VAS in subjects that learned

(N = 53) and failed to learn (N = 47) the amphetamine

discrimination. (Redrawn from Chait et al. 1989.)

placebo, whereas they identified 2 mg lorazepam as DZ. Likewise, the

subjective effects of 2 mg diazepam were virtually identical to those of placebo,

as illustrated in figure 4 for the POMS Confusion scale. In contrast, the

subjective effects of 2 mg lorazepam were similar to the subjective effects of

DZ. Again, this is illustrated in figure 4 for the PCAG scale of the ARCI. Thus,

drugs that were identified as placebo had placebo-like effects, whereas drugs

that were identified as DZ had DZ-like subjective effects. This convergence of

effects is particularly striking in the case of AMP, which was tested in

DZ-trained subjects and identified as placebo. As shown in figure 5, under

these conditions, the subjective effects of AMP were similar to those of placebo.

In contrast, in previous experiments using AMP as the training drug, the

subjective effects of AMP were different from placebo and typical of stimulant

drugs (e.g., increases in the High rating; see figure 3). These contextual effects

on alterations in mood clearly need to be evaluated in future studies.

A fourth strategy that can be used to evaluate the relationship between DS and

subjective effects is to compare the subjective effects of the same test

compound that is identified as placebo by some subjects but identified as the

training drug by other subjects. For instance, in the DZ experiment, 64 percent

of the subjects identified 50 mg pentobarbital as DZ, whereas the remaining

191
FIGURE 4. Representative subjective effects of a drug that was identified as

placebo (2 mg diazepam) and a drug that was identified as drug

(2 mg lorazepam) in subjects trained to discriminate placebo from

10 mg diazepam.

subjects identified it as placebo. The subjective effects of this test drug might be

expected to differ in these two groups of subjects. To test this assumption, a

three-way ANOVA was performed, with an added between-group factor. As

shown in figure 6, the subjective effects of pentobarbital differed significantly in

these two groups of subjects. In subjects that identified PB as DZ, its effects

were more similar to DZ than to placebo. Likewise, in the other group of

subjects, the subjective effects of PB were more like those of placebo. Similar

group differences were reported in a study by Chait et al. (1986a).

Approximately half of the subjects in that study identified 40 mg fenfluramine as

AMP, whereas the remaining subjects identified it as placebo. Correspondingly,

in the former group, fenfluramine produced significant increases on the BG

192
FIGURE 5. The subjective effects of 10 mg d-amphetamine on the Arousal

scale of the POMS in contrast to 10 mg diazepam (DZ) and

placebo in subjects trained to discriminate between DZ and

placebo.

scale of the ARCI, the Vigor scale of the POMS, and the Stimulated and High

rating on the VAS relative to placebo; in the other group, there were no

increases.

In summary, the four strategies used to evaluate the relationship between DS

and subjective effects reveal that the correspondence between these two

measures is remarkably robust. Reliable differences were found between the

subjective effects of the training drug and placebo, and the effects of drug were

similar to those that have been reported previously following acute

administration of diazepam and amphetamine. Likewise, in the AMP studies in

which an appreciable number of subjects did not learn the discrimination, the

nondiscriminators failed to report reliable differences in the subjective effects of

AMP and placebo. Test compounds that were identified as drug in the DZ

experiment produced subjective effects that were similar to those reported

following DZ. Likewise, the subjective effects of the drugs that were identified as

placebo showed placebo-like changes in mood. Finally, the subjective effects

reported by subjects that identified a drug as DZ were similar to those of DZ,

whereas among other subjects that identified the same drug as placebo, the

subjective effects were like those of placebo.

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FIGURE 6. Representative changes in subjective effects (Arousal scale of the

POMS) produced by 10 mg diazepam (squares), placebo

(circles), and 50 mg pentobarbital (triangles) in subjects that

identified 50 mg pentobarbital as placebo (closed symbols) or DZ

(open circles).

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in baboons: Cross-drug generalization and interaction with Ro 15-1788. J

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Chait, L.D.; Uhlenhuth, E.H.; and Johanson, C.E. The discriminative stimulus

and subjective effects of phenylpropanolamine, mazindol, and

d-amphetamine in humans. Pharmacol Biochem and Behav 24:1685-1672,

1986b.

Chait, L.D.; Uhlenhuth, E.H.; and Johanson, C.E. Individual differences in the

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16:451-460, 1989.

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properties of benzodiazepines, barbiturates, and pharmacologically related

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37:113-123, 1976.

de la Garza, R.; Evans, S.; and Johanson, C.E. Discriminative stimulus

properties of oxazepam in the pigeon. Life Sci 40:71-79, 1987.

de la Garza, R., and Johanson, C.E. The discriminative stimulus properties of

intragastrically administered d-amphetamine and pentobarbital in rhesus

monkeys. Journal of Pharmacology 243:955-962, 1987.

Evans, S.M., and Johanson, C.E. The discriminative stimulus properties of

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of Docs., U.S. Govt. Print. Off., 1987. pp. 214-220.

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midazolam in the pigeon. J Pharmacol Exp Ther 248:29-38, 1989.

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18:73-80, 1983.

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Balster, R.L., eds. Transduction Mechanisms of Drug Stimuli.

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Spealman, R.D. Discriminative-stimulus effects of midazolam in squirrel

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Psychopharmacology 86:1-11, 1985.

AUTHOR

Chris-Ellyn Johanson

Department of Psychiatry

Uniformed Services University of Health Services

4301 Jones Bridge Road

Bethesda, MD 20814

196
Tolerance to Drugs Acting as Discriminative

Stimuli

Alice M. Young, Ph.D.

The initial effects of many drugs diminish during repeated administration of an

initially effective dose, so that larger doses become necessary to produce the

original effect, an outcome termed tolerance (Johnson and Fleming 1989). In

contrast, sensitivity to drugs acting as discriminative stimuli remains strikingly

consistent over extended training periods during which subjects receive regular

doses of their training drug. Whether or not this long-lasting sensitivity implies

that discriminative stimulus effects of drugs are insensitive to tolerance

processes is a topic of debate (reviewed by Colpaert 1978a; Young and

Sannerud 1989). The present paper reviews studies of whether tolerance

develops to the discriminative stimulus effects of drugs, focusing on studies of

whether the doses required for maintenance or generalization of stimulus

control can be altered by changes in the dose, frequency, duration, or type of

repeated drug treatment.

An individual’s initial sensitivity to the stimulus effects of a training drug is a

product of both the potency of the drug and the behavioral conditions under

which it is presented. The doses required for generalization of stimulus control

are determined jointly by the dose employed for training and the conditions of

differential reinforcement. Generally, subjects trained with higher doses of a

drug require higher doses for stimulus control than do subjects trained with

lower doses (Beardsley et al. 1987; Colpaert et al. 1980; Shannon and

Holtzman 1979; White and Appel 1982). For individual subjects, increasing or

decreasing the training dose produces a corresponding increase or decrease in

the dose required for stimulus control (Gauvin and Young 1989). Sensitivity to a

training drug is also modulated by prevailing reinforcement contingencies. For

example, the doses of a drug required for stimulus control can be changed by

arranging conditions so that the relative frequency of reinforcement following

injections of a drug differs from the frequency of reinforcement following

injections of saline (De Vry et al. 1984; Koek and Slangen 1982; McMillan and

197
Wenger 1984). Changes in sensitivity to a drug stimulus that result from such

changes in training dose or reinforcement contingencies are not viewed as

instances of tolerance.

Once established, stimulus control by a particular drug is generally maintained

over extended periods of training without increases in training dose.

Additionally, the range of doses required for generalization can be remarkably

consistent over repeated observations. For example, Colpaert and colleagues

(1978a, 1978b) reported that the range of doses of fentanyl or cocaine required

for discriminative control does not systematically increase or decrease over

extended training periods. In experiments with fentanyl, 2 weeks of daily

training with 0.04 mg/kg fentanyl and saline separated each of five test

sequences in which selected doses of fentanyl were tested on successive days.

In individual subjects, the doses of fentanyl or morphine required for

fentanyl-like stimulus effects oscillated by twofold to fourfold over successive

tests but did not progressively increase or decrease. Dose-response functions

were similar throughout the 17-week test sequence (Colpaert et al. 1976).

These results mirror the general observation that sensitivity to the stimulus

effects of drugs is maintained over extended periods of repeated drug exposure

without evidence of tolerance.

It may be, of course, that tolerance does develop during extended training

periods but is obscured as continued differential reinforcement acts to transfer

stimulus control to progressively lower doses (Hirschhorn and Rosecrans

1974). If so, the doses required for stimulus control would be expected to

decrease following a drug-free interval. However, experiments in which training

and its correlated drug exposure are halted for a time have shown few changes

in the range of doses required for stimulus control (McMillan 1987; Sannerud

and Young 1987). Although few drugs have been examined systematically after

drug holidays, the available data support the suggestion (Colpaert et al. 1976)

that the regular drug administration required by most training protocols does not

confer tolerance to drug stimulus control.

Tolerance can develop to the discriminative stimulus effects of drugs, however,

if the discriminative relationship between a drug stimulus and reinforced

behavior is suspended during repeated treatment with appropriate doses of the

training drug itself or related compounds. Under such procedures, stimulus

control by drug dose is first established with conventional training procedures,

initial dose-effect functions are obtained, and then training is discontinued while

a high dose of drug is administered repeatedly. Finally, during continued

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absence of training, dose-effect functions are redetermined in order to assess

changes in the doses required for stimulus control. Under such procedures,

tolerance, defined by an increase in the dose required for stimulus control, has

been reported for a wide variety of training stimuli, including d-amphetamine,

caffeine, cocaine, fentanyl, morphine, and nicotine, suggesting that

susceptibility to tolerance may be a common characteristic of

drug-discriminative stimuli. Such tolerance to drug-discriminative stimuli is

determined jointly by the conditions of repeated drug treatment, the training

dose, and the behavioral conditions imposed during repeated treatment (Young

1990; Young and Sannerud 1989). The following sections use our recent

studies of tolerance to the discriminative stimulus effects of morphine to

illustrate some of these characteristics of tolerance to drugs acting as

discriminative stimuli.

GENERAL METHODS

In all experiments, saline and a dose of 3.2 mg/kg morphine were established

as discriminative stimuli for food-reinforced behavior of rats, as described by

Young et al. (1990). Briefly, training sessions were divided into several discrete

trials, each consisting of a 15-min timeout (TO) component followed by a 5-min

ratio component. At the start of each trial, the rat was administered an injection

of saline or morphine and placed in a darkened chamber. At the end of the TO

component, stimulus lamps were illuminated and lever presses were reinforced

under a fixed ratio (FR) 15 schedule of food delivery. Following saline

administration, responses on the right lever produced food; following morphine

administration, responses on the left lever produced food. Each response on

the incorrect lever reset the ratio counter. A trial ended, and the chamber was

darkened, at the end of 5 min or after delivery of 50 pellets, whichever occurred

first. At the end of 5 min, the subject was removed and administered an

injection, and the next TO component was initiated. Training sessions varied in

the sequence of trials conducted, so that a similar number of drug and saline

training trials were conducted each week.

Training sessions were conducted daily until discriminative control was

established. Then, several tests of generalization to cumulative doses of

morphine were conducted in each subject in order to determine initial

sensitivity. During test sessions, saline or an increasing dose of morphine was

administered at the start of successive TO components, and completion of 15

consecutive responses on either lever produced food during each ratio

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component. At least three training sessions were conducted between

successive tests.

To examine development of tolerance, training sessions were halted, and each

rat was administered daily injections of morphine and returned to the home

cage. Several groups of subjects were tested, each exposed to a different dose

or frequency of morphine treatment. In order to study the development and loss

of tolerance, cumulative dose-response tests were conducted at various times

during repeated treatment and after treatment ended. Training was not resumed

until sensitivity to the stimulus and rate-aftering effects of morphine returned to

initial levels.

RESULTS

Development and Loss of Tolerance to Stimulus Effects of Morphine

As shown in figure 1 (left panels), repeated treatment with morphine produced a

dose-dependent tolerance to the stimulus effects of morphine, and magnitude

of tolerance increased with maintenance dose. Repeated treatment with daily

injections of 10 mg/kg or twice daily injections of 10 or 17.8 mg/kg increased

the dose of morphine required for stimulus control by twofold, fourfold or

fivefold, respectively (Sannerud and Young 1987; Young et al. 1990; cf.

Shannon and Holtzman 1976). Daily treatment with saline or the training dose

did not change the dose of morphine required for stimulus control,

demonstrating that the loss of sensitivity to the stimulus effects of morphine was

not the result of suspended training. Other experiments have shown that

treatment with daily doses of 20 or 110 mg/kg increases the dose required for

stimulus control by only twofold or fourfold in rats trained with higher doses of

5.6 or 10 mg/kg morphine (Miksic and Lal 1977; Young et al. 1990), suggesting

that the proportional relation between maintenance dose and training dose may

control the magnitude of tolerance. A similar proportional relation has been

reported for caffeine. Repeated treatment with 60 mg/kg/day increases the dose

of caffeine required for stimulus control by fourfold in rats trained with a dose of

10 mg/kg caffeine, but only by threefold in rats trained with a higher dose of 30

mg/kg (Holtzman 1987).

As shown in the right panel of figure 1, sensitivity to the stimulus effects of

morphine recovered within 3 to 5 days after termination of repeated treatment.

Because discrimination training did not resume until these tests were

completed, it appears that development of tolerance to the stimulus effects of

200
FIGURE 1. Dose-response functions for stimulus control by morphine in rats

before, during, and after repeated treatment with saline or

selected doses of morphine. Morphine (3.2 mg/kg) and saline

were established as discriminative stimuli for food-reinforced

performances in rats. Open circles represent the mean ± 1 SEM

for two to four tests conducted in each of five to nine subjects

before repeated treatment, and are replotted in both panels.

Closed circles represent the mean ± SEM for one observation in

each subject during the second week of treatment (left panel) and

3 to 5 days after treatment ended (right panel). Abscissae: dose of

morphine, fog scale. Ordinates: responses to morphineappropriate lever, as percentage of total session responses. (Data

replotted from Sannerud and Young 1987 and Young et al. 1990.)

201
morphine reflected a pharmawdynamic process rather than establishment of

control by a higher training dose (cf. Colpaert 1978b), inasmuch as the effects

of establishing a higher training dose would be expected to persist after the end

of treatment.

A direct relationship between maintenance dose and magnitude of tolerance

also has been reported for cocaine (Wood and Emmett-Oglesby 1986, 1987;

Wood et al. 1984, 1987). In one series of experiments, rats were trained to

discriminate saline and a dose of 10 mg/kg cocaine. Generalization functions

were determined before and after repeated treatment with doses of 5, 10, or 20

mg/kg cocaine administered three times a day for approximately 1 to 2 weeks.

The magnitude of tolerance to the stimulus effects of cocaine varied with

maintenance dose. Repeated treatment with a daily dose of 15 mg/kg did not

change the dose of cocaine required for generalization, whereas treatment with

daily doses of 30 and 60 mg/kg increased the dose required twofold. In other

subjects treated with 60 mg/kg, stimulus control by the original training dose

reappeared within 18 days after treatment ended without resumption of training,

again suggesting that the loss of sensitivity to the stimulus effects of cocaine

was not the result of shifting control to a higher training dose. The longer

recovery time for cocaine, as compared to morphine (figure 1), may reflect

differences in the physiological processes underlying altered sensitivity to the

drugs.

Time Course of Tolerance Development

The time wurse of tolerance development was examined by comparing

sensitivity to the stimulus effects of morphine at various times after initiation of

treatment with twice daily doses of 10 mg/kg morphine (figure 2, left panels).

Consistent with the findings of Shannon and Holtzman (1976), an

approximately twofold tolerance to the stimulus effects of morphine developed

within 3 days, The magnitude of tolerance increased when the duration of

treatment was extended to 1 week, but did not increase markedly after a

second week of treatment. Other studies showed that one or two acute

treatments with 10 mg/kg morphine do not alter sensitivity to the stimulus

effects of morphine 12 hr later (Sannerud and Young 1987; Young et al.

1991 b). Thus, it appears that tolerance to the stimulus effects of morphine

develops slowly, over a period of days. The gradual onset of tolerance with

repeated injections parallels that reported for continuously infused opioids.

Emmett-Oglesby et al. (1989) have shown that continuously infused fentanyl

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FIGURE 2. Time course of development of tolerance to stimulus effects of

morphine (left panel) or morphine-like stimulus effects of

methadone (right panel). Rats trained to discriminate 3.2 mg/kg

morphine and saline were treated for various times with twice

daily doses of 10 mg/kg morphine. Abscissae: dose of drug, log

scale. Ordinates: responses to morphine-appropriate lever as

percentage of total session responses. Open circles represent the

mean ± 1 SEM for three or four tests conducted in each of five or

six subjects before repeated treatment. Closed circles represent

the mean ± SEM for one observation in each subject during

repeated treatment. (Data replotted from Young et al. 1990,

Young et al. 1991a, and unpublished observations, 1990.)

203
evokes stimulus control within 8 hr of the onset of a continuous infusion, and

that tolerance to these effects begins to develop within 12 to 48 hr.

Cross-Tolerance

The tight panel of figure 2 illustrates cross-tolerance to the morphinelike

stimulus effects of a second mu agonist, dl-methadone. Repeated treatment for

3 days with twice daily injections of 10 mg/kg morphine did not markedly alter

stimulus control by methadone, but treatment for 1 or 2 weeks increased the

dose of methadone required for stimulus control by approximately twofold to

threefold. In agreement with an earlier report by Shannon and Holtzman (1976)

repeated morphine treatment evoked less tolerance to methadone than to

morphine itself. These differences in magnitude of tolerance may reflect

differences in agonist activity. The lesser tolerance to methadone is consistent

with reports that under certain conditions, repeated treatment with morphine

produces less tolerance to the rate-altering and stimulus effects of both l- and

dl-methadone than to similar effects of morphine (e.g., Craft et al. 1989;

McMillan et al. 1980; Shannon and Holtzman 1976), although similar tolerance

has also been reported (e.g., Leander et al. 1975). Such differences may result

from differences in agonist efficacy or drug disposition (Lange et al. 1983;

Werling et al. 1988; Young et al. 1991a).

Tolerance and cross-tolerance to the stimulus effects of opioids appear

pharmacologically specific. Tolerance develops during repeated treatment with

appropriate doses of morphine or fentanyl but not during treatment with doses

of pentobarbital sufficient to induce tolerance to the hypnotic effects of

pentobarbital itself (Emmett-Oglesby et al. 1988; Miksic and Lal 1977; Shannon

and Holtzman 1976; Young et al. 1990). Moreover, repeated treatment with

doses of morphine sufficient to induce tolerance to the stimulus effects of

morphine itself does not induce cross-tolerance to the stimulus effects of

cocaine (Wood and Emmett-Oglesby 1986).

Tolerance to the stimulus effects of cocaine is also pharmacologically specific,

developing during repeated treatment with appropriate doses of amphetamine,

apomorphine, or cocaine, but not during treatment with morphine (Wood and

Emmett-Oglesby 1986, 1987). Repeated treatment with d-amphetamine also

produces cross-tolerance to the cocaine-like stimulus effects of amphetamine,

and treatment with cocaine produces cross-tolerance to the cocaine-like

stimulus effects of methylphenidate, phenmetrazine, and phentermine (Wood

and Emmett-Oglesby 1986, 1988). Interestingly, cocaine treatment produces an

204
insurmountable tolerance to the cocaine-like stimulus effects of apomorphine

and diethylpropion (Wood and Emmett-Oglesby 1987, 1988). Further

characterization of such cross-tolerance should provide useful information

about suggested commonalities in the neuronal systems mediating the

cocaine-like stimulus effects of these drugs. The patterns of cross-tolerance

between caffeine and methylphenidate also suggest a common neuronal

system (Holtzman 1987). Methylphenidate evokes generalization to a caffeine

training stimulus, and repeated treatment with either drug confers tolerance to

the caffeine-like stimulus effects of the other, supporting suggestions of

catecholaminergic involvement in the stimulus effects of caffeine.

Modulation of Tolerance by Behavioral Processes

The magnitude of tolerance to the stimulus effects of a drug can be diminished

by continuing discrimination training during the period of repeated drug

treatment. This effect was demonstrated in experiments that assessed whether

continuing training during repeated drug treatment would modulate the

development of tolerance to the stimulus effects of morphine (Sannerud and

Young 1987). Rats were trained as described above, and sensitivity to

morphine was assessed before and after repeated treatment with twice daily

injections of 17.8 mg/kg morphine. In one condition, training was suspended

during repeated treatment. In a second condition, training was continued during

treatment. When training was suspended, repeated treatment increased the

dose of morphine required for stimulus control to 10 mg/kg in seven of the nine

subjects (figure 3, upper right panel). However, in agreement with earlier

studies (Colpaert et al. 1978c; Hirschhom and Rosecrans 1974), tolerance was

diminished when discrimination training was continued throughout repeated

treatment (lower right panel). When training was continued, the training dose of

3.2 mg/kg continued to evoke stimulus control in three subjects, and 5.6 mg/kg

evoked control in the remaining six.

A similar pattern was observed when the experiment was repeated using a

lower treatment dose of morphine (figure 3, left panels; Young et al. 1990; A.M.

Young et al. unpublished observations 1990). Repeated treatment with twice

daily injections of 10 mg/kg morphine produced a fourfold tolerance to the

stimulus effects of morphine when training was suspended during treatment

(upper left panel) but no tolerance when discrimination training sessions were

continued throughout treatment (lower left panel). Although they have yet to be

replicated with drugs from other pharmacological classes, these results suggest

that conditioning and pharmacodynamic processes interact to regulate

205
FIGURE 3. Modulation of tolerance to the stimulus effects of morphine by

exposure to discrimination training sessions. Morphine (3.2

mg/kg) and saline were established as discriminative stimuli for

food-reinforced performances in rats. Open circles represent the

mean ± 1 SEM for two to four tests conducted in each of five to

nine subjects before repeated treatment. Closed circles represent

the mean ± SEM for one observation in each subject after 2

weeks of repeated treatment with morphine. Training was

suspended during treatment in the experiments summarized in

the upper panels. Training with saline and 3.2 mg/kg morphine

was continued during treatment in the experiments summarized in

the tower panels. See figure 1 for other details. (Data reploted

from Sannerud and Young 1987, and Young et al. 1990 and

unpublished observations, 1990.)

206
sensitivity to drug stimulus control. When an individual regularly encounters a

discriminative relation between a drug and opportunities for reinforcement,

development of tolerance to the drug stimulus is minimized. Tolerance does

develop, however, if the individual simultaneously encounters frequent high

doses of a drug and behavioral contingencies that limit opportunities for

discriminative learning.

SUMMARY AND CONCLUSIONS

The experiments described above highlight the ways behavioral and

pharmacodynamic processes interact to modulate the development of tolerance

to the discriminative stimulus effects of drugs. These studies suggest that

frequent drug exposure does not lead inevitably to the development of tolerance

to a drug’s discriminative effects. Rather, the interplay between a drug stimulus

and reinforcement opportunities shapes the sensitivity of discriminative

performances over successive episodes of drug exposure.

Maintaining a discriminative relation between a drug and behavior strengthens

the likelihood that an initially effective dose will maintain discriminative control.

Development of tolerance requires exposure to both treatment regimens

appropriate to the agent under study and behavioral contingencies that limit an

individual’s ability to learn a new discrimination. When both requirements are

met, tolerance does develop to drugs acting as discriminative stimuli.

When training is suspended during a period of chronic drug treatment, the dose

of drug required to evoke stimulus control can be increased by treatment with

appropriate maintenance doses of the training drug or a closely related drug.

Tolerance is proportional to maintenance dose, develops relatively slowly, and

disappears after termination of repeated drug treatment. Tolerance appears

pharmacologically specific and can be accompanied by cross-tolerance to other

drugs that evoke cross-generalization with the training drug. Finally, tolerance

can be diminished markedly by continuing training with the original training

dose. Taken together, these patterns suggest that development of tolerance to

drugs acting as discriminative stimuli is the result of joint actions of conditioning

and pharmacodynamic processes.

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ACKNOWLEDGMENTS

Preparation of this chapter was supported in part by USPHS grant DA03796

from the National Institute on Drug Abuse to A.M. Young, who is the recipient of

NIDA Research Scientist Development Award K02 DA00132. Malath M.

Makhay and Coni Burke provided excellent technical support for studies of

tolerance to drug stimulus control. J.B. Smith provided critical comments on a

prior version of the manuscript.

210
AUTHOR

Alice M. Young, Ph.D.

Professor

Department of Psychology

71 West Warren Avenue

Wayne State University

Detroit, Ml 48202

211

Tolerance: Role of Conditioning Processes

Shepard Siegel

The display of tolerance is greatly affected by cues present at the time of drug

administration, and a complete account of tolerance must acknowledge the role

of such cues. Demonstrations of the importance of such predrug cues have

inspired accounts of tolerance that emphasize learning principles. We will first

review the evidence that predrug cues modulate tolerance, and then discuss

analyses of tolerance that have been presented to account for these findings.

ENVIRONMENTAL CUES AND TOLERANCE

Tolerance to a variety of effects of many drugs is environmentally specific. That

is, tolerance to the last of a series of drug administrations is more pronounced if

this final administration occurs in the same environment as the prior drug

administrations.

Demonstrations of Environmental Specificity of Tolerance

The details of the designs of experiments demonstrating environmental

specificity of tolerance differ, but all incorporate two groups of subjects, both

receiving the drug a sufficient number of times for tolerance to develop during

the initial, tolerance-development phase of the experiment. The effect of the

drug is evaluated in a subsequent tolerance-test phase. For one of the two

groups, this test is conducted following the same cues that signaled the drug

during the tolerance-development phase (same-tested). For the second group,

the tolerance test is conducted following cues other than those that signaled the

drug during the tolerance-development phase (different-tested).

In most experiments concerned with environmental specificity of tolerance,

subjects have equal experience with both environments prior to the tolerance

test; thus, it is not the case that one of the environments is more novel or

stressful than the other. One frequently used design has been termed

“discriminative control of tolerance” (Siegel 1979). During the tolerance213
development phase of such an experiment, all subjects receive a number of

injections of the drug and a number of injections of physiological saline.

Different environmental cues are associated with each substance, such that all

drug injections take place in one environment (ED), and all saline injections take

place in a different environment (ES). Thus, on some days the subject is

injected with the drug in ED, and on other days it is injected with saline in Es.

Finally, the effect of the drug is tested for all subjects, with same-tested

subjects receiving this test in ED, and different-tested subjects receiving this test

in ES.

In addition to including same- and different-tested groups, the design of

tolerance environmental-specificity studies typically includes a third group. This

control group permits evaluation of the drug-elicited response in subjects

receiving the drug for the first time. Results obtained during the tolerance test in

a number of experiments using this procedure are summarized in figure 1.

Figure 1A summarizes results reported by Crowell et al. (1981) in their study of

tolerance to the hypothermic effect of ethanol. The figure depicts the change in

colonic temperature (postinjection minus preinjection) following a test-session

injection of 1.3 g/kg ethanol. Same-tested rats (“SAME”) received 20 pretest

injections of ethanol, each in the same environment as that accompanying the

test injection. Different-tested rats (“DIFF”) also received 20 pretest injections of

the drug, but in a distinctively different environment. Control rats (“CONTL”)

received their first injections of ethanol on the test session. As can be seen by

comparing control rats with same-tested rats, tolerance to the hypothermic

effect of the drug was apparent: control rats were hypothermic, but same-tested

rats were not. However, results obtained from different-tested rats indicate that

hypothermic tolerance is not the inevitable result of repeated ethanol

administrations. Different-tested rats had the same pharmacological history as

same-tested rats (i.e., they received the same dose of ethanol, equally often,

and at the same intervals), but different-tested rats were as hypothermic as

control rats. There are other reports that tolerance to the thermic effects of

ethanol in rats exhibits environmental specificity. In addition, environmental

specificity of tolerance has also been demonstrated with respect to both the

narcotizing effect of ethanol in mice and the cardiac and cognitive-performance

effects of ethanol in humans (see summary by Siegel 1987).

The environmental specificity of tolerance has been demonstrated with many

other drugs. Figures 1B through 1E summarize the results of other experiments

that have demonstrated that the display of tolerance is more pronounced in

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FIGURE 1. Summary of results of experiments demonstrating environmental specificity of tolerance to:

(A) the hypothermic effect of ethanol in rats (Crowell et al. 1981), (B) the sedative effect of

pentobarbital in rats (Hinson et al. 1982), (C) the cataleptic effect of haloperidol in rats

(Poulos and Hinson 1982), (D) the ambulatory activity-suppressive effects of midazolam in

rats (King et al. 1987), and (E) the analgesic effect of morphine in snails (Kavaliers and Hirst

1986).
same-tested than in different-tested subjects. Figure 1B summarizes results

reported by Hinson et al. (1982) concerning tolerance to the sedative effect of

pentobarbital. During the tolerance-acquisition phase of this experiment, rats

were injected with gradually increasing doses of the barbiturate (30-45 mg/kg).

On the tolerance-test session, following injection of 45 mg/kg of the drug, each

subject’s “sleeping time” was scored from videotape records. As can be seen,

tolerance to the sedative effect of pentobarbital (figure 1B), like tolerance to the

hypothermic effect of ethanol (figure 1A), displays environmental specificity.

Same-tested rats were less sedated by the drug (displayed shorter sleeping

times) than equally drug-experienced different-tested rats. Indeed,

different-tested rats were as behaviorally narcotized as control rats that

received the drug for the first time on the tolerance-test session. This

experiment by Hinson et al. (1982) confirmed a previous report of

environmental specificity of pentobarbital tolerance (Cappell et al. 1981).

Environmental specificity of tolerance is found not only with sedatives, such as

ethanol and pentobarbital, but also with many other types of drugs. Figure 1C

summarizes results reported by Poulos and Hinson (1982) concerning tolerance

to the cataleptic effect of the neuroleptic drug haloperidol. Catalepsy was

measured with a standard bar-hanging procedure; both the rat’s paws were

placed on a bar, and the amount of time that the animal remained hanging was

scored. During both the tolerance-acquisition and tolerance-test phases of the

experiment, the dose of haloperidol was 3 mg/kg. Tolerance to the cataleptic

effect of the drug was seen in same-tested rats but not in different-tested rats.

As can be seen in figure 1C, these different-tested rats were about as cataleptic

as control animals.

Environmental specificity of tolerance has also been demonstrated with respect

to a variety of benzodiazepines (Siegel 1986). Figure 1D summarizes data

reported by King et al. (1987) concerning tolerance to the ambulatory

activity-suppressive effects of the short-acting benzodiazepine midazolam.

Same-tested rats were significantly more active on the tolerance-test session

than were different-tested or control rats.

Although environmental specificity of tolerance has been demonstrated with

many effects of a variety of drugs, most experiments concerning this

phenomenon have evaluated tolerance to the analgesic effect of morphine.

Environmental specificity of such tolerance has been demonstrated with many

analgesia-assessment procedures, and in a variety of species, including

humans (Siegel and MacRae 1984). The generality of the phenomenon is

216
illustrated in figure 1E, which summarizes data reported by Kavaliers and Hirst

(1986) concerning morphine tolerance in the terrestrial gastropod snail Capaea

nemoralis. During each tolerance-development session, snails were injected

with 1 μg/kg morphine. Analgesia was assessed by measuring the latency of

the “foot”-lifting response when the snails were placed on a 38.5° surface. As

apparent in figure 1 E, same-tested snails responded more quickly to the

thermal stimulation (i.e., were more tolerant to the analgesic effect of morphine)

than were different-tested snails. The fact that environmental specificity of

tolerance can be seen even in invertebrates suggests that such specificity “may

be a general phenomenon having an early evolutionary development and broad

phylogenetic continuity” (Kavaliers and Hirst 1986, p. 1201).

Environmental Specificity of Tolerance to the Lethal Effect of Drugs

Perhaps the most dramatic evidence for environmental control of tolerance

comes from demonstrations that tolerance to the lethal effect of some drugs

demonstrates environmental specificity. The results of experiments

demonstrating such environmental modulation of drug-induced mortality are

summarized in figure 2.

The data summarized in figure 2A demonstrate that environmental cues

modulate tolerance to the lethal effect of ethanol (Melchior and Tabakoff 1982).

In this experiment, mice were injected with 3.5 g/kg ethanol twice daily for 4

days. On the 5th day different subgroups were injected with 4.5-7.0 g/kg

ethanol in either the drug-associated or a novel environment. The LD50 dose of

the drug was determined. As can be seen in figure 2A, the LD50 dose is

smallest for control mice, who had no pretest exposure to ethanol, but the

effects of environmental cues are still evident: The LD50 dose is significantly

lower for different-tested than for same-tested mice.

Figure 2B summarizes data reported by Siegel et al. (1982) indicating

environmental control of tolerance to the lethal effect of heroin. In this

experiment, rats prepared with chronically implanted intravenous cannulae

received a series of heroin infusions (with the dose gradually increased from 1

to 8 mg/kg). Finally, on the tolerance-test session, these rats were administered

15 mg/kg of the opiate in either the same environment as that previously

associated with the drug or an alternative environment. As can be seen in figure

2B, both these groups with pretest experience with sublethal doses of heroin

were more likely to survive than control animals, which received heroin for the

first time on the tolerance-test session. These results indicate that tolerance

217
FIGURE 2. Summary of results of experiments demonstrating environmental specificity of tolerance to

the lethal effect of: (A) ethanol (Melchior and Tabakoff 1982). (B) heroin (Siegel et al. 1982).

and (C) pentobarbital (Vila 1989).
resulted from pretest injections independently of the environment associated

with these injections. However, mortality was significantly higher in

different-tested than same-tested rats, indicating, once again, that identical

pretest pharmacological histories do not necessarily result in equivalent

tolerance to the lethal effect of heroin. Case report data suggest that

environmental cues similarly modulate tolerance to the lethal effect of heroin in

human heroin addicts (Siegel 1984) and in patients receiving medically

prescribed morphine (Siegel and Ellsworth 1986).

Figure 2C depicts the results of an experiment by Vila (1989). The design of this

experiment concerning pentobarbital overdose was similar to that of Siegel et

al. (1982) concerning heroin overdose. Experimental group rats were

intraperitoneally injected with pentobarbital (30 mg/kg) in one environment and

saline in another. After 20 administrations of each substance, the rats were

injected with a high dose of the barbiturate (95 mg/kg) in either the

drug-associated (same-tested) or saline-associated (different-tested)

environment. Control rats had no prior exposure to pentobarbiial priir to

injection of the large dose on the test session. As is apparent in figure 2C,

environmental control of tolerance to the lethal effect of the drug was complete;

drug-experienced rats challenged with the drug in the context of the usual drug

cues displayed lower mortality than drug-experienced rats challenged with the

drug in the context of alternative cues. Indeed, in the latter case there was no

evidence of tolerance-mortality among different-tested rats was near that of

control rats who received pentobarbital for the first time on the final test session.

