Pharmacology

and Toxicology

of Amphetamine

and Related

Designer Drugs

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol Drug Abuse and Mental Health Administration
Pharmacology and Toxicology

of Amphetamine and Related

Designer Drugs

Editors:

Khursheed Asghar, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

Errol De Souza, Ph.D.

Addiction Research Center

National Institute on Drug Abuse

NIDA Research Monograph 94

1989

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

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, DC 20402

Pharmacology and Toxicology

of Amphetamine and Related

Designer Drugs
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from a technical

review on pharmacology and toxicology of amphetamine and related

designer drugs that took place on August 2 through 4, 1988, in Bethesda,

MD. The review meeting was sponsored by the Biomedical Branch,

Division of Preclinical Research, and the Addiction Research Center,

National Institute on Drug Abuse.

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 matieral

in this volume except quoted passages from copyrighted sources is in the

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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 U.S. Department of Health and Human

Services.

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

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

context of the studies reported herein.

DHHS publication number (ADM)89-1640

Printed 1989

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.

iv
Contents

Page

Preface i x

Structure-Activity Relationships of MDMA-Like Substances 1

David E. Nichols and Robert Oberlender

Self-Injection in Baboons of Amphetamines and Related

Designer Drugs 30

CA. Sannerud, J.V. Brady, and R.R. Griffiths

Stimulus Properties of Hallucinogenic

phenalkylamines and Related Designer Drugs:

Formulation of Structure-Activity Relationships 43

Richard A. Glennon

Amphetamines: Aggressive and Social Behavior 68

Klaus A. Miczek and Jennifer W. Tidey

Neurochemical Mechanisms Involved in

Behavioral Effects of Amphetamines and Related

Designer Drugs 101

Lisa H. Gold, Mark A. Geyer, and George F. Koob

Neuronal Actions of Amphetamine in the Rat Brain 127

Philip M. Groves, Lawrence J. Ryan, Marco Diana,

Stephen J. Young, and Lisa J. Fisher

v
Page

Methamphetamine and Related Drugs: Toxicity and

Resulting Behavioral Changes in Response to

Pharmacological Probes

Lewis S. Seiden and Mark S. Kleven

146

Role of Dopamine in the Neurotoxicity Induced

by Amphetamines and Related Designer Drugs

James W. Gibb. Donna M. Stone, Michel Johnson,

and Glen R. Hanson

. 161

Acute and Long-Term Neurochemical Effects of

Methylenedioxymethamphetamine in the Rat 179

Christopher J. Schmidt

Effects of MDMA and MDA on Brain Serotonin Neurons:

Evidence from Neurochemical and Autoradiographic Studies 196

Errol B. De Souza and George Battaglia

Characterization of Brain Interactions With

Methylenedioxyamphetamine and

Methylenedioxymethamphetamine 223

Robert Zaczek, Stephen Hurt, Steven Culp, and

Errol B. De Souza

Pharmacologic profile of Amphetamine Derivatives at

Various Brain Recognition Sites: Selective Effects

on Serotonergic Systems

George Battaglia and Errol B. De Souza

240

Effects of Amphetamine Analogs on Central Nervous

System Neuropeptide Systems

Glen R. Hanson, Patricia Sonsalla, Anita Letter,

Kalpana M. Merchant, Michel Johnson, Lloyd Bush,

and James W. Gibb

Effects of Neurotoxic Amphetamines on Serotonergic

Neurons: Immunocytochemical Studies

Mark E. Molliver, Laura A. Mamounas, and

Mary Ann Wilson

Studies of MDMA-Induced Neurotoxicity in Nonhuman

Primates: A Basis for Evaluating Long-Term Effects

in Humans

George A. Ricaurte

259

270

306

vi
Page

Dose- and Time-Dependent Effects of Stimulants 323

Everett H. Ellinwood, Jr., and Tong H. Lee

Recommendations for Future Research on Amphetamines

and Related Designer Drugs 341

Ray W. Fuller

List of NIDA Research Monographs 358

vii

Preface

The abuse of amphetamines is of national concern from a public health

perspective. Review of this subject is timely and important, because the

problem of amphetamine-like drugs has recently been amplified by the

introduction of designer drugs in the illicit market. There has been an

increasing number of attempts by chemists in clandestine laboratories to

synthesize structurally altered congeners that might intensify the moodaltering property of this class of compounds. While attention over the last

few decades has been centered on research related to amphetamine,

methamphetamine, and clinically prescribed amphetamine derivatives

including fenfluramine, recent attention has focused on a variety of

amphetamine-related designer drugs. These designer drugs include ringsubstituted derivatives of amphetamine and methamphetamine such as

3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA “ecstasy”), respectively. MDMA has been the focus of

a great deal of recent attention, since it represents one of a number of

“designer drugs” that is being increasingly abused among certain segments

of the population, especially among college students. This popularity is

ascribed to the drugs’ mixed central nervous system (CNS) stimulant and

hallucinogenic effects. Furthermore, MDMA has been the subject of recent

scientific and legal debate, as several psychiatrists have reported that

MDMA may “enhance emotions” and “feelings of empathy” and thus serve

as an adjunct in psychotherapy. While the psychotherapeutic usefulness of

this drug remains to be determined, a great deal of research has been

carried out on the abuse liability, behavioral effects, and neurotoxic effects

of the amphetamine-related designer drugs.

A technical review meeting entitled “Pharmacology and Toxicology of

Amphetamine and Related Designer Drugs” was held at the National

Institutes of Health on August 2-4, 1988. The purpose of the technical

review was to bring together scientists who have been carrying out research

in the area to (1) summarize the research findings, (2) understand the

neuronal mechanisms through which the amphetamines produce their effects,

and (3) develop a consensus regarding future directions that may lead to

better characterization of the effects of these drugs on various physiological

parameters. An understanding of the mechanisms is critical to the

development of therapeutic approaches for the treatment of intoxication,

addiction, and adverse effects. The proceedings of this meeting are

presented in the following chapters.

ix
Khursheed Asghar, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

Rockville, MD

Errol B. De Souza, Ph.D.

Neurobiology Laboratory

Neurosciences Branch

Addiction Research Center

National Institute on Drug Abuse

Baltimore, MD

x
Structure-Activity Relationships of

MDMA-Like Substances

David E. Nichols and Robert Oberlender

INTRODUCTION

There is virtually no one who is involved in drug abuse research, or who

studies the properties of recreationally used drugs, that is not by now familiar with 3,4-methylenedioxymethamphetamine (MDMA) (figure 1). Over

the past 4 years this substance, usually referred to in the popular press as

“Ecstasy,” has received widespread media attention, This chapter will relate

recent fmdings with respect to the potential dangers attendant on the use of

MDMA and explore its pharmacological properties.

MDMA (1)

FIGURE 1. MDMA

As the title implies, MDMA has pharmacological properties that set it apart

from other classes of drugs. This is one of the most intriguing aspects of

MDMA, largely overlooked as researchers examined the potential risks to

health of MDMA use. Basic questions of how drugs work and why some

are pleasurable and some are not are fundamental to our understanding of

why humans use drugs. Although much of the popularity of MDMA can

no doubt be attributed to curiosity following media attention, the drug itself

must have some rewarding qualities.

MDMA typifies a central problem with the substituted amphetamine-type

substances: The fact that we know so little about any of these kinds of

drugs. What does MDMA actually do? What are the psychopharmacological properties that make it attractive for recreational use? Is it “just another

hallucinogenic amphetamine,” as some have asserted? In the following

1
discussion, an attempt will be made to address some of these issues, and to

put the questions into a broader perspective.

MDMA was patented in 1914 by a German pharmaceutical firm and evaluated as an appetite suppressant (Shulgin 1986). In that sense, MDMA is

not a “designer drug.” Its rediscovery in the late 1970s probably had little

to do with the fact that it was, technically speaking, a legal drug. There

were a variety of legal psychoactive drugs, many of which could probably

have been synthesized and marketed with greater economic profit than

MDMA, a substance with unremarkable quantitative potency, being only two

to three times more active on a weight basis than mescaline (Shulgin and

Nichols 1978). Nonetheless, no other substituted amphetamines with the

popularity of MDMA have appeared. The explanation seems to be that

MDMA has psychopharmacological properties that are deemed especially

rewarding to the user.

MDMA is believed to have unique psychoactive properties that clearly

distinguish it from hallucinogenic or psychostimulant phenethylamines. Not

only have MDMA users consistently reported this distinctiveness, but

subsequent studies of MDMA and similar compounds, in many laboratories.

have shown that they do not fit within the structure-activity relationships

that presently are understood to define the hallucinogenic amphetamines.

STRUCTURAL FEATURES OF MDMA

One of the structural features of MDMA that is somewhat unusual is the

fact that it is 3,4-disubstituted. Both 3,4-methylenedioxyamphetamine

(MDA) (figure 2) and MDMA possess the 3,4-methylenedioxy function, and

there apparently are no other active compounds known that fall within the

MDA (2)

FIGURE 2. MDA

substituted amphetamine class and have substituents only in the 3 and 4

positions. The largest group of substituted amphetamines with significant

haIlucinogenic potency possess either 3,4,5- or 2,4,5- trisubstitution patterns.

The parent compound MDA, although classified as a hallucinogenic amphetamine and available on the illicit market for about 20 years, had gained a

reputation as the “love drug” (Weil 1976). It had been recognized for many

years by both recreational drug users and clinicians (Turek et al. 1974) that

2
MDA had unique psychoactive properties that were different from hallucinogens such as LSD or mescaline. While MDA in high doses appears to

be hallucinogenic or psychotomimetic, it seems not to have been used for

this effect, but rather for its effects on mood: production of a sense of

decreased anxiety and enhanced self-awareness. Even early reports

described the desire of MDA users to be with and talk to other people

(Jackson and Reed 1970). MDA is also the only substituted amphetamine

that received serious clinical study as an adjunct to psychotherapy (Yensen

et al. 1976).

A second structural feature of MDMA that distinguishes it from hallucinogenic amphetamines is the fact that it is a secondary amine. That is, the

basic nitrogen is substituted with an N-methyl, while hallucinogenic

amphetamines are most potent as primary amines. In either 3,4,5- or

2,4,5-substituted phenethylamine derivatives, N-methylation decreases

hallucinogenic potency by up to an order of magnitude (Shulgin 1978).

When MDA is ingested, the hallucinogenic effects are long lasting, typically

10 to 12 hours, similar to the duration of LSD or mescaline. By contrast,

MDMA has a much shorter action, with perhaps a 3- to 5-hour duration of

effects. There is no evidence that typical doses of MDMA lead to hallucinogenic effects in a significant proportion of users, although in high doses

hallucinogenic effects have been reported (Siegel 1986). Thus, the simple

addition of the N-methyl group limits the temporal course of the action to

less than half that of MDA and attenuates or abolishes the hallucinogenic

effects that occur with MDA itself.

A third important difference between MDMA and the hallucinogenic

amphetamines is the reversal of stereochemistry that occurs in MDMA. In

every substituted hallucinogenic amphetamine that has been studied, the

isomer with the R absolute configuration in the side chain is more potent in

animal models, in a variety of in vim assays, and in man (figure 3). The

two isomers differ in potency by a factor of 3 to 10, depending on the

assay system (Nichols and Glennon 1984). By contrast, the S isomer of

MDMA is more potent (figure 4). This was first reported in experiments

with rabbits and in clinical studies (Anderson et al. 1978), and it has

recently been confirmed in other animal models (Oberlender and Nichols

1988; Schechter 1987).

It is difficult to trivialize the significance of this argument, since the

stereospecificity of biological receptors is accepted as a basic tenet of

pharmacology. There is no rationale or experimental precedent for believing

that the 3,4-methylenedioxy substitution should do anything that would

cause the receptor(s) involved to accommodate a side chain stereochemistry

reversed from that for phenylisopropylamines with other aromatic

subtituents.

3
FIGURE 3. The more active R-(-)-enantiomer of the hallucinogenic

amphetamine DOM

S-(+)-MDMA

FIGURE 4. The more active S-(+)-enantiomer of MDMA

Several studies have now clearly shown that the R enantiomer of MDA has

the hallucinogenic effects of the racemate, while the S enantiomer possesses

more potent MDMA-like properties than the R in animals models (Anderson

et al. 1978; Shulgin 1978; Glennon and Young 1984a; Nichols et al. 1982;

Nichols et al. 1986; Oberlender and Nichols 1988). Further, although

(+)-MDA appears similar to amphetamine in the drug discrimination assay

in rats (Glennon and Young 1984a), it is not generally realized that the

effects of (+)-MDA in humans qualitatively resemble those of MDMA,

rather than those of amphetamine (Shulgin, personal communication, 1985).

This is a unique situation. Both enantiomers of MDA are active, having

nearly equal quantitative potencies, but differing in qualitative effect.

N-methylation of the racemic material dramatically and selectively attenuates

the hallucinogenic effects of the R enantiomer, while essentially leaving

intact the properties of the S enantiomer.

In earlier proposals (Anderson et al. 1978), based on this stereoselectivity

for the S enantiomer of MDMA, it was suggested that, rather than having a

direct effect at serotonin receptors, perhaps MDMA was a neurotransmitterreleasing agent, acting in a fashion similar to amphetamine, for which the S

enantiomer is also more active than the R enantiomer. A subsequent study

4
in our laboratory indicated that the S isomers of MDA and MDMA were

indeed potent releasers of [3H]serotonin from prelabeled rat brain

synaptosomes (Nichols et al. 1982). Recently, it was repotted that MDA

and MDMA were potent releasers of serotonin from superfused hippocampal

slices prelabeled with [3H]serotonin (Johnson et al. 1986). In all studies to

date, whether of release of monoamines from synaptosomes or brain slices,

or of the inhibiting of monoamine reuptake into synaptosomes (Steele

et al. 1987), the S enantiomer of MDMA is either equipotent to the R

isomer or more potent.

THE ENTACTOGENS

As a consequence of these and other studies that have indicated that

MDMA has a pharmacology different from the hallucinogenic amphetamines, and in view of the reports by certain psychiatrists (Greer and

Tolbert 1986; Wolfson 1986) that MDMA could facilitate the process of

psychotherapy, it was hypothesized that MDMA and related compounds

represent a new pharmacological class, with as yet unexplored potential as

psychiatric drugs (Nichols 1986; Nichols et al. 1986). This class of drugs

has been called entactogens. Recently, efforts have been directed toward

understanding the mechanism of action of MDMA and related compounds

and testing the hypothesis that entactogens are a novel pharmacological

class, distinct both from hallucinogenic agents and from central stimulants

such as amphetamine or cocaine.

Important support for this hypothesis came from the discovery that the

alpha-ethyl homolog of MDMA, MBDB (figure 5) possessed MDMA-like

S-(+)-MBDB (3)

FIGURE 5. The S-(+)-enantiomer of the alpha-ethyl homologue of MDMA,

MBDB

properties in man and in the drug-discrimination paradigm in rats (Nichols

et al. 1986; Oberlender and Nichols 1988). It was known that homologation of the alpha-methyl of the hallucinogenic amphetamines completely

abolished hallucinogenic activity (Standridge et al. 1976). For example, the

alpha-ethyl homolog of R-DOM, BL-3912A (figure 6) was evaluated by a

major pharmaceutical firm and found to lack hallucinogenic activity at doses

more than a hundredfold higher than those effective for DOM (Winter

1980). This additional feature of the entactogens, that the alpha-ethyl

5
homologs retained activity, was a final and most powerful argument that

MDMA, and certainly MBDB, could not lit within the well-established

structure-activity relationships of the hallucinogenic amphetamines.

R-(-)-BL3912A (4)

FIGURE 6. The nonhallucinogenic alpha-ethyl homologue of DOM,

BL-3912A

STUDIES OF STRUCTURE-ACTIVITY RELATIONSHIPS

EEG Studies

Recently, Dr. W. Dimpfel has used quantitative radioelectroencephalography

in the rat to characterize the electroencephalograph (BEG) “fingerprint” of

hallucinogenic amphetamines, MDMA, and MBDB. In this technique, four

bipolar stainless steel electrodes are chronically implanted in each of four

brain regions in rats: the frontal cortex, the hippocampus, the striatum, and

the reticular formation (Dimpfel et al. 1986). The rats are freely moving;

transmission of field potentials is accomplished using a telemetric device.

The EEG is analyzed by Fourier analysis; power density spectra are

computed for periods of 4 seconds, segmented into six frequency bands, and

averaged on each channel over timeblocks of 15 minutes.

Using this method, a variety of hallucinogenic and nonhallucinogenic compounds were examined. As previously reported (Spüler and Nichols 1988),

hallucinogens produce a marked increase of power in the a, frequency

(7.0 to 9.50 Hz) in the striatum. The ability to increase power in this

region of the EEG has been observed for other classes of serotoninergic

drugs, including the 5-HT1A agonists ipsapirone, gepirone, and buspirone,

and with serotonin-uptake inhibitors (Dimpfel et al. 1988). With 5-HT1A

agonists, however, an increase in power is recorded only from the frontal

cortex and hippocampus.

Doses of DOM, DOB, or DOI of 0.2, 0.1, and 0.1 mg/kg, respectively,

produced a pronounced and long-lasting increase in a, power recorded from

the striatum. By contrast, doses of (+)-MDMA and (+)-MBDB up to

1.6 mg/kg did not elicit this characteristic feature in the EEG. Thus, in this

6
sensitive quantitative EEG procedure, neither MDMA nor MBDB elicited an

EEG fingerprint (four electrodes by six frequency bands per electrode) that

resembled that produced by the hallucinogenic amphetamines DOM, DOB,

DOI, or LSD. These data are consistent with the results obtained in other

models and further support the hypothesis that MDMA and MBDB are not

hallucinogenic phenethylamines.

Thus, for this class of psychoactive agent, preliminary structure-activity

relationships are being formulated. Currently, four structural features

contrast the structure-activity relationships of entactogens with those of

hallucinogenic amphetamines.

(1)

(2)

(3)

(4)

Ring substitution at only the 3,4- positions does not give active

hallucinogens, except for MDA. However, this substitution is active

for entactogenic agents.

N-methylation greatly attenuates hallucinogenic activity, but has no

significant effect on potency of entactogens. N-ethylation also seems

to allow compounds to retain entactogenic activity.

The more active stereochemistry of the entactogens is S, while that of

the hallucinogenic amphetamines is R.

Extension of the alpha-methyl to an alpha-ethyl abolishes

hallucinogenic activity, but has only a minor effect on entactogens.

Drug Discrimination Studies

At the present time these contrasts seem sufficient to distinguish between

the two drug classes. The stereochemical argument and the effects of

alpha-ethylation are extremely powerful. A significant problem with the

hypothesis remained: showing that entactogens differed from another structurally related class, the central nervous system (CNS) stimulants. Several

studies have characterized MDMA as an amphetamine-like or cocaine-like

agent, based on its stimulus properties or its self-administration in primates

(Beardsley et al. 1986; Lamb and Griffiths 1987; Evans and Johanson 1986;

Kamien et al. 1986). It is well known that both amphetamine and cocaine

have powerful effects on dopamine pathways in the brain, and it seems

likely that drugs that release dopamine, or stimulate dopamine receptors,

have reinforcing properties that lead to self-administration and dependence

liability (Wise and Bozarth 1987).

It could not be anticipated that the extension of the alpha-methyl of MDMA

to an alpha-ethyl would also attenuate the effects of the compound on

dopaminergic pathways in the brain. In contrast to MDMA, MBDB has no

significant effect either on inhibition of uptake of dopamine into striatal

synaptosomes (Steele et al. 1987) or on release of dopamine from caudate

7
slices (Johnson et al. 1986). In subsequent drug discrimination experiments

in rats, the dopaminergic properties of MDMA were evident, while MBDB

seemed to have a pharmacologically “cleaner” discriminative cue.

To characterize further the behavioral pharmacology of MDMA and MBDB,

extensive drug discrimination studies were carried out using rats trained to

discriminate saline from LSD, saline from (+)-amphetamine, saline from

(±)-MDMA, and saline from (+)-MBDB. Table 1 summarizes the results of

those experiments. As is the case with hallucinogens, the drug discrimination paradigm should not be considered, in strict terms, an animal model for

entactogen activity. Yet, data from these experiments can provide a good

initial behavioral evaluation of the qualitative and quantitative effects of a

variety of compounds of interest.

It is clear from these results that, in MDMA- or MBDB-trained rats, complete generalization of the training cue to the typical hallucinogenic drugs

LSD, DOM, and mescaline does not occur. Furthermore, transfer of the

training stimulus does not occur to MDMA or MBDB in animals trained to

discriminate LSD from saline (Nichols et al. 1986). Although MDMA has

been shown to substitute for mescaline (Callahan and Appel 1987).

(+)-MBDB-trained rats did not recognize the mescaline cue as similar to the

training drug. These results are consistent with the conclusion that MDMA

and MBDB are not hallucinogenic, as discussed earlier.

These data clearly illustrate the enantioselectivity of the (+)-isomers of

MDA, MDMA, and MBDB in producing an MDMA-like stimulus and

underscore the fact that in vitro studies of the biochemical pharmacology of

these substances should reveal similar selectivity, once the primary

pharmacological process underlying the interoceptive cue is identified. The

data also indicate that (+)-MDA is the most potent of all the drugs tested in

MDMA- or in (+)-MBDB-trained animals. The fact that (+)-MDA does not

substitute in amphetamine-trained animals in our studies supports the

argument that the pharmacology of this enantiomer of MDA is MDMA-like

and is not like amphetamine.

Although amphetamine substitutes for MDMA in our studies, this occurs

only at doses that disrupt a significant number of animals. Furthermore, the

large ED50 for amphetamine substitution in MDMA-trained rats is certainly

not consistent with the known potency of amphetamine in measures of its

stimulant activity. That is, in man, or in animal assays of its activity as a

CNS stimulant, amphetamine is perhaps 10 times more potent than MDA or

MDMA. Thus, its large ED50 relative to that of the enantiomers of MDA

or MDMA seems to suggest strongly that the primary discriminative cue of

MDMA cannot simply be “amphetamine-like.” Although some investigators

have reported stimulus transfer with MDMA in animals trained to discriminate amphetamine from saline, in our paradigm no substitution occurred.

8
TABLE 1. Results of drug discrimination transfer tests in LSD, (+)-amphetamine, (±)-MDMA, or (+)-MBDB-trained rats (ED50

expressed in micromoles per kilogram of body weight)

Substitution

Drug

LSD

LSD

0.025

AMP

Training Drug

MDMA

NS PS1

(+)-MBDB

PS2

DOM 0.61 NS NS NS

(+)-AMP NS 1.68 4.22 NS

(+)-MDA NS NS 1.63 1.43

(-)-MDA 2.94 NS 2.27 3.09

(+)-MDMA NS NS 1.92 1.67

(-)-MDMA NS NS 5.03 3.09

(+)-MBDB NS NS 3.67 3.28

(-)-MBDB NS NS 6.71 6.51

Cocaine NT 20.0 13.9 PS3

Mescaline 33

Fenfluramine PS4

NSb NT NS

NS NT 2.01

KEY: NS=no substitution occurred; PS=partial substitution; NT=not tested.

NOTE: Training doses: LSD tartrale 0.186 μmol/kg; (+)-amphetamine sulfate

5.43 μmol/kg; racemic MDMA.HCl 7.63 μmol/kg; and (+)-MBDB.HCI 7.19 μmol/kg.

1

78% at 0.372 μmol/kg; 257% at 0.186 μmol/kg; 363% at 29.42 μmol/kg; and 47l% at

4.68 μmol/kg.

SOURCES: Stolerman and D’Mello 1981; Schechler and Rosecrans 1973.

Differences in experimental design or in numbers of animals and doses

tested may account for this discrepancy. In our experiments, symmetrical

transfer did not occur between MDMA and amphetamine.

These results show that the MDMA cue is complex and may have some

similarity to amphetamine. However, suggestions that the pharmacology of

(+)-MDMA is essentially the same as that of amphetamine are clearly not

warranted by the data, This partial amphetamine-like action is believed to

9
be reflective of the effect that MDMA has on dopaminergic pathways

(Johnson et al. 1986; Steele et al. 1987). Other workers have reached

similar conclusions (Gold and Koob 1988).

Similarly, self-administration of MDMA in monkeys trained to selfadminister amphetamine (Kamien et al. 1986) or in monkeys or baboons

trained to self-administer cocaine (Beardsley et al. 1986; Lamb and Griffiths

1987) probably reflects a dopaminergic component to the pharmacology of

MDMA. This would be consistent with current theories of dopamine

involvement in the mechanism of action of drugs with dependence liability

(Wise and Bozarth 1987).

In vitro studies have also shown that the alpha-ethyl congener MBDB lacks

significant effects on dopamine systems in the brain. The drug discrimination data support this idea, and amphetamine does not substitute in

(+)-MBDB-trained rats. Furthermore, while cocaine fully substitutes in

MDMA-trained rats. it produces partial substitution in (+)-MBDB-trained

rats. This is further evidence of the decreased effect of MBDB on

catecholaminergic systems. If the data have been interpreted correctly, this

might suggest that MBDB would not be self-administered in animal models

of dependence behavior, and, hence, might have low abuse potential. It has

been found, however, that (+)-MBDB produces serotonin neurotoxicity in

rats, although MBDB is somewhat less toxic than MDMA (Johnson and

Nichols, unpublished).

To summarize the data in table 1, neither MDMA nor MBDB has hallucinogen-like discriminative stimulus properties. Symmetrical transfer of the

MDMA and MBDB stimulus indicates that their primary discriminative

stimulus effects are very similar. For both MDMA and MBDB, there is

enantioselectivity for the S isomer, with about a twofold eudismic ratio.

Finally, the substitution of (+)-amphetamine and cocaine in MDMA-trained

rats may indicate that MDMA has some psychostimulant-like properties,

while MBDB seems to lack this activity.

Effect of the Side Chain Alpha-Ethyl

It seemed likely that an alpha-ethyl moiety would attenuate the ability of

other phenethylamines to interact with dopaminergic systems. To test this

hypothesis, the alpha-ethyl homolog of methamphetamine was synthesized.

This compound (figure 7) was also tested in the drug discrimination

paradigm in (+)-amphetamine trained rats, and compared with (+)-methamphetamine. While (+)-methamphetamine was found to have an ED50 of 1.90

micromoles per kilogram (μmol/kg), the racemic alpha-ethyl homolog only

produced full substitution at high doses, and had an ED50 of 19.62 μmol/kg,

making it approximately one-tenth the potency of (+)-methamphetamine.

This confirmed our speculation, and illustrated that the alpha-ethyl group

10
was effective in reducing the effect of phenethylamines on catecholamine

pathways.

FIGURE 7. The alpha-ethyl homologue of methamphetamine

Thus, for structure-activity studies of MDMA-like substances, emphasis has

been placed on the use of (+)-MBDB as the training drug, since it seems to

possess a primary psychopharmacology similar to that of MDMA, but lacks

the psychostimulant component of MDMA. That is, MBDB is pharmacologically less complex.

Table 2 is a summary of drug discrimination testing data for drugs that

completely substitute in rats trained to discriminate saline from

(+)-MBDB-HCl (1.75 mg/kg; 7.19 μmol/kg). These data are arranged in

order of decreasing relative potency.

It is clear that the (+)-isomers of MDA and MDMA are the most potent in

producing an MBDB-like cue. Furthermore, the stimulus produced by

(+)-MDA is probably unlike that produced by amphetamine, based on the

data presented in the earlier table. Thus, if the psychopharmacology of

(+)-MDA is like that of MDMA, then N-methylation has little effect on the

entactogenic properties of the molecule, but serves primarily to attenuate the

hallucinogenic activity of (-)-MDA. Nevertheless, (-)-MDA also substitutes,

and the psychopharmacology of racemic MDA might be viewed as comprised of the hallucinogenic and entactogenic properties of the (-)-isomer

and the entactogenic and psychostimulant properties of the (+)-isomer. This

illustrates why detailed studies of the mechanism of action of psychoactive

compounds should be done on the pure optical isomers.

But what is the effect of MBDB or MDMA? We have been attempting to

define this through the use of drug discrimination assays, with rats trained

to a variety of drugs. Through the use of appropriate agonists and antagonists, we may be able to define the pharmacology of MBDB. Although

there are some exceptions (e.g., fenfluramine), most of the substituted

phenethylamines described in the literature can be categorized as hallucinogens or as stimulants. The psychopharmacology of MDMA perhaps

represents a third category, and it is possible that other phenethylamine and

amphetamine derivatives may possess similar pharmacology,

1 1
TABLE 2. Compounds that completely substitute for (+)-MBDB in drug

discrimination tests in rats

Test Drug ED50(μmol/kg)

95% Confidence

Interval

S-(+)-MDA

S-(+)-MDMA

Fenfluramine

(±)-MDA

(±)-MBDB

R-(-)-MDMA

R-(-)-MDA

S-(+)-MBDB

(±)-MDMA

R-(-)-MBDB

1.43 0.9 - 2.29

1.67 0.98 - 2.86

2.01 1.30 - 3.09

2.09 1.36 - 3.21

2.92 2.17 - 3.92

3.09 1.80 - 5.32

3.09 1.88 - 5.07

3.28 2.15 - 5.01

3.35 2.35 - 4.77

6.51 4.54 - 9.34

In view of the apparent pleasurable effects of MDMA, it becomes of considerable interest to understand the mechanism of action of substances with a

similar effect. Major efforts have been directed toward the study of agents

that have an effect on serotonin pathways, since that is the neurotransmitter

system that seems most implicated in the mechanism of action of MDMA.

This hypothesis is further reinforced by the observation that MDMA substitutes for fenfluramine (Schechter 1986). and fenfluramine substitutes for

MBDB (Oberlender and Nichols, unpublished). The substitution data for

(+)-amphetamine and cocaine in (+)-MBDB-trained rats are also similar to

the data for substitution of these agents in fenfluramine-trained rats (White

and Appel 1981).

However, the specific serotonin uptake inhibitor fluoxetine failed to produce

an MBDB-like cue and failed to block the stimulus effects of MBDB when

it was given prior to a training dose of MBDB. Table 3 summarizes results

of fluoxetine testing in MBDB-trained rats. In other exploratory studies,

pretreatment of MDMA-trained rats with either methysergide or ketanserin

failed to block completely the MDMA-discriminative stimulus.

12
Based on the modest ability of the (+)-isomers of MDMA and MBDB to

inhibit the reuptake of norepinephrine (NE) into hypothalamic synaptosomes

(Steele et al. 1987). it seemed possible that noradrenergic pathways might

be involved in the cue. In another series of drug discrimination experiments designed to test this hypothesis, the specific NE uptake inhibitor

(-)-tomoxetine was tested for stimulus transfer in doses up to 10 mg/kg in

MDMA-trained rats. At 5 mg/kg, 67 percent of the animals responded on,

the drug lever. However, pretreatment with tomoxetine in six rats trained

to discriminate MDMA from saline had no effect on the discrimination of a

subsequent dose of MDMA.

TABLE 3. Results of tests for fluoxetine substitution in (+)-MBDB.HCltrained (1.75 mg/kg) rats

Dose of

Fluoxetine N

Percentage

Selecting

Drug Lever

7.23 μmol/kg 8 38%

14.46 μmol/kg 8 50%

29.92 μmol/kg 7 43%

At the present time, a variety of other pharmacological agents are being

tested for their ability either to antagonize or to potentiate the effect of

MDMA in these animals. There is hope that appropriate pharmacological

manipulations will eventually be found that will give useful information

about the mechanism of action for entactogens.

ANALYSIS OF STRUCTURE-ACTIVITY RELATIONSHIPS

Medicinal chemists have a distinct advantage in pursuing mechanism-ofaction studies because it is possible to synthesize a series of structurally

related congeners and measure their biological activity. A correlation

between activity and particular structural features not only helps to identify

the pharmacophore, or active moiety imbedded within the molecule, but also

may establish critical requirements or complementarity for the biological

target or receptor for the particular drug class.

When a particular behavioral pharmacology is associated with a specific

biochemical action within a series of congeners, it is likely that the

biochemistry is a functional component of the observed behavioral activity.

This is not necessarily the case if only one or a few molecules are available

for study; they may well possess ancillary biochemical pharmacology that is

13
unrelated to the behavioral phenomenon being observed. However, the

larger the series of structurally diverse molecules in which the two activities

are associated, the stronger the basis for believing that a cause-effect

relationship exists.

In designing studies of the structure-activity relationships of MDMA and

related substances, there are at least three areas for structural modification.

First, the nature of the amine substituents can be varied: other N-alkyls can

be studied, or the nitrogen can be incorporated into a ring system. A

second point for structural modification is the side chain. As already

demonstrated, the alpha-methyl can be extended to an alpha-ethyl. Other

modifications of the side chain would include incorporation into a variety of

ring systems, or dialkylation. Finally, the nature and location of the

ring substituents can be modified.

N-Alkylation

A number of investigators have examined the N-ethyl congener of MDMA,

MDE (or MDEA), which has also gained popularity on the illicit market.

Braun et al. (1980) have reported that, of the N-substituted MDA derivatives that were studied for analgesic action and human psychopharmacology,

only the N-methyl, N-ethyl, and N-hydroxy compounds were active. The

latter compound, the N-hydroxy, in all probability serves merely as a

prodrug for MDA, being metabolically reduced to the primary amine, as has

been observed for para-chloramphetamine (PCA) (Fuller et al. 1974). Since

the range of modification of N-substitution seems so limited, it appears

unlikely that studies of N-substituted MDA analogs will offer significant

insight into mechanism of action. However, different N-alkyl groups may

affect regional brain distribution and pharmacokinetic properties. For

example, Boja and Schechter (1987) have found that the N-ethyl analog

MDE has a much shorter biological half-life than does MDMA.

Ring Substituents

Little is presently known about requirements for particular aromatic ring

substituents enabling a compound to possess MDMA-like activity. The

3,4-ethylidenedioxy and 3,4-isopropylidenedioxy compounds (figures 8 and

9) have been examined for ability to substitute in LSD- or MDMA-trained

rats in the drug discrimination paradigm. Both compounds gave full

substitution in rats trained to either drug. Those results and comparison

data for MDA are given in table 4. Addition of steric bulk to the dioxole

ring reduces CNS activity, whether defined as LSD-like or MDMA-like.

Fenfluramine also produces a cue that is similar to both MDMA and

MBDB, in that complete substitution occurs and does so at a relatively low

dose of fenfluramine. This would seem to imply that the dioxole ring is

14
FIGURE 8. The dioxole-ring methylated homologue of MDA, EDA

FIGURE 9. The dioxole-ring dimethylated homologue of MDA, IDA

not essential, and many workers have drawn comparisons between the

neurotoxicity of fenfluramine and that of MDMA. However, the psychopharmacology of fenfluramine is quite different from that of MDMA.

TABLE 4. ED50 values for substitution in LSD-trained or MDMA-trained

rats, in the drug discrimination paradigm

Compound LSD ED50 (mg/kg) MDMA ED50 (mg/kg)

MDA (figure 2) 0.97 0.88

EDA (figure 8) 3.07 1.86

IDA (figure 9) 7.12 5.21

NOTE: LSD tartrate=0.08 mg/kg, IP; (±)-MDMA.HCl=1.75 mg/kg, IP.

While MDMA produces CNS stimulation and euphoria, fenfluramine is

more of a sedative and dysphoric. A detailed comparison of the

pharmacology of fenfluramine and MDMA may be necessary to understand

exactly how MDMA works.

Another study underway has begun to examine the effect of paramethoxyamphetamine (PMA) in MDMA-trained rats. After testing a few doses, it

appears that full substitution may occur and that the S enantiomcr of PMA

15
is more potent. This result would also be consistent with a mechanism of

action for MDMA where serotonin release is important, since PMA is a

potent releasing agent of serotonin both in vitro (Tseng et al. 1978) and

in vivo (Tseng et al. 1976; Nichols et al. 1982). PMA is also a potent

releaser of NE in peripheral tissues (Cheng et al. 1974) but the blockade of

its behavioral effects by chlorimipramine (Tseng et al. 1978) suggests that

serotonin release may be important in the mechanism of action. PMA did

make a brief appearance on the illicit market in the early 1970s but was

responsible for several deaths (Cimbura 1974), and its use subsequently

declined.

One might also speculate that PCA would have an effect similar to MDMA.

Indeed, the early clinical data for PCA suggested that it possessed

antidepressant activity (Verster and Van Praag 1970). This would suggest

that the human psychopharmacology of PCA may well be closer to that of

MDMA than fenfluramine, but it is unlikely that clinical studies can be

carried out to study this.

Side-Chain Modifications

A variety of side-chain modified analogs of MDMA and MBDB have begun

to be examined. Very early studies were of the dimethyl analog,

3,4-methylenedioxyphentermine (figure 8a) and its N-methyl derivative

(figure 10). This latter compound proved to lack MDMA-like activity

(Shulgin, unpublished). Interestingly, this compound also lacked the ability

to stimulate the release of [

3H]serotonin from prelabeled rat brain

synaptosomes (Nichols et al. 1982).

Recently the tetralin and indan analogs of MDA have been examined

(figures 11 to 14). It was previously shown that when hallucinogenic

amphetamine derivatives were incorporated into similar structures, the

hallucinogen-like activity in animal models was lost (Nichols et al. 1974).

Thus. one might anticipate that a similar strategy with MDMA would lead

to congeners that would lack MDA-like hallucinogenic effects. Furthermore,

by examination of the two methylenedioxy positional isomers, one could

infer the binding conformation of MDMA itself at the target site. As

shown in table 5, one positional isomer is clearly preferred for MDMA-like

activity. Furthermore, the indan derivative, figure 12, has a potency at least

comparable to that of MDMA. This series has begun to define some of the

conformational preferences of the receptor or target sites with which

MDMA interacts, at least in producing its discriminative cue.

NON NEUROTOXIC ENTACTOGENS?

Although the problem of MDMA abuse has generated great interest because

of MDMA’s potential neurotoxicity, it is possible that nonneurotoxic

entactogens can be developed. As in most areas of technology, this is a

16
R=H

R=CH3

FIGURE 10. The -dimethyl homologues of MDA(a) and MDMA(b)

FIGURE 11. Nonneurotoxic tetralin analogue of MDMA

FIGURE 12. Nonneurotoxic indan analogue of MDMA

two-edged sword. A major concern might be that a nonneurotoxic entactogen could become popular as a recreational drug. A major deterrent to

widespread use of MDMA should be the consideration by potential MDMA

users that there is the possibility of neurotoxicity with unknown

consequences, perhaps delayed for years before the consequences become

manifest. On the other hand, researchers must give serious attention to the

fact that any possible clinical utility for MDMA-like substances cannot be

explored until the issue of neurotoxicity is resolved. Hence, a nonneurotoxic MDMA congener would allow clinical testing of the assertion that

these compounds are useful adjuncts to psychotherapy.

Undoubtedly, nonneurotoxic entactogens can and will be discovered. Sufficient evidence already exists to support this hypothesis. We know, for

example, from the work of Schechter (1986) that the discriminative stimulus

properties of MDMA are largely dissipated within 4 hours of drug

administration. On the other hand, Schmidt (1987) has shown that MDMA

17
FIGURE 13. Tetralin analogue of MDMA that lacks MDMA-like effects

FIGURE 14. Indan analogue of MDMA that lacks MDMA-like effects

TABLE 5. Drug discrimination results: Substitution tests in

Compound

Figure 11

Figure 12

Figure 13

MDMA-trained rats

Result in MDMA-Trained Rats

CS ED50=1.29 mg/kg

CS ED50=0.59 mg/kg

PS (75% drug responding @ 1.75 mg/kg)

Figure 14 PS (67% drug responding @ 0.5 mg/kg)

KEY: (±)-MDMA.HCl, 1.75 mg/kg IP, CS=complete substitution; PS=partial substitution.

has a biphasic depleting effect on cortical serotonin, with the later phase

(more than 6 hours) associated with the long-term toxicity, a toxicity

blocked by fluoxetine. Schmidt and Taylor (1987) administered the

serotonin uptake inhibitor fluoxetine to rats 3 hours after treatment with

MDMA and were able to prevent neurotoxicity. These workers suggested

that the unique neurochemical effects of MDMA are independent of the

long-term neurotox-icity. In our own studies, cited above, we have shown

that fluoxetine does not antagonize the MDMA cue. Battaglia et al. (1988)

reported that acute MDMA treatment decreased brain serotonin and 5-HIAA

18
levels, but that multiple MDMA treatments were required to decrease the

number of 5-HT uptake sites, the latter presumably a reflection of neuron

terminal degenera-tion. These studies indicate that the acute pharmacology

can be dissociated from the long-term neurotoxic effects of MDMA.

Further, it is also known from work with the neurotoxin PCA that some

structural congeners have an acute depleting effect on brain 5-HT, but lack

the long-term neurotoxicity that is characteristic of PCA (Fuller et al. 1977).

Since the psychopharmacological effects of MDMA have a relatively rapid

onset and, in rodents, are largely dissipated at a time when a serotonin

uptake inhibitor can still block neurotoxicity, it seems quite clear that

molecules can be developed that will probably possess human psychopharmacology similar to MDMA, but will lack serotonin neurotoxicity. When

this is accomplished we can look forward to a clearer definition of the

primary pharmacology of entactogens. One would hope that, at that time,

clinical studies with such a compound would be possible, to determine

finally whether entactogens represent a new technology for psychiatry.

DISCUSSION

QUESTION: What are the criteria that you used for these newer compounds in order to classify these newer drugs as either sympathomimetic or

hallucinogenic?

ANSWER: We are basically forced to deal with a variety of models. First

of all, we have LSD-trained rats, and we have used that as our general

screen for hallucinogen-like activity. If you are familiar with the drug

discrimination literature, you can get false-positives, and perhaps

Professor Glennon will correct me if I am wrong, but I am not aware of

false-negatives. There are no cases where, in the drug discrimination

paradigm, an animal has said this drug is not hallucinogenic when, in fact,

in humans it is known that it is. So my feeling with drug discrimination is

that we are detecting false-positives.

We are using I-125-labeled DOI as a radioligand and that has been shown,

particularly by Professor Glennon and his coworkers, to be a good model

for hallucinogenic activity. I think 5-HT2 agonists, in terms of biochemical

pharmacology, are the clearest indication that a compound is hallucinogenic.

We are routinely screening compounds for ability to displace I-125 DOI

from frontal cortex homogenates. As far as the CNS stimulant effects,

differentiating from psychostimulants, the present model we are using is

substitution in amphetamine-trained rats, in drug discrimination. We have

used synaptosomes and looked at their effect on dopamine release and

reuptake. But basically they are correlative models.

19
And it is certainly true that these compounds could well be hallucinogenic

but fall outside what we understand the structure-activity relationships of

these compounds to be. For example, it may well be that MBDB in

humans at some dose is hallucinogenic and is acting by some mechanism

that is totally different from what we understand to be the mechanism of

mescaline, DOM, or LSD. But at the present time, based on what we

understand about structure-activity relationships, it should not be. That

remains to be seen.

COMMENT: It might be advisable to stick to more operational definitions

in talking about these compounds. One runs a risk if compounds have not

been tested in people, and to refer to a compound as hallucinogenic when it

is operative. A drug discrimination test might lead you to certain

assumptions about the drug that are not true.

RESPONSE: Generally, it is safest to say there is LSD-like activity in

drug discrimination profiles. Similarly, with these so-called entactogens, the

name we have given them, we do know that we find in the tetralins and

indans, for example, that a particular amino-indan we tested has fairly high

potency in substituting for MDMA or MBDB. But we do not know what

its effects would be in humans. There is no way to test that. Basically we

are trying to develop correlative models based on what we know from the

clinical data. But, again, it is speculative in the absence of clinical studies.

COMMENT: I would not rule out the possibility that MDMA or MDA

produces effects at serotonin-2 receptors. Some of the data that I believe

Dr. Battaglia has accumulated shows that of the 20 brain recognition sites

that we have looked at, using standard radioligand binding procedures,

MDMA has the highest affinity at serotonin-2 receptors as labeled by

tritiated DOB. But I must qualify that. If you compare MDMA to

something like DOI, it is about a hundredfold weaker. But its affinity is

still 100 nanomolar in terms of an IC50, concentration, which is still

relatively potent considering the concentrations that may be achieved in

brain at some of the doses used in animals.

RESPONSE: I have tended to think that things do not have affinity unless

we see low nanomolar affinity. I think the EEG studies are fairly revealing

in that regard. The fact that we see this increase in alpha-l power in the

striatum is a characteristic of 5-HT2 agonists. And we are clearly seeing

EEG effects at doses that are not increasing that power in the alpha-l

frequency. I tend to think that 5-HT2 agonist effects are not that important

in the action of these compounds.

COMMENT/QUESTION: I was very intrigued by your substitution data

from the drug discrimination paradigm. But my question is not unlike

Lou Seiden’s. For with substances that are characterized by tremendously

qualitatively different effects, biphasic in nature, and in many functional

20
assessments, I feel it may be premature to zero in on one selected training

dose and give that a label.

I would like to know whether or not you have explored minimal discriminational doses of MDMA or MBDB and whether you have contrasted them

with higher doses and have done experiments that are reminiscent of the

Appel and White type approach where different mechanisms kick in at

different dose ranges of the drug. Do we cover the relevant qualitatively

different effects with that technique and with that approach, where one is

zeroing in on one amphetamine dose and one MDMA dose?

I also have another question. When you compare release data from a slice

preparation where it is in one application with discrimination data, are you

comparing a creature that has received hundreds of injections every other

day, on the average? I do not know what your protocol looks like, but I

presume every other day is a drug and every other day is a control

condition. Here you have an acute preparation and the relationship, of

course, is quite tenuous.

ANSWER: Yes. We have looked at the lower doses of MDMA; the

1.75 mg/kg is the dose that gave us the best discriminability. We tried

initially to train with 1 mg/kg but could not. We continued to increase the

training dose by increments until we found the dose where we got reliable

discrimination. It was 1.75 mg/kg. At least in our paradigm, I do not see

how we could go much lower.

We have not explored all of the dose-response relationships. And with

respect to the nature of the cue, we have studies underway now with a

variety of serotonin agonists and antagonists, for example, fluoxetine. And

have looked at MDMA. We cannot block the cue with fluoxetine. We are

also looking at 8-hydroxy-DPAT, buspirone. PCPA pretreatment is on the

way. So there are a variety of manipulations that we have in process.

The treatments are all randomized, so a lot of them are only half finished,

and no one can say what is happening. But in terms of pinning it down, I

think that needs to be done.

We are looking at biochemical models as really pointing us in a particular

direction. They are not rigorous; I recognize that. If we focused all our

attention on drug discrimination we could do some complete studies. My

emphasis in medicinal chemistry is to explore structure-activity relationships

and synthesize tools to explore how the drugs work So we basically, more

than focusing on pharmacological rigor, have tried to find quick screens that

would point us in a direction so we could synthesize a drug to test this

hypothesis.

Ultimately, these compounds will require a good deal of pharmacological

evaluation, and we are in the early stages of that. In accordance with

21
Dr. Gibb’s hypothesis regarding dopamine involvement, we thought that

perhaps MBDB would not be neurotoxic because of a lack of effect on

dopamine. But, in fact, it is neurotoxic as well, measured by whole-brain

serotonin 5-HIAA and tritiated paroxetine binding sites. It is perhaps

two-thirds the toxicity, on a molecular weight basis, of MDMA, but it is

toxic.

A number of the studies that we have done are not completely rigorous, but

their purpose is to see whether neurotoxicity is related to the nature of the

cue. Your questions are well taken, but it has really been a choice between

economy and rigor so that we could find the chemical structure to

synthesize.

COMMENT/QUESTION: You have answered the first question, which was

on the issue of whether or not MBDB produced long-term effects on the

amine system. The second question has to do with the nature of the cue.

We have talked with people who participated in our study over the last

year. As you know, many of them have experimented with a wide variety

of psychoactive drugs, including MBDB. When asked about MBDB their

response seems to be lukewarm in terms of how it compares to subjective

effects, and whether these effects are comparable to those of MDMA. Is

that accurate?

ANSWER: When we decided to make MBDB we felt the alpha-ethyl

would attenuate hallucinogenic activity. Dr. Shulgin made that compound

because he was looking at things that had a stimulant effect. He had made

it but had not evaluated it at effective doses. After a discussion, he

evaluated it in the group of people that worked with him.

Basically, the consensus was that the psychopharmacology was similar but

that the compound lacked the ability to produce the kind of euphoria

produced by MDMA. And he reported that there were at least one or two

individuals who felt they never wanted to take the compound again.

My own bias is toward the therapeutic potential. I do not care whether

anything we develop produces euphoria or dependence potential. I think

from the point of view of a drug abuse problem or a dependence liability

that the alpha-ethyl probably does not have the reinforcing qualities and is

not as pleasurable as MDMA.

COMMENT: My question to these people would be directed toward this

quality they regard as unique for MDMA--this rush. They admit that that

is not the main reason for taking it. They do seem to be able to make that

distinction. They do not dispute the fact that they enjoy the rush from an

MDMA dose. Whatever this other quality is, they recognize it. And it is

that quality that was less apparent in MBDB than in MDMA.

22
RESPONSE: I think this is an area where you would have to do detailed

double-blind crossover studies and some fairly extensive testing to map out

what the nature of that effect is.

In the drug discrimination assay we get symmetrical transfer. They seem to

be the same. And the consensus, at least from Dr. Shulgin’s group, is that

it generally has the same kind of effect. Obviously it has not become a

problem on the street. And I think if it was a very desirable compound we

might well have heard something about it.

QUESTION: Have you done any studies of the metabolism of these

compounds? As you probably know, there have been reports that MDMA

is very quickly metabolized into MDA. Have you looked at MBDB to see

if the ethyl group gets cleaved so that you essentially have an MDMA

compound after you are through?

ANSWER: There is no chemical precedent for that kind of transformation.

I really cannot think of an enzyme system that would cleave that down to

the alpha-methyl. I think the effect is due to the alpha-ethyl.

In terms of other sites of metabolism, we are looking at the metabolism in

the dioxole ring and in dealkylation. We have seen some interesting things,

but I could not comment on this right now. With respect to the alphaethyl, I think that the parent compound is probably the one that is active.

QUESTION: I have two questions about your MBDB discrimination

studies. It sounds as though you are doing experiments to investigate

whether neuronal stores of serotonin are required for MBDB to be recognized. You mentioned that fluoxetine did not prevent the recognition.

Does it prevent the release of serotonin in vitro? In other words. is that a

carrier-dependent release by MBDB as it is, for example, in the case of

p-toluylamphetamine?

My second question is this: You mentioned fenfluramine. I presume you

used the racemic mixture. which would mean that in the brain you would

have both R and S fenfluramine and R and S norfenfluramine present. And

since these differ widely in their effects on dopamine versus serotonin

neuronal systems, have you studied individual enantiomers of either

fenfluramine or norfenfluramine?

ANSWER: Actually we used synthesized (+)-fenfluramine. The fluoxetine

story is not clear. It does not block the discriminative cue, but other

workers have shown that it blocks the neurotoxicity. We have not looked

at it in enough detail or at any of the in vitro models to see whether it

blocks or releases serotonin.

23
COMMENT: It seems as though that might be a good tool to determine

whether the discrimination really does relate to serotonin release because,

clearly, it has been shown to block the serotonin release in vivo. If it does

not block the drug discrimination it seems that it is not consistent with the

idea that that is a consequence of serotonin release.

RESPONSE: When you are in this business, you get letters from many

strange people. I received an unsolicited letter from a fellow in Geneva,

Switzerland, about a year ago, who told me that he had taken fluvoxamine,

which I believe is available clinically in Geneva, and had subsequently

taken MDMA. He said that the fluvoxamine had no effect on the action of

the MDMA. I think this is an interesting question which, at least in one

anecdotal account, suggests that there is a difference.

The biochemical followup would be interesting if it does prevent the

release. And maybe the serotonin is a red herring. But that is the only

thing we have seen consistently at this point

COMMENT [DR. SCHUSTER]: I am extremely pleased to see the sophistication of the animal studies and the medicinal chemistry studies. I lament

the current lack of sophistication with regard to the available data in

humans. It is feasible, as my colleagues and others have shown, to train

drug discrimination in humans-to do as precise quantitative work there as

is done in animals. In fact, probably more precise.

As far as subjective effects are concerned, and people’s responses regarding

why they take drugs, I have to say that I have a fair degree of skepticism

that people are reporting in any way what is relevant. It may be, it may

not be. But I can assure you that the contingencies that shape verbal

behavior may be very different from the contingencies that shape the

drug-taking behavior. And as a consequence there may not be any

necessary correlation.

It is unfortunate, and this is a real deficit in this field, that we cannot do

the very human studies that I know you would all like to do and, therefore,

we rely upon whatever evidence we have to reach conclusions. But we

have to be wary of the fact that the human data are weak in comparison.

QUESTION: Have you made any attempt to antagonize the MBDB stimulus with serotonin antagonists?

ANSWER: We have tried ketanserin, but it did not antagonize the

stimulus. I do not believe we have tested fluoxetine in the MBDB-trained

animals. It has only been tested in the MDMA-trained animals. We have

not found an antagonist to the cue yet.

24
QUESTION: Did you measure tryptophan hydroxylase or just the

5-HT/5-HIAA depletion?

ANSWER: We used it in the 20 mg/kg twice a day for a 4-day regimen

with MDMA, and then corrected for molecular weight and used an

equimolar dose of MBDB, sacrificed the animals 2 weeks later, and then

measured. We used basically HPLC and used serotonin and 5-HIAA from

one hemisphere and then measured tritiated pyroxetine from the other

hemisphere. And we got something like 60 percent depletion of serotonin,

and the pyroxetine binding site Bmax, decreased by about 60 percent. With

MBDB it was decreased by about 40 percent. It was a clear and significant

decrease, but not quite to the extent that we had. But we have not looked

at tryptophan hydroxylase.

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1987.

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Nichols, D.E.; Barfknecht, C.F.; Long, J.P.; Standridge, R.T.;

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ACKNOWLEDGMENTS

This research was supported in part by U.S. Public Health Service grant DA

02189 from the National Institute on Drug Abuse and Biomedical Research

Support Grant 2-S07-RR05586-18 from the Division of Research Resources.

28
AUTHORS

David E. Nichols, Ph.D.

Professor of Medicinal Chemistry

Robert Oberlender, Ph.D.

Research Assistant

Department of Medicinal Chemistry

and Pharmacognosy

School of Pharmacy and Pharmacal Sciences

Purdue University

West Lafayette, IN 47907

29
Self-Injection in Baboons of

Amphetamines and Related

Designer Drugs

C.A. Sannerud, J.V. Brady, and R.R. Griffiths

INTRODUCTION

Recent controversy about the recreational abuse and potential therapeutic use

of “designer drugs” has focused attention on MDA (methylenedioxyamphetamine HCl) and structurally related phenylisopropylamine compounds,

including MDMA (d,l-3,4-methylenedioxymethamphetamine HCl, “ecstasy”).

These compounds are structural analogs of the psychomotor stimulant

amphetamine and the hallucinogen mescaline, and produce stimulant and/or

hallucinogenic effects (Shulgin 1978).

In humans, MDA and MDMA have been reported to produce positive mood

changes, enhanced emotional awareness, and improved interpersonal

communication (Greer and Tolbert 1986; Downing 1986; Shulgin 1986;

Peroutka et al. 1988). Because of these psychotropic effects, MDA and

MDMA have been used in psychotherapeutic situations (Naranjo et al. 1967;

Yensen et al. 1976; Grinspoon and Bakalar 1986). In addition, presumably

because of the same positive subjective effects, recreational use of MDMA

on college campuses has increased in recent years (Peroutka et al. 1988).

Recreational abuse of “designer drugs” poses a major problem. Evidence

concerning the safety of these drugs has shown that MDA and MDMA are

toxic to serotonergic neurons in rodent (Ricaurte et al. 1985; Stone et al.

1987; O’Heam et al. 1988) and primate brains (Ricaurte et al. 1988).

MDMA has also been associated with toxicity in humans. To date, there

have been five cases reported in which MDMA has contributed to death in

recreational users (Dowling et al. 1986).

Based in part on the neurotoxicity and recreational abuse, the Drug

Enforcement Administration (DEA) has placed MDA, MDMA, and other

“designer drug” analogs of stimulant/hallucinogens on Schedule I, used for

drugs with high abuse potential and no recognized therapeutic usefulness.

30
Although MDA and MDMA were recently brought under legal regulation by

scheduling under the Controlled Substances Act, other derivatives of these

compounds can be synthesized easily, and these new “designer drugs” have

begun to be used recreationally. Evaluation of these substituted phenylethylamine compounds for abuse liability should require an assessment of

the reinforcing effects of the drugs and a comparison to structurally similar

compounds, to determine relative potency and structure-activity relationships

(SARs). This chapter will summarize previously published drug selfadministration research with a variety of substituted amphetamine compounds, comparing the self-administration of stimulant/hallucinogenic analogs

of MDA to standard anorectic phenylethylamine compounds in baboons.

METHOD FOR ASSESSING REINFORCING EFFECTS OF DRUGS

The use of nonhuman primates to assess abuse liability of test compounds

is indicated, since there is a good correlation between the drugs that are

abused by man and those that maintain self-injection behavior in animals

(Schuster and Thompson 1969; Griffiths et al. 1980). Of the many different

types of procedures developed to determine whether a drug will maintain

self-injection, the substitution is the most common and reliable. The

procedure involves establishing self-injection using a dose of a standard

drug that is known to maintain reliable self-injection behavior. After this

behavioral baseline is stable, a dose of test drug is substituted for the

standard compound to determine whether the test drug will maintain

self-injection.

PROCEDURE

The methods and procedures used to evaluate self-injection of these

compounds were similar to those previously described by Griffiths and

colleagues (Griffiths et al. 1976; Griffiths et al. 1979). Eighteen male

baboons (Papio cynocephalus) weighing between 15 and 30 kg were used as

subjects. Each animal was adapted to either a standard restraint chair

(Findley et al. 1971) or a harness tether restrainmg system (Lukas et al.

1982). The chaired animals were housed individually in sound-attenuated

chambers. The tethered animals were housed in standard stainless steel

primate cages surrounded by a sound-attenuating, double-walled plywood

external enclosure.

An aluminum “intelligence panel” used in self-injection studies has been

previously described (Griffiths et al. 1975). Briefly, the panel containing

levers and associated stimulus lights (approximately 1 cm in diameter) was

mounted on the inside of the chamber (chaired animals) or on the rear wall

of the cage (tethered animals). A Lindsley lever (lower left of panel), a

leaf lever (lower right of panel), and a food hopper with stimulus light

(lower left of panel) were mounted on the panel. A 5x5 cm translucent

Plexiglas panel that could be transilluminated was mounted on the aluminum

31
panel in the upper left comer. A speaker for delivery of white noise and

tones was mounted behind the panel. A feeder for delivering food pellets

into the food pellet tray was mounted on the top of the wooden enclosure.

Baboons were surgically prepared with chronically indwelling silastic

catheters implanted in either femoral or jugular veins under pentobarbital or

halothane anesthesia using methods described in detail by Lukas et al.

(1982). All baboons had served in studies of intravenous self-injection with

a variety of drugs. They had continuous access to water via a drinking

tube and to food pellets (as described below) and received two pieces of

fresh fruit and a multivitamin daily.

The infusion system was similar to that described by Findley et al. (1972).

The catheter was attached to a valve system that allowed slow continuous

administration (55 to 60 mL in 24 hours) heparinized saline (5 units/mL)

via a peristaltic pump to maintain catheter patency. Drug was injected into

the valve system by means of a second pump and then flushed into the

animal with 5 mL of saline from a third pump. This system necessitated a

delay of approximately 20 seconds between the onset of drug delivery and

actual injection into the vein. Drugs were delivered within a 2-minute

period.

Food was available 24 hours per day under a fixed ratio 30 (FR 30)

response schedule on the leaf lever; i.e. every thirtieth response delivered a

1 g banana-flavored food pellet and produced a brief flash of the hopper

light.

Animals were trained to self-inject cocaine (0.4 or 0.32 mg/kg/injection)

under an FR 160 response schedule on the Lindsley lever. Drug injections

were available every 3 hours and were signaled by a 5-second tone,

followed by the illumination of the jewel light over the Lindsley lever.

When the jewel light was illuminated, each response on the Lindsley lever

produced a brief feedback tone. Upon completion of the FR requirement,

the jewel light was extinguished and the 5 mL drug injection was begun,

followed by a 5 mL flush injection. Following the completion of the

injections, the 5x5 cm translucent panel was illuminated for a 1-hour period,

and the 3-hour timeout period was begun. There was no time limit for the

completion of the response requirement.

When criterion cocaine self-injection performance (six or more injections

per day for 3 consecutive days) was obtained a dose of drug or vehicle

was substituted for cocaine for 12 to 15 days. Occasional equipment

malfunction necessitated extending the period of substitution beyond

15 days. Cocaine self-injection performance was reestablished, and when

criterion performance was obtained (typically in 3 to 5 days), another dose

of drug was substituted This procedure of replacing cocaine with drug was

continued through the study of a range of drug doses and their vehicles.

32
The order of exposure to different doses was either a mixed or an

ascending sequence. The drug vehicle was generally examined immediately

before or after the series of doses.

Drugs and Doses Tested. Drug solutions were prepared by dissolving the

drug in physiological saline (0.9 percent sodium chloride) and were filter

sterilized (Millipore). Drug doses (mg/kg/infusion) were calculated on the

basis of the salt. The following drug doses were tested: d-amphetamine

sulfate (0.01, 0.05, 0.1, 0.5); l-3,4-methylenedioxyamphetamine sulfate

(MDA) (0.1, 0.5, 1.0, 2.0, 5.0); 4-methoxyamphetamine hydrochloride

(PMA) (0.001, 0.01, 0.1, 0.1 1.0); 2,5-dimethyoxy-4-methylamphetamine hydrochloride (DOM) (0.001, 0.01, 0.1, 1.0); 2,5-dimethyoxy-4-ethylamphetamine

hydrochloride (DOET) (0.001, 0.01, 0.1, 0.32. 1.0); d,l-3,4-methylenedioxymethamphetamine HCl (MDMA) (0.1, 0.32, 1.0, 3.2); phentermine hydrochloride (0.1, 0.5, 1.0); diethylpropion hydrochloride (0.1, 0.5, 1.0, 2.0);

phenmetrazine hydrochloride (0.1, 0.5, 1.0); phendimetrazine tartrate (0.1,

0.5, 1.0, 2.0); benzphetamine hydrochloride (0.1, 0.5, 1.0, 3.0); l-ephedrine

hydrochloride (0.3, 1.0, 3.0, 10.0); clotermine hydrochloride (0.1, 1.0, 3.0,

5.0); chlorphentermine hydrochloride (0.1, 0.5, 2.5, 5.0); and fenfluramine

hydrochloride (0.02, 0.1, 0.5, 2.5).

Chemical Structures. Figure 1 shows the chemical structures for 14

phenylethylamine compounds. Nine of these compounds are used clinically

as anorectics (d-amphetamine, phentermine, diethylpropion, phenmetrazine,

phendimetrazine, clotermine, chlorphentermine, benzphetamine, and

fenfluramine). Four of these compounds are not approved for clinical use

and are reported to have hallucinogenic properties (MDA, PMA, DOM, and

DOET). The final compound (l-ephedrine) is used clinically for bronchial

muscle relaxation, cardiovascular, and mydriatic effects. Figure 2 shows the

chemical structure for MDMA, the methyl analog of MDA. MDMA is not

approved for clinical use and has been reported to produce both LSD-like

and cocaine-like effects.

RESULTS

Figure 3 presents the mean levels of self-infusion for the 14 phenylethylamines shown in figure 1. Of all the drugs tested, d-amphetamine was the

most potent, maintaining levels of drug self-injection above saline levels at

doses of 0.05 and 1.0 mg/kg/mfusion. Phentermine, diethylpropion, phenmetrazine, phendimetrazine, benzphetamine, and MDA maintained levels of

self-injection above saline at doses of 0.5 and 1.0 mg/kg/infusion. The

compounds l-ephedrine, clotermine, and chlorphentermine were the least

potent substances that maintained performance; self-injection rates were

33
FIGURE 1. Chemical structure of 14 of the 15 phenylethylamines tested to

determine whether they maintain drug self-administration

SOURCE: Griffiths et al. 1979, copyright 1979, Academic Press.

above saline control levels at doses of 3.0 and 10 mg/kg/infusion for

l-ephedrine, 3.0 and 5.0 mg/kg/infusion for clortermine, and 2.5 and

5.0 mg/kg/infusion for chlorphentermine. In contrast to the other phenylethylamines tested, fenfluramine, PMA, DOM, and DOET did not maintain

self-injection at levels greater than saline at any dose tested.

34
FIGURE 2. Chemical structure of MDMA

Figure 4 shows that MDMA maintained responding above vehicle level in

the three baboons tested, the highest levels of self-injection were maintained

by 0.32 or 1.0 mg/kg/injection; lower levels were maintained by 0.1 and

3.2 mg/kg/injection. During self-injection of cocaine, vehicle (saline), and

low doses of MDMA, there were no unusual changes in gross behavior of

the baboons. While self-injecting higher doses of MDMA, however, all

three baboons engaged in notably unusual behaviors. Two animals appeared

to track nonexistent visual objects (suggesting hallucinations), were uncharacteristically aggressive toward laboratory personnel, and engaged in

repetitive scratching/self-grooming behavior.

Similar MDMA self-injection findings have been reported in rhesus

monkeys (Beardsley et al. 1986). In three of the four animals trained to

self-administer cocaine, substitution of at least one dose of MDMA resulted

in rates of self-injection that exceeded vehicle rates; two animals selfadministered MDMA at rates higher than cocaine rates.

CORRESPONDENCE OF BEHAVIORAL EFFECTS IN HUMANS

AND ANIMALS

In a summary of the human abuse literature on anorectic phenylethylamines,

Griffiths et al. (1979) found there was a good correlation between the

results of self-administration studies in animals and information about the

subjective effects and abuse in man. Specifically, amphetamine, diethylpropion, and phenmetrazine have been associated with numerous clinical

case reports involving abuse, and these three compounds as well as benzphetamine and l-ephedrine have shown similar subjective effects in drug

abuser populations (Griffiths et al. 1979). In addition, fenfluramine was

associated with low incidence of abuse in humans and did not maintain

self-injection responding in animals. Chlorphentermine was similarly

associated with low incidence of abuse in man, but did not maintain selfinjection uniformly in animals (Griffiths et al. 1979).

35
FIGURE 3. Mean number of injections per day with 14 phenylethylamines

for the last 5 days of drug or saline substitution under a

160-response T.0. 3-hour schedule of intravenous injection

NOTE: The vertical axis represents the number of injections per day. The horizontal axis

represents doses of drug (log scale). The points above “C" represent the mean of all 3-day

drug dose or saline substitution. The points above "S” represent the mean of the last 5 days

periods of cocaine HCI (0.4 mg/kg/infection) availability that immediately preceded every

obtained during saline substitution (2 saline substitutions in each of 15 animals). Vertical

bars indicate ranges of individual animal's means. Drug data points represent the mean of 

the last 5 days during substitution of a drug dose for individual animals.

SOURCE: Griffiths et al. 1979, copyright 1979, Academic Press.

36
FIGURE 4. Mean number of injections per day for the last 5 days of

MDMA and MDMA vehicle (saline) substitution under a

160-response T.O. 3-hour schedule of intravenous injection

NOTE: The points above “C” represent the grand mean of the 3 days of concaine HCl(0.32

mg/kg/injection) availability that preceded each MDMA dose or saline substitution. The

points above “V” represent the mean of the last 5 days obtained during vehicle substitution.

Vertical bars indicate ranges of individual animals' means.

SIMILARITIES AMONG AND DIFFERENCES BETWEEN

PHENYLETHYLAMINE COMPOUNDS

A comparison of MDMA to d-amphetamine, MDA, and DOM can provide

an understanding of the pharmacology of MDMA and its abuse liability.

While there are differences between MDMA and amphetamine in the

subjective effects in humans (Shulgin and Nichols 1978). the similarities in

the self-injection and preclinical pharmacology profile between MDMA and

d-amphetamine suggest that MDMA has abuse liability. Both MDMA and

d-amphetamine maintain self-injection behavior above vehicle control levels,

and high doses of both drugs are associated with a cyclic pattern of selfinjection over days (Lamb and Griffiths 1987; Griffiths et al. 1976). At

doses larger than those needed to maintain self-injections, both MDMA and

d-amphetamine suppressed food intake and food-maintained behavior (Lamb

and Griffiths 1987; Griffiths et al. 1976) and produced similar changes in

gross behavior, such as tracking nonexistent visual objects and repetitive

self-grooming (Lamb and Griffiths 1987; Lamb and Griffiths, unpublished

observations). Both MDMA and amphetamine also sham discriminative

37
stimulus properties with d,l-MDA or amphetamine, but not with DOM, in

rat drug-discrimination paradigms (Glennon and Young 1984a; Glennon and

Young 1984b; Glennon et al. 1983).

MDMA has both similarities to and differences from l-MDA. MDMA and

l-MDA are self-injected in baboons and share stimulus properties with MDA

in the rat drug-discrimination paradigm (Glennon and Young 1984a). In

addition, MDMA, but not l-MDA, shares discriminative stimulus properties

with amphetamine in the rat drug-discrimination paradigm (Glennon and

Young 1984a). Consistent with the reports of lesser hallucinogenic effects

of MDMA as compared to MDA or LSD (Shulgin 1978), l-MDA, but not

MDMA, shares discriminative stimulus properties with DOM in the rat

drug-discrimination paradigm (Glennon et al. 1982; Glennon et al. 1983).

Although the substituted phenylethylamine compounds that have hallucinogenic properties in man (e.g., DOET, DOM, PMA, MDA, and MDMA) are

commonly abused by humans, only MDA and MDMA maintained selfinjection behavior in baboons. This suggests that this animal self-injection

procedure may not be useful in predicting hallucinogenic drug effects. In

addition, it suggests that the reinforcing properties of MDA and MDMA in

baboons may be unrelated to the fact that these drugs produce hallucinogenic effects. Some phenyl-substituted phenylisopropylamines, such as

MDA, PMA, and MDMA, have pharmacological properties distinct from

those of amphetamine or DOM. Therefore, predictions about the abuse

liability of these compounds based on their similarities to or differences

from classic stimulants (such as cocaine or amphetamine) or hallucinogens

(such as LSD or DOM) may provide inappropriate results.

STRUCTURE-ACTIVITY RELATIONSHIPS AMONG

PHENYLETHYLAMINE COMPOUNDS

A comparison between the chemical structures of substituted

phenylethylamine compounds and their potency in producing behavioral

effects reveals an inverse relationship between the size of the substituent

and central activity (Braun et al. 1980). Similarly, reports of SARs among

phenylethylamine compounds have suggested that the size of the ring

substitution in general may decrease potency of the phenylethylamines for

maintenance of self-injection behavior. Research with a series of N-ethylaminates substituted at the meta position of the phenyl ring has

demonstrated that the potency of these compounds, either to increase

locomotor behavior in mice (Tessel et al. 1975) or to maintain self-injection

behavior in rhesus monkeys (Tessel and Woods 1975; Tessel and Woods

1978). was inversely related to the size of the meta-substituted constituent.

These findings indicate that the failure of fenfluramine (meta-trifluromethylN-ethyl-amphetamine) to maintain self-injection behavior is attributable to its

meta-trifluromethyl group.

38
An examination of the SAR, comparing figures 1 and 3, also supports the

suggestion that ring substitutions may decrease potency for maintaining

self-injection behavior. The seven compounds shown in the right column of

figures 1 and 3 have substitutions on the phenyl ring; these compounds

were generally less potent in maintaining self-injection than were the

compounds in the left columns of these figures, which do not have ring

substitutions. In addition, phentermine differs structurally from both

chlorphentermine and clotermine, which have the addition of a Cl at either

the para or ortho positions of the phenyl ring; however, chlorphentermine

and clotermine appear to be less potent than phentermine in maintaining

self-injection behavior.

A similar SAR was found between side-chain substitutions and behavioral

effects of phenylethylamines. A study using a series of d-N-alkylated

amphetamines, synthesized in a series up to and including d-N-butylamphetamine, found that, for substitutes larger than ethyl, potency for maintaining

drug self-administration in rhesus monkeys and for disrupting milk-drinking

activity in rats of the d-N-alkylated amphetamines was inversely related to

the N-alkyl length (Woolverton et al. 1980).

The pharmacological properties of phenylethylamines that control selfadministration are complex. The effects of phenylethylamines on a variety

of pharmacological measures do not appear to predict the reinforcing effects

of these drugs, as measured by the cocaine substitution procedure in

primates. Specifically, none of the following behavioral effects of these

compounds accurately predict the results of self-administration experiments

within the phenylethylamine class (Griffiths et al. 1976; Griffiths et al.

1979): the ability to suppress food intake (Griffiths et al. 1978); the ability

to produce rate-dependent effects on schedule-controlled behavior (Harris et

al. 1977; Harris et al. 1978); the ability to produce discriminative stimulus

properties similar to amphetamine, DOM, or MDA (Glennon et al. 1982;

Glennon et al. 1983; Glennon and Young 1984a; Glennon and Young

1984b Glennon et al. 1985; Glennon et al. 1988). Self-injection testing

should remain an integral part of a continued analysis of abuse liability of

these compounds.

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ACKNOWLEDGMENTS

Preparation of this paper was supported by grant DA 01147 from the

National Institute on Drug Abuse and contract no. 271-86-8113 from the

National Institute on Drug Abuse.

AUTHORS

Christine A. Sannerud, Ph.D.

Joseph P. Brady, Ph.D.

Roland R. Griffiths, Ph.D.

Department of Psychiatry and Behavioral Sciences

Department of Neuroscience

Johns Hopkins University School of Medicine

Traylor Building 624

720 Rutland Avenue

Baltimore, MD 21205

42
Stimulus Properties of

Hallucinogenic Phenalkylamines

and Related Designer Drugs:

Formulation of Structure-Activity

Relationships

Richard A. Glennon

INTRODUCTION

The purpose of the studies with phenalkylamine derivatives is severalfold:

(1) to classify these agents by their primary effect; (2) to understand the

structure-activity relationship (SAR) for each type of activity; and (3) to

elucidate the mechanisms of action of these agents. Armed with information on SAR, one can, theoretically make predictions about the activity of

agents yet to be synthesized; an understanding of the mechanisms of action

can aid in the development of potential antagonists that could be useful for

reversing the effects of these substances. Obviously, before one can investigate SAR and mechanisms of action, it is important to have some reliable

method of classification. In the course of the studies, several different

procedures have been used to examine the actions of these agents. Perhaps

the most useful is the drug discrimination procedure. In this paradigm,

animals are trained to recognize (or discriminate) the stimulus effects of a

particular dose of a given agent; once trained, the animals can be

administered doses of a test compound (i.e., a challenge drug) to determine

if the challenge drug produces stimulus effects similar to those of the

training drug. In such tests, referred to as tests of stimulus generalization,

the animals essentially indicate whether or not a similarity exists between

the actions of a new agent and those of a reference agent. Dose:response

curves can be obtained and ED50 values determined. Thus, the procedure

provides data that are both qualitative and quantitative. Needless to say,

there are occasions when such studies produce results that are less than

straightforward and are difficult to interpret. In other words, although drug

discrimination studies provide very useful information on similarity of

effect, potency, timecourse of action, mechanism of action, activity of

metabolites, and other data, they cannot be used by themselves to characterize completely the pharmacological effects of a given agent. Reviews on

the drug discrimination paradigm, particularly as it applies to the study of

43
phenalkylamines, appear in the following publications: Glennon

et al. (1983), Glennon (1986), and Young and Glennon (1986).

In drug discrimination studies, groups of rats were trained to discriminate

either the stimulant phenalkylamine (+)amphetamine (AMPH) or the hallucinogenic phenalkylamine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane

(DOM) from saline. Other structurally related training drugs that have been

used include the iodo and bromo analogs of DOM, i.e., DOI and R (-)DOB,

as well as methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA). Such investigations, coupled with the results of

radioligand binding studies, have permitted the classification of a number of

phenalkylamines (Glennon et al. 1983; Glennon 1986; Young and Glennon

1986) and have allowed proposal of a mechanism of action for the

hallucinogenic phenalkylamines (Glennon et al. 1986a). The present review

describes in detail some of the SARs that have been formulated on the

basis of drug discrimination studies. This discussion of results is not meant

to be comprehensive. Because species of animal, schedule of reinforcement,

presession injection intervals, doses of training drugs, and other conditions

have remained constant throughout the studies, it should be possible to

make stricter comparisons than if data were compared across different

laboratories. This SAR analysis is based, for the most part, on the results

of discrimination studies already published.

Some of the agents used in the present study have not been previously

reported in the literature. These agents were prepared in our laboratories,

and details of their synthesis will be published elsewhere. However, three

of these agents are potential metabolites of MDMA and are described here

to the extent that such information might be helpful to other investigators

studying the metabolism of MDMA. All three were isolated as their white,

crystalline hydrochloride salts, and all were analyzed correctly for carbon,

hydrogen, and nitrogen. Melting points and recrystallization solvents (in

parentheses) are provided. N-methyl-1 -(4-hydroxy-3-methoxyphenyl)-2-

aminopropane (N-Me 4-OH MMA): 210-212 °C (isopropanol/ ether); Nmethyl-1-(3-hydroxy4-methoxyphenyl)-2-aminopropane (N-Me 3-OH PMA):

164-165 °C (isopropanol); N-methyl-1-(3,4-dihydroxyphenyl-2aminopropane

(N, dimethyl dopamine or N-Me 3,4-diOH AMPH): 116-118 °C

(isopropanol/ether).

Examination of the stimulus properties of a large number of phenalkylamines and related derivatives shows many can be characterized as producing

either AMPH-like stimulus effects or DOM-like stimulus effects. The

structures of some of these agents are shown in figure 1. Certain other

agents could not be reliably classified as either AMPH-like or DOM-like

because, at the highest dose tested, they either produced vehicle-appropriate

(i.e., saline-appropriate) responding or resulted in disruption of behavior.

44
FIGURE 1. Chemical structures of some of the agents employed in the

present study

Representative potency data (ED50 values) are presented in tabular form;

data in these tables are given as μmol/kg so that direct potency comparisons can be made within a series. However, data presented in figures are

given in mg/kg for the purpose of convenience.

Various phenallcylamines were shown to produce either DOM-like or

AMPH-like stimulus effects; the structure-activity requirements for these

activities are different from the standpoints of aromatic substitution patterns,

terminal amine substituents. and optical activity. Thus, it has been possible

to formulate two distinct SARs. It should be realized, however, that

phenalkylamines need not produce only one of these two types of effects;

certain phenallcylamines can produce pharmacological effects like neither

DOM nor AMPH. Moreover, they can produce effects that are primarily

peripheral, not central, in nature (Glennon 1987a). The fact that an agent

produced DOM- or AMPH-like effects does not imply that it cannot

produce an additional effect; conversely, if an agent does not produce either

DOM- or AMPH-like stimulus effects, it is not necessarily inactive.

45
DOM-LIKE STRUCTURE-ACTIVITY RELATIONSHIPS

Aromatic Substituents

Substituents on the aromatic ring play a critical role in determining

whether or not phenalkylamines possess DOM-like activity. Hallucinogenic

activity is commonly associated with methoxy-substituted derivatives

(Shulgin 1978); for this reason, much of this work has focused on these

types of agents.

Methoxy-Substituted Derivatives. Phenalkylamines lacking aromatic substituents do not produce DOM-like stimulus effects. None of the three

possible monomethoxy derivatives, 2-methoxy-(OMA), 3-methoxy-(MMA),

or 4-methoxyphenylisopropylamine (PMA), produce DOM-like effects. Of

the six dimethoxy analogs (DMAs) (i.e., 2,3-DMA, 2,4-DMA, 2,5-DMA,

2,6-DMA, 3,4-DMA, and 3,5-DMA), only the 2,4- and 2,5-dimethoxy

derivatives 2,4-DMA and 2,5-DMA, respectively, are active. These two

agents are essentially equipotent and are approximately one-tenth as potent

as DOM. For purposes of comparison, the potencies of 2,5-DMA and

DOM are 23.8 and 1.8 μmol/kg. Five trimethoxy analogs (TMAs) have

been examined: 2,3,5-TMA is approximately one-third as potent as

2,5-DMA, and 2,3,4-TMA and 3,4,5-TMA are equipotent with 2,5-DMA.

The other two, 2,4,5-TMA and 2.4.6-TMA, are about twice as potent as

2,5-DMA. None of the three possible tetramethoxy analogs has been

investigated, and the pentamethoxy analog does not produce DOM-like

stimulus effects. From these studies, it is apparent that the 2,4- and 2,5-

dimethoxy substitution pattern plays an important role; certain 2.6-dimethoxy

derivatives are also active, depending upon what substituents are present at

the 4-position.

2,5-Dimethoxy Analogs. It should come as no surprise that methoxy

groups at the 2- and 5-positions are important, when it is realized that

DOM is a 2,5-dimechoxy-substituted derivative. Data for some representative 2,5-DMA analogs are provided in table 1. Removal of either one of

the methoxy groups abolishes DOM-like stimulus effects. Introduction of a

methyl group at the 4-position of 2,5-DMA, to produce DOM, enhances

potency by more than an order of magnitude. Homologation of this alkyl

group to ethyl (DOET) and n-propyl (DOPR) produces an increase in

potency; further homologation to n-butyl (DOBU) decreases potency, and to

amyl (DOAM) results in an agent that does not produce DOM-like stimulus

effects. The relative potencies of these agents, as compared to 2,5-DMA,

are: 2,5-DMA (1)<DOM (13)<DOET (27)<DOPR (43)>DOBU (7).

Branching of this alkyl chain has varying effects. The isopropyl analog

DOIP is eight times more potent than 2.5-DMA but is only about one-fifth

as potent as its nonbranched counterpart DOPR. The tertiary butyl

derivative DOTB does not produce DOM-like effects. In fact, it has been

46
TABLE 1. Results of stimulus generalization studies using racemic DOM as

a training drug

ED50

Agent Optical Isomer R4 R R' (μmol/kg)

2,5-DMA (±)

(-)

2,4,5-TMA (±)

DOM (±)

(-)

(+)

N-Me DOM (±)

(-)

-desMe DOM

DOET (±)

(-)

(+)

DOPR

DOIP (±)

DOBU

(±)

(±)

DOF (±)

DOC (±)

DOI (±)

(-)

(+)

DOB (±)

(-)

(+)

N-Pr DOB (±)

-desMe DOB

4-OH 2,5-DMA (±)

4-COOH 2,5-DMA (±)

DOTB (±)

DOAM (±)

H

H

OCH3

CH3

CH3

CH3

CH3

CH3

CH3

C2H5

C2H5

C2H5

nC3H7

iC3H7

nC4H9

F

C1

I

I

I

Br

Br

Br

Br

B r

OH

COOH

tC4H9

nC5H11

DOBZ (±) CH2

-C6H5 H CH3 NSG

H

H

H

H

H

H

CH3

CH3

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

nC3H7

H

H

H

H

H

CH3 23.8

CH3

CH3

14.0

13.7

CH3

CH3

0.8

CH3

1.8

6.9

CH3 15.3

CH3 10.0

H 5.6

CH3

CH3

0.9

0.3

CH3 3.3

CH3 0.6

CH3 2.9

CH3 3.2

CH3 5.8

CH3

CH3

1.2

1.2

CH3 0.6

CH3 2.6

CH3

CH3

0.6

0.3

CH3

2.6

CH3 13.4

H 2.2

CH3 NSG

CH3

CH3

NSG

NSG

CH3 NSG

KEY: NSg=no stimulus generalization at the highest dose tested.

NOTE: Training drug=DOMHCl (1.0 mg/kg, IP) administered 15 minutes prior to testing. In test of

stimulus generalization, a presession injection interval of 15 minutes was employed. ED50

values are given where stimulus generalization occured.

47
found that DOTB acts as a partial agonist and can antagonize the stimulus

effects of DOM (Glennon 1987b).

Certain polar substituents at the 4-position of 2,5-DMA render the

compounds inactive; for example, the 4-COOH and 4-OH derivatives do not

produce DOM-like effects. On the other hand, 4-halogenated compounds

result in relatively potent derivatives. The 4-fluoro derivative DOF is

4 times more potent than 2,5-DMA. whereas the 4-chloro (DOC) and 4-iodo

(DOI) analogs are about 20 times more potent than 2,5-DMA. The most

potent halogenated derivative is the 4-bromo analog DOB, which is about

40 times as potent as 2,5-DMA.

The location as well as the nature of these substituents is important. For

example, moving the methyl group of DOM, or the bromo group of DOB,

from the 4-position to the 3-position (to produce isoDOM and isoDOB,

respectively) results in agents that do not produce DOM-like stimulus

effects. IsoDOB (or SL7161). for example, produces saline-appropriate

responding at 100 times the ED, dose of DOB.

2,4-Dimetboxy Analogs. 2,4-DMA is approximately equipotent with

2,5-DMA. Introduction of a 5-methyl or 5-bromo group, to produce

5-methyl-2,4-DMA and 5-bromo-2,4-DMA, results in active agents, but they

are not significantly more potent than 2,4-DMA itself. It seems that the

methyl and bromo substituents are tolerated at the 5-position, but they do

not produce the increase in activity seen in the 2,5-DMA series.

Terminal Amine Substituents

A primary (i.e., unsubstituted) amine appears to be optimal for DOM-like

activity. Simple N-monomethylation of DOM results in a tenfold decrease

in potency. Larger N-alkyl substituents produce an even greater decrease in

potency; for example, N-n-propyl DOB is approximately one-thirtieth as

potent as DOB itself (Glennon et al. 1986b). Using animals trained to

discriminate R (-)DQB from saline, racemic DOB is 10 times more potent

than N-monomethyl DOB, which, in turn, is 10 times more potent than

N,N-dimethyl DOB (Glennon et al. 1987). The quaternary analog

N,N,N-trimethyl DOB iodide (QDOB) is inactive.

Alpha-Methyl Group

The u-methyl group is important, but not usually necessary for activity.

For example, the desmethyl analogs of DOM and DOB are both about

one-third as potent as their parent agents. The ademethylation of

3,4,5-TMA, to produce mescaline, results in a similar (i.e., 2.5-fold)

decrease in activity. Although these desmethyl analogs produce stimulus

effects similar to those of DOM, there is some evidence that the spectrum

of effects produced by these agents, in rats and in humans, is not

48
necessarily identical with that (spectrum) of their parent compounds (Shulgin

and Carter 1975; Glennon et al., in press).

Optical Isomers

Due to the presence of the a-methyl groups, these agents exist as optical

isomers. Both isomers usually produce DOM-like effects, and the R

(-)isomers constitute the eutomeric series. In this regard then, the effects of

these agents are stereoselective, but not stereospecific. In general, the R

(-)isomers are twice as potent as their racemates and about 5 to 8 times

more potent than their S (+)enantiomers. Some representative data are

provided in table 1.

AMPHETAMINE-LIKE STRUCTURE-ACTIVITY RELATIONSHIPS

Aromatic Substituents

An unsubstituted aromatic ring appears to be optimal for AMPH-like

stimulus effects. Using animals trained to discriminate 1.0 mg/kg of

(+)amphetamine sulfate from saline (ED50=1.8 μmol/kg), no aromaticsubstituted derivative has yet been found to be more potent than AMPH

itself. For example, each of the monomethoxy-substituted derivatives, i.e.,

OMA, MMA, and PMA, produce AMPH-appropriate responding but are 4

to 15 times less potent than AMPH itself (table 2). The (+)AMPH stimulus

does not generalize to any of the above-mentioned DMAs or TMAs (or, for

that matter, to any of the agents listed in table 1); however, several of these

agents (notably 2,4-DMA, 2,5-DMA, 2,4,5-TMA, 2,4,6-TMA, and

3,4,5-TMA) result in partial generalization (40 to 50 percent AMPHappropriate responding) suggesting that they may be capable of producing

some AMPH-like activity in addition to their DOM-like effects (Glennon

et al. 1985). The 4-OH derivative (parahydroxyamphetamine, or Paradrine),

which is the O-desmethyl analog of PMA, produces saline-appropriate

(2 percent drug-appropriate) responding at greater than 50 μmol/kg. The

N-ethyl-3-trifluoromethyl derivative of AMPH, fenfluramine, produces

saline-like effects at doses up to about 20 μmol/kg and disruption of

behavior at doses greater than or equal to 24 μmol/kg. Complete reduction

of the aromatic nucleus of AMPH does result in retention of activity,

although potency is significantly decreased; that is, propylhexedrine produces

AMPH-like stimulus effects (ED50=15.5 μmol/kg).

Terminal Amine Substituents

In contrast to what was observed for DOM-like activity, N-monomethylation

of AMPH-like agents does not decrease their AMPH-like character. MethAMPH (i.e., N-monomethylamphetamine) is slightly more potent than

amphetamine; likewise, methcathinone (N-monomethylcathinone) is twice as

potent as cathinone. N-methylation of DOM-like agents does not convert

49
TABLE 2. Results of stimulus generalization studies using (+)amphetamine

as a training drug

ED50

Agent Isomer X R (μmol/kg) x R R'

Amphetamine

Methamphetamine

Phenethylamine

Cathinone

Methcathinone

N-OH AMPH

(+)N-Et AMPH

OMA

MMA

PMA

PMMA

4-OH AMPH

Fenflummine

3,4-DMA

N-Me 3,4-DMA

2,4-DMA

N-Me 2,4-DMA

2,5-DMA

N-Me 2,5-DMA

(±)

(-)

(+)

(±)

(+)

(±)

(-)

(+)

(±)

(±)

(+)

(±)

(±)

(±)

(±)

(±)

(±)

(±)

(±)

(±)

(±)

(±)

(±)

H2

H2

H2

H2

H2

H2

0

0

0

0

H2

H2

H2

H2

H2

H2

H 2

H2

H2

H2

H2

H2

H2

H2

H

H

H

H

H

H

H

H

H

H

H

H

2-OCH3

3-OCH3

4-OCH3

4-OCH3

4-OH

3-CF3

3,4-di OCH3

3,4-di OCH3

2,4-di OCH3

2,4-di OCH3

2,5di OCH3

2,5-di OCH3

H

H

H

CH3

CH3

H

H

H

H

CH3

OH

C2H5

H

H

H

CH3

H

C2H5

H

CH3

H

CH3

H

CH3

CH3 2.6

CH3 5.3

CH3 1.8

CH3 1.5

CH3

1.2

H NSG

CH3 3.8

CH3 1.6

CH3 23.4

CH3 1.8

CH3 1.1

CH3 4.3

CH3 38.7

CH3 17.0

CH3 9.5

CH3

CH3

NSG

CH3

NSG

NSG

CH3 NSG

CH3

CH3

NSG

CH3

NSG

NSG

CH3 NSG

CH3 NSG

KEY: NSG=no stimulus generalization at the highest dose tested.

NOTE: Rats trained to dircriminate (+)amphetamine sulfate (1.0 mg/kg) from saline administered

15 minuted prior to testing.

them to AMPH-like agents; for example, see N-Me 2,4-DMA and N-Me

2,5-DMA (table 2). Homologation of the methyl to an ethyl group results

in retention of AMPH-like activity, although potency is somewhat reduced;

50
for example, (+)N-Et AMPH is 2.5 times less potent than (+)AMPH itself.

Although there have been no systematic investigations of N-alkylation,

certain AMPH analogs bearing larger substituents are active. Mefenorex,

the N-(3-chloro-n-propyl) analog of AMPH, produces saline-appropriate

behavior at about 5.5 μmol/kg and disruption of behavior at 6

μmol/kg.The N-hydroxy analog of AMPH (i.e., N-OH AMPH), a metabolite of

AMPH, also produces AMPH-like effects and is about twice as potent as

AMPH (table 2).

Alpha-Methyl Group

Removal of the a-methyl group of AMPH results in phenethylamine (PEA).

PEA does not produce AMPH-like effects. Likewise, removal of the

a-methyl group of cathinone, resulting in desmethylcathinone, also results

in an agent that does not produce AMPH-like stimulus effects. Huang and

Ho (1974a) have demonstrated that pretreatment of the animals with a

monoamine oxidase inhibitor prior to administration of PEA does lead to

stimulus generalization, suggesting that the adesmethyl analogs may simply

lack protection from metabolism.

Beta-Substituents

Very few ~-substituted analogs of AMPH have been investigated.

Ephedrine, for example, produces weak AMPH-like activity (Huang and Ho

1974b). (+)Norpseudoephedrine (cathine) also produces AMPH-like stimulus

effects. The oxidized analogs of norephedrine and ephedrine, cathinone and

methcathinone, respectively, however, are potent AMPH-like agents

(table 2).

Optical Isomers

Both optical isomers of AMPH are active (Schechter 1978). In general, for

the few isomeric pairs that have been examined, the S isomers of

AMPH-like agents are slightly more potent than the racemates and about 3

times more potent than the R isomers (Young and Glennon 1986).

S(+)AMPH, for example, is 3 times more potent than R(-)AMPH (table 2);

S(-)cathinone is 2.5 times more potent than racemic cathiione, but

(unexpectedly) is nearly 15 times more potent than R(+)cathinone.

Miscellaneous Analogs

Certain agents with AMPH-related structures also produce AMPH-like

stimulus effects. Agents in which the terminal amine has been incorporated

into a cyclic structure, such as methylphenidate (Huang and Ho 1974b

D’Mello 1981), phenmetrazine, and phendimetrazine, are active. These

agents might be considered as N-alkyl substituted phefialkylamines.

Aminorex is another agent that falls into this category and is essentially

equipotent with AMPH.

51
METHYLENEDIOXY-SUBSTITUTED PHENALKYLAMINES

Methylenedioxy-substituted phenalkylamines are considered separately,

because it has been shown that the parent 3,4-methylenedioxy analog of

AMPH, MDA, is capable of producing both DOM-like and AMPH-like

stimulus effects. Its ED, value in DOM-trained rats is 7.8 μmol/kg and in

(+)AMPH-trained rats, 10.6 μmol/kg. The DOM-like properties reside

primarily with the R (-)isomer (ED50=3.8 μmol/kg), whereas the AMPH-like

activity resides with the S (+)isomer (ED50=4.2 μmol/kg) (figures 2 and 3).

To this extent, 3,4-MDA is not a particularly potent agent; it is approximately one-sixth as potent as (+)AMPH and less than one-third as potent as

DOM. A positional isomer of 3,4-MDA. 2,3-MDA, produces neither DOMnor AMPH-like stimulus effects. The 2-methoxy analog of 3,4-MDA (i.e.,

2-methoxy 4,5-MDA or MMDA-2) produces weak DOM-like effects

(ED50=13.7 μmol/kg), but does not produce AMPH-like stimulus effects.

3,4-MDA is unique. Not only does it produce both types of effects, but it

seems to conflict with some of the above-mentioned SARs. For example,

aromatic-substituted phenalkylamines such as the 3-methoxy and 4-methoxy

derivatives MMA and PMA arc only weak AMPH-like agents, and the

3.4-dimethoxy analog 3,4-DMA (which is structurally very similar to

3,4-MDA) does not produce AMPH-like effects. The 3-OH, 4-OMe, and

the 3-OMe 4-OH analogs of amphetamine are also inactive. Thus, it is

surprising that 3,4-MDA possesses AMPH-like character. Likewise, neither

MMA, PMA, nor 3,4-DMA produce DOM-like effects; yet 3,4-MDA does.

2-Methoxy 4,5-MDA (MMDA-2) and 2,4,5-TMA share a common substitution pattern; interestingly, these agents are essentially equipotent in

producing DOM-like stimulus effects. Table 3 displays selected results.

CONTROLLED SUBSTANCE ANALOGS (“DESIGNER DRUGS”)

One application of SARs is to make predictions concerning new agents.

Assuming that the new agents are producing one of the above-mentioned

effects, it should be possible to make approximate predictions of both

activity and potency. Over the past decade, several new agents have

appeared, and their activities and/or potencies have been consistent with

these SARs. Some of these agents have been mentioned. Also encountered

were some agents that do not fit the foregoing SAR; it is probably

worthwhile considering these agents in depth. For example, PMMA, the

N-monomethyl analog of PMA, should produce AMPH-like effects with a

potency several times that of PMA itself. In fact, PMMA produces neither

AMPH-like nor DOM-like effects. The animals’ behavior, however, was

disrupted at very low doses (<1 μmol/kg) suggesting that it may produce a

central effect that is other than (or in addition to) AMPH-like or DOM-like.

52
TABLE 3. Results of stimulus generalization studies with MDA analogs

H

H

H

H

OCH3

OCH3

H

H

H

H

H

H

H

H

Agent Isomer R2

ED, Values (μmol/kg)

R R' AMPH-Like DOM-Like

3,4-MDA (MDA) (±)

(-)

(+)

HPA

2-OMe 4,5-MDA (±)

N-Me 2-OMe

4,5MDA (±)

MDMA (±)

(-)

(+)

MDE (±)

(-)

(+)

MDP (±)

N-OH MDA (±)

H CH310.6

H CH3 NSG

H CH3

 4.2

H H NSG

H CH3 NSG

CH3 CH3 NSG

CH3 CH3

CH3 CH3

7.1

NSG

CH3 CH3 2.6

C2H5 CH3

C2H5 CH3

NSG

C2H5 CH3

NSG

NSG

C3H7 CH3 NSG

OH CH3 NSG

7.8

3.7

NSG

NSG

13.7

NSG*

NSG*

NSG*

NSG

NSG

NSG

NSG

NSG

*Partial generalization (i.e., 40 to 55 percent drug-appropriate responding, followed, at slightly higher

doses, by disruption of behavior. NSG=no stimulus generalization at the highest dose tested.

NOTE: AMPH-like rats were trained to discriminate 1.0 mg/kg of (+)amphetamine sulfate from saline;

DOM-like rats were trained to discriminate 1.0 mg/kg of DOM-HC1 from saline.

The N-monomethyl analog of 3,4-MDA is MDMA (XTC, “Ecstasy,”

“Adam”). It would be anticipated that N-monomethylation of MDA would

reduce DOM-like character by at least an order of magnitude, simuitaneously enhancing the AMPH-like character. Thus, the AMPH stimulus

should generalize to the racemate and to the S (+)isomer (with the latter

being the more potent, and somewhat more potent than S(+)MDA), and the

R (-)isomer might have, at best, some weak DOM-like character. Indeed,

the (+)AMPH stimulus generalizes to racemic MDMA (ED50=7.1 μmol/kg)

(figure 4) and to S(+)MDMA (ED50=2.6 μmol/kg), but not to R(-)MDMA.

53
FIGURE 2. The effects of racemic 3,4-MDA (MDA) and its optical isomers

in rats trained to discriminate DOM from saline

KEY: DOM=effect produced by the training dose, 1 mg/kg. of DOM; S=effect produced by

1 mL/kg of 0.9 percent saline.

NOTE: Doses of S(+)MDA greater than 1.5 mg/kg resulted in disruption of behavior. Results not

shown for all doses evaluated. Where stimulus generalization did not occur, result of highest

nondisruptive dose of test compounds is shown; slightly higher doses produced disruption of

behavior.

More recently, others (Evans and Johanson 1986; Kamien et al. 1986) have

published similar results with racemic MDMA. The DOM stimulus does

not generalize to racemic MDMA or to either isomer. To this extent, the

results appear to be consistent with established SARs.

MDE (MDEA, “Eve”) is the N-ethyl analog of MDA. SARs would suggest

that this agent should possess little, if any, DOM-like character and that it

should be a rather weak AMPH-like agent, Interestingly, neither the

racemate (figure 4) nor either optical isomer (figure 5) produces

(+)AMPH-appropriate responding. As expected, DOM stimulus generalization does not occur with racemic MDE or with either of its optical isomers

(figure 6). Another inconsistency is encountered with the N-hydroxy analog

of MDA (i.e., N-OH MDA). Because N-hydroxylation of AMPH has relatively little effect on its stimulus properties, it was anticipated that N-OH

MDA might behave in a manner similar to that of MDA. Figure 7 shows

that N-OH MDA produces neither AMPH-like nor DOM-like stimulus

effects. It should be noted, however, that the optical isomers of N-OH

MDA have not yet been examined.

54
DOSE (mg/kg) DOSE (mg/kg)

FIGURE 3. Effect of R(-)MDA and S(+)MDA in rats trained to

discriminate S(+)AMPH from saline

KEY: AMPH=effect of the training dose. 1 mg/kg. of S(+)amphetamine sulfate; S=the effect of

1 mL/kg of 0.9 percent saline.

NOTE: Results not necessarily shown for all doses that were examined. Where stimulus

generalization did not occur, result of highest nondisruptive dose is shown; evaluation of a

slightly higher dose resulted in disruption of behavior.

At this point, the unexpected results cannot be readily explained with

PMMA, MDE, or N-OH MDA. This is particularly confounding in view of

the report that MDMA and MDE apparently produce similar psychopharmacological effects in humans (Braun et al. 1980). There are several possible

explanations: (1) these agents may produce effects in rats that are different

from those produced in humans; (2) these agents may produce in rats a

central effect that somehow masks or obscures AMPH-like effects that

might have otherwise been observed at higher doses had disruption of

behavior not occurred at lower doses; and (3) some of these agents might

be capable of producing a stimulus effect distinct from those produced by

either AMPH or DOM (Glennon et al. 1988). The recent results of

Oberlender and Nichols (1988) would tend to support the latter possibility.

To gain further insight into the stimulus properties of these agents, a group

of rats was trained to discriminate MDA (1.5 mg/kg) from saline. Consistent with the generalization results described above, the MDA-trained

animals recognized both racemic AMPH and DOM (table 4). MDA

stimulus generalization also occurred with both isomers of MDA, with

S(+)MDA (ED50=2.4 μmol/kg) being about twice as potent as R(-)MDA

55
TABLE 4. Results of stimulus generalization studies using rats trained to

discriminate 1.5 mg/kg of racemic MDA from saline

Agent Optical Isomer

ED50

(μmol/kg)

MDA

AMPH

DOM

MDMA

3,4-DMA

2,3-MDA

Cocaine

LSD

(±)

(-)

(+)

(±)

(±)

(±)

(±)

(±)

(+)

(+)

3.0

5.5

2.4

7.7

2.5

4.2

23.2

13.4

17.3

0.07

(ED50=5.5 μmol/kg). Because MDA produces both AMPH-like and DOMlike stimulus effects, it would be expected that MDA-trained animals would

recognize both cocaine and LSD, this was found to be the case (Glennon

and Young 1984a). Interestingly, the MDA stimulus also generalized to

3,4-DMA and 2,3MDA, agents to which neither the AMPH or DOM

stimulus generalizes. These results suggest that MDA is indeed producing

both AMPH-like and DOM-like effects and that it may also produce some

other stimulus effect.

Next trained was a group of rats to discriminate racemic MDMA from

saline. It was found that MDMA-trained animals (MDMA, ED50=2.2

μmol/kg) recognize both S(+)MDMA (ED50=1 μmol/kg) and R(-)MDMA

(ED50=4.3 μmol/kg). Thus, both isomers of MDMA appear to be active,

with S(+)MDMA being 4 times more potent than R(-)MDMA (Glennon

et al. 1986c). More recently, Schechter (1987) has reported an enantiomeric

potency ratio of about 2, whereas Oberlender and Nichols (1988) obtained a

ratio of 2.6. All three studies agree that the S (+)isomer is the more active

isomer, and two of the three studies find that it is twice as potent as the

racemate. Schechter (1987), on the other hand, has found that the racemate

is twice as potent as the S (+)isomer.

It is probably important to note that although there may be differences

between the effects produced by MDMA and MDE, there are also significant similarities. For example, preliminary data using MDMA-trained

animals suggest that racemic MDMA and MDE produce similar stimulus

56
FIGURE 4. Effect of racemic MDMA, MDE, and MDP in animals trained

to discriminate (+)AMPH from saline

KEY: AMPH=effect of the training dose., 1 mg/kg, of S(+)amphetamine sulfate; S=the effect of

1 mL/kg of 0.9 percent saline.

NOTE: Results not necessarily shown for all doses that were examined. Where stimulus

generalization did not occur, result of highest nondisruptive dose is shown; evaluation of a

slightly higher dose resulted in disruption of behavior.

effects, with MDE being slightly less potent than MDMA. At 8.2 μmol/kg.

MDE produces stimulus effects comparable to those of 6.5 μmol/kg of

MDMA. These results are consistent with those of Boja and Schechter

(1987), who used animals trained to discriminate MDE from saline. On the

other hand, whereas both MDMA and MDE are significantly less potent

than (+)AMPH in increasing locomotor activity in mice, S(+)MDMA and

S(+)MDE are about an order of magnitude more potent than their

R (-)enantiomers, and S(+)MDMA is at least several times more potent than

S(+)MDE (Patrick and Glennon, unpublished data).

Several recent reports allay fears that some progress is being made. For

example. whereas MDA (Glennon and Young 1984b; Evans and Johanson

1986; Kamien et al. 1986). S(+)MDA (Glennon and Young 1984b), MDMA

and/or S(+)MDMA (Glennon and Young 1984b; Evans and Johanson 1986;

Kamien et al. 1986; Glennon et al. 1988) produce AMPH-like effects,

S(+)MDA produces cocaine-like effects (Broadbent et al. 1987), and

although MDMA-trained animals recognize S(+)AMPH (Oberlender and

Nichols 1988), there are additional reports that AMPH-trained animals fail

57
DOSE (mg/kg) DOSE (mg/kg)

FIGURE 5. Effect of R(-)MDE and S(+)MDE in animals trained to

discriminate (+) AMPH from saline

KEY: AMPH=effect of the training dose, 1 mg/kg, of S(+)amphetamine sulfate S=the effect of

1 mL/kg of 0.9 percent saline.

NOTE: Results not necessarily shown for all doses that were examined. Where stimulus

generalization did not occur, result of highest nondisruptive dose is shown; evaluation of a

slightly higher dose resulted in disruption of behavior.

to recognize S(+)MDA (Broadbent et al. 1987). MDMA, S(+)MDMA. and

R(-)MDMA (Oberlender and Nichols 1988). Furthermore, Appel and

coworkers have reported in one study that LSD-trained animals recognize

both isomers of MDA (Broadbent et al. 1987) and, in another study, that

LSD-trained animals recognize R(-)MDA but not S(+)MDA, R(-)MDMA, or

S(+)MDMA (Callahan and Appel 1987). Consistent with results in DOM

animals, Nichols and coworkers (1986) have found that LSD-trained animals

recognize racemic MDA and R(-)MDA. In the latter study, half the animals

tested also recognized R(-)MDMA. and 78 percent of a group of rats

trained to discriminate MDMA from saline selected the drug-appropriate

lever when administered LSD. However, R(-)MDA, S(+)MDA, R(-)MDMA,

and S(+)MDMA all produced drug-appropriate responding in rats trained to

discriminate mescaline from saline (Callahan and Appel 1987). These

inconsistencies might be due to procedural differences, or they might be of

greater significance. It is believed that R(-)MDA produces primarily, but

not exclusively, DOM-like (or hallucinogenic) effects, and that S(+)MDA

produces primarily, but not exclusively, AMPH-like effects. N-monomethylation of MDA enhances AMPH-like character and decreases DOM-like

properties.

58
FIGURE 6. Effect of MDE and its optical isomers in rats trained to

discriminate DOM from saline

KEY:

NOTE:

DOM=effect produced by the training dose, 1 mg/kg of DOM; S=effect produced by

1 mL/kg of 0.9 percent saline.

Doses of S(+)MDA (greater than 1.5 mg/kg resulted in disruption of behavior. Results not

shown for all doses evaluated. Where stimulus generalization did not occur, result of highest

nondisruptive dose of test compounds is shown; slightly higher doses produced disruption of

behavior.

Evidence further suggests that MDMA (possibly MDA), and particularly

MDE and MDP, can produce effects that are distinct from (or that are in

addition to, but mask) AMPH-like and/or DOM-like effects.

Recent work shows that, in rodents, MDMA is metabolized, at least in part,

to MDA, and that racemic MDMA is preferentially metabolized to

S(+)MDA (Fitzgerald et al. 1987). The extent to which MDMA metabolites

might contribute to the stimulus properties of MDMA is unknown at this

time. Because S(+)MDA is capable of producing AMPH-like stimulus

effects, involvement of this metabolite might explain some of the different

results reported for MDMA (particularly if different animal species and

various presession injection intervals were employed). In contrast, certain

other potential metabolites of MDMA, such as 3-hydroxy-PMA, 4-hydroxyMMA, 3,4-dihydroxy-AMPH methyldopamine), N-methyl-3-hydroxyPMA, N-methyl-4-hydroxy-MMA, N-methyl-3,4-dihydroxy-AMPH

(N-methyl- methyldopamine) do not produce AMPH-like stimulus effects,

but may be capable of producing other, distinct types of central activity or

may somehow interfere with potential AMPH-like effects.

59
FIGURE 7. Effect of N-OH AMPH and N-OH MDA in rats trained to

discriminate (+)amphetamine from saline

KEY: AMPH=effect of the training dose, 1 mg/kg. of S(+)amphetamine sulfate; S=the effect of

1 mL/kg of 0.9 percent saline.

NOTE: Results not necessarily shown for all doses that were examined. Where stimulus

generalization did not occur, result of highest nondisruptive dose is shown; evaluation of a

slightly higher dose resulted in disruption of behavior.

CONCLUSION

The drug discrimination paradigm is a powerful tool for studying centrally

acting agents of the phenalkylamine type. It has been used to classify a

large number of agents as being either AMPH-like or DOM-like, and it

allows for the formulation of SARs. Though not discussed here, drug

discrimination studies have proven invaluable in understanding the

mechanisms of action of many of these agents (Glennon et al. 1986a;

Young and Glennon 1986; Glennon 1988). The SAR can also be used to

predict the activity (AMPH-like or DOM-like) and potency of novel agents.

Two significant exceptions to established SARs have been encountered

PMMA and the MDA analogs. Findings with PMMA were wholly unexpected. MDA analogs probably represent a special case; because no

methylenedioxy analogs were included in the data set used to formulate the

initial SARs, it may not be wholly valid to attempt extrapolation to these

types of agents. Nevertheless, there are instances where the SARs correctly

predict the activity and potency of certain analogs (e.g., MMDA-2,

MDMA). However, other MDA analogs (i.e., N-OH MDA, MDE, MDP)

seemingly lack all regard for the established SARs. Although differences in

60
A

B

FIGURE 8. Phenalkylamine analogs appear to produce central stimulus

effects along AMPH-like to DOM-like continuum, depending

upon the substituent groups present

NOTE: (A.) MDA produces both types of stimulus effects. (B.) Trifurcated model is presented to

account for a possible third, as yet undefined, type of central effect. Certain phenalkylamines

may exert effects better described by the MDA/X component of this model than by either

pure AMPH-like or DOM-like action.

metabolism and distribution may account for some of the observed results,

it is entirely possible that some of these agents might also produce a unique

effect that is neither AMPH-like nor DOM-like. Future drug discrimination

studies, using the MDA analogs as training drugs, should be of immense

benefit in understanding their stimulus effects.

Phenalkylamines are capable of producing a wide variety of pharmacological

effects; prominent among the central stimulus effects produced by these

agents are AMPH-like effects and DOM-like (or DOB-like) effects. Nearly

a decade ago, the author proposed that such agents exist along an

AMPH/DOM continuum and that the nature and location of pendent substituents determine where along this continuum an agent may lie (Glennon

et al. 1980). It seems likely that MDA is positioned somewhere near the

center of this continuum, because it produces both AMPH-like and

DOM-like effects. Current evidence suggests the need for a revised model

to explain the activity of MDE and MDP and the finding that 2,3-MDA and

3,4-DMA produce MDA-like, but not AMPH-like or DOM-like stimulus

61
effects. Certain of these agents may produce non-AMPH/non-DOM-like

effects (with or without a certain amount of residual AMPH-like or

DOM-like character); to account for this, a trifurcated continuum as shown

in figure 8 is proposed. Agents such as MDE and MDP may exist

somewhere along the MDA/X segment of this new model. The presence of

the methylenedioxy group may not be a prerequisite for agents to exist

along this arm of the model if, for example, agents such as PMMA can be

shown to produce effects similar to those of MDE. In contrast, the amine

substituents may play an important role. Obviously, additional studies will

be necessary to support this working model. The model, as simplistic as it

may be, accounts for the fact that certain of these agents possess some

AMPH-like or DOM-like character but, at the same time, do not seem to

follow the established SAP. The “X-like” activity (which could, in reality,

consist of several different actions) may be a consequence of direct or

indirect actions on dopamine and/or serotonin receptors (or populations of

these receptors not normally involved in the actions of AMPH or DOM) or

may represent actions at entirely different types of receptors.

DISCUSSION

COMMENT: I was surprised by the results with the N-hydroxy compounds, because a number of years ago N-hydroxy-p-toluylamphetamine was

studied, and it was found that it was identical to ptoluylamphetamine in its

properties because it was actually rapidly and essentially quantitatively

converted to p-toluylamphetamine. You are finding that the N-hydroxy

analog of MDMA is not MDMA-like in its properties. These data suggest

that they certainly are not metabolized in a similar way, perhaps.

RESPONSE: I did not show that the N-hydroxy analog of MDA is not

MDMA-like. I showed that it is not amphetamine-like.

COMMENT: That makes it not MDMA-like.

RESPONSE: You can extrapolate. I have problems with those

extrapolations.

COMMENT/QUESTION: I was not using MDMA-like in the sense that

you were using it--as a substitute. I am simply saying it did not have the

pharmacologic effect that MDMA had; namely, substitution for amphetamine, which obviously must mean that the N-hydroxy compound is not

converted to MDMA to the same extent at least as the p-toluylamphetamine

analog was. I would like to know if you have any information about the

metabolism of those N-hydroxy compounds.

RESPONSE/QUESTION: None whatsoever. We are looking at the

metabolism of some of the other compounds, but we have not looked at the

62
N-hydroxy. Has the N-hydroxy MDA been identified as a metabolite of

MDA from the earlier studies?

ANSWER: Not that I can remember.

RESPONSE: We have not looked at that at all. But it is surprising. I

expected to see either hydroxylation or dehydroxylation, so I expected to

see similar activities. But this is what we see. If you compare the

activities on a milligram per kilogram basis, the N-hydroxy amphetamine

appears to be equipotent. However, when you look at the molecular

weights (it is a different salt), it is in fact twice as potent. I cannot explain

that.

QUESTION: With respect to the N-hydroxy MDA, have you done a

timecourse to see whether at longer times you might pick it up if it is a

metabolic induction?

ANSWER: No, we have not. It is an idea.