Summary of Research Demonstrating Environmental Specificity of Tolerance

Many studies have demonstrated environmental specificity of tolerance. Such

environmental specificity occurs with respect to tolerance to a variety of effects

(including the lethal effect) of many drugs. It should be noted that often such

environmental specificity is not absolute, i.e., some tolerance is noted in

different-tested subjects compared to control subjects receiving the drug for the

first time. Furthermore, although environmental specificity of tolerance is a very

general observation, there are occasional reports to the contrary. For example,

as summarized above (figure 1D), King et al. (1987) reported clear

environmental specificity of tolerance to the sedative effect of midazolam;

however, Griffiths and Goudie (1986) reported no environmental specificity of

tolerance to this drug’s hypothermic effect (see also Siegel 1989). However, the

general finding, obtained with many drugs, dosages, species, and procedural

variations, is that tolerance is more pronounced when the drug is given in the

219
usual drug administration environment than when it is given elsewhere.

Speaking casually, tolerance is greater when a drug is expected than when it is

unexpected.

PAVLOVIAN CONDITIONING AND TOLERANCE

To incorporate the contribution of drug expectation to drug tolerance, several

investigators have suggested that learning contributes to tolerance. That is,

tolerance is due, in part, to an association between drug-predictive cues and

the systemic effect of the drug. The study of such associations is the study of

Pavlovian conditioning.

The Pavlovian Conditioning Situation

In the Pavlovian conditioning situation, a contingency is arranged between two

stimuli; typically, one stimulus reliably predicts the occurrence of the second

stimulus. The second of these paired stimuli is usually termed the unconditional

stimulus (UCS). The UCS, as the name implies, is selected because it elicits

relevant activities from the outset (i.e., unconditionally), prior to any pairings.

Responses elicited by the UCS are termed unconditional responses (UCRs).

The stimulus signaling the presentation of the UCS is “neutral,” (i.e., it elicits

little relevant activity prior to its pairing with the UCS), and is termed the

conditional stimulus (CS). The CS, as the name implies, becomes capable of

eliciting new responses as a function of (i.e., conditional upon) its pairing with

the unconditional stimulus.

Drugs as Unconditional Stimuli

A wide range of exteroceptive and interoceptive stimuli have been used in

Pavlovian conditioning experiments. Drugs constitute a particularly interesting

class of UCSs. After some number of drug administrations, each administration

reliably signaled by a CS, pharmacological conditional responses (CRs) can be

observed in response to the CS.

The Pharmacological Conditional Response. Most pharmacological

conditioning research has been greatly influenced by Pavlov’s theory of CR

formation. According to this theory, the CR is a replica of the UCR, and, indeed,

much drug conditioning work has demonstrated CRs that mimic the drug effect

(Stewart and Eikelboom 1987). In contrast, in 1937 Subkov and Zilov reported

that dogs with a history of epinephrine administration (each injection eliciting a

220
tachycardiac response) displayed a conditional bradycardiac response. The

authors cautioned against “the widely accepted view that the external

modifications of the conditional reflex must always be identical with the

response of the organism to the unconditional stimulus” (Subkov and Zilov

1937, p. 296). Subsequent research has suggested that the characteristics of

the pharmacological CR depend very much on the nature and mechanism of

the drug effect (Eikelboom and Stewart 1982; Siegel 1989). For many effects of

many drugs, the CR is an anticipatory compensation for the drug effect. For

example, the subject with a history of morphine administration (and its

analgesic consequence) often displays a CR of hyperalgesia (Krank 1987;

Krank et al. 1981; Siegel 1975). Similar compensatory CRs have been reported

with respect to the thermic, locomotor, behaviorally sedating, and

gastrointestinal effects of morphine. The CR seen with many nonopiate drugs is

similarly opposite to the drug effect, e.g., atropine, chlorpromazine,

amphetamine, methyldopa, lithium chloride, haloperidol, ethanol, caffeine, and

several immunostimulatory and immunosuppressive drugs (MacQueen et al. in

press; Siegel 1989).

The Pharmacological Conditional Response and Drug Tolerance.

Drug-compensatory CRs would be expected to be a feature of normal drug

administration procedures. In those cases in which the same drug is repeatedly

administered, with discrete environmental stimuli signaling each drug

administration, drug-compensatory CRs should function to increasingly

attenuate the drug effect. A decreasing response to a drug over the wurse of

successive administrations defines tolerance.

Pharmacological Conditioning and Tolerance’s Environmental

Specificity. The observation that there often is pronounced environmental

specificity to the display of tolerance is readily interpretable by an analysis of

tolerance that incorporates Pavlovian conditioning principles. If the repeatedly

drugged organism is administered the drug in the context of normal predrug

cues, the compensatory CR partially cancels the drug effect; thus, tolerance is

apparent. However, if this organism is administered the drug in the context of

cues not previously associated with the drug, there would be no CR attenuating

the drug effect, and tolerance attributable to such a CR would not be observed.

Other Conditlonlng Accounts of Tolerance. A substantial amount of data

have been presented supporting the compensatory-CR analysis of tolerance.

There are also data that have been interpreted as contrary to this analysis

(Siegel 1989), and some investigators have attempted to develop theories that

221
will accommodate evidence that tolerance is clearly affected by drug-associated

cues, yet not necessarily through a compensatory-CR mechanism. These

explanations are known as “habituation” theories of tolerance. One such

alternative associative framework for tolerance, based on Wagner’s information

processing theory of memory (e.g., Wagner 1976), has been suggested by

Siegel (1977) and elaborated by Baker and Tiffany (1985). According to this

view, drug-associated environmental cues “prime” the drug effect in short-term

memory, causing a decrease in the effectiveness of processing of the

pharmacological stimulation.

Although there are some important theoretical distinctions between the two

associative accounts of tolerance (Siegel 1989), both make many similar

predictions. For example, both predict that an alteration in predrug cues should

attenuate the display of tolerance in drug-experienced subjects.

OTHER SIGNALS FOR DRUGS

There are potentially many cues, in addition to the environment of drug

administration, that can signal the effect of a drug, and there is evidence that a

variety of drug-associated stimuli can influence the display of tolerance.

Magnetic Fields as Signals for Drugs

Results of an experiment by Kavaliers and Ossenkopp (1985) suggest that

quite subtle cues, present at the time of drug administration, may contribute to

the development of tolerance. In this experiment, mice received 10 daily

injections of morphine, either in the presence or absence of rotating magnetic

fields (2-35 gausses). Analgesia was assessed following each of these pretest

drug administrations. Subjects were then tested for analgesic tolerance in both

the presence and the absence of magnetic stimuli.

The results of this experiment indicated that magnetic fields per se affect the

development of tolerance. During the pretest phase of the experiment, mice

repeatedly administered morphine in conjunction with magnetic field exposure

were relatively retarded in the acquisition of tolerance to the analgesic effect of

morphine. In addition, the field also functioned as an effective cue for the drug.

In the test session, tolerance was more pronounced following drug

administration in the presence of the same magnetic stimuli (either presence or

absence of field) that prevailed during the pretest drug administrations than

following drug administration in the presence of the alternative magnetic stimuli.

222
According to by Kavaliers and Ossenkopp (1985), their resub extend the

conditioning analysis of tolerance. The finding that magnetic field exposure

during pretest sessions attenuated the acquisition of tolerance is congenial with

suggestions that such exposure detrimentally affects the acquisition of learned

responses (Kavaliers and Ossenkopp 1985). The further finding that magnetic

fields can become associated with morphine and influence the display of

tolerance suggests that magnetic stimuli may play a heretofore unappreciated

role in the development of tolerance. The authors suggest that their results may

be relevant to reported circadian differences in morphine effects in mice.

Thermal Signals for a Drug

As discussed previously, Kavaliers and Hirst (1986) demonstrated

environmental specificity of tolerance in the terrestrial snail Capaea nemoralis.

These investigators also demonstrated that thermal stimuli can serve as

drug-associated cues that control the display of tolerance in this species. Snails

were repeatedly injected with morphine in the presence of one of two thermal

cues: either ambient temperature (22%) or a higher temperature (35°C). In a

test session, tolerance to the analgesic effect of morphine was seen when

snails were injected in the presence of the thermal cue previously associated

with the drug, but not when they were injected in the presence of the alternative

thermal cue.

According to Kavaliers and Hirst (1986) the higher temperature used in their

experiment is normally avoided by morphs of Capaea; thus, it is unclear

whether the effective morphine-associated signal was the temperature per se,

or the stress induced by this thermal stimulation. in any event, the results of this

experiment further suggest that even in the snail, a variety of stimuli can

become associated with a drug and contribute to the development of tolerance.

Pharmacological Signals for Drugs

Another category of predrug signals that contribute to tolerance are

pharmacological cues. That is, if a given drug (D1) is repeatedly administered

before a second drug (D2), a pharmacological CR is elicited by D1. Such

drug-drug associations may contribute importantly to the development of

tolerance (Krank and Bennett 1987; Taukulis 1986). In fact, because the early

effect of a drug almost invariably signals a later effect, responses made to

pharmacological cues may be a very common feature of tolerance.

223
Pharmacological Signaling of One Drug by Another Drug. Taukulis (1986)

demonstrated that a drug can serve as a signal for another drug and control the

expression of tolerance to the signaled drug. In his experiment, atropine sulfate

was routinely injected prior to pentobarbital injection. Tolerance to the

hypothermic effect of the barbiturate was much more pronounced when it was

preceded by atropine than when it was presented without the signal provided by

atropine. That is, same-tested rats (receiving the barbiturate subsequent to the

usual signal provided by the antichoiinergic) were more tolerant than

different-tested rats (receiving the barbiturate in the absence of the usual signal

provided by the anticholinergic).

Results of recent research concerning the effects of pentobarbital on morphine

tolerance are readily interpretable as a result of an association between a

pharmacological signal (generated by the interoceptive effects of the

barbiturate) and morphine. Terman and colleagues (1983, 1985) reported that

rats with a history of morphine administration, administered pentobarbital prior

to a final injection of morphine, do not display the analgesic tolerance seen in

nonanesthetized rats. That is, the barbiturate apparently blocks morphine

tolerance. Some interpretations of pentobarbital blockage of morphine tolerance

have postulated direct pharmawdynamic interaction between the barbiturate

and the opiate (Pontani et al. 1985). Results of a recent experiment, however,

indicate that “state-dependent learning,” rather than pharmacodynamic

interaction, best accounts for such barbiturate-opiate effects (Siegel 1988).

There is a considerable amount of evidence that drug states in general, and the

state generated by barbiturates in particular, can serve as salient stimuli (see

Järbe 1986). That is, learned responses acquired when the subject is not under

the influence of a centrally acting drug, such as pentobarbital, may fail to be

displayed subsequently when the subject is tested while under the influence of

this drug. To the extent that tolerance to the analgesic effect of morphine is

mediated by learning, it might be expected that tolerance will display such drugstate dependency-pharmacological cues, such as those generated by

pentobarbital, may function very much like environmental cues in affecting the

display of tolerance. in other words, just as there is environmental specificity of

morphine tolerance (because of associations between morphine-signaling

environmental cues and the opiate), there might also be state specificity of

morphine tolerance (because of associations between morphine-signaling

pharmacological cues and the opiate).

224
Siegel (1988) confirmed the finding that pentobarbital interferes with the

expression of morphine tolerance in rats that had not previously received

barbiturate-opiate pairings. Additionally, the results supported the

state-dependency interpretation of this interference.

Pharmacological Signaling of a Drug by itself. A drug may serve not only

as a cue for another drug but also as a cue for itself, and this association may

contribute to tolerance (Greeley et al. 1984). In this Greeiey et al. (1984) study,

rats in one group (paired) consistently received a low dose of ethanol (0.8 g/kg)

60 minutes prior to receiving a high dose (2.5 g/kg). Another group of rats

(unpaired) received the low and high doses on an unpaired basis. When tested

for the tolerance to the hypothermic effect of ethanol, paired subjects, but not

unpaired subjects, displayed tolerance. Moreover, if the high dose of ethanol

was not preceded by the low dose, paired rats failed to display their usual

tolerance. This tolerance, dependent on an ethanol-ethanol pairing, was

apparently mediated by an ethanol-compensatory thermic CR; paired rats, but

not unpaired rats, demonstrated a hyperthermic CR (opposite to the

hypothermic effect of the drug) in response to the low dose of ethanol.

Results of this study provide convincing evidence that a small dose of a drug

can serve as a signal for a larger dose of the same drug. Because a gradual

increase in systemic concentration is an inevitable consequence of most drug

administration procedures, such drug-drug associations may play a hitherto

unappreciated role in the effects of repeated drug administrations.

Cues Accompanying Self-Administration

The Pavlovian conditioning analysis emphasizes the contribution of predrug

cues to the development of drug tolerance and dependence. In the discussion

thus far, these cues have been conceptualized as environmental (i.e., the

physical location of drug administration), pharmacological (i.e., one drug

signaling another, or the early effect of a drug signaling the later effect), or other

detectable stimuli (magnetic fields). Often, of course, drugs are

self-administered. It might be expected that interoceptive cues accompanying

self-administration (e.g., cognitive-volitional or proprioceptive signals for the

systemic effect of the drug) similarly contribute to the effects of repeated

pharmacological stimulation.

Self-administration cues have been evaluated in experiments that compare the

effects of drugs in animals that self-administer the drug with effects in animals

225
yoked to these self-administering animals. Typically, the self-administering

subject is prepared with a chronic jugular cannula, allowing for repeated

intravenous injection. The subject can self-inject an opiate by pressing a lever

in an experimental chamber. Yoked animals are similarly cannulated, and

placed in a similar chamber, but lever presses have no consequence. Rather,

the yoked subject receives the drug at the same time as the self-administering

subject. Thus, yoked animals have no control over drug delivery, but rather

receive the same doses of the drug, equally often, and est session, tolerance

was more pronounced following drug administration in the presence of the

same magnetic stimuli (either presence or absence of field) that prevailed

during the pretest drug administrations than following drug administration in the

presence of the alternative magnetic stimuli.

According to by Kavaliers and Ossenkopp (1985), their results extend the

conditioning analysis of tolerance. The finding that magnetic field exposure

during pretest sessions attenuated the acquisition of tolerance administration

are the most obvious type of predrug signals, there are many other potential

cues for the effect of a drug. Some of these are quite subtle yet are important

contributors to the display of tolerance. Thus, the CR that mediates tolerance

may be elicited by any of a variety of exteroceptive or interoceptive stimuli

routinely present at the time of drug administration.

OTHER EVIDENCE FOR THE CONDITIONING ANALYSIS OF TOLERANCE

The fact that any of a variety of drug-signaling stimuli contribute to the display of

tolerance to many effects of many different drugs provides the strongest

evidence for a conditioning account of tolerance. There are, in addition, many

other findings that implicate conditioning in tolerance. Generally, these

additional findings demonstrate that nonpharmacological manipulations of

drug-predictive cues have similar effects on tolerance and CRs. Thus,

tolerance, like other learned responses, is subject to extinction, partial

reinforcement effects, inhibiiory learning, higher order conditioning, stimulus

generalization, disruption by novel stimuli (“external inhibition”), and compound

conditioning effects (“overshadowing” and “blocking”) (Siegel 1989).

In summary, although an appreciation of pharmacodynamic and

pharmacokinetic principles is of great importance in understanding drug

tolerance, a complete account of the phenomenon must acknowledge the

important contribution of conditioning principles.

226
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ACKNOWLEDGMENT

Research from the author’s laboratory summarized in this chapter was

supported by grants from the Natural Sciences and Engineering Research

Council of Canada.

AUTHOR

Shepard Siegel

Department of Psychology

McMaster University

Hamilton, Ontario

Canada L8S 4K1

229

Responding to Drug-Related Stimuli in

Humans as a Function of Drug-Use History

Ronald N. Ehrman

Steven J. Robbins

Anna Rose Childress

A. Thomas McLellan

Charles P. O’Brien

INTRODUCTION

In the last 30 years there has been much interest in the role played by classical

conditioning in the elicitation of physiological and subjective responses by

drug-related stimuli. It has been suggested that environmental events that

signal drug use elicit conditioned responses that influence the drug-seeking and

self-administration behavior of human drug abusers. Clinically, these

conditioned responses are thought to play an important role in the relapse

process.

Figure 1 shows how a conditioning model applies to substance abuse.

The conditioned stimulus, a drug-related event such as the sight of a “shooting

gallery,” elicits a drug-related conditioned response. Different theoretical models

(e.g., Siegel 1979; Stewart et al. 1984; Wikler 1965) hypothesize that this

response can be either drug-like or drug-opposite in nature. Regardless of its

form, this conditioned response is thought to motivate the individual to perform

instrumental responses such as seeking out and self-administering the drug.

The reinforcer or reward in this paradigm is the effect of the drug.

The purpose of this paper is to examine whether the responsivity brought about

by drug-related stimuli in human drug abusers is the product of classical

conditioning. The focus will be on data collected by our group at the

Philadelphia Veterans Affairs Medical Center (VAMC)/University of

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CS

(Shooting Gallery)

CR

(Conditioned Withdrawal-Like / Craving State)

Instrumental Response

(Drug Seeking & Self-Administration)

Reward

(Drug Effect)

FIGURE 1. Hypothesized role of conditioning processes in human drug use.

Pennsylvania Addiction Research Center in studies with patients who abuse

opiates and cocaine.

We have used two approaches to study conditioning in human drug abusers.

Cur first experiments attempted to establish conditioned responses in the

laboratory using stimuli such as tones and flavors that bear no intrinsic relation

to drug use (e.g., O’Brien et al. 1977; Ternes et al. 1982). We quickly realized,

however, that such studies do not shed much light on conditioning outside the

laboratory. Although robust conditioning might occur under the carefully

controlled conditions used in the lab, addicts are probably not exposed to ideal

conditioning parameters in their natural environments.

Because of the artificial nature of laboratory conditioning studies, our recent

work has looked at responding to cues that are assumed to be associated with

drug use in the abusers’ natural environment. These studies have been referred

to as naturalistic stimulus studies. The stimuli used in these experiments

232
consist of some combination of audiotapes, videotapes, and manual tasks with

either drug- or nondrug-related content. A number of designs utilizing this basic

procedure are possible.

TWO STIMULUS-ONE GROUP DESIGN

The most common design employing naturalistic stimuli exposes a single

homogeneous group of drug-abusing subjects to both drug and nondrug stimuli.

The studies discussed here examined either opiate abusers or cocaine

abusers. In our two stimulus-one group design, stimulus sessions contained a

videotape and a manual task. During drug-stimulus sessions, the videotape

depicts a typical scene of drug use. For example, a heroin injector would view a

tape in which a drug user is shown buying, preparing, and actually injecting a

substance resembling heroin. The task in such a session would require the user

to perform his usual drug preparation ritual but stop prior to the actual injection.

Control stimuli are typically nonarousing and nondrug-related. In our

experiments, subjects watch a nature documentary and play the pong video

game. Typically, both drug-related and nondrug-related stimuli are presented in

a single experimental session.

A variety of physiological and self-report measures are taken both before and

after presentation of the stimuli. The purpose of such studies is to examine

whether drug-related stimuli produce greater changes in responding than do

nondrug-related stimuli compared with baseline levels.

In designing these studies, we have created a standard set of stimuli for each

type of drug use. Because these stimuli do not match perfectly with the

experience of any individual drug abuser, the level of responding observed may

actually underestimate responses to drug-related events in the addicts’ real

environments.

At the start of a session the subject is attached to a polygraph that is used to

continuously monitor his heart rate, galvanic skin response (GSR), and skin

temperature. He is then seated in the recording chamber for the remainder of

the session. The initial portion of the session is a 10- to 15-min baseline period

during which no events occur. Toward the end of the baseline perii, self-report

measures are taken on the subject’s state of high, craving, and withdrawal

relative to his drug of abuse. Next, the subject views a 10-min videotape and

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then performs a manual task. Following the completion of the task, self-report

measures are again obtained.

This sequence of baseline, videotape, and task is then repeated during the

remainder of the session. Half of the session consists of drug-related stimuli

and the other half to nondrug-related stimuli.

Changes from baseline scores for each physiological measure used in these

studies are analyzed. These scores are calculated by subtracting mean

measures of responding during the last 5 min of the baseline period from those

in the last 5 min of the video stimulus and the last 5 min of the task. In this way,

change scores are calculated for each stimulus modality for both the

drug-related and nondrug-related stimulus types.

Self-report scores in these studies consist of subjects’ ratings of high, craving,

and withdrawal on a scale of 1 to 10, where a score of 1 represents no effect

and a score of 10 represents a maximum possible effect. The change in these

ratings between the baseline and poststimulus periods are then analyzed.

Two diiferent kinds of analysis are performed on the change score data. First,

we examine whether there is a difference between change scores during drug

stimuli and change scores during nondrug stimuli. Second, we analyze whether

individual change scores differ reliably from zero. This second analysis shows

whether a signfficant change from baseline has occurred.

Responding to naturalistic stimuli has been assessed in our center using both

heroin and cocaine abusers (e.g., Childress et al. 1988; O’Brien et al. 1990).

The overall purpose of these studies was to evaluate various combinations of

substance abuse treatments. The data shown here represent the pretreatment

responses of the patients.

The three subject groups in this design were all inpatients at the Philadelphia

VA Hospital. The groups were composed of 89 methadone-maintained opiate

users, 15 drug-free opiate users, and 51 drug-free cocaine users. Table 1

depicts the results of these studies. The asterisks indicate cases where change

from baseline scores significantly differed between the drug and nondrug

stimulus presentations.

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TABLE 1. Significant differences in responding to drug-related and

nondrug-related stimuli in three populations

As shown in table 1, temperature and GSR are the most reliable physiological

measures; there was a significant difference on these two measures between

the drug- and nondrug-related stimuli for all three groups. Both skin temperature

and GSR significantly decreased relative to baseline in response to the

drug-related stimuli.

Craving is the most consistent subjective measure distinguishing the

drug-related and nondrug-related stimuli in each of the groups. Relative to

baseline, craving is significantly increased following the presentation of

drug-related stimuli. In the methadone-maintained and cocaine groups,

withdrawal is also significantly increased by the drug-related stimuli, as is the

self-reporf of high in the cocaine subjects. It may seem inconsistent that both

high and withdrawal scores increase in the cocaine patients. However,

inspection of the data reveal that individuals who show the greatest self-report

changes typically respond to only one of the questions.

Unfortunately, there is a general problem with interpreting the results of these

studies. Because the stimuli that are brought into the laboratory already elicit

responding, it is impossible to determine whether these responses represent

conditioned or unconditioned effects. For example, a typical video stimulus

shown to opiate users depicts an individual injecting himself with the drug. We

235
know from interviews with subjects in these studies that the scene showing the

actual injection is considered arousing or unpleasant per se. Consequently, it is

possible that the differential responding to drug-related and nondrug-related

stimuli represents an unconditioned response and is unrelated to learned

associations between the drug stimuli and actual drug use.

TWO GROUP-TWO STIMULUS DESIGN

A more elaborate naturalistic stimulus design would control for the

unconditioned arousing nature of the drug stimuli. For example, drug-related

and nondrug-related stimuli could be shown not just to drug users but also to

individuals lacking a drug history. Any difference in responding to the two types

of stimuli in the drug-naive group would then reflect unconditioned effects

because this group lacks a conditioning history with the drug-related stimuli.

The optimal result in such a study would be to see greater responding to the

drug-related stimuli than to the nondrug-related stimuli only in the drug-using

group. However, as tong as the drug-using group shows a larger difference in

responding to the different stimuli than does the drug-naive group, evidence of

conditioning exists.

Although such a design is clearly an improvement on the first procedure, it

unfortunately contains a more subtle confound. The problem with this design is

that the two subject groups may generally differ in their responsiveness to the

unconditioned effects of arousing stimuli. Although we are unaware of any

studies that have examined the general arousability of opiate or cocaine

abusers, there are data showing that alcoholics demonstrate more

unconditioned responsiveness to nonalcohol-related salient stimuli than do

social drinkers (e.g., Chandler et al. 1975). It may also be the case that drug

users are more aroused by the disturbing drug videotape than are the

drug-naive subjects, simply because drug users are more easily aroused in

general. This possibility is especially likely given that these two subject groups

are entirely self-selected.

THREE GROUP-THREE STIMULUS DESIGN

A better design for examining the role of conditioning in responding to

drug-related stimuli involves exposing two different groups of drug users to cues

for different kinds of drugs. The idea is to see if each group responds more to

cues related to their drug of abuse than to cues related to a drug that they have

236
not previously used. More specifically, such a study might employ two

drug-abuse groups, such as opiate abusers and cocaine abusers, and a third

group of drug-naive individuals. All subjects would be exposed to three types of

stimuli: stimuli related to use of one drug, e.g., opiates; stimuli related to use of

a second drug, e.g., cocaine; and nondrug-related stimuli.

Additionally, it is important that the two drug-using groups administer their drug

by different routes. This protocol is necessary to minimize generalization of

conditioned responding across the two types of drug stimuli resulting from

common stimulus elements.

The outcome of interest in this study involves showing that drug users are

maximally responsive to drug cues relevant to their own drug-use histories.

That is, drug 1 users should respond more to drug 1 cues than to either drug 2

or nondrug cues. Conversely, drug 2 users should show more responding to

drug 2 cues than to drug 1 stimuli or nondrug cues. Drug-naive individuals

should show equal responding to the three sets of stimuli.

This crossover pattern of results would deal with the kinds of problems

associated with the other naturalistic stimulus designs, that is, that the groups

being compared differ in their arousability or that the stimuli being compared

differ in their unconditioned effects. One would be unable to argue that results

are the consequence of different levels of arousability in the two drug groups

because each group reacts to the appropriate drug stimuli. Similarly, one could

not argue that one set of drug cues was intrinsically more arousing, because

each type of stimulus would evoke responding in the appropriate group.

Therefore, seeing this crossover data pattern in the two drug groups would

provide a strong argument that responding to the drug stimuli is a specific

function of a past conditioning history.

An implementation of this design, consistent with previous studies performed in

our laboratory, would include cocaine-only users, opiate-only users, and

drug-naive subjects. Cocaine-only users are individuals who are currently using

cocaine and have no history of opiate use. Such subjects might well have an

extensive history of alcohol or marijuana use, however. Similarly, opiate-only

users are individuals using opiates who have no history of using cocaine.

Unfortunately, our patient population at this time includes few individuals who

qualify for the opiate-only group. Consequently, we examined only two groups

of subjects-cocaine users with no history of opiate use and subjects with no

237
history of using either cocaine or opiates. This reduced design prevents us from

looking at the full crossover data pattern discussed above. However, it does

allow us to determine whether differential responding to drug stimuli occurs in

the cocaine group as a function of that population’s history. In addition, the

inclusion of a drug-naive group provides a better assessment of the

unconditioned effects of our stimuli than has so far been available.

Method

Subjects. Male patients with a history of smoking cocaine and no history of

opiate use were recruited from the inpatient drug dependency ward of the

Philadelphia VAMC. Fifteen cocaine subjects have tested so far in this ongoing

study. Nine drug-naive control subjects, employees of the hospital of similar

ages as the inpatients, have also been tested.

Design. The subjects in this study were exposed to three kinds of stimuli.

Cocaine-related stimuli were presented in one session, opiate-related stimuli in

a second session, and nondrug-related stimuli in a third session. The order of

the three sessions was counterbalanced across subjects.

The variables were physiological measurements of GSR, heart rate, and skin

temperature; subjective self-reports of drug-specific high, craving, and

withdrawal were also collected. Change from baseline scores were calculated

for each of the measures in a manner similar to the one discussed earlier.

Statistical tests were performed both to determine whether a stimulus

presentation caused a significant change from baseline responding and to

determine whether responsiveness differed between the stimuli. Although

physiological data was collected during the entire session, only the

physiological data collected during the video stimulus will be presented.

Procedure. After subjects had recording electrodes attached and were seated

in the recording chamber, they were asked for their self-reports of high, craving,

and withdrawal relative to both cocaine and opiate use.

Physiological recording was initiated at this point. After a 15-min baseline

period during which no events occurred, subjects listened to an audiotape,

watched a videotape, and performed a manual task. Within any one session, all

three stimuli were either cocaine related, opiate related, or nondrug related. The

videotape stimulus for the opiate session shows an individual buying, preparing,

and injecting heroin. During the cocaine session, the videotape depicts two

238
users free-basing and smoking cocaine. The neutral videotape is a scene from

a nature documentary. Following the completion of the manual task, subjects

were again asked about their level of high, craving, and withdrawal related to

the two drugs.

RESULTS AND DISCUSSION

Cocaine Subjects

Figure 2 depicts the physiological responding in the cocaine-only users-the

mean change from baseline and standard error produced by the videotape

stimulus for each physiological dependent measure in each session. The mean

change in baseline level of cocaine craving is also depicted. The particular

session associated with each data bar is indicated on the abscissa. In general,

it should be noted that cocaine stimuli are more arousing in these subjects than

are the other two sets of cues.

Specifically, cocaine users showed a significant heart rate increase to the

cocaine stimuli when compared to prestimulus baseline responding (t(13)

= 3.09, p < .01). By contrast, the opiate-related videotape did not produce a

change in heart rate (p > .05), and the neutral videotape actually caused a

significant decrease in heart rate (t(13) = -2.98, p < .05). Comparisons of

responding between pairs of stimuli revealed a significantly greater increase in

heart rate in response to the cocaine-related videotape than to either the

opiate-related videotape (t(13) = 4.48, p .01) or the neutral videotape (t(13)

= 4.09, p < .01).

For the GSR measure, the cocaine-related videotape caused a significant

decrease from baseline (t(14) = -2.98, p < .01). The opiate- and

nondrug-related videotapes had no significant effect (p > .05). Comparisons

between stimuli showed that the cocaine-related videotape had a greater

impact on GSR than the opiate-related videotape (t(14) = -2.11, p = .05) but

failed to lower GSR significantly more than the neutral videotape (p > .05).

For skin temperature, both the cocaine- and opiate-related stimuli caused a

significant decrease from baseline (t(14) = -4.27, p < .01 for the cocaine-related

videotape; (t(14) = -3.22, p < .01 for the opiate-related videotape). However, the

decrease in response to cocaine stimuli was significantly greater than the

decrease to the opiate cues (t(14) = -3.11, p < .01). Responding to the

cocaine-related and neutral videotapes differed as well (t(14) = -3.84, p < .01).

239
FIGURE 2. Change from baseline scores during each of the three stimulus sessions (cocaine, opiate,

neutral) for the cocaine-only group.
Once again, this pattern of data is consistent with a conditioning interpretation

of the response. Furthermore, the responsiveness to the opiate cues may

reflect unconditioned arousal to injection scenes, as was mentioned earlier.

All analyses of the subjective data were performed using nonparametric tests.

Data are presented only for craving because there were no significant changes

in results for high or withdrawal. Figure 2 shows changes in the subjective state

of cocaine craving produced by the stimuli. Craving to use cocaine increased

significantly only in the cocaine session (Wilcoxon T(10)=0, p < .01). By

comparison, no significant craving for opiates was reported by the cocaine

subjects in any session (p > .05).

Drug-Naive Subjects

In contrast to the pattern of responding observed in the cocaine subjects, the

pattern among drug-naive subjects was characterized by a lack of differential

responsiveness to the three stimuli across all the dependent measures (see

figure 3). This pattern is consistent with a conditioning interpretation of the

responding shown by cocaine subjects to the cocaine stimuli.

For example, while neither the cocaine nor the neutral stimuli caused a

significant change in heart rate (p > .05), the opiate cues actually caused a

significant decrease on this measure (t(9) = -2.90, p < .05). It should be noted

that the direction of the effect of the opiate cues on heart rate is opposite that of

the cocaine cues on heart rate in the cocaine group. The drug-naive subjects

showed no significant changes in GSR to any of the cues (p .05).

With respect to skin temperature, it is of some interest to note that the

drug-naive subjects showed a near significant (p = .057) decrease in skin

temperature in response to the opiate cues but exhibited no change in response

to the cocaine-related or neutral videotapes (p > .05). This decrease in

temperature brought about by the opiate cues is similar to that seen in the

cocaine subjects and supports the idea that the change in temperature may

partially represent an unconditioned effect.

Not surprisingly, drug-naive subjects reported neither cocaine nor opiate

craving in any of the three sessions (p > .05).

Although we were unable to test an opiate-only group in this design, the data

discussed above from previous naturalistic stimulus studies are relevant to

241
FIGURE 3. Change from baseline scores during each of the three stimulus sessions (cocaine, opiate,

neutral) for the drug-naive group.
interpreting the current results. The present studies employed the same opiate

cues that we have used previously. Consequently, the results of those earlier

studies demonstrate that opiate-only subjects are responsive to the identical

opiate cues that had a minimal impact on the cocaine and drug-naive subjects

in the present study (see table 1).

For example, opiate-only subjects showed significant decreases in skin

temperature and GSR in response to opiate stimuli but not to neutral stimuli.

Similarly, these subjects reported significant increases in opiate craving in

response to opiate stimuli but not to neutral cues (Childress et al. 1988). These

results indicate that the opiate cues used in the current study are indeed

evocative among individuals with the appropriate drug-use history.

This pattern of data reinforces the idea that the differential responsiveness to

the drug stimuli observed in the cocaine-only group was not simply the result of

unconditioned differences in evocativeness between the cocaine- and

opiate-related stimuli.

CONCLUSION

The data represented here are the best evidence to date that responding to

naturalistic stimuli results from a history of conditioning. Additional data

comparing responsivity to nondrug-related arousing stimuli between groups of

drug users and drug-naive individuals might also prove useful. Such data would

provide an additional assessment of potential differences in arousability that

may exist between these groups. In general, the current results encourage

efforts to develop treatment strategies based on conditioning principles.

REFERENCES

Chandler, B.C.; Parsons, O.A.; and Vega, A. Autonomic functioning in

alcoholics: A study of heart rate and skin conductance. J Stud Alcohol

36:566-577, 1975.

Childress, A.R.; McLellan, A.T.; Ehrman, R.; and O’Brien, C.P. Classically

conditioned responses in cocaine and opioid dependence: A role in relapse?

In: Learning Factors in Drug Dependence. National Institute on Drug Abuse

Research Monograph 84. DHHS Pub. No. (ADM)88-1576. Washington,

DC.: Supt. of Docs., U.S. Govt. Print. Off., 1988. pp. 25-43.

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O’Brien, C.P.; Childress, A.R.; McLellan, T.; and Ehrman, R. Integrating

systematic cue exposure with standard treatment in recovering drug

dependent patients. Addict Behav 15:355-365, 1990.

O’Brien, C.P.; Testa, T.; O’Brien, T.J.; Brady, J.P.; and Wells, B. Conditioned

narcotic withdrawal in humans. Science 195:1000-1002, 1977.

Siegel, S. The role of conditioning in drug tolerance and addiction. In: Keehn,

J.D., ed . Psychopathology in Animals: Research and Treatment Implications.

New York: Academic Press, 1979. pp. 143-168.

Stewart, J.; deWit, H.; and Eikelboom, R. The role of unconditioned and

conditioned drug effects in the self-administration of opiates and stimulants.

Psych Rev 91:251-268, 1984.

Temes, J.W.; O’Brien, C.P.; and Testa, T.T. Rapid acquisition of conditioned

responses to hydromorphone in detoxified heroin addicts. Psychopharmacol

Bull 18:215-219, 1982.

Wikler, A. Conditioning factors in opiate addiction and relapse. In: Wilner, D.I.,

and Kassenbaum, G.G., eds. Narcotics. New York: McGraw-Hill, 1965.

pp. 85-100.

ACKNOWLEDGMENTS

This work was supported by the VA Medical Research Service and USPHS

grant DA 03008.