QUESTION: I noticed that my group is the major one that disagrees with

the amphetamine-like activity of MDMA. And when you look at the MDE

and MDP you lose that. Is it possible that this MDMA-like activity is

really an artifact? That is, that the rats are saying it seems to be

amphetamine-like but, in fact, it really is not. And when you go ahead and

put the ethyl or propyl, you do not see the amphetamine-like activity

because that simply is not what it is?

ANSWER: Obviously no one knows what the rats are thinking. My

opinion, based on what we have done so far, is that MDE and MDP may

be doing something different. We may have a third wheel on this

continuum, it may be a three-way continuum with MDA in the middle.

Maybe there is a third kind of effect that MDA is capable of producing, but

this is grossly overshadowed by its amphetamine-like or DOM-like activity.

If we start making analogs of MDA like the N-methyl, that moves it a little

off center. It still retains some amphetamine-like activity. It may, at high

doses, have DOM-like activity. We certainly do not see it. And then we

have this third type of effect if we go even farther out to the ethyl homolog

or to the types of compounds that you are making. You may have now

gotten far enough from center that these compounds no longer have the

amphetamine-like or the DOM-like character. But what we see with

MDMA is that it is amphetamine-like.

QUESTION: Where does parachloroamphetamine fit in here?

ANSWER: We have never looked at parachloroamphetamine itself.

63
QUESTION: Substitution of lipophilic moieties on the phenyl ring of DOM

makes the compounds more potent. Substitution of ionic-type moieties. like

hydroxyl anions, makes them less potent. Is that a good generalization, that

making the phenyl ring more lipophilic makes the compounds more potent

in the DOM series?

ANSWER: No. There appears to be an optimal potency beyond which the

compounds are no longer active as agonists but can, in fact, act as

antagonists. So we have analogs of DOM that can antagonize the effects of

DOM, because we have passed this optimal lipophilicity. The idea of

lipophilicity at the four position is not new, and a number of investigators

have looked at this over the years with regard to hallucinogenic activity.

Recently we have been looking at it with regard to binding at 5-HT2 sites.

And we see this correlation fits very well. As we get up to a certain point

though, it stops. It appears that, in terms of discrimination and in humans,

the propyl compound appears to be optimal. Once we get beyond that,

lipophilicity continues to increase, 5-HT2 receptor affinity continues to

increase. The compounds start decreasing in potency and, in fact, they

become inactive. So it may be that some of these are partial agonists, and

ultimately we get out to antagonists of DOM. So it is not a strictly linear

relationship.

QUESTION: Is there any evidence that a common effect of amphetamine

and MDMA is mediated through a common biochemical mechanism; for

example, antagonism studies in haloperidol?

ANSWER: No, we have not done anything in that regard in terms of drug

discrimination.

QUESTION: You seemed to have looked through all of the various

substituents in your amphetamine structure, with one exception. You did

not touch the benzene ring itself. What would happen if you saturate the

benzene ring and make a saccharide derivative of amphetamine?

ANSWER: It retains amphetamine-like activity.

RESPONSE: This is what, propylhexedrine? We have looked at

propylhexedrine, and it does retain amphetamine-like activity, but it is less

potent. In rats trained to discriminate 1.5 mg/kg of racemic MDMA from

saline (ED50=0.76 mg/kg), the ED50 values for stimulus generalization to

MDE and N-OH MDA are 0.73 and 0.47 mg/kg, respectively (Glennon and

Misenheimer, unpublished observations).

64
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in pigeons. Drug Alcohol Depend 18:159-164, 1986.

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Rosecrans, J.A. Identification of 3,4-methylenedioxyamphetamine (MDA)

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Glennon, R.A. Synthesis and evaluation of amphetamine analogs. Proceedings of the Joint WHO/Drug Enforcement Administration Conference on

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Drug Stimuli. Berlin: Springer-Verlag, 1988. pp. 16-31.

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effects similar to those of 3,4-DMA, LSD and cocaine. Eur J Pharmacol

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66
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ACKNOWLEDGMENTS

Work supported in part by Public Health Service grant DA 01642 and by

funding from the World Health Organization, the U.S. Drug Enforcement

Administration, the VCU Grant-in-Aid Program, and the A.D. Williams

Fund.

AUTHOR

Richard A. Glennon, Ph.D.

Department of Medicinal Chemistry

School of Pharmacy

Medical College of Virginia

Virginia Commonwealth University

Richmond, VA 23298-0581

67
Amphetamines: Aggressive and

Social Behavior

Klaus A. Miczek and Jennifer W. Tidey

INTRODUCTION

The potential of sudden, intense acts of violence is one of the most

attention-getting facets of amphetamine action. Hippies of the 1960s

warned: “Speed kills.” At that time, reports from law enforcement

personnel, psychiatrists, and drug abusers themselves could be viewed to

indicate that “amphetamines, more than any other group of drugs, may be

related specifically to aggressive behavior” (Ellinwood 1972). Neurotoxic

effects of amphetamines and, more recently, their designer derivatives on

neurons containing dopamine and serotonin--two neurotmnsmitters of paramount significance in neurobiological mechanisms of aggressive, defensive,

social, and sexual behavior--have added a new dimension to the current

wave of stimulant abuse (Seiden and Vosmer 1984; Ricaurte et al. 1985).

In fact, amphetamines may be associated with extreme changes in aggressive and social interactions: intense and sudden acts of aggression as well

as total withdrawal from any social intercourse. These striking, seemingly

opposite shifts in social and aggressive behavior under the influence of

amphetamines and related substances are the product of numerous pharmacological, behavioral, and environmental, as well as genetic determinants.

Another paradox about amphetamines and related psychomotor stimulants is

their calming effect on excessively aggressive children and adolescents

diagnosed with attention deficit disorder. The neurobiological mechanisms

for the multiple effects of amphetamines on aggressive behavior have been

most often related to those relevant to the motor-activating and motorarousing effects of these drugs. Yet, mechanisms of amphetamine action

specific to their effects on aggressive and social behavior have eluded a

satisfactory delineation.

AMPHETAMINES AND HUMAN AGGRESSIVE AND SOCIAL

BEHAVIOR

Case Reports and Surveys

Case reports and survey data provide a complex account of the link

between amphetamines and aggressive behavior, leading to sharply differing

68
opinions on the severity and nature of the problem. As recently reviewed

(Miczek 1987), a series of clinical observations and surveys of institutionalized drug abusers and delinquents point to greatly varying representation of amphetamines in these individuals during the commission of violent

and criminal behavior. For example, several descriptions of murders and

other intense violent behavior attribute these seemingly unpredictable and

drastic changes in behavior to amphetamine abuse (Ellinwood 1971;

Siomopoulos 1981). Frequently, clinical analyses suggest that chronic

amphetamine intoxication, particularly by the intravenous route, produces a

psychotic paranoid state, including frightening delusions that may result in

aggressive acts (Kramer 1969; Angrist and Gershon 1969; Ellinwood 1971;

Siomopoulos 1981).

Some surveys found sizable proportions of prison populations and juvenile

delinquents to have committed their crimes of violence while intoxicated by

amphetamines (Hemmi 1969; Simonds and Kashani 1979); conversely,

others reported rare cases and very small percentages of juvenile delinquents

and excessively hostile individuals as having abused amphetamine

(Tinklenberg and Woodrow 1974; Tinklenberg et al. 1977; Gossop and Roy

1976). The reliability of several of these surveys is compromised by the

lack of adequately matched samples in highly selected populations of institutionalized individuals. Reliability is also compromised by reliance on

notoriously variable verbal reports for the details of the dose and frequency

of amphetamine intake, as well as on the exact nature of the drug. It may

very well be that the unusual and intense violent acts are more prominent

among chronic high-dose abusers than they are among occasional amphetamine abusers. This possible distinction needs to be investigated

systematically. So far, no reports have been published showing that

substituted amphetamines are linked to a high incidence of excessively

violent behavior or other offensive social behavior.

Attention Deficit Disorders

Reductions in aggressive behavior after treatment with amphetamine and

other psychomotor stimulants are seen in children and adolescents who have

been diagnosed with hyperkinesis or attention deficit disorder. There is

considerable disagreement about these diagnostic categories and about

whether the violent outbursts and uncontrolled episodes of aggressive

behavior are limited to the early developmental period or continue into

adulthood (Mendelson et al. 1971; Minde et al. 1972).

The early report by Bradley (1937) on beneficial treatment effects with

amphetamine in aggressive, destructive, irritable, and hyperactive boys was

repeatedly confirmed by double-blind, placebo-controlled studies, Significant

reductions in aggressive behavior and improvements in social interactions

were found after treatment with 10 to 40 mg/day of d- or l-amphetamine

for boys and girls, 5 to 14 years of age, who had been diagnosed as

69
hyperkinetic, autistic, explosive, unsocialized, or emotionally disturbed

(Conners 1969; Conners 1972; Winsberg et al. 1972; Winsberg et al. 1974;

Arnold et al. 1973; Maletzky 1974).

Experimental Studies on Human Aggression

Earlier experimental studies on amphetamine and human behavior focused

on performance measures as well as on eating and sleep disorders. None of

these studies identified an increase in aggressive behavior as a problematic

side effect (Leventhal and Brodie 1981; Laties and Weiss 1981). As a

matter of fact, controlled studies on amphetamine and human social

behavior, acute doses of d-amphetamine (5 to 30 mg) were found to

increase socializing and speaking with no indications of aggressive acts

(Griffiths et al. 1977). However, antifatigue and endurance-enhancing

effects of amphetamines may contribute to the effects of these substances

on aggressive behavior.

In an experiment that exposes a human subject to a competitive task leading

to prize money, acute amphetamine doses (5 and 10 mg) increased aggressive responses such as delivering blasts of noise or subtracting money from

the presumed competitor (Cherek et al. 1986). At the higher dose (20 mg),

the rate of aggressive behavior declined, but the rate of money-winning

responses increased, further indicating a dissociation between amphetamine

effects on aggressive and nonaggressive responses. In contrast to amphetamine, acute administrations of caffeine only decreased aggressive responses,

regardless of whether the subject was strongly or moderately provoked by

loss of prize money (Cherek et al. 1983). This experimental approach to

the study of human aggressive behavior under controlled laboratory conditions fulfills the demands for accurate, objective, and reliable behavioral

measures. It is unclear, however, whether or not this experimental preparation is a valid model of clinically significant problem behavior. Future

studies with hyperaggressive individuals or those prone to stimulant-induced

aggressive behavior will be needed to validate the laboratory situation.

AMPHETAMINES AND AGGRESSION IN NONHUMAN SUBJECTS

Amphetamine Aggressiveness

More than four decades ago, Chance (1946a; Chance 1946b) observed episodes of rapid running, audible vocalizations, upright postures, biting, and,

eventually, increased lethality after administration of near-toxic doses of

amphetamine (greater than 10 mg/kg) to mice that were housed in groups.

This so-called “amphetamine aggressiveness or rage,” most often studied in

laboratory rats and mice, but also in chicks, consists of fragmented agonistic

acts and postures embedded in stereotyped motor routines (Randrup and

Munkvad 1969; Hasselager et al. 1972). The phenomenon of amphetamine

aggressiveness in otherwise placid laboratory rats or mice has limited

70
behavioral validity and appears to be primarily of pharmacological or

toxicological interest; like motor stereotypies, the so-called amphetamine

aggressiveness is reduced by experimental compromises of the nigrostriatal

dopamine system such as synthesis inhibitors, receptor antagonists, and

neurotoxic or electrolytic lesions in this region.

Traditional Research Methodologies

Amphetamine, cocaine, and other psychomotor stimulants have been

examined with traditional research methodologies involving isolation-induced

aggression in mice; pain-induced aggression in mice, rats, or squirrel

monkeys; brain stimulation-induced aggression in cats; or mouse killing by

rats. The results show an inconsistent mixture of increases, decreases, or

no effects. Among the most important determinants of amphetamine effects

on aggressive and defensive responses are the stimulus situation, species,

prior experience with these types of behaviors (table 1) and, most critically,

dosage and chronicity of drug exposure.

TABLE 1. Doses of amphetamines for modulating behavior

Aggression Nonaggressive

Increases Decreases Motor Activity References

Isolation-Induced Aggression in Mice

None

None

None

2.0 IP

None

2.0 IP

None

4.0 IP

None

None

None

10.0 IP

ED50 > 3 IP

5.0 IP

> 2.0 IP

4.0 IP

6.0 IP

10.0 IP

8.0 IP

8.0 IP

0.25-1 PO

5 IP

10.0 IP

ED50 3 IP

N/S

> 2.0 IP

4.0 IP

N/S

N/S

4.0, 8.0 IP

8.0 IP

> 1.0 PO

N/S

Melander 1960

DaVanzo et al. 1966

Valzelli 1967

Charpentier 1969

Le Douarec and

Broussy 1969

Welch and Welch

1969

Scott et al. 1971

Hodge and Butcher

1975

Miczek and

O’Donnell 1978

Krsiak 1979

Essman and Valzelli

1984

71
TABLE 1. (Continued)

Aggression

Increases Decreases

Nonaggressive

Motor Activity References

Pain-Induced Aggression in Mice

8.4 PO None

0.1 IP None

0.5 PO None

None 5.0 PO

Pain-Induced Aggression in Rats

None 3.0 IP

0.25-1 IP 4.0 IP

1.0 IP 3.0 IP

3.48 IP N/S

None > 2.5 IP

9.3 PO

N/S

> 0.5 PO

2.5 PO

N/S

N/S

N/S

N/S

N/S

Stille et al. 1963

Kostowski 1966

Hoffmeister and

Wuttke 1969

Tedeschi et al. 1969

Lal et al. 1968

Crowley 1972

Powell et al. 1973

Mukherjee and

Pradhan 1976

Sheard 1979

Pain-Induced Aggression in Squirrel Monkeys

None

0.125-1 SC

0.125-1 SC

0.3, 1 IM

2.0 SC

2.0 SC

0.03-1 IM

> 2 SC

> 2 SC

DeWeese 1977

Hutchinson

et al. 1977

Emley and

Hutchinson 1972;

Emley and

Hutchinson 1983

Extinction-Induced Aggression in Rats

0.1 IM 0.5, 1.0 IM 0.1-1.0 IM

Brain Stimulation-Induced Aggression in Rats

None 2.0 IP 2.0 IP

Brain Stimulation-Induced Aggression in Cats

5-7.5/cat IP

None

None

10/cat IP

>4 IP

0.3, 0.8 IP

N/S

N/S

N/S

0.125-0.5 IP 1-1.5 IP N/S

0.5-3 IP N/S N/S

Miczek 1974

Panksepp 1971

Sheard 1967

Baxter 1968

MacDonnell and

Fessock 1972

Marini et al. 1979

Maeda et al. 1985

72
TABLE 1. (Continued)

Aggression

Increases Decreases

Nonaggressive

Motor Activity References

Drug-Induced Aggression in Mice

l-dopa

2.0 IP N/S N/S Lal et al. 1970

Drug-Induced Aggression in Rats (Withdrawal from Opiates)

2.0 IP N/S

ca. 3-11/day PO N/S

1-4 IP N/S

2.0 IP N/S

2.0 IP N/S

2.0 IP N/S

Mouse Killing in Rats

N/S

N/S

N/S

N/S

N/S

N/S

None

None

2-15 IP 4-5 IP

ED50 1.5 IP ED50 6.6 IP

None

None

None

0.5-2 IP

ED50 0.8 IP

ED50 1.8 IP

> 2 IP

ED50 4.2 IP

1-3 IP

None 5.0 IP N/S

None

None

None

None

None

None

None

None

None

None

2, 4 IP

ED50 0.18 IP

1.5 IP

ED50 0.6 IP

0.75-3 IP

2.0 SC

2 IP

ED50 1.15 IP

0.5-2 IP

1-3 IP

1, 1.5 IP

> 0.18 IP

N/S

N/S

N/S

N/S

N/S

N/S

N/S

2-3 IP

Florea and Thor 1968

Thor 1971

Lal et al. 1971

Carlini and

Gonzalez 1972

Puri and Lal 1973

Gianutsos et al. 1975

KarLi 1958

Horovitz et al. 1965;

Horovitz et al.

1966

Kulkarni 1968

Sofia 1969

Salama and Goldberg

1970; Salama and

Goldberg 1973

Valzelli and

Bemasconi 1971

Vergnes and

Chaurand 1972

Malick 1975

Gay et al. 1975

Malick 1976

Gay and Cole 1976

Posner et al. 1976

Barr et al. 1976

Barr et al. 1977

Barr et al. 1979

Russell et al. 1983

NOTE: All doses are expressed in mg/kg; N/S=Data not specified, PO=oral injection.

SOURCE: Miczek 1987.

73
Low acute amphetamine doses enhance pain-induced aggressive/defensive

reactions in mice, rats, and squirrel monkeys (Kostowski 1966; Hoffmeister

and Wuttke 1969; Crowley 1972; Powell et al. 1973; Emley and Hutchinson

1972; Emley and Hutchinson 1983). For example, squirrel monkeys subjected to electric shocks to their tails, bite a rubber hose more frequently

after being administered amphetamine (0.06 to 1.0 mg/kg, SC) (Emley and

Hutchinson 1972; Emley and Hutchinson 1983; Hutchinson et al. 1977). In

rats, these pain-induced aggressive/defensive responses increase with doses

of 0.1 to 1.0 mg/kg (Crowley 1972).

Intermediate to higher amphetamine doses routinely decreased or disrupted

isolation- and extinction-induced aggressive behavior and pain-induced

aggressive/defensive reactions in mice, rats, and squirrel monkeys while

increasing nonaggressive motor activity (Melander 1960, DaVanzo

et al. 1966, Miczek 1974; Hodge and Butcher 1975; Krsiak 1979). It may

also be mentioned that amphetamines, as well as other psychomotor stimulants, reliably block mouse-killing behavior in selected laboratory rats

(Horovitz et al. 1965; Kulkami 1968; Malick 1976; Russell et al. 1983). In

this screening test for antidepressant drugs, the antimuricidal effect of

amphetamines may be considered a false positive (Howard and Pollard

1983).

This complicated pattern of amphetamine effects in the traditional models of

aggression, each relying usually on a single index, may be conveniently

interpreted to reflect how amphetamine’s effects on aggression depend on

the particular measurement technique. Yet, such conclusions are not

heuristic. More recently, an ethological approach to the study of drug

action on aggression has focused on biologically valid test situations and

detailed behavioral measurements, in an effort to gain insight into causative

and functional determinants of aggressive, defensive, submissive, and flight

behaviors (Miczek et al. 1984). In the following, an examination of the

most important pharmacological and behavioral determinants of amphetamine effects on aggressive and defensive behavior in several animal species

will emphasize the lawful, systematic nature of these drug behavior interactions and, at the same time, highlight their social and environmental

constraints.

BEHAVIORAL DETERMINANTS OF AMPHETAMINE EFFECTS ON

AGGRESSION

Differentiation Between Attack, Defense, Submission, and Flight

In animal species commonly used in laboratory research, social aggregation

and dispersion are achieved by agonistic behavior patterns with various acts,

postures, movements, and signals. Confrontations between a territorial

resident and an intruder, between a dominant and lower-ranking group

member, between rival males or females, between a lactating female and a

74
potential threat to her offspring can be reproduced and studied under

controlled laboratory conditions. Amphetamine differentially alters attack

and threat behaviors vs. defensive and flight reactions.

In situations of social conflict, amphetamine increases the frequency of

escape and defensive responses to threats and attacks by a stimulus animal

in mice, rats, cats, rhesus monkeys, and squirrel monkeys in a doscdependent manner (Hoffmeister and Wuttke 1969; Crowley et al. 1974;

Miczek and O’Donnell 1978; Miczek 1979; Schlemmer and Davis 1981;

Haber et al. 1981). Even in the absence of a distinctive behavioral stimulus

from an opponent, amphetamine induces escape and defensive responses in

mice. Krsiak considered these unprovoked defensive and escape responses

as signs of “timidity” (Krsiak 1975; Krsiak 1979; Poschlova et al. 1977).

Amphetamines decrease attack and threat behavior by dominant animals

toward lower-ranking group members, by territorial residents toward an

intruder, by lactating females defending their litter, and play fighting by

juveniles, mainly due to distortions in the perception of socially significant

signals and the disruption of integrated sequences of threat and attack

behavior (Miczek and Gold 1983; Miczek et al. 1989). Large and intense

increments in aggressive behavior after amphetamine administration may

occur suddenly in mice, rats, cats, and several primate species, under

limited conditions. Several determinants for these infrequent but important

amphetamine effects have begun to be identified, such as the base rate of

aggressive behavior before any amphetamine administration, previous

experiences with aggressive and defensive behavior, and the level of

habituation to an aggression-provoking situation.

Baseline

Studies of amphetamine effects on behavior, mainly shaped and controlled

by schedules of reinforcement, have led to the general principle of rate

dependency; low rates of behavior tend to be increased by amphetamine-like

drugs, intermediate rates are less altered, and high rates are decreased

(Dews and Wenger 1977). This principle applies only rarely to the effects

of amphetamines on aggressive behavior (Miczek and Krsiak 1979). In

isolated mice, amphetamine increased the incidence of aggressive behavior

only in those subjects that were selected for their near-zero levels during

vehicle control tests. Amphetamine decreased aggressive behavior in

animals with high rates during vehicle control tests (Krsiak 1975; Krsiak

1979). These results lend themselves to a rate-dependency interpretation.

Comparisons between separate groups of subjects, one displaying a low rate

of aggressive behavior, the other a high rate, however, are less persuasive

evidence for rate dependency of amphetamine effects than is the demonstration of differential drug effects on low and high rates of behavior within

the same subject.

75
A minute-by-minute analysis of rates of attack behavior during either a

5- or 28-minute confrontation between a resident and an intruder shows a

high rate of aggression in the initial phase of the encounter and a gradual

decline in the later phase (figure 1). This decrement from high to low rates

FIGURE 1. Effect of d-amphetamine on the frequency of attack bites by a

male resident mouse toward a male intruder during 28-

minutes (left) or 5-minutes (right) confrontations

NOTE: The resident mouse was adminstered an acute dose of amphetamine 30 minutes before

confrontation. Frequency of attacks is minute-by-minute average.

of aggression could be due to fatigue, habituation, or changes in the

stimulus qualities of the intruder animal. Contrary to the effects of drugs

such as alcohol, there was no evidence that amphetamine increased either

the high attack rates in the early phase of the encounter or the lower rates

76
of attack in the later phase (Miczek, unpublished observations). Also,

higher amphetamine doses that decreased attack behavior at the start of an

encounter did not lead to any rebound in the later phases, even during

28-minute encounters. Apparently, once an aggressive interaction has been

initiated, and the opponent reacts with defensive and flight responses,

amphetamine does not increase further the rate of aggressive behavior

within the same encounter.

Habituation

A substantial increase in aggressive behavior is seen when amphetamine is

administered to animals that are repeatedly confronting an intruder (Winslow

and Miczek 1983). Specifically, during 2-hour sessions, resident male mice

pursued, threatened, and attacked intruders 10 times, each 5-minute

encounter being separated from the next by 5 minutes. ‘The threat and

attack behavior exponentially declined over the course of the 10 consecutive

encounters; half of all aggressive behavior was displayed during the first

3 encounters, and the remaining 7 encounters were characterized by very

low levels of aggressive behavior (Winslow and Miczek 1984). It is in this

later phase of the habituation process that amphetamine more than doubled

the rate of attack behavior (figure 2). These amphetamine effects on attack

and threat behavior were dissociated from those on elements of motor

activity such as walking, rearing, or grooming, in terms of timecourse and

dose-effect curve. This pattern of effects suggests a direct action of

amphetamine on the habituation process, an elementary form of learning, in

addition to the well-known antifatigue effects of amphetamine.

Burst-Like Pattern of Aggressive Behavior

Amphetamine substantially alters the characteristic temporal pattern of

agonistic behavior (Miczek 1983; Miczek et al. 1989). Normally, epochs or

bursts of intense and frequent threat and attack behavior alternate with

periods of relative behavioral quiescence, as, for example, in confrontations

between a resident mouse and an intruder. The intervals that separate

consecutive attacks are exponentially distributed, with 70 to 80 percent of

all intervals being very short and constituting the steep portion of this

distribution; the remaining long intervals represent the gaps that separate

bursts of attacks. Amphetamine, at doses that did not alter the frequency or

duration measures of aggressive behavior, increased the size of the

aggressive bursts, and at higher doses abolished the characteristic burst

pattern (figure 3).

Sequences of aggressive behavior that are composed of characteristic acts

and postures following each other rapidly are disrupted. These disorganizing effects parallel the analysis of amphetamine effects on other intricately

patterned behaviors such as feeding, maternal care, play behavior, or

reproductive interactions. For example, amphetamine suppresses play

77
FIGURE 2. Effects of d-amphetamine and methysergide on the cumulative

frequency of attack bites and sideways threats (top) and

walking duration (bottom) during the initial and later

resident-intruder confrontations

NOTE: Confrontations were in a sequence of 10 consecutive 5-minute trials. each trial seperated

from the next by a 5-minute interval.

SOURCE: Winslow and Miczek 1983.

behavior in juvenile rats, an effect that is not antagonized by dopamine or

norepinephrine receptor antagonists (Beatty et al. 1984). Similarly, maternal

care is severely disturbed in female vervet monkeys under the influence of

amphetamine (Schirring and Hecht 1979). These findings and those of

others emphasize the disintegrative effects of amphetamine on patterns of

78
NOTE: Superimposed on the histograms are curves of a mixed exponential distribution and the

component distributions. The length of attack bouts is estimated from the intersection of

the component distributions. The intervals between attacks that represent the gaps between

bouts are shaded.

SOURCE: Miczek et al. 1989.

FIGURE 3. Frequency historgrms of interval length between

consecutive attack bites by a resident mouse toward an

intruder after saline control, 2.5, or 5.0 mg/kg

d-amphetamine (n=20). B. Number of interattack intervals

surviving to increasing durations from single encounters

under saline control conditions, 1.25, 2.5, and 5.0 mg/kg

d-amphetamine.

social interaction (Kjellberg and Randrup 1971; Kjellberg and Randrup

1973; Garver et al. 1975; Miczek 1981b).

PHARMACOLOGICAL DETERMINANTS

Dose

Dose-dependent biphasic effects on aggressive behavior may be seen in

several, but not all animal species and situations (Miczek and Krsiak 1979;

79
Miczek 1987). The paramount importance of dosage for amphetamine

effects on aggressive and social behavior is illustrated by experiments in

male rats confronting an opponent, either in a competitive situation or as an

intruder into their homecage, showing aggression-enhancing effects at low

acute doses (Miczek 1974; Miczek 1979). On occasion, increases in

aggressive behavior after administration of low acute amphetamine doses

have also been seen in fish, mice, and selected rhesus and stumptail

macaque monkeys (Weischer 1966; Haber et al. 1981; Winslow and Miczek

1983; Smith and Byrd 1984; Kantak and Miczek 1988). A much more

consistent observation, however, is the amphetamine-related increase in

defensive, submissive, and flight reactions, which systematically increase

with dose, up to a level at which motor stereotypies begin to interfere with

the display of these behaviors (Hoffmeister and Wuttke 1969; Miczek 1974;

Miczek and O’Donnell 1978).

Ongoing experiments with methylenedioxymethamphetamine (MDMA) show

a systematic dose-dependent decrease in attack and threat behavior in mice

confronting an intruder into their homecage (Miczek et al., unpublished

observations). The decrement in aggressive behavior appears to be

behaviorally specific; it is obtained at MDMA doses (0.3, 1, 3 mg/kg) that

are lower than those necessary to decrease measures of conditioned

performance under the control of schedules of positive reinforcement.

Because of species-dependent neurotoxicity, MDMA’s effects on aggressive

behavior need to be explored in other species, including primates.

Chronicity

Tolerance or sensitization may result from repeated exposure to amphetamines, depending on the interval between consecutive amphetamine administrations (Segal et al. 1980; Robinson and Becker 1986). with continuous

drug exposure resulting most often in tolerance, and intermittent

administration in behavioral sensitization. Most of the evidence on the

determinants of tolerance and sensitization to amphetamine derives from

studies on the motor-activating effects of these drugs as measured in

situations promoting locomotion, circling, or stereotyped movements.

Unfortunately, only a few experimental studies have focused on the effects

of repeated amphetamine administration on aggressive and social behavior,

although it is precisely this condition that is associated with the most

troubling clinical experiences. Methamphetamine, given in daily increasing

doses. decreased aggressive behavior in seven different mouse strains and

genera, except for grasshopper mice (Richardson et al. 1972). Daily

administration of d-amphetamine or cocaine for 2 to 4 weeks to resident

mice confronting an intruder failed to shift the dose-effect function for these

drugs’ effects on any element of threat and attack behavior, while

augmenting the stereotypy-inducing effects (O’Donnell and Miczek 1980).

Slow-release amphetamine capsules, implanted subcutaneously in rats that

80
lived in large all-male colonies, produced hyperactivity and social

withdrawal in the initial phase of drug exposure; after about a week a high

incidence of startle, threat, and defensive responses was seen (Ellison 1978;

Eison et al. 1978). Similar, chronically implanted amphetamine capsules in

vervet monkeys again resulted in hallucinatory-like grooming, grasping, and

head movements, and disrupted social interactions without evidence for

tolerance development (Nielsen and Lyon 1982). These progressively more

pronounced social withdrawal and motor stereotypies are also seen in groups

of macaques or marmosets that are administered amphetamine daily (Garver

et al. 1975; Ridley et al. 1979). So far, neither tolerance nor sensitization

to amphetamine’s effects on withdrawal from all social and aggressive

interactions has been seen in the very few studies that either examined

changes in the ongoing rate of these behaviors during the course of repeated

amphetamine administration or that tested for shifts in dose-effect functions

before, during, and after chronic amphetamine exposure.

The only evidence on chronic amphetamine administration and heightened

aggressiveness derives from the studies, discussed earlier, on group-housed

placid laboratory rats or mice. The behavioral validity of these phenomena

under near-toxic dosage conditions, however, needs to be resolved.

Opiate Withdrawal

Amphetamine effects on aggression are markedly modulated by opiates and

opioid peptides. Withdrawal from prolonged exposure to opiates may lead

to increased defensive and aggressive responses in mice and rats and

increased hostility in humans (Lal et al. 1971; Gossop and Roy 1976;

Kantak and Miczek 1986). Amphetamine and cocaine, as well as dopaminergic agonists, increase further the already high levels of defensive

responses in aggregated rats undergoing withdrawal from opiates, leading in

extreme cases to the death of the subjects (Lal et al. 1971; Puri and Lal

1973).

Locomotor-activating effects of amphetamine have previously been linked to

dopamine release (Iversen 1977), and it has been suggested that the

aggression-enhancing effects may be mediated by a similar mechanism

(Gianutsos and Lal 1976). Enhancement of aggression by treatment with a

combination of l-dopa and d-amphetamine can be blocked with the

dopamine receptor antagonist haloperidol (Lal et al. 1975); aggression

induced by challenge with amphetamine during morphine withdrawal is

blocked by either haloperidol or alpha-methyl-para-tyrosine (Lal 1975; Puri

and Lal 1973).

The dramatic heightening of aggressive behavior in morphine-withdrawn

animals may be due to dopamine receptor upregulation (Gianutsos

et al. 1975; Lal et al. 1975). Morphine and methadone inhibit dopamine

receptors in the central nervous system (CNS) suggesting possible disuse

81
supersensitivity and hyperactivity of the receptor during withdrawal (Puri

and Lal 1973; Martin and Takemori 1986). Further enhancement of

morphine-withdrawal aggression by amphetamine has been interpreted to

reflect stimulation of supersensitive dopamine receptors (Puri and Lal 1973;

Kantak and Miczek 1988).

Recently, it was found that single-housed mice that had been undergoing

withdrawal for 48 hours (after removal of a subcutaneously implanted

75-mg morphine pellet) showed an elevation of attack and threat behavior

that was doubled when these mice were challenged with amphetamine,

cocaine, l-dopa, or apomorphine (figure 4) (Kantak and Miczek 1986;

FIGURE 4. The frequency of attack, threat, walking, and grooming (mean

±SEM per 5 minutes) following saline or 0.1, 0.5, 1.0, or

25 mg/kg d-amphetamine

p<0.05 compared to vehicle control.

NOTE: These doses were administered to male resident mice implanted with either placebo pellets

(open circles) or morphine pellets (solid circles) subsequently withdrawn 48 hours prior to

testing.

SOURCE: Kantak and Miczek 1988.

Kantak and Miczek 1988). Similarly, Lal et al. (1971) and Thor

et al. (1970) found that in aggregated rats, amphetamine enhances defensive

82
upright postures and audible squeals most strongly about 72 hours after

termination of a chronic morphine injection schedule. Mice that have been

in withdrawal for 5 hours, however, do not show this enhancement when

challenged with amphetamine (Miczek and Tidey, unpublished observations).

This difference in the reaction to amphetamine may reflect changes in

sensitivity of dopamine receptors over time: shortly after withdrawal from

opiates, a lessened sensitivity to amphetamine’s heightening effects on

aggression is seen; later a supersensitivity emerges.

To assess this possibility, selective dopamine receptor agonists were

administered to mice 5 hours after subcutaneous morphine pellet removal

(Miczek and Mohazab 1987). Challenge with either quinpirole, a selective

D2 agonist, or SKF 38393, a selective D1 agonist, or a combination of both

did not result in heightened aggression. In fact, the studies with combined

administration of D1 and D2 agonists indicate that, in the presence of D1

receptor activation by a small dose of SKF 38393 (3.0 mg/kg), very large

doses of D2 receptor agonists are necessary to modify aggressive behavior

in these mice, suggesting a subsensitivity of D2 receptors. This particular

timecourse relates solely to the aggression-enhancing effects; the authors and

others (Bläsig et al. 1973; Lal 1975; Kantak and Miczek 1988) have noted

that different autonomic and somatic opiate withdrawal signs emerge at

earlier times after morphine pellet removal or termination of a chronic

injection schedule.

The sub- and supersensitivity to amphetamine’s aggression-modulating

effects during withdrawal from morphine depend on the time since the last

exposure to opiates; it will be intriguing to determine how the relevant

opioid and dopamine receptor populations are altered at these behaviorally

critical phases of opiate withdrawal. The display of aggressive, defensive,

and submissive behavior is accompanied by marked changes in the functioning of brain opioid peptides in the absence of any drug exposure (Miczek

et al. 1986); it will also be interesting to determine how amphetamine’s

effects in individuals with differential experiences with aggressive or

submissive behavior may involve alterations in brain opioid peptides and

their receptors.

ANTAGONISM OF AMPHETAMINE EFFECTS ON SOCIAL AND

AGGRESSIVE BEHAVIOR

The most consistent and potent antagonism of amphetamine effects on

increased motor activity and stereotyped movements is obtained with

antagonists at dopamine receptors of the D2 subtype (Creese et al. 1982).

This is not the case with amphetamine’s disruptive effects on social and

aggressive behavior, So far, no antagonists have been identified that reverse

amphetamine’s disruption of sexual, play, maternal, or aggressive behavior.

In many ways, this situation parallels the clinical experiences, in being

83
unable to reverse the negative symptoms of both amphetamine-induced and

endogenous psychoses with classic neuroleptics (Crow 1985).

Dopamine Receptor Antagonists

Haloperidol and chlorpromazine potently decrease aggressive and social

behavior as well as many other behavioral functions in various animal

species and humans. The marked potency and long-lasting nature of the

antiaggressive effects of the neuroleptics with dopaminergic receptorblocking properties may be the reason why these types of drugs are most

frequently used in treating pathologically violent individuals (Itil 1981;

Leventhal and Brodie 1981; Sheard 1984; Tupin 1985). The poor

behavioral specificity of their antiaggressive effects, however, renders the

phenothiazines, butyrophenones, or thioxanthines as less than ideal choices;

this pattern of effects is already apparent in preclinical studies (Malick

1979; Miczek and Winslow 1987).

Recently, the effects of more selective dopamine receptor antagonists on

aggressive behavior were explored. In resident mice confronting an intruder

into their homecage; quinpirole (0.1 to 1.0 mg/kg) potently reduced pursuit,

threat, and attack behavior; however, it also reduced concurrent motor

activity. This pattern of effects paralleled haloperidol effects in the same

species and situation. However, the D1 receptor agonist SKF 38393 more

selectively, although less potently, decreased aggressive behavior by resident

mice, in the absence of concurrent changes in motor functions. These

studies highlight the problem of identifying a dopamine antagonist that

could be useful in the blockade of amphetamine effects, but would not

suppress behavior on its own.

Dopaminergic receptor antagonists do not antagonize the disruptive effects

of amphetamine on aggression. In squirrel monkeys, d-amphetamine

(1.0 mg/kg) disrupted agonistic and social behavior; haloperidol pretreatment

did not prevent this disruption (figure 5, right) (Miczek and Yoshimura

1982). Similarly, d-amphetamine decreased attack and threat behavior in

resident mice confronting an intruder haloperidol pretreatment failed to

reverse this disruption, but further decreased aggressive behavior in

amphetamine-treated mice (figure 5, left) (Miczek 1981a). By contrast, the

large activation of motor activity, as evidenced by increased time spent in

locomotion, was effectively antagonized by haloperidol in mice as well as

in squirrel monkeys (figures 5). Similarly, play fighting in juvenile rats is

profoundly disrupted by amphetamine, and this disruption is not reversed by

haloperidol or chlorpromazine (Beatty et al. 1984). By contrast, in those

situations where low, acute doses of amphetamine enhance aggressive

behavior, dopaminergic receptor antagonists attenuate this enhancement.

These observations suggest differential mechanisms for the aggressionheightening effects of amphetamine as distinct from the disruptive actions

on social and aggressive behavior. The neurobiological mechanisms for

84
amphetamines’ disruption of social and aggressive behavior remain to be

elucidated.

FIGURE 5. Mice: Frequency of attack bites (A.) and the duration of

walking across cage (B.) by resident male mice after administration of d-amphetamine alone (open circles), and after

pretreatment with haloperidol (0.25 mglkg, solid circles).

Squirrel monkeys: Frequency of aggressive behavior (A.)

and walking (B.) by dominant squirrel monkeys in established social groups following administration of amphetamine alone (open bars), and combined with haloperidol

(0.25, 0.5 mg/kg, IM, solid bars).

KEY: Vertical lines at each data point represent ± 1 SEM

Noradrenergic Receptor Antagonists

Antagonism of several characteristic effects of amphetamine and cocaine by

the alpha adrenergic receptor antagonist prazosin is a most recent example

of noradrenergic mechanisms in the actions of psychomotor stimulants

(Tessel and Barrett 1986). We investigated whether or not prazosin may

attenuate the disruptive effects of amphetamine on social and aggressive

behavior in mice and squirrel monkeys (Miczek, unpublished observations).

Pretreatment with prazosin (0.4 mg/kg) attenuated the disruption of attack

85
bites and sideways threats in resident mice treated with higher doses of

amphetamine, but no such attenuation was found of amphetamine-disrupted

aggressive behavior by dominant squirrel monkeys after prazosin

pretreatment (figure 6). By contrast, amphetamine’s hyperactivity, measured

FIGURE 6. Left: Frequency of attack bites (A.) and duration of walking

across cage (B.) by resident male mice after administration

of d-amphetamine alone (open circles), and after pretreatment with 0.4 mglkg prazosin (solid circles). Right:

Frequency of aggressive behavior (A.) and walking (B.) by

dominant squirrel monkeys in established social groups

following administration of amphetamine alone (open

circles), and after pretreatment with 0.4 mg/kg prazosin, IM

(solid circles).

KEY: Vertical lines at each data point represent ± 1 SEM.

as time spent in locomotion, was attenuated by prazosin pretreatment both

in mice and squirrel monkeys. Previously, we have observed that

pretreatment with phenoxybenzamine or propanolol did not attenuate the

suppression of aggressive behavior in amphetamine-treated resident mice

(Miczek 198la). In juvenile rats, the suppression of play fighting by

amphetamine was also not reversed by phenoxybenzamine or propranolol

(Beatty et al. 1984). Again, although the evidence is limited to a few

86
receptor antagonists and to laboratory rodents, so far there is no evidence

pointing to the possible attenuation or reversal of amphetamine’s disruptive

effects on social and aggressive behavior by noradrenergic receptor

antagonists. The negative evidence from efforts to antagonize

amphetamine’s effects on aggressive behavior with noradrenergic receptor

antagonists suggests that these amphetamine effects do not involve

noradrenergic mechanisms.

Opioid Antagonists

Opioid receptor antagonists have been found to modulate brain dopaminemediated behavioral and cellular functions such as motor activity, drug selfadministration, and brain stimulation reward (Koob and Bloom 1988).

Naloxone has been found to attenuate the increased motor activity in rats

and guinea pigs after amphetamine administration (Holtzman 1974; Haber

et al. 1978; Hitzemann et al. 1982; Andrews and Holtzman 1987). Similarly, opiate antagonists reduced the enhancement of rewarding electrical brain

stimulation by amphetamine and cocaine (Bain and Kometsky 1987), and

intracerebral injections of opiate antagonists into the nucleus accumbens

selectively blocked heroin self-administration and motor activation in rats

(Amalric and Koob 1984; Vaccarino et al. 1985). Although independent

studies have found marked changes in social, aggressive, defensive, and

submissive behavior after either opiate antagonists or psychomotor

stimulants, the potential antagonism of amphetamine effects on these

behaviors by opiate receptor antagonist has not been investigated until

recently.

In experiments with mice and squirrel monkeys, we confirmed and extended

the antagonism of amphetamine-induced motor hyperactivity by naltrexone;

at the same time, however, amphetamine’s disruption of aggressive and

social behavior was not reversed by naltrexone (Winslow and Miczek, in

press). Specifically, in mice, the resident’s attack and threat behavior

toward an intruder was even further reduced by amphetamine after

naltrexone pretreatment (figure 7). Squirrel monkeys that are dominant

within their social group exhibit significantly lower levels of aggressive

display toward other group members and initiate fewer social interactions

after amphetamine treatment; naltrexone did not block these effects. The

interactive effects of amphetamine and naltrexone on locomotor behavior are

consistent with the proposed modulation of dopamine-mediated functions by

opioids; however, the interaction between amphetamine and naltrexone on

social behavior appears to involve a different mechanism.

SUMMARY

Clinical case reports and survey data point to incidences of intense violence

in certain individuals self-administering high doses of amphetamine via the

87
FIGURE 7. Left: Frequency of attack bites (A.) and duration of walking

across cage (B.) by resident male mice after administration

of d-amphetamine alone (open circles), and after pretreatment with 1.0 mg/kg naltrexone (solid circles). Right:

Frequency of aggressive behavior (A.) and walking (B.) by

dominant squirrel monkeys in established social groups

following administration of amphetamine alone (open

circles), and after pretreatment with 1.0 mg/kg, IM,

naltrexone (solid circles).

KEY: Vertical lines at each data point represent ± 1 SEM. 

SOURCE: Winslow and Miczek 1988.

intravenous route. It is unclear how common this amphetamine effect is,

what circumstances promote its occurrence, and which characteristics

predispose an individual to exhibit this effect,

Amphetamine may engender a dose-dependent biphasic effect on aggressive

behavior in experimental situations, both with human and animal subjects,

as, for example, in subjects that have habituated to an aggression-provoking

stimulus. Most often, however, amphetamines disrupt social, sexual,

matemal, and aggressive behavior patterns in a dose-dependent manner;

88
neither tolerance nor sensitization appears to develop to these disruptive

effects.

Amphetamine consistently enhances defensive and flight reactions in various

experimental situations and animal species. This effect appears to be

mediated by brain dopaminergic systems. So far, no dopaminergic,

noradrenergic, or opioid antagonists have been found that attenuate, reverse,

or prevent the disruptive effects of amphetamines on social and aggressive

behavior. The evidence from opioid-withdrawn subjects strongly suggests a

profound modulatory influence by opioid peptides on the aggression-altering

effects of amphetamines.

DISCUSSION

QUESTION: You know the serine compound is very potent. Have you

tried lower doses on a rate-decreasing effect of the stimulant drug?

ANSWER: I tried 0.3 and 1.0. In mice, 0.3 does not have an effect in

itself. In rats, 0.3 could be quite disruptive. So there is quite a bit of a

species difference. The range of dose is very different in mice and rats.

QUESTION: What do you think causes the aggressive decreasing effects?

Are the mice stereotyping or perseverating on some other object?

ANSWER: In the studies we did in mice, rats, and monkeys, we looked

carefully at motor changes that might intrude into the behavior and prevent

the animals from showing the behavior, not in this dose range. They are

nonoverlapping dose ranges. You have to go to higher doses to see

stereotypic and motor-activating effects.

In fact, Cherek made that point in one of the very first studies. You

cannot see further increases in monetary reinforced behavior. But you see a

decline in aggressive behavior. And that is true in other species and

humans, too. So the most significant point is that the disruptive effects are

due to the intrusion into the repertoires of other repetitive routines.

COMMENT: One of the first studies that was done with SCH compound

23390 showed that it had pronounced antiaggressive effects. This was a

Canadian study of people who were in backward, isolated conditions. It

had a fairly pronounced effect there.

I think one of the things that is confusing in the aggressive homicide

literature is the fact that at low doses, i.e., 10, 20, 30 milligrams for a

70-kilogram person, there is a calming effect. This was one of the things

that we used to see with hyperactive children. Many of those hyperactive

children were indeed aggressive-hyperactive children, and the amphetamines

89
had a very pronounced effect on that. This probably represents a low-level

activity.

In really aggressive people who have taken amphetamines a long time, you

see what is called the reactive phase of aggressiveness.

Let me give you an example of this, which is particularly true in homicides.

The individual is engaged in an activity and suddenly misinterprets

something. He wakes up in the back of a car and smells poison gas and

hits someone over the head with a pipewrench. Or he is robbing a store

and someone smiles. There is a sudden impulse and he kills an individual.

If you look at the court records, you see that story repeatedly, i.e., this

reactive component. And you can see the same thing in chronic animals.

You do have to take them out to a 3- or 6-month period to see those

effects. During long-term chronic use, the dopamine at that point is

markedly depleted. We are talking about animals that have 20 or

30 percent of the original dopamine levels a month or so after they have

been given the last dose of amphetamine.

So I think we are talking about two or three different phenomena, and I

think it is very important that we make those distinctions.

RESPONSE: I left aside the hyperactivity issue because that is a literature

study in itself. It is also limited to adolescents, children, and juveniles,

although there are some reports in adults as well. But there the therapeutic

range for amphetamine is 20, 30, or 40 milligrams, and for methylphenidate

it is slightly higher, which is actually the preferred agent.

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99
ACKNOWLEDGMENTS

This review and research were supported in part by U.S. Public Health

Service research grant DA 02632 and AA 05122. Mr. J.T. Sopko provided

expert assistance in preparing the illustrations, the computerized

bibliographic data base, as well as in conducting the experimental work.

AUTHORS

Klaus A. Miczek, Ph.D.

Jennifer W. Tidey, B.S.

Tufts University

Medford, MA 02155

100
Neurochemical Mechanisms

Involved in Behavioral Effects of

Amphetamines and Related

Designer Drugs

Lisa H. Gold, Mark A. Geyer, and George F. Koob

INTRODUCTION

The psychoactive drug 3,4-methylenedioxymethamphetamine (MDMA) has

become increasingly popular as an abused substance (Beck and Morgan

1986; Peroutka 1987). Biochemically, MDMA is thought to release

serotonin and to a lesser extent dopamine (Johnson et al. 1986; Nichols

et al. 1982; Schmidt et al. 1987), while structurally, MDMA resembles both

mescaline and amphetamine (Nichols et al. 1986; Shulgin 1978). MDMA is

the N-methylated form of 3.4-methylenedioxyamphetamine (MDA), another

substituted phenylethylamine with psychotropic properties that may have

contributed to its popular name, “the love drug.” MDA is considered to be

frankly hallucinogenic and has been found to be highly toxic to serotonergic

neurons (Ricaurte et al. 1985). Recently, long-term depletions of serotonergic markers have also been observed following single and multiple

injections of MDMA in experimental animals, indicating a neurotoxic

potential similar to that associated with MDA (Mokler et al. 1987; Schmidt

1987; Stone et al. 1986).

Interestingly, some psychotherapists have been using MDMA to enhance the

psychotherapeutic process and to promote easy emotional communication in

their patients (Grinspoon and Bakalar 1986). MDMA is characterized as

evoking an altered state of consciousness with emotional and sensual

overtones (Shulgin and Nichols 1978). This state is described as a pleasant

state of introspection, a highly controllable experience that invites

intensification of feelings (Grinspoon and Bakalar 1986) and greatly

facilitates interpersonal communication (Nichols et al. 1986). Encouraged

by these properties, the advocates of MDMA-assisted therapy argue that

MDMA is a useful therapeutic tool. Unfortunately, sympathomimetic side

effects are occasionally mentioned (Barnes 1988; Grinspoon and Bakalar

1986; Shulgin and Nichols 1978), and concern over a potential to induce

arrhythmias in individuals with underlying cardiac disease has been

expressed (Dowling et al. 1987).

101
To better understand the behavioral effects of MDMA, this drug and various

analogs have been tested in several behavioral procedures in animals.

Significant abuse potential for MDMA was demonstrated by animal selfadministration of MDMA (Beardsley et al. 1986, Lamb and Griffiths 1987)

and a lowering of self-stimulation thresholds by MDMA (Hubner

et al. 1988). MDMA has also been reported to generalize to amphetamine

in drug discrimination studies, indicating that MDMA may have subjective

effects similar to those of amphetamine (Evans and Johanson 1986; Kamien

et al. 1986; Oberlender and Nichols 1988). A more complex mechanism of

action has been suggested by one report of generalization to the serotonin

agonist fenfluramine (Schechter 1986) and another report that described

drug-like responding following MDMA in rats trained on mescaline

(Callahan and Appel 1987). Indeed, several authors have concluded that

MDMA may produce discriminative stimulus effects that are different from

both stimulants and hallucinogens (Glennon et al. 1988; Oberlender and

Nichols 1988).

LOCOMOTOR ACTIVITY AND PSYCHOSTIMULANT EFFECTS

Locomotor activity has historically been used as an index of psychostimulant effects. Simple assessment of amount of locomotor activity can

provide the basis for anatomical as well as pharmacological analysis of the

neural substrates that mediate the behavioral expression of stimulant action.

More sophisticated behavioral measurement systems can record multiple

measures of activity and describe spatial and temporal patterning of locomotion. In such systems, qualitative aspects of behavioral activation can be

evaluated by examining the entire activity profile. A comparison of the

effects of novel drugs with those produced by well-characterized substances

may lead to a better understanding of their mechanisms of action and

subjective properties.

Neural Substrates of Psychostimulant Locomotion

The neural substrates of locomotor activation produced by psychomotor

stimulants have been linked for some time to dopamine function in the

nucleus accumbens. An early finding reported that direct injection of

dopamine into the nucleus accumbens produced enhanced locomotor activity

in rats (Pijnenburg and Van Rossum 1973), and the unconditioned motor

activation produced by amphetamine was shown to be blocked by dopamine

receptor antagonists (Pijnenburg et al. 1975). Destruction of dopamine

terminals within the nucleus accumbens with 6-hydroxydopamine (6-OHDA)

was found to attenuate the locomotion produced by indirect sympathomimetics (Joyce and Koob 1981; Kelly et al. 1975; Kelly and Iversen 1976)

but not to disrupt the locomotor-activating properties of caffeine, scopolamine (Joyce and Koob 1981) (figure 1). corticotropin-releasing factor (CRF)

(Swerdlow and Koob 1985). or heroin (Vaccarino et al. 1986) (figure 2) in

rats. Thus, the locomotor stimulation produced by psychostimulant drugs

102
has been hypothesized to result from release of dopamine from the

mesolimbic dopamine terminals in the region of the nucleus accumbens, but

other drugs with locomotor-activating properties may interact with other

parts of the limbic-nucleus accumbens-ventral pallidal circuitry known to be

important for psychostimulant activation (Swerdlow et al. 1984; Swerdlow

et al. 1986).

Neural Substrates of Psychostimulant Reinforcement

The locomotor-activating properties of psychomotor stimulants have been

hypothesized to be one aspect of their reinforcing properties (Mucha

FIGURE 1. Effects of amphetamine, scopolamine, caffeine, and saline on

locomotor activity in rats with 6-OHDA lesions of the

nucleus accumbens or sham-operated controls

(n=8 rats/group)

*Refers to a signficant group effect.

**Refers to a significant difference between the groups at 10 minutes postinjection, simple main effects.

KEY: Values in upper right corner of each panel represent mean ± SEM for the total activity

over the 2-hour drug test.

SOURCE: Joyce and Koob, 1981, Copyright 1981, Springer-Verlag.

et al. 1982; Spyraki et al. 1982; Swerdlow and Koob 1984). Animals will

learn to prefer an environment previously associated with drugs that produce

hyperactivity, and pharmacological or surgical manipulations that block the

locomotor-activating properties of psychomotor stimulants block this place

preference. The nucleus accumbens, which has been demonstrated to be

103
FIGURE 2. Effects of 6-OHDA lesions of the nucleus accumbens on the

locomotor response ajier SC injection of heroin (0.5 mg/kg)

or amphetamine (0.25 mg/kg)

*Significantly different from sham group. p<0.05.

NOTE: Rats were habituated to the photocell cages for 90 minutes. after which they were injected.

Inserts show the mean ± SEM total counts for 180 minutes for eight rats in the sham and

lesion group, respectively. 

SOURCE: Vaccarino et al. 1986, Copyright 1986, Pergamon Press.

104
involved in a variety of the behavioral actions of stimulants and opiates,

may act as a bridge between the limbic system and the extrapyramidal

motor system, integrating limbic influences and motor activity (Mogenson

and Nielson 1984; Swerdlow et al. 1986).

The reinforcing properties of psychomotor stimulants have also been linked

to the activation of central dopamine neurons and their postsynaptic receptors. When the synthesis of catecholamines is inhibited by administering

alpha-methyl-para-tyrosine, an attenuation of the subjective effects of

euphoria associated with psychomotor stimulants occurs in man (Jonsson

et al. 1971), and a blockade of the reinforcing effects of methamphetamine

occurs in animals (Pickens et al. 1968). Furthermore, low doses of dopamine antagonists will increase response rates for intravenous injections of

d-amphetamine (Risner and Jones 1976; Yokel and Wise 1975; Yokel and

Wise 1976).

Noradrenergic antagonists such as phenoxybenzamine, phentolamine, and

propranolol had no effect on stimulant (amphetamine) self-administration

(DeWit and Wise 1977; Risner and Jones 1976; Yokel and Wise 1976).

Wise and coworkers hypothesized that a partial blockade of dopamine

receptors produced a partial blockade of the reinforcing effects of

d-amphetamine. Thus, animals were thought to compensate for decreases in

the magnitude of the reinforcer by increasing their self-administration

behavior. Similar results have been observed with alpha-flupenthixol

(Ettenberg et al. 1982) and many other dopamine receptor antagonists,

including haloperidol, chlorpromazine, metoclopramide, thioridazine, and

sulpiride (Roberts and Vickers 1984). Recently, the selective D-1 antagonist

SCH 23390 was shown to increase cocaine self-administration at doses that

did not impair motor function (Koob et al. 1987a), whereas spiperone, a

D-2 selective compound, produced only small increases in responding at

doses close to those that produced motor dysfunction. These results suggest

that dopamine receptor blockade, particularly D-l receptor blockade, may be

involved in the reinforcing effects of psychomotor stimulants in rats. It

should be noted, however, that the SCH 23390 compound failed to produce

this action consistently when administered intravenously to rhesus monkeys

self-administering cocaine (Woolverton 1986).

The role of dopamine in the reinforcing properties of psychomotor stimulants was extended by the observations that 6-OHDA lesions of the nucleus

accumbens produce extinction-like responding and a significant and longlasting reduction in self-administration of cocaine and d-amphetamine over

days (Lyness et al. 1979; Roberts et al. 1977). These effects were thought

to be due largely to the depletion of dopamine, since rats pretreated with

desmethylimipramine before the nucleus accumbens lesion (to protect norepinephrine neurons from destruction with the 6-OHDA) showed an identical

extinction-like response (Roberts et al. 1980). Similar results were obtained

following 6-OHDA lesions of the ventral tegmental area (Roberts and Koob

105
1982). Subsequent studies have shown that 6-OHDA lesions of the frontal

cortex (Martin-Iverson et al. 1986) and corpus striatum (Koob et al. 1987b)

do not significantly alter cocaine self-administration. Interestingly, lesions

of specific subsets of the dopamine forebrain projections have been

associated with facilitated acquisition of amphetamine self-administration

(Deminiere et al. 1984; Deminiere et al. 1988), suggesting that some

specific neuropathology within the dopamine system could sensitize

individuals to the reinforcing actions of psychostimulants.

These results, showing facilitated acquisition of psychostimulant selfadministration with lesions of subsets of the dopamine projections, emphasize the need for other measures of reinforcement besides a continuous

reinforcement schedule. To this end, rats that had been trained to selfadminister cocaine intravenously were subjected to a progressive-ratio

procedure following 6-OHDA lesion to the nucleus accumbens or corpus

striatum. The rats with a lesion of the nucleus accumbens showed a significant decrease in the highest ratio for which they would respond to obtain

cocaine (figure 3) (Koob et al. 1987b). Complementary results have been

obtained using a similar progressive-ratio procedure in which rats with

6-OHDA nucleus accumbens lesions increased significantly the highest ratios

for which they would self-administer apomorphine (Roberts and Vickers

1988). This motivational probe thus avoids many of the problems associated with measuring local rates of responding. For example, the rats with

6-OHDA lesions showed a decrease in cocaine self-administration while on

a continuous reinforcement schedule that superficially could be interpreted

as either a decrease or increase in the reinforcing value of cocaine. The

results in the progressive-ratio test suggest that this decrease in local rates

of responding, previously observed with lesions to the region of the nucleus

accumbens, does in fact represent a motivational deficit.