AUTHORS

Ronald N. Ehrman, Ph.D

Steven J. Robbins, Ph.D

Anna Rose Childress, Ph.D

A. Thomas McLellan, Ph.D

Charles P. O’Brien, M.D., Ph.D

Treatment Research Center

University of Pennsylvania

3900 Chestnut Street

Philadelphia, PA 19104-6178

244
State Dependency as a Mechanism of Central

Nervous System Drug Action

Francis C. Colpaert

INTRODUCTION

A response acquired in a given state may not occur when the subject is in a

different state. The phenomenon is referred to as state dependency (StD) (e.g.,

Overton 1983) and means that engrams that are stored in memory in a given

state are often more accessible for recall in the same state than they are in a

different state (e.g., Weingartner 1978).

The StD paradigm typically involves subjects that are trained to emit some

classically conditioned or operant response while in a given (e.g., drug-induced)

state; the StD of the acquired response is subsequently examined by

determining to what extent the response occurs in different states (e.g.,

following the injection of a different drug or of the vehicle). Experiments using

this paradigm have demonstrated the existence of StD with a wide variety of

states that were induced either pharmacologically or otherwise (Overton 1982a).

A major problem arising with the StD paradigm is that it is difficult to obtain

reproducible, robust evidence of the phenomenon in either human (Weingartner

1978) or animal subjects (Overton 1966). The recognition of this methodological

problem, in the early seventies, was followed by decreased interest in the

typical StD paradigm and increased use of the drug discrimination paradigm

(Stolerman and Shine 1985). In the latter paradigm, subjects typically are

trained to emit one response after drug administration, and to emit-under

otherwise identical conditions-an alternative response after vehicle treatment

(e.g., Colpaen et al. 1976). However, while some authors (Overton 1983)

assume that the two paradigms define a single phenomenon, others have

argued that the processes underlying drug discrimination and StD may differ

(Colpaert et al. 1976).

245
We have recently described an StD procedure in which drug-to-saline or

saline-to-drug state changes yielded robust and quantifiable response

decrements following relatively low doses of chbrdiazepoxide (CDP) in rats

(Colpaert 1986). The studies summarized here document the dose dependency

of the CDP state and examine its possible relationship to tolerance,

dependence, and the anxiolytic action of benzodiazepines. The data provide

initial evidence that StD may in fact constitute the very mechanism of the

central nervous system (CNS) actions of the benzodiazepines.

EXPERIMENTAL PROCEDURES

The state-dependency procedure and details of the experimental conditions

have been described elsewhere (Colpaert 1990). Briefly, rats were trained in a

food-rewarded, lever-pressing task until they could complete a fixed ratio (FR

10) requirement within the first 120 sec of the session, and were tested for the

retention of this response requirement after having reached this criterion. The

pharmacological treatment instituted at the time of tests was either the same as

(same-state condition) or different from (different-state condition) the treatment

used during acquisition.

In one further series of experiments, a conflict procedure was used in which rats

were placed in a new environment containing a probe and allowed to explore

(Meert and Colpaert 1986). Exploration of the probe is reduced when the probe

is electrified, and anxiolytics disinhibit the behavior.

BENZODIAZEPINE STATE DEPENDENCY

Drug Effects on Acquisition

In the course of different experiments, a total of 188 rats were trained with

saline injections that were given via various routes and at various time intervals.

Six of the rats died prior to reaching criterion, and 15 failed to attain criterion in

fewer than 20 sessions. The remaining 167 animals reached criterion after a

median number of 10 sessions (95 percent CL (confidence unit), 8-14).

Analysis of the acquisition data that were obtained with the other treatments

with which animals were trained failed to reveal any reliable effect on

acquisition of any of these treatments, with the exception of cocaine. That is,

after receiving 10 mg/kg of cocaine, as many as 22 rats exceeded the 20

acquisition sessions cut-off before 7 were found that reached criterion in less

246
than 20 sessions; acquisition with 10 mg/kg of cocaine thus required more

sessions (p < .01; Mann-Whitney U test) than with saline (Siegel 1956).

Same-State Operant Performance

Control data collected throughout these experiments provided extensive

evidence that rats trained to complete the FR 10 schedule of bar press

responses for food reward within 120 set will reliably do so in test sessions if

there is no state change between acquisition and test. That is, all 52 rats trained

and tested with saline (subcutaneous [SC] over 30 min) completed the schedule

within 120 set during the test session; that was also the case with 29 of 30 rats

that were both trained and tested with 40 mg/kg of CDP (SC over 30 min). The

response thus failed to occur within 120 set in only 1 of a sample of 82 animals

(i.e., less than 5 percent) in which no state change was implemented.

These data therefore indicate that the completion of the FR schedule within 120

set during the test constitutes a satisfactory criterion of transfer. Transfer will

henceforth be defined as the completion of 10 lever press responses within 120

sec during a test session. The data described below will be expressed in terms

of the percentage of animals in which transfer (as defined above) occurred.

Chlordiazepoxide: Dose-Response Studies

Figure 1 shows that only a few failures to transfer occurred in rats trained with

0.16 to 2.5 mg/kg doses of CDP and tested with saline or 0.16 to 40 mg/kg

CDP. Note that the 40 mg/kg test dose of CDP depressed overall response rate

in rats trained with 0.16 or 0.63 mg/kg, but not in animals trained with 2.5 mg/kg

or higher doses of CDP.

In rats trained with 10 mg/kg CDP, complete transfer occurred with test doses

of 40 and 10 mg/kg; transfer then decreased, however, with test doses lower

than 10 mg/kg. Transfer in 10 mg/kg trained rats was thus dose dependent, as

indicated by Litchfield and Wilcoxon ED50 results of 0.96 mg/kg (95 percent CL,

0.34-2.7; Tallarida and Murray 1987). Test doses of CDP lower than 10 mg/kg,

as well as saline, also depressed response rate in a manner that related

inversely to the dose.

In rats trained with 40 mg/kg of CDP, transfer was similarly reduced in a

dose-dependent manner at test doses smaller than 40 mg/kg; the ED50 of CDP

for transfer in rats trained with 40 mg/kg CDP was 9.8 mg/kg (95 percent CL:

247
248
2.9-32). Test doses smaller than 40 mg/kg, again, depressed rate in a

dose-dependent manner, and rate depression was virtually complete with saline.

In rats trained with saline, failure to transfer from saline to drug occurred in a

dose-dependent manner at test doses of 10 to 160 mg/kg [ED50, 29 mg/kg (95

percent CL: 11-75)].

Figure 1 (insert) shows how transfer in tests with saline varied as a function of

the CDP dose used during acquisition. The ED50 acquisition dose of CDP at

which drug-to-saline transfer failed to occur was thus found to be 5.0 mg/kg (95

percent CL: 2.9-8.6). This ED50 dose was also significantly lower (p < .05) than

the dose (i.e., 29 mg/kg) at which saline-to-CDP transfer failed to occur

(Tallarida and Murray 1987).

The following are notable features of the total number of responses that were

emitted in the course of the 15-min test sessions (figure 1). First, in tests in

FIGURE 1. Results of transfer tests in rats that acquired a response in a

given pharmacological condition and that were tested in the same

or another pharmacological condition. Rats were trained until they

completed a fixed ratio (FR 10) schedule of lever-press responses

for food reward within 120 set after the beginning of a daily

15-min session. Training sessions occurred 30 min after

subcutaneous (SC) injection of either saline (dose 0) or one of

five different acquisition doses of chlordiazepoxide (CDP). Once

trained, animals were given one test session 30 min after SC

injection of saline or of one of the test doses of CDP. Upper

panels: Percentage of transfer, i.e., the percentage of animals that

completed the FR 10 schedule within 120 sec. during the test

session. lower panels: meat ± 1 SEM of total number of

responses made in the 15-min test session. Asterisks indicate

significant differences from the group tested with the treatment

that was also used during training (two-tailed p < .05;

Mann- Whitney U test; Siegel 1956). The insert reiterates data that

were obtained in tests with saline in animals that acquired the

response with one of the different doses of CDP. Lower right

panel: Data obtained in another series of experiments in which

rats were trained with saline and tested with either saline or one of

several different doses of haloperidol (SC over 30 min). Each data

point is based on n = 7.

249
which the test treatment was the same as that used in acquisition, total

responding was similar (i.e., an average of about 700 responses), irrespective

of whether the treatment was saline or any dose of CDP. Second, response

rate generally correlated with transfer in experiments using CDP.

However, this pattern is not a general feature of the paradigm, because in

saline-trained rats, 0.0025 to 0.16 mg/kg doses of haloperidol caused

dose-dependent suppression of rate while leaving transfer intact (figure 1,

bottom right). Third, saline lowered total responding in rats trained with 40

mg/kg CDP just as much as did 40 mg/kg CDP in rats trained with saline.

Chlordiazepoxide: Transfer After Extended Food Deprivation

In this experiment, rats were trained to criterion with CDP (40 mg/kg SC over 30

min) and were tested with either saline or CDP (40 mg/kg SC over 30 min) after

1, 2, 3, or 4 days of food deprivation. Seven animals were used with each test

condition. The results are summarized in figure 2.

Rats tested with 40 mg/kg of CDP transferred after 1 day of food deprivation,

and other rats also did so after 2 or 3 days of deprivation. Rats tested with

saline failed to transfer regardless of the length of food deprivation preceding

the test.

Six animals that were scheduled to be tested after 4 days of deprivation died

before the tests were conducted, most likely as a result of malnutrition and

dehydration. This outcome was entirely unexpected based on observations of

wildlife (Hart 1980), indicating that rats can go without food for up to 10 days. At

any rate, all experiments involving extended food deprivation were immediately

discontinued at that point.

Thus, it appears that food deprivation, even when it is very extreme, did not

overcome the failure of transfer of the food-rewarded response that occurred

when the response was acquired in the 40 mg/kg CDP state and tested in the

saline state.

Prolonged Testing In Switched State Conditions

In this experiment, two groups of seven rats each were trained to criterion with

either saline or 40 mg/kg of CDP (SC over 30 min). The animals were then

tested (1) for 1 session in the condition that was the same as that of training,

250
FIGURE 2. Transfer in rats trained to criterion with 40 mg/kg of

chlordiazepoxide (CDP) and tested with saline (open symbols) or

40 mg/kg CDP (closed symbols) after 1, 2, or 3 days of food

deprivation. Percentage of transfer is the percentage of animals

that completed the FR 10 schedule within 120 sec during the test

session. Each data point is based on n = 7.

(2) for 10 sessions in the different-state condition (i.e., 40 mg/kg of CDP or

saline, respectively), (3) for 5 sessions again in the same state condition, and

(4) for another 5 sessions in the different state condition.

The first switch from the same to the different state caused failure to transfer to

occur in all seven CDP-trained rats and in four of seven saline-trained rats

(figure 3). Continued testing or training in the different state resulted in complete

transfer after about five sessions. The reinstitution of the same state had no

apparent effect in CDP animals but caused a transient disruption in two

251
FIGURE 3. Rats were trained to criterion with either saline (n = 7, open symbols} or 40 mg/kg CDP (n =

7, closed symbols) and then tested succesively in the "same" and the "different” state (i.e.,

saline or 40 mg/kg CDP, respectively). The number of subsequent sessions in which

animals were tested in each phase and state is apparent from the abscissa. Ordinate:

Percentage of transfer is the percentage of animals that completed the FR 10 schedule

within 120 sec during the test session.
saline-trained rats. Performance after switch 3 indicated that by then the

response was, for all animals, retrievable in both states.

It is noteworthy that the data from this experiment further suggest that

relearning after a saline-to-CDP state change requires fewer sessions (median,

2 sessions) than acquisition in experimentally naive animals (median, 10

sessions; see above).

Tolerance to CDP State Dependency

In this experiment, rats were trained to criterion with 40 mg/kg of CDP (SC over

30 min) and training was continued for 5, 10, 20, or 40 additional sessions

before a test was carried out with either saline or 40 mg/kg of CDP.

Rats tested with 40 mg/kg of CDP continued to transfer. Rats tested with saline

continued to fail to transfer, regardless of the number of additional training

sessions that they had undergone with 40 mg/kg of CDP (figure 4).

In the preceding experiments, the performance of rats that were trained with

saline and tested for transfer to 40 mg/kg of CDP on 10 consecutive sessions

can, of course, also be viewed as conventional data on the development of

tolerance, possibly through relearning, to depressant effects of CDP on the rate

of operant behavior. The data (figure 4, insert) show CDP to markedly (p < .01;

Wilcoxon test) decrease rate in the first test (retraining) session. The

depressant effect of CDP was no longer significant (p > .05) in the second

session, and disappeared entirely after three injections. Such rapid

development of apparent tolerance stands in marked contrast with the main

data in figure 4 showing that lack of CDP-to-saline transfer persisted after a

total number of CDP injections that, including the approximately 10-session

training phase, amounted to as many as 50.

CDP: State Dependence and Physical Dependence

Rats were trained to criterion with 40 mg/kg CDP (SC over 30 min) and then

tested, in 10 consecutive sessions, with either saline (SC over 30 min; n = 7) or

40 mg/kg CDP (SC over 30 min; n = 7). Transfer and total responding were

monitored, and rats were weighed 30 min before each daily session.

As in previous experiments (figures 1 and 4), the CDP-to-saline state change

caused a failure to transfer, which now appeared to require five sessions to

253
FIGURE 4. Transfer in rats that were trained to criterion with 40 mg/kg of CDP and given an additional 5

to 40 sessions of training before being tested (SC over 30 min) with either saline (open

symbols) or 40 mg/kg CDP (closed symbols). Transfer was defined as the percentage of

animals that completed the FR 10 schedule within 120 sec during the test session. Each

data point represents one test session in n = 7 rats. The insert shows data from the

preceding experiment in which 7 rats were trained to criterion with saline and then tested

(retrained) with 40 mg/kg CDP in 10 consecutive sessions. Data points represent the

mean ± 1 SEM number of responses per 15-min session on the last saline session (open

symbol) as well as on the 10 CDP injections (closed symbols).
FIGURE 5. Rats were trained to criterion with 40 mg/kg CDP and fesfed, in 10

consecutive sessions, with either 40 mg/kg CDP (n = 7, closed

symbols) or saline (n = 7, open symbols). Transfer, total

responding, and body weight were monitored daily. Body weight

was expressed for each rat as a percentage of the body weight on

the day criterion was reached; data points represent the mean ± 1

SEM. The asterisk indicates p < .05 for the difference between the

two groups.

255
recover (figure 5). Saline also depressed responding, and this depression, too,

required about five sessions to disappear.

Relative to their weight on the day that criterion was reached, animals that

continued to receive CDP steadily gained weight during the 10 days that test

sessions took place. Animals receiving saline showed a slight decrease on Day

3 of testing. However, the weight of saline-treated rats failed to differ either from

their own criterion weight (one-tailed p > .05; Wilcoxon test) or from that of

animals that received CDP (one-tailed p > .05; Mann-Whitney U test). Note also

that the drop in the weight of animals receiving saline on Day 3 failed to

coincide with their maximal drop of transfer, which occurred on Day 1 (figure 5).

Benzodiazepine State Dependency: Receptor Specificity

In these experiments, several groups of rats were trained to criterion with two

injections and tested with two injections of either the same or different

injections; the two injections were SC and intraperitoneal (IP) and both were

given 30 min before the session, as specified in table 1.

Table 1 shows that diazepam caused a dose-dependent failure to transfer from

saline; its IP ED50 was 9.7 mg/kg (95 percent CL: 2.9-33).

Rats trained with 10 mg/kg of IP diazepam showed complete transfer with 40

mg/kg of SC CDP, but not with saline. Rats trained with 40 mg/kg of SC CDP

failed to transfer either with saline or with a 0.63 mg/kg dose of IP diazepam.

However, transfer did occur and increased dose-dependently as the diazepam

test dose was increased; diazepam’s ED50 (IP) in substituting for 40 mg/kg of

CDP was 4.0 mg/kg (95 percent CL: 1.2-13).

Following Ro 15-1788, a benzodiazepine-receptor blocker that itself was

ineffective in saline-trained animals, transfer failed to occur in CDP-trained rats.

The compound antagonized the state induced by 40 mg/kg of CDP (table 1).

Finally, the following compounds (SC over 30 min) failed to induce transfer in all

rats (n = 7 per test compound) that were trained with 40 mg/kg of CDP:

yohimbine (10 mg/kg), haloperidol(0.16 mg/kg), cocaine (10 mg/kg), morphine

(10 mg/kg), apomorphine (0.16 mg/kg), ± propranolol (10 mg/kg), clonidine

(0.16 mg/kg), and methoxamine (2.5 mg/kg).

256
TABLE 1. Transfer test results in rats (n = 7) trained with SC and IP

injections over 30 mina

Acquisition Treatment (mg/kg) Test Treatment (mg/kg) Percentage of

Transfera

SC

Saline

Saline

Saline

Saline

Saline

Saline

Saline

CDP (40)

CDP (40)

CDP (40)

CDP (40)

CDP (40)

CDP (40)

Saline

CDP (40)

CDP (40)

IP

Saline

Saline

Saline

Saline

Saline

Diazepam (10)

Diazepam (10)

Saline

Saline

Saline

Saline

Saline

Saline

Saline

Saline

Saline

SC

Saline

Saline

Saline

Saline

Saline

Saline

CDP (40)

Saline

Saline

Saline

Saline

Saline

Saline

Saline

CDP (40)

CDP (40)

IP

Saline 100

Diazepam (0.53) 100

Diazepam (2.5) 84

Diazepam (10) 43

Diazepam (40) 14

Saline

Saline

Saline

Diazepam (0.63)

Diazepam (2.5)

Diazepam (10)

Diazepam (40)

Ro 15-1788 (10)

Ro 15-1788 (10)

Saline

Ro 15-1788 (10)

0

100

0

0

43

57

100

0

100

100

0

NOTE: COP = chlordiozepoxide

a

The percentage of animals that completed the FR 10 schedule within 120 sec during the test

session.

State Dependency With Nonbenzodiazepine Compounds

Rats that were trained to criterion with yohimbine and were tested with saline

showed a failure to transfer that was proportional to the yohimbine dose used in

acquisition (ED50 = 1.5 mg/kg; (95 percent CL: 0.68-3.1). (See table 2.) Failure

to transfer also occurred in rats that were trained with saline and tested with

257
0.63 to 10 mg/kg doses of yohimbine (ED50 = 4.9 mg/kg (95 percent CL:

2.9-8.3).

In contrast, no failure to transfer occurred in either drug-saline or saline-drug

transfer tests involving 2.5 and 10 mg/kg doses of cocaine (table 2). In rats

trained with saline, tests with 0.0025 to 0.16 mg/kg doses of haloperidol also

did not reveal any evidence of StD. These doses did, nonetheless, cause a

dose-dependent depression of the total number of responses made during the

15 min sessions (figure 1, bottom right).

BENZODIAZEPINES: ANXIOLYTIC ACTION AND STATE DEPENDENCY

Out of 38 control rats given SC saline, 3 failed to initiate the session. The

mean±SEM number of responses made during the session was 16±2.5 in

unpunished SC saline controls (n = 15) and 4.1±0.66 in the 20 animals that

served as punished SC saline controls. The number of rats that failed to initiate

the session before 9 were found per dose that did, was 1, 0, 0, and 2 with CDP

doses of 0.63, 2.5, 10, and 40 mg/kg, respectively. CDP (SC) increased

punished responding in a dose-dependent manner, the effect being statistically

reliable at 10 and 40 mg/kg (figure 6). The CDP dose at which the distance,

along the Y axis, between punished and unpunished controls was covered

halfway, was estimated by linear interpolation to be 30 mg/kg.

Out of 37 IP saline control rats, 4 failed to initiate the session. The 13

unpunished IP saline controls emitted a mean of 17±2.2 responses; the 20

punished IP saline controls averaged 3.1±1.6. The number of rats that failed to

initiate the session before 9 were found per dose that did, was 1, 2, and 15 with

diazepam doses of 0.63, 2.5, and 10 mg/kg, respectively. Diazepam (IP)

increased punished responding dose dependently; its effects were significant at

2.5 and 10 mg/kg. The dose at which diazepam covered the distance between

punished and unpunished controls halfway was 7.8 mg/kg.

The doses at which CDP and diazepam exerted anticonflict effects are

important for at least two reasons. First, the 30 mg/kg dose at which CDP had a

50 percent anticonflict effect was considerably higher than the dose necessary

to produce drug-to-saline transfer failure (i.e., 5.0 mg/kg; figure 1). The 50

percent anticonflict dose of diazepam (i.e., 7.8 mg/kg) was also higher than the

dose (i.e., 4.0 mg/kg; table 1) at which diazepam substituted for CDP in

CDP-trained animals. These data indicate that both benzodiazepines produced

state dependency in drug-to-saline transfer tests at doses that were lower than

258
TABLE 2. Transfer test results in rats trained to criterion with various

pharmacological treatments

Acquisition Treatment Test Treatment

Compound Dose Route Time Compound Dose Route Time Transfera

(mg/kg) (min) (mg/kg)

Saline - IP 15 Saline - IP 15 100

Saline - IP 15 Yohimbine 0.63 IP 15 100

Saline - IP 15 Yohimbine 2.5 IP 15 66

Saline - IP 15 Yohimbine 10.0 IP 15 14

Yohimbine 0.04 IP 15 Saline - IP 15 100

Yohimbine 0.16 IP 15 Saline - IP 15 66

Yohimbine 0.63 IP 15 Saline - IP 15 86

Yohimbine 2.5 IP 15 Saline - IP 15 29

Yohimbine 10.0 IP 15 Saline - IP 15 0

Saline - SC 30 Saline - SC 30 100

Saline - SC 30 Cocaine 2.5 SC 30 100

Saline - SC 30 Cocaine 10.0 SC 30 100

Cocaine 2.5 SC 30 Saline - SC 30 100

Cocaine 10.0 SC 30 Saline - SC 30 100

a

The percentage of animals that completed the FR 10 schedule within 120 sec during the test

session.

the doses that produced anticonflict effects. Second, comparing the

saline-to-drug failure-to-transfer data (figure 1; table 1) with anticonflict activity

reveals the two curves to be mirror images with both CDP and diazepam (figure

6). In fact, the anticonflict doses of CDP and diazepam (i.e., 30 mg/kg and 7.8

mg/kg, respectively) were virtually identical to ED50 in causing a failure to

transfer in saline-to-drug tests (i.e., 29 and 9.8 mg/kg, respectively; figure 1 and

table 1).

GENERAL DISCUSSION

The present data indicate that the completion of an FR 10 schedule of

food-rewarded lever presses within 120 sec constitutes a conditioned operant

response requirement with which drug state changes can be shown to yield

robust, reliable response decrements.

259

Three types of state changes involving CDP were analyzed in the present

studies.

1. Drug-to-saline transfer tests in rats trained with one of several doses of

CDP and tested with saline indicated StD occurred at an ED50

(acquisition) dose as low as 5.0 (95 percent CL: 2.9-8.6) mg/kg. Note that

this dose is low indeed relative to the doses at which CDP exerts

behaviorally depressant effects (figure 1, lower panel). These findings

argue against the earlier view that StD effects can be obtained only at

behaviorally toxic doses of drugs (Overton 1983).

2. Dose-to-dose transfer tests indicated lower doses of CDP substituted in a

dose-dependent manner for a higher CDP dose used in acquisition (figure 1).

3. Saline-to-drug transfer tests revealed CDP disrupted the transfer of the

saline-acquired response only at a dose (i.e., 29 [95 percent CL: 18-75]

mg/kg) that was six-fold higher than that at which drug-to-saline transfer

was disrupted. The interpretation of saline-to-drug transfer data has so far

been hampered by the confounding effects that drugs may have on the

rate of behavior (Overton 1983). These data indicate that it is arbitrary to

ascribe any transfer failure in saline-to-drug tests simply to behaviorally

depressant drug effects and the methodology presented, which clearly

differentiates memory deficits due to state change from drug effects on the

rate of behavior. Specifically, saline caused as large a rate-depressant

effect in rats trained with 40 mg/kg CDP as did 40 and 160 mg/kg doses of

CDP in saline-trained animals. Also, up to 0.16 mg/kg test doses of

FIGURE 6. Anticonflict and state-dependency effects of chlordiazepoxide

(CDP) and diazepam in rats. Upper left panel: Effects of CDP on

punished responding. Data points represent the mean± 1 SEM of

n = 9 with each dose ( ); n = 15 for unpunished (o, upper) and 20

for punished (o, lower) saline controls. All injections were SC.

With diazepam, data points are also based on n = 9 per dose;

there were 13 unpunished and 20 punished saline controls;

injections were made IP. Asterisks indicate one-tailedp to be <.05

(*) or < .001 (***) for the difference with punished saline controls

(Mann-Whitney U test; Siegel 1956). Lowerpanels: Plot of

saline-to-drug transfer data obtained with SC CDP (data from

figure 1) and IP diazepam (data from table 1).

261
haloperidol profoundly depressed response rates in saline-trained rats

without causing any transfer failure (figure 1).

Another series of experiments substantiated the pharmacological specificity of

the benzodiazepine state; the benzodiazepine receptor blocker Ro 15-1788

(Hunheler et al. 1981) antagonized CDP in producing the state, whereas the

typical receptor agonist diazepam substituted for CDP (table 1). In contrast,

various nonbenzodiazepine drugs that act on the CNS did not substitute for

CDP in producing the benzodiazepine state. Data obtained with yohimbine

(table 2) indicate that the StD procedure used here can also demonstrate StD

with nonbenzodiazepine compounds. Cocaine, however, failed to produce StD

in conditions that were otherwise similar to those in which cocaine is highly

discriminable (Colpaett and Janssen 1982). The latter finding deserves further

analysis in view of a longstanding theoretical controversy about whether StD

and discriminative drug effects reflect the same phenomenon (Colpaert et al.

1976; Overton 1982b).

The data shown in figure 2 are dramatic evidence of the extent to which the

memory for the response was locked into the CDP state; prolonged food

deprivation, even to the point of starvation, failed to make the food-rewarded

response transfer from the CDP to the undrugged state. Further evidence

(figure 3) showed that it is nonetheless possible to render the response

retrievable in both states by having it conditioned separately in the CDP and the

undrugged state. Without such conditioning, however, the response, if acquired

in the CDP state, remained unretrievable in the undrugged state, even when

subjects were overtrained by up to 40 sessions (figure 4). Clearly, these data

fail to satisfy the operational definition of tolerance, which requires that the

repeated administration of the drug leads to a demonstrable loss of its initial

effect (Carlton 1983). That state dependency persisted after as many as 50

injections in all of 40 mg/kg CDP is all the more remarkable as only 3 injections

were sufficient (figure 4, insert) to cause an apparently complete tolerance to

CDP’s known depressant effects on behavior (Cook and Sepinwall 1975). A

similar loss of saline’s behaviorally depressant effects developed at a similar

rate in rats trained with 40 mg/kg of CDP (figure 5). Note that these data also

add to previous evidence demonstrating that the learning (or relearning) of the

engram can account for apparent tolerance to drug effects on behavior

(Colpaert and Shearman 1988). Using body weight as a measure of physical

dependence (Martin et al. 1963) no evidence was obtained in the present

experiments that CDP-StD was associated with physical dependence (figure 5).

262
Our data suggest that there may be a relationship between CDP-StD and drug

dependence. Drug dependence can be defined as a situation in which the

physiological or psychological integrity of the subject is conditional upon a drug

(Balster 1985; Tatum et al. 1929). That CDP-trained animals require CDP to be

able to emit vital, food-reinforced behavior (figure 2) can thus be taken to

suggest that StD can operate as a mechanism of drug dependence. The

marked resistance to tolerance to the CDP state (figure 4) makes it possible for

this potential mechanism of benzodiazepine dependence to be extremely

powerful and persistent.

Further experiments explored the possible relationship between StD and the

anxiolytic action of benzodiazepines. Anxiolytic activity was assessed in rats

using a modification (Meert and Colpaert 1986) of the conflict paradigm (Geller

and Seifter 1960) that has predictive validity for the anxiolytic potency of

benzodiazepines in humans (Cook and Davidson 1973; Sepinwall and Cook

1978). The data indicated (figure 6) that the prototypical benzodiazepines, CDP,

and diazepam produce anticonflict effects at doses (i.e., 30 and 7.8 mg/kg,

respectively) that, if anything, were higher than those at which drug-to-saline

transfer failure occurs (i.e., 5.0 and 4.0 mg/kg, respectively) and that were

essentially identical to those at which saline-to-drug transfer failure occurs (i.e.,

29 and 9.7 mg/kg, respectively).

Two important considerations would seem to arise from these findings. One is

that any use of benzodiazepines as anxiolytics may be associated with StD;

some storage of information in memory, or perhaps much of the storage that

takes place while subjects are on anxiotytic doses of benzodiazepines, may fail

to transfer to the normal state. Thus, StD provides a possible explanation for

what is conventionally referred to as widespread retrograde amnesia produced

by the benzodiazepines (Lister 1985; Weingartner 1985). Second, the finding

that benzodiazepines produce anticonflict activity at precisely the doses at

which saline-to-drug transfer fails makes it possible to consider StD as the very

mechanism of the benzodiazepines’ anxiolytic activity. Unlike such primary

drives as hunger and thirst, anxiety has been characterized by behavioral

theorists (Miller 1955; Mowrer 1939) as a “chiefly secondary,” or an acquired,

drive; anxiety is progressively learned, and stored in memory, in the course of

ontogeny. The implication is that for anxiety to be operative at a mature stage,

the subject must retrieve what it has learned about the anxiety drive in its

undrugged past. Our data show that an undrugged-to-drugged state change

produced with benzodiazepines can make such retrieval fail. An interesting

prediction from this StD mechanism of benzodiazepine anxiolysis is that the

263
dose at which anxiolysis occurs is the same dose at which saline-to-drug

transfer failure occurs; the anxiolytic dose, therefore, should not vary with

specific behavioral contingencies. We have shown previously (Meert and

Colpaert 1986) that CDP’s anxiolytic dose is indeed constant and does not

change with varying levels of shock intensity in a rat conflict procedure. It is

interesting to ask whether similar mechanisms may apply to engrams that are

acquired phylogenetically.

In summary, the research discussed here indicates that the retrieval from

memory of a hunger-driven operant response can be made drug-state

dependent; robust retrieval failures occurred in drug-to-saline and in

saline-to-drug tests for transfer involving benzodiazepines. Although the learned

behavioral response was reinforced by food, food deprivation to the point of

starvation was unable to overcome the retrieval failure that occurred with a

drugged-to-undrugged state change. Up to 50 injections of CDP also failed to

prevent the drugged-to-undrugged retrieval failure, indicating that the CDP state

is remarkably resistant to tolerance. A new theory of benzodiazepine drug

action results: the state dependency of memory retrieval may constitute a

parsimonious, integrative explanation for, and mechanism of, the anxiolytic and

untoward (amnesic, drug dependence) actions of these drugs.

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New York: Academic Press, 1978. pp. 363-383.

AUTHOR

Francis C. Colpaert

Centre de Recherche Pierre Fabre

17, avenue Jean Moulin

F-81 106 Castres Cedex

France

266
State-Dependent Learning With Social Drugs

Geoff Lowe

INTRODUCTION

Drug-induced state-dependent learning (SDL) is now a well-established

phenomenon (Overton 1978). The term is used to describe the finding that

behavior learned in one drug state is better remembered when retention is

tested in the same drug state. This refers to an ability to access information

under the same or a different set of retrieval cues and, indeed, might be more

accurately described as state-dependent retrieval. Even drinking immediately

after sober learning may affect consolidation and result in recall deficits if there

is drug-state dissociation between storage and retrieval phases (Lowe 1982).

In studies with human subjects, alcohol (Lowe 1981), marijuana (Darley et al.

1974) barbiturate and amphetamine (Bustamente et al. 1970), methylphenidate

(Swanson and Kinsboume 1976) and nicotine (Peters and McGee 1982) have

all been shown to produce SDL effects. In some cases, the effect was

asymmetrical. That is, recall tended to be poorest when the acquisition stage

was under a drug (D) state but the retrieval stage was drug free (ND). With

drug-free acquisition and drug-state retrieval (ND-D), recall is also poor but less

so than under D-ND conditions. However, in studies reporting asymmetrical

effects, it is worth noting that there were slight drug-induced deficits in Day 1

learning, compared with drug-free learning. When equivalence of original

learning between D and ND states is obtained, symmetrical SDL effects have

been observed, at least in the case of alcohol (Lowe 1981).

The combined use of alcohol and cigarettes, and subsequently caffeine, is a

common occurrence (Carmody et al. 1985), and concern has been shown for

the disproportionate increase in susceptibility to disease in populations where

the incidence of joint consumption is high (e.g., Keller 1979, 1977; Walton

1972). However, few researchers have examined the psychopharmacological

interactions of alcohol, nicotine, and caffeine, particularly in relation to SDL.

267
The interaction between alcohol and stimulant drugs is generally regarded as

complex, with antagonistic (e.g., Knott and Venables 1979, 1977) synergistic

(e.g., Lee and Lowe 1980; Leigh 1982), and negligible or mixed (e.g., Tong et

al. 1974) effects being reported. Since both alcohol (A) and nicotine (N) have

separately been shown to produce SDL in man, it is reasonable to expect that

the A+N combination should produce similar state-dependent effects. Although

caffeine (C) has not yet been shown to produce SDL in man, cognitively

disruptive effects of alcohol-caffeine combinations have been observed (Lee

and Lowe 1980) and, thus, SDL effects would also be expected from the A+C

combination. However, in terms of the possible antagonistic/synergistic

interaction, of particular interest is the recall performance of those subjects

given a single drug on Day 2 after previously undergoing the acquisition phase

under the influence of an alcohol-nicotine/caffeine combination. Recall

decrements (or otherwise) on Day 2, when either alcohol or nicotine/caffeine is

absent, should shed more light on the nature of these drug combination

interactions, at least in relation to the phenomenon of SDL.

EXPERIMENT 1: ALCOHOL AND NICOTINE

Method

Twenty-four undergraduate students, ages 18 to 28, volunteered as subjects.

There were 12 females and 12 males, all of whom were smokers who regularly

combined use of alcohol and cigarettes. Their smoking histories ranged from

8 months to 7.5 years (mean, 2.84 yr). Their mean consumption was

9.2 cigarettes per day and 24.5 units of alcohol per week (a “unit” is defined as

one measure of spirits or one half-pint of beer or equivalent).

The alcoholic beverage consisted of vodka (37.5 percent alcohol per volume)

mixed with an equal volume of Schweppes Russchian aromatic tonic water.

The subject was given 4.4 ml of this mixture for every kg of body weight, so the

alcohol dose was moderate (0.66 g/kg), and designed to produce a

blood-alcohol concentration (BAC) of around 80 mg/100 ml (the United

Kingdom’s legal limit for driving). In the placebo drink, the vodka was replaced

by an equivalent amount of water, and the rim of the drinking glass was

smeared with vodka to give an identical initial olfactory cue. Nicotine

administration consisted of subjects smoking two Benson & Hedges Pure Cold

middle tar cigarettes within a 12-min period. These cigarettes have a rated

average nicotine delivery of 1.4 mg. In the placebo condition, two Honeyrose

268
herbal cigarettes (nicotine-free) were given. Although puff frequency was

monitored, no direct measure of inhalation was possible.

Design and Procedure

On Day 1 (learning), all subjects received the same drug treatment (A+N)

immediately prior to the acquisition stage. On Day 2 (retrieval) male and female

subjects were randomly allocated to one of four conditions to receive either

(1) both drugs (A+N); (2) nicotine plus placebo (N+O); (3) alcohol plus placebo

(A+O); or (4) no drugs (O+O). There were three males and three females in

each subgroup.

Subjects were asked to refrain from smoking and from consuming caffeinated

beverages for at least 3 hr before each daily session. They were also required

to consume no alcohol for at least 18 hr before the experiment. Upon arrival in

the laboratory, each subject was weighed and required to complete a

questionnaire on smoking and drinking history. The subjects were then tested

with an Alcolmeter AE-D1 (Lion Laboratories) in order to check that they were

alcohol-free and to familiarize them with this instrument.

Pairs of subjects consumed their alcoholic beverages and smoked two

cigarettes within a 20-min period. Approximately 40 min after starting drinking,

subjects were retested with the Alcolmeter before undertaking the learning task,

which consisted of a simplified geographical map visually displayed with a

19-item set of auditory instructions about a particular route (Lowe 1981). An

arbitrary criterion of at least 14 correct items was adopted as a learning

measure. If after four learning trials, the subject had not attained this level, then

the highest number correct in any of the four trials was taken as the learning

performance score. This learning session lasted approximately 5 min, and was

followed by another Alcolmeter reading. Day 1 ended with a final Alcolmeter

reading, approximately 50 min after commencing the drinking session.

The procedure for Day 2 (recall) was the same as that for Day 1 (learning)

except for the different drug conditions of the subgroups and the fact that there

was only one recall trial. The number of correctly recalled items from the Day 1

presentation of the route map was recorded as the recall performance score.

269
Results and Discussion

Table 1 shows the mean number of items correct from the route map for

learning (Day 1) and recall (Day 2) sessions, and for each condition. Mean

BACs during route map performance are also shown. A one-way analysis of

variance on the learning scores of Day 1 revealed no significant difference

among the four subgroups (F[3,20] = 1.053, p > .05). Table 1 shows the mean

decrements between learning and recall for each condition. Apart from the

slightly increased recall performance on Day 2 in the A+N condition, all other

conditions resulted in significant recall decrements due to alcohol (F[1,20] =

23.09, p < .0001) and to nicotine (F[1,20] = 6.08, p < .05). Although the nicotine

effect was more marked in the alcohol condition, the A+N interaction was not

statistically significant (F[1,20] = 3.36, p > .05).

The lack of recall decrement when subjects ingested both alcohol and nicotine

on Day 2 supports the notion that SDL was induced by an alcohol-nicotine

combination. Both the double placebo (O+O) and nicotine only (O+N)

conditions resulted in the largest dissociation decrements from the (A+N)

learning state. There was just as much dissociation when nicotine was ingested

on Day 2 as when no drugs were taken. This suggests that the major SDL

effect was due to alcohol. However, it seems likely that nicotine had some

influence because when nicotine was absent during the alcohol session on Day

2 (A+O), the dissociation decrement was about halfway between that of the

combination (A+N) and nicotine only (O+N) conditions. In other words, the

alcohol-alone state was insufficient to prevent a recall decrement on Day 2; the

TABLE 1. Alcohol and nicotinea

Condition Items correct Mean Recall Mean BAC

Day 2) Day 1 Day 2 Decrements Day 1 Day 2

(Learning) (Recall)

A+N 12.25 12.75 -0.5b69.5 80.1

A+O 12.75 8.00 4.75c87.4 73.0

O+N 15.00 6.67 8.33c74.2 -

O+O 12.00 4.00 8.00c

81.4 -

aBAC = blood alcohol content; A = alcohol; N = nicotine; O = placebo.

bNot significant.

c

p<.01.

270
absence of nicotine led to a partial dissociation from the Day 1 (A+N) learning

state.

If, as Leigh (1982) suggests, the combination of alcohol and nicotine can result

in a synergistic interaction, it seems likely that the nicotine-alone state is

discriminatively different from the combination, and much more so than the

alcohol-alone state. But the difficulty of assessing the relative degree of SDL

produced by a mixture of two drugs when each is tested at only one dose must

be acknowledged, especially in the case of nicotine when no biological markers

are taken.

EXPERIMENT 2: ALCOHOL AND CAFFEINE

Method

Sixteen undergraduate students, ages 18 to 28 yr, volunteered for this

experiment. There were eight males and eight females, and all were regular

alcohol and coffee drinkers.

The experimental design and general procedure were identical to those of

Experiment 1 except that the nicotine conditions were replaced by caffeine

conditions. Subjects drank two cups of Gold Blend (Nescafe) coffee, each

containing 1.5 5-ml teaspoons, approximating a total dose of 300-375 mg

caffeine. This was consumed within a 10-min period subsequent to the

alchol/placebo drinking period. In the placebo condition, Gold Blend

decaffeinated coffee was administered at the same concentration.

On Day 1 all subjects received the same drug treatment (A+C) immediately

prior to the acquisition stage. On Day 2 (retrieval) subjects were randomly

allocated to one of four conditions to receive either (1) both drugs (A+C); (2)

placebo plus caffeine (O+C); (3) alcohol plus placebo (A+O); or (4) no drugs

(O+O). See table 2.

Results and Discussion

A one-way analysis of variance on the learning scores of Day 1 revealed no

significant differences among the four subgroups (F[3,12] 0.196, p > .05).

Dependent t-tests (comparing Day 1 with Day 2) were computed for the

performance scores for each condition. In the caffeine conditions (A+C; O+C)

271
TABLE 2. Alcohol and caffeinea

Condition

(Day 2)

A+C

A+O

O+C

O+O

Items correct Mean Recall Mean BAC

Day 1 Day 2 Decrements Day 1 Day 2

(Learning) (Recall)

14.25 14.00 0.25b78.0 84

14.25 10.75 3.55c79.5 81

14.75 13.75 1.OOb79.0 -

13.75 8.25 5.50c80.5 -

aBAC = blood alcohol content; A = alcohol; C = caffeine; O = placebo.

bNot significant.

C

p < .01.

there were no significant differences. But whenever caffeine was absent on Day

2 (A+O; O+O), significant recall decrements were observed.

The lack of recall decrement when subjects ingested both alcohol and caffeine

on Day 2 supports the notion that SDL was induced by an alcohol and caffeine

combination. Both the double-placebo (O+O) and alcohol-only (A+O) conditions

resulted in the largest dissociation decrements from the (A+C) learning state.

There was almost as little dissociation when only caffeine was ingested on Day

2 as when both drugs were ingested. This finding suggests that the major SDL

effect was due to caffeine, although the relatively high dose used could be the

major determinant. The alcohol-alone state was insufficient to prevent a recall

decrement on Day 2; the absence of caffeine led to a partial but significant

dissociation from the Day 1 (A+O) learning state.

If, as Waldeck (1974) suggests, the combination of alcohol and caffeine can

result in a synergistic interaction, it appears that the caffeine-alone state is not

discriminatively different from the combination, whereas the alcohol-alone state

does seem to be. This finding contrasts somewhat with the results of

Experiment 1 using alcohol-nicotine combinations, which showed alcohol to be

the dominant drug.

272
GENERAL DISCUSSION

These studies offer some initial insights and confirm the complex nature of

social drug combinations in an area of cognitive behavior (SDL) in which these

particular drug combinations have not previously been applied. Demonstrations

of state-dependent effects of social drugs are of practical interest only if they

involve dosages and combination practices typically used outside the

laboratory. Our results indicate that SDL can occur not only in carefully

designed animal laboratory experiments but also under more naturalistic

conditions of moderate social drinking, smoking, and caffeine use. The clinical

implications of the phenomenon of state-dependent learning are potentially

large. There is anecdotal evidence from drug users suggesting that state

dependence may play a role in their use of drugs. Users often report a

continuity of desirable thoughts, perceptions, and feelings from one occasion of

drug use to another and relative amnesia for the pleasant feelings and thoughts

experienced in between. Overton (1978) has outlined and reviewed several

models linking state dependence with drug abuse.

One theory proposes that an altered repertoire of drug-state responses

develops with repeated drug use. If the drug-specific behaviors are more

reinforcing than normal “undrugged” behaviors, then the user may ingest drugs

to gain access to the drug-response repertoire rather than because of any

intrinsically reinforcing drug effects. For instance, other people may treat a

person differently (and possibly more favorably) when he is intoxicated than

when he is sober. Alternatively, drug effects may alter the user’s sensitivity to

social reinforcement so that reinforcing contingencies are effectively changed

even in the absence of any real change in the external environment (Lowe

1984).

Another theory treats the discriminative effects of psychoactive drugs as a

conditioned stimulus that evokes a reinforcing effect. Yet another theory

proposes that SDL is a direct causal factor in drug dependence and alcoholism.

The notion is that the dissociative barrier prevents recall in the sober or nondrug

state for many of the negative consequences of drug use. Only the initial

(usually positive) effects of ingestion are well recalled. Evidence supporting this

proposal has been obtained by Tamerin et al. (1971), who showed that

alcoholics selectively fail to remember much of the dysphoric content of their

drinking episodes.

273
A final point relates to cognitive anticipation involved in drug effects. It is a

well-established fact that people in Western cultures have distinct expectations

about the effects of social drugs on behavior and feelings (e.g., Brown et al.

1980; Southwick et al. 1981). A prominent hypothesis assumes that many acute

effects of alcohol, for instance, are mediated by such expectations alone

(Marlatt and Rohsenow 1980), and a recent meta-analysis indicates substantial

empirical support for this contention (Hull and Bond 1986).

The extent of such cognitive influences should not be underestimated, as was

amply illustrated in Marlatt and Rohsenow’s review of balanced-placebo-design

studies (1980). In the majority of studies, expectations alone produced certain

behaviors, but pharmacological effects alone did not. Hence, there is

convincing evidence that some alcohol-related behaviors are actually the

consequence of cognitive factors or of other factors that have no basis in

pharmacology. Whether such cognitive factors influence the outcomes of

state-dependent learning studies reported in the present paper remains to be

seen. We are currently running a series of experiments designed to explore

such possibilities.

In conclusion, a significant proportion of everyday forgetting could be due to

SDL effects, and drug combinations offer an increased range of dissociation

possibilities. It would thus be helpful if people became more aware of the

conditions that can limit the potential range of retrieval cues available when

recall of information is required.

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State-dependent learning in humans. Physiol Behav 5:793-796, 1970.

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W.E. Co-occurrent use of cigarettes, alcohol, and coffee in healthy,

community-living men and women. Health Psychol 4:323-335, 1985.

Darley, C.F.; Tinklenberg, J.R.; Roth, W.T.; and Atkinson, R.C. The nature of

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smoking and smoking deprivation. Psychophysiology 14:150-156, 1977.

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perceptual-motor task. IRCS Med Sci 8:420, 1980.

Leigh, G. The combined effects of alcohol consumption and cigarette smoking

on critical flicker frequency. Addict Behav 7:251-259, 1982.

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Lowe, G. Alcohol-induced state-dependent learning: Differentiating stimulus and

storage hypotheses. Curr Psychol Res 2:215-222, 1982.

Lowe, G. Alcohol and alcoholism. In: Sanger, D.J., and Blackman, D.E., eds.

Aspects of Psychopharmacology. London: Methuen, 1984. pp. 84-109.

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Expectancy and the balanced placebo design. In: Mello N.K., ed. Advances

in Substance Abuse: Behavioural and Biological Research. Greenwich,

Conn.: JAI Press, 1980. pp. 159-199.

Ovetton, D.A. Major theories of state dependent learning. In: Ho, B.T.;

Richards, D.W.; and Chute, D.L., eds. Drug Discrimination and State

Dependent Learning. New York: Academic Press, 1978. pp. 283-318.

Peters, R., and McGee, R. Cigarette smoking and state dependent memory.

Psychopharmacology 76:232-235, 1982.

Southwick, L.; Steele, C.; Marlatt, A.; and Lindell, M. Alcohol-related

expectancies: Defined by phase of intoxication and drinking experience.

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Swanson, J.M., and Kinsboume, M. Stimulant related statedependent learning

in hyperactive children. Science 192:1354-1357, 1976.

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Alcohol and memory: Amnesia and short-term memory function during

experimentally induced intoxication. Am J Psychiatry 127:1659-1664, 1971.

Tong, J.E.; Knott, V.J.; McGraw, D.J.; and Leigh, G. Alcohol, visual

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128:1455-1456, 1972.

AUTHOR

Geoff Lowe

Department of Psychology

University of Hull

Hull HU6 7RX, UK

276
Discriminative Stimulus Effects of Drug

Mixtures in Rats

I.P. Stolerman, E.A. Mariathasan, and H.S. Garcha

INTRODUCTION

It is widely recognized that the discriminative stimuli that many drugs produce

may have a compound nature. In dealing with exteroceptive stimuli it is often

easy to generate simple, unitary stimuli, such as light of a defined wavelength.

Drugs are different. Almost all compounds have multiple effects, both at the

level of receptors and in terms of subjective effects, and recognition of this fact

has led to the widespread use of the term “discriminative stimulus complex” in

such work. The processing of drug-produced stimuli may therefore be more like

the processing of compound than of simple exteroceptive stimuli. Few studies

have addressed the implications of this situation, although there are several

cases in which the complex nature of stimuli have been unraveled to various

extents. For example, cyclazocine produces discriminative stimuli with multiple

components that seem to parallel the effects of this drug at both the mu and the

kappa subtypes of opioid receptors (reviewed by Holtzman 1985). Other results

suggest that clonidine has both cocaine-like and noncocaine-like activity (Wood

et al. 1985) and imply that chlorphentermine acts on both dopamine and

serotonin mechanisms (Young 1988). There are also cases of generalization to

mixtures of drugs in circumstances when the component drugs failed to

generalize fully; Holloway et al. (1985) and Gauvin et al. (1989) have reported

full generalization from amphetamine or cocaine to certain mixtures of caffeine

with phenylethylamines. The present experiments represent an attempt to use

mixtures of drugs to generate compound interoceptive stimuli, the components

of which may be manipulated independently by varying the doses of drugs in

the mixtures.

Figure 1 shows some examples of situations in which mixtures of drugs may

serve as discriminative stimuli. The different paradigms are shown according to

the conceptualization of Järbe and Swedberg (1982) in comparison with a

277
FIGURE 1. Schematic outline of four different drug discriminations produced

by extending the conceptualization of Järbe and Swedberg

(1982). Upper left section shows a drug versus nondrug

discrimination. RA = response associated with a particular dose of

the training drug. RN = response associated with absence of

drug Upper right section shows basic mixture discrimination in

which RA is associated with particular doses of drug A and drug

B. lower left section extends this conceptualization to

discriminate explicitly between mixture and component drugs by

making RB = response associated with drug A or drug B. Lower

right shows one of several further extensions, in this case to a

three-choice paradigm with responses associated with and, or,

and nondrug states.

simple form of discrimination based on a single drug. In the latter case (figure 1,

upper left section), one response is associated with the presence of the drug

and a second response is associated with its absence. Figure 1 (upper right

section) shows an equivalent concept of a discrimination based on the effects

of a mixture of two dissimilar drugs. Here, one response is associated with the

simultaneous presence of both drugs (i.e., drug A and drug B) and a second

278
response is associated with the absence of both drugs; this procedure is

subsequently called the “AND” discrimination. Figure 1 shows only two of the

many other possible concepts involving mixtures of drugs. The lower left section

of figure 1 shows the particularly interesting case of an “AND-OR”

discrimination where one response is associated with a mixture of drugs A and

B and the second response is associated with the effects of either drug A or

drug B. Figure 1 (lower right section) shows an elaborated form of the AND-OR

discrimination in which a third response is associated with the absence of both

drugs. (Two different responses, one for each component drug, could be used

in a further elaborated model.)

These conceptual models are important because they focus attention on

functional differences between the different paradigms; drug discriminations

developed in the different paradigms may well exhibit different characteristics

as a result of these varying functional relationships. This paper is limited mainly

to studies of the AND paradigm for mixtures (figure 1, upper right), plus results

for some preliminary studies with an AND-OR discrimination (figure 1, lower

left); it also expands the concepts and data put forward by Stolerman et al.

(1987a,b).

A number of earlier investigations also shed light on the discrimination of drug

mixtures, but usually in ways rather different from the studies reviewed here.

The earliest study that has come to light is that of Overton (1966), who

developed an AND discrimination based on a mixture of atropine and

pentobarbitone using a shock-escape procedure in a T-maze. Subsequently,

Järbe and Johansson (1976) studied the AND discrimination with a mixture of

the anticholinesterase physostigmine and the muscarinic blocking mixture

Ditran (which was itself a mixture). However, studies with drugs that partly

cancel each other’s effects are difficult to interpret unless the characteristics of

noninteracting drugs are understood. The present studies therefore attempt first

to define the main features of a discrimination based on a mixture of nicotine

and midazolam. Discriminations based on these drugs given singly had

previously been studied in some depth under similar conditions and there was

no reason to expect interactions through pharmacological mechanisms. The

studies conceptually closest to the present experiments seem to be those of

Hanlin and Appel(1985), but these have appeared only in abstract form.

Witkin et al. (1980) carried out some work with a mixture of amphetamine and

pentobarbitone. One group of pigeons was trained to discriminate a mixture of

amphetamine and pentobarbitone from amphetamine alone (an AND

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discrimination within a drug versus drug design). Other pigeons were trained to

discriminate a mixture of amphetamine and pentobarbitone from pentobarbitone

alone. In previous behavioral experiments, amphetamines and barbiturates

were found to interact in additive, synergistic, or antagonistic ways, depending

on behavioral baselines and other factors (Branch 1974; Rushton and Steinberg

1983). In drug discrimination experiments, such mixtures might exhibit unusual

characteristics that could contribute to understanding why they are particularly

subject to abuse; studies directed to this end have been carried out. Preliminary

work with mixtures of pentazocine and tripelennamine (T’s and blues), caffeine

and phenylpropanolamine (formerly an over-the-counter substitute for

amphetamine), and morphine and nicotine has also been performed to test the

generality of findings to date.

Snoddy and Tessel (1983) studied mixtures of amphetamine and nisoxetine,

but there was cross-generalization between these drugs in simple drug

discrimination experiments, and studies on such mixtures can make a limited

contribution to understanding compound interoceptive stimuli. Glennon and

Young (1984) studied the discrimination of a drug mixture without really

intending to do so. In the wurse of investigations in which racemic methylene

dioxyamphetamine (MDA) was used in training, it became apparent that the (-)-

and (+)-isomers in the mixture generated qualitatively distinct stimuli. It was

suggested that (-)-MDA acted mainly through serotonergic receptors whereas

(+)-MDA acted on dopaminergic mechanisms. Many other drug discriminations

based on racemic mixtures may also involve multiple mechanisms, and a full

understanding of such discriminations will therefore depend on knowledge of

how compound stimuli produced by drugs are processed.

The main questions to be answered about the processing of such compound

interoceptive stimuli include the following: Can such complex procedures yield

orderly, readily interpretable results? Are stimuli produced by mixtures of drugs

perceived and processed in terms of the component drugs or as new

homogeneous entities? What are the effects of altering the doses and relative

amounts of drugs used for training? Does the use of mixtures for training

influence the specificity of the discriminations obtained? What are the effects of

antagonist drugs that selectively affect responses to the individual drugs used

to establish the discrimination? What is the importance of psychological

processes such as overshadowing and blocking, which have been found to play

key roles in discrimination of compound exteroceptive stimuli? Answers to

some of these questions are emerging from the work summarized here.

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METHODS

Animals

Male, Lister hooded rats were housed individually in rooms maintained at about

20°C with a regular light-dark cycle (light from 8 a.m. to 8 p.m.). Initially, the rats

weighed 210-280 g, but throughout the experiments they were fed restricted

amounts of food to maintain their weights at about 80 percent of normal. Water

was available in the living cages at all times.

Apparatus

Standard experimental chambers (Campden Instruments, London, UK) were

contained in sound-insulated, ventilated enclosures. The chambers were fitted

with two retractable response bars separated by a recess in which 45 mg

pellets of food could be presented. White noise was present at all times to mask

external sounds. The experiments were controlled by programs written in

ONLIBASIC, running on CUBE microcomputers (Control Universal, Cambridge,

UK).

Training Procedure

The procedure was based on that described previously for establishing

discriminations based on single drugs (Garcha et al. 1985; Pratt et al. 1983).

Rats were trained to press bars for food reinforcers before any injections were

given. Then, after injections of two drugs, animals were reinforced for pressing

one of the two bars; presses on the other bar were reinforced in sessions after

saline injections. The dose of drugs, the route by which they were administered,

and the interval between injections and training depended on the drugs used,

as detailed below. Training sessions were of 15 min duration. Drug and saline

training sessions took place in random order, with the restriction that no more

than three sessions with the same treatment occurred in succession. The final

schedule of food reinforcement was tandem variable-interval 1 min fixed-ratio

10; under this schedule, food was presented following the tenth consecutive

response on the correct bar after a randomly determined interval (mean = 1

min). The doses of drugs were based on preliminary experiments and previous

work.

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Generalization Test Procedure

Discriminative effects of drugs and drug mixtures were determined with groups

of 8-12 rats in 5-min extinction tests. Such tests took place twice weekly, with

training sessions continuing on the intervening days. Tests with different doses

of the training drugs or with other drugs normally took place in random order.

The index used to assess discriminative effects was the number of responses

on the bar appropriate for the mixture of drugs used for training, expressed as a

percentage of the total number of responses on both bars. A minimum of 10

responses was required for this index to be calculated.

Variations In Training Doses

In some experiments, the relative amounts of drugs in mixtures used to

maintain discriminations were varied. After first establishing discrimination with

a particular mixture and carrying out tests for stimulus control as detailed below,

the dose of one of the drugs used to maintain the discrimination was altered.

The rats were trained for at least 12 further drug and saline sessions (in random

order), and then stimulus control was examined again by means of extinction

tests. Several different dose ratios were used in succession to maintain

discriminations in this way, as detailed in the Results section.

Drugs

Nicotine bitartrate (BDH, Poole, Dorset, UK), midazolam maleate (Hoffman-La

Roche, Basle, Switzerland), (+)-amphetamine sulfate (Smith Kline and French,

Welwyn Garden City, UK), sodium pentobarbitone, pentazocine, tripelennamine

hydrochloride, phenylpropanolamine hydrochloride (all from Sigma, Poole,

Dorset, UK), morphine hydrochloride (May and Baker, Dagenham, UK), and

quipazine maleate (Miles, Slough, UK) were dissolved in isotonic saline.

Caffeine (Sigma) and phencyclidine hydrochloride (NIDA) were dissolved in

distilled water. All injections were given in volumes of 1 ml/kg, and all doses

were calculated as those of the base. Pentazocine and tripelennamine were

administered 30 min before sessions. All other drugs were administered 15 min

before sessions except as noted below.

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RESULTS AND DISCUSSION

Stimulus Control With Compound Drug Stimuli and Their Elements

A variety of different mixtures engendered strong stimulus control in a manner

generally similar to single drugs. Such results have been obtained in rats

trained with mixtures of nicotine and midazolam, amphetamine and

pentobarbitone, morphine and nicotine, pentazocine andtripelennamine, and

caffeine and phenylpropanolamine. Rats acquired strong discriminative

responses cued to the drug states with an accuracy of at least 85 percent. The

results with the mixtures were compared with results of component drugs tested

separately at the doses used to maintain discriminations. Such tests with single

drugs serve as tests of stimulus control by elements of a compound

interoceptive stimulus.

Examining first results for rats trained with nicotine (0.4 mg/kg SC) and

midazolam (0.2 mg/kg SC), figure 2 shows that these drugs separately

produced about 60 percent drug-appropriate responding, as compared with 88

percent after the mixture and 8 percent after saline (Stolerman et al. 1987).

These results set a pattern that was repeated with only minor variations in the

studies with mixtures of other drugs. Figure 3 shows that in tests with

amphetamine (0.4 mg/kg SC) or pentobarbitone (10 mg/kg SC), there was

about 75 percent drug-appropriate responding in rats trained to discriminate a

mixture of these drugs from saline. Generally similar results were obtained with

pentazocine (8 mg/kg IP) and tripelennamine (8 mg/kg SC), with morphine (3

mg/kg SC) and nicotine (0.4 mg/kg SC), and with caffeine (20 mg/kg IP) and

phenylpropanolamine (20 mg/kg IP) in rats trained with mixtures of each pair of

drugs at the doses stated. In these experiments only, morphine was

administered 30 min before sessions.

These experiments suggest that in rats trained with mixtures of dissimilar

psychoactive drugs, the separate drugs at the doses used for training engender

considerable amounts of mixture-appropriate responding. This principle may be

very generalizable because such findings have been obtained with five different

mixtures.

Dose-Response Studies With Drugs Used for Training

Full dose-response data have been obtained only for mixtures of nicotine and

midazolam and amphetamine and pentobarbitone. Figure 4 shows the results of

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FIGURE 2. Tests of responses to the training drugs in rats trained to

discriminate a mixture of nicotine and midazolam from saline

(AND discrimination). Results are shown for a group of 8-9 rats

tested with saline (SAL), nicotine and midazolam (MIX),

midazolam alone (MDZ). or nicotine alone (NIC) at doses shown.

All data were obtained in 5-min extinction tests and are expressed

as mean percentages of drug-appropriate responding ± SEM.

(Data from Stolerman et al. 1987).

these studies in rats trained to discriminate nicotine (0.4 mg/kg SC) and

midazolam (0.2 mg/kg SC). Administering different amounts of the mixture,

while keeping constant the ratio of the doses of the component drugs, yielded a

progressive increase in the percentage of drug-appropriate responding

(Stolerman et al. 1987). The dose-response curve resembled typical curves for

subjects trained on single drugs. Nicotine administered alone, up to the amount

in the mixture used for training (i.e., in doses of 0.04-0.4 mg/kg), increased the

percentage of drug-appropriate responding in a dose-related manner. Figure 4

284
FIGURE 3. Tests of responses to the training drugs in four groups of rats

trained to discriminate different mixtures of drugs from saline (n =

8-10, AND discrimination). In each case, results are shown for

saline (SAL), mixtures of drugs (MIX), and component drugs, as

follows. Upper left: amphetamine (0.4 mg/kg, AMP) and

pentobarbitone (10 mg/kg, PEN). Upper right: morphine (3 mg/kg,

MOR) and nicotine (0.4 mg/kg, NIC). Lower left: pentazocine (8

mg/kg, PZ) and tripelennamine (8 mg/kg, TR). Lower right:

caffeine (20 mg/kg, CA) and phenylpmpanolamine (20 mg/kg, PP).

285
FIGURE 4. Dose-response curves from rats trained to discriminate a mixture

of nicotine (0.4 mg/kg) and midazolam (0.2 mg/kg) from saline (n

= 9, AND discrimination). Results are shown for discriminative

effects of mixtures with the dose ratio for component drugs held

constant for nicotine alone (X), and for midazolam alone

Responding after saline is also shown (O). After large doses of

drugs some animals did not respond sufficient/y for discriminative

effects to be assessed and data are not shown where n < 7. All

data are shown as means ± SEM, with overlapping SEM omitted

for clarity. (Data from Stolerman et al. 1987.)

shows that the scores were slightly below those for the mixture. Midazolam

(0.02-0.2 mg/kg) also increased drug-appropriate responding in a dose-related

manner, and again the scores were below those for the mixture. However,

increasing the dose of midazolam to 0.36 mg/kg (i.e., above the dose in the

training mixture) further increased drug-appropriate responding, which then

286
approached the highest levels obtained after administering mixtures. Data for

nicotine at doses above that used for training could not be obtained because

such doses reduced response rates too severely. Both nicotine and midazolam

reduced response rates in these experiments; their effects in mixtures were not

additive, but were between those seen with either drug separately. Stolerman et

al. (1987) have described the changes in response rates in detail.

Similar experiments have been carried out in rats trained to discriminate

amphetamine (0.5 mg/kg SC) and pentobarbitone (12 mg/kg SC) from saline.

Figure 5 shows that the mixture increased drug-appropriate responding in a

dose-related manner. Amphetamine (0.05-0.73 mg/kg SC) and pentobarbitone

(1.2-18 mg/kg SC) given separately also increased drug-appropriate

responding, although in each case the effect was less than for amounts of the

mixture containing the same dose of each drug. When either of these drugs

was given at a dose above that used for training, drug-appropriate responding

approached the highest levels seen after administering the mixture. However,

the number of rats for which these data could be obtained was reduced

because the large doses of the drugs severely reduced overall response rates.

These experiments confirm that rats trained to discriminate mixtures of drugs

can respond to the discriminative effects of the component drugs when they are

given separately. Furthermore, the responses are dose related and, in some

instances, there is almost complete generalization from mixtures to components.

Studies With Antagonists

A further potentially powerful approach to analyzing discriminative effects of

mixtures of drugs utilizes specific pharmacological antagonists. Stolerman et al.

(1987) examined the effects of a nicotine antagonist and a benzodiazepine

antagonist in rats trained with a mixture of nicotine and midazolam. These

animals were trained to discriminate a mixture of nicotine (0.4 mg/kg SC) and

midazolam (0.2 mg/kg SC) from saline. Tests with the benzodiazepine

antagonist flumazenil (Ro 15-1788) and the nicotine antagonist mecamylamine

showed that there was no generalization to the antagonists, either when they

were administered separately or together as a mixture (figure 6). Administering

either flumazenil or mecamylamine reduced the discriminative effects of the

mixture of nicotine and midazolam, but the percentage of drug-appropriate

responding did not fall below about 60-70 percent. However, administering the

antagonists together completely blocked the discriminative effect of the mixture.

The doses of antagonists used had previously been shown to be more than

287
FIGURE 5. Dose-response curves from rats trained to discriminate a mixture

of amphetamine (0.5 mg/kg and pentobarbitone (12 mg/kg) from

saline (n = 10, AND discrimination). Results are shown for

discriminative effects of mixtures with the dose ratio for

component drugs held constant for amphetamine alone (X),

and forpentobarbitone alone ( ). Responding after saline is also

shown (0). After large doses of drugs some animals did not

respond sufficiently for discriminative effects to be assessed and

data are not shown where n < 4. All data are shown as means ±

SEM, with overlapping SEM and those smaller than diameters of

symbols omitted for clarity.

sufficient to fully block the discriminative effects of their respective agonists in

rats trained in conventional, single-drug discrimination procedures (Stolerman

et al. 1983, 1966).

288
FIGURE 6. Tests with antagonists in rats trained to discriminate a mixture of

nicotine (0.4 mg/kg) and midazolam (0.2 mg/kg) from saline (AND

discrimination). Left section shows results of tests for

generalization with saline (SAL), flumazenil (20 mg/kg, FL),

mecamylamine (1 mg/kg, ME), and both antagonists at these

doses (FL + ME). Right section shows tests for antagonism of

training mixture by the same doses of flumazenil and

mecamylamine. All data were obtained in 5-min extinction tests

and are expressed as mean percentages of drug-appropriate

responding ± SEM. (Data from Stolerman et al. 1987.)

It is notable that neither the benzodiazepine antagonist nor the nicotine

antagonist alone produced more than a marginal and unconvincing block of the

discriminative effects of the mixture, despite the fact that each antagonist alone

blocked an important element of the stimulus complex. Such marginal degrees

of antagonism are commonly interpreted as negative outcomes in pretreatment

289
studies. The present findings suggest that when a drug produces a compound

interoceptive stimulus, weak degrees of antagonism may reflect real effects that

should not always be ignored. Appel et al. (1978) reported that the

discriminative effects of pentazocine were fully blocked by a mixture of a

dopamine antagonist and a narcotic antagonist, and that either drug alone

produced only partial blockade.

Much more extensive work will be needed to develop fully the insights that may be

attainable through the use of antagonists in such studies. The data available are

very restricted; even with the benzodiazepine and nicotine antagonists employed in

the study detailed above, there are no dose-response data available for mixture

experiments. Such work will need to include cross-antagonism tests to determine

whether antagonists are fully selective in their effects on the appropriate agonists.

The effects of antagonists in cases where the training drugs interact may be

particularly complex, and they may also depend on the methods used to establish

the discrimination in the first place.

Role of Training Dose Ratio

Results are presented for three studies with generally similar designs but

involving different mixtures of drugs. The first study examined discrimination of

mixtures of nicotine and midazolam and the sequence of training doses studied

is shown in table 1; the doses of both drugs were varied in the course of the

study. In this experiment, midazolam was injected 5 min before sessions.

Discrimination between saline and the mixtures was well maintained throughout

and was not greatly dependent on the dose ratio used to maintain the

discrimination (Garcha and Stolerman 1989).

TABLE 1. Sequence of training doses used to maintain discrimination of a

nicotine-midazolam mixture in rats (n = 8).a

Test Set Nicotine (mg/kg) Midazolam (mg/kg) Ratio

1 0.20 0.10 2.0

2 0.32 0.10 3.2

3 0.32 0.06 5.3

4 0.32 0.04 8.0

5 0.32 0.10 3.2

aA set of extinction tests was carried out after a minimum of 12 sessions of discrimination training

with each mixture and saline. All drugs were given by subcutaneous injection.

290
At first, the discrimination was maintained by a mixture in which the ratio of the

doses (nicotine:midazolam) was 2:1. Extinction tests with the drugs given

separately showed that the response to the midazolam was much greater than

that to the nicotine (figure 7). Thus, at these doses, midazolam seemed to

produce a more salient stimulus than nicotine. When the dose ratio was

increased to 3.2:1, there was a strong discriminative response to each drug.

Further increases in the dose ratio enhanced the response to nicotine and

decreased the response to midazolam. With a dose ratio of 8:1, the

discrimination of the mixture could be attributed mainly to the nicotine and the

role of the midazolam was minimal. Thus, a fourfold change in dose ratio, from

2:1 to 8:1, was sufficient to reverse the relative contributions of the two drugs to

stimulus control by the mixture. The final set of extinction tests was carried out

after reinstating a training dose ratio of 3.2:1. As in the initial tests with this dose

ratio, the responses to the nicotine and midazolam were approximately equal in

magnitude, suggesting that the nonrandom sequence of training doses was not

a major confounding factor (Garcha and Stolerman 1989).

The next experiment examined discriminations maintained by mixtures of

amphetamine and pentobarbitone. In this study, the dose of one drug,

pentobarbitone, was varied while the dose of the other drug, amphetamine, was

held constant at 0.4 mg/kg (SC). At first, discrimination was maintained by a

mixture in which the ratio of the doses (pentobarbitone:amphetamine) was

12.5:1; extinction tests with the drugs given separately showed that the

response to the amphetamine was much greater than that to the pentobarbitone

(figure 8). Thus, at these doses, amphetamine seemed to produce a more

salient stimulus than pentobarbitone. When the dose ratio was increased to

25:1, there was a strong discriminative response to each drug. Further

increases in the dose ratio enhanced the response to pentobarbitone and

decreased the response to amphetamine. With a dose ratio of 50:1, the

discrimination of the mixture could be attributed mainly to the pentobarbitone

and the role of the amphetamine was minimal. Thus, a fourfold change in dose

ratio, from 12.5:1 to 50:1, was sufficient to reverse the relative contributions of

the two drugs to stimulus control by the mixture.

The final experiment of this type examined discriminations maintained by

mixtures of morphine and nicotine. In this study, the dose of one drug, nicotine,

was varied while the dose of the other drug, morphine, was held constant at 3

mg/kg (SC). At first, the discrimination was maintained by a mixture in which the

dose of nicotine was 0.1 mg/kg (SC); extinction tests with the drugs given

separately showed that the response to the morphine was much greater than

291
FIGURE 7. Discriminative stimulus effects of nicotine and midazolam in rats

trained to discriminate mixtures of these drugs from saline (n = 8).