Both amphetamine and cocaine have also been reported to support intracranial self-administration in the mesolimbic/mesocortical dopaminergic

system. Rats will self-administer cocaine into the medial prefrontal cortex

(Goeders and Smith 1983). while amphetamine is self-administered into the

orbitofrontal cortex of rhesus monkeys (Phillips and Rolls 1981) and the

nucleus accumbens of rats (Hoebel et al. 1983; Monaco et al. 1981). These

data indicate that the mesolimbic/mesocortical dopaminergic system is

involved in the initiation of stimulant reinforcement processes, and this work

suggests that the region of the nucleus accumbens, more specifically the

mesolimbic dopamine system, may be an important substrate for reinforcing

properties of several psychomotor stimulant drugs.

Behavioral Profile of MDMA

Both quantitative and qualitative aspects of the behavioral profile of motor

activation produced by MDMA and methylenedioxyethylamphctamine

106
FIGURE 3. Effects of 6-OHDA lesions to the nucleus accwnbens and

corpus striatum on responding for rats self-administering

cocaine

*Significantly different from sham group. p<0.05 Newman-Keuls test.

KEY:

NOTE:

H.=2 times the normal 0.75 mg/kg/injection dose; M=middle dose range,

0.75 mg/kg/injection; L=1/2 the 0.75 mg/kg/injection dose.

Top panel shows continuous reinforcement data averaged over the first 3 days postlesion

(means ± SEM). Sham, vehicle-injected (0.1 mg/mL ascorbic acid in saline) controls.

Caudate, rats receiving 8 μg in 2 μL of 6-OHDA injected into the corpus striatum. N.

Acc rats receiving 8 μg in 2 μL of 6-OHDA injected into the nucleus accumbens. Middle panel shows the dose-effect functions for each group. Bottom panel shows the mean

rewards and mean highest ratio obtained by each group on the progressive ratio probe

SOURCE: Koob et al. 1987b. Copyright 1987, Raven Press.

107
(MDE) have been characterized and, the effects of these drugs compared

with those of classic stimulants and hallucinogens (Gold et al. 1988).

Exploratory activity was monitored in eight separate behavioral pattern

monitor (BPM) chambers, each consisting of a 30.5 by 61 cm black Plexiglas holeboard with three floor holes, seven wall holes, and a steel

touchplate 15 cm above the floor that detected rearings against the wall

(figure 4) (Geyer et al. 1986). The frequency of photobeam breaks was

used as a general measure of motor activity, and the number and duration

of holepokes and rearings were cumulated.

FIGURE 4. Diagrammatic representation of the behavioral pattern monitor

chamber. The positions of the seven wall and three floor

holes are shown in each diagram

KEY: a. Infrared photobems are arranged in a Cartesian coordinate system on 7.6-cm centers

and are sampled five times per second

b. Sectors are equal 15-cm squares and are used to define crossovers, a measure of

horizontal locomotion.

c. Regions are unequal in size and are used primarily to define entries into the center

region and for the CV9 analysis of spatial patterns of locomotion.

SOURCE: Geyer et al. 1987. Copyright 1981. Pergamon Press.

MDMA significantly altered the behavioral activity profile of rats.

Figure 5A illustrates the timecourse of the effects of MDMA on crossovers

resolved into 30-minute blocks across the 2-hour test session. Doses of

1.25, 2.5, 5.0, and 10.0 mg/kg produced significant increases in crossovers,

which remained elevated at the end of the session at the two highest doses

studied. Interestingly, during the first 10 minutes in the chamber (10 to 20

minutes postinjection), doses of 2.5 to 10 mg/kg did not significantly

increase crossovers.

108
Concomitant with this total increase in horizontal locomotion, MDMA (1.25

to 10.0 mg/kg) caused alterations in measures of investigatory behavior.

MDMA had a profound effect on the distribution of investigatory holepokes

over time. Whereas the control animals exhibited a decrease in the number

of investigatory holepokes as they habituated to the chambers during the

session, the MDMA-treated rats demonstrated an initial decrease in the

number of holepokes, followed by the tendency to increase investigatory

holepoking over time (figure 5B). Similarly, MDMA (1.25 to 10.0 mg/kg)

dramatically reduced the amount of rearing behavior measured. The amount

and duration of this suppression of rearing was related to the dose of

MDMA studied (figure 5C). Rearing in rats heated with MDMA was

markedly reduced compared to control rats during the first 30 minutes.

More descriptive measures of the animals’ behavior were provided by

cumulating entries into and time spent in each of nine unequally sized

regions, which included the center and the four comer regions (Geyer

et al. 1986). Accompanying these changes in the amount of rearing and

investigatory holepoking was an observable avoidance of the center of the

experimental chamber. Thus, a significant decrease in average duration of

center entries for the first 30 minutes was obtained following MDMA doses

of 1.25 to 10.0 mg/kg.

MDE, the N-ethyl derivative of MDA, produced a behavioral profile similar

to that described for MDMA. MDE increased the number of crossovers

measured during a 1-hour experimental session (figure 5A). A transient

decrease in the number of crossovers during the first 10 minutes in the

chambers (0:549.1, 1.0:645.7, 3.0:313.1, 10.0:284.4) was noted for MDE at

doses of 3.0 and 10.0 mg/kg. As with MDMA, the two highest doses of

MDE tested (3.0 and 10.0 mg/kg) significantly decreased the total number

of holepokes for the first 30 minutes. Rearing was also suppressed by

these doses of MDE over a similar timecourse (figures 5B and 5C). At the

10 mg/kg dose of MDE, avoidance of the center was again observed as a

significant decrease in the average duration of center entries.

For spatial pattern analyses, the data were reduced to sequences of X,Y

positions as described elsewhere (Geyer et al. 1986). These sequences were

used to produce video displays of the animal’s position, rearings, and

holepokes, which could be viewed from 1 to 20 times teal-time speed. The

transition frequency between any of five areas (two ends, center, and two

long wall areas) was calculated, as was the coefficient of variation (CV) for

the relative transition frequencies (Geyer 1982). A related but slightly

different procedure evaluated the sequence of position changes by calculating the number of occurrences of each of the 40 transitions among any of 9

specified regions. As an animal preferentially repeats certain transitions, the

CV increases, while a more random pattern produces a lower CV. T’he CV

thus reflects the extent to which the animal establishes a preferred pattern

of locomotor activity over time.

109
FIGURE 5. Timecourse effects of MDMA (n=7 to 8 rats/group) and MDE

(n=9 to 12 rats/group) on A (crossovers), B (total

holepokes), and C (rearings) per 30 minutes in the BPM

p<0.05.

NOTE: Animals were injected 10 minutes before being placed in the chambers. Effects of selected

doses are shown as group means ±SEM.

110
Both MDMA and MDE caused some obvious qualitative changes in the locomotor patterns of rats. At moderate to high doses of MDMA, a definite

avoidance of the center of the experimental chamber was frequently seen,

and circling around the perimeter was the dominant behavior. This thigmotaxis is similar to that previously observed with apomorphine or scopolamine (Geyer et al. 1986). Although most rats had a predominant direction

of rotation, occasionally the rats reversed direction for one or more

revolutions. The impression of a disruption in locomotor patterns described

above was corroborated by a significant change in the spatial CV measure.

Both MDMA and MDE increased the spatial CV, which suggests a more

perseverative nature of locomotor patterns (table 1). In contrast, doses of

amphetamine (AMPH) that produced similar increases of horizontal

locomotion tended to induce highly varied patterns of directional changes,

which were reflected in a reduced spatial CV (Geyer et al. 1986).

Neural Substrates for the Psychostimulant Actions of MDMA

The neurochemical mechanisms for the stimulant properties of MDMA were

examined in a photocell cage apparatus following pharmacological and

neurochemical manipulations. Locomotor activity was measured in a bank

of 16 wire cages 20 cm by 25 cm by 36 cm, each cage with two horizontal

infrared beams across the long axis 2 cm above the floor. Total photocell

beam interruptions and crossovers were recorded by a computer every 10

minutes. Before the drug series, each rat was habituated to the photocell

cages overnight, and, prior to drug injection, the rats were habituated again

to the photocell cages for at least 90 minutes.

A role for serotonin in the stimulant actions of MDMA was tested by

examining the effects in rats of the serotonin antagonist methysergide on

MDMA activation (Gold and Koob 1988). The locomotor-activating properties of MDMA. amphetamine, and methysergide are seen in figure 6. Drug

doses for amphetamine and MDMA were selected to produce similar increases in activity, although MDMA appears to have a longer duration of

action (Gold et al. 1988). Once the rats were habituated to the photocell

apparatus, saline injection produced only transient arousal (lasting less than

20 minutes) followed by relative inactivity (figure 6C). MDMA at

10 mg/kg produced an increase in beam interruptions that lasted for at least

2 hours (figure 6A). Methysergide (2.5, 5, 10 mg/kg) significantly

potentiated the locomotor hyperactivity produced by MDMA (10 mg/kg)

when compared to MDMA injection alone (figure 6A). This enhancement

of MDMA’s locomotor effects was evident within the first 10 minutes and

lasted for the full 2-hour session. In contrast, methysergide only slightly

and nonsignificantly increased the locomotor hyperactivity produced by

0.5 mg/kg of amphetamine (figure 6B). Methysergide alone at these doses

had no effect on locomotor activity (figure 6C).

111
TABLE 1. Effects of MDMA, MDE, and AMPH on spatial CV

Dose (mg/kg) CV5 CV9

0 .485 ± .02

MDMA 1.25 .709 ± .l

MDMA 2.5 .730 ± .17

MDMA 5.0 1.063 ± .21*

MDMA 10.0 .995 ± .13*

0

MDE 1.0

MDE 3.0

MDE 10.0

0 .522 ± .04

AMPH 0.25 .379 ± .06*

AMPH 0.5 .376 ± .03*

AMPH 1.0 .373 ± .04*

AMPH 2.0 .506 ± .03

1.723 ± .02

1.737 ± .05

2.209 ± .18*

2.007 ± .ll

*p<0.05, Dunnett's t-test

NOTE: Group means ± SEM are shown: an increase in spatial CV indicates a more repetitive pattern

of movements in the BPM: a decrease indicates a more highly varied pattern.

The role of the mesolimbic dopamine system, which is known to be critical

for amphetamine-stimulated locomotion, was investigated in MDMA-treated

rats with 6-OHDA lesions of the nucleus accumbens (Gold et al., in press).

Rats received bilateral injections of 6-OHDA (8 μg/2 μl, expressed as the

free base) dissolved in saline containing ascorbic acid (0.1 mg/mL; lesion

group) or injections of saline-ascorbic acid vehicle alone (sham group).

Approximately 9 days following surgery, rats were injected with saline, and

locomotor activity was measured for 120 minutes. In one study, rats (sham:

n=8, lesion: n=8) were injected on the following day with 5 mg/kg

MDMA, 3 days later with 0.5 mg/kg AMPH, and again with saline. Locomotor hyperactivity produced by MDMA was attenuated in the group with

6-OHDA lesions (figure 7A). When the rats were injected with 0.5 mg/kg

AMPH, the sham-operated group showed a large increase in locomotor activity; this effect was significantly reduced in the rats with lesions.

Moreover, the hyperactivity seen in the sham rats was somewhat greater

than that usually observed following this dose of AMPH, suggesting a

cross-sensitization between MDMA and AMPH. The day after the AMPH

injections, all the rats were reinjected with saline. At this time there was

no significant difference between the sham- and lesion-operated rats.

112
FIGURE 6. Locomotor activity during 120-minute test session in the

photocell cage apparatus

*Significantly different from 0 methysergide dose, Newman-Keuls test following significant ANOVA

main effect

KEY: Values in the upper right corner of each panel represent mean ±SEM for the total activity

over the 2-hour drug test.

NOTE: Following a habituation period, rats were injected with methysergide (0-10 mg/kg, SC; C)

and 2 minutes latex by (A) MDMA (10 mg/kg. SC). (B) amphetamine (0.5 mg/kg. SC).

SOURCE: Gold and Koob 1988, Copyright 1986, Pergamon Press.

113
FIGURE 7. Effects of 5 mg/kg MDMA, 0.5 mg/kg AMPH, or 5 mg/kg

MDMA plus 25 mg/kg methysergide on locomotor activity

in rats with 6-OHDA or sham lesions of the nucleus

accumbens

*p<0.05.

**Significant interaction.

†p.055.

KEY: Total photobeam interruptions, measured in photocell cage apparatus, for 120-minute test

session, shown as group means ± SEM.

NOTE: A. The significant difference between sham-operated rats and those with 6-OHDA lesions

following saline injection was attributed to a reduced response to the injection procedure in

the lesion-operated group. Note that means for the two groups were almost identical

(sham=576±84, lesion=524±55) for the 90-minute habituation period preceding saline

injection. Sham group, n=8; lesion group, n=8. B. Sham group. n=8; lesion group, n=6.

SOURCE: Redrawn from Gold et al., in press.

114
In an additional study, following recovery and saline injection, rats (sham:

n=8, lesion: n=8) were injected with 0.5 mg/kg AMPH on day 9 or 10 and

5 mg/kg MDMA 3 days later. On day 16 or 17 these rats received two

injections: a serotonin antagonist, 2.5 mg/kg methysergide; and 5 mg/kg

MDMA. Rats were injected with 0.5 mg/kg AMPH on the next day and,

as in previous experiments, the locomotor hyperactivity produced by AMPH

was attenuated in the group with 6-OHDA lesions (figure 7B). The

mean ± SEMs per 120 minutes for the sham and lesion groups were

1,995.9±389.3 and 906±132, respectively. In the first experiment described

above, the means for these groups were 3.111.9±421.8 and 1,176.5±248.1,

respectively. When the rats were injected with 5 mg/kg MDMA 3 days

later, the sham-operated group showed a large increase in locomotor activity; this effect was significantly reduced in the rats with lesions. The

mean ± SEMs for the sham and lesion groups were 1,368±249.5 and

754.1±107.4, respectively. These values were not different from those

described in the first experiment (sham: 1,401.3±257.8, lesion:

745.2±81.7). Therefore, a cross-sensitization from AMPH to MDMA was

not evident. On the next day, locomotor activity was measured following

injections of a serotonin antagonist and MDMA. Here, methysergide

potentiated the effects of MDMA. Both the effects of surgery and the

influence of methysergide were observed. However, a log transformation of

the data eliminated the significant interaction between the two, which

suggests that the interaction effect was due to scaling differences. Thus, the

response of both the sham rats and the rats with lesions was increased by

the serotonin antagonist.

Biochemical analyses of 6-OHDA-injected animals revealed a 93 percent

depletion of dopamine. The tissue was assayed using electrochemical

detection following separation by high-pressure liquid chromatography

(Felice et al. 1978). recorded as ng/mg protein in the nucleus accumbens

and compared to control rats with sham lesions (sham=65.5±4.4,

lesion=4.9±1.5; t(39)=23.4). A lesion was defined as complete if 75 percent

or more of the dopamine was determined to be depleted from the nucleus

accumbens compared to mean sham group values.

SUMMARY

The motor activation produced by psychomotor stimulants has been long

associated with the midbrain dopamine systems. While focused stereotyped

behavior produced by high doses of indirect sympathomimetics is blocked

by removal of dopamine terminals in the corpus striatum (Creese and

Iversen 1975), the locomotor activation produced by low doses of indirect

sympathomimetics is blocked by removal of dopamine terminals in the

region of the nucleus accumbens (Kelly et al. 1975). This dopaminergic

substrate for psychostimulant effects appears selective for the indirect

sympathomimetics in that dopamine lesions to the region of the nucleus

115
accumbens do not block caffeine, scopolamine, heroin, or CSF-induced

locomotor activation (Swerdlow and Koob 1985; Vaccarino et al. 1986).

The neurochemical sites for psychomotor stimulant reward are likely to be

the presynaptic dopamine terminals located in the region of the nucleus

accumbens, frontal cortex, and other forebrain structures that originate in the

ventral tegmental area. Note, however, that intracranial self-administration

of cocaine is elicited from the frontal cortex, but not from the nucleus

accumbens (Goeders and Smith 1983). Thus, concomitant activation of

structures other than the nucleus accumbens may be an important part of

the circuitry involved in initiation of cocaine intravenous self-administration,

as has been hypothesized for the opiates (Smith and Lane 1983; Smith

et al. 1982).

In addition, these neuropharmacological studies provide evidence to show

that, in the rat, the neural/neurochemical substrates for processing the

reinforcing and stimulant properties of psychomotor stimulants may be

similar, if not identical. Parallel manipulations using dopamine receptor

antagonists and 6-OHDA lesions produce parallel results. How far this

parallelism continues in further processing is under current investigation;

however, such an overlap brings additional impetus to earlier hypotheses

relating reinforcement and motor function (Glickman and Schiff 1967).

The motor activation produced by MDMA and MDE has similarities to

classic psychostimulants, but also some important differences. In the BPM

system, the stimulant-like properties of these drugs were reflected in

significant increases in horizontal locomotor activity measured across a wide

dose range. Interestingly, medium to high doses of MDMA or MDE

produced a transient decrease in horizontal locomotion for the first 10

minutes, followed by a sustained increase. The increase in holepokes and

rearings that typically accompanies the increase in ambulation seen with

amphetamine itself or other indirect sympathomimetics (Geyer et al. 1986)

was not observed with MDMA or MDE. Instead, initial decreases in holepokes and rearings and a tendency to avoid the center were evident, a

behavioral profile that is characteristic of hallucinogenic indoleamine or

phenylethylamine derivatives (Adams and Geyer 1985a; Adams and Geyer

1985b; Geyer et al. 1979).

MDMA and MDE also produced locomotor patterns that differed significantly from other stimulants. Previous studies in rats have demonstrated

that amphetamine-induced hyperactivity involves complex patterns of widely

distributed locomotion with frequent directional changes (Geyer et al. 1986;

Geyer et al. 1987). In contrast, similar levels of behavioral activation

produced by scopolamine or apomorphine are associated with relatively

smooth locomotor paths, in which the same movement patterns are frequently repeated. Other stimulants, such as caffeine or nicotine, increase the

amount of locomotor activity without significantly altering its pattern (Geyer

116
et al. 1986). With LSD and other hallucinogens, the behavioral profile is

characterized by an increase in the diversity of locomotor patterns and a

concomitant suppression of the exploration of novel and open areas (Adams

and Geyer 1985a). When evaluated on this basis, MDMA and MDE are

similar to hallucinogens in producing an avoidance of the center. This

effect is particularly notable in light of the simultaneous increases in the

total amount of locomotor activity. Unlike LSD, however, MDMA produced a scopolamine- or apomorphine-like increase in perseverative and

thigmotactic patterns of locomotion, reflected by increases in the spatial CV

measures. The MDMA profile was also similar to that of apomorphine

insofar as both drugs reduced holepoking and rearing, behaviors that are

increased by scopolamine. However, relative to apomorphine, the MDMAinduced rotational patterns were less strictly unidirectional, and the

reductions in investigatory responses were less complete. Rather, most

animals injected with MDMA changed directions and exhibited investigatory

responses at least occasionally, effects similar to those observed following

various doses of scopolamine (Geyer et al. 1986). Hence, the behavioral

profile engendered by MDMA and MDE appears to be unique among the

various drugs that have been so characterized to date.

Investigation of the neurochemical substrates for the psychostimulant effects

of MDMA suggests a role for the mesolimbic dopamine system. Destruction of dopamine terminal fields in the nucleus accumbens significantly

attenuated the locomotor activation produced by MDMA. A similar blockade of amphetamine-induced locomotor hyperactivity is known and was

observed following amphetamine injection in these same rats. Such results

support the hypothesis that at least one component of MDMA-induced

hyperactivity is dopamine mediated and suggest that mesolimbic dopamine

specifically is the critical substrate. In this way, MDMA resembles other

classical psychostimulants like amphetamine and cocaine. Interestingly,

evidence for functional cross-sensitization was suggested in the study in

which an injection of amphetamine followed MDMA injection.

The stimulant properties of MDMA were enhanced by the presence of a

serotonin antagonist, methysergide. Thus, following serotonin-receptor

blockade, profound locomotor hyperactivity was observed. This result can

be viewed as a disinhibition of the dopamine neurons from serotonin modulation. These data are consistent with the hypothesis that MDMA acts

predominantly as a serotonin agonist with weak dopamine activity. In this

study, methysergide did not potentiate the effect of amphetamine. However,

Hollister et al. (1976) reported that methysergide potentiated locomotion

produced by 2 mg/kg amphetamine intraperitonealIy. In fact, an

enhancement of an amphetamine response after prior exposure to MDMA

has also been observed. It is possible that previous exposure to MDMA

may have resulted in neurotoxic damage to some serotonin neurons.

Depletions of serotonin and its metabolites have been repotted following

single injections of MDMA (Mokler et al. 1987; Schmidt 1987; Stone

117
et al. 1986). A decrease in serotonergic tone would also result in a

disinhibition of dopamine neurons and may explain the enhanced

amphetamine response. Indeed, evidence for such a “functional lesion” has

been reported in an operant procedure in which MDMA-induced serotonin

depletion was found to potentiate its psychomotor stimulant effects (Li

et al. 1986). In the case where amphetamine was given first, followed by

MDMA, no change in responsiveness would have been expected.

The stimulation of locomotor activity by MDMA and the importance of

mesolimbic dopamine in this response reflect similarities with the prototype

phenylethylamine stimulant, amphetamine. It is important to note that these

parameters are frequently associated with rewarding aspects of drugs and

drug abuse. Additionally, the behavioral profiles of MDMA and MDE

share certain characteristics with hallucinogen-like agents. This unique

mixture of stimulus properties and neurochemical actions may contribute to

a dangerous behavioral toxicity and neurotoxic potential for drugs like

MDMA.

DISCUSSION

QUESTION: Can you get animals to self-administer cocaine into the

nucleus accumbens?

ANSWER: No, I never tried that, but the literature there is complicated, as

you know. Animals will, however, self-administer cocaine into the frontal

cortex. Amphetamine is self-administered into the nucleus accumbens.

You have to know that we take out most of the mesocorticolimbic dopamine system with that lesion; we are not just taking out the nucleus

accumbens dopamine projection. I am very careful to put the region of the

nucleus accumbens on my slide.

I think the way Jim Smith and I have discussed this paradox is a follows:

He thinks that the frontal cortex has something to do with initiation of

cocaine self-administration, and I think probably the whole system may be

involved in maintaining the behavior once the animals have learned it.

QUESTION: Have you tried MDMA into the nucleus accumbens?

ANSWER: No, we haven’t tried self-administration of MDMA. I am not

sure we would get rats to switch from cocaine to MDMA.

QUESTION: Have you had the opportunity to look at the impact of

methysergide pretreatment on MDMA’s effects on exploration and rearing?

ANSWER: No, we just put that on the books. We would really like to

look in the behavioral pattern monitoring system. I predict that the lesion

118
is going to block the crossovers. But I don’t know what methysergide

would block. I believe it would be incredibly interesting if it would turn

those rats into amphetamine-like rats, and they would explore and show less

thigmotaxis and more nosepokes.

QUESTION: How can you dissociate the locomotor effects from the

reinforcing effects? It has been agreed that lesions of the mesolimbic

system affect locomotor activity and shown by Eberson with respect to the

dopaminergic system. How do you know you don’t have a rat that is

motorically compromised and can’t press the lever to get the cocaine? How

can you dissociate that from the reinforcement efficacy?

ANSWER: I think the only thing I can really argue strongly is that we

have made similar lesions in rats the lever pressed for heroin, so they are

lever pressing in the exact paradigm as a reinforcer, and continue to take

heroin, although the cocaine self-administration extinguishes. I have a slide

of a rat who keeps plugging along on heroin self-administration and at the

same time every other day is tested on cocaine. The paradigm was heroin

on Monday, cocaine on Tuesday, heroin on Wednesday, cocaine on

Thursday, His cocaine self-administration was completely extinguished, yet

his heroin self-administration continued. This is one piece of evidence that

looks like a real dissociation. The animals can lever press for another

reinforcer, but they choose not to lever press for cocaine.

And the other part would be that they show locomotion with other drugs.

It is just the indirect sympathomimetics where locomotion is blocked.

It could be said that the reason they are not pressing the lever for cocaine

is that it doesn’t do anything for them. And then you get into a circuit

where it is the psychostimulant effects that produce the reinforcing effects.

COMMENT: Your data showed that, at least with that one model rat, there

was a good extinction pattern and high levels of activity. I would consider

that to be a better piece of evidence.

RESPONSE: Yes, it means that the animal is capable of moving around

for apomorphine. But then it gets subtle. In people, too, there is initially a

high level of activity to exhaust residual dopamine stores and then the

activity goes down to a very low level. The apomorphine can reinstate

some locomotion. I think the most convincing evidence is the heroin. It is

not true motor activity.

QUESTION: Do you have any explanation for sensitization within the

MDMA or the methysergide? There has been evidence of serotonergic

inhibition.

119
ANSWER: I think there are two ways to look at it I would say one

reason is MDMA doesn’t look like amphetamine. Why doesn’t MDMA

look like amphetamine without any other drugs added on? The animal has

the psychedelic repertoire, whatever that is, interfering. This is what Klaus

Miczek and I were discussing. The animal perhaps has another behavioral

repertoire interfering with the expression of motor activity, and that happens

to be that the animal is hanging close to the side of the cage and for whatever reason, if it is LSD-like, he doesn’t want to go into the center because

it is frightening. He is hallucinating. I am speculating here. And if you

then take away that competing behavior or competing brain Gestalt of

psychedelic activity, then you are turning the drug into basically amphetamine. That is one way of looking at it.

Another way of viewing it would be as levers going off. Serotonin is

inhibitory, dopamine is excitatory. That is naive, but there is evidence to

suggest that in the neurochemistry of those compounds there has been a

kind of yin and yang.

QUESTION: How do you explain MDMA sensitization for amphetamine?

ANSWER: There is evidence that even one exposure of MDMA at

10 mg/kg can cause some serotonin neurotoxicity. Dr. Seiden has shown in

DRL procedures with repeated exposure that there is more of an

amphetamine-like effect after some of the serotonin has been depleted.

QUESTION: Had you looked at that at all?

ANSWER: In terms of the biochemistry itself, no, not at all.

COMMENT: I am not sure the serotonin is inhibitory and dopamine

excitatory is all that naive. There were clinical studies published where

they showed that serotonin agonists could completely suppress the CNS

stimulant effects of amphetamine clinically in humans. So you may be

seeing the same thing. I am not sure that it has to be a psychedelic

activity superimposed. It may simply be some kind of a synergistic

attenuation.

COMMENT: Both Campbell and Harvey, in independent experiments, have

shown if you take away the serotonin input you can exacerbate the

psychomotor stimulant effects of amphetamines.

RESPONSE: Yes, and there is some recent study too that was done by

Lyness showing that if the serotonin system is destroyed, toxicity and

self-administration of amphetamines are increased. There is a lot of

evidence that some of this interaction does occur at some level, but we

don’t know where yet.

120
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ACKNOWLEDGMENTS

This work was supported in part by National Institute of Mental Health

Research Scientist Development Award MH00188, National Institute on

Drug Abuse Awards DA 02925 and DA 04398, and a Parkinson’s Disease

Summer Fellowship.

AUTHORS

Lisa H. Gold, B.S.

Predoctoral Fellow

Division of Preclinical Neuroscience and Endocrinology

Department of Basic and Clinical Research

Research Institute of Scripps Clinic

La Jolla, CA 92037

Mark A. Geyer, Ph.D.

Associate Professor of Psychiatry

Department of Psychiatry, T-004

University of California, San Diego

La Jolla, CA 92093

George F. Koob, Ph.D.

Associate Member

Division of Preclinical Neuroscience and Endocrinology

Department of Basic and Clinical Research

Research Institute of Scripps Clinic

La Jolla, CA 92037

126
Neuronal Actions of Amphetamine

in the Rat Brain

Philip M. Groves, Lawrence J. Ryan, Marco Diana,

Stephen J. Young, and Lisa J. Fisher

INTRODUCTION

Amphetamine and related designer drugs have widespread actions on neuronal activity in the brain. This is believed to be due in part to the

enhanced release and blockade of reuptake of catecholamines (Kuczenski

1983). The sites of action of such stimulant drugs of abuse include presynaptic neurons, by virtue of the action of released catecholamines on

autoreceptors (Tepper et al. 1985), and postsynaptic targets of catecholamine

axons, including neurons in the cerebral cortex, basal ganglia, cerebellum,

reticular formation, and other neuron systems of the brainstem (Groves and

Rebec 1976).

The consequences of amphetamine administration include widespread neuronal pathology in the brains of experimental animals (Groves et al. 1987;

Seiden and Kleven, this volume; Gibb et al., this volume) and significant

changes in the pattern and intensity of neuronal activity throughout the

brain. One particularly useful approach to understanding the sites and

mechanisms of action underlying the behavioral effects of amphetamine has

been to record the electrophysiological consequences of amphetamines

administration in the rat brain (Groves and Rebec 1976; Groves and Tepper

1983).

EFFECTS OF AMPHETAMINE ON CATECHOLAMINERGIC

NEURONS

One of the most widely known electrophysiological actions of amphetamine

on the brain is to decrease the firing rate of dopaminergic and noradrenergic

neurons recorded in vivo from the rat brain (Bunney et al. 1973; Graham

and Aghajanian 1971). The underlying mechanisms include possible inhibition by afferent systems and by local inhibitory mechanisms involving local

release of catecholamine (Groves et al. 1975). In the case of dopaminergic

neurons, this release occurs from dendrites, whereas, in noradrenergic nuclei,

release occurs from axonal collateral innervation as well as from dendrites

(Groves et al. 1979). The effect of amphetamine on monoamine neurons is

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dose dependent. Noradrenergic neurons of the locus coeruleus are most

sensitive, with a mean dose for 50 percent inhibition of firing of 0.25

mg/kg, IV (Engberg and Svenson 1979; Ryan et al. 1985). Dopamine neurons are less sensitive, requiring a mean dose of approximately 1.6 mg/kg,

IV (Bunney et al. 1973). Serotonin neurons are the least sensitive,

requiring a mean dose for 50 percent inhibition of 3.0 mg/kg, IV (Rebec

et al. 1982).

The inhibition of firing of catecholamine neurons resulting from amphetamine administration is likely due to activation of somatodendritic

autoreceptors. This causes a hyperpolarization of the somatodendritic

membrane of both locus coeruleus noradrenergic and substantia nigra

dopamine neurons, probably as a consequence of an increase in potassium

conductance (Lacey et al. 1987; Williams et al. 1985).

Because the cell body is hyperpolarized by autoreceptor stimulation, it

seemed plausible that stimulation of autoreceptors located on the synaptic

endings of such neurons might similarly lead to a decrease in excitability.

Over the past several years, we have determined the excitability of the

axonal synaptic endings of single monoaminergic neurons in vivo by electrical stimulation of the axon while recording the antidromic responding of

the neuron at the level of the cell body. It is now apparent that, in

addition to causing a decrease in the amount of transmitter released by each

action potential (Langer 1977), stimulation of autoreceptors located at the

terminal field results in a decrease in terminal excitability in all

monoaminergic neuron systems that have been tested (Tepper et al. 1985).

The effects of amphetamine on catecholamine terminal excitability are very

similar to the effects of direct-acting agonists. Nigrostriatal dopamine

neurons, for example, show a decrease in terminal excitability following the

systemic administration of doses of amphetamine ranging from 0.25 to

5.0 mg/kg, IV (Groves et al. 1981). Direct infusions of amphetamine into

the terminal fields also decrease excitability. A similar decrease in

excitability is seen after the administration of the direct-acting D2 agonist

apomorphine (Tepper et al. 1984) and the D1 agonist SKF 38393 (unpublished data). The action of amphetamine may be blocked by dopamine

antagonists, including haloperidol, fluphenazine, and sulpiride, as well as by

pretreatment with the dopamine synthesis blocker metbylparatyrosine

(Tepper et al. 1984). These actions of amphetamine occur only in regions

of the dopamine axon containing presynaptic autoreceptors; infusions of

amphetamine into the medial forebrain bundle were without effect, whether

excitability was tested by stimulation at the terminal field or along the axon

in the medial forebrain bundle.

The activation of presynaptic autoreceptors, as revealed by changes in terminal excitability, suggests that amphetamine releases dopamine at every tested

dose. This observation is consistent with recent direct demonstrations using

128
dialysis, which show that amphetamine induces dopamine release in the neostriatum in a dose-dependent manner (Hemandez et al. 1987; Impcrato and

Di Chiara 1984). Amphetamine-induced release is opposed, to some extent,

by the action on autoreceptors of released dopamine. Activation of somatodendritic autoreceptors decreases dopamine neuron firing, which, in

combination with dopamine terminal autoreceptor activation, will decrease

impulse-dependent release. Thus, the net effect of amphetamine on catecholamine release will be a compromise between release-inducing actions

and release-diminishing actions.

Indeed, at some doses, amphetamine may actually reduce norepinephrine

release from terminals of locus coeruleus neurons, Amphetamine is a less

potent releaser of norepinephrine than it is of dopamine, and norepinephrine

release is provoked only at high doses (Kuczenski 1983). Since locus

coeruleus neurons are very sensitive to inhibition of neuronal firing, the net

effect of amphetamine at low doses may be to reduce impulse-dependent

release enough that amphetamine overwhelms the amphetamine-induced

nonimpulse-dependent release. Huang and Maas (1981). for instance,

observed a biphasic dose effect of amphetamine on hippocampal neuron

firing, which they interpreted as indicating that amphetamine reduced

norepinephrine release at low doses. A biphasic dose effect on

amphetamine has been observed on the terminal excitability of locus

coeruleus axons in frontal cortex, which we also interpreted in this manner

(Ryan et al. 1985). Thus, amphetamine may have quite different dosedependent effects on noradrenergic and dopaminergic neurons.

EFFECTS OF AMPHETAMINE ON NEOSTRIATAL NEURONS IN

BEHAVING RATS

Amphetamine can alter neostriatal unit activity directly by enhancing the

release of dopamine from terminals of the midbrain dopamine projections of

the substantia nigra and, at higher doses, by increasing serotonin release in

the neostriatum. It may also indirectly alter neostriatal activity by changing

activity in systems that project into the neostriatum, including the neocortex.

thalamus, and amygdala. The net effect of amphetamine on neostriatal activity will be determined by the relative magnitudes of these various

influences.

In anesthetized rats, amphetamine causes dose-dependent changes in

neostriatal unit activity. Spontaneously active neostriatal cells are uniformly

inhibited at low (<2.0 mg/kg, IP) doses. At intermediate doses, an initial

excitation precedes the inhibition, and, at high doses (>5.0 mg/kg, IP), the

predominant effect is excitation (Groves and Rebec 1976). Regional

differences in the direction, magnitude, and duration of the response of

neurons in the neostriatum exist (Rebec and Curtis 1983).

129
In contrast. initial studies of neostriatal unit activity in unanesthetized,

behaving animals suggested that neostriatal units were excited by even small

doses of amphetamine (Hansen and McKenzie 1979; Rebec and Gardiner

1985; Trulson and Jacobs 1979; Warenycia and McKenzie 1984a;

Warenycia and McKenzie 19848; Warenycia and McKenzie 1984c;

Warenycia et al. 1984; West et al. 1985). and that the firing rate did not

change with the transition between amphetamine-induced locomotion and

stereotypies (Hansen and McKenzie 1979). These studies, though. did not

report the behavioral correlates of the unit activity recorded in the predrug

period. Since the firing of neostriatal neurons can vary widely with

different behaviors, the observed changes in neuronal firing following

amphetamine could reflect solely the change in behavior produced by

amphetamine. Furthermore, in these studies, primarily spontaneously active

neurons were recorded from, either as single- or multiple-unit responses.

Since the majority of neostriatal neurons are very slowly firing, rapidly

firing neurons were undoubtedly overrepresented in these studies.

In our studies, we examined how amphetamine altered the firing of

identified neostriatal projection neurons during specific pre- and postdrug

behaviors. Neurons were identified as projection neurons by antidromic

activation from the substantia nigra, using criteria that we previously

established (Ryan et al. 1986b). Of 41 antidromically identified neostriatal

cells, only 1 fired faster than 1 Hz during any of the four behaviors that we

analyzed, namely locomotion: face washing; quiet, nonmoving waking; and

sleep. The median firing rate during locomotion was 0.02 Hz (Ryan et al.,

in press). An additional group of 24 nonantidromically activated neurons

was also studied. Most of these neurons also fired infrequently; the median

rate during locomotion was also 0.02 Hz. Indeed, with the exception of

two cells that fired over 6 Hz, the nonantidromically activated cells

resembled the antidromic cells in all respects. Many antidromic and

nonantidromic neurons showed tenfold or greater changes in rate across the

four different behaviors.

The effect of amphetamine on these neostriatal neurons was relatively

uniform. Four doses of amphetamine were tested: 0.25, 1.0, 2.5, and 5.0

mg/kg, SC. At all four doses, amphetamine reduced the firing rate of both

antidromic and nonantidromic neurons during the initial drug-induced period

of locomotion as compared to the rate during predrug locomotion (figure 1).

At the higher doses, several stages of stereotyped behaviors were seen. The

transition from amphetamine-induced locomotion to locomotion associated

with stereotyped side-to-side head movements was accompanied by a further

reduction in firing rate. In those animals in which focused stereotypy was

observed following this period of locomotion plus head movements, neurons

showed a still further reduction in firing rate (figure 2).

130
FIGURE 1. Change in firing rate of both antidromically and

nonantidromically activated neostriatal neurons following

amphetamine administration in the freely moving, behaving

rat

NOTE: Firing rate during predrug locomotion compared to firing rate during initial drug-induced

locomotion shows the majority of neurons inhibited by amphetamine. Some cells fired no

action potentials either pre- or postdrug.

These effects were seen for both antidromic and nonantidromic neurons.

However, of the three most rapidly firing neurons, two showed an acceleration in firing rather than a reduction, much as has been previously reported

for spontaneously active neurons (Hansen and McKenzie 1979; Rebec and

Gardiner 1985; Trulson and Jacobs 1979; Warenycia and McKenzie 1984a;

Warenycia and McKenzie 1984b Warenycia and McKenzie 1984c;

Warenycia et al. 1984; West et al. 1985). Thus, amphetamine may induce

a divergence in firing rate, exciting rapidly firing neurons and inhibiting

slowly firing neurons.

Both the degree and pattern of neostriatal activity are altered by amphetamine. Since identified striatonigral projection neurons are inhibited by

amphetamine, the inhibitory control of the substantia nigra pars reticulata

may be decreased. The rapid tonic firing of these neurons may be enhanced, ultimately resulting in increased inhibition of the targets of the pars

reticulata, namely the ventromedial thalamus, the superior colliculus, and the

pendunculopontine (PPN) nucleus. Thus, amphetamine may cause the inhibition of these structures, thereby locking in a particular behavioral pattern.

131
FIGURE 2. Change in firing rate of activated neostriatal neurons, data

combined across all doses

NOTE: Amphetamine-induced inhibition of neostriatal unit firing during postdrug compared to predrug

locomotion may be clearly observed. As locomotion gives way to locomotion with

stereotyped side-to-side head movements. there is a further decline in firing rate. When

locomotion with head movements was followed by focused stereotypies. there was a still

further decrease in firing rate.

EFFECTS OF AMPHETAMINE ON TERMINAL EXCITABILITY OF

STRIATONIGRAL PROJECTION NEURONS IN BEHAVING RATS

Amphetamine may exert its effects not only by altering the firing rate of

neurons, but also by altering the coupling between action potentials and

neurotransmitter release, by acting on presynaptic terminal receptors.

Dopamine receptor activation has been shown to increase GABA release in

the substantia nigra (Reubi et al. 1977; Starr 1987). It is possible that this

modulation of GABA release occurs by dopamine acting on D1 receptors

known to reside on the presynaptic terminals of striatonigral projection

neurons (Altar and Hauser 1987; Aiso et al. 1987). If so, amphetamine

may activate these receptors by inducing the local release of dopamine

within the substantia nigra (Groves et al. 1975), and this change may be

detected by measuring the electrical excitability of the axon terminal

(Groves et al. 1981). We have recently shown that, in urethane-anesthetized

rats, local infusions of the specific D1 agonist SKF 38393 (10 μM) into the

substantia nigra decrease the electrical excitability of suiatonigral neuron

terminals (unpublished data). In contrast, amphetamine, at doses ranging

from 0.5 to 5.0 mg/kg, SC, did not alter terminal excitability in either

unanesthetized, freely moving rats (Ryan et al., in press) or in

132
urethane-anesthetized rats (unpublished data). Thus, it seems unlikely that

amphetamine alters GABA release in the substantia nigra by acting at

presynaptic D1 receptors on striatonigral terminals.

EFFECTS OF AMPHETAMINE ON SUBSTANTIA NIGRA PARS

RETICULATA NEURONS IN BEHAVING RATS

The substantia nigra pars reticulata represents one of the major targets of

the neostriatum. This projection has been demonstrated anatomically

(Grofova 1979) and has been shown electrophysiologically to inhibit the

firing of its target neurons (Deniau et al. 1976). Thus, since amphetamine

inhibits the firing of neostriatal neurons, it is plausible that peripheral

administration of amphetamine could alter the tonic activity of substantia

nigra pars reticulata cells across behavioral states. It is, therefore, of some

interest to study the effects of amphetamine on the activity of pars reticulata

neurons.

In anesthetized animals, the iontophoretic application of dopamine increases

the firing of pars reticulata neurons (Matthews and German 1986; Ruffieux

and Schultz 1980; Waszczak and Walters 1983), and dopamine attenuates

the inhibitory effects of GABA, which is the transmitter used by some of

the striatonigral projection. Little is known, though, about how amphetamine changes activity in this structure in freely moving animals. One

recent study by Olds (1988) suggested that the activity of nondopamine

neurons of the substantia nigra increases after amphetamine administration.

However, the behavioral mix of the 90-minute predrug period was uncontrolled; since, as we have observed, the firing rate of these neurons varies

widely with behavior, it is unclear whether this action reflects the effects of

amphetamine on these neurons or the behavioral activation induced by

amphetamine. We have recently begun a series of experiments to elucidate

the relationship between firing rate of pars reticulata neurons and specific

behaviors and to demonstrate how amphetamine alters these correlations.

Preliminary data suggest that the firing rate of pars reticulata neurons during

several behaviors is increased by amphetamine, compared with the same

preamphetamine behavior. The activity of a single pars reticulata neuron

during pre- and postamphetamine locomotion and face washing is shown in

figure 3.

In this cell, the tonic firing of the neuron during this behavior is increased

by amphetamine. This result is consistent with our finding that neostriatal

units projecting to the pars reticulata are inhibited by amphetarninc. The

resulting disinhibition of these nigral units may, in turn, increase their tonic

inhibitory control over their target structures, such as the deep layers of the

superior colliculis (Chevalier et al. 1985). the thalamus (Deniau and

Chevalier 1985) and the PPN. These output structures are known to affect

the motor behaviors that amphetamine influences (Di Chiara et al. 1979).

133
FIGURE 3. Amphetamine increases the firing rate of a substantia nigra

pars reticulata neuron in the chronically implanted,

behaving rat

NOTE: Action potentials are represented as a pulse output from a spike-height discriminator: Each

vertical line represents one action potential. The firing rate of this neuron during similar preand postdrug behaviors is increased by injection of 1.0 mg/kg, SC, amphetamine. This

increase occurs for both locomotion (from a mean of 28.3 to 39.0 spikes/sec) and face

washing (from a mean of 43.7 to 46.5 spikes/sec).

EXTRASTRIATAL EFFECTS OF AMPHETAMINE ON THE

CORTICALLY EVOKED STRIATAL RESPONSE IN

ANESTHETIZED RATS

The rat neostriatum receives massive input from the cerebral cortex and

thalamus (Chung 1979; Kemp and Powell 1971; Somogyi et al. 1981).

134
Neostriatal processing may not be solely influenced by effects of amphetamine on intrastriatal dopamine systems but may also be influenced by

actions within these other major afferent systems. Thus, amphetamine,

which produces, among other effects, increased firing of mesencephalic

reticular neurons (Boakes et al. 1971), depression of response in locus

coeruleus (Graham and Aghajanian 1971). and cortical desynchronization

(Arushanian and Belozertsev 1978) may have marked effects on activity

within the neostriatum. As an approach to understanding the contribution of

these extrastriatal actions on striatal functioning, the effect of amphetamine

on cortically evoked intracellular events and field potentials in the

neostriatum is being studied.

Electrical stimulation of the neocortex evokes a regular sequence of

intracellular and extracellular potentials in the neostriatum (Liles 1973; Hull

et al. 1973). In intracellular striatal recordings, single-pulse stimulation of

cortical afferents elicits an initial depolarizing postsynaptic potential (DPSP),

which is followed by

depolarization (figure 4).

a long-lasting afterhyperpolarization and a rebound

FIGURE 4. Illustration of the correspondence between components of the

cortically evoked neostriatal intracellular (top, positive up)

and field potential (bottom, negative up) response

NOTE: Time calibration: 5 milliseconds for both traces. Intracellular amplitude. calibration: 10 mV.

135
A correspondence between these intracellular events and components of the

cortically evoked neostriatal field potential (Ryan et al. 1986a) have recently

been demonstrated. As shown in figure 4, the initial extracellular positive

wave Pl is associated with the intracellular DPSP. The negative wave N2,

which might reflect intrastriatal collateral inhibition, occurs during the initial

period of intracellular hyperpolarization. The later period of hyperpolarization, which has been attributed to a loss of tonic excitation from cortical

and thalamic inputs (Wilson et al. 1983). is seen to overlap with the second

extracellular positive wave P2. A late negative wave, N3, occurs in phase

with the rebound depolarization. These correspondences between intracellular and extracellular events encouraged employment of the cortically

evoked field potential as an index of striatal population response to the

effects of amphetamine. Systemic amphetamine (0.5 to 5.0 mg/kg) has

been found to reduce the amplitude of Pl and dramatically decreases the

latency to N3. Interestingly, these effects appear to be due, at least in part,

to the action of amphetamine at extrastriatal sites, since they could be

mimicked by high-frequency, low-current stimulation of thalamic afferents in

the mesencephalic reticular formation (Ryan et al. 1987a). Further, these

changes in the cortically evoked neostriatal field potential following either

systemic amphetamine or high-frequency reticular stimulation were abolished

by kainic acid lesion of the medial thalamus. The pharmacological

characterization of this response supports the extrastriatal origin of these

effects of amphetamine. Dopaminergic antagonists, such as haloperidol and

fluphenazine, do not block or reverse the effects of amphetamine on wave

Pl or N3. In contrast, amphetamine’s actions are potentiated by the 2

noradrenergic autoreceptor antagonist yohimbine and are reversed by the

beta antagonists propranolol and metoprolol (Ryan et al. 1987b). In

addition, the latency to a positive wave recorded in the region of somatosensory cortex overlying the neostriatum and temporally coincident with the

neostriatal wave N3 is reduced by amphetamine by the same amount as is

wave N3. These temporally similar actions in neostriatum and neocortex

also indicate an extrastriatal site of action for amphetamine. To characterize

further the extrastriatal effects of amphetamine, cortically evoked neostriatal

field potentials and intracellular responses have been examined after either

local application of amphetamine or high-frequency stimulation of the reticular formation (RF) (Fisher et al. 1987). Brief, low-intensity (0.1 mA) 60

Hz stimulation of the PPN produces a depolarization of cell-restingmembrane potential and a resulting decrease of the DPSP. This depolarization is reflected in a parallel reduction of the Pl wave in the evoked field

potential. In addition, a decrease in the latency of the rebound depolarization is observed following RF stimulation, corresponding to a shift in N3 in

the extracellular response. Notably, a local infusion of amphetamine into

the PPN (10-6M, total volume 0.2 uL over 4 minutes) produces alterations in

the intracellular and extracellular evoked responses, similar to those

observed with RF stimulation (figure 5) and systemic amphetamine

administration.

136
FIGURE 5. Cortically evoked intracellular (A, positive up) and field

potential (B, negative up) response in the neostriatum at

increasing times following local infusion of amphetamine

(10-6M) into the pedunculopontine reticular nucleus

NOTE: Following amphetamine, the intracellular postsynaptic potential (PSP) is reduced as is P1 in

the field potential. A reduction in the latency to and a modification in components of the

rebound potential can also be observed. Time calibration: 50 milliseconds. Intracellular

amplitude: 5 mV.

Activation of the RF presumably alters neostriatal functioning via its effects

on thalamocortical pathways. These alterations may affect striatal excitability and timing. Results suggest that important alterations in striatal functioning can result from extrastriatal actions of amphetamine.

CONCLUSION

The actions of amphetamine are widespread throughout the brain.

Amphetamine’s immediate effect is to alter the release of monoamines in a

dose-dependent manner that is specific for each monoamine transmitter

neuronal system. The net effect of amphetamine on monoamine release is

complex, with some mechanisms tending to increase monoamine release

(e.g., blockade of reuptake and nonimpulsedependent release), and several

mechanisms tending to diminish release (e.g., activation of somatodendritic

and terminal autoreceptors).

It is important to consider that the behavioral outcome of amphetamineinduced alterations in monoamine release is determined by changes induced

in postsynaptic targets of monoamine neurons. The consequences of

137
amphetamine on the activity of these target cells reflects both direct actions

of monoamines and changes in the pattern and intensity of interactions of

afferents and intrinsic neurons. In the neostriatum, for instance,

amphetamine affects neostriatal cells directly by altering the release of

dopamine and serotonin, and indirectly by changing activity patterns in

cortical, thalamic, and amygdalar afferents. One of the greatest challenges

facing neuropharmacologists is to dissect these multitudinous influences to

understand how amphetamine and related designer drugs produce their

important behavioral consequences.

DISCUSSION

QUESTION: Have you looked at the globus pallidus yet?

ANSWER: We have looked at the globus pallidus in anesthetized animals,

which appears to be uniformly increased by amphetamine administration. I

might mention that Jean Walters has also looked at the globus pallidus in

the behaving animals, and it is routinely increased by amphetamine

administration.

QUESTION: Do you think that there is a similar feedback regulation

system of the reticulata part for the nigra?

ANSWER: I think that is controversial right now. Yes, I think there is a

projection system that runs from the pars reticulata to pars compacta, but

the consequences of activation of that system and how you would get

access to it are not well understood at this time.

QUESTION: Is there a parallel to the reticulata component of the striatum,

the dorsal striatum in the ventral tegmental area?

ANSWER: I wish I knew that. It certainly seems so, and we are trying to

record nucleus accumbens at this time. It seems that nucleus accumbens is

similar to neostriatum, but ventral tegmental area has a much greater

heterogeneity of nerve cells than does substantia nigra. It may be that the

nondopaminergic neurons of the ventral tegmental area are just sprinkled

through and that, of course, is the neurophysiologist’s nightmare because

you don’t know where electrodes pick up, although we think there are

neurophysiological criteria. Ultimately, that question will be amenable to

analysis. I wish I could answer it now.

QUESTION: Are the effects of amphetamine in the anesthetized

preparation confounded by the anesthetic agent?

ANSWER: I am not entirely sure, but people in my lab believe that the

effects are related to two different populations of nerve cells, that those

excited by amphetamine administration represent a different population, and

138
there are only five or seven or so different types of nerve cells in the

neostriatum, with 95 percent of them in one morphological class.

But there are those in my lab who believe that the excitation is being seen

by a bias toward large cells and that they represent a large cell population

in the neostriatum. I don’t necessarily believe that. I don’t know why, in

the anesthetized animal, you can flip a nerve cell that is inhibited by

amphetamine by increasing the dose. It has been postulated that the

excitation is related to the occurrence of both the stereotyped behaviors, and

that this may be provoked at doses that produce neurotoxicity. We have

also done a number of studies looking at the neurotoxicity of amphetamine

administration in animals, most of which replicate Lou Seiden’s work.

QUESTION: Do you find a population of autoreceptors at the axonal

endings using your techniques?

ANSWER: I think it is very clear that there is a population of

autoreceptors at the axonal end, but there is no population of autoreceptors

in the axon that passes through the medial forebrain level. The antidromic

stimulation is up there, and it looks as if amphetamine and related

dopamine agonists cause a decrease in excitability of the terminal field in

the same way that they cause a decrease in excitability in the cell body.

And we believe that this fact, only recently shown by Allan North’s group,

is that they cause an increase in potassium conductance and as a

consequence hyperpolarization of the cell body.

QUESTION: Does dopamine do it?

ANSWER: Yes.

QUESTION: Whose paper is that?

ANSWER: That is Lacey et al., Allan North’s group. It was published in

the Journal of Physiology last year. It was also an abstract in the Society

2 years ago. It is the consequence of that application of the agonists,

recording intracellularly in the slice of the dopamine neuron. He gets the

same thing by virtue of application of norepinephrine agonists to noradrenegic slice preparation. That is a conventional way to create a hyperpolarization of the cell, to increase the potassium conductance, and so forth.

This is presumably the way that much of the polarization of the cells

occurs.

QUESTION: Do you think it is necessary to have an intact cell to have

this hyperpolarization?

ANSWER: I believe very strongly that you need an intact system. Now

there are certain questions that you can answer in a slice. You deprive the

139
cells of all their normal afferents and in many cases you cut their axons.

You cut the axon of the very cell you are recording, but you are unaware

that has occurred. But there are many different traumatic events that take

place when you extract the slice. The behavior of the cells is quite

abnormal if you look at it carefully.

QUESTION: If you slice the medial forebrain bundle, is there a change in

the resistance at the terminal area? Which way does it go?

ANSWER: I don’t know. There is a change at the cell body; the

resistance goes way up, and it will set itself into a repetitive firing mode,

which is quite an abnormal looking mode of firing, but the dopamine

neuron or the norepinephrine neuron or virtually any neuron in slice will go

into this bizarre repetitive mode of firing.

Still, activation of autoreceptors causes an inhibition of the activity. So,

there are certain qualitative similarities that lead us to believe that the slice

is a useful preparation for entering certain types of neuropharmacologic

questions. But as far as answering how it is that the brain works, I don’t

think the slice is going to solve it for us.

COMMENT: You were showing your reticulata cell showing an increased

activation. Some people think that they may have this feedback mechanism.

Several years ago Koob and others were postulating that this was indeed

part of an outfIow system. And if you put GABA or GABAergic drugs

into that outflow system, that you could also produce behaviors.

RESPONSE: Yes.

QUESTION: Could it be either/or?

ANSWER: Well, I don’t think it is either/or.

QUESTION: Do you think it is a feedback system?

ANSWER: I don’t think it is a feedback system per se. I think the

feedback system goes a long way. I think Steve Bunney and

George Aghajanian believe that it is a feedback system and that it routes

itself from the neostriatum through the pars reticulata and that the pars

reticulata then causes the inhibition of firing in the dopamine system. Now,

as far as feedback is concerned, I think it is much more likely. for

example, that the globus pallidus causes an inhibition of firing of the

dopamine neuron when amphetamine is injected because amphetamine

causes this dramatic increase in firing of globus pallidus, and we know

anatomically that the globus thalamus projections end up in substantia nigra

pars compacta. So it is much more likely that the globus pallidus is

140
influencing the activity of the dopamine neuron by virtue of an afferent

system.

And there are others who believe that dopamine is indirectly or directly

influencing pars reticulata by virtue of being released from dendrites and

exciting the pars reticulata neuron, so that is another theoretical approach

that has not, in my opinion, been adequately tested.

QUESTION: Do you find autoreceptors in mesolimbic structures?

ANSWER: Yes, we do. As far as our evidence goes, we have tested

many hundreds of mesolimbic neurons, and there is a theory that a certain

group of mesolimbic dopamine, cortically projecting neurons lack autoreceptors. We have studied the cortically projecting tegmental area of

dopamine neurons ad nauseam, and in our hands they look exactly the same

as the substantia nigra pars compacta. They have autoreceptors and they

are influenced by low doses of amphetamine, which we know is used in the

ventral tegmental area, operated by virtue of autoreceptor activation. They

are influenced by low doses; both the excitability at the terminal and the

excitability of the cell body are inlluenced by autoreceptor activation

through the tegmental area in the cortically projecting cells. So our

evidence does not agree with that point of view.

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adrenoceptors which increase potassium conductance in rat locus coeruleus

neurons. Neurosci 14:95-101, 1985.

Wilson, CJ.; Chang, H.T.; and Kitai, S.T. Disfacillation and long-lasting

inhibition of neostriatal neurons in the rat. Exp Brain Res 51:227-235,

1983.

ACKNOWLEDGMENTS

This work was supported by grants DA 02854 and DA 00079 from the

National Institute on Drug Abuse and a grant from the Office of Naval

Research.

AUTHORS

Philip M. Groves, Ph.D.

Lawrence J. Ryan, Ph.D.

Marco Diana, M.D.

Stephen J. Young, Ph.D.

Lisa J. Fisher, Ph.D.

Department of Psychiatry

University of California, San Diego

La Jolla, CA 92093

145
Methamphetamine and Related

Drugs: Toxicity and Resulting

Behavioral Changes in Response to

Pharmacological Probes

Lewis S. Seiden and Mark S. Kleven

INTRODUCTION

Some substituted phenethylamines are toxic to certain neurons in the brain.