Responses to nicotine, midazolam, and mixtures are shown for

four sets of tests carried out while stimulus control was

maintained by mixtures containing different doses of drugs (AND

discrimination). Responding after saline is also shown. Abscissa,

ratio of dose of nicotine to that of midazolam, with data shown for

ratios of 2:1, 3.2:1, 5.3:1, and 8:1 (doses of both drugs were

varied, as detailed in table I). Ordinate, responses on

drug-appropriate bar expressed as percentage of total responses

on both bars. All data were obtained in 5-min extinction tests.

Results are means ± SEM, with overlapping SEM and those

smaller than diameters of symbols omitted for clarity. (Data from

Garcha and Stolerman, 1989.)

282
FIGURE 8. Discriminative stimulus effects of amphetamine and

pentobatbitone in rats trained to discriminate mixtures of these

drugs from saline (n = 8). Responses to these drugs are shown

for five sets of tests carried out while stimulus control was

maintained by mixtures containing different doses of

pentobarbitone (AND discrimination). Dose of amphetamine was

0.4 mg/kg throughout. Responding after saline is also shown.

Abscissa: dose of pentobarbitone. Ordinate: responses on

drug-appropriate bar expressed as percentage of total responses

on both bars. All data were obtained in 5-min extinction tests.

Results are means ± SEM, with overlapping SEM and those

smaller than diameters of symbols omitted for clarity.

that to the nicotine (figure 9). Thus, at these doses, morphine seemed to

produce a more salient stimulus than nicotine. When the dose of nicotine was

increased to 0.4 mg/kg, there was a strong discriminative response to each

drug. A further increase in the dose of nicotine enhanced the response to

293
FIGURE 9. Discriminative stimulus effects of morphine and nicotine in rats

trained to discriminate mixtures of these drugs from saline (n = 8).

Responses to these drugs are shown for four sets of tests carried

out while stimulus control was maintained by mixtures containing

different doses of nicotine (AND discrimination). Dose of morphine

was 3 mg/kg throughout. Responding after saline is also shown.

Abscissa: dose of pentobarbitone. Ordinate: responses on

drug-appropriate bar expressed as percentage of total responses

on both bats. All data were obtained in 5-min extinction tests.

Results are means ± SEM, with overlapping SEM and those

smaller than diameters of symbols omitted for clarity.

nicotine and decreased that to morphine, but there was still a marked response

to the morphine alone. Thus, in these experiments, even an eightfold increase

in dose ratio was not sufficient to completely reverse the relative contributions

of the two drugs to stimulus control by the mixture.

294
These experiments demonstrate the critical importance of the ratio between the

training doses of drugs in mixtures as a determinant of the characteristics of the

cue obtained. Relatively small fourfold shifts in dose ratio were sufficient to

entirely reverse the dominant component in cues produced with mixtures of

nicotine and midazolam and amphetamine and pentobarbitone. With mixtures

of nicotine and morphine, even an eightfold change in the dose ratio had a less

pronounced effect, although similar trends were apparent. The reason for this

difference is unclear. The studies reported should be regarded as only a

preliminary exploration of the role of training doses in discrimination of mixtures.

At least three major manipulations remain unexamined. These include (1)

dose-response determinations by means of extinction tests at different training

dose ratios and (2) the consequences of training with relatively large or small

amounts of mixtures with the dose ratio held constant. It can also be expected

that (3) profiles of generalization to other compounds will be critically linked to

training dose ratios and amounts.

Specificity of Cues Based on Mixtures

Koek and Slangen (1982) and Overton (1982) have shown that under certain

conditions the specificity of discriminations based on single drugs may be

influenced by training the alternative response not simply with vehicle but with

multiple drugs (in different sessions). Although the functional parallel is inexact,

this raises the possibility that training a discrimination with two or more drugs in

a mixture may influence specificity. Studies to test this issue have been carried

out in rats trained to discriminate mixtures of nicotine and midazolam and of

amphetamine and pentobarbitone from saline.

Figure 10 shows the results of dose-response (generalization) tests with

amphetamine, morphine, and quipazine in rats trained to discriminate nicotine

(0.32 mg/kg SC) and midazolam (0.1 mg/kg SC) from saline. In this experiment,

midazolam was injected 5 min before sessions. The test drugs did not increase

the percentage of drug-appropriate responding above the level associated with

injections of saline. The responses to saline and to the mixture of nicotine and

midazolam confirmed the persistence of stimulus control by the training mixture

throughout the period of tests for specificity. Amphetamine, morphine, and

quipazine all reduced the total numbers of responses in a dose-related manner

(Garcha and Stolennan 1989).

Similar experiments have been carried out in rats trained to discriminate

amphetamine (0.5 mg/kg SC) and pentobarbitone (12 mg/kg SC) from saline.

295
FIGURE 10. Tests of responses to novel drugs in rats trained to discriminate a

mixture of nicotine (0.32 mg/kg) and midazolam (0.1 mg/kg) from

saline (n = 8, AND discrimination). Results are shown for different

doses of amphetamine, quipazine, and morphine. Abscissa: dose

of drug. Ordinates: responses on drug-appropriate bar expressed as

a percentage of total responses. Control data above 0 on the

abscissa show responses to saline and the mixture used for training

as means for three sets of such tests carried out during work with the

novel drugs. (Data from Garcha and Stolerman, 1989.)

The results were compared with those obtained in two additional groups of rats

trained to discriminate the same doses of amphetamine or pentobarbitone from

saline in a conventional, single-drug discrimination procedure. Tests for

generalization to morphine, quipazine, and phencyclidine were carried out.

Generalization to a test drug was never greater in rats trained on the mixture

than in rats trained on the component drugs. All drugs were tested at doses that

markedly reduced overall rates of responding.

296
The results of the tests to date suggest that there is no general loss of

specificity that is necessarily associated with the use of mixtures for training

discriminations. The possibility of certain unusual profiles of generalization with

particular mixtures of drugs that interact cannot be precluded. It would also be

reasonable to expect generalization to many drugs that normally share stimulus

properties with either training drug, possibly resulting in generalization to a

wider range of compounds than would typically be the case for single-drug

discriminations.

Another Paradigm for Mixtures: The AND-OR Discrimination

The findings outlined above may be related to the use of one particular

paradigm for training the preceding discriminations based on drug mixtures.

This paradigm is laid out in figure 1 (upper right) according to the scheme of

Järbe and Swedberg (1982). Several other paradigms are also possible and the

lower left section of figure 1 illustrates one on which work has begun. In this

procedure, subjects are trained to discriminate the simultaneous presence of

two drugs (AND) from the presence of either drug alone (OR), called the

AND-OR discrimination. According to this nomenclature, the preceding work

with mixtures may be called the AND discrimination. It may be noted that OR

discriminations have been established previously and in these cases OR

referred to one of the following situations: either of two drugs was associated

with one response and vehicle with another response (Colpaert and Janssen

1982); one of several drugs was associated with one response and just one

drug with another response (Overton 1982); or three doses of one drug were

associated with one response and vehicle with the other response (Stolerman

et al. 1984). Therefore, it seemed that there would be no fundamental difficulty

in establishing the OR element of the discrimination, but there appeared to be

no previous report of an AND-OR discrimination.

The first question about the AND-OR discrimination was simply whether it could

be established. This question has been answered positively because rats have

been trained to discriminate amphetamine (0.4 mg/kg SC) and pentobarbitone

(10 mg/kg SC) from the same doses of amphetamine or pentobarbitone. This

AND-OR discrimination was acquired more slowly than AND discriminations

studied previously with the same drugs, but mean accuracy for all 10 rats

reached 83 percent after 48 training sessions. Dose-response curves for

amphetamine, pentobarbitone, and the mixture were then determined in the

eight most accurate rats and figure 11 shows the main findings. Different

amounts of the mixture (with the ratio between the doses of the component

297
drug held constant) produced increasing amounts of mixture-appropriate

responding, up to a maximum of 90 percent. None of the tested doses of either

amphetamine or pentobarbitone produced any appreciable amount of

mixture-appropriate responding. Thus, the capacity of the rats to perform the

AND-OR discrimination was confirmed and the distinction between mixture and

component drugs was maintained even when the doses of component drugs

were increased slightly above those used for training. Figure 11 shows another

very intriguing characteristic of this AND-OR discrimination: Saline was

generalized with the single drugs since it failed to produce any appreciable

amount of mixture-appropriate responding.

From the preceding results, it would appear that rats trained in an AND-OR

discrimination procedure can treat the effects of drug mixtures as distinctly

different from those of any dose of the component drugs. It is debatable

whether the mixture should be considered as producing a novel, distinct

stimulus under such conditions, but operationally the rats behaved as if it did.

Whether most drug mixtures can function in such a way remains to be

determined; amphetamine-barbiturate mixtures can produce unusual patterns of

behavioral effects under other conditions (Branch 1974; Rushton and Steinberg

1968) and this form of drug interaction may be contributing to the acquisition

and performance of the AND-OR discrimination.

After completion of the dose-response determinations described above, the

training procedure for all 10 rats was changed to the AND procedure used in all

the earlier studies. The doses of amphetamine and pentobarbitone associated

with one response were held constant as before, but the second response was

associated with saline. After 22 training sessions under these conditions, the

dose-response determinations were repeated. Discrimination between mixture

and saline remained strong and dose related, as before (96 percent accuracy).

Amphetamine alone failed to increase mixture-appropriate responding

appreciably (maximum of 24 percent). Pentobarbitone slightly increased

mixture-appropriate responding, but to no more than 46 percent. Thus, a

previous history of training under AND-OR conditions greatly influenced the

characteristics of a discrimination based on a drug mixture, as compared with

its characteristics in rats trained and maintained on an AND discrimination

throughout their experimental history (as described earlier). Barrett and

Olmstead (1989) have described how testing history influences the

characteristics of a discrimination based on spiroxatrine, a drug that acts as a

dopamine antagonist and a serotonin agonist. Studies are now needed to

298
FIGURE 11. Dose-response curves from rats trained to discriminate a mixture

of amphetamine (0.4 mg/kg) and pentobarbitone (10 mg/kg) from

either drug separately (n = 8, AND-OR discrimination). Results

are shown for discriminative effects of mixtures with the dose ratio

for component drugs held constant amphetamine alone (X),

and pentobarbitone alone Responding after saline is also

shown (0). After large doses of drugs some animals did not

respond sufficiently for discriminative effects to be assessed and

data are not shown where n < 4. All data shown as means ± SEM,

with overlapping SEM and those smaller than diameters of

symbols omitted for clarity. These results should be compared

with those for similar experiments in rats trained in an AND

discrimination with the same drugs.

investigate the role of behavioral history in the discrimination of drug mixtures in

a more systematic manner.

299
DISCUSSION

Orderly Form of the Data

The most surprising feature of the results is their orderly nature despite the

complexity of the experiments and the use of several mixtures of drugs thought

to have complex, interactive effects. Thus, readily interpretable findings have

been obtained in tests of generalization to component stimuli, in dose-response

studies, in pilot studies with antagonists, in tests of generalization to novel

drugs, and when training dose ratios have been altered. This orderliness bodes

well for the further use of drugs when analyzing the processing of compound

interoceptive stimuli.

The AND Discrimination

In all cases of the AND discrimination studied to date, involving nine drugs in

five different mixtures, rats trained with the mixtures have responded strongly to

the component drugs. Rats therefore seem to identify and process the mixture

stimulus on the basis of its elements rather than as a new and homogeneous

entity. This result was obtained first with drugs that are not abused as a mixture,

which on theoretical and empirical grounds are thought to produce different,

nonoverlapping stimuli, and which do not interact pharmacologically (i.e.,

nicotine and midazolam; Stolerman et al. 1987). These findings have now been

extended to include drugs that are abused as mixtures and are thought to

interact in complex ways that await full definition (amphetamine and

pentobarbitone, pentazocine and tripelennamine, caffeine and

phenylpropanolamine). All these consistent results have been obtained with the

AND discrimination (drug A and B versus vehicle); this functional relationship

may be crucially important.

The AND-OR Discrimination

Results for amphetamine and pentobarbitone in rats trained in an AND-OR

discrimination suggest a radically different picture. In this case, total

discrimination between a mixture and its elements was obtained, even in

dose-response studies. There was no generalization of responding between

mixture and component drugs regardless of the dose of the latter. Such a result

is, perhaps, not surprising in view of the training conditions imposed.

Nevertheless, this complex discrimination was learned with little difficulty, and it

supports the view that under particular conditions of use mixtures may have a

300
unique profile of behavioral effects. It will be important to determine the

generality of this finding with regard to other classes of drugs, to examine yet

other paradigms involving mixtures, and to test whether training that yields

different discriminative effects also influences other effects of the drugs (e.g., on

response rates) and the receptors on which they act.

History

Different behavioral and pharmacological histories can markedly influence

reactions to drugs (Barrett et al. 1989). In a more limited context, this principle

also applies to discriminative stimulus effects (Barrett and Olmstead 1989). The

present experiments demonstrate the role of behavioral pharmacological history

in a novel, orderly, and predictable manner. Rats transferred from AND-OR

discrimination to AND discrimination responded to amphetamine and

pentobarbitone in a manner radically different from rats maintained on an AND

discrimination throughout their experimental history. The characteristics of the

AND-OR discrimination persisted to a great extent after the programmed

contingencies were changed. The limits of this phenomenon need to be fully

explored. Within the context of drug abuse, it could imply that the manner in

which the effects of mixtures are perceived will depend on the precise

circumstances surrounding their previous use. It is noteworthy that possibly the

first demonstration of a history effect in behavioral pharmacology also used an

amphetamine-barbiturate mixture (Steinberg et al. 1961).

Overshadowing and Blocking

Interactions between drugs are normally interpreted in pharmacological terms,

by means of pharmacodynamic and pharmacokinetic processes. There are

complementary mechanisms based on behavioral constructs such as stimulus

masking, overshadowing, and blocking (Mackintosh 1974). These constitute a

rich new field with enormous potential for adding to our knowledge of

interactions between both clinically used and abused mixtures of drugs.

Stimulus masking has not appeared to be a pervasive phenomenon in drug

discrimination experiments, but Gauvin and Young (1989) have demonstrated

its occurrence with amphetamine and morphine in certain conditions. Järbe et al

(1983a, 1983b, 1989) have clearly demonstrated mutual overshadowing and

blocking of drug-produced discriminative stimuli by conditioning with more

salient exteroceptive stimuli. No studies, however, seem to have explicitly

looked for overshadowing and blocking between pairs of drug-produced stimuli

301
(i.e., with drug mixtures). The present experiments contain indications that such

phenomena can occur and are worthy of systematic study.

Normally, rats can reliably discriminate nicotine in doses of 0.1-0.2 mg/kg from

saline (Stolerman et al. 1984). However, very little stimulus control accrued to

these doses of nicotine when conditioning occurred in the presence of

midazolam (figure 7) or morphine (figure 9). Similarly, figure 8 shows that little

stimlus control accrued to amphetamine (0.4 mg/kg) in the presence of

pentobarbitone (20 mg/kg), and figure 9 shows that nicotine (0.8 mg/kg)

weakened stimulus control by morphine (3 mg/kg). It is suggested that

overshadowing may be the common mechanism underlying these diverse

interactions. Overshadowing (Mackintosh 1974) is shown as weakening of

conditioning to a normally adequate stimulus by conditioning with a compound

stimulus in which there is another, more salient element. It is thought unlikely

that pharmacological interactions could account for the observations because

the drugs used in the mixtures are not agonist-antagonist pairs and are thought

to act through diierent receptor mechanisms. Extensive tests for

pharmacological interactions between nicotine and midazolam have been

carried out in drug discrimination experiments under conditions similar to the

present work, and they have yielded negative results (Stolerman et al. 1987).

There are, however, some indications that amphetamine may weaken a

pentobarbitone stimulus and that pentobarbitone may weaken an amphetamine

stimulus (Kline and Young 1986; Mariathasan et al. 1990). These are rather

fragile interactions with limited generality and they may reflect either a

pharmacological mechanism or stimulus masking. Fully controlled

overshadowing and blocking experiments like those carried out with

exteroceptive discriminative stimuli are needed to clarify the issues raised by

the present findings.

Overshadowing may also be a key factor in the acquisition of discriminations

produced by single drugs with multiple effects; it may contribute to the general

tendency for such discriminations to be based on the major effects of the drugs,

rather than on an extremely complex mixture of main effects and side effects.

Clinically Used Mixtures of Drugs

The complexities and uncertainties of psychiatric diagnosis, coupled with the

limited clinical efficacy of many psychoactive drugs, lead many clinicians to

prescribe mixtures of drugs. Often, little psychopharmacological study has been

done on such mixtures because there were no compelling reasons to expect

302
any exceptional interactions between their components. The coexistence of

depressive symptoms with those of schizophrenia and anxiety is common,

leading to the combined use of neuroleptics, antidepressants, and anxiolytics in

different permutations. Studies of the discriminative stimulus properties of

mixtures of such drugs may contribute to understanding the effects of such

widely used but poorly understood mixtures.

CONCLUSIONS

There are numerous areas of application for discriminations based on mixtures

of drugs; the phenomenon is not a laboratory curiosity without practical

relevance. It provides an approach to identifying general principles that underlie

the perception and processing of compound interoceptive stimuli. It may help us

understand why certain mixtures of drugs are particularly prone to abuse as

mixtures, which is one aspect of the pervasive phenomenon of polydrug abuse

that has often been neglected in laboratory studies. Drug discrimination based

on mixtures of drugs may shed new light on certain clinically used mixtures of

drugs, and it will provide the methodology needed to test the roles of

overshadowing and blocking in ways that may extend and complement classical

pharmacological accounts of drug interactions. It also provides a novel

approach to testing how behavioral and pharmacological histories of subjects

may combine to produce marked individual variations in response to mixtures of

drugs. Finally, much of what has been said about mixtures of drugs may also

apply to single drugs with multiple effects.

ACKNOWLEDGMENT

These studies were carried out with support from grant DA-05543 from the

National Institute on Drug Abuse.

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Authors

I. P. Stolerman, Ph.D.

E.A. Mariathasan, Ph.D.

H.S. Garcha, I.A.T.

Department of Psychiatry

Institute of Psychiatry

De Crespigny Park

London SE5 8AF UK

306
Application of Drug Discrimination With

Drugs of Abuse To Develop New Therapeutic

Agents

Theo Frans Meert

INTRODUCTION

The drug discrimination test procedure is a behavioral technique that can

preclinically characterize the subjective effects of known and new substances.

The test is based on a classical conditioning paradigm in which a drug is used

as a conditioning stimulus. The internal effects (or cueing properties) evoked by

a drug are used as discriminative stimuli for different ways of responding.

Typically, in order to obtain food pellets, food-deprived rats are required to

press one lever after treatment with the training drug and another lever after

treatment with saline (or the vehicle).

The drug discrimination procedure has several advantages compared to

classical behavioral tests, in that it is completely standardized with fixed

parameters and well-defined conditions. The procedure is not limited to

interactions at one particular neurotransmitter system. Depending on the

training drug employed, responding may represent interactions at various

systems including serotonin, dopamine, acetylcholine, noradrenaline, and

opiate. Also, compounds acting at different receptor subtypes of one

neurotransmitter system can be studied and differentiated. Furthermore,

complex stimuli without any obvious behavioral reaction and often not limited to

a single neurotransmitter system also fall within the scope of this procedure.

Often, drugs of abuse are used as training drugs. For several of these

compounds, a correlation has been demonstrated between the similarity of the

discriminative stimulus properties of these drugs in rats and their specific

subjective effects in humans (Colpaert and Slangen 1982). For instance, it was

demonstrated that stimulus generalization with lysergic acid diethylamide (LSD)

307
in rats represents a necessary, albeit insufficient, condition to predict

hallucinogenic activity in man (Appel et al. 1982; Glennon et al. 1984). Drug

discrimination studies with drugs of abuse in animals, and also in humans, may

have some predictive value for the abuse potential of new chemical

substances. Typically, generalization experiments are performed to test

whether a new compound is recognized by rats trained on standard drugs of

abuse. The same drug discrimination training also allows the characterization of

new therapeutic agents. In antagonism studies, compounds are tested for their

ability to attenuate the stimulus effects of the drugs of abuse. This process can

identify antidotes for the drugs of abuse and also clinically relevant new

compounds.

In order to demonstrate the usefulness of the drug discrimination test

procedure, examples using new neuroleptic and antiepileptic agents will be

presented. Ritanserin, a selective serotin-2 (5-HT2) antagonist (Awouters et al.

1988) with abuse-reducing properties in rats (Meert et al. 1990a, 1990b) will

also be considered.

In all studies reported, rats used a two-lever, food-reinforced drug discrimination

test procedure. The equipment, training, and test procedures have been

described in detail in previous papers (Meert et al. 1989, 1990c).

USE OF DRUG DlSCRIMlNATlON TO DETECT NEW NEUROLEPTIC

AGENTS

All clinically active neuroleptics, such as haloperidol, have the ability to

antagonize central dopamine (DA) receptor activation (Niemegeers and

Janssen 1979). Clinically, these drugs are effective against the positive but not

the negative symptoms of schizophrenia. They all have the risk of inducing

extrapyramidal side effects (EPS) (Berger 1978). Selective 5-HT2 antagonists

such as ritanserin, given in combination with neuroleptics, can overcome the

negative symptoms of schizophrenia and reduce EPS (Gelders 1989;

Reyntjens et al. 1986). Blockade of 5HT2 receptors seems to increase

therapeutic activity and reduce the side effects of D2 receptor blockade.

Risperidone is a potent serotonin 5-HT2 and D2 antagonist. Its serotonin

antagonism is more pronounced than its antidopaminergic activity (Janssen et

al. 1988). Clinically, risperidone has been shown to be active against both the

positive and negative symptoms of schizophrenia, with a very low incidence of

EPS (Casteleao et al. 1989; Mew et al. 1989; Mesotten et al. 1989). Recently,

R 79 598, a benzisoxazolyl derivative (Janssen et al. 1990), was characterized

308
as a potent neuroleptic with equipotent 5-HT2

 and D2 antagonist properties. In

rats, R 79 598 was at least as potent as haloperidol and several times more

potent than risperidone in inhibiting dopaminergic overactivity. In dogs, R 79

598 was even more potent than haloperidol. In order to further characterize the

pharmacological profile of R 79 598 and to preclinically confirm its possible

neuroleptic activity, the drug was compared with risperidone and haloperidol in

the drug discrimination test procedure. The selected training drugs included

LSD, 1-(2,5-dimethoxy-4-methyphenyl)-2-aminopropane (DOM), cocaine, and

d-amphetamine.

The serotonin agonist DOM was selected because it is a 5-HT2 agonist that can

produce hallucinations in humans (Glennon et al. 1983, 1984). LSD produces

cardiovascular changes and feelings of tension, anxiety, and depressed mood

(Cohen 1967; Freedman 1968, 1969) as well as hallucinations, especially at

high doses (Isbell 1959). Potent LSD antagonists such as risperidone (Meert et

al. 1989) have been clinically shown to possess antipsychotic activity. Both

d-amphetamine and cocaine produce stimulatory effects with psychotic-like

symptoms in man (Beamish and Kiloh 1960; McLellan et al. 1979). There are,

however, basic differences in the stimulus properties of cocaine and

&hetamine. Whereas D2 antagonists such as haloperidol can antagonize

the discriminative stimulus effects of d-amphetamine, these compounds only

partially antagonize the cocaine cue (Meert et al. 1990c). The selected training

conditions in this study included a discrimination of either 0.63 mg/kg DOM

(intraperitoneal [IP]; time [T]-15 min), 0.16 mg/kg LSD (IP; T-15 min), 10.0

mg/kg cocaine (IP; T-15 min), or 1.25 mg/kg d-amphetamine (subcutaneous

[SC]; T-30 min) versus saline.

Within each training group, complete dose-response functions for stimulus

generalization and antagonism were established for haloperidol, risperidone,

and R 79 598. The stimulus generalization experiments were performed after

1 hr whereas for the antagonism studies, time intervals between 1 and 8 hr

were used. Also, the ED50 values for stimulus generalization of the training

drugs with the training condition were established.

In terms of stimulus generalization (table 1), all training drugs produced a

complete stimulus generalization 1 hr after subcutaneous treatment. The ED50

value of DOM in 0.63 mg/kg DOM-trained rats was 0.17 mg/kg. For 0.16 mg/kg

LSD, 10.0 mg/kg cocaine, and 1.25 mg/kg d-amphetamine, the corresponding

ED50 values were 0.26, 1.55 and 0.51 mg/kg, respectively. Except for LSD, the

ED50 values for stimulus generalization were 2.45 (d-amphetamine) to 6.45

309
TABLE 1. ED50 values (plus 95 percent confidence limits) of the training

drugs, haloperidol (HALO), risperidone (RISP), and R 79 598, for

stimulus generalization with the training conditionsa

Training Training DrugC HALOd RISPd R 79 598d

Conditionb

(dose m mg/kg)

DOM 0.17 0.63 0.63 0.16

(063;IP;T-15) (0.11-0.25)

LSD 0.26 0.63 0.63 0.16

(O.16;IP;T-15) (0.16-0.41)

Cocaine 1.55 0.63 0.63 0.16

(10.0;IP;T-15) (1.03-2.31)

d-Amphetamine 0.51 0.63 0.63 0.16

(1.25;SC;T-30) (0.34-0.76)

aAII generalization experiments, including those of the training bugs with the mining conditions,

were performed 1 hr after subcutaneous treatment

bDOM = 1-(2,5dimethoxy-4-methylphenyl)-2-aminopropane; LSD= lysergic acid diethylamide; IP =

intraperitoneal; SC = subcutaneous.

cDoses in mg/kg.

dAll values are "greater than."

(cocaine) times lower than the training dose. For 0.16 mg/kg LSD, the ED50 for

stimulus generalization was 1.63 times higher. Two elements can account for

the higher ED50 of LSD. First, LSD may have a relatively short duration of

action. As a consequence, the dose of LSD must be increased to obtain a

generalization when the pretreatment time is increased from 15 to 60 min.

Second, the alteration in route of administration (IP for the training and SC for

the stimulus generalization conditions) may cause a difference in ED50 values.

Neither R 79 598, risperidone, nor haloperidol resulted in stimulus

generalization to any of the training drugs (table 1). Thus, up to doses that

severely reduced response rate, no selection of the drug lever occurred.

The antagonism studies are summarized in table 2. In terms of DOM

antagonism, the ED50 of R 79 598 to antagonize the stimulus properties of 0.63

mg/kg DOM after 1 hr was 0.0027 mg/kg. For risperidone, the corresponding

ED50 was 0.024 mg/kg. Haloperidol, at doses up to 0.63 mg/kg, which clearly

reduced response rate, did not antagonize DOM. A similar result was obtained

310
TABLE 2. Antagonism studies of R 79 598, risperidone (REP), and

haloperidol (HALO) on DOM, LSD, cocaine, and d-amphetaminea

Training Drug R 79 598c RISPC HALOC

Treatmentb

Time (hr)

DOM (0.63

mg/kg;lP/T-15)

LSD (0.16

mg/kg;lP;T-15)

4

8

Cocaine (10.0 1

mg/kg;lP;T-15) 2

d-Amphetamine 1

(1.25mg/kg;SC;T-30)

2

0.0027 0.024 > 0.63

(0.0018-0.0040) (0.016-0.037)

0.097 0.028 > 0.63

(0.072-0.13) (0.015-0.051)

0.021 0.028 > 0.63

(0.01-0.043) (0.015-0.051)

20% at 0.63 0.064

(0.037-0.12)

0.45

(0.23-0.85)

> 0.31 > 2.50 40% at 0.63

60% at 0.63 40% at 2.50 > 0.63

40% at 0.31 0.34 0.13

(0.21-0.54) (0.086-0.19)

0.043

(0.0264069) (0.24-.81) (9.091-0.14)

aED50 values (plus 95% confidence limits) are presented over time for antagonism of the training

condition or the maximal percentage of blockade measured.

bDOM = 1-(2.5-dimethoxy-4-methylphenyl)-2-aminopropane; LSD = lysergic acid diethylamide; IP

= intraperitonsal; T = time in min; SC = subcutaneous.

cDoses in mg/kg, administered subcutaneously.

with haloperidol against LSD after both 1 and 2 hr. On the contrary, risperidone

completely antagonized LSD with a lowest ED50 of 0.028 mg/kg after both 1 and

2 hr. Even after 8 hr, there was still a complete antagonism with an ED50 of 0.45

mg/kg. Risperidone is therefore characterized as a potent and long-acting LSD

antagonist (Meert et al. 1989). The lowest ED50 of R 79 598 for LSD

antagonism was 0.021 mg/kg after 2 hr. After 4 hr, however, only a partial

antagonism of 20 percent was obtained at 0.63 mg/kg. Therefore, R 79 598 was

as potent as risperidone in terms of the lowest ED50, but the drug had a shorter

duration of action. The data presented here on the antagonism of LSD confirm

the idea that the discriminative stimulus properties of LSD are both serotonin311
(especially 5HT2

) and catecholamine-(D2 and noradrenaline) mediated (Meert

et al. 1989, 1990c).

None of the tested compounds completely antagonized the discriminative

stimulus properties of 10.0 mg/kg cocaine. A partial antagonism of 40 percent

was obtained with 0.63 mg/kg haloperidol (1 hr) and with 2.50 mg/kg

risperidone (2 hr). At 0.63 mg/kg R 79 598, there was a partial antagonism of 60

percent (2 hr). These results confirm earlier studies indicating that the

discriminative stimulus properties of 10.0 mg/kg cocaine cannot simply be

antagonized by either D2 or mixed D2/5-HT2 antagonists (Meert et al. 1989,

1990c).

In terms of an antagonism of the discriminative stimulus properties of 1.25

mg/kg d-amphetamine, a complete antagonism was obtained with all three test

compounds. The ED50 values of haloperidol after 1 and 2 hr were 0.13 and 0.11

mg/kg, respectively. For risperidone the corresponding values were 0.34 and

0.44 mg/kg. R 79 598 only partially antagonized the cueing properties of 1.25

mg/kg d-amphetamine after 1 hr. At 0.31 mg/kg R 79 598, a dose with

rate-reducing effects, only a 40 percent antagonism was obtained. After 2 hr,

however, the response rate reductions were less pronounced, and a lowest

ED50 of 0.043 mg/kg was measured. Thus, in terms of lowest ED50 for

d-amphetamine antagonism, R 79 598 was 2.56 and 7.91 times more potent

than haloperidol and risperidone, respectively. Globally, these results on the

antagonism of d-amphetamine indicate that D2 antagonists are able to

antagonize the discriminative stimulus properties of 1.25 mg/kg d-amphetamine

(Meert et al. 1989).

In terms of response rate, a second parameter measured in the drug

discrimination studies, haloperidol by itself reduced response rate at doses

20.16 mg/kg. In the antagonism studies, doses 0.04 mg/kg were active.

Similar results were obtained with R 79598. However, for R 79 598, the

rate-reducing effects disappeared over time. After 2 hr, an antagonism without

effects on response rate was measured. It is therefore possible that the rate

reducing effects of R 79 598 did mask the antagonist properties, especially at

short time intervals. For risperidone, reductions in response rate were observed

in both the generalization and antagonism studies at doses 0.16 mg/kg. In

addition, response rate reductions disappeared over time, and, at intervals

2 hr, an antagonism without any rate reduction could be obtained.

312
Globally, the results presented here indicate that R 79 598 is a potent DOM,

LSD, and d-amphetamine antagonist. The data confirm earlier studies indicating

that R 79 598 has 5-HT2 and D2 antagonist properties. The antagonist

properties of R 79 598 were most pronounced after 2 hr when the effects of the

drug on response rate were minimal. As compared to haloperidol and

risperidone, lower ED50 values were obtained with R 79 598 for the antagonism

of DOM and d-amphetamine. Because DOM and d-amphetamine are

respectively, 5HT2- and D2-mediated, R 79 598 was clearly more potent than

both other drugs in terms of 5-HT2 and D2 antagonism. With regard to LSD, R

79 598 was equipotent to risperidone but showed a shorter duration of activity.

Effectiveness against both the positive and negative symptoms of

schizophrenia with a low rate of extrapyramidal side effects can be predicted for

this new compound for the following reasons: R 79 598 has D2 and 5-HT2

antagonist properties; the drug antagonizes the stimulus properties of various

compounds with psychotomimetic symptoms in man; and R 79 598 to some

extent resembles risperidone.

DRUG DISCRIMINATIVE ANALYSIS OF THE ANTIEPILEPTIC AGENT

LORECLEZOLE

Loreclezole was introduced recently as an antiepileptic agent (Wauquier et al.

1990). It is a triazole derivative with a broad spectrum of activity in different

animal species and is unrelated to prototype antiepileptic drugs. The compound

is active against audiogenic seizures and chemically or electrically induced

seizures. An antagonism of both tonic and clonic seizures was obtained.

Loreclezole increased the threshold for both behavioral and

electroencephalographic seizures. The drug was found to be nonteratogenic in

rats (Wauquier et al. 1990).

In order to further characterize the pharmacological profile of foreclezole, the

drug was compared with the antiepileptic agents clobazam and carbamazepine

and the benzodiazepine chlordiazepoxide in rats trained to discriminate either

5.0 mg/kg chlordiazepoxide (IP, T-15 min) or 20.0 mg/kg metrazol (IP, T-15

min) from saline. All generalization and antagonism studies with the four

compounds were performed 1 hr after oral treatment. For the generalization

experiments of the training drug with the training condition, data on

subcutaneous injections after 1 hr are presented.

313
The ED50 value of chlordiazepoxide (SC; 1 hr pretreatment) in 5.0 mg/kg trained

rats was 3.08 mg/kg. For metrazol the corresponding ED50 value in the 20.0

mg/kg metrazol group was 9.36 mg/kg.

In terms of generalization to 5.0 mg/kg chlordiazepoxide, a dose-related

substitution was observed with chlordiazepoxide, clobazam, and loreclezole

(figure 1). The corresponding ED50 values for stimulus generalization 1 hr after

oral treatment were 3.55, 24.73, and 18.76 mg/kg, respectively. No

generalization was observed with carbamazepine at doses up to 40.0 mg/kg. A

significant reduction in response rate was present only at 80.0 mg/kg

loreclezole.

In terms of substitution for metrazol (figure 2), none of the tested compounds

produced a stimulus generalization at doses 40.0 mg/kg after oral treatment.

Reductions in response rate were present at 10.0 and 40.0 mg/kg

chlordiazepoxide, 40.0 mg/kg carbamazepine and clobazam, and 40.0 mg/kg

loreclezole in metrazol-trained rats. In chlordiazepoxide-trained animals, rate

reductions were measured only at 80.0 mg/kg loreclezole. Thus, the training

condition appears to codetermine the degree of response rate reduction

observed with the various compounds.

Regarding the antagonism of the discriminative stimulus properties of 20.0

mg/kg metrazol (figure 3), a complete antagonism after oral treatment was

obtained with chlordiazepoxide and loreclezole. The ED50 of metrazol

antagonism was 2.69 mg/kg for chlordiazepoxide and 8.17 mg/kg for

loreclezole. Clobazam revealed a partial antagonism. At 40.0 mg/kg, the

highest dose tested, a partial antagonism (60 percent) was obtained.

Carbamazepine at doses up to 40.0 mg/kg was inactive. No rate-reducing

effects were observed for any of the four compounds at the doses tested.

The results for loreclezole in the drug discrimination test procedure indicated a

complete generalization with 5.0 mg/kg chlordiazepoxide and an antagonism

with 20.0 mg/kg metrazol. A comparable effect was obtained with the

benzodiazepine chlordiazepoxide at lower doses. Clobazam had a somewhat

similar profile. Carbamazepine, on the contrary, was completely different. These

drug discrimination data, together with the results obtained in animal models of

anxiety and in procedures measuring side effects of the benzodiazepines

(Meert et al. 1990d), support the idea that loreclezole possesses a

pharmacological profile different from catbamazepine and, to some extent,

comparable to the benzodiazepines clobazam and chlordiazepoxide.

314
FIGURE 1. Generalization experiments in 5.0 mg/kg chlordiazepoxide (IP, over 15 min) trained rats. Number

of rats selecting the drug lever and response rate are expressed as a percentage of last saline

session. Drugs were given orally 1 hr prior to testing. At each dose, results for five rats are given.

Doses and ED50 values are given in mg/kg. Statistical differences were evaluated with the

Wilcoxon test (two-tailed; * = p < .05).
FIGURE 2. Generalization experiments in 20.0 mg/kg metrazol (IP, over 15 min) trained rats. See also

legend to figure 1.
FIGURE 3. Antagonism studies in 20.0 mg/kg metrazol (IP, over 15 min) trained rats. Number of rats

selecting the drug lever and the response rate are expressed as a percentage of last drug

session. Compounds were given orally at 1 hr before testing and at 45 min before intraperitoneal

injection with metrazol. See also legend to figure 1.
Globally, the studies with both the neuroleptics and the antiepileptfc drugs

illustrate how drugs of abuse can be used to select and characterize therapeutic

agents. Besides the selection of new compounds, the drug discrimination test

procedure can also help to clarify the mechanism of action of therapeutic

agents. Furthermore, in an early phase the procedure can reveal information

about the abuse liability of a new substance. The following example illustrates

this.

DRUG DISCRIMINATIVE ANALYSIS OF RITANSERIN: A

PHARMACOLOGICAL APPROACH FOR ADDICTION

Ritanserin is a specific 5-HT2 antagonist belonging to a series of piperidine

derivatives (Awouters et al. 1988). In rats, low doses on the order of 0.1 mg/kg

antagonize the behavioral effects induced by serotonergic stimulants such as

tryptamine, mescaline, or 5-hydroxytryptophan (5-HTP). Similar doses increase

deep slow wave sleep (Dugovic et al. 1989) and markedly disinhibit behavior

induced by natural aversive stimuli (Meert and Janssen 1989). At somewhat

higher doses, an activity is observed in models used to screen antidepressants

(Meert and Janssen 1989). Normal behavior in rats is not affected up to high

doses, which indicates that central 5-HT2 antagonism by itself is

neurobehaviorally silent (Awouters et al. 1988; Leysen et al. 1989). In humans,

rftanserin also markedly increases slow wave sleep (ldzikowski et al. 1986) and

improves mood and drive in dysthymic patients (Reyntjens et al. 1986). It has

been suggested that restoration of physiological sleep promotes improvement

of neurotic symptoms (Janssen 1987, 1988). Occasional observations in the

past 5 years also indicated that ritanserin may be of value in subjects

withdrawing from drugs of abuse, who may experience problems similar to

dysthymic patients in coping with everyday activities. Therefore, experiments

were performed to test the effectiveness of rftanserin against drug abuse. A

typical example of these experiments is given in figure 4. The effects of

rftanserin versus 3 percent alcohol, 0.1 mg/ml cocaine, and 0.02 mg/ml fentanyl

are presented.

In these three experiments, rats were allowed access to a drug solution of

either alcohol, cocaine, or fentanyl as the only available drinking fluid for 10

days. On Day 11, the rats were randomly assigned to treatment groups

receiving subcutaneous injections of either vehicle or one of the various doses

of rftanserin between 0.01 and 2.5 mg/kg. Both drug solution and water were

made available in identical graduated bottles immediately following treatment.

The injections were given on 5 consecutive days at 8 a.m. Individual

318
FIGURE 4. Mean fluid intake (percent of total SEM) of drug solution or wafer during the period of treatment

with vehicle or various doses of ritanserin. Statistical differences from corresponding vehicle

controls: (Mann-Whitney u-test, two-tails) * = p < .05; ** = p < .07.
consumption of drug and water was measured every 24 hr following each

injection. The results of the treatment are shown in figure 4. Total fluid

consumption, set at 100 percent for the control rats, did not change in the

ritanserin dose range, which produced marked changes in the drinking pattern.

Drug consumption was 71 to 80 percent of total fluid intake in the control rats of

the three sessions. Alcohol intake decreased significantly at 0.04 mg/kg, and at

0.16 mg/kg a further pronounced fall was observed. The decrease in cocaine

consumption was very similar within the same dose range of ritanserin. For

fentanyl, reductions in drug intake were measured at doses 0.63 mg/kg. In all

three experiments, water consumption was the mirror image of drug

consumption. Because ritanserin was active against three pharmacologically

different drugs of abuse, the effects of ritanserin might reflect rather a general

mechanism of action rather than a direct interaction with one of the drugs of

abuse.

To rule out the possibility that ritanserin substitutes for the drugs of abuse or

that the drug directly potentiates the activity of the substances of abuse,

riianserin was tested against different drugs of addiction in the drug

discrimination procedure. Different groups of rats were trained to discriminate

either 40.0 (SC, T-30 min) or 5.0 (IP, T-15 min) mg/kg chlordiazepoxide, 0.16

mg/kg LSD (IP, T-15 min), 0.04 mg/kg fentanyl (SC, T-30 min), 10.0 mg/kg

cocaine (IP, T-15 min), 1.25 mg/kg d-amphetamine (SC, T-30 min), 0.63 mg/kg

DOM (IP, T-15 min), or 1,000 mg/kg ethanol (IP, T-15 min) from saline. With

ritanserin, at doses up to 40.0 mg/kg, no stimulus generalization was observed

in any of these training groups. Furthermore, for those tested, no shift in the

dose response functions of the training drugs was observed. In other studies

using different doses of ritanserin (e.g., 1.25 mg/kg given IP at T-15 min or 10.0

mg/kg given SC at T-60 min), it was demonstrated that rats could not be trained

to discriminate between the presence and absence of riianserin (Meert and

Janssen 1989). These studies thus indicate that riianserin does not substitute

for any of the current types of drugs of abuse and that the drug is devoid of

internal stimulus properties. It may be that ritanserin is devoid of an abuse

potential. Furthermore, in rats, ritanserin is not self-administered (Meert and

Janssen 1989). Because ritanserin is a drug with a high safety, lacks an abuse

potential, and reduces the abuse of various drugs of addiction (Meert et al.

1990a, 1990b), the compound may be a supportive pharmacotherapy for

subjects who wish to abstain from drugs.

320
CONCLUSION

The three examples presented here indicate that the drug discrimination

procedure with drugs of abuse as training stimuli is a valuable behavioral

technique. The procedure could be used with almost any drug of abuse and the

variety of experiments that could be conducted would all have typical outcomes.

In generalization experiments, the drug discrimination procedure can be used to

determine whether substances substitute for a particular drug of abuse. Results

from these studies, together with information on discriminative stimulus

properties, can provide some insight into the abuse liability of novel agents. In

antagonism studies, direct antagonists of specific drugs of abuse can be

tracked. These antagonists might be useful as antidotes and, especially those

of drugs of abuse that can introduce symptoms resembling psychiatric states,

might be useful in psychiatric disorders. Finally, mechanisms of action of drugs

can be identified through these discrimination studies. The drug discrimination

test procedure is thus a valuable and widely applicable behavioral technique,

the outcomes of which can contribute to addiction research as well as to clinical

medicine.

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serotonin S2 receptor blocker. Drug Dev Res 8:205-211, 1986.

Wauquier, A.; Fransen, J.; Melis, W.; Ashton, D.; Gillardin, J.M.; Lewi, P.J.; Van

Clemen, G.; Vaught, J.; and Janssen, P.A.J. Loreclezole (R-72063): An

anticonvulsant chemically unrelated to prototype antiepileptic drugs. Drug

Dev Res 19:375-392, 1990.

AUTHOR

Theo Frans Meert, Ph.D.

Janssen Research Foundation

Dept. Neuropsychopharmacology

B 2340 Beerse, Belgium

323

Intracranial Stimulation as Reinforcer for

Neuropeptide Discrimination

Muriel Gewiss, Christian Heidbreder and Philippe De Witte

INTRODUCTION

In establishing drug discrimination behavior, at least three variables must be

controlled: the pharmacological class of the drug; the animal’s behavior; and the

unconditioned stimulus that reinforces the behavior.

These variables are not strictly independent. Drugs can produce motor effects

that interfere with discrimination acquisition, and they can also possess strong

reinforcing properties of their own. Therefore it would seem useful to establish a

method by which discrimination and reinforcing properties of drugs could be

assessed at the same time. Classical rewarding brain stimulation can be used

as an unconditioned stimulus to evaluate the rewarding value of numerous

drugs. Intracranial self-stimulation gives direct access to the reward centers in

the brain and any modification of the activity in the reward centers by drugs

becomes measurable by the rate of bar presses per brain stimulation. Because

the discrimination of drugs results in access to a reward that reinforces the

correct behavior, reinforcing brain stimulation could conceivably be used in a

classical drug discrimination paradigm to study some qualities of reinforcement

and discriminative properties of drugs at the same time.

The purposes of this study were to describe a design using brain stimulation as

the reinforcer in a drug discrimination paradigm; to study the effects of varying

the intensity of the brain reinforcer; and to test the use of a neuropeptide as

discrimination stimulus to obtain the reward, i.e., reinforce brain stimulation.

325
DESIGN AND ADVANTAGES FOR DRUG DISCRIMINATION USING BRAIN

STIMULATION REINFORCER

Male albino Wistar rats from a random breeding, individually housed under

standard laboratory conditions (12 hr light/dark cycle, temperature 22-24°C

humidity 55 percent), and maintained ad lib with laboratory food pellets, were

used. The animals weighed 300 g at the time of surgery and were implanted

with a monopolar nickel-chrome electrode (0.25 mm) insulated except for the

cross section of the tip. The electrode was implanted stereotaxically according

to the following coordinates: A-3.5 mm behind bregma, L-1.2 mm, and

H-8.3 mm below the skull surface (lateral posterior hypothalamus). This brain

area was chosen for the consistent rate of self-stimulation that can be obtained

there. The indifferent electrode was placed 2 mm in front of the bregma. The

animals were allowed 1 week to recover and were then trained to self-stimulate.

They were allowed to self-stimulate in modified Skinner boxes using brain

stimulations of 0.2 sec duration, 0.2 msec negative pulses being delivered at a

frequency of 100 Hz. Threshold for self-stimulation behavior ranged from 60 to

200 μA with a mean of 130 μA. Rats were given daily training sessions until the

bar-pressing rate became steady (1200 bar presses per 15-min session).

The drug discrimination procedure began with injection of drug or vehicle.

Animals were placed in an illuminated and sound-attenuating chamber

containing two levers. The appropriate lever was connected to the brain

stimulator which automatically delivered one stimulation train after the required

number of lever presses had been made; this number varied from 1 to 10 in the

acquisition process of the fixed ratio (FR) requirement. The electrical

parameters of the rewarding brain shock were always monitored by means of

an oscilloscope, and the number of bar presses on both levers was recorded.

Subjects were placed in the test chamber for a 15-min session 10 min after an

intraperitoneal injection of ethyl alcohol (1 g/kg) or saline. Injections of alcohol

(A) or vehicle (V) were given according to two weekly alternating sequences,

i.e., Week 1: V-A-A-V-A and Week 2: V-V-A-V-A. The right lever was

associated with alcohol and the other lever with saline.

Discrimination training began simultaneously with training on the FR 10

schedule. The following data were recorded: number of bar presses on the

incorrect lever before the first reward was delivered, number of bar presses for

the entire session, and number of rewards received during each session (De

Witte 1982).

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FIGURE 1.

A correct choice was defined as a mean percentage of correct responses

beyond 82 percent (and no more than two presses on the incorrect lever before

obtaining the first reward). The criterion of acquisition was set at 8 such correct

choices out of 10 consecutive training sessions. Animals (six out of eight)

acquired the FR 10 lever press response for brain stimulation after a mean

period of 9 weeks. Figure 1 shows that response rates initially increased as the

FR requirement increased. The mean percentage of correct responses

increased from 56 percent at the end of the FR 10 learning period to attain the

criterion of 82 percent in the 15th week, and from this week on, the percentage

always remained above criterion.

This experiment shows that electrical brain stimulation can serve as a reward in

a drug discrimination paradigm. While comparison to published results (Chipkin

et al 1980, Kubena and Barry 1969, Schechter 1978, Winter 1977) on alcohol

discrimination remains difficult, the use of brain stimulation as a reinforcer

327
TABLE 1. Number of responses on the appropriate levera

Brain Electrical lntensitybLow Dose (0.5 g/kg) High Dose (2 g/kg)

Threshold 611 (±53) < 900 (±102)c

+20μA 877 (±112) = 932 (±83)d

+40μA 1604(±188) > 1057 (±124)c

a

15-min session.

bMean number of bar presses (± standard deviation) for brain stimulation at the threshold and after

increasing the intensity (+ 20 and + 40 μA).

cSignificantly (p < .01) greater performance, using Student’s f-test for dependent samples.

dNot significant.

would seem to delay the FR 10 learning (9 weeks). Nevertheless, once animals

learned the task, discrimination remained stable for the entire experiment.

The main advantages of our method of using direct intracranial brain stimulation

as the reward include the following. (1) Because rats are nondeprived, they

have normal body weight. (2) The high number of responses (about 2000 bar

presses per session) enables stabilization of the discriminative conditioning.

(3) In addition to the analysis of the discriminative properties of drugs, the

effects of the drug on the brain reward centers are estimated by the number

of brain stimulations. After lever selection had occurred, rats self-stimulated,

using FR 10 procedure, for the remainder of the 15-min session.

VARIATIONS OF THE BRAIN REINFORCER AND EFFECTS ON THE

DISCRIMINATIVE PROPERTIES

Drug self-administration procedures have clearly shown that drugs can possess

strong reinforcing effects in addition to strong discriminative properties. In a

drug discrimination procedure this reinforcing property of the drug by itself,

added to the reinforcer used to discriminate, could differentially affect the drug

and saline sessions. Indeed, classically, an asymmetry was found to reach the

discrimination criterion, and animals reached this criterion more rapidly under

drug training sessions than under saline sessions (Colpaert et al. 1980).

The use of intracranial stimulation allows study of the effects of variations of

magnitude of the reinforcement itself on the generalization gradients and the

performance on the appropriate lever in a drug-drug discrimination (alcohol

328
2 g/kg vs. alcohol 0.5 g/kg). After estimating the generalization gradient, the

intensity of the electrical reward was increased by 20 μA for each rat. The level

of self-stimulation increased and was computed for 2 weeks of normal drug

training (table 1). Then the generalization gradient was estimated using the

same test doses. Following this, the intensity of the electrical brain reinforcer

was raised another 20 μA (i.e., + 40 μA from the threshold intensity) and the

generalization gradient was again estimated for the same doses (figure 2). The

resulting generalization gradients diverged concurrently with the frequency of

reinforcement. Cur results show that the obtained ED50 progressively slipped to

the right; i.e., the greater the intensity of the brain reinforcer, the flatter the

gradients became (De Witte and Gewiss 1986).

In a classical two-lever discrimination paradigm, the reinforcement used was

food delivered to deprived animals and given asymmetrically and equally for

responding on the two levers. Reinforcement rates were different between drug

and saline training sessions in a way that animals received more

reinforcements during drug sessions than during saline sessions (Colpaert and

Janssen 1981; Koek and Slanger 1982).

By using electrical brain stimulation as the reinforcer and manipulating the

intensity of the electrical stimulation, we have shown that the absolute number

of reinforcements received varies under low- and high-dose training sessions

(table 1). The number of reinforcers obtained using brain stimulation increased

along with the increase in current intensity only for the low training dose; the

performance remained constant at high ethanol dosage training sessions. By

manipulating the intensity of the brain stimulation, we can control the rate of

reinforcement, thus obtaining the same frequency of reinforcement delivered on

both levers (i.e., the case of + 20 μA in table 1).

Cur experiment shows that, independent of the action of alcohol on brain

reward systems, the use of brain reinforcing stimulation as reward in a

conditioned paradigm produces a quantitative generalization gradient with the

delivery of the same number of rewards after low dose or high dose of the

pharmacological substance injected. This represents an important variable to

control before studying qualitative generalization to other substances in the

research of the pharmacological profile when using a two-lever drug

discrimination paradigm.

329
FIGURE 2. Mean sample score on the drug lever (ordinate) after injection of

different test doses of alcohol (abscissa). Different gradients are

obtained successively depending on the intensity of the brain

electrical stimulation. This figure also shows the mean ED50 for

each obtained gradient.

NEUROPEPTIDE AS DISCRIMINATION STIMULUS

In a drug discrimination procedure, drugs can induce internal unconditioned

stimuli, which can serve as discriminative stimuli for responses that lead to

reinforcement. We used a procedure in which the discrimination of the presence

of the neuropeptide caerulein (CER), a cholecystokinin analog (CCK-8), was

the condition to obtain the reward (a reinforcing brain stimulation, which

reinforced the organism when performing the correct discrimination between

CER and its vehicle). The use of direct electrical stimulation in a rewarding

brain center producing short (0.2 sec) and highly powerful repetitive rewards

without satiety offers the advantage of training animals of normal body weight

without food deprivation. This last characteristic is essential since CER and

CCK-8 are neuropeptides acting on feeding (i.e., inducing satiety). We can thus

suppose that these peptides will be differently perceived under a deprived state

than under a normal steady state. Animals reached the criterion of acquisition of

the discrimination 12 to 18 weeks of training. At this point the experiment shows

that while a neuropeptide such as CER can serve as a cue in a drug

330
discrimination paradigm, more sessions are required to reach the discrimination

criterion with CER than with classical pharmacological substances.

It is known that drugs producing only peripheral action are harder to

discriminate than centrally acting drugs (Lal 1977), but the mechanism by which

CCK and CER exert their wide-ranging effects on the CNS remains

controversial. It is also unclear whether CCK peptides cross the blood-brain

barrier. In this regard, immunoreactivity of CCK-8 is not observed in rabbit CSF

following IV injection. However, behavioral and biochemical studies show that

peripherally administered peptides may indeed alter central dopamine (DA)

mediated processes. These actions could occur through an effect mediated by

the vagus without necessarily having to cross the blood-brain barrier. Thus, the

neural information from peripheral to central sites implicates the ascending

sensory fibers of the vagus nerve to the nucleus tractus solitarius (NTS)

(Crawley and Schwaber 1984). From this subdivision of the NTS, projections

ascend to the lateral hypothalamus. In turn, the lateral hypothalamus returns

projections to the NTS and then to the efferent motor fibers of the vagus nerve

to the gut (Schwaber et al. 1982).

A few years ago, cholecystokinin carboxyl terminal octapeptide immunoreactive

neuronal cell bodies were discovered in the rat substantia nigra and ventral

tegmental areas. They were present in such large numbers that coexistence of

CCK with DA was suggested in these neurons (Hökfelt et al. 1980). Following

this discovery of DA-CCK-8 coexistence, a large core of behavioral data

suggested that CCK-8 had neuroleptic-like properties (De Witte it al. 1985; Van

Ree et al. 1963; Zetler 1983). Moreover, clinical data suggested a potential

involvement of this peptide in several psychiatric disorders, particularly

schizophrenia. Nevertheless, the origins of the neuroleptic-like properties of

CCK-8 remain unknown and the potential role of dopaminergic systems in the

discriminative properties of the neuropeptide cholecystokinin has not yet been

studied experimentally. In this context, the search for common cueing

properties between CER, a CCK-8 analog, and unsulfated CCK; DA agonists,

such as amphetamine (AMPH) and apomorphine (APO); and antagonists, such

as chlorpromazine (CPZ) and haloperidol (HAL) could help define the possible

pharmacological role of CCK (table 2).

CCK-8 in its sulfated form shares properties with the training drug CER, while

CCK-4 and the unsulfated form of CCK-8 present no pharmacological

properties similar to CER. These results thus confirm previous work using other

331
TABLE 2. Results of statistical analyses of treatment effects with chi-square test

Training Condition Number Responding on Drug Mean Responses on Selected

Lever/Number Tested Lever (15-min session)

Training Drug

Caerulein

3 μg/kg 1/6* 1,088

Test Drugs

Caerulein

1 μg/kg 1/6* 922

2μg/kg 5/6 731

Cholecystokinins

CCK-4

10 μg/kg 0/6* 1,122

20 μg/kg 0/6* 1,373

200 μg/kg 0/6* 1,412

CCK-8

5 μg/kg 0/6* 987

10 μg/kg 3/6* 1,011

20 μg/kg 6/6 932

CCK-8 (unsulfated)

10 μg/kg 0/6* 1,267

20 μg/kg 0/6* 962

200 μg/kg 1/6* 817

Dopamine Agonists

d-Amphetamine

0.32 mg/kg 0/6* 1,673

0.64 mg/kg 0/6* 2,370

1.28 mg/kg 0/6* 1,655

Apomotphine

0.05 mg/kg 0/6* 978

0.10 mg/kg 1/6* 251

0.20 mg/kg 0/6 300

0.60 mg/kg 0/6* 0

Dopamine Antagonists

Chlorpromazine

0.5 mg/kg 2/6* 728

1.0 mg/kg 2/6* 473

2.0 mg/kg 5/6 182

Haloperidol

0.03 mg/kg 5/6 621

0.05 mg/kg 5/6 268

0.20 mg/kg 0 0

*Probability of difference from training drug (caerulein 3 μg/kg) being due to chance; p < .05

chi-Square test.

332
behavioral techniques showing differential effects between those forms of

cholecystokinin (De Witte et al. 1987; Hsiao et al. 1984).

Both DA agonists AMPH and APO are generalized to the vehicle lever. Animals

generalize HAL 30 μg/kg to CER, possibly the result of the neuroleptic-like

properties of CER. The other DA antagonist, CPZ, is also completely

generalized to CER but at a different dosage than HAL, i.e., 2 mg/kg CPZ

versus 30 μg/kg HAL. Thus, a 67 times greater dose of CPZ is required to

obtain an equi-active cue with HAL similar to the training CER. These

equi-active doses of HAL and CPZ are in the range of those obtained using the

APO-antagonism test in dogs and in clinical use. The large difference in dosage

between HAL and CPZ in the generalization to the CER may be the result of the

different pharmacological properties of these two DA antagonists.

Thus our experiment clearly shows that peripheral injection of HAL and CPZ

induces pharmacological cues similar to those of CER, while injections of

AMPH and APO do not share pharmacological cues with CER. Peripherally

injected CCK-8 is thus generalized with HAL. Similarly, the neuroleptic effect of

peripherally administered CER in the rat is similar to the effect of haloperidol.

However, since CCK behaves like a direct DA agonist when injected directly

into the caudal part of the accumbens (De Witte et al. 1987), the effect of

intraperitoneally or intraventricularly injected CER might be an indirect

consequence of CCK-8 excitation of some projections to the caudal accumbens

known to antagonize the effect of DA in that nucleus. According to this

assumption, CCK-8 differs in many actions from neuroleptics and this could

explain the discrepancies observed in the studies of its clinical

pseudoneuroleptic effect on human beings (De Witte et al. 1988).

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Vanderhaeghen, J.J. Psychopharmacological profile of cholecystokinin using

the seif-stimulation and the drug discrimination paradigms. In:

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York: Annals of the New York Academy of Sciences, 1985. pp. 470-487.

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Evidence for coexistence of dopamine and CCK in mesolimbic neurons.

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Hsiao, S.; Katsura, G.; and Itoh, S. Cholecystokinin tetrapeptide, proglumide

and open-field behavior in rats. Life Sci 34:2165-2168, 1984.

Koek, W. and Slanger, J.L. Effect of reinforcement differences between drug

and saline sessions on discriminative stimulus properties of fentanyl. In:

Colpaert, F.C., and Slanger, J.L., eds. Drug Discrimination: Applications in

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Kubena, R.K.; and Barry, H.E. Ill. Generalization by rats of alcohol and atropine

stimulus characteristics to other drugs. Pychopharmacologia

15(6):195-206, 1969.

Lal, H. Discriminative Stimulus Properties of Drugs. New York: Plenum Press,

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Schwaver, J.S.; Kapp, B.S.; Higgins, G.A.; and Rapp, P.R. Amygdala and basal

forebrain direct connections with the nucleus at the solitary tract and the

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Pharmacol 93:63-78, 1983.

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ACKNOWLEDGMENT

This work was supported by Fonds de la Recherche Scientifique Médicale

3.4579.90 and Fonds de Développement Scientifique 1989-1991.

AUTHORS

Muriel Gewiss, Ph.D.

Christian Heidbreder

Philippe De Witte, Ph.D.

Laboratoire Psychobiologie, Université de Louvain

1, Place Croix du Sud,

B-1348 Louvain-la-Neuve

Belgium

335

Drug Discrimination Used To Study Drug

Withdrawal

M. W. Emmett-Oglesby and G.A. Rowan

INTRODUCTION

Withdrawal from the long-term use of psychoactive drugs produces a variety of

readily discernible phenomena in humans. In clinical terms, these phenomena

can be classified as those that have an objectively verifiable basis (signs) and

those that are subjective in nature (symptoms). Symptoms of drug withdrawal

have been described as mixtures of anxiety, dysphoria, and drug craving, and

at least some of these symptoms are shared across classes of drugs (Marks

1978; Wikler 1980). Even when drugs produce obvious signs of withdrawal, the

symptoms continue long after signs have abated. Thus, symptoms of drug

withdrawal may play a significant role in promoting the continuation of drug

dependence: they provide motivation for continued drug use, and they occur

prior to or in the absence of physical signs of withdrawal. In order to clarify the

role of subjective symptoms of withdrawal in drug dependence, animal models

have been developed that may address this problem (for reviews see

Emmett-Oglesby et al. 1990; File 1990). Clinically, these models may be

particularly important for testing new treatments for withdrawal and studying the

biological basis of drug dependence.

This paper will focus on efforts to study withdrawal using drug discrimination

methodology. Two approaches have been reported for these drug

discrimination models of withdrawal. The first stems from the observation that

withdrawal in humans frequently causes anxiety. Thus, if animals could be

taught to discriminate an anxiogenic drug, it is conceptually plausible that such

a discrimination might generalize to drug-withdrawal stimuli. The discrimination

of pentylenetetrazole (PTZ) has been reported to have predictive validity for

identifying both anxiogenic and anxiolytic drugs, and, in subjects trained to

detect PTZ, withdrawal from benzodiazepines substitutes for this stimulus.

These data have been extensively reviewed elsewhere (e.g. Emmett-Oglesby

337
et al. 1987, 1990) and in general support the idea that the discrimination of PTZ

may be useful for detecting withdrawal from a variety of drugs of dependence,

particularly those of the sedative-hypnotic class. For example, withdrawal from

benzodiazepines or ethanol substitutes fully for PTZ, and withdrawal from

cocaine, morphine, or nicotine substitutes partially for the stimulus properties of

PTZ.

A second approach to drug discrimination models involves training animals to

detect directly the stimulus effects arising during withdrawal, and the primary

aim of this chapter is to review and evaluate the utility of this paradigm. These

experiments maintain subjects on a baseline of chronic drug of dependence

such as morphine and attempt to train the stimulus properties of an antagonist

such as naloxone. In general, subjects are trained to respond on one of two

levers and food is presented for correct responses. They are then maintained

chronically on a drug of dependence, and withdrawal is presumably precipitated

by using an antagonist (e.g., Holtzman 1985a; Valentino et al. 1983). Thus,

responding on one of the levers is reinforced following antagonist

administration, and responding on the other lever is reinforced following vehicle

administration.

When a discrimination is trained based on drug versus vehicle, it has been well

established that only some drugs will substitute for the training drug, and a

significant correlation exists between drugs having stimulus properties

comparable to the training stimulus and drugs that humans describe as having

the same class of subjective effects (Schuster and Balster 1977). To date only

discriminations based on precipitated withdrawal of the opiates and

benzodiazepines have been reported using this method. We will review data

showing that it is difficult to specify what stimulus is controlling behavior in

these discriminations. Although experimenters attempt to establish withdrawal

as the controlling stimulus, because the animals are maintained on a baseline

of chronic drug, the discrimination may be based, at least in part, on the direct

effects of the drug of dependence. To alleviate this problem we will describe

data expanding the traditional two-lever discrimination to a three-lever choice

procedure that includes the drug of dependence, the drug producing

withdrawal, and saline.

TRAINING TO DETECT STIMULUS EFFECTS OF WITHDRAWAL

In a typical drug discrimination procedure, subjects are presented with a simple

choice task in which they are differentially reinforced for responding in the

338
presence or absence of the drug. Either vehicle or a dose of drug is injected

prior to training, and animals are allowed to respond while the drug is producing

its effects. If the experimental contingencies are arranged so that one of the

responses will be reinforced in the presence of the drug effect and the other

response will be reinforced in the absence of the drug effect, then the drug can

serve as a discriminative stimulus; i.e., its presence or absence will differentially

control responding. Although two training conditions exist to the experimenter,

drug versus vehicle, it is important to note that apparently it is only the presence

of the drug stimulus that controls responding. The animal does not learn drug

versus "vehicle” per se; instead, the discrimination appears to be based on

“drug-specific stimuli” versus “all other stimuli.” This distinction is apparent when

testing animals that are trained in a two-choice task using a drug versus vehicle

procedure. When tested with other drugs, only those known to produce similar

subjective effects in humans will substitute for the training stimulus (Altman et

al. 1977). For example, in animals trained to detect morphine, only mu agonists

reliably produce full drug-lever selection, whereas kappa agonists, or drugs

from other classes, produce vehicle-lever responding (Colpaert 1978; Holtzman

1985b; Lal et al. 1978). Since these other drugs can produce their own stimulus

effects, this pattern of results is generally interpreted to mean that the training

stimulus serves as a reference stimulus, and responding on the drug versus

vehicle lever is controlled by how closely the test stimulus matches the stimulus

produced by the training drug. This distinction may become particularly relevant

when attempting to train the stimulus properties associated with withdrawal.

Initial attempts to train the stimulus properties of pure opiate antagonists were

not successful. When given in nondependent rats these drugs (naloxone and

naltrexone) either had low efficacy or did not function as discriminative stimuli

(Lal et al. 1978; Overton 1982; Weissman 1978). In contrast, if animals were

maintained on a chronic baseline of opioid dependence, the antagonists were

more readily trained as discriminative stimuli (Gellert and Holtzman 1979;

Hoitzman 1985a; Valentino et al. 1983). Discriminations based on this

procedure have been reported for rats (Gellen and Holtzman 1979; Holtzman

1985a), pigeons (France and Woods 1987; Valentino et al. 1983), and monkeys

(France and Woods 1989), and the data clearly indicate that the opioid

antagonist given in this paradigm can function effectively as a discriminative

stimulus. The critical question becomes, What is the nature of the stimulus that

is controlling behavior?

339
Two-Choice Procedures

In the traditional “drug versus no-drug” discrimination procedure, subjects

experience only the stimulus produced by the training drug, but in the opioid

antagonist experiments this may not be the case. The problem arises because

subjects are maintained on a chronic baseline of opioid drugs, which have been

shown to produce discriminative stimuli of their own (Colpaert 1978; Herling et

al. 1980; Holtzman 1985b). Although the experimenter intends that the

discrimination training involve the effects associated with nattrexone as a

reference stimulus, two other possibilities exist (table 1). The first is that the

saline injection allows the opiate drug to exert a discriminative stimulus and the

antagonist simply turns off the opioid stimulus. A second possibility is that the

subject learns a distinctive stimulus associated with withdrawal and a second

stimulus associated with the direct effects of the opiate. In this case, instead of

training drug versus no-drug, it is possible that drug A versus drug B is trained.

Based on these alternatives, what would be acceptable evidence that

precipitated withdrawal is actually the controlling stimulus? Perhaps the easiest

way to answer this question is to terminate the chronic administration of the

drug of dependence. Initially, physical signs of withdrawal should occur, and

during this period vehicle should substitute for the antagonist. When

spontaneous withdrawal has dissipated, if what the animals have learned is a

precipitated-withdrawal stimulus, both the vehicle and the antagonist should

now produce vehicle lever responding. Using this approach, Holtzman (1985a)

has provided evidence that morphine withdrawal has indeed been trained as a

discriminative stimulus.

More recently, France and Woods (1989) trained rhesus monkeys that were

maintained on morphine (1.78 or 3.2 mg/kg daily) to discriminate the stimulus

properties of naltrexone (0.01 mg/kg) from saline injections. When chronic

morphine was terminated, monkeys switched their responding from the saline to

the naltrexone lever between 8 and 27 hr post morphine injection, and

naltrexone responding occurred when signs of opioid withdrawal were maximal.

Another interesting aspect of this study was that doses of naltrexone that

produced responding on the naltrexone lever also produced effects typically

seen during opioid withdrawal, including salivation, miosis, occasional

vocalizations, and irritability. These results indicate that dependence developed

to the once daily morphine injections and that the naltrexone discriminative

stimulus may have been related to the withdrawal-precipitating actions of

naltrexone. Still, using this two-lever procedure it is unclear whether

340
dependence has developed and what the animals have actually learned. As

noted by France and Woods (1989), “although the results of substitution studies

support the notion that dependence has developed and monkeys were showing

signs of withdrawal when the naltrexone lever was selected, these results might

also be interpreted in a context of morphine (saline injection) vs. non-morphine

(naltrexone injection) discrimination; the dose effects and time-effects are fully

consistent with either hypothesis and neither can be conclusively rejected with

the present paradigm” (p. 942). As described above, had these subjects been

maintained free of morphine for an extended period of time, it may indeed have

been possible to reject one of these hypotheses.

Using this procedure, in preliminary studies from this laboratory we have

attempted to train rats to detect withdrawal from chlordiazepoxide (CDP)

(Emmett-Oglesby et al. 1990). In these experiments, subjects were maintained

on a baseline of 100 mg/kg of CDP per day, given in two doses of 50 mg/kg as

part of a nutritionally balanced liquid diet (Idemudia and Lal 1989). They were

trained with the benzodiazepine antagonist flumazenil (Ro 15-1788) 20 mg/kg,

and saline on a two-lever choice task using food as a reinforcer, with daily

training sessions occurring 6 hr after the morning feeding of CDP. The

discrimination was rapidly acquired with error rates less than 5 percent within

30 training sessions. In tests with flumazenil, doses from 2.5 to 20 mg/kg all

produced flumazenil-lever responding, with the dose eliciting 50 percent

flumazenil selection (ED50) of approximately 0.64 mg/kg. When chronic CDP

was terminated, results from a variety of drug substitution tests suggested that

stimulus control in this discrimination was actually maintained by both the direct

effects of CDP and the effects of precipitated withdrawal by flumazenil. For

example, during this drug-free period, CDP produced vehicle-lever responding,

and PTZ produced flumazenil-lever responding, but both flumazenil and vehicle

produced mixed responding on both levers. These results are consistent with

the hypothesis that the stimulus trained was associated with both flumazenil

and CDP, because the saline baseline failed to return to its prewithdrawal

baseline.