In view of this neurotoxicity, we will review some data relevant to this

process. First, we will review data showing that methamphetamine

(METH), a prototypic psychomotor stimulant, which has been widely used

for nonmedical purposes at doses often a good deal higher than therapeutic

doses, is neurotoxic to dopamine (DA) and serotonin (5-hydroxytryptamine

(5-HT)) systems. Second, we will examine the evidence that other

substituted phenethylamines are also neurotoxic to certain transmitter

systems. Last, we will examine the behavioral and pharmacological

consequences of neurotoxicity that result from exposure to some of these

amphetamine-related drugs.

Phenethylamines can be ring- and/or side chain-substituted, and many of

these derivatives show potent pharmacological effects (Weiner 1985). Of

phenethylamines without ring substitutions, pharmacologically active

compounds tend to be mainly psychomotor stimulants, possessing

sympathomimetic, antifatigue, and reinforcing effects in humans using the

drugs. The antifatigue and reinforcing properties are likely to be

responsible for their abuse potential. In very high doses, such as those used

by human amphetamine abusers, the amphetamine-type drugs can lead to a

psychotic state, which has paranoid delusional symptoms that are very often

indistinguishable from an acute psychotic episode seen in patients with

schizophrenia (Jonsson and Gunne 1970). Outbreaks of METH epidemics

have occurred in several countries including the USA, Sweden. and Japan

(Kramer et al. 1967; Inghe 1969; Brill and Hirose 1969).

The prototype amphetamine enhances release and blocks reuptake of DA,

norepinephrine (NE), and 5-HT and is also a monoamine oxidase inhibitor.

As a result of these effects, drugs in this class are potent indirect agonists

at monoaminergic receptors. In experimental animals, amphetamine

146
stimulates locomotor activity at low doses and causes stereotypic activity at

higher doses; amphetamine also interferes with food and water consumption.

These behavioral effects are related to amphetamine’s actions on DA, NE,

and 5-HT systems (Lewander 1977; Moore 1978).

Amphetamine-related drugs such as 3,4-methylenedioxyamphetamine (MDA)

or 3,4-methylenedioxymethamphetamine (MDMA), with substitutions on the

benzene ring, tend to show hallucinogenic activity at lower doses and

psychomotor stimulation at higher doses (Climko et al. 1986). In contrast

to the behavioral effects of ring-substituted amphetamines, side-chain

analogs show psychomotor stimulation at low doses and hallucinogenic

activity at higher doses. Their abuse potential raises the question of

whether amphetamine-like drugs may have long-lasting and/or toxic effects

on the central nervous system (CNS). The data show that some of these

ring-substituted compounds have toxic effects on the DA and 5-HT systems,

whereas others seem to be toxic mainly to the 5-HT system. Also of

interest are some structure-activity relationships, the toxicity to DA and/or

5-HT fibers, and a possible mechanism by which these drugs are toxic.

Data from behavioral tests using pharmacological probes show that these

neurotransmitter systems are compromised.

NEUROTOXIC EFFECTS OF AMPHETAMINE-RELATED DRUGS

Although the symptoms produced in humans suggested that the amphetamine type of drugs may engender a neurotoxic response in the CNS, there

were no signs of brain pathology until the early 1970s. Koda and Gibb

(1973) reported that 18 hours after a dosing regimen in which METH was

injected every 5 hours, the Vmax for tyrosine hydroxylase was reduced.

Seiden et al. (1975-76) reported that, in monkeys administered amphetamine

for several months and sacrificed 3 to 6 months after the last injection, the

levels of DA in the brain were reduced. This long-term reduction in brain

DA suggested but did not prove that METH was toxic to DA cells.

Neurotoxicity of METH was shown to occur in rats by virtue of the facts

that: (1) levels of DA and activity of the enzyme that is rate limiting for

DA synthesis were decreased for a long period after cessation of drug

treatment (Ricaurte et al. 1980; Ricaurte et al. 1982; Hotchkiss et al. 1979);

(2) the number of reuptake sites for DA were reduced (Ricaurte et al. 1980;

Ricaurte et al. 1982); and (3) there was shown to be neuronal degeneration

in DA-rich areas of the brain (Ricaurte et al. 1982; Ricaurte et al, 1984).

It is important to stress that these three criteria must be met before

neurotoxicity can be established. Similar effects upon 5-HT levels, reuptake

sites, and morphology must also be observed before it can be concluded

that 5-HT neurotoxicity has occurred. In this regard, multiple doses of

METH have been shown to produce long-lasting reductions in tryptophan

hydroxylase activity (Hotchkiss et al. 1979) as well as 5-HT content and

uptake sites (Ricaurte et al. 1980) in the rat brain.

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The criteria for neurotoxicity have been met for the hallucinogenic

amphetamines MDA and MDMA (table 1). Levels of 5-HT were depleted

as long as 8 weeks following a repeated administration of either MDA or

MDMA (Ricaurte et al. 1985; Schmidt et al. 1986; Schmidt 1987a; Stone

et al. 1987a; Stone et al. 1987b). Additionally, both MDA and MDMA

reduced numbers of 5-HT uptake sites (Ricaurte et al. 1985; Commins

et al. 1987; Schmidt 1987a), depressed tryptophan hydroxylase activity

(Stone et al. 1986; Schmidt and Taylor 1987) and produced signs of

neuronal degeneration (Ricaurte et al. 1985; Commins et al. 1987). It is

clear from these data that neurotoxicity is directed primarily toward the

5-HT system. Much higher doses are required to produce significant

reductions in the levels of DA or its metabolites.

TABLE 1. Effects of amphetamine-related compounds on

monoaminergic neurons

Drug

Amphetamine

Cathinone

METH

MDA

MDMA

Fenfluramine

Dethylpropion

Mazindol

PPA

Cocaine

MPH

KEY: METH=methamphetamine PPA=phenylpropanolamine; MPH=methylphenidate.

Levels Uptake Sites FinkD A N E 5-HT DA 5-HT Heimer

More recently, the N-ethyl analog of MDA (MDE) was examined for

possible neurotoxic effects (Stone et al. 1987a; Schmidt 1987b). In

comparison to MDA and MDMA, MDE was much less potent in causing

depletions of 5-HT 2 weeks after a multiple dose regimen (10 mg/kg every

6 hours for five consecutive intervals). This regimen also failed to reduce

tryptophan hydroxylase activity; additionally, 5-HT uptake sites were not

reduced 1 week after a single 20 mg/kg injection, unlike the reduction

found with similar doses of MDA and MDMA (Schmidt 1987b). However,

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as observed with MDA and MDMA, no effects of MDE on DA neurons

were reported in these studies.

Fenfluramine, like MDMA and MDA, is a ring-substituted amphetamine

derivative that has been found to meet all the criteria for neurotoxicity.

When administered in doses higher than 12 mg/kg/day, depletions of 5-HT

and 5-hydroxyindoleacetic acid (5-HIAA) last up to 6 months after cessation

of drug treatment (Harvey and McMaster 1975; Harvey et al. 1977;

Clineschmidt et al. 1978; Steranka and Sanders-Bush 1979; Schuster

et al. 1986; Kleven et al. 1988). Other long-lasting effects of fenfluramine

include a decrease in 5-HT uptake sites (Schuster et al. 1986) and

tryptophan hydroxylase activity (Steranka and Sanders-Bush 1979).

Previous studies have also indicated that fenfluramine produces signs of

neuronal degeneration (Harvey and McMaster 1975; Harvey and McMaster

1977; Harvey et al. 1977). Recent immunohistochemical studies also

indicated that fenfluramine produced morphological damage to 5-HT

terminal fields (Appel and De Souza 1988). Collectively, the neurochemical

and histological data support the idea that fenfluramine is neurotoxic to

5-HT.

Cathinone and phenylpropanolamine are side-chain-substituted amphetamines

that have thus far met only some of the criteria for neurotoxicity. Like

d-amphetamine, cathinone releases and, at very high concentrations, blocks

uptake of DA (Wagner et al. 1982). Similarly, cathinone mimics

d-amphetamines long-lasting toxic effects on DA levels and uptake sites

(Wagner et al. 1982). The possibility that neuronal damage occurs

following neurotoxic doses of cathinone has not been examined. Neurotoxic

effects of phenylpropanolamine are unlike those reported for other

substituted phenethylamines. When very high doses (200 mg/kg by

injection) are administered, slight decreases in frontal cortex DA have been

observed (Woolverton et al. 1986). with no effects on other monoamine

systems.

Several substituted phenethylamines, such as methylphenidate (MPH) and

mazindol, are notably lacking in toxic effects on DA or 5-HT (Wagner

et al. 1980). However, mazindol does produce a slight decrease in NE,

following repeated administration. None of the other neurotoxic

amphetamine derivatives have been found to have long-lasting effects on the

NE system. Both amphetamine and MPH are potent monoamine-releasing

agents; however, MPH appears to act primarily on a pool that does not

depend upon recent synthesis (Scheel-Kruger et al. 1977). The newly

synthesized transmitter pool seems to be required for the neumtoxicity

produced by METH, since inhibition of DA synthesis blocks the

neurotoxicity of METH (Commins and Seiden 1986; Wagner et al. 1983).

The similarities in acute neurochemical effects of cocaine and amphetaminelike compounds raise the possibility that repeated exposure to cocaine might

149
produce long-term neurotoxic changes similar to those produced by METH.

Most previous studies have examined effects of repeated doses of cocaine

within 24 hours after the last injection (Roy et al. 1978; Taylor and Ho

1976; Taylor and Ho 1977). These studies reveal that exposure to cocaine

for up to 45 days causes only small decreases in DA and/or 5-HT levels.

Longer lasting consequences of repeated exposure to cocaine have only

recently been examined (Trulson and Ulissey 1987; Trulson et al. 1986;

Trulson et al. 1987). For example, it has been reported that repeated

cocaine administration to rats reduced striatal and mesolimbic tyrosine

hydroxylase activity 60 days after the last injection (Trulson et al. 1986;

Trulson et al. 1987), a finding that suggests that prolonged exposure to

cocaine might produce long-lasting damage to DA-containing neurons. A

reduction in tyrosine hydroxylase activity for several days or weeks is

consistent with toxicity to catecholamine neurons. However, using

neurochemical methods, we have not found such evidence of neurotoxicity

(Kleven et al., in press). Repeated injections of either moderate

(20 mg/kg/day) or high doses (100 mg/kg/day) of cocaine for 10 days failed

to produce long-term reductions in the concentration of monoamines or

metabolites in any of the brain regions examined, including the striatum.

Similarly, increasing exposure to 21 days of continuous infusion of a high

dose of cocaine (100 mg/kg/day) failed to significantly deplete DA and

5-HT in the striatum or other regions examined. Higher doses of cocaine

were lethal within 4 days of exposure. Therefore, long-lasting depletions of

monoamines do not seem to occur following cocaine administration.

The data reviewed above indicate that amphetamines and a number of their

analogs are neurotoxic to DA and/or 5-HT. Although both ring- (MDA,

MDMA, MDE) and side-chain-substituted amphetamines (METH, cathinone,

mazindol) have been found to produce neurotoxicity, some differences are

apparent. First, side-chain-substituted phenethylamines are primarily toxic to

DA neurons, with effects on 5-HT appearing at higher doses. On the other

hand, ring-substituted phenythylamines are selectively toxic to 5-HT

neurons, with effects on DA evident only at much higher doses. Second,

these two classes of substituted phenethylamines may also differ in terms of

potency, either absolute or relative to other behavioral effects.

Table 2 summarizes results of neurotoxicity studies that have utilized the

same regimen of drug injections (twice daily for 4 days) and survival times

(2 weeks). In addition, the ability of these drugs to suppress milk intake in

rats is also presented. It is clear that ring-substituted amphetamines are

more potent in terms of absolute dose required to reduce amine content than

is the parent compound amphetamine. With regard to relative potency,

METH is toxic to DA and 5-HT neurons at doses that are more than

tenfold higher than doses that produce anorexia, whereas fenfluramine,

MDA, and MDMA are toxic to 5-HT neurons at doses that are only three

150
TABLE 2. Relationship between behavioral and neurotoxic potency

Drug

METH

Cathinone

PPA

Dethylpropion

MDA

MDMA

Fenfluramine

Neurotoxicity*

Dose†† DA-striatum 5-HT-hippo Anorectic ED50

†

100 65% 57%

100-200 50%

100-200 64% >100

25 67% 10.0

10 41% 2.5

20 30% 2.8

6.25 43% 5.0

1.6

3.9

*Levels of transmitter % of control.

†Reduction of intake of sweetened condensed milk during 15-minute sessions.

††mg/kg injected twice daily for 4 days, rats were sacrificed 2 weeks after the last injection.

KEY: PPA=phenylpropanolamine; no effect.

to four times higher than those needed to suppress milk-drinking activity in

rats. These data suggest that ring substitution increases neurotoxic potency

to a greater extent than increasing behavioral potency.

BEHAVIORAL EFFECTS OF NEUROTOXIC AMPHETAMINES

The most robust behavioral change that has been observed after a brief

regimen of amphetamine-related drugs is altered sensitivity to subsequent

administration of the same or a related drug. The underlying effect is not

apparent until it is unmasked by pharmacological challenge. Thus, longlasting behavioral effects of neurotoxic amphetamines may be more subtle

than those produced by monoamine neurotoxins such as 6-hydroxydopamine

(6-OHDA) or 5,6-dihydroxytryptamine (5,6-DHT). Perhaps this is because,

with the toxic amphetamines, levels of neurotransmitter are usually depleted

to about 50 percent of normal. Studies in which monoamine neurons are

lesioned using 6-OHDA or 5,6-DHT show behavioral deficits when levels

are depleted to 80 or 90 percent of normal. Even in these studies, the most

common finding is a change in sensitivity to pharmacological probes

(Heffner and Seiden 1979; Levine et al. 1980).

The original observation of long-term depletions of DA in the rhesus

monkey was made during a study of the development of tolerance to the

effects of daily injections of METH (Fischman and Schuster 1977). In this

study, it was found that behavioral tolerance to METH on a differentialreinforcement-of-low-rate (DRL) task persisted long after the repeated

METH regimen. In a similar study conducted later, monkeys treated with

repeated METH showed reduced sensitivity to apomorphine and increased

151
sensitivity to haloperidol (Finnegan et al. 1982). Increased sensitivity to

haloperidol and tolerance to METH’s effects on locomotor activity of rats

(Lucot et al. 1980) and a force-lever task in rhesus monkeys (Ando et al.

1985) have also been reported following a regimen of METH. Since, in

each of these studies, repeated administration of METH produced substantial

decreases of DA, tolerance to subsequent METH injections is most likely

related to selective destruction of DA terminals.

In contrast to studies in which tolerance to METH was observed, sensitivity

to the effects of MDMA on DRL performance in rats has been found to

increase as a consequence of a neurotoxic regimen of MDMA (Li et al., in

press). Acute administration of MDMA at 2, 4, and 6 mg/kg increased the

response rate and decreased the reinforcement rate of rats performing under

a DRL 72-second schedule, similar to that observed with other psychomotor

stimulants. Repeated administration of MDMA for 4 days (6 mg/kg, SC,

twice daily), to rats performing on the DRL schedule produced a shift to

the left of the MDMA dose response curve and also increased the maximal

response to MDMA at all dosages. Since levels of 5-HT but not NE or

DA were significantly depleted following the MDMA regimen, the

behavioral results suggest that 5-HT neurons normally exert an inhibitory

action upon the psychomotor stimulant effects of MDMA. Since the

psychomotor stimulant effects of amphetamines appear to be mediated

primarily by the DA system, these results provide evidence that 5-HT and

DA may represent opposing systems insofar as they play a role in DRL

schedule-controlled behavior.

It has recently been found that sensitivity to the analgesic effects of

morphine is altered in rats previously treated with a neurotoxic regimen of

MDMA (Nencini et al. 1988). Rats were injected twice a day for 4 days

with 20 mg/kg MDMA or saline, and, after 14 days, nociception was

determined by measuring reaction time to the tail immersion in heated water

(55 °C). After determining baseline reaction times, rats were randomly

assigned to four groups receiving saline or morphine (2.5, 3.55, or 5 mg/kg,

SC), and the nociceptive test was repeated at various times after drug or

saline administration. Morphine administration produced an analgesic effect

that was more potent and prolonged in MDMA- than in saline-pretreated

rats. These data indicate that morphine was more potent as a consequence

of a neurotoxic regimen of MDMA.

During evaluation of neurotoxicity produced by fenfluramine, an apparent

transitory depletion of 5-HT was observed, with recovery of levels occurring

at 16 weeks for most regions except the hippocampus. It was of interest to

examine this finding in greater detail because of previous work that had

suggested irreversible effects of the drug. Tolerance to the anorectic effects

of fenfluramine was observable 2 but not 8 weeks following a standard

4-day regimen of fenfluramine (6.25 mg/kg. twice daily). Because levels of

5-HT are apparently returning toward control values by 8 weeks in striatum

152
and hypothalamus, it is possible that tolerance to fenfluramine’s anorectic

effect is due to 5-HT depletion. Thus, sensitivity to the anorectic effect

may be related to existing levels of 5-HT. Rats previously allowed to drink

sweetened condensed milk during daily 15-minute sessions were treated with

fenfluramine (6.25 mg/kg twice daily for 4 days) or saline. After 2 to 8

weeks, rats were administered fenfluramine acutely, tested for milk intake,

and sacrificed 2 hours later. Acute adminitration of fenfluramine produced

a dose-related decrease in milk intake and 5-HT levels in various brain

regions. The milk intake data indicated that tolerance to the anorectic effect

of fenfluramine occurred as a result of prior exposure to fenfluramine.

However, levels of 5-HT were also depleted 2 and, to a lesser extent,

8 weeks after the fenfluramine regimen. There was apparent tolerance to

the acute 5-HT-depleting effect of fenfluramine as a result of the 4-day

fenfluramine regimen; partial recovery of this neurochemical tolerance was

observed at 8 weeks. The results suggest that tolerance to the anorectic

effects of fenfluramine may be due to a selective depletion of 5-HT.

CONCLUSION

The results of behavioral studies reviewed are summarized in table 3. It is

clear that a neurotoxic regimen of METH produced tolerance to the effects

of subsequent injections of METH on either conditioned or unconditioned

behaviors. The regimen of METH produced long-lasting depletions of DA

in each of these studies. Similarly, repeated administration of fenfluramine

also produced decreases in 5-HT and tolerance to the anorectic effects of

fenfluramine. Repeated administration of MDMA to rats performing a DRL

schedule resulted in sensitization to the effects of MDMA. This latter

finding is interpreted as being due to the effects, MDMA on DA release, in

TABLE 3. Evidence of neurotoxin-induced behavioral changes in response

to pharmacological probes

Drug Regimen Test Effect Reference

METH 0.5-16 mg/kg/day DRL MA Fischman et al. 1977

Haloperidol

METH 1-32 mg/kg/day DRL MA Finnegan et al. 1982

Haloperidol

METH 100 mg/kg/day * 4 Locomotor MA Lucot et al. 1980

Haloperidol

METH 4-40 mg/kg/day * 4 Force Lever MA Ando et al. 1985

MDMA 6.0 mg/kg/inj * 8 DRL-40 sec MDMA

MDMA 20 mg/kg/inj * 8 Tail Flick

Li et al., in press

Morphine Nencini 1988

FEN 6.25 mg/kg/inj * 8 Milk Intake FEN Kleven et al. 1988a

Key: METH=methamphetamine; FEN=fenfluramine; =tolerance; =sensitization.

153
the absence of effects on 5-HT release, as a consequence of the prolonged

depletions. Each of these studies has utilized pharmacological techniques to

unmask the behavioral deficits produced by the neurotoxic regimen of drug.

It should be noted that persisting behavioral effects of the chronic regimen

of drug, in the absence of such pharmacological challenges appear not to

have been reported. While pharmacological probes reveal an underlying

change in DA and/or 5-HT function, the nature of behavioral deficits in the

absence of drug challenge remains to be determined.

DISCUSSION

QUESTION: Have you or anyone else had the opportunity to look at the

changes in the neurochemical parameters in animals that self-administer

some of the amphetamines?

ANSWER: No, not to my knowledge. What we have done is look at what

the consequences are on self-administration from these chronic regimes.

In other words, we do not have them self-administering these toxic doses.

We have done it with some rhesus monkeys that were self-administering

methamphetamine. If you give them a regime that depletes the dopamine

and serotonin, and then see what alterations there are in self-administration,

it does go down, but we have not looked at that.

QUESTION: Do you get bigger effects on some of the behavioral

parameters after the amphetamine treatments if you pretreat the animals with

a low dose of alpha methyltyrosine during that period, during the postamphetamine period? In the old days, when we had a partial lesion, we

gave a low dose of AMFT, and it would reexpose the lesion. Have you

tried that?

ANSWER: No. It is a good idea, though.

QUESTION: I find the technique of challenging the animals with various

“typic” agents quite intriguing for assessment after prolonged exposure to

MDMA or amphetamine. How long do those changes last? I saw in your

slide something on the order of 2 weeks or a few days after the MDMA

treatment. If you would come back a few months later, would that

supersensitivity still exist?

ANSWER: We are not sure. We have not systematically looked at later

times; we have done so accidentally, however. Sometimes we are not ready

to do an experiment, and we have repeated the MDMA experiment, We

have also, by chance, done tests 6 weeks later, and we have gotten

essentially the same results.

154
QUESTION: Is that reasonably concordant then with the depleting effects

of these treatments?

ANSWER: For MDMA, it is. It certainly would not be for fenfluramine.

But we haven’t looked functionally yet.

QUESTION: Because by 8 weeks you already see some sort of recovery?

COMMENT: I would like to show a slide because I believe the data are

interesting. It has to do with the strategy of looking for a functional

change after serotonin is lost following fenfluramine treatment.

There is a recent clinical report by Emil Coccaro and colleagues that I think

might be relevant to the kind of thing you have done in rats. They have

been looking at endocrine responses to fenfluramine in humans as a marker

of central serotonergic function. And they have observed an increase in

serum prolactin concentration, which is felt to be due to serotonin release.

They reported that, in subjects who received a second dose of fenfluramine

within 12 days after the first dose, that there was a blunted response to

serum prolactin.

There are probably a multitude of explanations, but clearly one would be a

possible persistent depletion of serotonin, the substrate whose release is

required for the acute response to prolactin.

So I think the accumulation of the additional data as you have presented in

rats and perhaps additional data like that in humans may help to clarify

whether there are functional consequences of that loss of serotonin

following fenfluramine.

RESPONSE: That is very interesting. I should mention that, for

fenfluramine, the toxic dose as for MDMA is very close to the therapeutic

dose.

I didn’t go in much detail into what the effects of different doses were, but

with fenfluramine we are getting toxicity in the range of 3, 6, and

10 mg/kg and to interfere with feeding behavior in the rat you arc dealing

with an order of 2.0 mg/kg. So there isn’t much of a window there.

Similarly, for MDMA, the neurotoxic dose range is 10, 20 mg/kg and a

human is taking approximately 2.0 mg/kg. Behaviorally effective doses are

in the neighborhood of 4, 5, and 6 mg/kg.

In contrast to methamphetamine, where we are dealing with behaviorally

effective doses that are in the range of 1 to 4 mg/kg, toxicity doses are in

the range of 50 to 100.

155
So you see, according to our thinking, some of these drugs are more

dangerous because the toxic doses are so very close to the behaviorally

active therapeutic doses.

COMMENT: Just a very brief comment that originally followed up on the

idea of pharmacologic challenge. It is a very powerful technique with

which one can detect underlying or covert neurochemical deficits,

The beauty of it is not only can it uncover an otherwise unapparent deficit,

but it is a technique that can be readily applied to humans.

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159
AUTHORS

Lewis S. Seiden, Ph.D., Professor

Department of Pharmacological and Physiological Sciences

and Department of Psychiatry

Mark S. Kleven, Ph.D., Research Associate (Assistant Professor)

Department of Pharmacological and Physiological Sciences

The University of Chicago

947 East 58th Street

Chicago, IL 60637

160
Role of Dopamine in the

Neurotoxicity Induced by

Amphetamines and Related

Designer Drugs

James W. Gibb, Donna M. Stone, Michel Johnson,

and Glen R. Hanson

INTRODUCTION

In 1971, extensive excitement about the increasing abuse of amphetamines

piqued the authors’ interest in the effects of amphetamine and its analogs

on biogenic amine metabolism; specifically, whether the biosynthesis of

biogenic amines may be altered. In the prior year, Mandell and Morgan

(1970) reported that methamphetamine (METH) produced an increase in

adrenal tyrosine hydroxylase (TH) activity. Fibiger and McGeer (1971) also

observed that chronic treatment with METH caused an increase in TH

activity in the adrenal gland and a decrease in enzyme activity in the

neostriatum.

METHAMPHETAMINE STUDIES

METH Effects on the Dopaminergic System

In an attempt to simulate in rats the dosage regimen commonly employed

by abusers of amphetamines, METH was administered (10 or 15 mg/kg

every 6 hours; four to six doses), after which the animals were killed (Koda

and Gibb 1971: Koda and Gibb 1973). TH activity and catecholamine concentrations were measured in various brain regions and in the adrenal.

Neostriatal TH activity was depressed in a dose-dependent manner and

reached its nadir at 36 hours. Dopamine (DA) and norepinephrine concentrations were initially elevated, but then decreased in parallel with TH

activity. Adrenal TH activity was elevated. presumably because of stress

associated with the toxic doses of METH.

It was later determined whether this was a generalized response to METH

of all transmitter systems or whether it was characteristic of specific

systems. In the dosage used, METH did not affect striatal choline

161
acetyltransferase or glutamic acid decarboxylase; however, tryptophan

hydroxylase (TPH) activity was dramatically decreased (Hotchkiss

et al. 1979). These observations suggested that METH selectively altered

the dopaminergic and serotonergic systems, but did not change the striatal

cholinergic or GABAergic systems.

METH Effects on the Serotonergic System

While there are significant similarities in the response of the dopaminergic

and serotonergic systems to METH, there are also qualitative and quantitative differences. Decreases that occur in the serotonergic parameters are

much larger than the changes in parameters of the dopaminergic system;

moreover, the doses required to obtain the response in the serotonergic

system are lower. Furthermore, the decrease in striatal TH activity is not

observed until approximately 12 hours after METH administration, while

there is a pronounced effect in the serotonergic system within 15 to 30

minutes. (figure 1). TPH activity and serotonin (5-HT) concentrations

declined rapidly, while concentrations of 5-hydroxyindoleacetic acid

(5-HIAA) were transiently elevated after a single dose of METH; tryptophan

content was also elevated after a single dose of METH. Similar responses

occurred with p-chloroamphetamine and amphetamine (Peat et al. 1985).

Since dopaminergic fibers are confined to fewer regions of the brain, while

the serotonergic system is present in many brain areas, the effect of METH

on the brain serotonergic system is more widespread than is the dopaminergic response (Hotchkiss and Gibb 1980).

When only a single dose of METH (10 mg/kg) was administered, TPH

activity returned to normal in all areas within 2 weeks after administering

the drug (Bakhit and Gibb 1981). However, with repeated administration of

METH (five doses, given every 6 hours), TPH activity in the neostriatum,

cerebral cortex, nucleus accumbens, and hippocampus recovered to some

extent but remained significantly depressed 110 days after the last dose of

the drug had been administered; neostriatal TH activity was also depressed

after 110 days.

These findings suggest that, although a single exposure to METH does not

result in permanent alteration of the serotonergic system, repeated

administrations of large doses of METH result in sustained damage. These

observations, together with the METH-induced morphological alterations

(Ricaurte et al. 1982) and the compromised uptake of DA (Wagner

et al. 1980), suggest that METH, given in repeated, large doses, is

neurotoxic to brain serotonergic and dopaminergic neurons.

Role of DA and Its Reactive Metabolites

After the response to METH had been relatively well characterized, the

mechanism responsible for the effect was still unidentified. Since DA

162
FIGURE 1. Effect of acute METH on serotonergic parameters

*Significantly different from control, p<0.05 by Student's t-test.

NOTE: A single dose of METH (10 mg/kg. SC) was administered and rats were killed 3 hours later.

TPH activity and concentration of tryptophan (TRP), 5-HT. and 5-HIAA in the neostriatum

were determined; saline control values (means ± SEM): 5-HT. 0.75±0.l; 5-HIAA, 0.72± 0.06,

TRP, 5.68± 0.17 ng/mg; TPH activity, 24.5± 1.4 nmol/g tissue/hr (n=6 or more).

antagonists had previously been reported to attenuate other effects of

amphetamines (Lasagna and McCann 1957; Randrup et al. 1963; Espelin

and Done 1968), the influence of chlorpromazine or haloperidol on the

METH-induced decreases in TH activity (Buening and Gibb 1974) was

163
investigated. Either drug prevented the response of TH to METH in the

neostriatum; moreover, at the dosage used, haloperidol prevented and chlorpromazine attenuated the elevation of adrenal TH activity. More recently,

it was found that the decrease in striatal TH activity is both a D1- and

D2-mediated event (Sonsalla et al. 1986).

Whether the serotonergic responses to METH could be attenuated by DA

antagonists was next examined (Hotchkiss and Gibb 1980). Surprisingly,

haloperidol, administered concurrently, prevented the METH-induced decrease in neostriatal TPH activity (figure 2). Similar responses were

observed in the cerebral cortex.

FIGURE 2. Effect of HA on METH-induced decrease in neostriatal

TPH activity

*p<0.05 compared to control.

†p<0.05 vS. METH done by Student’s t-test

NOTE: METH (15 mg/kg, SC) was administered four times at 6-hour intervals and rats were killed 5

days after the first administration. HA (3 mg/kg. IP) was administered on the same schedule

(n=4 to 10).

Subsequent experiments revealed that DA is necessary for METH to cause

neurotoxicity; when DA synthesis was inhibited with -methyl-p-tyrosine

(MT), the usual decrease in striatal TH activity observed after METH was

prevented (figure 3A) (Kogan and Gibb 1979). Reinstatement of DA synthesis by administering, l-dopa. which circumvents the inhibited TH step,

returned the METH-induced decreased in TH activity.

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FIGURE 3. A. Inhibition of DA synthesis on the METH-induced decrease

in neostriatal TH activity. B. Inhibition of DA synthesis on

the METH-induced &crease in neostriatal TPH activity

*p<0.05 vs. Control

†p<0.001 vs. METH alone, by Student’s t-test

NOTE: A. METH (15 mg/kg, SC) was administered four times at 6-hour intervals, and rats were

killed 5 days after the first administration. MT (60 mg/kg, lP) was administered on the same

schedule. B. METH (IS mg/kg. SC) was administered five times at 6-hour intervals, and rats

were killed 18 hours after the first administration. MT (60 mg/kg. IP), l-dopa (50 mg/kg. IP),

and RO 44602 (25 mg/kg, IP) were administered concurrently (n=4 to 10).

More fascinating was the response of the serotonergic system to MT and

METH. When DA synthesis was interrupted by concurrent administration

of MT, TPH activity remained normal after METH; however, when DA

synthesis was reinstated by administering concurrently l-dopa, a peripheral

165
dopa decarboxylase inhibitor (RO 44602). MT, and METH, the METHinduced depression of TPH activity was again observed (figure 3B).

It was thought that additional evidence for involvement of DA in the

serotonergic response to METH could be obtained by selectively destroying

the dopaminergic input to the neostriatum with bilateral injections of the

neurotoxin 6-hydroxydopamine (6-OHDA) into the substantia nigra. METH

was then administered to determine whether the decrease of TPH activity

caused by METH would be absent in the neostriatum but present in the

other regions. 6-OHDA was injected bilaterally into the substantia nigra

11 days prior to METH administration. In the neostriatum, deprived of

dopaminergic input, there was no decrease in TPH activity. In the frontal

cortex and hippocampus, however, the METH-induced decrease in TPH

activity still occurred (Johnson et al. 1987).

In summary, three different approaches were used to examine the role of

DA in the METH response: first, blockade of DA receptors by haloperidol

or more specific DA antagonists prevented the METH-induced alteration of

the dopaminergic and serotonergic systems, suggesting that DA may be

involved in these alterations; second, when DA synthesis was inhibited, the

METH-induced changes were prevented in both monoaminergic systems;

finally, when dopaminergic input to a specific brain region was interrupted,

the METH-induced decrease in TPH activity in that brain region was selectively abolished.

OTHER DRUG STUDIES

Effect of MDMA on Serotonergic and Dopaminergic Systems

In 1985, Seiden and his coworkers reported that 3,4-methylenedioxyamphetamine (MDA) caused a decrease in brain 5-HT and 5-HIAA concentrations; 5-HT uptake was also compromised (Ricaurte et al. 1985). We

compared the effects of the methylenedioxy derivatives of METH and

amphetamine on the serotonergic and dopaminergic parameters previously

demonstrated as altered by METH administration (Stone et al. 1986).

When 3,4-methylenedioxymethamphetamine (MDMA) or MDA was administered to rats in a single dose, TPH activity was markedly depressed.

Multiple doses of MDMA or MDA resulted in a further decline in TPH

activity (figure 4). In contrast to METH, however, neither MDA nor

MDMA altered neostriatal TH activity. The decrease in TPH activity was

accompanied by a dramatic decrease in 5-HT and 5-HIAA concentrations;

these changes in TPH activity and in 5-hydroxyindole content also occurred

in other serotonergic terminal areas such as the hippocampus and cerebral

cortex. Both neostriatal DA and homovanillic acid (HVA) were initially

elevated 3 hours after a single dose of MDMA, but had returned to normal

166
by 24 hours. Dihydroxyphenylacetic acid (DOPAC) concentrations were

initially decreased (15 minutes) and had recovered by 24 hours.

Subsequent experiments were designed to characterize further the response

of the serotonergic system to MDMA. When a single low dose (5 mg/kg)

of MDMA was administered, there was an initial decrease in TPH activity

and concentrations of 5-HT and 5-HIAA. These serotonergic parameters

FIGURE 4. Effect of multiple-dose drug treatment on neostriatal TH and

TPH activity

**p<0.01 vs. saline control, by Student’s t-test.

NOTE: Rats were administered five SC doses of MDA (10 mg/kg). MDMA (10 mg/kg). or METH

(15 mg/kg), one dose every 6 hours. and killed 18 hours after the last dose. Results are

presented as the means ± SEM, expressed as a percent of saline control. Control values

were: TH, 2645± 163 nmol tyrosine oxidized/g tissue/hr and TPH, 45.0± 3.5 nmol 14CO2

liberated/g tissue/hr

returned toward control and were essentially normal 2 weeks after the single

dose. If, however, higher doses of MDMA (10 mg/kg, given every 6

hours) were administered and the serotonergic parameters were monitored

for varying periods of time after discontinuing treatment, neostriatal TPH

activity and concentrations of 5-HT and 5-HIAA remained significantly

depressed for at least 110 days (figure 5) (Stone et al. 1987). The

timecourse of recovery was similar in other serotonergic terminal regions

examined.

167
Role of DA in MDMA-Induced Changes in the 5-HT System

The possible role of DA in the MDMA-induced alterations of the

serotonergic system was then examined. Techniques previously used in

studying the role of DA in the METH-induced neurochemical effects were

employed. When DA synthesis was inhibited with MT, the effect of

multiple doses of MDMA on TPH activity (figure 6) and concentrations of

5-HT and 5-HIAA was attenuated. The degree of protection with MT

seemed to be a function of the size and number of doses of MDMA used

as well as a function of the serotonergic parameter that was measured.

FIGURE 5. Long-term recovery of neostriatal serotonergic parameters

after multiple doses of MDMA

*p<0.01 vs. time-matched saline, by Student’s t-test

NOTE: Rats were administered live doses of MDMA (10 mg/kg). one dose every 6 hours, and killed

at specified times thereafter. Results are the means ± SEM (n=6 to 10). expressed as a

percent of time-matched saline-treated control.

Moreover, the early transient response to a single dose of MDMA was less

attenuated by MT than was the persisting response that occurred after

multiple doses of MDMA.

DA was then depleted by using a different drug to determine whether the

response to MDMA would be attenuated. When a single high dose of

MDMA (20 mg/kg) was administered after reserpine, MT, or MT plus

reserpine pretreatment, the usual decrease in neostriatal TPH activity

168
remaining 3 days after MDMA treatment was completely prevented (figure

7). Additionally, when animals were injected with 6-OHDA bilaterally into

the substantia nigra and a single dose of MDMA (10 mg/kg) was

administered 11 days later, the acute (3 hours) MDMA-induced decline in

neostriatal, but not hippocampal or cortical, TPH activity was significantly

attenuated (figure 8).

FIGURE 6. Effect of concurrent MT on the neostriatal TPH deficit induced

by multiple doses of MDMA

**p<0.01 vs. vehicle-saline.

†p<0.05.

††p<0.01 vs. corresponding vehicle-MDMA group, by two-way ANOVA and Newman-Keuls multiple

comparisons test.

NOTE: Rats were administered multiple doses of MDMA (2.5, 5, or 10 mg/kg, SC, five doses, one

every 6 hr). Concurrent with each MDMA dose, MT (60 mg/kg) or saline vehicle was

administered IP. Rats were killed 18 hours after the last dole. Results are the means of ± SEM

(n=6 to 8). expressed as a percent of control (vehicle-saline).

It was previously demonstrated that amfonelic acid, a DA-uptake blocker,

partially prevented the METH-induced decrease in TPH activity (Schmidt

et al. 1985). Recently, effects were investigated of a specific DA-uptake

blocker, GBR 12909, on the MDMA-induced response in the serotonergic

169
FIGURE 7. Effect prior DA depletion on the neostriatal TPH deficit

induced by a single high dose of MDMA

**p<0.01 vs. vehicle-saline

†p<0.05.

††p<0.01 vs. vehicle-MDMA by two-way ANOVA and Newman-Keuls multiple comparisons test.

NOTE: Rats were pretreated IP with MT (120 mg/kg. 90 min before ), reserpine (5 mg/kg. 12 hr

before) or a combination (60 mg/kg MT + 5 mg/kg reserpine, 90 min and 12 hr before,

respectiely). A single dose of MDMA (20 mg/kg) was administered after the specified time

following pretreatment, and rats killed 3 days later. Results are the means ± SEM (n=6 to 7),

expressed as a percent of control (vehicle-saline).

system. GBR 12909 (20 mg/kg) was administered 15 minutes prior to

MDMA (20 mg/kg), and rats were killed 3 days later. The DA-uptake

blocker significantly attenuated the usual MDMA-induced decrease in striatal

TPH activity (figure 9) as well as the decrease in neostriatal 5-HT and

5-HIAA content

These experiments provide evidence that DA and/or its reactive metabolites

are likely involved in MDMA-induced changes in the serotonergic system.

When DA synthesis was inhibited with MT, or when DA innervation was

interrupted by 6-OHDA lesions, the effects of MDMA were prevented or

attenuated. Depletion of DA with reserpine, or inhibition of DA uptake

with GBR 12909, also attenuated the effects of MDMA on the serotonergic

system.

170
FIGURE 8. Effect of prior substantia nigral lesions on the immediate

MDMA-induced decreases in regional TPH activity

**p<0.01 vs. sham-saline.

†p<0.05.

††p<0.01 vs. sham-MDMA by two-way ANOVA and Newman-Keuls multiple comparisons test.

NOTE: Lesions were induced bilaterally by local injection of 4 μg 6-OHDA/ 8 μL 0.1% ascorbate.

vechile/side. Control rats received sham lesions of ascorbate vehicle alone. After an 11-day 

recovery period, acute MDMA (10 mg/kg) was adminstered SC and rats killed 3 hours later.

Results are the means ± SEM, expressed as a percent of sham-saline (n=22 for sham-saline

group, n=14 for 6-OHDA-saline group, n=6 to 8 for MDMA-treated groups). Because

6-OHDA itself significantly elevated TPH activity, values from MDMA-treated rats were

expressed as a percentage ± SEM of their respective saline-treated control mean: in the 

neostriatum, TPH activity for the 6-OHDA-MDMA group was 67.6±5.1% vs. 37.5±2.3% for

sham-MDMA, p<0.01 by Student’s t-test. When similarly expressed, no significant differences

were found between sham-MDMA and 6-OHDA-MDMA groups in the hippocampus or

frontal cortex. 

171
It is premature to define the exact mechanism by which DA is involved in

the response to METH or MDMA. It is known- that these drugs release

large quantities of DA and that DA can be readily oxidized to reactive

metabolites, which could possibly cause destruction of nerve terminals

(Graham 1978; Maker et al. 1986). Moreover, these effects could be

enhanced by inhibition of monoamine oxidase, which is known to occur

with these drugs (Susuki et al. 1980). The possibility that 6-DOHA is

formed and subsequently destroys the nerve terminals, as suggested by

Seiden and Vosmer (1984), also requires investigation.

FIGURE 9. Effect of DA-uptake inhibition on the neostriatal TPH deficit

induced by a high single dose of MDMA

**p<0.01 vs. vehicle-saline.

††p<0.01 vs. vehicle-MDMA by two-way ANOVA and Newman-Keuls multiple comparisons test.

NOTE: GBR 12909 (20 mg/kg, IP) or vehicle was administered 15 min prior to a single dose of

MDMA (20 mg/kg. SC); rats were killed 3 days later. Results are the means ± SEM (n=5 to

6). expressed as a percent of control (vehicle-saline).

172
CONCLUSIONS

Observations made over the last 18 years concerning the effects of METH

on the dopaminergic and serotonergic systems, comparisons of the monoaminergic responses to METH and MDMA, and the studies of the possible

role of DA and/or its reactive metabolites as mediators in the alterations

observed with these drugs provide evidence that DA is necessary for the

effects to occur. Further studies are indicated to define more precisely the

mechanism(s) responsible for the neurotoxic effects of these drugs. These

studies may help to elucidate the potential neurotoxic effects of amphetamine and its related congeners in persons who ingest these agents, and may

also have important implications in understanding the etiology of

Parkinsonism and mental psychoses.

DISCUSSION

QUESTION: You have dopamine reuptake blockade, you have the SCH

23390 blocking, specifically blocking this tryptophan hydroxylase effect.

Do you have a mechanism? How would you see that interaction taking

place?

ANSWER: We have thought about that and I think one of the major

challenges we have as a group is to determine the mechanism by which this

occurs.

I think the results are compatible with the idea that Dr. Seiden has, that it

is 6-hydroxydopamine. I think that it could be a 6-hydroxydopamine or

some other reactive metabolite of dopamine that is causing the effect.

I think that probably dopamine is taken up and in some way oxidized and

therefore may cause the destruction of the nerve terminals. But there are

other possibilities as well that need to be explored to find out whether that

indeed is the case. I think the jury is still out as to the mechanism that

might occur.

COMMENT: That is the reason for mentioning the SCH 23390 being

specific for blocking it and the sulpiride is not blocking it--

RESPONSE: In the serotonergic system.

COMMENT: Right, and it wouldn’t explain a 6-hydroxydopamine

mechanism. It is fascinating that there is that D1, D2 relationship.

RESPONSE: Yes, those data are a bit confusing.

I think the fact that haloperidol and all the dopamine antagonists work

doesn’t bring much clarity to the situation. I think it muddies the water.

173
But it is there, and I think it is important that we show it here so that the

total picture is presented.

COMMENT: I totally agree with the AMT. We have also found that

AMT blocks the serotonergic depletion, so we are in agreement.

Furthermore, and we haven’t reported it yet, PCPA would also be expected

to block the serotonergic depletion. It does nothing to the serotonin

toxicity. So you are right, it is somewhat of a mystery as to what is going

on.

COMMENT: You have presented some evidence that dopamine may be

involved in the neurotoxic action of methamphetamine in terms of

dopaminergic neurons, and you presented evidence suggesting that it may be

involved in not only the dopamine system but also the serotonin system.

I think one has to be very careful, and it goes back to something that

Dr. Seiden raised earlier, that if you are going to speak of the ncurotoxicity,

I really think you have to look at a wide array of neurochemical changes,

not only at 1 or 2 days but at 2, 3, or 4 weeks. Then the changes should

be correlated with morphological changes.

I have no doubt that the bearing on these data is that you have shown the

pharmacological effects on tyrosine and tryptophan hydroxylase activity. I

am not sure that I can equate those with effects on actual neurotoxicity.

The main reason for my suspicion is that in the experiment with AMT

where the effect of methamphetamine on tyrosine hydroxylase activity can

be blocked and then reinstituted by coadministering l-dopa, one would

predict that if one is really talking about neurotoxic effects, then one ought

to be able to observe the same changes 2 weeks later.

We have attempted that experiment, And while it is true that on an acute

basis we can indeed restore an effect on neurochemical parameters at a day,

that is not the case at 2 weeks. This leads me to suspect that one is really

dealing with pharmacological effects of methamphetamine, reinstitution of

these pharmacological effects with l-dopa. But that may not equate with a

neurotoxic action of the drug. I think it is important because it opens up

the issue of whether dopamine does in fact mediate a neurotoxic action of

MDMA and these other compounds rather than some acute pharmacological

effects of these drugs.

RESPONSE/QUESTION: I think that your point is well taken, but I would

ask: Have you and Dr. Seiden demonstrated your uptake blockade after 2

weeks or so as well? And then I would address the question to you or

him--have you done the Fink-Heimer work at longer periods of time?

174
ANSWER: No, we haven’t done the Fink-Heimer work at the longer

periods with the blocking agents. We have done the AMT protection

experiment 2 weeks later. That seems to work on the 5-HT neurons.

QUESTION: What we really need is to do some morphological studies

with something that is very selective for those particular neurons, and I

agree with you that that is something we need to strive for. Are you doing

that at the Addiction Research Center?

ANSWER: We are trying to look at it using some of the neuroanatomical

techniques that you described in terms of localizing uptake sites.

COMMENT: Let me make something clear. We don’t need any new

techniques. The experiments are very simple; they just have to be done at

2 weeks rather than at 18 hours. And it may be that you are entirely

correct, But until those experiments are performed at longer timcpoints, I

don’t think we know if we are dealing with pharmacology or toxicology.

QUESTION: In your experiments, have you ever looked at the hippocampus? It is interesting to note that there is very little dopamine in the

hippocampus. And if the theory of the necessity for dopamine is correct,

then you should not see the depletion of serotonin in the hippocampus.

ANSWER: Good point. We have looked at the hippocampus and have

found that it is protected. We think that not only dopamine is involved, but

probably other catecholamines as well.

QUESTION: How do you imagine that both a receptor antagonist and an

uptake inhibitor would block the effects? It would seem that if dopamine is

involved, it would either be acting on a membrane receptor or inside, but

not both. I would also like to ask a more specific question. You showed

that the alpha MT protected effect could be reversed by dopa. And I think

you imagined that that was because of dopamine formation. But have you

tried dopamine agonists to see if they would antagonize either the protective

effect of alpha methyltyrosine or, particularly, the protective effect of the

dopamine antagonists to try to verify that those protective effects really

have to do with blockade of a dopamine receptor as opposed to some other

possibility?

ANSWER: That is a good question. I haven’t.

COMMENT: I think that one thing that we are not dealing with effectively

is the degeneration in the cortex, which, in our hands, is quite extensive.

Somatosensory cortical, pyramidal cells die at a very high rate with chronic

administration. It seems to me that the involvement of dopaminc in that is

175
less likely, and the postulation that a 6-hydroxydopamine mechanism that

doesn’t account for the sparing that one sees in certain regions.

RESPONSE: We do have experiments in progress that will address that

cortex issue because it is one that needs to be resolved.

COMMENT: We feel that it is due to the formation of 5,6-DHT in the

cortex. These cells are indeed innervated by serotonin cells and, as a

matter of fact, we have an experiment that is being published in Brain

Research where we show that if we inject 5,6-DHT into the ventricles, we

can produce exactly the same type of degeneration in the pyramidal cells,

due to the formation of the 5,6 from the 5-hydroxytryptamine. We are

exploring the possibility of it being another catecholamine in addition to

dopamine, so I think both of those may be helpful in answering your

question.

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ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service grants DA 00869

and DA 04222. The National Institute of Drug Abuse contributed the

methamphetamine hydrochloride. MDMA, and MDA, NOVO Industrials is

acknowledged for contributing GBR 12909.

AUTHORS

James W. Gibb, Ph.D.

Donna M. Stone, Ph.D.

Michel Johnson, Ph.D.

Glen R. Hanson, D.D.S, Ph.D.

Department of Pharmacology and Toxicology

University of Utah

Salt Lake City, UT 84112

178
Acute and Long-Term

Neurochemical Effects of

Methylenedioxymethamphetamine

in the Rat

Christopher J. Schmidt

INTRODUCTION

Administration of a single dose of methylenedioxymethamphetamine

(MDMA) to rats at doses above 10 mg/kg produces a biphasic pattern of

serotonin (5-HT) depletion in the central nervous system (CNS) shown for

the cerebral cortex in figure 1. Much of the work on MDMA in our laboratory has involved the characterization of these two phases of transmitter

depletion following MDMA. Our results indicate that these two periods of

depletion are unique with respect to their mechanism, timecourse, and

stereochemical requirements. The acute effect of MDMA, which is maximal between 3 and 6 hours following drug administration, involves a

disruption of 5-HT synthesis coupled with an increase in transmitter turnover. These early effects of MDMA on the serotonergic neuron appear to

be ultimately reversible. The second phase of depletion develops several

days after the administration of MDMA and is associated with a decrease in

the number of serotonergic nerve terminals. It is the latter decrease in

transmitter concentrations that corresponds to the neurotoxic effect of

MDMA. Attempts have been made to compare these in vivo effects of

MDMA with some of its in vitro activities to gain insight into the

mechanism(s) responsible for the complex neurochemical response elicited

by this drug.

ACUTE EFFECTS

Characteristic of its amphetamine-like structure, MDMA is a potent

monoamine-releasing agent as demonstrated both in vitro (Nichols et al.

1982; Johnson et al. 1986; Schmidt et al. 1987) and in vivo (Yamamoto and

Spanos 1988). This release occurs through a carrier-mediated, Ca2+-

independent mechanism typical of the phenethylamines (Schmidt et al.

1987). Figure 2 shows the concentration-dependent, MDMA-induced transmitter release from preloaded rat striatal slices superfused in vitro. From

the figure, it is apparent that MDMA behaves similarly to the selective

179
serotonergic neurotoxin p-chloroamphetamine (PCA) as a releasing agent.

Both PCA and MDMA show a greater potency for [

3H]5-HT release as

compared to another neurotoxic amphetamine, methamphetamine. However,

methamphetamine is a more potent releasing agent for [

3H]dopamine (DA)

than either MDMA or PCA, which again appear very similar. These results

are interesting in that, in contrast to the selective serotonergic neurotoxicity

of PCA and MDMA, methamphetamine has been shown to be neurotoxic to

both dopaminergic and serotonergic neurons in the rat brain (Gibb et al.,

this volume).

FIGURE 1. Timecourse of changes in 5-HT concentrations and TPH

activity following a single dose of MDMA

NOTE: All data presented as mean ± SEM.

This similarity between MDMA and PCA is also observed in vivo in that

PCA produces both an acute and long-term depletion of 5-HT (Fuller et al.

1975; Steranka et al. 1977). Like PCA, the acute decrease in 5-HT concentrations produced by MDMA is associated with a decrease in the activity of

the rate-limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (TPH).

The timecourse of this change in cortical enzyme activity is also shown in

figure 1. More detailed analysis of this acute effect of MDMA and kinetic

analysis of TPH activity reveals that the decrease in enzyme activity actually precedes the decline in transmitter levels and is due to a reduction in

the Vmax activity of the enzyme (Schmidt and Taylor 1987; Schmidt and

Taylor 1988). As shown for the cortex in figure 3, the decrease in 5-HT

180
also follows a transient spike in the concentration of 5-HIAA before the

metabolite concentrations also begin to decline. These data indicate that an

increase in transmitter release and a decrease in TPH activity are both

required to produce the large depletion produced acutely by MDMA.

FIGURE 2. Comparison of the MDMA-, PCA-, and methamphetamineinduced release of tritiated monoamines from superfused

striatal slices in vitro

**p<0.01

***p<0.001 compared to MDMA.

†p<0.001 compared to MDMA and p<0.02 compared to PCA.

NOTE: All data presented as mean ± SEM.

The above conclusion is supported by the results shown in figure 4. Just as

inhibitors of the 5-HT uptake carrier can antagonize MDMA-induced

[

3H]5-HT release in vitro, coadministration of MDMA with an uptake inhibitor such as citalopram can completely block the acute depletion of 5-HT.

Although citalopram also antagonized the MDMA-induced decrease in TPH

activity, there was still a significant loss of enzyme activity when compared

to control. This implies that if MDMA requires access to the interior of

the nerve terminals to affect TPH activity, it does not require the activity of

the uptake carrier to gain entrance. Hence, these results are consistent with

the outcome of synaptosomal uptake experiments with [

3H]MDMA (Schmidt

et al. 1987), which show that MDMA is not actively concentrated by a carrier system. Furthermore, it is apparent that the loss of enzyme activity

alone is not sufficient to reduce 5-HT concentrations, but that release via

the carrier must occur simultaneously, to deplete the terminal once synthetic

capacity is reduced.

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FIGURE 3. Detailed timecourse of serotonergic changes in the cerebral

cortex after the administration of 10 mg/kg MDMA

*p<0.05 compared to control.

**p<0.01 compared to control.

***p<0.001 compared to control.

NOTE: All data presented as mean ±SEM.

In attempting to determine the mechanism responsible for this loss of enzyme activity, a number of possibilities have been examined. Direct addition of MDMA to brain homogenates was without effect on TPH activity

(Schmidt and Taylor 1987) as has been demonstrated previously for a number of amphetamine analogs, including PCA (Knapp et al. 1974). The

results of attempts to incubate P2 synaptosomes with MDMA were deemed

unreliable due to a large decrease in the activity of synaptosomal TPH upon

incubation. The activity of the control synaptosomes incubated for 2 hours

at 37 °C was consistently 40 to 50 percent of the activity measured in

unincubated synaptosomes. Although, under these conditions, MDMA

increased 5-HT release, as evidenced by a decrease in synaptosomal 5-HT

concentrations, there was no further effect on TPH activity (table 1). The

use of superfused cortical slices was found to stabilize TPH activity in prolonged incubations compared to synaptosomes; however, MDMA was still

without effect on enzyme activity even after exposure to concentrations as

high as 250 μM for 2 hours (Schmidt and Taylor 1988). In a final attempt

to reproduce the effect of MDMA on TPH activity in vitro, we selected a

182
FIGURE 4. Effect of inhibition of the 5-HT uptake carrier by citalopram

on the MDMA-induced changes in cortical TPH activity and

5-HT concentrations 3 hours post-MDMA

***p<0.001 compared to control.

††p<0.01 compared to MDMA.

†††p<0.001 compared to MDMA.

NOTE: All data presented as mean ± SEM.

mouse mast cell line, P815, to test the effects of MDMA in a cell culture

system. These cells have been characterized as containing both 5-HT and a

high level of TPH activity. The inset of figure 5 shows this activity was

easily measured in our assay. However, as also shown in the figure,

incubation of P815s with a high concentration of MDMA (250 μM) for

18 hours had no measurable effect on TPH activity in the cells (Schmidt

and Taylor 1988).

The inability to demonstrate an effect of MDMA on TPH activity in vitro

seemed to point to a requirement for intact neuronal circuitry or in vivo

metabolism of the drug. We therefore attempted to determine if a direct

effect of MDMA in the rat CNS could be achieved by local administration

of the drug directly into the brain. The injection sites were selected to

include the most likely sites of action in the brain, Figure 6 shows enzyme

activity was not altered in the right cerebral cortex 3 hours after 300 μg of

MDMA were stereotaxically injected into the right cerebral ventricle. Cortical 5-HT and 5-HIAA concentrations were also unaffected by this treatment,

as were transmitter concentrations and enzyme activity in the striatum and

hippocampus. Injections of the same dose of MDMA into the substantia

nigra and near the serotonergic cell bodies of the dorsal raphe yielded similar results (Schmidt and Taylor 1988). These injections were performed

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FIGURE 5. Lack of effect of MDMA on TPH activity in the mouse

mastocytoma cell line, P815

NOTE: Cells were incubated for 18 hours in 250 μM MDMA prior to assay. Enzyme activity was

linear with respect to time under the culture conditions used, as shown at the right. Data

presented as mean ± SEM.

under halothane to allow rapid recovery from the anesthesia and observation

of the animals. Surprisingly, there were no obvious behavioral differences

between saline- and MDMA-injected rats. In the absence of any behavioral

effect of MDMA, the results from these experiments were considered

inconclusive as evidence for or against a direct central effect of MDMA.

This prompted us to set up experiments using a constant intracerebroventricular infusion of MDMA to insure that brain concentrations of the drug

were maintained for a behaviorally relevant period of time. In the

established design, conscious rats were continuously infused with either

MDMA or saline for a l-hour period, after which they were observed for

an additional 2 hours prior to sacrifice. Using this approach, doses as low

as 300 μg produced significant changes in regional TPH activity (figure 7).

The latter quantity, corresponding to a dose of approximately 1 mg/kg, was

without effect on TPH activity in any of the three brain regions examined,

when given peripherally by the subcutaneous route (Schmidt and Taylor

1988). Although this seems a high dose for direct central administration, it

is consistent with data reported by Marquardt et al. (1978) showing that

10 percent of a peripherally administered dose of methylenedioxyamphetamine (MDA) was present in the rat brain within 30 minutes of injection.