Based on these preliminary studies, the parameters of the experiment were

altered in an attempt to train subjects to detect withdrawal from dependence on

benzodiazepines against a baseline of minimal CDP-like effects. The two major

parameters that were altered included the dose of flumazenil used for training

and the feeding schedule of chronic CDP. A much lower dose of flumazenil was

used, 2.5 instead of 20 mg/kg, since the initial study revealed that these doses

were essentially interchangeable, i.e., both produced full generalization to the

341
training dose of 20 mg/kg. Also, the morning dose was lowered to 25 mg/kg and

the dose after testing was raised to 75 mg/kg. The rationale for this lower

morning dose was to reduce the amount of CDP on the receptor at the time of

training. During “vehicle” training, this change would presumably result in a

decrease in the stimulus produced by CDP.

Following acquisition of this discrimination, flumazenil was substituted for the

training stimulus in a dose-dependent manner (0.64-2.5 mg/kg), with an ED50

of 0.94 mg/kg. The animals were then taken off CDP, and in testing 8 days

later, saline produced 67 percent flumazenil-lever selection, perhaps indicating

similarity between spontaneous withdrawal at this time and the flumazenil

stimulus that was trained under chronic CDP. In contrast, in testing 14 days

after termination of CDP, the response to vehicle was not significantly different

than before withdrawal of the vehicle. Thus, only flumazenil acting as an

antagonist produced a controlling stimulus, rather than both flumazenil and

CDP producing controlling stimuli.

After obtaining the time course data, these animals were placed back on

chronic CDP and trained for an additional 3 months. Subsequently, the

dose-effect curve for flumazenil was redetermined (figure 1); this curve (ED50 of

approximately 0.64) was not significantly different than the initial dose-effect

curve. CDP at doses of 5, 20, and 80 mg/kg did not substitute for the flumazenil

stimulus. However, PTZ (5-40 mg/kg) did produce a dose-dependent

substitution for flumazenil in these animals (figure 2). As described at the start

of this review, in animals trained to discriminate PTZ, flumazenil substitutes for

PTZ only after chronic administration of a benzodiazepine (Emmett-Oglesby et

al. 1983, 1987). Thus, there is symmetrical generalization between these

compounds that occurs only when flumazenil is tested in subjects maintained

on a chronic baseline of benzodiazepine administration. Indeed, when subjects

are trained to detect the effects of flumazenil directly (not while being

administered a benzodiazepine), PTZ does not generalize to flumazenil (Rowan

and Lucki 1989).

The data from this series of experiments suggest that subjects learn a stimulus

that is associated with flumazenil, is PTZ-like in nature, and is similar to

spontaneous withdrawal from the benzodiazepine CDP. Thus, these data are

consistent with having trained precipitated withdrawal.

Although benzodiazepine and morphine withdrawals appear to have been

trained using two-choice technology, these discriminations are intrinsically

342
FIGURE 1. Stability of flumazenil dose-effect curve. The dose-effect curve for

flumazenil (Ro 15-1788 [RO]) was determined in rats (n = 12)

trained to detect the stimulus properties of RO (2.5 mg/kg) while

receiving chronic administration of chlordiazepoxide (CDP, 100

mg/kg/day) in the form of a nutritionally balanced liquid diet. On

the ordinate, 100% RO lever selection would indicate that all

animals selected the RO lever, while 0% RO lever selection would

indicate that the animals selected the vehicle (saline) /ever. Rats

were trained 6 hr after the morning CDP feeding (25 mg/kg) and

received the remainder of the CDP dose (75 mg/kg) after training.

The initial dose-effect curve produced an ED50 of approximately

0.64 mg/kg. When the animals were retested 3 months later, a

similiar ED50 was obtained. These data indicate the stability of this

discrimination across a 3-month training period.

unsatisfactory. Proving that the stimulus produced by the antagonist is in fact

related to withdrawal requires long periods when the subjects are drug free.

Similarly, proving that the antagonist provides the only controlling stimulus also

343
FIGURE 2. Substitution of pentylenetefrazole for flumazenil. These animals (n

= 12) were trained to detect the stimulus properties of flumazenil

(RO, 2.5 mg/kg), while being maintained on chronic

chlordiazepoxide (CDP, 100 mg/kg/day). They were then tested

for substitution of COP (5, 20, and 80 mg/kg) and

pentylenetetrazole (PTZ; 5, 20, and 40 mg/kg). CDP did not

substitute for RO at any of the doses tested, while PTZ did

produce a dose-dependent substitution for RO. These data

support the hypothesis that withdrawal from benzodiazepines

shares common stimulus properties with PTZ.

requires this test. Moreover, few guidelines are available to decide how much of

the drug of dependence should be given daily or how long after administration

of the drug of dependence training should occur. Finally, several weeks of

training must occur before the discrimination is learned, and only then can the

critical drug-free test be conducted.

344
lntroducing a Third Choice

Some of the problems that arise in a two-choice procedure may be readily

resolved by the addition of a third choice that actively trains the drug of

dependence as a controlling stimulus. There have been several reports of

three-lever (rats) or three-key (pigeon) discriminations. These experiments

usually attempt to train the animals to discriminate either the stimulus properties

of two different drugs or different doses of the same drug versus saline (France

and Woods 1987; Gauvin and Young 1989; White and Holtzman 1981, 1983).

These experiments have demonstrated that the discriminative stimulus effects

of drugs in a three-lever procedure can be very similar to training these

compounds in a two-choice procedure. For example, this laboratory has been

successful in training a three-lever discrimination between PTZ, midazolam,

and saline. The acquisition data for this discrimination are illustrated in figure 3.

As in a two-lever discrimination involving midazolam (Garcha et al. 1985;

Woudenberg and Slangen 1989), diazepam and chlordiazepoxide produced

midazolam-lever selection.

The particular advantage of this procedure in a drug of dependence study is

that by training withdrawal versus the drug of dependence, the vehicle choice

now can encompass all stimuli that are not similar to these two choices. Thus,

the vehicle choice functions as a non-drug A or non-drug B choice but,

interestingly, not necessarily as a “neutral” choice (Järbe and Swedberg 1982).

Conceptually, it may be inconsequential whether any distinct stimulus is

associated with the vehicle: the critical determinant is that the stimulus

associated with this choice should be less intense than the other two stimuli

trained. This assertion is made within the context of evidence from dose-dose

discriminations that stimulus control resides entirely in the more intense of two

stimuli. There are several experiments demonstrating that animals can learn to

discriminate two doses of the same drug (Colpaert and Janssen 1982a, 1982b;

DeVry and Slangen 1986; Young et al. 1989). In experiments such as these,

when the magnitude of difference between the two doses trained is large, the

discrimination approximates a drug versus saline discrimination. Hence, in a

three-choice discrimination involving drug dependence, the nature of the

stimulus associated with the vehicle may not be important as long as it is less

intense than the other two stimuli being trained.

There are three likely configurations that could be trained in a three-choice

discrimination involving a drug of dependence, its antagonist, and saline. Based

on the stimulus condition associated with vehicle administration, these

345
FIGURE 3. Acquisition of three-choice discrimination in non-dependent

animals. The animals were trained to detect the stimulus

properties of pentylenefetrazole (20 mg/kg), midazolam (1.0

mg/kg), and saline. The acquisition of the discrimination is

presented in terms of percent correct lever responding for the

three compounds. The discrimination developed differentially for

the three choices, as determined by animals reaching a 75%

correct-lever selection criterion.

possibilities can be conceptualized in the following ways (see table 1):

antagonist versus vehicle (neutral) versus agonist; antagonist versus vehicle

(low-dose agonist effect) versus agonist (highdose agonist effect); antagonist

(intense withdrawal) versus vehicle (mild withdrawal) versus agonist. An

experiment that illustrates the difficulties of specifying which of these cases

occurs involved training naltrexone, morphine, and saline in

morphine-dependent pigeons. France and Woods (1987) attempted to show

346
TABLE 1. Possible Controlling Stimuli: 2-Choice Discrimination1

Withdrawal Neutral

Neutral Drug of dependence

Withdrawal Drug of dependence

Possible Controlling Stimuli: 3-Choice Discrimination?

Withdrawal Neutral Drug of dependence

Withdrawal Drug of dependence L Drug of dependence H

Withdrawal H Withdrawal L Drug of dependence

1Stimuli in the left panel arise following antagonist treatment whereas stimuli in the right panel follow

vehicle administration. The first theoretical possibility for the stimuli controlling the two-lever

discrimination is the antagonist versus vehide. A second possibility is that the animals simply learn

the onset and offset of the agonist treatment. In the third case, during vehicle training the animals

learn to discriminate the stimulus properties of the antagonist versus the agonist (drug of

dependence).

2Stimuli in the left panel arise following antagonist treatment; stimuli in the center panel arise

following vehicle administration; and stimuli in the right panel arise following administration of the

chronic agonist. The first possibility trains the drug of withdrawal (antagonist) versus a neutral

stimulus (vehicle) versus the stimulus properties of the drug of dependence. A second possibility is

that during vehicle training the animals learn to discriminate the stimulus properties of the lower

dose of the drug of dependence than is trained on the third choice lever (Drug of dependence H). In

the third case, the animals simply learn the withdrawal stimulus in a high and low form versus the

drug of dependence.

that naltrexone produced a withdrawal-related stimulus in this three-key

discrimination. When morphine administration was terminated and subjects

were retested at various intervals, responding occurred on the morphine key at

1 hr, the saline key at 6 hr, and the naltrexone key at times up to 30 hr.

Unfortunately, no test results beyond this time were reported, and thus it is

impossible to know if these subjects would have eventually arrived at a point in

time where naltrexone produced saline-key responding. France has examined

this problem (personal communication) and notes that naltrexone did produce

saline responding, but the baseline appeared to be unstable. Hence, it is

possible that these animals have learned a discrimination in which withdrawal is

one of the controlling elements, but the degree of control is difficult to specify.

347
Based on these experiments we attempted to expand the two-lever flumazenil

(2.5 mg/kg) experiment to include another lever for CDP. Animals were

maintained on chronic CDP, 100 mg/kg/day, fed in divided doses, 25 mg/kg in

the a.m. and 75 mg/kg in the p.m, as described for the two-lever discrimination

above. The animals were trained to detect the stimulus properties of flumazenil

(2.5 mg/kg), CDP (20 mg/kg), and saline. The acquisition of the discrimination

developed differentially for the three compounds, as determined by reaching a

75 percent correct-lever selection criterion. The discrimination of flumazenil

developed in approximately 12 trials, while the CDP and saline discrimination

took closer to 36 and 46 trials, respectively (figure 4). The animals also showed

dose-dependent generalization for both flumazenil and CDP (figures 5 and 6).

In the flumazenil generalization, there was no responding on the CDP lever. As

the dose of flumazenil increased, the animals switched their responding from

the saline lever to the flumazenil lever. It is also interesting to note that the

generalization curve for flumazenil is very similar to that obtained in the

two-lever flumazenil discrimination. In the CDP generalization curve the animals

demonstrated dose-dependent increases in CDP-lever selection as the dose of

CDP increased. The animals switched from saline to CDP-lever responding as

the dose increased with little flumazenil-lever selection.

Problems With Three-Choice Procedures for Studying Withdrawal

A three-choice discrimination involving a baseline of chronic drug administration

presents several technical problems. Many of these difficulties are shared with

the two-choice discriminations reviewed above. For example, what dose of the

antagonist should be used for training, and how frequently and in what dose

should the drug of dependence be administered each day? Also, how long after

a maintenance injection of the drug of dependence should training occur? The

answers to these questions are generally arrived at empirically and are based

on the goals described previously for two-choice discriminations.

We suggest that subjects be trained on as large a dose of the drug of

dependence as possible. There are three reasons for this suggestion. First,

bigger doses of drugs are easier to learn and provide better discriminative

control than smaller doses (Colpaert and Janssen 1982b; Overton 1982, 1987);

thus, the discrimination training conditions should be readily distinguishable for

the subject.

A second reason for using large doses of drugs arises from the unknown nature

of the stimulus baseline induced by chronic administration of the drug of

348
Blocks of 4 training sessions

FIGURE 4. Acquisition of three-choice discrimination in animals dependent on

CDP. The animals were trained to detect the stimulus properties

of flumazenil (RO, 2.5 mg/kg), chloroiazepoxide (CDP, 20 mg/kg),

and saline, while maintained on a liquid diet containing CDP (100

mg/kg/day). Acquisition of the discrimination is presented in terms

of percent correct lever responding for the three compounds. The

discrimination developed differentially for the three choices, as

determined by animals reaching a 75% correct lever selection

criterion. The RO discrimination developed in approximately 12

trials, while the CDP and saline took closer to 36 and 46 trials,

respectively.

dependence. Several scenarios are possible, at least one of which requires

large training doses of the drug of dependence. For example, suppose that

subjects are trained at 6 hr after administration of chronic drug. If the drug has

not completely cleared the body (which is presumably the case if withdrawal is

to be precipitated when an antagonist is trained), then it is likely that this drug is

349
FIGURE 5. Generalization of flumazenil in a three-choice discriminatrion.

The dose-effect curve for flumazenil (Ro 15-1788 [RO]) was

determined by testing independent doses in rats (n = 9) trained to

detect RO (2.5 mg/kg), chlordiazepoxide (CDP, 20 mg/kg), and

saline in a three-lever choke task. Animals were maintained

chronically on CDP 100 mg/kg/day. On the ordinate, 100% lever

selection indicates that all animals initially responded on the lever

shown by the symbol to a fixed ratio of 10 lever presses. As

percent selection of the RO lever increased in a dose-dependent

manner, saline-lever selection decreased. No CDP-lever selection

was evident during the test.

exerting a stimulus effect. Thus, the problem for the subject will be to

differentiate between the stimulus effects of chronic drug versus the stimulus

effects of this same drug given in a larger dose for training purposes. The closer

these two stimuli are in magnitude, the more difficult this discrimination will be

(Colpaert and Janssen 1982a, 1982b). Consequently, when administered for

350
FIGURE 6. Generalization of chlordiazepoxide in a three-choice

discrimination. Dose-effect curve for chlordiazepoxide (CDP) was

determined by testing independent doses in rats (n = 9) trained to

detect flumazenil (Ro 15-1788 [RO], 2.5 mg/kg), chlordiazepoxide

(CDP, 20 mg/kg), and saline in a three-lever choice task. Animals

were maintained chronically on CDP, 100 mg/kg/day. On the

ordinate, 100% lever selection indicates that all animals initially

responded on the lever shown by the symbol to a fixed ratio of 10

lever presses. As percent selection of the CDP lever increased in

a dose-dependent manner, saline-lever selection decreased. Little

RO-lever selection was evident during any test.

training purposes, the dose of the drug of dependence should be large enough

to maximize the difference between these two stimuli.

351
A third reason for using large doses is based on experiments demonstrating

tolerance to the stimulus effects of drugs. When subjects are trained to detect

drugs such as morphine, cocaine, or midazolam, if the training drug is

administered several times daily in doses significantly higher than the training

dose, tolerance is seen. Typically the dose-effect curve for the detection of the

training drug shifts two- to fourfold to the right. (For a review of this literature,

see Young and Sannerud 1989.) This degree of tolerance is usually seen

following periods of drug administration lasting up to 10 days, and we are

unaware of any studies that have assessed whether a greater degree of

tolerance develops if chronic administration is continued for the extended

periods that drug-withdrawal studies entail.

This type of tolerance is likely to be a significant problem in training the drug of

dependence. For example, when our subjects that are trained to detect the

benzodiazepine midazolam are tested acutely with CDP, it substituted for

midazolam with an ED50 of approximately 2.25 mg/kg. In contrast, when training

on the discrimination was hatted and rats were injected with CDP, 20 mg/kg

every 8 hr for 7 days, the ED50 for CDP substitution increased to approximately

5.0 mg/kg (unpublished observations). Thus, as little as 60 mg/kg/day of CDP

can produce greater than twofold tolerance to the benzodiazepine training

stimulus, and this finding suggests that in our drug withdrawal studies in which

subjects receive CDP, 100 mg/kg/day, the degree of tolerance should be at

least this profound. Similarly, when rats are used as subjects in a

morphine-withdrawal discrimination, at least twofold tolerance is likely to

develop to the stimulus effects of opioids if the dose of morphine used

chronically to produce dependence is on the order of 15 mg/kg/day or greater

(Emmett-Oglesby et al. 1988; Shannon and Holtzman 1976).

The combination of factors, including dosedose discriminations and tolerance,

is likely to produce significant difficulties in learning a three-choice

discrimination based on drug withdrawal. If the difference between vehicle

(residual drug effects from chronic administration) and the training dose is to be

distinguished, a training dose may be necessary that is at least twofold, and

perhaps as much as fourfold, higher than that necessary for training in the

absence of chronic drug administration.

Duration and Timing of Training

As described above, a critical test in a drug-withdrawal discrimination occurs

when subjects are taken off the chronic regimen and allowed to remain drug

352
free for several days or weeks. When spontaneous withdrawal has subsided,

tests with both the antagonist and its vehicle should produce vehicle-choice

responding. This test has the disadvantage of being carried out after long

periods during which animals are not trained on the discrimination, and, given

the other problems with maintaining stimulus control in a three-choice

discrimination, it seems likely that this particular test may be even more difficult

to run than in two-choice discriminations.

One procedure that may help to minimize this difficulty is to overtrain the

animals on the three-choice discrimination. Suppose a “learning” criterion is

adopted such that animals must initially choose the correct lever before

receiving a reinforcer for 9 of 10 consecutive sessions. Data from experiments

with exteroceptive stimuli show that overtraining results in sharper

generalization gradients and a longer duration of stimulus control (Mackintosh

1974). Since this latter feature is what is desired in a drug-withdrawal

experiment, it seems likely that either setting a very stringent criterion or training

for many sessions beyond the minimum criterion will result in sufficient stimulus

control to test during prolonged periods when training is not possible.

How frequently should the drug of dependence be given chronically, and how

long after administration should discrimination training occur? Data are not

available to answer either question definitively, but the literature supports the

following suggestions. Regarding frequency of dosing, the general observation

in drug-dependence studies is that maximum dependence occurs when high

concentrations of the drug are given frequently (e.g., Kalant et al. 1971). The

limiting case appears to occur with constant drug infusion (for discrimination

training this might be achieved with pumps or pellet implants), and there are

reasons to believe that this would facilitate learning a three-choice

discrimination. Among other points, a constant drug infusion would minimize the

stimulus produced during chronic treatment with the drug of dependence (no

peaks would occur in plasma drug concentration) and would prevent

spontaneous withdrawal (no valleys would occur in plasma drug concentration).

Although this approach suffers from not modeling human drug taking behavior,

it is attractive because it provides stable baselines for both dependence and the

training of discriminative stimuli.

If drug is absorbed constantly, the question of how long after chronic

administration to wait before training becomes moot. If chronic drug is given

intermittently, the answer to this question depends on the pharmacokinetics of

the drug of dependence. At the time training occurs, enough of the drug should

353
be on the receptor that an antagonist will precipitate withdrawal, but not so

much drug should be present that the vehicle condition is not sufficiently

different from the drug of dependence condition. We suggest that it is perhaps

better to err on the side of too much drug still present than on the side of too

little. In the latter case, withdrawal will be difficult to train because the

antagonist cannot precipitate as intense a stimulus, and because the antagonist

stimulus may be difficult to differentiate from spontaneous withdrawal occurring

under the vehicle condition.

CONCLUSIONS

Three-choice drug discriminations are conceptually interesting and offer more

potential for studying withdrawal phenomena than their two-choice

counterparts. Unfortunately, little information is available concerning

fundamental parameters of three-choice learning. Moreover, this technology

has been used only recently to study drug withdrawal. As more data

accumulate from these experiments, it should be possible to specify optimal

parameters for training the discrimination. More importantly, it should be

possible to unequivocally determine whether withdrawal-related stimuli can be

studied using drug discrimination methodology.

One caveat should perhaps be added. The withdrawal-related stimuli of most

interest are probably those arising from spontaneous withdrawal, for it is this

type of withdrawal that humans usually experience. Whether a three-choice

discrimination can be extended to encompass training of spontaneous

withdrawal is unknown, but such a discrimination would probably be even more

difficult to establish than one using antagonists. On the other hand, in other

tests spontaneous and precipitated withdrawal are usually seen to differ only in

their magnitude and duration of signs and symptoms. Thus, it is likely that data

acquired from studies of precipitated withdrawal will have direct application to

human phenomena. In addition, it should be possible to test to what extent

spontaneous withdrawal substitutes for the antagonist in these discriminations,

which should also serve as a proof that similar events are being measured.

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morphine in the rat. J Pharmacol Exp Ther 198:54-65, 1976.

356
Valentino, R.J.; Herling, S.; and Woods, J.H. Discriminative stimulus effects of

naltrexone in narcotic-naive and morphine-treated pigeons. J Pharmacol Exp

Ther 224:307-313, 1983.

Weissman, A. The discriminability of naloxone in rats depends on concomitant

morphine treatment. In: Colpaert, F.C., and Rosecrans, J.A., eds. Stimulus

Properties of Drugs: Ten Years of Progress. Amsterdam: Elsevier-North,

1978. pp. 209-214.

White, J.M., and Holtzman, S.G. Three-choice drug discrimination in the rat:

Morphine, cyclazocine, and saline. J Pharmacol Exp Ther 217:254-262,

1981.

White, J.M., and Holtzman, S.G. Further characterization of the three-choice

morphine, cyclazocine, and saline discrimination paradigm: Opioids with

agonist and antagonist properties. J Pharmacol Exp Ther 224:95-99, 1983.

Wikler, A. Opioid Dependence: Mechanisms and Treatment. New York: Plenum

Press, 1980.

Woudenberg, F., and Slangen, J. Discriminative stimulus properties of

midazolam: Comparison with other benzodiazepines. Psychopharmacology

79:466-470, 1989.

Young, A.M., and Sannerud, C.A. Tolerance to drug discriminative stimuli. In:

Goudie, A.J., and Emmett-Oglesby, M.W., eds. Psychoactive Drugs:

Tolerance and Sensitization. Clifton, NJ: Humana Press, 1989. pp. 221-278.

Young, A.M.; Walton, M.; and Perkins, A. Characterization of a discrimination

among two doses of morphine and saline in pigeons. Drug Dev Res

16:163-168, 1989.

ACKNOWLEDGMENTS

Supported by grants DA RO1 03521 and DA F32 05367 from the National

Institute on Drug Abuse.

AUTHORS

M. W. Emmett-Oglesby, Ph.D

G. A. Rowan, Ph.D.

Department of Pharmacology

Texas College of Osteopathic Medicine

Fort Worth, TX 76107-2690

USA

357

Schedule-Induced Self-Injection of Drugs

George Singer

INTRODUCTION

In our laboratories we have combined a self-injection technique with a food

delivery schedule and developed the method of schedule-induced

self-injections (SISI). We have been able to classify drugs according to

acquisition patterns and have shown that the stimulus complex necessary for

self-injection to occur varies with the type of drug presented. The presence of a

food delivery schedule and the state of nutrition interact with the drug in the

acquisition and maintenance of drug-intake behaviors.

This model can be used in traditional pharmacological studies to investigate

specific and general blockers of drug intake. Other studies have shown the

importance of intact dopaminergic neurons in the nucleus accumbens septum

for the acquisition and maintenance of drug intake behavior. Biochemical

studies with the SISI model have shown that an increase in corticosteroid levels

is associated with schedule-induced behaviors. The theoretical implications as

well as implications for therapeutic programs are included in the following

discussion.

REINFORCEMENT VERSUS PHYSICAL DEPENDENCE

Drug and alcohol research and treatment programs are usually based on one of

two assumptions. The first is the psychological concept of “reinforcement” and

its major role in the acquisition and maintenance of drug intake behavior, and

the second is the pharmacological concept of physical dependence.

Those who subscribe to the notion of reinforcement believe that a drug is either

initially reinforcing or acquires reinforcement value after repeated exposure.

The facts that most naive organisms generally reject drugs initially because of

This paper is an update of a paper presented as the Fifty-first Beattie Smith Lecture in the

Department of Psychiatry at the University of Melbourne in 1985.

359
aversive taste or aversive after-effects, and that, even in the later stages of drug

intake, aversive after-effects remain are ignored. In addition, it is often

postulated that drugs create a new “need state” and that the satisfaction of this

need is reinforcing. Because this need state cannot be measured independently

from the drug intake behavior, it is not a useful concept.

Those who subscribe to the notion of physical dependence regard physical

dependence as synonymous with withdrawal and believe that organisms take

drugs and become dependent on them in order to relieve the discomfort of

withdrawal. Experimental evidence does not confirm this thesis and shows that

relief of withdrawal is neither a necessary nor a sufficient condition for either the

acquisition or the maintenance of drug intake.

This is not an attempt to deny that physiological changes occur in the central

nervous system (CNS). Rather, it is the role of these changes as a major

driving force to take drugs that is questioned. These remarks do not apply to

drug discrimination and should be confined to theory and experimentation

concerned with voluntary drug intake.

The purpose of this paper is to review recent research, including 10 years of

drug research in our laboratories, in order to provide guidance for research and

treatment programs.

METHOD

Using a combination of self-injection techniques and the scheduled delivery of

food pellets to animals with reduced body weight (schedule-induced

self-injection), we have produced a classification of psychoactive drugs based

on behavioral factors shown in table 1.

The scheduled food delivery, in which one pellet is delivered every 60 sec to a

chronically hungry animal, causes frustration and stress, which are reflected in

increased plasma corticosterone levels. This is shown in table 2. These data

suggest that the initiation of drug intake behavior is dependent on the

interaction of the following three factors: (1) the presence of a drug molecule in

the injection solution; (2) reduced body weight; and (3) the use of a food

delivery schedule. In common parlance, these factors are drug, nutrition, and

stress. These factors form a stimulus complex that is necessary for the initiation

of drug intake behavior in animals. (The effect of the combination of factors,

however, varies with the type of drug.)

360
TABLE 1. A classification of drugs according to the acquisition of intake

patternsa

Group 1 Group 2 Group 3 Group 4

Alcohol

Some opiates

(e.g., heroin)

Self-injections

occur without the

schedule but are

enhancedby

reduced body

weight and the

schedule

Amphetamine

Cocaine

Phentermine

-THCb Haloperidol Saline

Nicotine

Methadone

Acetaldehyde

Benzodiazepines

Barbiturates

Self-injections are Self-injections

greatly enhanced occur in the

with reduced body presence of the

weight schedule plus

reduced body

weight

Currently

unsuccessful in

inducing

self-injection

a

From Singer and Wallace 1984a.

bDelta-9-tetrahydrocannabinol.

TABLE 2. Mean plasma corticosterone levels (μg/100 mL after

schedule-induced) drinking behaviora

Mean Plasma Corticosterone Levels

Day Scheduled Animals

2 274

10 265

20 270

Nonscheduled Animals

134

111

125

a

From Finlay and Wallace 1981.

In human drug intake behavior the variety and complexity of factors that interact

are probably much greater, and the total stimulus complex is more difficult to

specify. One observation from this research is that the interaction of reduced

body weight and CNS stimulants, which are also anorectics, leads to increased

361
stimulant drug intake. This phenomenon has also been noted by other

researchers (Papasava and Singer 1985; Papasava et al. 1981, 1985a, 1985b;

Takahashi et al. 1978). Thus, when anorectic drugs are effective in producing

weight loss, their addiction potential increases (Papasava et al. 1985a, 1985b).

Cur research also shows that stress enhances the intake of most, but not all,

drugs of abuse-a point sociologists have made for some time. In another

series of experiments we have shown that the factors that are important in the

initiation of drug intake are not always essential for its maintenance if a

relatively stable pattern of intake has developed. Some of this work is shown in

table 3.

Of particular relevance in the present context are the findings of an experiment

conducted by Madden et al. 1979. In this experiment, it was shown that after 20

days of heroin self-injection, rats will self-inject saline at high rates, a behavior

that does not normally occur. Moreover, it was shown that rats that were

allowed to self-inject saline after self-injecting heroin had fewer withdrawal

symptoms than rats that were withdrawn from heroin without the opportunity to

self-inject saline. This finding supports earlier conclusions that drug

dependence involves many factors and shows that this is also true for

withdrawal symptoms.

Kalant (1982) has recently shown that the occurrence and degree of tolerance

can be conditioned to the environment. However, this phenomenon, which has

been called behaviorally conditioned tolerance, becomes less

environment-dependent as the drug dose is increased.

In an elegant presentation at the conference Dr. Siegel has also argued that

tolerance is dependent on the environment.

STIMULUS COMPLEX INTERACTION MODEL

Collectively, these data show that dependence, withdrawal, and tolerance result

from the interaction between environment and pharmacological factors and that

all these phenomena can be explained in terms of a stimulus complex

interaction model. The hypothesis on which this model is based raises a

number of interesting questions about treatment programs. First and foremost is

the efficacy of combining pharmacological and psychological therapies.

Although the use of Antabuse and methadone in conjunction with

environmental or psychological treatments is an application of this principle, this

combination is rare in regard to drug and alcohol treatment programs. The

362
TABLE 3. Maintenance patterns of drug self-injectiona

Drug Intake and Response

Schedule removal

Heroin

Nicotine

Cocaine

Alcohol

Body weight restitution

Nicotine

Cocaine

THCC

Substitution of saline (vehicle) for drug

Heroin

Antagonism of drug effect

Heroin and naloxone

Intake drops

After 5 days of schedule, intake drops.

After 14 days, intake maintained. Will

initiate intake without schedule if body

weight is 80%, but acquisition is very

slow (>22 days).

Intake high if body weight low

Intake drops

Intake maintained if recovery period

occurs with nicotine intake under SISl.b

Intake maintained if only saline

available during recovery.

Intake drops immediately

Intake drops

Responding maintained

Responding for drug drops to saline

pretreatment levels

aSee review by Singer and Wallace 1984a for individual references.

bSchedule-induced self injection.

cDelta-Stetrahydrocannabinol.

question is whether it is better to totally change the stimulus complex in

treatment procedures or to remove one component of the stimulus complex and

keep everything else constant.

363
For example, a total change in the stimulus complex occurred when

drug-addicted Vietnam War veterans were discharged and returned to their

home towns. Everything changed, including friends, job, home conditions, and

food and reports indicate that most of these veterans were able to withdraw

from drugs (Jaffe 1980).

The alternative to a total environment change is removing the drug gradually.

We have observed outpatients on a methadone maintenance program in which

all features of the stimulus complex were maintained except that methadone

was substituted for heroin. This procedure was based on Madden’s (1983)

laboratory work and has shown some early promise in a clinical setting.

However, further clinical research is needed to clarify these findings. Table 4

shows this experimental paradigm.

The experiments that follow outline some of the research that was designed to

specify more precisely the relationship between environment, drug intake

behavior, neurochemical substrates, and neuroanatomical loci. First, we

conducted traditional pharmacological experiments using the schedule-induced

self-injection procedure. These experiments were directed toward elucidating

the neurochemical substrates that mediate the intake of specific drugs. For

instance, phentermine has both dopaminergic and noradrenergic effects. The

question is whether one or both of them are involved in phentermine intake. The

method used was to let animals self-inject the drug until a stable baseline was

established. When this occurred, animals were injected with various blocking

agents known to interfere with neurotransmitter function. It should be noted that

conditions that control for the effect of activity changes due to treatment

compounds are essential in these experiments.

Table 5 shows the data from a range of pharmacological blocking studies. It can

be seen that while phentermine has both noradrenergic and dopaminergic

effects, the dopaminergic effects maintain self-injection. An example is shown

in figure 1.

A further step in this research is to identify specific neuroanatomical loci

involved in self-injection. This has been achieved by introducing neurotoxins

directly into specific brain areas.

Table 1 indicated that nicotine self-injection is contingent on the presence of the

schedule. Furthermore, the schedule increases the level of plasma

corticosterone. When dopaminergic neurons in the nucleus accumbens septum

364
TABLE 4. Experimental paradigm

Phase 1 Phase 2 Results

Methadone is given once Orange juice Patients are unable to

daily in an orange juice administration is estimate methadone

vehicle to heroin-free continued without content of orange juice

outpatients. methadone. and show few withdrawal

symptoms.

TABLE 5. Traditional pharmacological experimentsa

Drug Self-Injected Pretreatment Effect

Phentermine Alpha blockers None

Beta blockers Stops self-injection

Haloperidol

(dopamine blocker)

Alcohol Naloxone Stops self-injection

Buprenorphine

Benzodiazapines Naloxone None

GABA blockers Stops self-inection

Benzodiazapine blocker Stops self-injection

aSee review by Singer and Wallace 1984a.

are depleted with the neurotoxin 6-hydroxydopamine, much nicotine

self-injection behavior is abolished (Singer et al. 1982), as are

schedule-induced increases in plasma corticosteroids (Wallace et al. 1983)

(See table 6).

Similar findings occur in the case of heroin self-injection (Singer and Wallace

1984b), which is also enhanced by the stress-producing schedule. These

examples show that both the stress factor and drug intake are regulated by

dopamine neurons and suggest that stress management, which leads to a

reduction in cortisol levels, could be a promising component in drug treatment

programs for some drugs, e.g., nicotine, alcohol, and opiates.

365
FIGURE 1.

TABLE 6. Mean plasma corticosterone levels for 6-OHDA-lesioned and

sham-lesioned ratsa

Lesion Schedule No Schedule

6-OHDA 12.34 19.19

(1.99) (3.64)

Sham 26.55 13.59

(2.37) (2.25)

aMeasured after 10 daily 1-hr sessions of scheduled food or food delivered in one presentation.

Corticosterone levels are μg/100 mL (±SEM).

366
CONCLUSION

The acquisition of drug self-administration is related to nutritional state and also

to neuroendocrine factors. These variables interact with different drug

molecules in different ways. Some of these environmental factors have been

neglected by those working in the field of drug discrimination. We have

identified an involvement of corticosteroids and dopaminergic neurons in the

nucleus accumbens septum. Some of our findings should be useful in the

interpretation of data and in the design of experiments in drug discrimination.

ACKNOWLEDGMENTS

The work of the following individuals in the research from our laboratories is

acknowledged: Drs. Meredith Wallace, Kim Ng, Tian Oei, Wendy Myers,

Michael Papasava, Reinaldo Takahashi, and Chris Madden and graduate

students Stephen Gibson, Rudy Pilotto, Don Jefferys, and Ann Martin.

REFERENCES

Finlay, J.D., and Wallace, M. Effect of scheduled food delivery on

corticosterone levels in the rat. Proc Aust Neurosci Soc 1:1981.

Jaffe, H.J. Drug addiction and drug abuse. In: Gilman, A.G.; Goodman, L.S.;

and Gilman, A., eds. The Pharmacological Basis of Therapeutics. 6th ed.

New York: Macmillan, 1980. pp. 535-84.

Kalant, H. Behavioral and neurochemical factors in ethanol tolerance. In: Collu,

R., ed. Brain Peptides and Hormones. New York: Raven Press, 1982. pp.

357-365.

Madden, C.C. “Opiate-seeking behavior in animals and humans.” Ph.D. Thesis,

La Trobe University, 1983.