Assuming that the distribution of MDA and MDMA are similar, this means

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TABLE 1. Effect of MDMA on TPH, 5-HT, and 5-HIAA

Frozen control

TPH

(nmol oxidized/mg/h)

0.530

5-HT 5-HIAA

(ng/mg) (ng/mg)

Incubated control (0.218± 0.052) (2.36 ± 0.02) (2.03 ± 0.14)

100 ± 23.9 100 ± 1.0 100 ± 6.9

1 μM MDMA 85.8 ± 7.3 49.6 ± 5.9 103 ± 12.8

10 μM MDMA 88.1 ± 2.3 25.0 ± 1.7 59.1 ± 3.9

NOTE: P2 synaptosomes were incubated in a Krebs Ringer bicarbonate buffer for 2 hours at 37 °C.

The frozen control was not incubated. Values for MDMA-treated samples are given as a

percent of the incubated control ± SEM.

that peripheral administration of 10 mg/kg of MDMA could be expected to

yield even higher brain concentrations of the drug than were achieved with

the infusion of 300 μg over 1 hour. These results therefore indicate that

the acute effect of MDMA on TPH activity in the rat is a centrally

mediated event requiring sustained, high brain concentrations of the drug.

The lipophilicity of MDMA apparently precludes maintaining such concentrations when the drug is rapidly administered directly into the brain.

Although these results exclude a peripheral metabolite of MDMA as the

causative agent in its acute effect on TPH activity, they do not eliminate a

role for a central metabolite. The ultimate cause of this effect of MDMA

and related drugs therefore remains to be determined.

LONG-TERM EFFECTS

A clear differentiation of the acute and long-term effects of MDMA was

first accomplished by comparing the neurochemical effects of the optical

isomers of MDMA at 3 hours and 1 week. As shown in figure 8, either

enantiomer of MDMA produced the acute depletion of 5-HT. but only rats

treated with the (+)isomer still showed depletion 1 week later. There was

also a significant reduction in the uptake of [

3H]5-HT into synaptosomes

prepared from the latter group of animals (Schmidt 1987a). Hence, the

neurotoxic effect of MDMA is primarily a property of the (+)stereoisomer,

while the acute effect of MDMA has less stringent stereochemical requirements. In addition, the results with (-)MDMA indicate that the acute effect

of the drug on 5-HT concentrations is not permanent, since in the absence

of neurotoxicity the depletion of the transmitter produced by (-)MDMA is

reversed by 1 week. In addition to comparing the enantiomers in vivo, their

effects on neurotransmitter release in vitro were also compared. As shown

in figure 9, (+)MDMA was more potent than (-)MDMA at releasing either

185
FIGURE 6. Lack of effect of intracerebroventricular MDMA (300 μg) on

serotonergic parameters in the cerebral cortex 3 hours after

administration by bolus injection

NOTE: Data presented as mean ± SEM.

[

3H]DA or [3H]5-HT, although the difference between the enantiomers was

less marked for the release of [

3H]5-HT.

A comparison of the acute and long-term effects of MDMA with those of

its homologs MDA and N-ethyl-methylenedioxyamphetamine (MDE) also

contrasts the acute and neurotoxic effects of these compounds. It has previously been demonstrated that all three drugs produce the acute depletion

of 5-HT measured at 3 hours (Schmidt 1987b). However, as shown in

figure 10, if the animals are allowed to survive for 1 week after drug

administration, only MDA- and MDMA-treated rats show the reduction in

5-HT concentrations and [

3H]5-HT uptake indicative of neurotoxicity.

Therefore, the depletion of 5-HT produced at 3 hours by MDE was completely reversible. These results are similar to our observations with the

186
FIGURE 7. Reduction in regional TPH activity 3 hours after the start of a

1-hour intracerebroventricular infusion of MDMA (300 μg)

*p<0.05 compared to control.

**p<0.01 compared to control.

NOTE: Data presented as mean ± SEM.

(-)stereoisomer of MDMA. In vitro, the three methylenedioxy analogs were

very similar in terms of [

3H]5-HT release, but differed in their potency for

releasing [

3H]DA. Here, the order from most to least potent was

MDA>MDMA>MDE (figure 11).

A final experiment demonstrating the distinction between the acute and

neurotoxic effects of MDMA is shown in figure 12. In this case, the 5-HT

uptake inhibitor fluoxetine was administered at various times after MDMA,

with all animals being sacrificed 1 week later. The results are shown as a

percentage of control cortical 5-HT concentrations. Simultaneous administration of an uptake inhibitor with MDMA completely blocked the decrease

in 5-HT concentrations measured 1 week later. However, administration of

the inhibitor 3 hours after MDMA still resulted in complete protection from

the neurotoxicity. Approximately 50 percent of the depletion could still be

blocked 6 hours after MDMA; by 12 hours, the administration of fluoxetine

no longer had any effect. Blockade of the neurotoxicity by an uptake

inhibitor 3 hours after MDMA clearly differentiates the acute and long-term

effects of MDMA, since at this point the acute depletion of 5-HT is already

at a maximum. The administration of fluoxetine to MDMA-treated animals

187
FIGURE 8. Comparison of the optical enantiomers of MDMA for the acute

(3 hours) and long-term (7 days) effects on the serotonergic

system

*p<0.05 compared to saline.

***p<0.001 compared to saline.

NOTE: Data presented as mean ± SEM.

at 3 hours could be considered analogous to the administration of MDE

alone, where an acute effect on 5-HT is observed without the subsequent

development of neurotoxicity. It is also apparent from these data that a

second or later carrier-mediated event is important in the production of the

neurotoxicity of MDMA. The critical phase of activity on the part of the

carrier leading to the neurotoxic response is occurring some time between

3 and 12 hours post-MDMA.

188
FIGURE 9. Comparison of the optical enantiomers of MDMA for the

release of tritiated monoamines from preloaded striatal

slices superfused in vitro

*p<0.05.

**p<0.01

***p<0.001.

NOTE: Data presented as mean ± SEM.

DISCUSSION

The results of the preceding set of experiments may identify several features

of the underlying mechanisms responsible for the acute and long-term

effects of MDMA. For example, it is apparent that the production of the

acute effect of MDMA has less stringent stereochemical requirements than

does the production of neurotoxicity. While both enantiomers of MDMA

cause the rapid depletion of transmitter concentrations as well as the depression in TPH activity (Schmidt and Taylor 1988). only the (+)stereoisomer

produces neurotoxicity at the doses used in these experiments. In a similar

manner, all three methylenedioxy compounds produce the acute interruption

of serotonin synthesis, yet only the two lower n-alkyl homologs caused the

long-term effect. Therefore, in selecting what ultimately leads to the acute

effect of the drug(s) on serotonergic neurons, those activities least

influenced by stereochemistry and affected equally by either the desmethyl

or N-ethyl homolog of MDMA would be the most likely candidates. The

lack of structural stringency characteristic of the acute effect is also

observed for the release of 5-HT in vitro. The role of carrier-mediated

189
5-HT release in the acute depletion of 5-HT has already been discussed. It

may be that a rapid increase in 5-HT elicited by MDMA and its analogs is

also involved in the inactivation of TPH. In contrast to the acute effect of

MDMA on 5-HT synthesis, the reduction in 5-HT concentrations and the

uptake of [

3H]5-HT measured at 1 week after drug administration is less

FIGURE 10. Comparison of the neurochemical effects of the three MDA

homologs 7 days after the administration of 20 mg/kg of

each drug to rats

**p<0.01 compared to saline.

**p<0.001 compared to saline

NOTE: Data presented as mean ± SEM.

likely to be a direct result of 5-HT release. since there is little difference in

the ability of either (+) or (-)MDMA to produce such release and virtually

no difference between the three methylenedioxy homologs. The long-term

effects of the three drugs do actually correspond to their potency for

producing DA release, however. Both the release of [

3H]DA and neurotoxicity follow the same rank order. Similarly, in comparing the

enantiomers of MDMA, the stereochemical specificity of the neurotoxicity is

the same as that of DA release.

Based upon the above considerations, it is hypothesized that the acute effect

of MDMA and its analogs is due to a rapid and sustained increase in the

190
FIGURE 11. Comparison of the three MDA homologs for the release of

tritiated monoamines from preloaded striatal slices

super-fused in vitro

*p<0.05 compared to MDE.

**p<0.01 compared to MDE.

**p<0.00l compared to MDE

†p<0.05 compared to MDMA.

†††pc<0.001 compared to MDMA.

NOTE: Data presented as mean ± SEM.

carrier-mediated release of 5-HT. In contrast, the neurotoxic effect of

MDMA and its analog, MDA, may be due to a sustained elevation in the

release of DA. In this regard, Sharp et al. (1986) have recently compared

the increase in extracellular DA and 5-HT measured by intrastriatal dialysis

after PCA administration. Their results show that the release of DA is

much more pronounced than that of 5-HT and has a much longer duration.

Extracellular DA concentrations 3 hours after injection were approximately

tenfold higher in PCA-treated (5 mg/kg) rats when compared to salineinjected controls. In contrast, by 3 hours, extracellular 5-HT concentrations

were only two to three times greater than control. Extrapolation of these

data to MDMA suggests that, during the period in which the neurotoxicity

of MDMA is developing, i.e., 3 to 12 hours after MDMA, extracellular DA

is still abnormally elevated. Yamamoto and Spanos (1988) have recently

demonstrated that DA release after a neurotoxic dose of MDMA (10 mg/kg)

is still elevated severalfold, 3 hours after drug administration.

191
Time Between Fluoxetine and

MDMA Administration

FIGURE 12. Timecourse for the antagonism of MDMA-induced neurotoxicity by the 5-HT uptake inhibitor fluoxetine

NOTE: The inhibitor (5 mg/kg) was administered at the indicated times after MDMA, and all animals

were sacrificed 7 days later. Data are presented as a percentage of the appropriate control

(saline or fluoxetine alone), mean ± SEM.

CONCLUSION

These studies have characterized both the acute and long-term neurochemical effects of a single administration of MDMA in the rat. The acute

depletion of 5-HT concentrations results from an as yet unexplained loss of

TPH activity in the serotonergic nerve terminals, coupled with a massive

carrier-mediated efflux of transmitter. The long-term depression of 5-HT

concentrations by MDMA is due to an apparent degeneration of serotonergic

nerve terminals. In its pattern of neurochemical effects, MDMA resembles

the selective serotonergic neurotoxin PCA, which may suggest a common

mechanism. Unfortunately, the mechanism responsible for the neurotoxicity

of PCA has been difficult to elucidate in spite of the number of studies that

have addressed this issue. MDMA therefore joins a well-studied group of

amphetamine analogs including amphetamine itself, methamphetamine, PCA,

and fenfluramine, which have in common an unexplained neurotoxic effect

on monoaminergic neurons in laboratory animals. It is hoped that the

increased interest in this area generated by MDMA and its well-publicized

abuse will provide the impetus to resolve the question of amphetamine

neurotoxicity.

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DISCUSSION

QUESTION: Did you try infusion for less than 1 hour? The acute TPH

changes occur as soon as 10 or 15 minutes after single administration, so

wouldn’t you expect to see changes following a 10- or 15-minute infusion?

ANSWER: You might, but I am not absolutely sure of that because the

timecourse of the levels of the drug in brain is going to differ from those

infusion paradigms, what you get with peripheral administration of the drug.

Based on the fact that at 3 hours with a very low dose of MDMA, I saw

an effect on the enzyme and no effect on the transmitter levels, we

probably can’t pay too much attention to that timecourse and expect to see

changes that are identical to what you see in the whole animal with

peripheral administration. That is what I would imagine. The experiments

could be done, though. They are not that difficult. You just stick the

cannulas in and infuse.

QUESTION/COMMENT: Is there any information on the half-lives of the

two enantiomers of MDMA in the brain? What about the half-lives of

MDA versus MDMA versus MDE?

My point is whether differing potencies, in regard to long-term effects,

might be accountable on the basis of the duration that the compounds

persist in the brain rather than intrinsic activities involving release of one or

another neurotransmitter.

ANSWER: Yes, that is a very good possibility. The difference between

PCA and amphetamine comes down to the fact that parachlorination of that

compound makes it persist in the brain longer and you go from something

like amphetamine, which has small neurotoxic effects on serotonin, to

parachloroamphetamine, which is very neurotoxic. So your point is well

taken and that is a possibility, I think.

QUESTION: To follow up on what you said about parachloramphetamine,

I think that the importance of that metabolism is even clearer if you compare 4-chloroamphetamine to 3-chloroamphetamine, where those compounds

are very different in their long-term effects, but become identical in rats

that are pretreated with drugs to block the ring hydroxylation. If you make

them equal metabolically, you make them equal in terms of their long-term

effects on serotonin. You can do that also by going to the guinea pig,

which doesn’t parahydroxylate. 3-chloroamphetamine and 4-chloroamphetamine are already equal metabolically and they are equal in terms of their

long-term effects, Does the same thing apply to the enantiomers of MDMA

and also the analogs of MDMA?

193
ANSWER: I think that is a very good possibility. It was that sort of

thinking that convinced us we really had to do infusion experiments. The

drug has to be there for a sufficient amount of time to have these effects.

QUESTION: Did you do those experiments with PCA?

ANSWER: Elaine Sanders-Bush has done those, and the enantiomers do

differ, but the enantiomers also differ in half-life, and it is probable that the

difference in long-term toxicity is simple because of that difference in halflife.

COMMENT: I know Larry Steranka did that comparison with amphetamine, but I don’t recall the results.

COMMENT: We have looked at tritiated MDA and MDMA clearance

from brain, and we haven’t seen much of a difference over a 24-hour

period. The tritium concentrations peaked in brain at about 45 minutes,

leveled off for a few hours, and then were gone by 24 hours. There was

some indication that more MDA got into the brain than MDMA, but these

are very preliminary experiments.

QUESTION: Where is your label?

ANSWER: The label for MDA was on the ring, and that was an important

point in terms of assuring that it was not just demethylation.

QUESTION: Were you measuring label or were you measuring specific

MDMA? Were you measuring radioactivity or MDMA itself?

ANSWER: We were measuring radioactivity. So far, we have looked at

MDA. We haven’t seen much metabolism of MDA to any other

metabolite. We haven’t looked at those experiments with MDMA yet.

REFERENCES

Fuller, R.W.; Perry, K.W.; and Molloy, B.B. Reversible and irreversible

phases of serotonin depletion by 4-chloroamphetamine. Eur J Pharmacol

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3H]serotonin and

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3H]dopamine release from superfused rat brain slices. Eur J Pharmacol

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3H]serotonin from rat brain synaptosomes. J Med Chem

25:530-535, 1982.

Ross, S.B., and Froden, O. On the mechanism of the acute decrease of rat

brain tryptophan hydroxylase activity by 4-chloroamphetamine. Neurosci

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Sanders-Bush, E., and Steranka, L. Immediate and long-term effects of

p-chloroamphetamine on brain amines. New York: Ann NY Acad Sci

305:208-221, 1978.

Schmidt, C.J. Neurotoxicity of the psychedelic amphetamine, methylene

dioxymethamphetamine. J Pharmacol Exp Ther 240:1-7, 1987a.

Schmidt, C.J. Acute administration of methylenedioxymethamphetamine:

Comparison with the neurochemical effects of its N-desmethyl and

N-ethyl analogs. Eur J Pharmacol 136:81-88, 1987b.

Schmidt, C.J.; Levin, J.A.; and Lovenberg, W. In vitro and in vivo neurochemical effects of methylenedioxymethamphetamine on striatal monoaminergic systems in the rat brain. Biochem Pharmacol 36(5):747-755,

1987.

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hydroxylase activity following the acute administration of methylenedioxymethamphetamine. Biochem Pharmacol 36(23):4095-4102, 1987.

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156:121-131, 1988.

Sharp, T.: Zetterstrom, T.; Christmanson, L.; and Ungerstedt, U. p-Chloroamphetamine releases both serotonin and dopamine into rat brain dialsates

in vivo. Neurosci Lett 72:320-324. 1984.

Steranka, L.; Bessent, R.; and Sanders-Bush, E. Reversible and irreversible

effects of p-chloroamphetamine on brain serotonin in mice. Comm

Psychopharmacol 1:447-454, 1977.

Yamamoto, B.K., and Spanos, L.J. The acute effects of methylenedioxymethamphetamine on dopamine release in the awake-behaving rat. Eur J

Pharmacol 148:195-203. 1988.

AUTHOR

Christopher J. Schmidt, Ph.D.

Merrell Dow Research Institute

2110 East Galbraith Road

Cincinnati, OH 45215

195
Effects of MDMA and MDA on Brain

Serotonin Neurons: Evidence from

Neurochemical and Autoradiographic Studies

Errol B. De Souza and George Battaglia

INTRODUCTION

The drugs 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) are ring-substituted derivatives of

methamphetamine and amphetamine, respectively. These methylenedioxysubstituted amphetamines have been reported to exhibit both stimulant and

psychotomimetic properties (Anderson et al. 1978; Braun et al. 1980;

Shulgin 1986). MDMA has received a great deal of attention recently,

since it represents one of a number of “designer drugs” that have been

increasingly abused among certain segments of the population, especially

college students. MDMA has been the subject of a recent scientific and

legal debate, as several psychiatrists have reported that MDMA may

“enhance emotions” and “feelings of empathy” and thus serve as an adjunct

in psychotherapy (Greer and Tolbert 1986). Recent data demonstrating that

MDMA is self-administered in nonhuman primates (Beardsley et al. 1986;

Lamb and Griffiths 1987) suggest that the drug may have high abuse

potential in man. These reports are particularly disturbing, as the authors

and others have recently demonstrated that MDMA is a potent neurotoxin

that appears to cause selective degeneration of brain serotonin neurons

(Battaglia et al. 1987; Battaglia et al. 1988; Commins et al. 1987; Schmidt

1986; O’Hearn et al. 1988) comparable to that reported for its structural

analog MDA (Battaglia et al. 1987; Ricaurte et al. 1985; Stone et al. 1986).

This chapter describes data on the neurotoxic effects of MDMA on brain

monoamine systems in rodents. Specifically, studies are described

examining the effects of in vivo administration of MDMA on brain

monoamine systems with respect to:

(1) the selective neurodegenerative effects on brain serotonin systems;

(2) the effects of dose and frequency of drug administration;

196
(3) the potential neurochemical mechanisms involved in the neurotoxic

effects of the drug;

(4) the characteristics and timecourse of recovery following destruction of

serotonin neurons;

(5) the relative sensitivity of various animal species; and

(6) the neuroanatomical and morphological specificity of MDMA- and

MDA-induced neurotoxicity.

MARKERS OF NEUROTOXICITY

Typically, neurotoxic effects of drugs on monoamine neurons have been

assessed from reductions in brain levels of monoamines and their metabolites, decreases in the maximal activity of synthetic enzymes activity, and

decreases in the active uptake carrier. In the present study, the traditional

markers described above have been used, including the measurement of the

content of monoamines and their metabolites in brain at several different

timepoints following drug administration. Since reports in the literature

have documented that MDMA and MDA can inhibit the activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis

(Stone et al. 1986; Stone et al. 1987). it is unclear whether MDMA-induced

reductions in the content of serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) may be due to suppressed neurotransmission in

otherwise structurally intact serotonin neurons or may represent the

consequence of the destruction of serotonin neurons and terminals.

Since monoamine uptake sites are highly concentrated on their respective

nerve terminals (Kuhar and Aghajanian 1973), the authors’ approach has

been to use selective radioligands to directly label these uptake sites in

brain and to assess the neurodegeneration of specific monoamine neurons,

by measuring the reductions in the density of their respective uptake sites.

For example, the authors have recently reported the feasibility of using the

measurement of [

3H]paroxetine-labeled serotonin uptake sites to quantify the

neurotoxic effects of MDA (Battaglia et al. 1987) and MDMA (Battaglia

et al. 1987; Battaglia et al. 1988) on serotonin neurons in homogenates of

various regions of rat brain. Visualization of MDMA- and MDA-induced

destruction of serotonin axons and terminals using antibodies directed

against serotonin (O’Hearn et al. 1988) and autoradiographic studies

demonstrating corresponding decreases in [

3H]paroxetine-labeled scrotonin

uptake sites in slide-mounted brain sections of MDA-treated rats (De Souza

and Kuyatt 1987) further validate use of changes in the density of serotonin

uptake sites as an appropriate index of serotonin neurodegeneration.

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IN VIVO EFFECTS OF MDMA: NEUROCHEMICAL STUDIES

The following studies were designed to assess and quantify both the neuro

chemical and neurodegenerative effects of short-term administration of

MDMA on monoamine neurons in rat brain.

Dose Dependence

A repetitive treatment regimen (sc injections twice daily for 4 consecutive

days) of MDMA at various doses up to 20 mg/kg resulted in dosedependent decreases in a variety of serotonergic markers in rat frontal

cerebral cortex, including serotonin, 5-HIAA, and the density of serotonin

uptake sites at 18 hours following the last injection (figure 1). At the

lowest dose of MDMA tested (5 mg/kg), serotonin content was markedly

reduced (45 percent), while only a small (14 percent), but statistically

significant, decrease in the density of serotonin uptake sites was observed; a

small decrease in 5-HIAA content was also observed at this dose, although

this change was not statistically significant. Higher doses of MDMA (10

and 20 mg/kg) resulted in comparable reductions in 5-HIAA levels (60 to

70 percent), while the decrease in serotonin was significantly greater at

20 mg/kg (90 percent) than at 10 mg/kg (80 percent). The density of

serotonin uptake sites decreased progressively as the dose of MDMA was

increased, with a maximal reduction of 90 percent observed at 18 hours

following administration of 20 mg/kg MDMA.

In contrast, following a treatment regimen of 20 mg/kg MDMA, there were

no significant differences in the density of [3H]mazindol-labeled norepinephrine (NE) uptake sites (fmol/mg protein) in the frontal cerebral cortex

between saline-treated (159±17) and MDMA-treated (152±5) animals. With

respect to the dose of MDMA, serotonin levels appeared to be more readily

decreased (45 percent reduction at 5 mg/kg), while comparable reductions in

5-HIAA levels and serotonin uptake sites were noted only at 10 or 20

mg/kg MDMA. This apparent discrepancy among the three serotonergic

markers measured in the present study may relate to effects of lower doses

of MDMA on synthetic enzyme activity (i.e., TPH), whereas the effects of

higher doses of MDMA in reducing all three markers may relate in part to

effects on TPH activity and in part to destruction of serotonin neurons as

evidenced by decreases in serotonin uptake sites.

The Effects of Single Versus Multiple Injections of MDMA

Since repeated systemic administration of 10 mg/kg MDMA caused marked

neurodegeneration of frontal cerebral cortex serotonin neurons, the authors

chose to investigate the neurodegenerative effects of single versus multiple

injections of MDMA at this dose. As shown in figure 2, increasing the

number of injections of MDMA (10 mg/kg, sc) resulted in significant and

progressively greater reductions in serotonin and 5-HIAA content. While

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FIGURE 1. The effect of repeated systemic administration of various doses

of MDMA on the content of serotonin (5-HT) and S-HIAA

and the density of 5-HT uptake sites in rat frontal cerebral

cortex

*Significantly different from control. p<0.05.

**Significantly different from control, p<0.01.

††Significantly different from all other MDMA-treated groups. p<0.01.

†††Significantly different from all other MDMA-treated groups, p<0.001.

NOTE: Rats were administered either saline or MDMA twice a day for 4 consecutive days and

sacrificed 18 hours after the last injection. Data represent the mean SEM from three to

live rats, expressed as a percent of values in control, saline-injected rats. Control values

for 5-HT and 5-HIAA levels were 387±61 and 251±20 pg/mg tissue, respectively. The

density of 5-HT uptake sites in the frontal cerebral cortex in controls was 396±15 fmol/mg

protein. Data were analyzed by one-way ANOVA and Duncan’s multiple range test.

SOURCE: Battaglia et al. 1988.

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one injection of MDMA was without effect on any of the serotonergic

parameters examined, two doses were sufficient to elicit a significant

reduction in serotonin content. As described above, these early effects of

MDMA on serotonin content may relate to MDMA suppression of TPH

activity. A significant reduction in 5-HIAA content (approximately

34 percent) was observed only after four injections of MDMA. Marked

reductions of 84 percent and 75 percent in serotonin and 5-HIAA,

NUMBER OF INJECTIONS OF MDMA (10mg/kg)

FIGURE 2. The effects of single and multiple injections of MDMA on the

content of serotonin (5-HT) and 5-HIAA and the density of

5-HT uptake sites in rat frontal cerebral cortex

*Significantly different from control, p<0.05.

††Significantly different from all other groups, p<0.01.

†††Signiftcantly different from all other groups, p<0.001.

NOTE: Rats were injected the specified number of times with either saline or 10 mg/kg MDMA

and sacrificed 18 hours after the last injection. Data represent the mean and SEN from

three to five animals, plotted as percent of respective values for each marker in control,

saline-injected rats. Control levels of 5-HT and 5-HIAA were 475±24 and 332±24 pg/mg

tissue, respectively. The density of 5-HT uptake sites was 349±24 fmol/mg protein in

control. Data were analyzed by one-way ANOVA and Duncan’s multiple range test.

SOURCE: Battaglia et al. 1988.

200
respectively, were observed following an eight-injection regimen of

10 mg/kg MDMA; the density of [

3H]paroxetine-labeled serotonin uptake

sites was also significantly decreased (approximately 64 percent) following

eight injections of MDMA at this dose. These data suggest that a longer

treatment regimen may be required for destruction of serotonin neurons,

while effects on the content of serotonin and 5-HIAA may occur following

fewer injections.

There was no change in the content of dopamine (DA) in any of the

experimental groups; however, a small, inconsistent decrease in NE content

(approximately 20 percent) was observed in all MDMA-treated rats. This

small change in NE following MDMA treatment was not accompanied by a

reduction in the density of [

3H]mazindol-labeled NE uptake sites.

Species Differences

Since amphetamines have been shown to be metabolized by different

pathways in rat, mouse, and guinea pig (Caldwell 1980). studies were

carried out to investigate whether MDMA induced neurotoxicity could be

demonstrated in other species such as mouse and guinea pig. Animals in

these studies were treated twice daily for 4 consecutive days with 20 mg/kg

MDMA. and levels of serotonin, 5-HIAA, and serotonin uptake sites were

measured at 7 days following the last injection, to assess the long-term

effects of the treatment. As shown in figure 3, MDMA caused comparable

and marked decreases in serotonin and 5-HIAA content and in the density

of serotonin uptake sites in rat and guinea pig cerebral cortex, but appeared

to be without effect on any of these serotonergic markers in the mouse.

Other studies (Stone et al. 1987) have also suggested that mice are less

susceptible to the neurotoxic effects of MDMA. Similar differences in the

sensitivity to the neurotoxic effects of parachloroamphetamine on serotonin

neurons have been observed in mouse when compared to its effects in rat

and guinea pig (Fuller 1978; Kohler et al. 1978; Sanders-Bush and Steranka

1978). The differential sensitivity may be due, in part, to species-dependent

differences in the half-life of the drug (Steranka and Sanders-Bush 1978).

Active neurotoxic metabolites or metabolic intermediates of parachloroamphetamine have been postulated previously as being responsible for its

neurotoxic effects on serotonin neurons (Gal and Sherman 1978; SandersBush and Steranka 1978), although to date no active neurotoxic species has

been identified. Although there has been no direct demonstration of a

neurotoxic metabolite of MDMA, some preliminary data suggest that an

active metabolite of MDMA may be responsible for eliciting its ncurotoxic

effects. The authors have previously reported that, in contrast to the

marked neurodegenerative effects on brain serotonin neurons following

systemic administration of MDMA or MDA, single, direct intracerebral

injections of MDMA or MDA were without effect on cerebral cortical

serotonin neurons, as visualized using immunohistochemistry (Molliver

et al. 1986). This observation of marked species differences and sensitivity

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FIGURE 3. The effects of repealed systemic adminisration of MDMA on

content of serotonin {5-HT) and 5-HIAA, and density of

5-HT uptake sites in rat, guinea pig, and mouse frontal

cerebral cortex

**Signifiantly different from control, p<0.001.

NOTE: Animals were treated with saline or 20 mg/kg MDMA twice a day for 4 consecutive days

and sacrificed 7 days after the last injection. Data represent ± SEM of five animals

per group, expressed as percent of saline-injected control values in respective species. In

rat, guinea pig, and mouse, control values of 5-HT were 275±41, 296±14, and 449±36

pg/mg tissue, respectively; control values of 5-HIAA were 345±40, 92±4. and 319±34

pg/mg tissue, respectively; control values of 5-HT uptake site were 397±10, 216±6, and

233±12 fmol/mg protein, respectively. Data were analyzed by Student’s t-test.

SOURCE: Battaglia et al. 1988.

202
to MDMA-induced serotonin neurotoxicity would be consistent with the

hypothesis of a peripherally produced neurotoxic metabolite of MDMA.

In more recent studies, it has also demonstrated that administration of 2.5 or

10 mg/kg MDMA twice daily for 4 consecutive days resulted in neurotoxic

effects in rhesus monkeys, with decreases in the density of scrotonin uptake

sites occurring at the higher dose (Johannessen et al. 1988). The neurotoxic effects of MDMA observed in primates included reductions in the

content of serotonin and 5-HIAA and marked reductions in the ccrebrospinal

(CSF) concentrations of 5-HIAA levels that were observed following drug

administration. These fmdings and other reports of neurotoxic effects of

MDMA in primates (Ricaurte et al. 1988) raise serious concerns for its

potential hazard in humans.

Potential Mechanisms

Since the neurotoxic effects of drugs such as parachloroamphetamine on

serotonin neurons can be prevented by serotonin uptake blockers (Ross

1976; Sanders-Bush and Steranka 1978). the possibility that serotonin uptake

carrier protein was likewise involved in the neurotoxic effects of MDMA

was investigated. As shown in figure 4, pretreatment of rats with the

selective serotonin uptake blocker citalopram (10 ml/kg), prior to each

injection of 10 mg/kg MDMA, resulted in nearly complete protection

against the neurotoxic effects of MDMA. Citalopram-pretreated rats

exhibited only a 15 percent decrease in serotonin uptake sites. No significant alterations in the content of serotonin and 5-HIAA were observed

following MDMA treatment, in comparison with 60 to 80 percent reductions

in the serotonergic parameters observed in rats treated with an identical dose

of MDMA alone.

The data described above demonstrate that destruction of serotonin axons by

MDMA involves the serotonin active uptake carrier and that administration

of citalopram, a selective serotonin uptake blocker, prior to administration of

MDMA, can prevent the decreases in serotonin markers elicited by MDMA

alone. These data are consistent with previous reports for other potent

serotonin neurotoxins, demonstrating that pretreatment with serotonin uptake

blockers can prevent the neurotoxic effects of parachloroamphetamine (Ross

1976; Sanders-Bush and Steranka 1978). Furthermore, it has been shown

that MDMA-induced neurotoxicity can be prevented or reversed if a serotonin uptake blocker such as fluoxetine is administered no later than

12 hours after MDMA treatment (Schmidt 1986).

In previous studies, it has been observed that, in contrast to the marked

serotonin neurodegenerative effects following systemic administration of

MDMA or MDA, single, direct intracerebral injections of MDMA or MDA

are without effect on cerebral cortical serotonin neurons, as visualized using

serotonin immunocytochemistry (Molliver et al. 1986). In addition, as

203
FIGURE 4. The effect of repeated systemic administration of 10 mg/kg

MDMA, MDMA plus 10 mg/kg citalopram, and MDMA plus

25 mg/kg SKF 525A on the density of serotonin (5-HT)

uptake sites in homogenates of rat frontal cerebral cortex

NOTE: Data are expressed as percent of values in control saline-treated rats and represent the mean

± SEM from four to six animals. Control levels of 5-HT uptake sites were 356±15 fmol/mg

protein.

described above, there appears to be a differential sensitivity to the neurotoxic effects of MDMA in different species, which metabolize amphetamines

by different pathways. These observations of marked differences in serotonin neurotoxicity to-centrally versus systemically administered MDMA and

differences in various species would be consistent with the hypothesis of a

peripherally produced neurotoxic metabolite of MDMA or MDA. Since

MDMA has been reported to interact with the hepatic microsomal enzyme

cytochrome P450 (Brady et al. 1986), the authors investigated whether

inhibition of this enzyme would alter the neurotoxic effects of MDMA was

investigated. In preliminary studies, it was found that, in rats pretreated

with the cytochrome P450 enzyme inhibitor SKF 525A, 45 minutes prior to

204
each administration of MDMA, there was neither potentiation nor attenuation of the neurodegeneration found following repeated administration of

10 mg/kg MDMA. As shown in figure 4, no changes in the density of

serotonin uptake sites were observed between rats treated with MDMA

alone and those treated with MDMA plus SKF 525A. Although these data

suggest that it is unlikely that the putative neurotoxic species is a

cytochrome P450-dependent metabolite of MDMA, the involvement of some

other peripheral and/or central metabolite of MDMA or the formation of an

MDMA-induced endogenous neurotoxin cannot be ruled out. Additional

studies are required to identify the mechanisms responsible for MDMAinduced neurotoxicity.

Regeneration of Serotonin Neurons

A detailed timecourse of recovery of affected serotonin neurons was carried

out to investigate whether serotonin neurons regenerate subsequent to their

destruction following MDMA treatment. As shown in figure 5, the

timecourse of neuronal regeneration was investigated by measuring the

recovery of serotonin uptake sites and serotonin levels in rat frontal cerebral

cortex at various timepoints up to 12 months following repeated systemic

administration (i.e., twice daily sc injections for 4 days) of 20 mg/kg

MDMA. At all timepoints up to 6 months during the recovery timecourse,

the density of serotonin uptake sites was significantly below the corresponding values in age-matched, saline-treated control rats. At the 6-month

timepoint, the density of serotonin uptake sites was only 75 percent of the

values of saline-treated controls, whereas by 12 months after MDMA

treatment, the density of serotonin uptake sites returned to control levels.

The shape of the recovery curve suggests that there may be a faster initial

rate of recovery of serotonin uptake sites occurring between 18 hours and

4 weeks, which is followed by a slower rate of recovery between 4 weeks

and 12 months. These data indicate that more than 6 months are required

for a complete recovery of serotonin uptake sites to control levels.

It was of interest that, in spite of the recovery of serotonin uptake sites to

control levels, the content of serotonin in the same brain region remained

markedly (40 to 50 percent) below age-matched controls for as long as

1 year after MDMA administration. It is unclear from these data whether

there is a regeneration of axons that have previously undergone degeneration

or whether the increased density of uptake sites is a consequence of

increased collateral sprouting of neurons unaffected by the drug treatment.

It is also possible that axonal regeneration and collateral sprouting arc

associated with considerably greater densities of uptake sites per neuron,

thereby making it more difficult to assess neuronal recovery from this

index. The fact that serotonin levels remain 40 to 50 percent below agematched controls for up to 1 year in spite of normal levels of serotonin

uptake sites indicates that, following lesion by MDMA, the serotonin

205
FIGURE 5. Timecourse of recovery of (A.) serotonin (5-HT) uptake sites

and (B.) 5-HT conten in rat cerebral cortex following

repeated systemic administration of MDMA

NOTE: Rats were treated with either saline or 20 mg/kg MDMA twice a day for 4 consecutive

days, then sacrificed at various times up to 12 months after the last injection of the drug.

Saline-injected control rats were killed at each of the timepoints; data represent the mean ±

SEM of five rats per group, plotted as percent of the value of age-matched saline-injected

control rats.

SOURCE: Adapted from Battaglia et al. 1988.

206
neurons that “recover” may not be functionally identical to those present in

age-matched control brains.

The persistent neurotoxic effects of MDMA on serotonin neurons is similar

to that observed following parachloroamphetamine administration, in which

marked reductions in serotonin have been observed up to 4 months after a

single injection of parachloroamphetamine (Fuller 1978; Kohler et al. 1978;

Sanders-Bush and Steranka 1978). Since neurochemical recovery of

serotonin uptake sites and serotonin content have been used, rather than

neuroanatomical indices of neuronal regeneration, it is unclear from the

present data whether there is actual regeneration of neurons that have

previously undergone axon or terminal degeneration or whether the

increased density of uptake sites is a consequence of increased collateral

sprouting of neurons unaffected by the drug treatment, It has previously

been reported that following 5,6-dihydroxytryptamine-induced axotomy,

axonal sprouting occurs within 4 to 5 days, and the appearance of new

axonal sprouts correlates with the recovery of [

3H]serotonin uptake

(Bjorklund et al. 1973). Evidence from both immunocytochemical data

(O’Hearn et al. 1988) and autoradiographic studies quantifying changes in

the density and distribution of [

3H]paroxetine-labeled serotonin uptake sites

(see figure 10) indicates that serotonin cell bodies appear to be insensitive

to the neurotoxic effects of repeated systemic administration of MDMA in

rats. The fact that serotonin cell bodies are unaffected by MDMA treatment

provides a mechanism by which terminal regeneration of MDMA-affected

neurons may occur in rats.

NEUROANATOMICAL AND MORPHOLOGICAL SPECIFICITY OF

THE EFFECTS OF MDMA AND MDA

The data described above clearly demonstrate the specific and marked

neurodegenerative effects of MDMA on serotonin axons and terminals in

the cerebral cortex. Two approaches have been taken to investigate whether

the effects of MDMA on brain serotonin neurons are ubiquitous or whether

the effects of MDMA show neuroanatomical specificity. The first approach

involved the measurements of various monoamines, their metabolites, and

monoamine uptake sites in homogenates of discrete areas of rat brain

following treatment with MDMA or MDA. Second, autoradiographic

techniques were used to visualize the effects of MDMA treatment on the

localization and density of [

3H]paroxetine-labeled serotonin uptake sites and

[

3H]mazindol-labeled NE and DA uptake sites in slide-mounted sections of

rat brain. In addition, to address further the neurochemical specificity of

the effects of MDMA on brain serotonin neurons, effects of MDMA and

MDA treatment on the content of DA, NE, and their respective mctabolites

and DA and NE uptake sites in homogenates of various brain regions will

be described. In some studies, the changes induced by the N-ethyl

derivative of MDA (MDE) have been investigated.

207
Effects of MDMA on Serotonin and 5-HIAA Content and

[

3H]Paroxetine-Labeled Serotonin Uptake Sites in Discrete Regions of

Rat Brain

The effects of repeated systemic administration of MDMA and MDA on

various serotonergic parameters were investigated at 2 weeks following the

last injection of the treatment regimen previously described (i.e., 20 mg/kg,

sc, twice daily for 4 days). As shown in figure 6, MDMA and MDA

produced marked decreases in the content of serotonin and 5-HIAA in

various brain regions. Both MDMA and MDA caused dramatic decreases

in 5-HIAA levels in cerebral cortex, hippocampus, striatum, and hypothalamus (figure 6B). In hypothalamus, the reduction in 5-HIAA levels elicited

by MDA was significantly greater (p<0.05) than that observed with MDMA

(figure 6B). When plotted as a percent of control values in the respective

brain regions, it was apparent that, while decreases in 5-HIAA content were

observed in all the brain regions examined. the reductions in cerebral cortex

and in hippocampus (40 to 60 percent) were greater than those observed in

striatum and in hypothalamus (30 to 40 percent). With respect to serotonin

levels, marked decreases were observed in cerebral cortex and hypothalamus

in both MDMA- and MDA-treated rats (figure 6A). While small decreases

were observed in hippocampal and striatal serotonin content following either

MDA or MDMA treatment, these reductions were found to be statistically

significant only in striatum (p<0.01) of MDMA-treated rats. Data calculated

as a percent of control serotonin levels in their respective brain regions

(figure 6A) indicate a more marked reduction in serotonin in cerebral cortex

(40 to 60 percent) than in hypothalamus (18 to 33 percent).

To determine whether changes in serotonin and/or 5-HIAA were a consequence of long-term suppression of serotonergic function in structurally

intact neurons or whether MDMA and MDA may be affecting a neurodegenerative process in each of the brain regions, we measured the density of

serotonin uptake sites in these brain regions. Both MDMA and MDA

caused substantial reductions in the densities of [

3H]paroxetine-labeled

serotonin uptake sites in all the brain regions examined. The densities of

serotonin uptake sites were calculated as a percent of the respective control

levels in cerebral cortex, hippocampus, striatum, hypothalamus, and midbrain

and are shown in figure 7. Significant reductions (all p<0.001) were

observed in cerebral cortex (60 to 70 percent), hippocampus (70 to

75 percent), striatum (50 percent), hypothalamus (40 to 50 percent) and

midbrain (50 to 60 percent). Interestingly, MDA produced a significantly

greater reduction in the density of serotonin uptake sites in cerebral cortex

than did MDMA. It has also been observed that MDE causes 40 percent

reductions in serotonin uptake sites in cerebral cortex with a comparable

treatment regimen, suggesting that this compound may be less toxic than

either MDA or MDMA. Scatchard analysis of [

3H]paroxetine saturation

208
FIGURE 6. Effect of repeated systemic administration of MDMA and MDA

on the concentration of (A) serotonin (5-HT) and (B)

5-HIAA in various brain regions

*Significantly different from control, p<0.05.

**Significantly different from control, p<0.01.

***Significantly different from control, p<0.001.

†Significantly different from MDMA-treated rats, p<0.05.

NOTE: Rats were injected subcutaneously twice daily for 4 days with drug (20 mg/kg) or saline

vehicle (1 mL/kg) and sacrificed 2 weeks after the last injection. 5-HT and 5-IIIAA levels

were measured using reversed phase HPLC. Data, plotted as percent of control values in

each brain region, represent the mean and SEM from four to six control and drug-treated

rats. Control values for 5-HT and 5-HIAA in each region were: cerebal cortex, 504±58

and 422±32; hippocampus, 4l0±67 and 684±89; striatum, 363±22 and 492±50; hypothalamus, 1605±55 and 997±42 pg/mg tissue, respectively. Data were analyzed by one-way

ANOVA and Duncan’s multiple range test.

SOURCE: Battaglia et al. 1987.

209
FIGURE 7. Effect of repeated systemic administration of MDMA and MDA

on the density of serotonin (5-HT) uptake sites in various

brain regions

***Significantly different from control. p<0.001.

†Significant difference between MDA and MDMA treatments, p<0.001.

NOTE: Rats were injected subcutaneously twice daily for 4 days with MDMA or MDA (20 mL/kg)

or saline vehicle (1 mL/kg) and sacrificed at 2 weeks a fter the last injection. Values were

determincd from saturation studies in each region except striatum and hypothalamus, where

the density of 5-HT uptake sites was assessed using a saturating concentration (0.25 nM) of

[

3H]paroxetine. No significant different from control KD values (10-20 pM) were

observed in either MDMA- or MDA-treated rats. Data, plotted as percent of the 5-HT

uptake site density observed in controls in each brain region, represent the mean and SEM

from three to six rats per group. Control values were: cerebral cortex, 338±10;

hippocampus, 360±17; striatum, 344±30; hypothalamus, 775±36; and midbrain, 570±16

fmol/mg protein. Data were analyzed by one-way ANOVA and Duncan’s multiple range

test

SOURCE: Battaglia et al. 1987.

data in control and drug-treated rats indicated that, in cerebral cortex,

[

3H]paroxetine binding was through a single population of binding sites as

indicated by the Hill coefficient values (1.02, 1.01, and 1.03 in control,

MDMA-. and MDA-treated rats, respectively). Furthermore, there were no

significant differences in the affimity of [

3H]paroxetine for the serotonin

uptake site (i.e., KD) between control and drug-treated rats (18.8, 20.8, and

17.9 pM in control, MDMA-, and MDA-treated rats, respectively).

Differences in the sensitivity in various brain regions to the effects of

MDMA and MDA are not unique, as previous data have demonstrated

differential sensitivity to the effects of methamphetamine (Ricaurte

et al. 1980) and parachloroamphetamine (Kohler et al. 1978; Fuller 1978)

on serotonergic systems in various brain regions. Biochemical and

210
histochemical data suggest that parachloroamphetamine primarily affects the

ascending serotonin systems, whereas the descending pathways are left intact

(Kohler et al. 1978; Fuller 1978).

Effects of MDA and MDMA on Catecholamine Neurons

In contrast with the marked and consistent effects of MDMA and MDA on

serotonergic systems, neither drug produced any widespread or consistent

changes in the levels of NE, DA, or their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) or homovanillic acid (HVA) in the various brain

regions examined (table 1). Small changes, however, were observed in

some brain regions. Both MDMA and MDA produced statistically significant increases in striatal DOPAC and cerebral cortical HVA content,

whereas only MDMA treatment resulted in an increase in hippocampal

DOPAC levels (table 1). Furthermore. neither MDMA nor MDA treatment

caused any significant reduction in the levels of [

3H]mazindol-labeled NE

uptake sites in cerebral cortex, hippocampus, or midbrain when compared

with the respective saline-treated controls (figure 8). Although a small

reduction was noted in NE uptake sites in hippocampus, this change was

not statistically significant. Similarly, no significant decreases were

observed in the number of [

3H]mazindol-labeled DA uptake sites in cerebral

cortex, hippocampus, striatum, and midbrain following treatment with MDA.

MDMA caused a statistically significant reduction (37 percent) in the

density of DA uptake sites only in midbrain.

The neurotoxic effects of MDMA and MDA appeared to be exerted preferentially on serotonergic neurons, as no widespread changes in a variety of

catecholamine markers were seen after chronic administration of these drugs.

The small increases in DOPAC and/or HVA that were seen in the cerebral

cortex, hippocampus, and striatum after chronic administration of these

MDA derivatives are comparable to similar increases in DA metabolite

levels that have been previously reported following both acute (Schmidt

et al. 1986) and chronic (Stone et al. 1986) administration of MDMA and

MDA. These alterations may reflect increases in DA turnover. Because

serotonin-containing terminals are present in high concentrations in midbrain

areas (substantia nigra and ventral tegmental area) and DA cell bodies

(Steinbusch 1983). the degeneration of serotonin terminals in these regions

after MDMA or MDA treatment may be responsible for the observed

changes in DA metabolites. Despite the small effects on DA turnover,

MDMA and MDA do not appear to produce any widespread destruction of

catecholaminergic terminals, as the only significant change observed was a

reduction in DA uptake sites in midbrain after administration of MDMA.

The reasons for the decrease in DA uptake sites are not clear at present.

Preliminary immunocytochemical data indicate that there are no changes in

the density or morphology of catecholamine axons after chronic

administration of MDMA or MDA (O’Hearn et al. 1988).

211
TABLE 1. Effect of repeated systemic administration of MDMA and MDA

on NE, DA, and DA metabolite levels in various regions of rat

brain

Brain Region NE

Concentration (pg/mg tissue)

DA DOPAC HVA

Cerebral Cortex

Control

MDMA

MDA

447 ± 53 59 ± 12 96 ± 14 19 ± 4

424 ± 1 3 72 ± 3 73 ± 5 32 ± 4*

404 ± 26 63 ± 4 94 ± 19 36 ± 5*

Hippocampus

Control

MDMA

MDA

528 ± 62 31 ± 9 39 ± 6 6±2

573 ± 34 1 3 ± 5 65 ± 12* 15 ± 5

608 ± 46 16 ± 4 32 ± 1 3 8 ± 52

Striatum

Control

MDMA

MDA

ND

ND

6,091 ± 596 3,212 ± 159 788 ± 58

6,974 ± 228 3,954 ± 320* 767 ± 46

ND 6,168 ± 569 3,669 ± 189* 890 ± 48

Hypothalamus

Control 3,320 ± 209 569 ± 40

MDMA 3,052 ± 159 475 ± 38

MDA 3,577 ± 148 585 ± 67

*Significant difference from saline-treated control rats at p<0.05.

228 ± 4 3 5 4 ± 4

200 ± 3 0 5 4 ± 5

229 ± 2 6 5 8 ± 3

KEY: ND=levels below the sensitivity of the assay. Data were analyzed by one-way ANOVA

and Duncan’s multiple range test.

NOTE: NE, DA, DOPAC, and HVA measured 2 weeks after administration of 20 mg/kg MDMA

or MDA, subcutaneously. every 12 hours for 4 consecutive days. Values represent the

mean ± SEM. n=4 to 6 rats.

SOURCE: Battaglia et al. 1981.

Autoradiographic Studies on [

3H]Paroxetine-Labeled Serotonin Uptake

Sites

In vitro autoradiographic studies of [

3H]paroxetine-labeled serotonin uptake

sites in control and MDMA-treated brains were carried out as previously

described (De Souza and Kuyatt 1987) to assess the neuroanatomic

localization of lesions induced by MDMA. Substantial reductions in

serotonin uptake sites (50 to 100 percent decreases) were observed in all

areas of cerebral cortex as early as 18 hours after a 4-day treatment

212
FIGURE 8. Effect of repealed systemic administration of MDMA and MDA

on the density of NE uptake sites in various brain regions

NOTE: Rats were injected subcutaneously twice daily for 4 days with MDMA or MDA (20 mg/kg)

or saline vehicle (1 mL/kg) and sacrificed 2 weeks after the last injection. NE uptake sites

were measured using 6 nM [

3H]mazindol in the presence of selective blockers as previously

described (Javitch et al. 1984). Data, plotted as percent of control values in each brian

region, represent the mean and SEM from six control. MDMA-, and MDA-treated animals.

Control values of NE uptake sites were: cerebral cortex. 164±6; hippocampus, 176±9;

midbrain, 157±13 fmol/mg protein.

SOURCE: Battaglia et al. 1987.

regimen (20 mg/kg MDMA twice daily); the reductions were maintained for

at least 2 weeks. As shown in table 2, cerebral cortical regions that

showed the most extensive destruction of serotonin uptake sites (i.e., more

than 90 percent) were the prefrontal (area 32), anterior cingulate (area 24).

entorhinal, and parietal cortex. Comparable decreases in serotonin uptake

sites were observed between day 0 (18 hours after last injection) and day

14 (14 days after last injection) in several regions of cerebral cortex such as

prefrontal, pyriform, frontal areas 8 and 10, entorhinal, and primary auditory

regions. In other areas of cerebral cortex, such as the sensory motor

regions, significant reductions in serotonin uptake sites were observed only

2 weeks after the treatment.

As shown in table 2 and figure 9, marked decreases in serotonin uptake

sites were observed following MDMA administration in all regions of

caudate putamen, olfactory tubercle, endopiriform nucleus, islands of Calleja,

and nucleus accumbens. Within the caudate putamen, some time-dependent

213
TABLE 2. Effects of repeated systemic administration of MDMA on the

regional decreases in [

3H]paroxetine-labeled serotonin

uptake sites

Brain Region Control MDMA

Cerebral Cortex

Prefrontal area 32

Cingulate area 24

Indusium griseum

Piriform

Frontal area 8

Frontal area 10

Sensorymotor

Parietal

Entorhinal

Primary auditory

Primary visual

Olfactory tubercle

Endopiriform nucleus

Islands of Calleja

2-

2

1

2

2

1+

2

l+

4

1

2+

4

3

3+

1

1

2

1

1+

ll0

1+

l2

2

2

1+

Basal Ganglia

Caudate putamen

dorsolateral

dorsomedial

ventrolateral

ventromedial

Nucleus accumbens

2 l1+ l3 2

2+ 1+

2 lSeptal Area

Medial septal nucleus

Lateral septal nucleus

Amygdala basolateral nucleus

4- 3+

3 2

4- 3+

Thalamus and Epithalamus

Anteroventral nucleus

Anteromedial nucleus

Anteroventral dorsomedial nucleus

Reuniens

Lateroposterior nucleus

Posterior nucleus

Posterioventromedial nucleus

Parafascicular nucleus

Lateral geniculate body

Medial geniculate body

Lateral habenula

3-

3

3-

4+

3

1

1

2

5-

2

3

0

0

0

1

1

1

1

l+

1+

1

l214
TABLE 2. (Continued)

2

Brain Region Control MDMA

Hypothalamus

Lateral nucleus 4 4-

Hippocampus

CA3 region

Dentate gyrus

Molecular layer

Parasubiculum

Presubiculum

2+ 1

2 1

2+ 1

3+

3 2

Midbrain

Inferior colliculus

Interpeduncular nucleus

Central gray

Superior colliculus

superficial layers

profundum

Substantia nigra

pars compacta

pars reticulata

paranigral nucleus

Ventral tegmental area

Dorsal raphe nuclei

Median raphe nuclei

3

3+

5+

3

2+

3+

3

2

2+

5+

5+

l3

5+

1

1

2

2+

3

2

5+

5+

Pons-Medulla

Locus coeruleus

Pontine reticular formation

5+ 5

2 2

Cerebellum (all lobules) 2 2

KEY: Relative density of [

3H]paroxetine binding sites: 1=0-50 fmol/mg tissue; 2=50-150 fmol/mg

tissue; 3=150-250 fmol/mg tissue; 4=250-400 fmol/mg tissue and 5=>400 fmol/mg tissue; +

and - values indicate the upper and lower limits, respectively, of each range.

NOTE: Data are based on observations from three animals per group. Rats were injected

subcutaneously twice daily for 4 days with MDMA (20 mg/kg) or saline (1 mL/kg) (control)

and sacrificed 14 days after the last injection. Anatomical terminology is derived from

Paxinos and Watson (1982). [

3H]Paroxetine binding sites were visualized by using a

saturating concentration (0.25 nM) of [

3H]paroxetine. Autoradiograms of rat brain were

generated using [

3H]Ultrofilm. Analysis of [3H]paroxetine-labeled serotonin uptake site

densities in the various brain regions was performed by computerized image analysis

densitometry. No correction for “grey-white” quenching of tritium was used.

215
SALINE

MDMA

FIGURE 9. Autoradiographic distribution of [

3H]paroxetine-labeled

serotonin uptake sites in coronal sections at the level of the

caudate putamen from (A) saline-treated and (B) MDMAtreated rats

NOTE: In these darkfield photomicrographs (tritium-sensitive Ultrofilm), autoradiographic grains (i.e.,

binding sites) appear as white spots; the tissue is not visible. The degree of nonspecific

binding defined in the presence of 2 μM citalopram was comparable for both treatments. In

(A), note the high density of serotonin uptake sites in cingulate cortex (CG), caudate putamen

(CPu), olfactory tubercle (Tu), islands of Calleja, and lateral septal nuclei (LS) in control

brains. In MDMA-treated animals (B), marked reductions were observed in most regions

except for the septal nuclei, which were relatively unaffected.

216
reductions in serotonin uptake sites were observed following MDMA

treatment. For example, equivalent decreases in serotonin uptake sites were

observed between day 0 and day 14 groups in the ventrolateral region,

while in dorsolateral and dorsomedial areas a significantly greater reduction

in serotonin uptake sites was observed only at the later timepoint. Other

brain regions that were sensitive to the neurodegenerative effects of MDMA

included various thalamic nuclei and regions of hippocampus. In contrast,

the dorsal and medial septal nuclei appeared to be less sensitive to the

neurotoxic effects of MDMA as the reductions (approximately 25 percent)

in serotonin uptake sites in these brain regions were not statistically

significant. Likewise, no significant reductions were observed in the

indusium griseum, which contains primarily serotonin axons of passage.

Within midbrain structures, regions containing serotonin projections appeared

to be more dramatically affected by MDMA than those containing scrotonin

cell bodies (figure 10). For instance, in both the superficial layers of

superior colliculus and profundum, serotonin uptake sites were reduced 85

to 90 percent, while in dorsal median raphe, central grey, and the ventral

tegmental region, there was little or no change after MDMA treatment.

Likewise, serotonin projections to substantia nigra pars compacta and

reticulata were markedly affected, while no changes in serotonin uptake sites

were observed in the interpeduncular nucleus and pontine reticular formation

up to 14 days following MDMA administration.

To assess the serotonergic selectivity of the neurodegenerative effects of

MDMA in brain, additional autoradiographic studies of NE and DA uptake

sites in brain regions containing catecholamine terminals and cell bodies

were carried out. NE and DA uptake sites were labeled using [

3H]mazindol

in the presence of specific blockers as previously published (Javitch

et al. 1985). With respect to NE uptake sites, no change was observed

from control levels of [

3H]mazindol binding sites in midbrain regions such

as locus coeruleus, interpeduncular nucleus, substantia nigra pars compacta,

or reticulata up to 14 days following MDMA treatment. In a number of

cerebral cortical regions that receive NE projections, [

3H]mazindol-labeled

NE uptake sites were not decreased 18 hours after treatment (i.e., day 0)

but rather appeared to be slightly increased at day 14. Consistent with the

minimal effects of MDMA on metabolic parameters associated with catecholamine neurons, there were no changes in the density of DA uptake sites,

when compared to levels in saline-treated rats, in either cell body regions

such as substantia nigra pars compacta and reticulata or terminal regions

such as caudate putamen, nucleus accumbens, and olfactory tubercle. These

results are therefore consistent with what has been observed in neurochemical studies in brain homogenates and indicate that the neurodegenerative

effects of MDMA appear to be confined primarily to serotonergic pathways,

since this treatment regimen did not reduce the density of uptake sites

associated with catecholamine-containing neurons. Additional studies are

necessary to assess further the neuroanatomic localization of any long-term

217
SALINE

MDMA

FIGURE 10. Autoradiographic distribution of [

3H]paroxetine-labeled

serotonin uptake sites in coronal sections at the level of

midbrain in (A) saline-treated and (B) MDMA-treated rats

NOTE: In (A), note the high density of serotonin uptake sites in control brain in regions containing

serotonin projections such as entorhinal cortex, superior colliculus (sc), presubiculum and

parasubiculum, as well as cell body regions such as dorsal (DR) and median (MR) raphe and

central grey. MDMA-treated animals exhibited marked reductions in [

3H]paroxetine binding

rites in presubiculum and parasubiculum, entorhinal cortex, and superior colliculus (sc), while

no changes in densities were observed in areas containing primarily serotonin perikarya such

as the raphe nuclei (DR and MR).

218
compensatory changes in NE projections or other neurotransmitter recognition sites that may occur as a consequence of MDMA lesion of scrotonin

pathways.

SUMMARY AND CONCLUSIONS

The data presented in this chapter provide strong evidence, from both

neurochemical and neuroanatomical studies, demonstrating that, following in

vivo administration of a number of methylenedioxy-substituted amphetamine

derivatives, there is widespread and long-lasting degeneration of scrotonin

neurons in brain, without any major or consistent effects on catecholamine

neurons. A detailed examination of the parameters involved in the

neurotoxic and neurodegencrative effects of MDMA on brain scrotonin

neurons indicates that:

(1) the severity of the lesion by MDMA is dependent on both the dose and

frequency of drug administration;

(2) the neurodegenerative effects of MDMA can be elicited in a number of

animal species including primates;

(3) the neurodegenerative effects on brain serotonin neurons can be

prevented by the serotonin uptake blocker, suggesting a role for the

active uptake of MDMA, a neurotoxic metabolite of MDMA, or an

unidentified endogenous neurotoxin; and

(4) the neurodegenerative effects of the drug are long-lasting (up to 1 year)

with respect to neuronal recovery, while functional recovery may be

permanently impaired.

In addition, the neurochemical and autoradiographic data suggest that there

is some neuroanatomical and morphological specificity to the neurodegenerative effects of MDMA and MDA, as evidenced by predominant reductions in serotonin uptake sites in brain regions containing primarily

serotonin terminals, while regions containing serotonin axons of passage and

cell bodies are relatively unaffected.

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ACKNOWLEDGMENTS

Drs. S.Y. Yeh, Thomas R. Insel, John Sharkey, and Michael J. Kuhar

contributed to various aspects of the work described. Supported in part by

the U.S. Food and Drug Administration.

AUTHORS

Errol B. De Souza, Ph.D.

Chief, Laboratory of Neurobiology

George Battaglia. Ph.D.

Assistant Professor

Department of Pharmacology

Loyola University Medical Center

Stritch School of Medicine

2160 South First Avenue

Maywood, IL 60153

222
Characterization of Brain

Interactions With

Methylenedioxyamphetamine and

Methylenedioxymethamphetamine

Robert Zaczek, Stephen Hurt, Steven Culp, and

Errol B. De Souza

INTRODUCTION

Methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine

(MDMA), like other amphetamine analogs, affect multiple in vitro monoamine parameters. These effects include stimulation of [

3H]serotonin and

[

3H]dopamine release (Johnson et al. 1986) and inhibition of serotonin,

dopamine, and norepinephrine uptake (Steele et al. 1987). In addition,

recent radioligand binding studies have demonstrated interactions of MDA

and MDMA at a variety of established postsynaptic brain recognition sites,

including serotonergic, adrenergic, and cholinergic receptors (Battaglia

et al. 1988).

Several studies have identified specific sites of interaction in brain that may

mediate the actions of amphetamine and its substituted analogs. Binding

sites for [

3H]amphetamine (Hauger et al. 1984) and [3H]fenfluramine

(Garattini et al. 1987) have been identified in discrete areas of rat brain.

These sites have similar characteristics in that both have high binding

capacities (amphetamine, Bmax=60 pmol/mg protein; fenfluramine, Bmax=63

pmol/g tissue), and binding to both sites is inhibited by sodium ions. To

date, two groups have studied interactions of [

3H]MDMA with rat brain.

Gehlert et al. (1985) reported high affinity specific binding (Kd=99 nM,

Bmax=31 fmol/mg protein) of [

3H]MDMA to rat brain membranes. However,

a subsequent study (Wang et al. 1987) suggested that these apparent binding

sites represent [

3H]MDMA association with glass fiber filter paper. The

present study reexamines the possibility of [

3H]MDMA as well as [3H]MDA

association with rat brain membranes. Also characterized is the nature of

[

3H]MDA association with brain membranes to evaluate the possible

importance of the binding site in mediating MDA’s neurochemical and

behavioral effects and to examine similarities between apparent [

3H]MDA

binding sites and those labeled by [

3H]amphetamine and [3H]fenfluramine.

223
Centrifugation assays were employed in all our studies to circumvent the

problem of [

3H]MDA and [3H]MDMA absorbing onto glass filters.

ASSOCIATION TO RAT BRAIN MEMBRANES

Assay for [

3H]MDA and [3H]MDMA Association

Assays were performed using a crude synaptosomal preparation of rat brain.

Male Sprague-Dawley rats were sacrificed by decapitation, and brains were

immediately dissected on ice for membrane preparation. Telencephalon

containing cerebral cortex, striatum, and hippocampus was used in all

experiments except in studies examining the regional distribution of the

binding sites. Brain areas were homogenized in 20 volumes of ice-cold

0.32 M sucrose using a smooth glass homogenizer equipped with a motordriven teflon pestle. The homogenate was centrifuged at 800 x g for

10 minutes at 4 °C to remove large particulate material, and the supematant

was removed and centrifuged at 20,080 x g. The resultant pellet was

resuspended in 20 volumes of the original wet weight in either ice-cold

50 mM Tris-HCl buffer (pH 7.1) alone or in the same buffer containing

0.27 M sucrose. Binding assays were performed in Beckman minivials

containing either 0.9 mL of 50 mM Tris-HCl (pH 7.1) or the same buffer

containing 0.27 M sucrose. The vials also contained 2 nM [

3H]MDA

(54 Ci/mmol) or [

3H]MDMA (74 Ci/mmol), competing drugs at various

concentrations, and 100 μL of each of the homogenates as indicated above.

Nonspecific binding was assessed by measuring the [

3H]MDA or

[

3H]MDMA incorporated into boiled tissue. All incubations were performed

for 90 minutes at 4 °C: unless indicated otherwise. Assays were terminated

by centrifugation at 32,008 x g at 4 °C. The vials were removed and the

supernatant fluid discarded. The pellets were superficially washed with

ice-cold water (7 mL three times), after which the excess water was wiped

from the inside of the vial and 5.0 mL of scintillation fluid was added to

each vial. The pellets were allowed to solubilize overnight, and the vials

were assessed for radioactivity by scintillation counting. Data from

saturation isotherms were analyzed by the nonlinear curve-fitting program

LIGAND (Munson and Rodbard 1980). Protein was measured by the

method of Lowry et al. (1951).

[

3H]MDA and [3H]MDMA Association to Rat Brain Membranes

Preliminary experiments to delineate the optimum conditions for [

3H]MDA

and [

3H]MDMA binding to rat brain membranes indicated that the best

signal-to-noise ratio was found using a crude synaptosomal preparation

incubated at 4 °C. While most experiments employed 50 mM Tris-HCl

(pH 7.1) as the incubation medium, we found that the addition of 0.27 M

sucrose to the incubation medium increased the specific binding approximately fivefold. Subsequent experiments were carried out under both

conditions. Other preliminary experiments suggested that [

3H]MDA and

224
[

3H]MDMA interacted with multiple sites in rat brain. A low affinity

[

3H]MDA binding site (apparent Kd>1.0 mM) was found to be resistant to

boiling of the synaptosomal preparation for 15 minutes. This site was

saturable, as indicated by a 30 percent inhibition of [

3H]MDA binding to

boiled synaptosomes by 1.0 mM MDA and a 56 percent inhibition of the

binding by 0.1 mM of the serotonin uptake blocker paroxetine. The indication of a saturable, nonspecific binding site for [

3H]MDA in boiled

membranes necessitated that we use boiled tissue to assess nonspecific

binding in all subsequent experiments.

The saturation profiles of [

3H]MDA and [3H]MDMA recognition sites in rat

brain synaptosomes in the presence of 0.27 M sucrose are shown in

figure 1. Both ligands exhibited shallow saturation curves, which extended

over 4 log units from approximately 100 nM to 1.0 mM MDA, suggesting

the presence of multiple binding sites. Data from [

3H]MDA saturation

experiments fit significantly (p<0.015) better to a two-site model, indicating

both high (Kd=887 nM, Bmax=23 pmol/mg protein) and low (Kd=45 μM,

Bmax=3.2 nmol/mg protein) affinity sites upon iterative nonlinear curvefitting analysis. Analysis of saturation data of apparent [

3H]MDMA binding

in the presence of sucrose also fit significantly better to a two-site model

(p<0.02; high affinity Kd=2.9 μM, Bmax=79 pmol/mg protein; low affinity

Kd=128 μM, Bmax=7.4 nmol/mg protein).

The effect of eliminating sucrose from the incubation medium of [

3H]MDA

binding assays is shown in figure 2. While the Eadee-Scatchard plot of the

data from [

3H]MDA saturation experiments performed in the absence of

sucrose was linear, suggesting [

3H]MDA binding to one population of sites,

the plot representing the data from experiments performed in the presence

of 0.27 M sucrose was curvilinear, consistent with [

3H]MDA binding to

multiple populations of sites (see above). Nonlinear curve-fitting analysis of

the data suggested a single apparent binding site for [

3H]MDA, when

incubated with synaptosomes in the absence of sucrose (Kd=2.8 μM,

Bmaxpmol/mg protein). Thus, removal of sucrose from the incubation

medium led to the elimination of low-affinity specific [

3H]MDA binding.

Similar to observations of [

3H]MDA binding, removal of sucrose from the

incubation medium led to a 61 percent decrease in the apparent specific

binding at 100 nM [

3H]MDMA.

The affinity (Kd values) observed for [

3H]MDA and [3H]MDMA binding

were similar to the effective doses (i.e., ED50 or K1 values) of MDA and

MDMA reported for various pre- and postsynaptic monoamine markers,

such as serotonin and dopamine release (Johnson et al. 1986), monoamine

transport (Steele et al. 1987), and multiple brain, ligand binding sites

(Battaglia et al. 1988).

225
FIGURE 1. Saturation of [3H]MDA and [3H]MDMA

brain synaptosomes

binding in rat

NOTE: lncreasing amounts of unlabeled MDA or MDMA were added to 1.0 mL of incubation buffer

containing 2 nM [

3H]MDA or 2 nM [3H]MDMA, respectively. Experiments were performed

on tissue in the presence of 0.27 M sucrose. Data are expressed as percent of [3H]MDA or 

[

3H]MDMA bound to tissue in the absence of added nonradioactive drug. Nonspecific

binding was assessed by measuring the amount of [

3H]ligand bound to boiled synaptosomes

incubated in the presence of 0.27 M sucrose.

The high capacities (i.e., Bmax value) of [

3H]MDA and [3H]MDMA binding

sites, as well as those that have been reported for [

3H]amphetamine binding

sites (60 pmol/mg protein) (Hauger et al. 1984) and [

3H]fenfluramine

binding sites (63 pmol/g tissue) (Garattini et al. 1987), argue against

bimolecular interactions of these drugs with monovalent protein-binding

sites. Although the mechanism by which sucrose acts to preserve low

affinity [

3H]MDA binding is yet to be determined, a similar phenomenon

has been observed for [

3H]amphetamine binding (Hauger et al. 1984). In

the latter study. a wash of tissue in isotonic sucrose prior to incubation was

reported to increase nearly threefold the specific binding over a wash with

50 mM Tris-HCl alone (Hauger et al. 1984).

Pharmacology of Specific [

3H]MDA Binding in Rat Brain

The pharmacology of [

3H]MDA binding was determined by examining the

effects of other monoamine reuptake blockers and related amphetamine

226
analogs on the inhibition of [

3H]MDA binding. The pattern of paroxetine,

desipramine (DMI), dimethoxymethamphetamine (DOM), and MDA inhibition of specific [

3H]MDA binding is shown in figure 3. Experiments were

performed in the presence of 0.27 M sucrose using 2 nM [

3H]MDA, conditions under which both high- and low-affinity [

3H]MDA binding sites are

labeled. Paroxetine was the most potent inhibitor (IC50= 1.6 μM) followed

by DMI (IC50= 5.9 μM), DOM (IC50 = 17 μM), and MDA (IC50 = 43 μM).

Analysis of paroxetine and DMI inhibition curves revealed Hill coefficient

values (nH) close to 1.00 (paroxetine, nH = 0.85; DMI, nH = 0.91). MDA and

DOM gave rise to much shallower inhibition curves (MDA, nH = 0.56; DOM,

nH = 0.53). providing additional evidence for the existence of multiple

apparent [

3H]MDA binding sites.

FIGURE 2. Eadee-Scatchard transformation of saturation data of

[

3H]MDA binding in rat brain synaptosomes

NOTE: Increasing amounts of unlabeled MDA were added to 1.0 mL of incubation buffer containing

2 nM [3H]MDA. Experiments were performed on tissue in the presence (open circles) and

absence (closed circles) of 0.27 M sucrose. Nonspecific binding was assesed by measuring

the amouut of [

3H]MDA bound to boiled synaptosomes incubated in the presence of 0.27 M

sucrose.

227
FIGURE 3. Inhibition of [

3H]MDA incorporation into synaptosomes

NOTE: [

3H]MDA binding assays were performed in 50 mM Tris-HCl (ph 7.1) containiag 0.27 M

sucrose and 2.0 nM [3H]MDA as described in the text. Results are expressed as percent

inhibition of specific [3H]MDA incorporation in the absence of inhibitors. Boiled tissue

blanks were performed at each concentration of drug.

The inhibition of [

3H]MDA binding by several other related compounds is

seen in table 1. All compounds were tested at a concentration of 10 μM

under conditions that favored the labeling of the high-affinity [

3H]MDA

binding site (zero sucrose, 2 nM [

3H]MDA) and at 100 μM concentration

under conditions designed to favor the study of the low-affmity [

3H]MDA

binding site (0.27 M sucrose, 3 μM [

3H]MDA). A significant positive

correlation (r2=0.80, p<0.01) between the relative inhibition potencies of the

test drugs at the high- and low-affinity [

3H]MDA binding sites was observed

upon linear regression analysis (figure 4A).

EFFECTS OF OSMOLARITY AND DETERGENTS

A possible explanation for the large capacity (i.e., high Bmax values) of

[

3H]MDA binding sites and stimulation of [3H]MDA binding by isotonic

sucrose is intrasynaptosomal internalization and sequestration of [

3H]MDA.

Studies of apparent chloride-dependent [

3H]glutamic acid binding (Pin

et al. 1984; Zaczek et al. 1987) have demonstrated this type of phenomenon

for labeled glutamate. This possibility was examined by measuring

[

3H]MDA binding in the presence of varying concentrations of sucrose and

228
estimating the relative synaptosomal volume by measuring [

3H]H2O

incorporation into synaptosomes in parallel experiments. As shown in

figure 5, decreasing the concentration of sucrose in the incubation medium

led to a decrease in the level of [

3H]MDA binding. This contrasted with an

increase in intrasynaptic volume, as indicated by an increase in the capacity

of the synaptosomes to retain [

3H]H2O. A significant negative correlation

(r2=0.84; p<0.02) was obtained when [

3H]H2O incorporation was correlated

with [

3H]MDA incorporation by linear regression analysis. These data argue

against a sequestration phenomenon, since the amount of [

3H]MDA binding

should increase with increasing vesicular volume, if intrasynaptosomal

internalization and sequestration was occurring.

TABLE 1. Pharmacology of inhibition of [

3H]MDA binding

Drug

Amphetamine

Mescaline

MDMA

N,N-DMT

PCA

DOM

Fenfluramine

Paroxetine

Desipramine

Imipramine

High-Affinity Low-Affinity

Binding Binding

47 ± 21 31 ± 13

36 ± 4 12 ± 6

48 ± 12 32 ± 3

59± 1 1 56 ± 20

84 ± 7 56 ± 8

70 ± 3 35 ± 8

63 ± 6 34 ± 9

100± 1 91 ± 8

96 ± 5 83 ± 9

96 ± 3 61 ± 15

NOTE: Inhibition of high-affinity [

3H]MDA binding was performed in 50 mM Tris-HCl (pH 7.1) in

the presence of 2 nM [3H]MDA to preferentially label the high-affinity site. Drugs were

tested at 10 μM concentrations for inhibition of high-affinity binding. Low-affinity

[

3H]MDA binding inhibition was performed in 50 mM Tris-HCl (pH 7.1) containing 0.27 M

sucrose in the presence of 3.0 μM [

3H]MDA to preferentially label the low-affinity site.

Inhibition was peformed using 100 μM concentrations of the drugs tested. Values represent

percent inhibition of specific [

3H]MDA binding (mean ± SEM) performed in the absence of

inhibiting drugs. Boiled tissue was no in simultaneous assays to assess nonspecific binding.

Another approach used to examine the possible existence of [

3H]MDA

sequestration into synaptosomes was to investigate the effects of the

detergents Triton X-100 and digitonin on the level of [

3H]MDA

incorporation into rat brain synaptosomes (table 2). Concentrations of

detergents lower than 0.01 percent did not affect specific [

3H]MDA binding.

Digitonin. at a concentration of 0.01 percent, caused a 30 percent decrease

(p<0.05) in the level of apparent [

3H]MDA binding as compared to control,

and 0.01 percent Triton caused a 71 percent decrease (p<0.01). These data

provide additional evidence against intrasynaptosomal internalization and

sequestration of [

3H]MDA since relatively high concentrations (0.01 percent)

229
FIGURE 4. Correlation between the relative inhibitory potencies of various

drugs at high- and low-affinity [

3H]MDA binding and

between drug lipophilicities and inhibition potencies of

[

3H]MDA binding

NOTE: Panel A represents the relationship between the relative inhibitory potencies of various drugs

at high- and low-affinity [3H]MDA binding. Percent inhibition by test drugs of low-affinity

[

3H]MDA binding is plotted vs. the inhibition of high-affinity binding. Panels B and C

represent the relation between the lipophilicity of test drugs and their ability to inhibit high

and low [3H]MDA binding, respectively. In both cases, the retention times of test drugs on

reverse-phase HPLC are plotted vs. percent inhibition of [

3H]MDA binding. Pearson’s

r values and levels of significance are derived from linear regression analysis of the data.

of the detergents were required to cause significant decreases in [

3H]MDA

incorporation into the tissue. Furthermore, these decreases were only

partial, which is in contrast to what is generally observed when labeled

230
substances am released from a membrane-intemalized pool by pore-forming

detergents, which abruptly release the total contents of membranesequestered compounds.

FIGURE 5. Effects of varying sucrose concentration on [

3H]MDA and

[

3H]water incorporation into rat brain synaptosomes

NOTE: Synaptosomes were prepared and incubated under standard procedures for [

3H]MDA

incorporation. The osmolarity of the incubation buffer was changed by the addition of

various concentrations of sucrose to 50 mM Ttis-HCl (pH 7.1). The incubation medium

contained either 100 nM [

3H]MDA or 2 million cpm of [3H]water. After a 90-min incubation

at 4 °C, the assays were terminated and assessed for radioactivity. Bars represent the percent

change in the level of radioactive H2O (shaded bars) end [

3H]MDA (open bars) incorporated

from the respective incorporation at 0.32 M surcrose.

The results of the osmolarity and detergent experiments indicate that MDA

is not internalized into synaptosomes to any appreciable degree. In addition, the [

3H]MDA binding assays were performed at 4 °C, demonstrating a

lack of dependence on physiological temperatures; this lack is characteristic

of membrane internalization phenomena. Since other investigators have

shown that [

3H]MDMA is not taken up into synaptosomes by a sodiumdependent mechanism (Wang et al. 1987), the internalization of MDA,

MDMA, and related compounds does not appear to be necessary for their

presynaptic actions to enhance the release and to inhibit the reuptake of

monoamines. Furthermore, the intraneuronal internalization of MDA or

231
MDMA is not likely to be involved in the ability of these compounds to

cause serotonin terminal degeneration. The preponderance of the evidence

supports the hypothesis that the association of [

3H]MDA with synaptosomes

is primarily with membrane elements.

TABLE 2. Effects of detergents on apparent [

3H]MDA binding

Detergent

Weight/Vol Percent

(Percent) Control

Triton X-100 .000l 95 ± 9

.001 98 ± 8

.01 37 ± 9**

Digitonin .0001 89 ± 9

.001 85 ± 11

.01 70 ± 4*

Difference significant at p<0.05 by ANOVA and Duncan’s multiple range test.

**Difference significant at p<0.01 by ANOVA and Duncan's multiple range test.

NOTE: Synaptosomes were incubated for 90 min at 25 °C in 50 mM Tris-HCl (pH 7.1) containing

0.27 M sucrose and 100 nM [3H]MDA. Results are expressed as percent of specific

[

3H]MDA binding in the absence of detergent. Results are the means ± SEM from three

seperate experiments done in duplicate.

Role of Lipophilicity in the Incorporation of [

3H]MDA Into Rat

Brain Synaptosomes

Compounds that were included in the pharmacologic profile of [

3H]MDA

binding were subjected to reverse-phase HPLC analysis to assess their

relative lipophilicity. Briefly, each compound (10 μg) was injected onto a

Waters Nova-Pak C18 column and eluted with a linear gradient from 95

percent buffer A:5 percent buffer B to 20 percent buffer A:80 percent

buffer B (buffer A=95 percent water, 5 percent acetonitrile, 0.1 percent

ammonium acetate; buffer B=20 percent water, 80 percent acetonitrile,

0.1 percent ammonium acetate). Detection was performed using a Waters

Model 441 UV detector at 214 nm. Figure 6 shows the reverse-phase

chromatographic elution pattern of MDA and related compounds, which are

listed in table 1. Since separation of compounds by reverse-phase chromatography is based upon the aqueous/organic partition coefficients of the

substances separated, this method gives an index of the relative lipophilicity of various compounds. The more lipophilic a compound is, the greater

its retention time on C18 material. The retention times, in minutes, of

the compounds tested in the present study were: amphetamine, 6.93;

mescaline, 6.93; MDMA, 10.6; N,N-dimethyltryptamine (N,N-DMT), 12.1;

232
FIGURE 6. Elution pattern of monoamine uptake blockers and

amphetamine analogs from reverse-phase HPLC

KEY: amph=amphetamine; mesc=mescaline; N,N-DMT=N,N-dimethyltryptamine

PCA=parachloroamphetamine; DOM=dimethoxymethamphetamine; DMI = desipramine; IMI=imipramine.

NOTE: HPLC detection of test drugs at 200 nm UV. Peak are named for the drugs they represent.

Peak identity was discerned by analyzing each drug individually and observing its retention

time and UV spectrum by photodiode array detection between 190 and 360 nm using a

Waters Model 990 PDA detector. Retention times are listed in the text.

parachloroamphetamine (PCA), 15.4; DOM, 18.8; fenfluramine, 23.4;

paroxetine. 30.9; DMI, 32.2; and imipramine, 34.7. Figure 4B and 4C

show the correlations that were found between the retention times of the

tested drugs and their levels of inhibition of high- and low-affinity

[

3H]MDA binding, respectively (table 1). A significant positive correlation

(r2=0.81, p<0.01) was observed between the HPLC retention times of the

tested drugs and their respective levels of inhibition of high-affinity

[

3H]MDA binding. Although a positive correlation was found between the

lipophilicity and the ability of test compounds to inhibit low-affinity

[

3H]MDA binding (r2=0.56), this correlation did not reach the level of

statistical significance.

As stated earlier, the primary site of association of [

3H]MDA with brain

synaptosomes is with membrane components, not with the intrasynaptic

space. While the phenolic ends of these compounds may enable them to

interact with hydrophobic environments of brain membrane components,

their polar side chains may inhibit the ability of these compounds to move

freely across the membranes, thus inhibiting internalization. The pKa of

233
amphetamine is 9.9, which indicates that over 99 percent of this drug and,

most likely, its structural analogs will be ionized at pH 7.4. The ability of

these compounds to enter the brain readily, which has been ascribed to their

supposed lipophilicity, may be due to the existence of a saturable transport

process for these drugs across the blood-brain barrier (Pardridge and Connor

1973). The high capacity for MDA and MDMA incorporation into brain

membranes could be explained by their absorption into a hydrophobic

membrane environment. We have shown positive correlations between the

lipophilicity of several monoamine uptake blockers and amphetamine

analogs and their relative inhibition potencies of both high- and low-affinity

[

3H]MDA binding (figure 4); however, this correlation is not perfect. For

instance, MDMA, which has a lower affinity (higher IQ for these sites of

interaction, is more lipophilic than MDA; fenfluramine, which is less potent

at inhibiting [

3H]MDA binding than PCA, is more lipophilic than the latter

compound. These data suggest that there may exist some level of structural

specificity beyond lipophilicity.

REGIONAL DISTRIBUTION OF APPARENT [

3H]MDA BINDING

The relative distribution of [

3H]MDA incorporation into p2

 preparations from

various regions of rat brain, liver, and kidney is shown in table 3.

Apparent [

3H]MDA binding had a heterogeneous regional distribution in

brain, being highest in neocortex and midbrain followed by medulla-pons,

striatum, and diencephalon. Brain levels of [

3H]MDA incorporation were

lowest in cerebellum, which had 50 percent fewer binding sites than

neocortex. Although [

3H]MDA incorporation was detected outside the brain,

levels were much lower than those found in most of the brain structures

studied. The level of [

3H]MDA binding in liver was 40 percent and that in

kidney 20 percent of that found in neocortex. The regional distribution of

[

3H]MDA incorporation in the absence of sucrose had a profile similar to

that performed in the presence of 0.27 M sucrose (data not shown). There

was a significant positive correlation between the regional levels of apparent

binding studied under the two conditions (r2=0.84, p<0.61). These data,

together with those indicating a significant correlation between the relative

inhibition potencies of MDA analogs at high- and low-affinity [

3H]MDA

binding, suggest an intimate relationship between the high- and low-affinity

[

3H]MDA binding sites. Although there exist some differences in the

pattern of regional distribution between [

3H]MDA binding found in the

present study and that of [

3H]amphetamine binding found by Hauger

et al. (1984). similarities also exist. For example, the lowest level of

binding in brain for both ligands is found in the cerebellum and low levels

of binding are found for both ligands in the periphery. The differences

among the regional distributions of [

3H]amphetamine, [3H]MDA, and

[

3H]fenfluramine (Garattini et al. 1987) binding may be attributed to the

various membrane preparation and assay procedures used in each study.

234
TABLE 3. Regional distribution of apparent [

3H]MDA binding

Region

[

3H]MDA Bound

(pmol/mg protein)

Neocortex 31 ± 4

Striatum 24±3

Diencephalon 24±5

Midbrain 29 ± 5

Medulla-Pons 26±5

Cerebellum 15 ± 3

Liver 10 ± 1

Kidney 6±2

NOTE: A p2 preparation was prepared from each dissected region and assayed for [

3H]MDA

incorporation as described in the text. Incubation was performed in 50 mM Tris-HCI

(pH 7.1) containing 0.27 M sucrose and 2.0 nM [

3H]MDA. [3H]MDA incorporation into

boiled tissue served as the measure of nonspecific binding. Values are expressed as specific

[

3H]MDA bound (mean ± SEM; pmol/mg protein) and are the results of three experiments

performed in triplicate.

Concentration of MDA in Brain After Systemic Administratian

To elucidate further the relevance of the high-nanomolar to low-micromolar

affinities of tbe [

3H]MDA binding sites, we determined the brain concentrations of MDA following systemic administration of behaviorally active doses

(20 mg/kg) of the drug to rats. Rats were injected subcutaneously with 20

mg/kg MDA containing 0.5 μCi of [

3H]MDA. Rats were sacrificed at

various times after injection, and the hippocampus was removed, weighed,

and solubilized overnight in Soluene 100 (Packard). Econoflour (5 mL)

(NEN) was then added to the solution, and radioactivity was assessed by

liquid scintillation counting. To evaluate the identity of the radioactivity, rats were sacrificed 45 minutes after injection, and brains were removed

and homogenized in 0.1 M sodium acetate using a polytron (10 seconds,

position 6). After centrifugation at 30,000 x g, the supernatant fluid was

collected and 4.0 mL was applied to a C18 Sep Pak cartridge (Waters).

The cartridge was washed with 3.0 mL of water, and radioactivity was

eluted in 1.0 mL of acetonitrile containing 0.1 percent trifluoroacetic acid.

After drying the organic eluate to approximately 200 μL under nitrogen,

50 μL of the extract was injected onto a Waters Nova Pak C18 column and

eluted at 1.0 mL per minute with a linear gradient from 3 percent acetonitrile:97 percent 50 mM potassium phosphate buffer (pH 6.4) to 80 percent

acetonitrile: 20 percent water over 30 minutes. Eluted compounds were

detected by UV absorbance at 214 nm. Fractions (1.0 mL) of the column

eluate were collected and added to vials containing 5.0 mL of formula 963,

which were then assessed for radioactivity by scintillation counting.

235
Accumulation and Clearance Findings

Figure 7A shows the timecourse of [

3H]MDA accumulation and clearance

from rat brain after a subcutaneous injection. Peak concentration, which

was reached at 45 minutes, was equivalent to 165 μM (36 μg/g). To

verify the identity of the radioactivity as [

3H]MDA, reverse phase chromatographic analysis was performed on brain extract from a rat 45 minutes

after MDA (20 mg/kg) injection (figure 7B). Radioactivity eluted as a

single peak at 14 minutes, which coincided with a peak having a retention

time of 13.8 minutes observed by UV detection. This peak, which was not

observed in naive animals, was isochromatographic with standard MDA.

Thus, the injected material reaching the brain at 45 minutes was MDA, not

a metabolite. The level of MDA found in brain indicates that the Kd values

of MDA’s interaction with rat brain synaptosomes are within the range of

the brain concentrations of MDA reached following administration of

behaviorally active doses of the drug.

SUMMARY

Brain recognition sites have been identified for [

3H]MDA and [3H]MDMA.

The dissociation constants of MDA and MDMA for these sites are similar

to the concentrations needed to affect several brain neurochemical

parameters and are in keeping with concentrations of MDA in brain

(165 μM) following administration of behaviorally active doses (20 mg/kg)

of the drug. While the characteristics of these binding sites suggest a

possible hydrophobic interaction with brain membranes, this interaction is

not without specificity, since it has a unique pharmacology and a

heterogeneous distribution in brain.

Similarities have been found between [

3H]MDA binding studied in the

present report and that of apparent [

3H](+)amphetamine binding studied by

Hauger et al. (1984). Both have extremely high Bmax values, are optimal in

p2 preparations, are stabilized by sucrose, and share similar patterns of

regional distribution. Measuring the specific binding of [

3H]amphetamine,

[

3H]fenfluramine, [3H]MDA, and related compounds under identical

conditions will be required to determine the possible relationships among

the interactions of these compounds with brain membranes. Further study is

also needed to determine the possible importance such interactions of

amphetamine and its substituted analogs may have with brain membranes in

relation to the pharmacology of these substances.

236
FIGURE 7. Determination of MDA concentration in rat brain following

administration of behaviorally active doses of the drug

NOTE Panel A shows concentration of hippocampal MDA at various timer after adminitration of

20 mg/kg MDA containing 0.5 uCi of [

3H]MDA. Each point represents the average MDA

concentration of four hippocampi from two animals. No regional variation was observed in

the analysis of MDA concentrations in other brain areas. Panel B shows the reverse-phase

chromatographic elution profile of radioactivity extracted from MDA-treated rats.

237
DISCUSSION

QUESTION: Do you know if there is cell activity at the site?

ANSWER: It has not been tested. We have not done much pharmacology

at all.

QUESTION: Do you know if your regional variations in this binding site

correlate at all with myelin?

ANSWER: No, I do not know offhand the concentration of myelin in

those areas. In fact, I did the profile to prove that it was not lipophilicity,

so I was not expecting that. I did not have the foresight to look at the

concentration of myelin.

REFERENCES

Battaglia, G.; Brooks, B.; Kulsakdinun, C.; and De Souza, E.B.

Pharmacologic profile of MDMA (3,4-methylenedioxymethamphetamine)

at various brain recognition sites. Eur J Pharmacol 149:159-163, 1988.

Garattini, S.; Mennini. T.; and Samanin, R. From fenfluramine racemate to

d-fenfhuamine. Ann NY Acad Sci 499:156-166, 1987.

Gehlert, D.R.; Schmidt, C.J.; Wu, L.; and Lovenberg, W. Evidence for

specific methylenedioxyamphetamine (ecstacy) binding sites in the rat

brain. Eur J Pharmacol 119:135-136, 1985.

Hauger, R.L.; Hulihan-Giblin, B.; Skolnick, P.; and Paul, S.M.

Characteristics of [

3H](+)amphetamine binding sites in the rat central

nervous system. Life Sci 34:771-782, 1984.

Johnson, M.P.; Hoffman, A.J.; and Nichols, D.E. Effects of the

enantiomers of MDA, MDMA and related analogues on [

3H]-serotonin

and [

3H]dopamine release from superfused rat brain slices. Eur J

Pharmacol 132:269-276, 1986.

Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; and Randall, R.J. Protein

measurement with folin phenol reagent. J Biol Chem 193:265-275, 1951.

Munson, P.J.. and Rodbard, D. LIGAND: A versatile computerized

approach for characterization of ligand binding systems. Anal Biochem

107:220-237. 1980.

Pardridge, W.M., and Connor, J.D. Saturable transport of amphetamines

across the blood-brain barrier. Experientia 29:302-304, 1973.

Pin, J.-P.; Bockaert, J.; and Recasen, M. The Ca2+/Cl-dependent

L-[3H]glutamate binding: A new receptor or a particular transport

process? FEBS Lett 175:31-36, 1984.

Steele, T.D.; Nichols, D.E.; and Yim, G.K.W. Stereochemical effects of

3,4-methylenedioxymethamphetamine (MDMA) and related amphetamine

derivatives on inhibition of uptake of [

3H]monoamines into synaptosomes

from different regions of rat brain. Biochem Pharmacol 36:2297-2303,

1987.

238
Wang, S.S.; Ricaurte, G.A.; and Peroutka, S.J. [

3H]3,4-methylenedioxymethamphetamine (MDMA) interactions with brain membranes and glass

fiber filter paper. Eur J Pharmacol 138:439-443, 1977.

Zaczek, R.; Arlis, S.; Markl, A.; Murphy, T.; Drucker, H.; and Coyle, J.T.

Characteristics of chloride dependent incorporation of glutamate into brain

membranes argue against a receptor binding site. Neuropharmacology

26:281-287, 1987.

AUTHORS

Robert Zaczek, Ph.D.

Steven Culp, B.S.

Errol B. De Souza. Ph.D.

Neurobiology Laboratory, Neuroscience Branch

Addiction Research Center

National Institute on Drug Abuse

P.O. Box 5180

Baltimore, MD 21224

Stephen Hurt, Ph.D.

Dupont Company

Boston, MA 02118

239
Pharmacologic Profile of Amphetamine Derivatives at Various Brain

Recognition Sites: Selective

Effects on Serotonergic Systems

George Battaglia and Errol B. De Souza

INTRODUCTION

The amphetamines have found widespread use in a number of clinical

conditions, including narcolepsy, manic-depressive psychosis, orthostatic

hypotension, nasal congestion, migraine, asthma, hyperactivity, and obesity.

Although amphetamine (phenylisopropylamine) is structurally a simple

molecule, modification of the compound at the aromatic ring, side chain, or

terminal amino group can considerably change the pharmacological specificity of the resulting compound. Amphetamine itself is a potent central

nervous system (CNS) stimulant and anorectic agent, which acts primarily

by blocking catecholamine uptake and causing neurotransmitter release. The

addition of a hydroxy group on the beta carbon atom reduces both the

stimulant and anorectic effects of the compound, while addition of a second

alpha methyl group to amphetamine preferentially attenuates the CNS stimulant properties. Anorectics with reduced stimulant and cardiovascular effects

can be created by insertion of groups onto the side chain, terminal amino

group, or aromatic ring. Aromatic ring substitution by a number of substituents, including methoxy groups, have been shown to markedly alter the

pharmacologic specificity of the drug, from a catecholaminergic agent to

one exerting effects primarily on serotonergic systems (Loh and Tseng

1971). For example, paramethoxylation of amphetamine was found to

increase greatly the blockade of serotonin uptake and increase the release of

[

3H]5-HT, while uptake and release of dopamine were found to be attenuated (Loh and Tseng 1971). Since a substantial amount of data have

implicated the involvement of brain serotonergic systems in the mechanism

of action of hallucinogenic agents (Downing 1964; Brawley and Duffield

1972; Freedman and Halaris 1978; Glennon and Rosecrans 1981; Glennon

1983), it would not be unexpected for a number of ring-substituted

psychotomimetic amphetamines to elicit their behavioral and/or subjective

effects via their preferential and potent interaction with central serotonin

recognition sites.

240
The psychotomimetic mono- and dimethoxyamphetamines have been

reported to produce a number of subjective effects similar to those elicited

by agents such as LSD and mescaline (Shulgin et al. 1969; Snyder

et al. 1969). Indeed, some of the most potent hallucinogens have been

ring-substituted structural analogs of amphetamine. Since the actions of

psychotomimetic amphetamines may be mediated at presynaptic serotonin

recognition sites, as well as at one or more of the postsynaptic serotonin

receptor subtypes (Titeler et al. 1987). it is important to develop a relative

pharmacologic profile for these drugs at the various serotonin binding sites.

This chapter will (1) elucidate the serotonergic sites of action of various

ring-substituted psychoactive derivatives of amphetamine with an emphasis

on derivatives of 2,5-dimethoxyamphetamine (2,5-DMA); (2) describe a

detailed pharmacological profile of the newer types of psychoactive

methylenedioxy-substituted amphetamine derivatives, the so-called “designer

drugs” such as 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-

methylenedioxyamphetamine (MDA); and (3) elucidate the role of various

serotonergic brain recognition sites in mediating some of the behavioral

and/or subjective effects of these methylenedioxy derivatives of

amphetamine.

INTERACTIONS OF 2,5-DMA DERIVATIVES WITH SEROTONIN

RECEPTORS

As mentioned, aromatic ring substituents can greatly enhance the serotonergic activity (Cheng and Long 1973; Cheng et al. 1974; Dyer et al. 1973;

Nair 1974) of the amphetamines. Substitution of methoxy groups in the 2,5

position and further substitution of substituents in the para position of the

phenyl ring of amphetamines markedly enhances their affinity for serotonin

receptors. The advent of the drug discrimination paradigm and its application to the study of such hallucinogenic agents (Hirschhorn and Winter

1971; Silverman and Ho 1980; Glennon et al. 1982; Glennon 1983; Appel

et al. 1982) has greatly enhanced our understanding of the putative sites of

action of hallucinogenic agents and of the similarities among various

hallucinogenic compounds. These studies have demonstrated a significant

correlation between the potencies of numerous agents in eliciting intero

ceptive hallucinogenic cues in animals and humans (Glennon et al. 1982;

Glennon 1983). Drs. Glennon, Titeler, and their collaborators have carried

out a series of behavioral and radioligand binding studies to elucidate the

serotonin receptor subtype(s) that may be primarily responsible for the

actions of these psychoactive agents. Specifically, these studies involved a

detailed determination of the affinities of various 2,5-dimethoxy derivatives

of amphetamine at 5-HT1 and 5-HT2 serotonin receptors (table 1) using

radioligand binding techniques to directly label these sites.

The structures of some of these agents are shown in figure 1. Compounds

listed in figure 1A are either hallucinogenic in man or produce

241
TABLE 1. Affinities of 2,5-DMA derivatives for 5-HT1

 and 5-HT2

serotonin receptors

5-HT2 Bindinga5-HT1 Bindingb

Ki(5-HT1)/

Agent Ki

(nM) (Hill Coefficient) Ki(nM) Hill Coeficient Ki

(5-HT2)

R(-)-DOI 9.9 2,290 (0.98) 230

(±)-DOI 18.9

(0.72)

(0.73)

S(+)-DOT 35 (0.66)

2,240 (0.86) 120

920 (0.73) 26

R(-)-DOM 60

63

(0.71) 3,550 (0.84)

(±)-DOB (0.80) 38,340 (0.77) 50

(±)-DOM 100 (0.71) 2,890 (0.82) 30

-demethyl DOM 110 (0.77) (0.72) 3

R(-)-DON 210 (0.75)

350

(0.79)

(0.91)

13,300 (0.85) 60

(±)-DON 300 14,100 (1.0) 45

R(-)-N-methyl DOM 260

(0.83)

4,300 (0.86) 15

(±)-N-methyl DOM 415 3,870 (0.86) 10

(±)-DOF 1,110

(±)-2,4,5-TMA 1,650

(0.76) 3,470 (0.97) 3

(0.68)

(0.77)

46,800 (0.49) 30

(±)-4-OEt2,5-DMA 2,220 35,500 15

(±)-4-Me PIA 3,360 (0.89) 14,800

(0.83)

(0.96)

(±)-2,5-DMA 5,200 (0.85) 1,020 (0.75)

4

< 1

(±)-PMA 33,600 (0.87) 79,400 (0.97) 2

(±)-3,4-DMA 43,300 (0.66) 64,600 (0.94) 1

NOTE: aKi values and Hill coefficients determined by competition experiments for 0.4 nM [3H]ketanserin-labeled

5-HT2 serotonin binding sites in rat frontal cortical homogenates. Data from Shannon et al. 1984.

bKi

 values and Hill coefficents determined by competition experiments for 1.0 nM

[

3H]LSD(+[10-3M]Ketanserin)-labeled 5-HT1

 sites in rat frontal cortical homogenates. Data from Shannon

et al. 1984.

cData from Glennon et al. 1986

hallucinogen-like responding in behavioral studies, while agents listed in

figure 1B do not generalize to a hallucinogen cue. As shown in table 1,

although 2,5-DMA itself exhibits higher affinity for 5-HT1 versus 5-HT2

serotonin receptors, all of the derivatives of 2,5-DMA exhibit substantially

higher affinity for 5-HT2 serotonin binding sites and appear to interact more

selectively with this site than do tryptamine agonists such as serotonin. The

selectivity of 2,5-DMA derivatives for 5-HT2 serotonin receptors is particularly marked for compounds with substituents in the para position. For

example, some of the para-halogenated compounds such as the iodinated

(DOI) and brominated (DOB) derivatives demonstrate an extremely high

affinity and degree of selectivity in their interactions with 5-HT2 serotonin

receptors.

Nearly all the derivatives of 2,5-DMA exhibited radioligand binding characteristics at 5-HT2

 serotonin receptors that were consistent with those of

serotonin and other tryptamine agonists. It has been demonstrated

242
R' R'' R2

 R4 R 5

(A) 2,5-DMA derivatives

(±)-2,5-DMA C H 3

 H OCH 3

 H OCH3

(±)-2,4,5-TMA C H 3

 H OCH 3

 OCH 3 OCH 3

(±)-4-OEt 2,5-DMA C H 3

 H OCH 3

 OC2 H 5 OCH 3

(±)-DOF C H 3

 H OCH 3

 F OCH3

(±)-DOB C H 3

 H OCH 3

 Br OCH3

(±)-DOI C H 3

 H OCH 3

 I OCH3

R(–)-DOI C H 3

 H OCH 3

 I OCH3

(±)-DON C H 3

 H OCH 3

 NO 2 OCH3

R(–)-DON C H 3

 H OCH 3

 NO 2 OCH 3

(±)-DOM C H 3

 H OCH 3 CH 3 OCH3

R(–)-DOM C H 3

 H OCH 3 CH 3 OCH3

-Demethyl DOM H H OCH 3

 CH 3 OCH3

(±)-N-Methyl DOM C H 3 CH 3 OCH3 CH3 OCH3

R(–)-N-Methyl DOM CH3 CH3 OCH3 CH3 OCH3

(B) Non-2,5-DMA derivatives

(±)-PMA C H 3

 H H OCH 3 H

(±)-3,4-DMA C H 3

 H H OCH 3 OCH3

(±)-4-Me PIA C H 3

 H H CH3

 H

FIGURE 1. Structures of a series of 2,5 DMA and non-2,5 DMA

derivatives

SURCE: Shannon el at. 1984.

previously that classical serotonergic agonists of the hyptamine class interact

with high- and low-affinity states of the 5-HT2 serotonin receptor (Battaglia

et al. 1984). Agonist-like properties of serotonin-related compounds were

243
initially revealed by Hill coefficient (nH) values of less than one in

radioligand binding studies. Values of less than one for nH suggest interaction of the compound with multiple binding sites or multiple states of the

receptor. As shown in table 1, nearly all the derivatives of 2,5-DMA

exhibited nH values of less than one at 5-HT2, serotonin receptors, suggesting

an agonist-like activity at this receptor. Data from competition experiments

can be further quantitated using a computer-assisted two-site analysis program (Munson and Rodbard 1980). Computer-assisted two-site analysis for

the interactions of a number of the 2,5-DMA derivatives with 5-HT2 serotonin receptors indicates that these compounds do indeed interact with highand low-affinity states of 5-HT2

 serotonin receptors, with the percentage of

binding sites in the high-affinity state for 2,5-DMA derivatives being

comparable to that observed for tryptamine agonists (table 2) (Battaglia

et al. 1984). The agonist high-affinity state of 5-HT2 serotonin receptors

has also been shown to be sensitive to divalent cations and guanine nucleotides (Battaglia et al. 1984; Titeler et al. 1985), as previously demonstrated

for agonists interacting with receptors coupled to a guanine nucleotide regulatory protein. Consistent with other agonist characteristics and, as shown

in figure 2, DOI, the 4-iodo-DMA derivative, exhibited guanine nucleotide

sensitivity. This is revealed by the decrease in overall affinity (K1 value)

and increase in nH closer to one in the presence of 5’-gyanylimidodiphosphate (Gpp(NH)p) (figure 2). Furthermore, derivatives such as DOI, DOB,

and DOM exhibited substantially higher overall affinities (K1) and higher

affinities at the high-affinity component (KH of 5-HT2 serotonin receptors

than did a number of tryptamine agonists at this site (table 2) (Battaglia

et al. 1984; Shannon et al. 1984). With respect to stereospecificity, the R(-)

isomers of DOI and other 2,5-DMA derivatives were the more potent isomers at 5-HT2

 serotonin receptors, while the S(+) isomers of methoxyamphetamines were more potent at presynaptic serotonin recognition sites. In

the last few years, [

125I]-DOI (Glennon et al. 1988) and [3H]-DOB (Titeler

et al. 1985; Lyon et al. 1987) have proven to be highly selective agonist

radiolabels for the high-affinity component of 5-HT2 serotonin receptors.

Subsequent studies investigating the affinities of these and additional

hallucinogenic phenylisopropylamines at 5-HT2 serotonin receptors have

clearly established a prominent role for 5-HT2 serotonin receptors in the

hallucinogenic process. Significant correlations were demonstrated between

the in vitro affinities of a series of amphetamine derivatives at 5-HT2

serotonin receptors and both their human hallucinogenic dose and their ED50

values in behavioral generalization to a hallucinogen cue (Glennon

et al. 1984; Titeler et al. 1988).

Although initial studies indicated that the various derivatives of 2,5-DMA

exhibited low affinity for 5-HT1 serotonin receptors (Shannon et al. 1984). it

was unclear from these studies what the affinities of the drugs were for the

respective subtypes of 5-HT1serotonin sites (i.e., 5-HT1A, 5-HTIB, and

5-HT1C receptors). In subsequent studies (Titeler et al. 1988), the affinities

244
TABLE 2. Two-site analysis of the interaction of tryptamine agonists and

2,5-DMA derivatives with 5-HT2 serotonin receptors

KH KL %RH KI

/KH

2,5 DMA Derivativesa

R(-)-DOI 1.5 ± 0.5

(±)-DOI 2.3 ± 1.0

R(-)-DOM 2.7 ± 1.6

(±)-DOB 2.4 ± 0.7

a-demethyl DOM 35 ± 11

R(-)-DON 68 ± 29

(±)-DON 137 ± 49

(±)-2,4,5-TMA 200 ± 60

(±)3,4-DMA 3,100 ± 950

30.0 ± 5.1

47.4 ± 16.8

190 ± 30

245 ± 90

100 ± 25

400 ± 110

900 ± 400

1,500 ± 730

6,250 ± 1,200

80,600 ± 19,000

40 ± 5 2 0

34 ± 9 21

22 ± 5 71

50 ± 9 17

19 ± 1 4 2

52 ± 2 12

55 ± 18 13

65 ± 19 11

41 ± 6 31

25 ± 5 26

Tryptamine Derivativesb

Serotonin 30 ± 3 1,173 ± 66 25 ± 4 39

5-Methoxytryptamine 130 ± 26 2,659 ± 550 45 ± 7 20

Bufotenine 96 ± 17 1,043 ± 220 35 ± 8 1 1

Tryptamine 302 ± 48 4,193 ± 570 15 ± 4 14

NOTE: Data were computer-analyzed with a two-site model (Munson and Rodbard 1980). KH

represents the dissociation constant of agonists clculated for the high-affinity component of

[3H]ketanserin binding. KL is the dissociation constant calculated for the low-affinity

component of [3H]ketanserin competition curves. KI/KHis the ratio of the two dissociation . .

constants. %RHrepresents the percentage of sites in a high-form for the agonist.

SOURCE: Shannon et al. 1984; Battaglia et al. 1984.

of a comparable series of psychoactive amphetamine derivatives were

compared at 5-HT1A, 5-HT1B, and 5-HT1C serotonin receptors. As shown in

table 3, all derivatives exhibited relatively low affinities at 5-HT1A and

5-HT1B serotonin receptors but markedly higher affinities at the 5-HT1C

serotonin receptor. Although these data suggest that 5-HT1C sites may

contribute to some of the effects of these psychoactive amphetamines, the

precise role of the 5-HT1C serotonin receptors in the hallucinogenic process

or other effects of these drugs remains unclear at the present time.

PHARMACOLOGIC PROFILE OF MDMA AT VARIOUS BRAIN

RECOGNITION SITES

Psychotomimetic amphetamines such as mescaline and DOM (STP) have

experienced periods of popularity during the last two decades. In recent

245
FIGURE 2. Competition curves of R(-)-DOI for [

3H]ketanserin binding to

5-HT2 serotonin receptors in rat frontal cortex membranes

in the presence and absence of the guanine nucleotide

analog Gpp(NH)p

NOTE: Lines represent the best fit of the data acording to a model for tow biding sites [in the

absence of Gpp(NH)p] and a model for one binding site [in the presence of Gpp(NH)p].

SOURCE: Shannon et al. 1984

years, a new class of designer drug, the methylenedioxyamphetamine derivatives, has received a great deal of attention. These compounds, which

include MDMA, MDA, and MDA’s N-ethyl derivative MDE, have been

reported to elicit both moderate “amphetamine-like” stimulant and weak

“LSD-like” hallucinogenic effects.

To elucidate the brain recognition sites through which MDMA might elicit

its various behavioral, psychotomimetic, and neurotoxic effects. an extensive

in vitro pharmacologic screening of MDMA was carried out at various brain

neurotransmitter receptors and recognition sites. The relative potencies of

MDMA at the various brain recognition sites were assessed from competition data in which affinities (Ki

 values) were determined using the nonlinear

curve-fitting program LIGAND (Munson and Rodbard 1980). Details of the

assay conditions and affinities of MDMA at the various recognition sites are

reported in table 4. The pharmacologic profile of MDMA demonstrates a

broad range of affinities of the drug for various brain recognition sites

(Battaglia et al. 1988a). MDMA had the highest affinity for serotonin

uptake sites (<1 μM) with lower but comparable affinities at 5-HT2

serotonin, adrenergic, M-1 muscarinic cholinergic and H-1 histamine

receptors (Ki values < 5μM). The rank order of affinities of MDMA at

246
TABLE 3. Affinities of 2,5-DMA derivatives at 5-HT1

 serotonin receptor

subtypes

Agent 5-HT1A 5-HT1B 5-HT1C

R(-)-DOB

DOI

DOB

DOPR

R(-)-DOM

DOET

DOM

S(+)-DOB

DOBU

2,4,5-TMA

MEM

2,5-DMA

2,4-DMA

3,4,5-TMA

2,332 ± 188

2,355 ± 77

3,770 ± 188

2,849 ± 170

4,004 ± 107

3,930 ± 115

5,122 ± 140

4,041 ± 156

4,178 ± 165

>10,000

>l0,000

1,131 ± 55

>10,000

>10,000

683 ± 46

1,261 ± 105

831 ± 37

2,330 ± 101

1,840 ± 172

2,451 ± 226

2,063 ± 112

883 ± 49

1,211 ± 86

>10,000

>10,000

8,435 ± 668

>10,000

>10,000

47 ± 10

30 ± 4

69 ± 16

14 ± 1

94 ± 17

101 ± 20

193 ± 20

81 ± 7

26±5

2,666 ± 76

2,278 ± 90

1,217 ± 89

3,152 ± 83

5,710 ± 150

NOTE: The 5-HT1A, 5-HT1B, and 5-HT1C receptors were labeled with ‘H-OH-DPAT, ‘H-5-HT, and ‘Hmesulergine respectively.

SOURCE: Titeler et al. 1988.

various brain receptors and uptake sites were as follows: serotonin uptake

> adrenergic = 5-HT2 serotonin = M-1 muscarinic = H-1 histamine >

norepinephrine uptake = M-2 muscarinic = -adrenergic = -adrenergic >

dopamine uptake = 5-HT1 serotonin >> D-2 dopamine > D-1 dopamine.

MDMA exhibited negligible affinities (>500 μM) at mu, delta, and kappa

opioid, central-type benzodiazepine, and corticotropin-releasing factor

receptors, as well as at choline uptake sites and at calcium channels.

Although not shown here, the affinities of MDA were comparable (< twofold difference) to those of MDMA at each of the respective brain recognition sites investigated. In general, the affinities of MDMA at the receptor

sites investigated could be classified as high-, moderate-, and low-affinity

interactions. These are summarized in table 5. MDMA appears to be most

potent at a number of serotonin recognition sites as well as adrenergic

and M-l muscarinic receptors with affinity constants (K1 values) in the high

nanomolar to low micromolar range. Affinities of MDMA in the micromolar range at the various recognition sites appear to be pharmacologically

relevant, since similar brain concentrations of the drug have been detected

in rats following systemic administration of a single dose of MDMA (20

mg/kg), which elicits behavioral as well as neurotoxic effects (Zaczek et al.,

unpublished observation).

247
TABLE 4. Pharmacologic profile of MDMA at various brain

recognition sites

Brain Recognition Affinity Brain Assay Time,

Site Ki

(μM) Radioligand/Displacer Region Tempeature Buffer

Uptake Sites

Serotonin 0.61 ± .05

Norepinephrine 15.8 ± 1.7

Dopamine 24.4 ± 1.9

Choline >500

Adrenergic Receptors

18.4 ± 1.2

3.6 ± 0.8

191 ± 21

Dopamine Receptior

D-1 148 ± 14

D-2 95 ± 15

Serotonin Receptors

5-HT1 23 ± 1.5

5-HT2 5.1 ± 0.3

Cholinergic Receptors

M-1 muscarinic 5.8 ± 0.3

M-2 muscarinic 15.1 ± 0.1

0.55nM[3H]paroxetinel 1

1μM citalopram

4.OnM[3H]mazindol/ 1

0.3μM desipramine

1.0nM[3H]GBR 12935/ 2

1μM mazindol

10nM[3H]hemicholinium-3/ 2

10μM hemicholinium-3

120 min, A

Rm T

90 min,

4 °C

A

60 min, A

Rm T

30 min, B

25 °C

0.5nM[3H]prazosin/ 1

10μM phentolamine

0.5nM[3H]para-aminocloni- 1

dine/10μM phentolamine

0.5nM[3H]dihydroalprenalol/ 1

1μM propranalol

30 min,

37 °C

30 min,

37 °C

30 min,

37 °C

0.2nM[3H]SCH 23390/ 2 30 min,

0.1μM flupenthixol 37 °C

0.2nM[3H]spiperone/ 2 30 min,

1μM (+)butaclamol 37 °C

2.5nM[3H]serotonin/ 1

10μM serotonin

0.4nM[3H]ketanserin/ 1

0.5μM cinanserin

30 min,

37 °C

30 min,

37 °C

0.1nM[3H](-)QNB/ 1

1μM atropone

0.1nM[3H](-)QNB/ 3

1μM atropine

90 min,

Rm T

90 min,

Rm T

2nM[3H]dihydromorphine/ 4

1μM levallorphan

4nM[3H]D-ala2-D-leu5- 4

enkephalin (30nM morphine)/

1μM levallorphan

1.6nM[3H]ethylketazocine 4

(30nM morphine + 100nM

D-ala2-D-leu5-enkephalin)/

1μM levallorphan

45 min,

25 °C

45 min,

25 °C

45 min,

25 °C

248

C

C

C

C

C

C

D

D

E

E

E

Opioid Receptors

Mu >500

Delta >500

Kappa >500
TABLE 4. (Continued)

Bran Recongnition Affinity Brain Assay Time,

Site KI

(μM) Radioligand/Displacer Region Temperature Buffer

Other Sites

H-1 histamine

Receptors

Benzodiazepin

receptors

Corticotropinreleasing factors

(CFR) receptors

Calcium channels

5.7 ± 2.4 2nM[3H]mepyramine/

1μM doxepin

>500 0.2nM[3H]flunitrazepam/

1μM clonazepam

>500 0.1nM125I-Tyr°-rat CRF/

1μM ovine CCRF

>500 0.2nM[3H]nitredipine/

0.1μM nifedipine

1 60 min, F

Rm T

1 60 min, G

Rm T

5 120 min, H

Rm T

1 60 min, G

Rm T

KEY: Assay buffers: A = 50 mM TRIS-HCl, 120 mM NaCl, 5 mM KCl (pH 7.4 at Rm T); B = 50 mM glycylglycine,

200 nM NaCl (pH 7.8 at 25 °); C = 50 mM TRIS-HCl, 10 mM MSO4, 0.5 mM K3HDTA (pH 7.4 aat 37 °C); D =

50 nM TRIS-HCl, 10 mM MGSO4 (pH 7.7 at Rm T) E = 0.17 M TRIS HCl (pH 7.6 at 25 °C); G = 50 mM TRISHCl (pH 7.7 at Rm T); F = 50 nM Na+K

+

 phospate (pH 7.4 at Rm T); H = 50 mM TRIS-CHl, 10 mM MgCl2, 2

mM EGTA 0.1% bovine serum albumin, 0.1 mM bacitracin sprotinin (100 KIU/mL) (pH 7.2 at 22 °C). Brain

regions: 1 = frontal contex; 2 = striatum; 3 = brain stem; 4 = whole brain; and 5 = olfactory bulb.