Madden, C.; Singer, G.; and Oei, T.P.S. The involvement of interoceptive

factors in the maintenance of heroin seeking behavior. Pharmacol Biochem

Behav 11:445-48, 1979.

Papasava, M.; Oei, T.P.S.; and Singer, G. Low dose cocaine self-administration

by naive rats: Effects of body weight and a fixed time one minute food

delivery schedule. Pharmacol Biochem Behav 15:485-486, 1981.

Papasava, M., and Singer, G. Self-administration of low-dose cocaine by rats at

reduced and recovered body weight. Psychopharmacology 65:414-425,

1985.

367
Papasava, M.; Singer, G.; and Papasava, C.L. Phentermine self-administration

in naive free-feeding and food-deprived rats: A dose response study.

Psychopharmacology 85:410-413, 1985a.

Papasava, M.; Singer, G.; and Papasava, C.L. Self-administration of

phentermine by naive rats: Effects of body weight and a food delivery

schedule. Pharmacol Biochem Behav 22:1071-1074, 1985b.

Singer, G., and Wallace, M. Schedule-induced self-injection of drugs: An animal

model of addiction. In: Bond, N.J., ed. Animal Models in Psychopathology.

New York: Academic Press, 1984a.

Singer, G., and Wallace, M. Effects of 6-0HDA lesions in the nucleus

accumbens on the acquisition of self-injection of heroin under schedule and

nonschedule conditions in the rat. Pharmacol Biochem Behav 20:807-809,

1984b.

Singer, G.; Wallace, M.; and Hall, R. Effects of dopaminergic nucleus

accumbens lesions on the acquisition or schedule induced self-injection of

nicotine in the rat. Pharmacol Biochem Behav 17:579-581, 1982.

Takahashi, R.N.; Singer, G.; and Oei, T.P.S. Schedule induced self-injection of

d-amphetamine by naive animals. Pharmacol Biochem Behav 9:857-861,

1978.

Wallace, M.; Singer, G.; Finlay, J.; and Gibson, S. The effect of 6-0HDA lesions

on the nucleus accumbens septum on schedule induced drinking, wheel

running and corticosterone levels in the rat. Pharmacol Biochem Behav

18:129-136, 1983.

AUTHORS

George Singer, Ph.D.

Director

Brain-Behavior Research Institute

Le Trobe University

Bundoora, Australia 3083

368
Use of Drug Discrimination in Drug Abuse

Research

James B. Appel

Lisa E. Baker

Rita L. Barrett

Julie Broadbent

EIizabeth M. Michael

Elizabeth E. Riddle

Bette J. Van Groll

INTRODUCTION

Because drug discrimination (DD) requires that organisms attend to the effects

of drugs to obtain reinforcement (contingent on the occurrence of an

appropriate response), it has been a reliable, sensitive, and, most important,

pharmacologically specific assay (Appel et al. 1982; Cunningham and Appel

1988). This quality has enabled investigators to classify and differentiate closely

related compounds (Barry 1974) and gather important information concerning

their mechanisms of action in vivo (Appel et al. 1978). The assay has been

especially useful in the study of abused drugs because the subjective effects of

most, if not all, such compounds are characterized by dramatic changes in state

(e.g., euphoria, dysphoria) that are readily discriminated by both humans and

other animals; indeed, drugs may well be abused because of the speed and

potency with which they cause such changes to occur.

Like previous international symposia, this meeting has been concerned with

important basic issues such as the mechanisms by which complex drug cues

are transduced (Philips 1990; Stolerman 1990), the laws governing the

development of tolerance to drug cues (Siegel 1990; Young 1990), and the

relationship between DD and related state-dependent phenomena (Overlon

1990). A relatively new focus was on how DD, which has involved primarily

animal research, can be used to further our understanding of important clinical

aspects of substance abuse in humans; in this regard, the papers on cocaine

369
craving (Ehrman 1990) and withdrawal from benzodiazepines and opiates

(Emmett-Oglesby 1990) were of particular interest.

However, it is impossible to comment more than superficially on all the uses of

DD reviewed in the past 2 days. Therefore, we will try to amplify three of

them-differentiation of neuronal mechanisms, receptor interactions, and

stereospecificity of discriminative stimulus effects-with data involving

hallucinogens, stimulants, and related compounds gathered over many years in

our laboratory.

NEURONAL MECHANISMS

DD has contributed to our understanding of the mechanisms subserving the

effects of (±) 2,5-dimethoxy-4-methylamphetamine (DOM), (+) lysergic acid

diethylamide (LSD), and related indoleamine and phenylethylamine

hallucinogens in a variety of ways. For example, Glennon (1990) reports that,

by training rats to discriminate DOM from saline, it has been possible to

(1) classify which agents produce DOM-like effects, (2) study the actions of

DOM metabolites, (3) formulate in vivo structure-activity relationships,

(4) demonstrate a correlation between stimulus generalization potencies and

human hallucinogenic potencies, and (5) investigate mechanisms of action.

We began using DD in the early 1970s to analyze the involvement of serotonin

(5HT) in the behavioral effects of LSD in animals, in part because other assays

(animal models) then in use had been yielding interesting but variable results

that often depended more on species, level of motivation or incentive, schedule

of reinforcement, and baseline response rate than on the particular dose of the

drug being given in a particular test situation (Appel 1968).

At that time, it was generally held that most, if not all, hallucinogens act

presynaptically by directly suppressing the characteristically slow, regular

activity (“firing rate”) of an inhibitory 5-HT system, the cell bodies of which are

located in the dorsal raphe (Aghajanian et al. 1968). However, evidence was

beginning to accumulate in our own (Appel et al. 1970) and other laboratories

(Trulson et al. 1976, 1981) that was at variance with this hypothesis and

suggested that hallucinogens act postsynaptically by both inhibiting and exciting

5-HT target neurons (Jacobs 1983). A somewhat later and particularly

compelling result was that low doses (0.005 mg/kg) of lisuride hydrogen

maleate (LHM), a structural congener of LSD that is not known to be

hallucinogenic in humans, was found to be even more potent than LSD in

370
suppressing raphe unit activity (White 1986). It thus became important to

differentiate the subjective effects of LSD and LHM in animals, a task that

proved to be difficult but possible with DD, in conjunction with extensive

antagonism testing and selective drug vs. drug training procedures (White and

Appel 1982a, 1982b, 1982c) and impossible in less sensitive assays such as

the limb flick (White et al. 1981).

Recently, we used an even more discriminating, three-lever drug vs. drug vs.

saline procedure similar to those described by Bigelow (1990) and

Emmett-Oglesby (1990) to reexamine the stimulus effects of LSD (0.08 mg/kg)

and LHM (0.04 mg/kg); we were again able to show that, in spite of their

structural similarities, these compounds are functionally quite different and,

more important, have different mechanisms of action.

Figure 1 shows that rats can discriminate quantitatively similar doses of LSD

and LHM with little confusion (Callahan and Appel 1990). That is, when given

substitution (dose-response) tests with LSD (0.02-0.16 mg/kg), animals

respond in a dose-related manner on either the LSD- or the saline-appropriate

lever; they never respond on the LHM lever (left panel). Similar effects occur

when the same animals are tested with LHM (0.005-0.08 mg/kg); that is,

responding occurs on either the LHM- or saline-appropriate lever but not on the

LSD lever (right panel).

One example of how the mechanisms underlying the effects of LSD and LHM

differ is suggested by the results of a generalization experiment (figure 2); when

tested with the dopamine (DA) agonist apomorphine, animals responded on

either the LHM- or the saline-appropriate lever (left panel); however, when

given the 5-HT2 agonist quipazine, they responded on the LSD-appropriate

lever (middle panel) and, following the convulsant pentylenetetrazol (which

does not act primarily through either DA or 5-HT systems) they pressed the

saline-appropriate lever (right panel). Thus, LHM appears to be considerably

more dopaminergic than LSD (White 1986), a result also seen during extensive

combination testing with DA and 5-HT antagonists (White et al. 1982b, 1982c).

Indeed, we have never been convinced that DA plays a significant role in the

stimulus effects of LSD. Although it is true that apomotphine sometimes

generalizes at least partially to LSD (in two-lever, apomorphine-vs.-saline

discriminations), we have never found that apomotphine substitutes for LSD in

three-lever (LSD-LHM-saline) discriminations or that the LSD cue generalizes

more than 40 percent to apomorphine in animals trained to discriminate LSD

371
FIGURE 1. Results of dose-response tests with LSD (left panel) and lisuride

(LHM; right panel) in rats (N = 9) trained to discriminate LSD (0.08

mg/kg) from LHM (0.04 mg/kg) from vehicle (saline). Solid lines

with closed triangles denote percent responding on the

LSD-appropriate lever; solid lines with closed squares denote

responding on the LHM-appropriate lever; broken lines with open

circles denote responding on the saline-appropriate lever. All

points are means ± SEM of eight or nine subjects that completed

at least 20 responses on any one of the three levers. (Reprinted

from Callahan and Appel (1990), with permission.)

from saline; moreover, this small amount of “generalization” is unrelated to dose

(Appel et al. 1978). More important, in experiments currently in progress, the

effects of other DA agonists including (+)amphetamine, cocaine, quinpirole (LY

171555) and SCH 2390 do not appear to resemble those of LSD. In addition,

no DA antagonist yet tested in combination with hallucinogens blocks the

effects of either LSD or mescaline (table 1).

372
FIGURE 2. Results of substitution (generalization) tests with apomorphine

(left panel), quipazine (center panel), and pentylenetetrazol (right

panel) in rats trained to discriminate LSD (0.08 mg/kg) from LHM

(0.04 mg/kg) from saline. Symbols as in figure 1. (Reprinted from

Callahan and Appel (1990), with permission.)

RECEPTOR INTERACTIONS

DD has also provided evidence that the same receptors and receptor subtypes

that mediate other physiological and biochemical effects of abused substances

are involved in their subjective properties and, hence, their abuse potential. This

point seems to be true with central nervous system (CNS) stimulants (Goudie

1990; Woolverton 1990) and other DA agonists (Appel et al. 1988)

cannabinoids (Järbe 1990), nicotine (Rosecrans and Villanueva 1990)

sedative-hypnotics (Barry 1990), opiates (Woods et al. 1990), and

phencyclidine-like agents (Balster 1990). The same point has been particularly

important in the case of hallucinogens, because the effects of these substances

have been so difficult to categorize with other procedures (Appel 1988).

373
TABLE 1. Results of combination (antagonism) tests in animals trained to

discriminate LSD (L) or mescaline (M) from saline

Training Putative Antagonist

Drugs(s)

Complete Antagonism ( 40%):

Reference

(L) (M)

(L)

(M)

(L) (M)

(L) (M)

(L) (W) LY-53857

(L) (M)

(L)

(L)

(L) (M)

Metergoline

Methiothepin

Methysergide

Pirenpirone

BC-105

Bromo-LSD

Cinanserin

Cyproheptadine

Ketanserin

(L)

(L)

Ritanserin

Trazodone

Colpaert et al. (1982)

Cunningham and Appel (1987)

Holohean et al. (1982)

Nielsen et al. (1985)

Winter (1978)

Cunningham and Appel (1987)

Browne (1978)

Browne and Ho (1975)

Winter (1975, 1978)

Browne (1978)

Browne and Ho (1975)

Kuhn et al. (1978)

White and Appel (1982c)

Appel and Callahan (1989)

Cunningham and Appel (1987)

Nielsen et al. (1985)

Appel and Callahan (1989)

Cunningham and Appel (1987)

Appel and Callahan (1989)

Cunningham and Appel (1987)

Kuhn et al. (1978)

Kuhn et al. (1978)

Colpaert et al. (1982)

Appel and Callahan (1989)

Cunningham and Appel (1987)

Nielsen et al. (1985)

Colpaert et al. (1985)

Cunningham and Appel (1987)

374
TABLE 1. Continued

Training Putative Antagonist

Drugs(s)

Partial Antagonism (41-79%):

(L) Bromo-LSD

(L) Cinanserin

(L) Cyproheptadine

(L) (M) Metergoline

(L) (M) Methysergide

(L) Metitepine

(L) Methiothepin

(L) Mianserin

(L) Naloxone

No Antagonism ( 80%):

(L) (M) Atropine

(L) (+) Butaclamol

(L) Chlorimipramine

(L) Cyprcheptadine

(L) Fluoxetine

(L) Fluphenazine

(L) (Alpha)-Fluphenthixol

Reference

Colpaert et al. (1982)

Kuhn et al. (1978)

Colpaert at al. (1982)

Kuhn et al. (1978)

Colpaert et al. (1982)

Kuhn et al. (1978)

Browne (1978)

Colpaert et al. (1982)

Browne and Ho (1975)

Colpaert et al. (1982)

Hirschhorn and Rosecrans (1974)

Colpaert et at. (1982)

White and Appel (1982c)

Colpaert et al. (1982)

Hirschhorn and Rosecrans (1974)

Browne and Ho (1975)

Hirschhorn and Rosecrans (1974)

Kuhn et al. (1978)

Kuhn et al. (1978)

Kuhn et al. (1978)

Hirschhorn and Rosecrans (1974)

Kuhn et al. (1978)

Kuhn et al. (1978)

Kuhn et al. (1978)

Appel and Callahan (1989)

375
TABLE 1. Continued

Training Putative Antagonist

Drugs(s)

(L) (M) Haloperidol

(L)

(L)

(L)

(L) (W)

(M)

(L)

(M) (L)

(L)

(L)

(M)

(L)

(L)

(L)

(L) (M)

8-OHDPAT

Naloxone

Phenoxybenzamine

Phentolamine

Pimozide

Promethazine

Propranolol

Pyrilamine

R-56413

Sch-23390

Spiperone

Trifluoperazine

L-tryptophan

Xylamidine

Reference

Colpaert et al. (1982)

Holohean et al. (1982)

Kuhn et al. (1978)

Nielsen et al. (1985)

White and Appel (1982c)

Cunningham and Appel (1987)

Colpaert et al. (1982)

Colpaert et al. (1982)

Browne and Ho (1975)

Kuhn et al. (1978)

Browne and Ho (1975)

Kuhn et al. (1978)

Browne and Ho (1975)

Colpaert et al. (1982)

Kuhn et al. (1978)

Colpaert et al. (1982)

Colpaert et al. (1985)

Appel and Callahan (1989)

Colpaert et al. (1982)

Cunningham and Appel (1987)

Kuhn et al. (1978)

Kuhn et al. (1978)

Appel and Callahan (1989)

Browne (1978)

Browne and Ho (1975)

White and Appel (1982c)

376
Hallucinogens and Serotonin

Although they may not be as convincing as the correlations with receptor

binding reported by Glennon (1990) and are certainly incomplete (because the

catalog of 5-HT receptor subtypes never seems to stop growing), results from

our laboratory suggest that the stimulus effects of both LSD (Cunningham and

Appel 1987) and mescaline (Appel and Callahan 1989) are, like those of DOM,

mediated primarily, if not exclusively, by 5-HT2 receptors. For example, in one

series of experiments, the effects of LSD were compared systematically to

those of 5-HT1 agonists.

Figure 3 shows that the stimulus effects of 0.08 mg/kg of LSD generalize to the

5-HT2 agonist quipazine as well as a sufficiently high dose of LSD but not to

compounds that act primarily at 5-HT1A or 5-HT1B receptor sites-namely,

8-hydroxy-2(di-n-propylamino)tetralin (8-OHDPAT) and

1-(m-triiluoromethylphenyl)piperazine (TFMPP), respectively (Cunningham and

Appel 1988). Moreover, LSD does not substitute either for 8-OHDPAT

(Cunningham et al. 1987) in animals trained to discriminate 0.4 mg/kg of this

5-HT1A agonist from saline (figure 4), or TFMPP (Cunningham and Appel 1986)

in animals trained to discriminate 0.8 mg/kg of this 5-HT1B agonist from saline

(figure 5).

Similarly, figures 6 and 7 show that the effects of mescaline generalize to other

hallucinogens (Callahan and Appel 1988) and, like those of LSD, are blocked

potently and completely by centrally acting, nonselective 5-HT and selective

5-HT2 antagonists (Appel and Callahan 1989) but not by either peripheral 5-HT

or DA antagonists (table 1).

Amphetamine, Cocaine, and Dopamine

Although DD has been less successful in delineating the mechanisms

underlying the subjective effects of CNS stimulants than those of hallucinogens

and other 5-HT receptor agonists, the papers of Goudie (1990) and Woolverton

(1990) indicate that this situation is changing rapidly. Studies involving this

assay are beginning to suggest, as we have argued elsewhere (Broadbent and

Appel 1990), that all stimulants may not be as similar as hitherto supposed and

that (+) amphetamine and cocaine may act differently at DA (Goudie 1990) as

well as other (5-HT?) receptor sites (Broadbent and Appel 1990).

377
FIGURE 3. Results of dose-response and substitution tests with putatively

selective 5-HT agonists in rats trained to discriminate LSD (0.08

mg/kg) from saline. Dots = performances during test sessions that

were significantly different from previous LSD training session.

(Reprinted from Cunningham and Appel (1987), with permission.)

378
FIGURE 4. Results of dose-response and substitution tests with the ergot

derivatives lisuride and LSD in animals trained to discriminate

8-OHDPAT (0.4 mg/kg) from saline. * = performances during test

sessions that were significantly different from the previous

8-OHDPAT training session. (Reprinted from Cunningham et al.

(1987), with permission.)

Our own work with (+) amphetamine has not been extensive. We showed some

time ago that, in amphetamine-trained animals, the effects of this CNS stimulant

are not at all like those of LSD and other hallucinogens (Kuhn et al. 1974) and,

more recently, replicated this result in rats trained to discriminate 0.08 mg/kg of

LSD from saline (above). We have also found, in agreement with Goudie

(1990) that D1 and, especially, D2 receptors are involved in the behaviorally

relevant actions of (+) amphetamine (Callahan et al. 1991; Kuhn et al. 1974). In

addition, and in response to Goudie, we have seen no evidence that the

stimulus effects of (+) amphetamine are altered significantly by compounds that

act (either presynaptically or postsynaptically) at noradrenergic (NE) receptor

sites.

379
FIGURE 5. Results of dose-response and substitution tests with Ru 24969,

LSD, and 8-OHDPAT in rats trained to discriminate

1-(m-trifluoromethylphenyl) piperazine (TFMPP) from saline.

Dots = performances during test sessions that were significantly

different from previous TFMPP training session. (Reprinted from

Cunningham et al. (1986), with permission.)

We have been more interested in the mechanisms underlying the stimulus

effects of cocaine and, like Woolverton (1990), have found that they involve DA

(especially DA uptake inhibition) in important and complex ways. Although

some directly acting DA agonists, particularly those that act at D2 receptors (LY

171555), appear to substitute at least partially for cocaine (Barrett and Appel

1989a; Callahan et al. 1991) other clinically effective dopaminergic

compounds, including bromocriptine and amantadine, apparently do not

(table 2).

Unfortunately, we have had great difficulty in blocking- the stimulus effects of

cocaine with both D1 and D2 antagonists (figure 8), although more potent,

nonselective DA antagonists such as cis-flupenthixol may be effective in this

regard.

As Woolverton (1990) points out, the stimulus effects of cocaine generalize

readily to DA uptake inhibitors such as bupropion, nomifensine, and GBR

380
FIGURE 6. Results of dose-response and substitution tests with other

hallucinogens in rats trained to discriminate mescaline (10 mg/kg)

from saline. Compounds tested, number of animals responding

(n), and number of animals tested (N) at each dose are shown at

top of figure. (Reprinted from Callahan and Appel (1988), with

permission.)

12909 (Broadbent et al. 1989b); we have also found that this generalization can

be blocked at least partially by cis-flupenthixol.

Further evidence that DA mediates the subjective effects of cocaine is provided

by results such as those illustrated in figure 9; in this experiment, the ability of

three DA agents to substitute for cocaine was potentiated significantly by a

subthreshold (nondiscriminable) dose of cocaine, which nevertheless inhibits

DA uptake (Barrett and Appel 1989b).

STEREOSPECIFICITY: DIFFERENTIATION OF THE EFFECTS

OF (+) AND (-) MDA

DD has provided important information about designer drugs such as

3,4-methylenedioxyamphetamine (MDA) and

3,4-methylenedioxymethamphetamine (MDMA), although the mechanisms

381
FIGURE 7. Results of tests with 5-HT2

 antagonists given 60 min prior to

experimental sessions to rats treated with mescaline (10 mg/kg)

15 min prior to testing. Compounds tested, number of animals

responding (n), and number of animals tested (N) at each dose

are shown at top of figure. (Reprinted from Appel and Callahan

(1989), with permission.)

mediating the effects of these abused neurotoxins (Insel et al. 1989; Johnson et

al. 1986; Schmidt 1987) continue to evade precise delineation. For example,

the optical isomers of MDA have been reported to have different, stimulant-like

or hallucinogen-like effects on the basis of findings that (-) MDA mimics DOM

but not (+) amphetamine, while (+) MDA mimics amphetamine but not DOM

(Glennon and Young 1984; Glennon et al. 1982).

We have been able to partially replicate and extend these results by training

animals to discriminate equivalent intraperitoneal doses (1.25 mg/kg) of the

enantiomers of MDA from saline (Appel et al. 1990). Figure 10 shows that the

stimulus effects of the (-) isomer generalize completely to those of LSD as well

as DOM, partially to mescaline, and not at all to stimulants such as

(+)amphetamine and cocaine.

382
TABLE 2. Drugs that do not substitute completely for cocaine (10 mg/kg, IP)

Drug Dose Percent Rate N

(mg/kg) (±SEM) (±SEM)

Dopaminergic agents

Amantadine (60 min)

NaCl

Cocaine

Amantadine

Bromocriptine (30 min)

NaCl

Cocaine

Bromocriptine

NE and 5-HT uptake inhibitors

Desipramine (15 min)

NaCl

Cocaine

Desipramine

Desipramine (30 min)

NaCl

Cocaine

Desipramine

lmipramine (30 min)

NaCl

Cocaine

lmipramine

383
TABLE 2. Continued

Drug

Nisoxetine (45 min)

NaCl

Cocaine

Nisoxetine

Dose Percent Rate N

(mg/kg) (±SEM) (±SEM)

- 10 ± 8 46 ± 6 12

10.0 100 ± 0 32 ± 9 12

4.5 9 ± 7 35± 7 12

9.0 12± 8 14 ± 2 12

13.5 19± 8 23 ± 14 12

18.0 44±14 20± 5 8

FIGURE 8. Results of combination tests with three DA antagonists in animals

trained to discriminate cocaine (10 mg/kg) from saline. (Reprinted

from Barrett and Appel (1989a), with permission.)

384
FIGURE 9. Potentiation of the cocaine-like effects of apomorphine,

bromocriptine, and L-DOPA by a low dose of cocaine (2.5 mg/kg).

The discriminative stimulus properties of the (+) isomer of MDA are less clear

than those of the (-) isomer in that they generalize completely to LSD and,

surprisingly, cocaine and only generalize partially to DOM, mescaline, and

(+)amphetamine (Fig. 11).

Moreover, the (-) MDA, but not the (+) MDA cue can be blocked by 5-HT2

antagonists such as pirenpirone, suggesting that, at least at the dose tested

(1.25 mg/kg), the (-) isomer is more serotonergic as well as LSD-like than the

(+) isomer (Appel et al. 1990).

CONCLUSIONS

Historically, the most important contribution DD has made to drug abuse

research (as well as to other aspects of psychopharmacology) has been in the

area of behavioral pharmacodynamics (Appel et al. 1978), which is concerned

385
FIGURE 10 Results of substitution tests with hallucinogens (LSD, DOM, mescaline) and stimulants ((+)

amphetamine, cocaine) in rats trained to discriminate (-) MDA (1.25 mg/kg) from saline. n/N

< 0.5 means that ratio of number of animals responding (n) to the number of animals tested

(N) was less than 0.5 (50%).
FIGURE 11. Results of substitution tests with hallucinogens (LSD, DOM, mescaline) and stimulants ((+)

amphetamine, cocaine) in rats trained to discriminate (+) MDA (1.25 mg/kg) from saline.

n/N < 0.5 means that ratio of number of animals responding (n) to number of animals

tested(N) was less than 0.5 (50%).
FIGURE 12. Results of substitution tests with the (+) and (-) enantiomers of

MDA and MDMA in rats trained to discriminate LSD (0.08 mg/kg)

from saline. Compounds tested, number of animals responding

(n). and number of animals tested (N) at each dose are shown at

top of figure. (Reprinted from Callahan and Appel (1988), with

permission.)

with delineating the neuronal and receptor mechanisms most relevant to the

overt and covert (subjective) effects of drugs in intact, behaving organisms.

With regard to the substances with which this review has been primarily

concerned (indoleamine and phenylethylamine hallucinogens, CNS stimulants,

and compounds that may have both hallucinogen-like and stimulant-like

effects), DD has (1) indicated that the most likely neuronal mechanism through

which DOM, LSD, and mescaline act in vivo is direct stimulation of postsynaptic

serotonin (5-HT2) receptors (although research still needs to be done with

substances that affect 5-HT1C, 5-HT3, and 5-HT4 mechanisms);

(2)demonstrated that CNS stimulants such as amphetamine and cocaine may

have different subjective effects that involve different DA mechanisms; and

(3) provided clues to the nature of the states induced by the optical isomers of

388
FIGURE 13. Results of substitution tests with (+) and (-) enantiomers of MDA and MDMA in rats

trained to discriminate LSD (0.16 mg/kg) from saline. n/N < 0.5 means that ratio of

number of animals responding (n) to number of animals tested (N) was less than 0.5

(50%).
designer drugs and, hence, why these dangerous, neurotoxic substances

continue to be abused (Downing 1986; Peroutka 1987).

However, DD can also be useful in other aspects of both preclinical and clinical

pharmacology. For example, if they do not have other side effects that preclude

this possibility, the fact that 5-HT2 antagonists block the discriminable effects of

DOM, LSD, and mescaline suggests that drugs like pirenpirone, ketanserin,

ritanserin, and especially risperidone (Meert 1990) ought to be useful in the

treatment of the bad trips induced by indoleamine and phenylethylamine

hallucinogens. The results of DD experiments also indicate that DA uptake

inhibitors (bupropion, nomifensine) ought to be more effective than NE uptake

inhibitors (desipramine, imipramine) in attenuating cocaine craving and relieving

the symptoms of cocaine withdrawal.

Finally, because of its correlation with many other diverse effects of abused

drugs ranging from receptor binding to reports of hallucinations, DD has been of

value in both the analysis of structure-activity relationships (Glennon 1990) and

the design and development of potential therapeutic agents (Balster 1990;

Meert 1990).

However, no pharmacological assay is free of problems, and DD certainly is no

exception. The technique is difficult and time-consuming to implement properly;

it therefore should not be used by investigators who have had little experience

conducting long-term behavioral experiments or who are interested in screening

large numbers of compounds for a given effect in a short period of time.

Another problem is that the assay involves the psychological process of

stimulus generalization, the interpretation of which is fraught with difficulties.

That is, the extent to which any stimulus generalizes to any other stimulus

obviously depends on stimulus or dimensional “similarity”-however that

concept may be defined (Herrnstein 1984)-but also on many other, less

obvious factors, including the organism’s experience, training history, and

sensory capacity (Domjan and Burkhard 1986). Thus, it should not be surprising

to learn that the results of DD experiments (e.g., the extent of generalization of

a compound to other, related agents) depend critically on stimulus (drug)

parameters such as training dose and nonstimulus events such as the

sensitivity of the particular training procedure used before testing begins (Järbe

1989).

390
As an example of how important one of these variables, training dose, can be,

we have shown that the effects of 0.08 mg/kg of LSD generalize to (-) MDA to a

much greater extent than they do to other designer drugs (figure 12)-a result

that supports those of other investigators (Nichols et al. 1986). However, this

effect does not always occur; for instance, it is not at all clear that the stimulus

properties of 0.16 mg/kg of LSD generalize any more than partially to the two

isomers of MDA (figure 13). Thus, it is premature to conclude from these data

that any isomer of any designer drug has or does not have LSD-like effects

although other results suggest that the effects of (-) MDA (1.25 mg/kg)

generalize convincingly to LSD (Appel et al. 1990). Similar effects occur with

other drugs of abuse including amphetamines (Stolerman and D’Mello 1981)

and cocaine (Broadbent et al. 1989a).

For all these reasons, the results of DD research should not be used

casually-for example, to provide quick answers to deceptively simple

questions such as whether compound X is a narcotic. Assuming we know what,

if anything, the term “narcotic” means pharmacologically, the best the assay can

do is (1) identify the extent to which the effects of a given range of doses of X

resemble those of prototypic members of a given class of opiates, e.g., mu or

kappa agonists, in a given species; and (2) suggest one or more likely

mechanisms whereby X might act in vivo: Are its subjective effects blocked by

naloxone?

In addition, as in any assay, the results of DD experiments depend on the

specificity (or, too often, lack of specificity) of the agents used by the

investigative team as pharmacological tools. Is SCH 23390 a selective D1

antagonist, and, if so, is the ability of this substance to partially block the effects

of cocaine (Woolverton 1990) due to this aspect of its neuropharmacology?

Finally, a number of technical problems continue to plague DD research. While

none of them may be of crucial importance to the success or failure of the

assay (which is still better than most others for all the reasons we have

discussed), debate about them continues to consume time that could probably

be spent more profitably in other ways. For example, there is still little

agreement on whether test data should be analyzed quantally or quantitatively

(Stolerman et al. 1990) and on what the criteria for substitution (generalization)

or antagonism ought to be: Should these concepts be defined statistically or by

some fixed percentage, as in psychophysics? A few of these problems might be

solved or at least diminished if some degree of standardization existed in the

field. To this end, it might be interesting to ask a select committee of members

391
of the Society for the Stimulus Properties of Drugs or the European Behavioral

Pharmacology Society to meet in Geneva to develop a set of standards to

guide all DD research. However, even if such standards could be established,

they would probably be ignored; how often do organic chemists,

pharmacologists, or, for that matter, politicians pay attention to the results of

Geneva conventions?

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396
ACKNOWLEDGMENT

Preparation of this manuscript was supported by U.S. Public Health Service

Research Grant R01 DA02543 from the National Institute on Drug Abuse.

AUTHORS

James B. Appel, Ph.D.

Lisa E. Baker, Ph.D.

Rita L. Barrett, M.A.

Julie Broadbent, Ph.D.’

Elizabeth M. Michael, B.A.

Elizabeth E. Riddle, B.A.

Bette J. Van Groll, B.A.

Behavioral Pharmacology Laboratory

Department of Psychology

University of South Carolina

Columbia, SC 29208

1Present address: Department of Physiology and Pharmacology, Bowman Gray

School of Medicine, 300 South Hawthorne Road, Winston-Salem, NC 27103.

397

Symposium Critique

L.S. Harris

The charge I was given by Dr. Glennon was to sum up and critique the meeting

from the point of view of an elder statesman who has a strong interest in the

effects of chemicals on the central nervous system (CNS) yet is not directly

involved in drug discrimination (DD) research. This role has been thrust on me

more and more lately and makes me feel like the chairman of the board who

can sit through a long, complex board meeting and brilliantly sum up and point

to new directions but 2 weeks later can barely remember what went on.

I was very appreciative of Drs. Schuster and Overton for providing a fine

historical perspective. After listening for the past 2 days, I am again impressed

by the breadth and depth of the progress in the DD field in the past few years.

The technique itself has much to commend it. However, many of its virtues

have led and may continue to lead to problems and difficulties. For instance,

although DD is the best animal procedure we have for predicting subjective

effects in humans, it leads, in many instances, to overinterpreting the data. The

problems of false positives and false negatives, which were alluded to by Drs.

Goudie and Bigelow, are not generally recognized. That the field is greatly in

need of additional human studies to provide validity for the animal studies is

beginning to emerge, as demonstrated by the presentations of Drs. Johanson

and Bigelow and other published reports. More, however, are needed.

Among the beauties of the DD technique are good selectivity, relative

operational ease, and reproducibility from laboratory to laboratory. These

qualities are also among its dangers, however. Testing for potential side effects

during drug development seems to be becoming a screening technique.

Standardization may lead to formalization of the problem of false positives and

false negatives and, in the long run, actually stifle originality in drug

development. An example from my own experience is the use of analgesic

tests. Over several decades we developed test procedures, such as the hot

plate and tail flick tests, that were highly predictive of analgesic activity in

humans. Unfortunately, they were also highly correlative with dependence

399
liability and thus were not very useful alone in developing new analgesics with

less or no abuse potential. We did have a clue from serendipitous studies in

humans with the opiate antagonist nalorphine, which had little or no analgesic

activity in the hot plate and tail flick tests. It antagonized morphine in these

procedures, yet in humans it proved to be equipotent with morphine as an

analgesic. In addition, it had little or no dependence potential in animals or

humans. Of course, we now know that nalorphine is a mixed

agonist-antagonist. In our development of other mixed agonist-antagonist

analgesics we discarded compounds that had strong activity in the tail flick and

hot plate tests. We developed tests to quantify opioid antagonism and selected

compounds for further evaluation on this basis. Thus pentazocine, cyclazocine,

and so on were sent out for clinical evaluation as analgesics with little evidence

for their efficacy in laboratory animals. Thus, from my experience, new

advances are more likely to be made by pursuing leads rather than

perseverating in formerly useful screening procedures.

On this note, let me point out that DD is not a test for dependence or abuse

liability. It does have the ability to classify drugs pharmacologically and, thus,

put in place one of the findings necessary for regulatory action. I would strongly

oppose the use of DD alone in making decisions about scheduling drugs under

either national or international control. Indeed, many people forget that under

the Psychotropic Convention, a substance must have pharmacological

properties similar to an already controlled substance and-I emphasize

and-have similar abuse potential and produce public health and social harm.

These effects cannot be predicted from discrimination data alone.

I was pleased to see the great attention being paid to the use of DD in

mechanistic studies. This is a very positive direction for the field to take.

However, along with Dr. Schuster, I would like to see more emphasis on the

role of behavioral and environmental factors in DD. For instance, has enough

been done to explore various schedules of reinforcement? I wonder whether the

theories of rate dependence would influence DD designs. Would schedules

engendering high output of behavior be more effective in revealing the

discriminative properties of CNS depressants and vice versa? It has been my

experience that, although behavioral techniques are useful in studying drugs,

we probably learn more by using drugs to study behavior.

Although dose-response studies are becoming standard in DD experiments, not

enough attention is being paid to biodispositional and pharmacokinetic

parameters. This lack of attention often leads to misinterpretation of results.

400
A word now about correlations, which continue to be used frequently in this

field. Correlations do not prove causal relationships. They are only suggestive.

When correlations are constructed, all data on each parameter-positive,

negative, and inconclusive-must be included. If they are not, your data are

likely to be misinterpreted.

Finally, a word about the development of medications for the treatment of the

chemical dependencies. I see DD techniques as playing a key role in this

development. Both animal and human data generated using discriminative

stimulus techniques will play an essential role in developing truly unique new

treatments for this massive worldwide public health problem.

I trust I have not stepped on too many toes and apologize for any feathers

ruffled. If I erred in any of my remarks, it was because of my lack of hands-on

experience in the field, and I hope you will forgive me.

AUTHOR

L.S. Harris, Ph.D.

Chairman

Department of Pharmacology and Toxicology

Medical College of Virginia

Virginia Commonwealth University

Richmond, VA 23298

401

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