NOTE: Data represent the mean ± SEM from three to five competition curves at each of the sites. Ki values were

determined using the nonlinear least-sqares curve-fitting program LIGAND.

SOURCE: Battaglis et al. 1988.

TABLE 5. Relative potencies of MDMA at various brain recognition sites

High Affinity Moderate Affinity Low Affinity

(0.6 to 6μM) (10 to 100 μM) (< 100 μM)

Serotonin uptake sites

5-HT2

 serotonin receptors

sites

-adrenergic receptors

Norepinephrine uptake sites

Dopamine uptake sites

5-HT1

 serotonin receptors

D-1 dopamine

receptors

Choline uptake

Mu, delta, and

kappa opioid

receptors

M-1 muscarinic receptors adrenergic receptors Benzodiaepine

receptors

D-2 dopamine

receptors

As shown in table 6, we have compared the affinities of a series of

methylenedioxy derivatives with those of the parent compounds (amphetamine and methamphetamine) at some of the recognition sites in brain at

which MDMA exhibited the highest affinities. These comparative studies

indicate that addition of the methylenedioxy substituent in the 3,4 position

increases their affinity at serotonin uptake, 5-HT2 serotonin, and M-1

muscarinic receptors, while the unsubstituted parent compounds appear to be

more potent at adrenergic receptors.

249
TABLE 6. Relative potencies of amphetamine derivatives at selected brain

recognition sites

Compound 5-HT Uptake 5-HT2 Serotonin adrenergic M-1Muscarinic

MDMA 1.0

MDA 1.8

MDE 0.4

Amphetamine 4.8

Meth- 3.4

amphetamine

1.0

0.5

3.5

2.6

2.4

1.0

0.5

3.3

0.09

0.61

1.0

1.4

1.8

4.8

3.6

NOTE: Comparison of the affinities (K1 values) of amphetamine derivates at serotonin(5-HT)

uptake sites, 5-HT2serotonin, adrenergic, and M-1 muscarinic receptors with respect to

the affinity of MDMA at these sites. Values smaller or larger than 1.0 indicate affinities

higher or lower, respectively, than those of MDMA.

SOURCE: Battaglia et al. 1988a.

Interestingly, the anxiolytic-like effects of MDMA do not appear to be

mediated through agonist actions at benzodiazepine receptors or antagonist

effects at corticotropin-releasing factor receptors as evidenced by the low

affinity of MDMA (>500 μM) at each of these receptors. In addition, the

reinforcing, analgesic, and mood-altering properties of the drug do not

appear to be mediated through interactions with any of the opioid receptor

subtypes, since MDMA has relatively low affinities for these receptors.

INTERACTIONS OF MDMA WITH SEROTONIN RECOGNITION

SITES

The previous data suggest that a number of the behavioral, psychotomimetic, and neurochemical effects of MDMA and other methylenedioxy

derivatives of amphetamine may be explained by interactions of MDMA at

multiple serotonin recognition sites in brain. MDMA may alter serotonergic

transmission in brain through direct actions at postsynaptic as well as

presynaptic serotonin recognition sites. As mentioned above, a number of

hallucinogenic phenylisopropylamine derivatives exhibit potent agonist-like

activity at brain 5-HT2 serotonin receptors (Shannon et al. 1984) and the in

vitro affinities of these hallucinogens at 5-HT2 serotonin receptors significantly correlate with both their behavioral potencies in animals in

generalization to other hallucinogens and with their human hallucinogenic

potencies (Glennon et al. 1984; Titeler et al. 1988). Similar to previous

observations for other ring-substituted amphetamines such as the derivatives

at 2,5-DMA, we found that MDMA and other methylenedioxy derivatives of

amphetamine also exhibited high-affinity agonist-like binding characteristics

at 5-HT2 serotonin receptors. The stereospecificity observed for methylene

dioxy derivatives at 5-HT2 receptors was consistent with that observed for

other hallucinogenic compounds at this receptor (Lyon et al. 1986; Battaglia

250
et al. 1986). In addition, it was reported that MDMA interactions with the

high-affinity state of 5-HT2 serotonin receptors were sensitive to the effects

of guanine nucleotides, similar to that observed for serotonin and other

classical tryptamine agonists at this site (Battaglia et al. 1984) (figure 3).

While the overall apparent affinity (K1 value) of MDMA for [

3H]ketanserinlabeled 5-HT2

 serotonin receptors is in the low micromolar range, the

authors have observed that the interactions of MDMA and other methylenedioxyamphetamines with the high-affinity state of 5-HT2

 serotonin receptors

labeled directly by [3H]DGB (Lyon et al. 1987) are much more potent

(K1<300 nM). Since this high-affinity component of 5-HT2 serotonin

receptors represents the most potent site of action for MDMA in brain, it is

likely that some of the “mood-altering” effects of MDMA may be mediated

by direct agonist actions at 5-HT2 serotonin receptors. A recent study

demonstrating that the serotonin receptor antagonist methysergide can

potentiate the MDMA-induced increases in locomotor activity (Gold and

Koob 1988) further supports the claim for direct actions of MDMA at

postsynaptic 5-HT2 serotonin receptors. A comparison of the relative

affinities of MDMA and MDA at postsynaptic 5-HT2 serotonin receptors

with those of other ring-substituted amphetamine hallucinogens suggests that

MDMA and MDA would be much weaker hallucinogens at this site than

would compounds such as DOM (STP) or DOI. This is not surprising, as

the methylenedioxy class of designer drugs has been reported to have

unique and subtle mood-enhancing subjective effects, rather than having the

more vivid and disorienting sensations commonly attributed to very potent

hallucinogens such as DOM or LSD.

In addition to the actions of MDMA and other derivatives at 5-HT2

serotonin receptors. some of the effects on serotonergic systems could be

mediated via 5-HT1A receptors, at which MDMA has a moderate affinity.

Direct agonist effects at this site might contribute to the mood-altering and

calming effects of the drug, since similar effects have been reported for

novel anxiolytics such as ipsaperone and buspirone, which interact with

5-HT1A serotonin receptors.

In addition to its relatively high affinity at postsynaptic 5-HT receptors,

MDMA exhibited high affinity for 5-HT uptake sites and has been shown

to increase the release of [

3H]5-HT and block [3H]5-HT uptake in vitro.

These data suggest that some of the actions of MDMA may be mediated at

presynaptic binding sites. With respect to [

3H]5-HT release, MDMA has

been reported to increase the release of [

3H]5-HT from brain synaptosomes

(Nichols et al. 1982) and hippocampal slices (Johnson et al. 1986). With

respect to uptake blockade, MDMA has been reported to competitively

inhibit 3H-5-HT uptake in vitro (Shulgin 1986). Furthermore, the neurotoxic

effects of in vivo administration of MDMA on serotonin terminals can be

blocked by concomitant administration of the 5-HT uptake blocker citalopram (Battaglia et al. 1988b; Schmidt and Taylor 1987). Additional

evidence in support of the hypothesis that MDMA produces some of its

251
FIGURE 3. Competition curves of (A) serotonin and (B) MDMA for [

3H]

ketanserin binding to 5-HT2 serotonin receptors in rat

frontal cortex membranes in the presence and absence of

the guanine nucleotide quanosine-5-triphosphate (GTP)

252
effects through presynaptic serotonergic mechanisms is provided by data

demonstrating that MDMA can generalize to a fenfluramine cue in stimulus

discrimination studies (Schechter 1986).

Classic a-adrenergic receptor antagonists such as phentolamine have been

reported to increase the release of [

3H]5-HT via effects on adrenergic

receptors (Timmermans and Van Zwieten 1982). Thus, one might speculate

that the serotonin-releasing effects of MDMA may be mediated, in part, by

high-affinity antagonist-like effects at adrenergic receptors localized to

presynaptic serotonin terminals. The relatively high affinity of MDMA at

the serotonin uptake site and adrenergic receptor may contribute, in part,

to the neurochemical, neurotoxic, and behavioral effects mediated at

presynaptic serotonin terminals.

While brain serotonin systems may play a key role in mediating some of

the effects of MDMA on analgesia and body temperature as well as in the

reported anxiolytic-like and mood-altering subjective effects of the drug,

additional neurotransmitter systems may contribute to some of the unique

subjective experiences reported for MDMA and other drugs in this class.

SUMMARY AND CONCLUSIONS

Ring-substituted psychoactive derivatives of amphetamine exhibited high

affinities for a number of serotonin recognition sites. Derivatives of

2,5-DMA exhibited preferential high affinity at 5-HT2 serotonin receptors

when compared to their relative affmities at 5-HT1 serotonin receptors.

Furthermore, 2,5-DMA derivatives exhibited agonist-like binding

characteristics at 5-HT2 serotonin receptors with the R(-) isomer being the

more potent isomer. There were significant correlations between the

in vitro affinities of 2,5-DMA derivatives at 5-HT2 serotonin receptors and

their human hallucinogenic potencies as well as with their potencies in

behavioral generalization studies, suggesting the importance of 5-HT2

serotonin receptors in mediating the hallucinogenic effects of the various

2,5-DMA derivatives.

A pharmacological profile of the methylenedioxy-substituted amphetamine

derivatives indicates that MDMA and MDA exhibited highest affinity for

serotonin uptake sites, 5-HT2 serotonin, arenergic and M-1 muscarinic

receptors. The methylenedioxy amphetamine derivatives exhibited an

inverse stereospecificity with respect to serotonin uptake sites versus

postsynaptic 5-HT receptors with the S(+) isomer being more potent at the

presynaptic recognition site, while the R(-) isomer was more potent at the

postsynaptic recognition sites. Similar to the 2,5-DMA derivatives, MDMA

and MDA exhibited agonist-like binding characteristics at 5-HT2 serotonin

receptors. Unlike 2,5-DMA derivatives, MDMA and MDA demonstrated

little selectivity for 5-HT2 versus 5HT1 subtypes of receptors. The

relatively weak hallucinogenic effects of the methylenedioxy-substituted

253
amphetamine derivatives (when compared to the 2,5-DMA derivatives) may

be mediated through actions at 5-HT2 serotonin receptors. In addition, the

neurotoxic, psychotomimetic, analgesic, temperature regulation, and moodaltering effects of MDMA and other methylenedioxy-substituted derivatives

may be mediated, in part, through their actions at other serotonin

recognition sites in brain, including serotonin uptake sites and 5-HT1A

serotonin receptors. Other behavioral. cardiovascular, and toxic effects of

MDMA and related derivatives may be mediated by actions at other central

and/or peripheral recognition sites, including muscarinic cholinergic receptors

and adrenergic receptors, for which these compounds exhibit relatively

high affinity. The precise mechanisms for the various effects of the

amphetamine derivatives remain to be determined.

DISCUSSION

QUESTION: In the absence of serotonin neurons, could MDMA still have

a direct agonist action at postsynaptic receptors or is that 5-HT?

ANSWER: Yes. From the present data, one would expect direct agonist

effects of MDMA at 5-HT2 receptors in the absence of serotonin neurons.

Based on the data that I showed you today, MDMA and other methylene

dioxy amphetamine derivatives exhibit agonist-like binding properties that

resemble those observed for 5-HT and other tryptamine agonists as well as

for other hallucinogenic amphetamines. We would expect the effects of

MDMA at 5-HT2 receptors to be somewhat weaker compared to those of

other amphetamine derivatives, since MDMA-like compounds exhibit substantially lower affinity than the 2,5-DMA derivatives at 5-HT2

 sites. In

addition, unlike what we Observe with the 2,5-DMA derivatives, MDMA

and the other methylenedioxy compounds do not exhibit the preferential

affinity for 5-HT2 sites over 5-HT1 subtypes as observed for the more potent

hallucinogens. The comparable affinity of MDMA for multiple 5-HT receptors may contribute to the comparatively weak hallucinogen-like properties

of this class of compounds. With respect to the second part of the

question, it would be expected that, in the presence of an intact serotonergic

system, MDMA-induced release of 5-HT via presynaptic sites of action

would also have some postsynaptic 5-HT2 receptor consequences. I inferred

that MDMA may have antagonist-like effects at alpha2 adrenergic receptors,

and this may be responsible for increased 5-HT release. However, the only

recognition site where we have tried to discern agonist versus antagonist

characteristics is at the 5-HT2 serotonin receptors.

QUESTION: Not the presynaptic?

ANSWER: No, only the postsynaptic 5-HT2 receptors.

COMMENT: It seems to me that, in the absence of the serotonin input,

the 5-HT2 serotonin receptors downregulate instead of upregulate. So if you

254
took away the serotonin input, you would expect to see a decreased

potency.

RESPONSE: Downregulation of 5-HT2 receptors, which has been observed

following treatment with antagonists, may be viewed as due to compensatory changes in response to tbe absence of 5-HT input. In order to assess

the hypothesis that there was modulation in the absence of serotonin, we

looked at 5-HT2 serotonin receptors following lesion with MDMA. We

chose to look at a time point 2 weeks after treatment in order to allow time

for postsynaptic receptor changes to occur. Although we did not see any

changes in the density of sites, we have not investigated whether there may

have been changes in second messenger systems coupled to these receptors.

QUESTION: Would you expect the direct effects of MDMA on the 5-HT2

receptor to have any significance in the presence of this massive 5-HT

release that it is causing?

ANSWER: When we are dealing with the effects of the methylenedioxysubstituted derivatives on serotonergic systems, we are dealing with a

multiplicity of effects.

Our data indicate that the racemates of these compounds most likely

mediate effects on both presynaptic as well as postsynaptic 5-HT sites.

Furthermore, there is an inverse stereospecificity associated with these

actions. For example, the dextro isomers of MDMA and other drugs in this

class exhibit higher affinity than the levo isomer for the presynaptic 5-HT

uptake and also appear to be more potent in causing 5-HT release. This is

the opposite of the isomer affinities at postsynaptic receptors. The levo

isomers of MDMA-like compounds exhibit preferentially higher affinity than

the dextro isomers for both 5-HT1 and 5-HT2 serotonin receptor subtypes.

Similar stereospecificity is observed with the parent compounds, amphetamine and methamphetamine, as well for the hallucinogenic 2,5-DMA derivatives. While we can discern the agonist properties of these compounds at

5-HT2 receptors, it is unclear whether these drugs are acting as agonists or

antagonists at the various subtypes of 5-HT1 receptors. With respect to the

original question, if MDMA exhibits simultaneous 5-HT2 agonist and 5-HT1

antagonist activity, then one may speculate that these effects can significandy influence the fmal response, even in the presence of massive 5-HT

release by these agents.

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ACKNOWLEDGMENTS

Dr. Richard Glennon and Dr. Milt Titeler permitted use of their data on the

effects of derivatives of 2,5-DMA at serotonin recognition sites.

AUTHORS

George Battaglia. Ph.D.

Assistant Professor

Department of Pharmacology

Loyola University of Chicago

Stritch School of Medicine

2160 South First Avenue

Maywood, IL 60153

Errol B. De Souza, Ph.D.

Chief, Laboratory of Neurobiology

Neuroscience Branch

Addiction Research Center

National Institute on Drug Abuse

Baltimore, MD 21224

258
Effects of Amphetamine Analogs on

Central Nervous System Neuropeptide Systems

Glen R. Hanson, Patricia Sonsalla Anita Letter,

Kalpana M. Merchant, Michel Johnson, Lloyd Bush,

and James W. Gibb

INTRODUCTION

Substantial efforts have been devoted to elucidating the effects of amphetamine analogs on central nervous system (CNS) monoaminergic pathways.

These agents enhance the activity of such neuronal systems by causing

release of their transmitter substances as well as by interfering with transmitter metabolism and reuptake. However, little is known about the consequences of the monoaminergic changes resulting from the administration of

these agents, i.e., the eventual effect of these drugs on transmitter systems

directly influenced by the monoaminergic pathways. Such effects are

important in transmitting the monoamine-initiated messages to those brain

regions that eventually mediate the drug-related behavioral changes. In

addition, these systems likely have important feedback functions on the

amphetamine-sensitive monoaminergic pathways. Consequently, druginduced changes in these feedback pathways might contribute to phenomena

such as tolerance and sensitization.

Of interest to the present work are the neuropeptide neuronal projections

associated with extrapyramidal structures and the responses of peptidergic

pathways to treatments with amphetamine analogs. These peptide systems

were selected for study because of their close association with the

mesostriatal dopaminergic neuronal circuitry, a system thought to contribute

to the locomotor and mood-altering effects of the amphetamine compounds.

For example, neurons containing substance P (SP), which originate within

the shiatum and terminate in the substantia nigra, are thought to serve an

excitatory feedback function on the mesostriatal dopamine (DA) pathway.

Thus, intranigral injections of SP cause striatal release of DA (Reid et al.

1988) and stimulate locomotion (Herrera-Marschitz et al. 1986). Nigral

administration of SP has no effect on locomotor activity in animals that

have received 6-hydroxydopamine lesions to their mesostriatal DA pathway

(Herrera-Marschitz et al. 1986).

259
The interactions between neurotensin (NT) pathways and the extrapyramidal

dopaminergic system are somewhat more complex. As the vast majority of

striatal and nigral NT receptors are associated with DA neurons (Quirion et

al. 1985). NT pathways certainly contribute to the regulation of

extrapyramidal DA activity. The overall CNS pharmacology of NT has

been compared to that of neuroleptic drugs (Nemeroff 1986), while

intraventricular administration of this peptide is reported to antagonize some

of the behavioral activity of amphetamine and cocaine (Skoog et al. 1986).

Finally, dynorphin (Dyn) A1-17 is associated with striatal-nigral neurons

that, like the SP pathway, have been postulated to be part of a feedback

system to the nigral-striatal DA neurons (Herrera-Marschitz et al. 1983).

However, such a feedback role for Dyn has been questioned recently, as the

locomotor activity induced by nigral Dyn injections is not impaired by

elimination of the nigral-striatal DA pathway (HerreraMarschitx et al.

1986).

EVALUATION OF NEUROPEPTIDE RESPONSE TO

AMPHETAMINE ANALOGS

This chapter discusses the responses of these extrapyramidal neuropeptide

systems to the amphetamine analogs methamphetamine (METH), methylene

dioxyamphetamine (MDA), and methylenedioxymethamphetarnine (MDMA).

These drugs were selected for this study because they represent somewhat

diverse mechanisms of action. While all three agents are able to enhance

extrapyramidal serotonergic activity (Schmidt et al. 1987). only METH has

been characterized as a substantial stimulant of the DA system. The effects

of MDA and MDMA on extrapyramidal DA systems have not been well

elucidated. Thus, evaluating and comparing the responses of the SP, NT,

and Dyn extrapyramidal systems to these drugs will help to determine the

nature of the DA responses to METH, MDA, and MDMA administrations.

Methods

Sprague Dawley rats (180 to 220 g) were treated with METH, MDA, and

MDMA generously donated by the National Institute on Drug Abuse. Following drug treatments, rats were sacrificed, brains removed, and the striatal

and nigral areas dissected out. Tissue samples were rapidly frozen and

stored until analyzed. The responses of these neuropeptide systems to treatments by the amphetamine analogs were assessed with radioimmunoassay

techniques by measuring drug-induced changes in the tissue content of

neuropeptidelike immunoreactivity. Highly selective and sensitive antibodies were used in the detection of SP (Hanson and Loveberg 1980), NT

(Letter et al. 1987). and Dyn (Hanson et al. 1987). The mean nigral contents of SP, NT, and Dyn for the control groups were 12 nanograms (ng),

595 picograms (pg), and 766 pg per mg protein, respectively. The mean

striatal contents for SP, NT, and Dyn for the control groups were

1,250,127, and 380 pg/mg protein, respectively. To characterize the

260
METH-induced changes in neuropeptide levels, selective D1

 (SCH 23390)

and D2 (sulpiride) dopaminergic receptor antagonists were coadministered.

The results are expressed as percent of control to facilitate comparisons;

each value represents the mean ± SEM of five to seven animals. Data

were subjected to either a Student’s r-test (figures 4 and 5) or ANOVA

analysis followed by a multiple comparisons test (figures 1, 2, and 3).

Significance was set at the .05 level.

FIGURE 1. Effects of METH on extrapyramidal SP content

*p<0.02 compared to corresponding groups. 

NOTE: METH was injected alone or concurrently with either SCH 23390 (SCH) (0.5 mg/kg/injection)

or sulpiride (sulp) (80 mg/kg/injection).

Results

Administrations of five injections of METH (15 mg/kg/injection; 6-hour

intervals between injections) caused substantial increases in the striatal and

nigral levels of all three neuropeptides examined in rats sacrificed 18 hours

following treatment. Figures 1 to 3 present the effects of blocking the D1

and D2 dopaminergic receptors on the responses by these peptide systems

261
FIGURE 2. Effects of METH on extrapyramidal NT content

*p<0.02 compared to corresponding control.

**p<0.01 compared to the corresponding METH- and sulpride (sulp)-treated groups, and p<0.001

compared to the corresponding control.

†p<0.05 compared to the corresponding METH-treated groups.

††p<0.01 compared to the corresponding METH-treated group.

NOTE: Animals were treated as described in figure 1.

to METH treatment. Figures 4 and 5 present the responses of SP, NT, and

Dyn extrapyramidal pathways to MDMA and MDA treatments, respectively.

Following METH administration, levels of SP were elevated to 150 percent

of control in the substantia nigra and 227 percent of control in the striatum

(figure 1). Blockade of either D1 or D2 receptors totally prevented the

METH-induced rise in nigral SP content.

In rats sacrificed 18 hours following METH treatment, nigral and striatal

levels of both NT (figure 2) and Dyn (figure 3) increased dramatically, to

200 to 400 percent of respective controls. However, the effects of D1

 and

D2 receptor antagonism on the METH-induced changes in these peptide

systems were somewhat different. Administration of sulpiride alone caused

an increase in striatal NT levels. METH administration in the presence of

262
FIGURE 3. Effects of METH on extrapyramidal Dyn A content

*p<0.02 compared to corresponding controls.

†p<0.05 compared to the corresponding METH-treated group.

NOTE: Animals were treated as described in figure 1.

this D2 blocker resulted in increases of striatal NT content approximately

equal to the summation of the effects of the two drugs when given individually. In contrast, the D1

 antagonist, SCH 23390, had no effect alone

and substantially attenuated the METH-induced striatal changes in NT

content. The SCH 23390 compound also completely blocked the METHmediated elevation of nigral NT levels, while sulpiride had no effect of its

own, nor did its presence significantly influence the response of the nigral

NT system to METH treatment.

Administration of sulpiride or SCH 23390 alone did not alter the striatal or

nigral content of Dyn. Blockade of D1 receptors substantially interfered

with the METH-induced changes in both striatal and nigral Dyn levels.

Blockade of D2 receptors by sulpiride appeared to attenuate the METHrelated changes in the Dyn levels, especially in the substantia nigra, but its

interference with the METH effects was less than that of the SCH 23390

compound.

263
FIGURE 4. Effects of MDMA on extrapyramidal neuropeptide contents

*p<0.05 compared to corresponding controls. 

**p<0.001 compared to corresponding controls.

NOTE: Animals were given multiple injections of MDMA (10 mg/kg/injection) and sacrificed 18

hours after treatment. Striatal and nigral content of SP, NT, and Dyn were examined

The effects of five injections of MDMA (10 mg/kg/injection) on striatal and

nigral neuropeptide content are presented in figure 4. Animals were treated

in a manner similar to that used for the METH experiments. Following

multiple MDMA administrations, the striatal levels of SP, NT, and Dyn

were elevated to 248 percent., 195 percent, and 148 percent, respectively, of

corresponding controls. Nigral content of these same peptides were

increased to 127 percent, 217 percent, and 157 percent, respectively,

compared to their controls. These effects resembled those observed with

METH treatment, Similar SP and NT responses were observed following

MDA treatment (figure 5).

CONCLUSION

These findings demonstrate that some neuropeptide systems associated with

mesostriatal dopaminergic projections are profoundly altered by treatment

with each amphetamine analog examined.Although the significance of

these drug-induced increases in striatal and nigral contents of SP, NT, and

264
FIGURE 5. Effects of MDA on extrapyramidal neuropeptide contents

**p<0.01 compared to corresponding controls.

NOTE: Animals were given multiple injections of MDA (10 mg/kg/injections) and sacrificed 18 hours

after treatment. Striatal and nigral levels of SP and NT were determined.

Dyn is not yet known, it is likely that such changes reflect variations in the

activity of the associated pathways. One possible explanation is that

increases in neuropeptide tissue levels are due to decreased release of the

transmitter, which diminishes the extracellular peptide metabolism and

results in accumulation of these peptide substances. Another possible

contributing factor is a drug-related alteration in neuropeptide synthesis. For

example, Bannon et al. (1987) reported that METH administration increased

the quantity of striatal messenger RNA for the SP precursor preprotachykinin. Thus, increases in peptide synthesis might contribute to increases in

peptide content caused by treatment with METH or the other amphetamine

analogs.

The dramatic responses to METH reported herein were most certainly a

consequence of drug-mediated changes in postsynaptic dopaminergic activity.

It is interesting that each neuropeptide response to METH treatment was

subtly unique. The increases in SP content cased by METH appeared to

occur primarily by activation of D2 receptors (figure 1). This conclusion is

based on previously reported findings that D2 agonists also increase nigral

SP levels, while D1 agonists actually cause a decrease in the nigral SP

265
concentration (Sonsalla et al. 1984). Even so. D1

 receptors appeared to play

a facilitatory role in this drug effect, as blockade of this receptor completely

prevented the METH effects. The effects of METH on the NT systems

appeared to be mediated completely by D1 receptors, as the presence of

SCH 23390 almost entirely blocked the METH-mediated changes in NT

levels, while sulpiride did not appear to interfere with the METH effects

(figure 2). Finally, these data suggest that the actions of METH on the

Dyn systems were mediated primarily by D1 receptors; even so, D2 receptors

also contributed to these effects as their blockade attenuated, although to a

lesser degree than D1 blockade, the METH-related increases in Dyn levels

(figure 3).

The present data demonstrate that the amphetamine analogs MDA and

MDMA influence the extrapyramidal neuropeptide systems in a METH-line

manner (figures 4 and 5). As already discussed, the METH effects on

these peptide systems are dopaminergically mediated, thus, it is likely that

the amphetamine designer drugs also influence SP, NT, and Dyn

extrapyramidal pathways by enhancing extrapyramidal dopaminergic activity.

In support of this conclusion, we have observed that blockade of D1

receptors with SCH 23390 completely blocks the increases in striatal NT

and Dyn induced by MDMA treatment (unpublished observation). This

finding is consistent with observations that MDMA and MDA stimulate the

release of striatal DA from tissue slices (Schmidt et al. 1987) and intact

animals (Yamamoto and Spanos 1988). In addition, Stone et al. (1986)

reported that treatments with MDA and MDMA resulted in increases in

striatal concentrations of homovanillic acid, a DA metabolite, which reflects

the extent of DA release.

While perhaps quantitatively different, each of the amphetamine analogs

examined had substantial effects on the extrapyramidal SP, NT, and Dyn

pathways. Thus. these peptide pathways likely contribute to the behavioral

effect of this group of agents in general; specifically, they might participate

in mediating the changes in locomotion or mood or the development of

psychotic disorders associated with administration of high doses of the

amphetamine analogs. More studies are necessary to identify specific

contributions ma& by each of these peptide systems to the pharmacological

profiles of these agents. In addition, these neuropeptide changes are of

interest as nemochemical markers for the effects of the amphetamine drugs

on postsynaptic dopaminergic activity and could be useful in the study of

such consequences of these drugs as tolerance and sensitization.

DISCUSSION

QUESTION: The last slide referred to postsynaptic actions of the drugs.

Do you mean postsynaptic consequences of their presynaptic actions?

266
ANSWER: Yes. We all know that the dopamine comes out The question

is: What happens after the dopamine comes out? We know that if nothing

occurred following the dopamine release as far as other transmitter systems

being influenced, them would be no behavioral effect. So downstream

systems like these peptides are probably involved in mediating those

monoaminergic messages to some other parts of the brain or playing

feedback roles and altering the way that the monoamine systems respond

So they may play roles in sensitization or tolerance by impacting on the

activity of those projections.

QUESTION: Did you mention that 6-hydroxydopamine blocks or elevates

neurotensin levels?

ANSWER: Yes, 6-hydroxydopamine by itself elevates neurotensin levels.

When you combine it with methamphetamine, you do not get any additivity.

It is just a 6-hydroxydopamine action. It is a bit complicated to interpret,

but it appears that it is still the nigral striatal dopamine pathway that is

mediating the methamphetamine effect.

QUESTlON: Have you had the opportunity to look at substance P,

possibly in the spinal cord? I am thinking about some of the work that

Dr. Seiden presented and potentially a role in analgesia.

ANSWER: We have not looked in the spinal cord at all for substance P.

Everything has been in the extrapyramidal and limbic systems.

COMMENT: Another reason why you should be looking at substance P in

the spinal cord is that, in spinal cord, substance P is cocontained in neurons

together with tyrosine hydroxylase.

RESPONSE: Right. And we have asked ourselves the question because of

the issue of coexistence, not only with substance P but with neurotensin and

probably dynorphin. Is this the reason these things are changing? Because

if they are coexisting with dopamine projections and there is some alteration

in dopamine, then maybe there is an intraneuronal action that results in the

peptide changes.

COMMENT: I was thinking about this, but I couldn’t remember if

substance P had been shown to be colocalized in the striatum.

RESPONSE: No, if there is any, it is very, very small coexistence of

substance P and tyrosine hydroxylase in the striatum. That is why we don’t

feel that that is the explanation for these changes.

267
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Regulation of mRNA and peptide levels by dopamine agonists and

antagonists. Mol Brain Res 3:31-37, 1987.

Hanson, G.R., and Lovenberg, W. Elevation of substance P-like immunoreactivity in rat central nervous system by protease inhibitors,

J Neurochem 35(6):1370-1374, 1980.

Hanson, G.R.; Merchant, K.M.; Letter, A.A.; Bush, L.; and Gibb, J.W.

Methamphetamine-induced changes in the striatal-nigraI dynorphin system:

Role of D1 and D2 receptors. Eur J Pharmacol 144:245-246, 1987.

Herrera-Marschitz, M.; Christensson-Nylander, I.; Sharp, T.; Staines, W.;

Reid, M.; Hokfelt, T.; Terenius, L.; and Ungerstedt, U. Striatonigral

dynorphin and substance P pathways in the rat: IL Functional analysis.

Exp Brain Res 64:193. 1986.

Herrera-Marschitx, M.; Hokfelt, T.; Ungerstedt, U.; and Terenius, L.

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injections into the substantia nigra reticulata of naive rats. Life Sci

33:555-558, 1983.

Letter A.A.; Merchant, K.; Gibb, J.W.; and Hanson, G.R. Effect of methamphetamine on neurotensin concentrations in rat brain regions.

J Pharmacol Exp Ther 241(2):443-447, 1987.

Nemeroff. C. The interaction of neurotensin with dopaminergic pathways in

the central nervous system: Basic neurobiology and implications for the

pathogenesis and treatment of schiiphrenia. Psychoneuroendocrinology

11(1):15-37, 1986.

Quirion, R.; Chiueh. C.; Everist, H.; and Pert, A. Comparative localization

of neurotensin receptors on nigrostriatal and mesolimbic dopaminergic

terminals. Brain Res 327:385-389, 1985.

Reid, M.; Herrera-Marschitz, M.; Hokfelt, T.; Terenius, L.; and

Ungerstedt, U. Differential modulation of striatal dopamine release by

intranigral injection of gamma-aminobutyric acid (GABA), dynorphin A

and substance P. Eur J Pharmacol 147:411-420, 1988.

Schmidt, C.J.; Levin, J.; and Lovenberg, W. In vitro and in vivo neurochemical effects of methylenedioxymethamphetamine on striatal monoaminergic systems in the rat brain. Biochem Pharmacol 36(5):747-755,

1987.

Skoog, K.; Cain, S.; and Nemeroff, C. Centrally administered neurotensin

suppresses locomotor hyperactivity induced by d-amphetamine but not by

scopolamine or caffeine. Neuropharmacology 25(7):777-782, 1986.

Sonsalla, P.K.; Gibb, J.W.; and Hanson, G.R. Opposite responses in the

striato-nigral substance P system to D1 and D2 receptor activation. Eur J

Pharmacol 105: 185-187, 1984.

Stone, D.M.; Stahl. D.; Hanson, G.R.; and Gibb, J.W. The effects of

3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) on monoaminergic systems in the rat brain. Eur J

Phannacol 128:41-48, 1986.

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Yamamoto, B.K., and Spanos, L.J. The acute effects of methylenedioxymethamphetamine on dopamine release in the awake-behaving rat. Eur J

Pharmacol 148:195-203, 1988.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service grants DA 00869

and DA 04222. The National Institute on Drug Abuse provided the

methamphetamine hydrochloride.

AUTHORS

Glen R. Hanson, Ph.D., D.D.S.

Anita Letter, Ph.D.

Kalpana M. Merchant, Ph.D.

Michel Johnson, Ph.D.

Lloyd Bush, M.S.

James W. Gibb, Ph.D.

University of Utah

Salt Lake City, UT 84112

Patricia Sonsalla, Ph.D.

Robert Wood Johnson Medical School

Piscataway, NJ 08854-5635

269
Effects of Neurotoxic Amphetamines on Serotonergic Neurons:

lmmunocytochemical Studies

Mark E. Molliver, Laura A. Mamounas, and

Mary Ann Wilson

INTRODUCTION

The goal of this chapter is to review recent morphologic studies in which

current anatomic methods have been used to characterize the neurotoxic

effects of psychoactive amphetamine derivatives. Several strategies

combining anatomic with biochemical data have been employed to analyze

the effects of selected drugs in this class. These studies show that

psychoactive drugs that have selective neurotoxic effects can be useful

experimental tools to study the neural mechanisms of elusive brain functions

such as affective state control and perceptual integration.

Serotonergic neurons appear to play an important role in higher mental

functions, especially in emotional expression, and in mediating many of the

effects of psychotropic or hallucinogenic drugs. The presence of serotonin

(5-HT) in the brain was first demonstrated in the 1950s employing assay

techniques based on the action of 5-HT on smooth muscle (Twarog and

Page 1953; Amin et al. 1954). Marked regional differences in brain levels

of 5-HT were later found in the dog and cat brain using spectrophotometry

(Bogdanski et al. 1957). High 5-HT levels in limbic areas of the brain led

these authors to speculate that 5-HT may be involved in emotional

expression. The observation that the hallucinogenic drug LSD antagonized

the contractile action of 5-HT on uterine muscle led Gaddum in 1953 and

1958 to propose that 5-HT may act as a central neurotransmitter with a

specific role in cerebral function. Based on the behavioral effects of LSD,

Woolley and Shaw (1954) postulated that 5-HT may be involved in

psychiatric disorders such as schizophrenia. LSD was then shown to

decrease 5-HT turnover in the brain and increase 5-HT levels (Freedman

1961), an effect that was presumably due to inhibition of 5-HT cells in the

dorsal raphe (DR) nucleus (Aghajanian et al. 1968; Aghajanian et al. 1970).

Subsequent physiologic studies have shown that the primary effects of LSD

and of phenethylamine hallucinogens (such as mescaline or 2,5-dimethoxy-4-

methylamphetamine (DOM) are exerted at serotonergic synapses in

270
forebrain (Aghajanian et al. 1970; Rasmussen and Aghajanian 1986, Trulson

et al. 1981; Jacobs 1984). Receptor binding studies have indicated that the

behavioral effects of several of these hallucinogenic drugs are blocked by

ketanserin. a 5-HT2 antagonist (Heym et al. 1984) and that the

hallucinogenic potency correlates roughly with the affinity of such

compounds for 5-HT2 binding sites (Glennon et al. 1984; Glennon 1985).

The studies cited above strongly implicate serotonergic synapses in

mediating hallucinogenic drug effects. Mom recent investigations have

supported the view that designer drugs that are substituted amphetamine

derivatives with psychotropic properties typically release 5-HT from 5-HT

axon terminals and, in some cases, may produce neurotoxic effects.

SURVEY OF SEROTONERGIC NEURONAL SYSTEMS IN THE

BRAIN

An overview of the anatomic organization of 5-HT projections in the brain

is useful as background for understanding the actions and toxicity of

psychotropic amphetamines. Important features of 5-HT neurons are the

diversity of cell types in multiple raphe nuclei and the specificity of their

organization. Serotonergic neurons, first demonstrated by the

histofluorescence method (Falck et al. 1962), are restricted to the brain

stem, where they are localized in multiple discrete clusters along the

midline, primarily within neuronal cell groups designated as the raphe nuclei

(Taber et al. 1960; Dahlstrom and Fuxe 1964). These serotonergic nuclei

extend from the midbrain to the caudal medulla and were originally

described as nine cell groups, named B1 to B9, by Dahlstrom and Fuxe

(1964). Serotonergic axon terminals have been found in widespread areas

of the forebrain (including cerebral cortex, striatum, and diencephalon)

(Fuxe 1965) and throughout the brain stem and spinal cord. A series of

studies employing small intracerebral lesions (Anden et al. 1966; Ungerstedt

1971) indicated that most 5-HT nerve terminals in the forebrain arise from

raphe nuclei in the midbrain and that the axons ascend through the lateral

hypothalamus within the medial forebrain bundle (Moore and Heller 1967;

Azmitia 1978; Conrad et al. 1974).

While most serotonergic cell bodies are located primarily in the midline

raphe of the brain stem, some 5-HT cells lie outside the boundaries of the

raphe nuclei, and not all raphe cells are serotonergic. Serotonergic axons

that innervate the forebrain arise from neurons within the mesencephalic

raphe nuclei. These cell groups are found primarily in the midbrain and

rostral pons and were originally classified as groups B6 to B9. The largest

group of serotonergic neurons is the DR nucleus (B7, DR), which lies within the periaqueductal gray matter. This nucleus extends from a level just

caudal to the oculomotor nucleus down to the rostral portion of the fourth

ventricle. The DR is continuous caudally with a smaller group of 5-HT

cells (B6) that lie along the midline and the floor of the fourth ventricle.

The median raphe (MR) nucleus (also designated central superior or B8)

271
lies within the central portion of the reticular formation in the midbrain

tegmentum (figure 1). The B9 cell group consists of a scattered group of

5-HT neurons that lie along the dorsal surface of the medial lemniscus in

the ventrolateral tegmentum. The other raphe nuclei, B1 to B5, contain

fewer serotonergic cells and are located along the midline in the midpons

and medulla. These more caudal cells give rise primarily to connections in

brain stem and spinal cord. Several more detailed reviews of the serotonergic cells have been published recently and should be consulted for further

information (Moore 1981; Wiklund et al. 1981; Consolazione and Cuello

1982; Jacobs et al. 1984; Molliver 1987). An account of serotonergic

pathways and ascending projections in the rat has been published by

Azmitia and Segal (1978). and a map of raphe cells and projections in the

primate is presented elsewhere (Azmitia and Gannon 1986).

SEROTONERGIC INNERVATION OF CORTEX

While it has been widely believed that 5-HT along with other monoamine

(MA) neurons have diffuse and nonspecific projections, numerous pieces of

evidence indicate that 5-HT projections, although widely distributed

throughout the forebrain, have a high degree of heterogeneity, specificity,

and organization. Recent studies have shown that all cortical areas are

innervated by 5-HT axons, which form a dense terminal arborization with

striking regional differences in the laminar distribution and density of axons.

The original histofluorescence studies were limited by weak fluorescence of

5-HT and rapid fading due to photodecomposition of fluorescent molecules.

The low sensitivity of histofluorescence did not permit detection of fine

axons in the forebrain, so that the density of innervation was initially

underestimated. It was not feasible to visualize the full extent of cortical

5-HT innervation until the advent of immunocytochemistry using 5-HT

antibodies developed by Steinbusch et al. (1978). which were used to depict

the distribution of 5-HT innervation in rat brain (Steinbusch 1981). An

antibody to 5-HT produced in this laboratory was used to analyze the 5-HT

innervation pattern of cerebral cortex (Lidov et al. 1980). Lidov and

colleagues demonstrated a high density of 5-HT-containing axons throughout

the cerebral cortex of the rat with marked regional differences in the density

of axons and the laminar pattern of innervation. A high density of axons

was found in frontal cortex with a gradual decrease in more caudal areas.

In that and subsequent studies (Kosofsky 1985; Blue et al. 1988a), a distinct

laminar pattern of innervation was found in somatosensory cortex, and a

quite different pattern in the cingulate cortex, hippocampus, and dentate

gyrus, where there are distinct bands of highly varicose axons. In the

primate, the 5-HT innervation of cerebral cortex is denser and more highly

differentiated among different architectonic and functional areas (Kosofsky

et al. 1984; Morrison et al. 1982; Morrison and Foote 1986; Wilson and

Molliver 1986; Wilson et al. 1989). For example, marked differences in the

density and distribution of 5-HT axons are found in the macaque on either

side of the central sulcus, in primary motor and somatosensory cortex: while

272
FIGURE 1. The locations of serotonergic cell bodies in midbrain

raphe nuclei

NOTE: The three raphe cell groups depictcd here are the source of serotonergic projections to most

parts of the forebrain. The DR is located in the central gray matter (cg) with many cells 

between and dorsal to the medial longitudinal fasciculus (mlf). The MR is a more

scattered group of 5-HT neurons located in the central portion of the midbrain tegmentum.

A small number of cells lies in the B9 cell group along the medial lemniscus (ml) and give

rise to a small number of cortical projections. This map was prepared by Dr. L

Mamounas based on a section prepared for 5-HT immunocytochemistry.

SOURCE: Mamounas and Molliver 1988, Copyright 1988, Academic Press.

motor cortex is sparsely innervated, somatosensory cortex is characterized

by a high density of 5-HT axons extending acres most layers, with subtle

changes seen within the subdivisions of somatosensory cortex. Primary

visual cortex (Area 17) has an exceptionally dense innervation with a

273
distinctive laminar distribution of 5-HT axons. In visual cortex, the density

in layer IV is particularly high but varies across sublayers, with a decrease

in laminae IVCß in the macaque (Kosofsky et al. 1984). Further primate

studies from this laboratory have revealed highly intricate, detailed

variations in innervation pattern and in the distribution of fine and beaded

axons (Wilson, in preparation; Wilson and Molliver 1989; Wilson

et al. 1989). In addition to these marked differences in innervation, subtle

differences in the laminar pattern of innervation are found between closely

related cortical areas, e.g., between the banks of the principal sulcus, the

divisions of the hippocampal formation, the anterior and posterior parts of

cingulate cortex, and among the subdivisions of somatosensory cortex

(Wilson, in preparation). The main point to be emphasized with regard to

cortical 5-HT innervation is that the characteristic regional differences may

reflect different functional influences of 5-HT neurons upon separate cortical

areas and variations in the effects of 5-HT projections upon particular

cortical cell types.

The intricacy of the 5-HT innervation of cortex is further emphasized by

differential cortical projections from the midbrain raphe nuclei. With the

development of new techniques, there has been progressive clarification of

the complex pattern of raphecortical innervation. Initial axon transport

studies suggested that the DR nucleus projects preferentially upon cerebral

neocortex and striatum while the MR innervates primarily hippocampus and

hypothalamus. Later studies using more sensitive methods demonstrated

that the projections were far more complex and that there is considerable

overlap in raphe projections to forebrain (O’Hearn and Molliver 1984; Imai

et al. 1986). Azmitia and Segal (1978) showed that the DR and MR nuclei

have direct projections to forebrain and give rise to multiple, anatomically

distinct ascending fiber bundles. The terminal distributions of the DR and

MR ascending projections converge, so that most areas of cerebral cortex

are innervated by both nuclei. with regional differences in the relative

contribution from each nucleus; evidence for differential but overlapping

raphe-cortical projections has been presented elsewhere (O’Hearn and

Molliver 1984) and summarized in a recent review (Molliver 1987).

Studies employing highly sensitive retrograde transport methods have shown

that calls within different regions of the raphe nuclei project topographically

to separate areas of cortex (Kohler and Steinbusch 1982; Jacobs et al. 1978;

O’Hearn and Molliver 1984; Waterhouse et al. 1986; Wilson and Molliver

1988). The functional significance of this complex topographic order is

indicated by evidence that individual zones of the raphe nuclei project to

functionally related parts of the brain (Kosofsky 1985; Imai et al. 1986).

Initial retrograde transport studies in the monkey reveal that there is a more

complex and intricate regional pattern of raphe-cortical projections in the

primate than in the rat (Wilson and Molliver 1988; Wilson, in preparation).

These topographic findings, although seemingly complex in detail, indicate

that the DR is heterogeneously organized and that particular zones of this

nucleus project to different cortical areas. The DR and MR nuclei have

274
overlapping projections with dissimilar patterns of organization, and they

terminate predominantly in different cortical layers and upon different cell

types.

DUAL 5-HT AXON TYPES

Further evidence for the specificity of 5-HT projections, of particular

relevance to amphetamine neurotoxicity, came from anterograde transport

studies of the lectin PHA-L conducted by Kosofsky (1985). While it was

known from several previous studies that 5-HT axon morphology is heterogeneous, Kosofsky made the unexpected discovery that there are consistent

morphologic differences between cortical axon terminals that arise from the

DR and MR nuclei, respectively (Kosofsky and Molliver 1987). 5-HT axon

terminals arising from the MR nucleus have large, spherical varicosities

(typically 2 to 3 μm in diameter), giving these axons a characteristic beaded

appearance (figure 2). In contrast, axons that arise from the DR nucleus

FIGURE 2. A schematic representation showing the two classes of

raphe-cortical axon terminals that were identified by

anterograde axon transport

NOTE: Axons that arise from cells in the DR nucleus are extremely fine with minute pleomorphic

or fusiform varicosities. Axons from the MR nucleus have a beaded appereance

characterized by large, spherical varicosities.

SOURCE: Adapted from Kosofsky and Molliver 1987. Copyright 1987, Alan R. Liss, Inc.

are of very fine caliber and typically have minute, pleomorphic varicosities

that are often granular or fusiform in shape. The fine axon terminals are

the most widespread and abundant type in cortex, while the beaded axons

have a more restricted and characteristic distribution. Distinctive beaded

5-HT axons have been described in other areas of forebrain, e.g., in the

entorhinal cortex (Kohler et al. 1980), in the olfactory bulb (McLean and

275
Shipley 1987), and in the hippocampus (Lidov et al. 1980; Zhou and

Azmitia 1986). In addition, two corresponding morphologic axon types

were found in the cat to form mutually distinct axon systems (Mulligan and

Tork 1987; Mulligan and Tork 1988). Moreover, similar, morphologically

distinct axon types have been described in neocortex and hippocampus in

the macaque monkey (Wilson et al. 1989). Preliminary reports state that

beaded 5-HT axons may form pericellular baskets around nonpyramidal

neurons in cortex of the marmoset (Homung et al. 1987) and that similar

5-HT axons may terminate upon GABA-positive cells in the cat (Tork and

Homung 1988).

SEROTONERGIC RECEPTORS IN CORTEX

Binding sites for 5-HT are present in high density throughout the brain, and

these receptors have been the subject of recent reviews by Altar et al. 1986,

Peroutka 1988, and Sanders-Bush 1988. One of the major discoveries in

5-HT pharmacology during the past decade has been the identification of

multiple 5-HT binding sites originally classified by Peroutka and Snyder

1979 and 1981, and designated as 5-HT1 and 5-HT2 receptor types. Similar

numbers of both types are found in cerebral cortex, yet each differs in its

anatomic distribution and in the specific second messenger that is activated

(Conn and Sanders-Bush 1987). Using a new ligand for detecting 5-HT2

receptors (

125I-MIL), which was developed by Dr. P. Hartig and Blue and

coworkers have compared the distribution of 5-HT axons in cortex with that

of 5-HT2 receptors. It was noted that the fine type of 5-HT axon terminals

(DR origin) was closely associated with 5-HT2 receptors, a relationship

especially evident in rat somatosensory cortex where both terminals and

receptors are extremely dense in the upper portion of layer V (Blue

et al. 1986, Blue et al. 1988b). These results raise the possibility that

5-HT2 receptors may be generally associated with fine axon terminals from

the DR and that separate 5-HT projections may form multiple and distinct

functional systems. The association of different classes of 5-HT axons with

different receptors and second messenger systems is further evidence of the

multiplicity and functional specificity of ascending 5-HT projections. The

association of fine axon terminals with 5-HT2 receptors is particularly

relevant to the action of certain psychotropic drugs, which are postulated to

act primarily at this receptor subtype (Glennon et al. 1984; Glennon and

Lucki 1988; Heym et al. 1984).

NEUROTOXICITY OF AMPHETAMINE DERIVATIVES

While neurotoxic effects of amphetamines upon MA neurons had been

reported in previous biochemical studies, a seminal paper from the

University of Chicago has stimulated a new wave of interest in the

neurotoxic effects of substituted amphetamines upon 5-HT projections.

Large doses of the ring-substituted amphetamine derivative

(±)3,4-methylenedioxyamphetamine (MDA) repeatedly administered to rats

276
by subcutaneous injection produced lasting reductions in biochemical

markers for 5-HT in forebrain. Brain levels of 5-HT, levels of the

metabolite 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT uptake into

synaptosomal suspensions were all substantially decreased 2 weeks after

drug treatment (Ricaurte et al. 1985). For example, 5-HT levels in striatum

and hippocampus were decreased more than 70 percent below control

values. These findings were extended and confirmed for both MDA and its

N-methyl analog 3,4-methylenedioxymethamphetamine (MDMA) by independent investigators at Chicago, the University of Utah, and elsewhere

(Commins et al. 1987; Schmidt 1987; Stone et al. 1986; Stone et al. 1987a;

Stone et al. 1987b). The latter study from Gibb’s laboratory showed that

the effects of MDA and MDMA were highly specific for 5-HT axons and

that repeated doses produced greater than 90-percent decreases of tryptophan

hydroxylase activity in cortex. These results were interpreted as indicating

that these hallucinogenic amphetamine derivatives may cause initial release

of 5-HT followed by lasting degeneration of 5-HT projections to forebrain;

they thus appear to be similar to parachloroamphetamine (PCA) in their

action (Schmidt 1987a).

IMMUNOCYTOCHEMICAL (ICC) STUDIES OF MDA AND MDMA

TOXICITY

Based on the biochemical studies that psychotropic amphetamines act largely

upon 5-HT neurotransmission and that prolonged exposure may be toxic to

5-HT neurons, it was of interest to examine the effects of MDA and

MDMA upon the morphology of 5-HT neurons, in order to determine

whether there may be evidence for structural damage to these cells or their

processes, Consequently, an ICC study of the neurotoxic effects of MDA

and MDMA was conducted in this laboratory by E. O’Hearn, in

collaboration with Battaglia, De Souza, and Kuhar from the National

Institute on Drug Abuse (NIDA) Addiction Research Center. In previous

studies, evidence for axon degeneration was reported in the striatum

following administration of MDA or MDMA (Ricaurte et al. 1985;

Commins et al. 1987b) using the Fink-Heimer method, a silver stain for

degenerating axons. However, because of low sensitivity for 5-HT axons,

the silver-staining methods do not accurately depict the full extent or

regional distribution of degenerating 5-HT axons, nor has any other

conventional anatomic method proven satisfactory for thii purpose. Due to

their limited sensitivity, the silver stains even fail to detect forebrain axon

degeneration of MA projections following lesions of the medial forebrain

bundle (MFB) (Moore and Heller 1967). At best, variants of the silver

methods stain a small fraction of degenerating 5-HT axons, primarily in

cingulate cortex, following raphe lesions (Hedreen 1973) or in hippocampus

(Conrad et al. 1974). In order to characterize the cytotoxic effects of MDA

and MDMA, it is important to determine whether there is morphologic

evidence for degeneration of specific monoaminergic axons following drug

administration. A central goal of this study was therefore to obtain

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anatomic evidence that would establish whether or not 5-HT neurons

degenerate following exposure to these drugs. Transmitter immunocytochemistry was employed for the visualization of 5-HT and catecholamine

axons in order to determine whether there is structural evidence for

degeneration, to identify the specific neuronal structures and neuronal

compartments that are damaged by the neurotoxic drugs, and to determine

the regional distribution of the effect 5-HT neurons, their axonal pathways,

and axon terminals were visualized by 5-HT immunocytochemistry using an

antibody to conjugated 5-HT, and cell bodies were examined in Nisslstained sections. Catecholamine axons and cell bodies were visualized using

an antibody to tyrosine hydroxylase (TH).

In initial ICC studies, animals were treated with MDA or MDMA using the

protocol described by Ricaurte et al. (1985). Adult Sprague-Dawley rats

(150 to 200 g) received subcutaneous injections of racemic MDA or

MDMA every 12 hours for 4 days. Each dose was equivalent to 20 mg/kg

of the free base. The rats were sacrificed by intracardiac aldehyde

perfusion 2 weeks after the final dose. In order to study subacute effects

for evidence of degeneration, additional rats received MDA every 12 hours

for 2 days and were sacrificed 24 hours after the last injection. Additional

experimental details are described elsewhere (O’Hearn et al. 1986; O’Hearn

et al. 1988). A series of animals treated identically and in parallel were

analyzed for changes in 5-HT levels and density of uptake sites using

paroxetine binding (Yeh et al. 1986; Battaglia et al. 1987).

The biochemical and pharmacologic results were largely in agreement with

previously reported effects of MDA and MDMA described above. The

main neurochemical results of these studies (see also Battaglia and De

Souza, this volume) confirm that, at 2 weeks after treatment, MDA and

MDMA produced marked reductions in the content of both 5-HT and its

metabolite 5-HIAA in most brain regions, with MDA causing a somewhat

more potent effect. For example, in frontal cortex, 5-HT and 5-HIAA

levels were reduced to 40 to 60 percent of control values; regional

differences are evident in that smaller reductions of approximately

30 percent were found in the hypothalamus. The density of 5-HT uptake

sites determined by paroxetine binding in homogenized tissue blocks showed

highly significant reductions in cerebral cortex (60 to 70 percent),

hippocampus (70 to 75 percent), and hypothalamus (40 to 50 percent)

(Battaglia et al. 1987). No significant changes were found in markers for

catecholamines. The above changes were closely matched by anatomic

changes found in ICC preparations, described below (O’Hearn et al. 1988).

Neurotoxicity of MDA and MDMA

It was previously shown that immunocytochemistry with an antibody

directed against 5-HT provides specific and highly sensitive visualization of

5-HT-containing cell bodies and nerve fibers throughout the central nervous

278
system (CNS) (Lidov et al. 1980, Lidov and Molliver 1982; Steinbusch

1981). The results of O’Hearn et al. (1988) showed that repeated doses of

MDA or MDMA cause, at 2 weeks survival, profound loss of serotonergic

axons throughout the forebrain, especially severe in neocortex, striatum, and

thalamus (figure 3). Catecholamine innervation was unaffected, since no

differences were seen between control and treated rats using TH immunocytochemistry. Both MDA and MDMA produce a similar pattern of denervation in cortex and other parts of the brain, but there is a smaller

reduction in 5-HT axon density following MDMA than after MDA. Therefore, both drugs have similar effects, but MDA is more potent at the

PCA CTRL MDA

FIGURE 3. Neurotoxic effects of psychotropic amphetamines upon 5-HT

axon terminals in rat neocortex

NOTE: Serotonin axons are visualized by 5-HT immunocytochemistry in parietal cortex. The central

panel shows the normal pattern of 5-HT innervation in a control animal. In the right panel,

there is a marked decrease in fine axon terminals 2 weeks following repeated systemic

injections of MDA (20 mg/kg). A similar loss of fine axons is seen in the left panle 2 weeks

following a single dose of PCA (10 mg/kg). Scale bar=100 μm. Darkfield photomicrograph.

If examined with high magnification brightfield microscopy, the spread axons in both treated

animals are all of the beaded type.

same dosage. The loss of 5-HT axons exhibits regional differences in

neurotoxic effects, which are exemplified by partial sparing of 5-HT axons,

particularly evident in hippocampus (figure 4), hypothalamus, basal

279
FIGURE 4. Serotonergic innervation of the dentate gyrus in rat

hippocampal formation

NOTE: Serotonin axons visualized by 5-HT immunocytochemistry in darkfield microscopy. A high

denisty of axons is seen in the control animal (central panel). Following multiple systemic

dosesof MDA (bottom panel) or two doses of PCA (top panel), most fine axon terminals 

degenerate, as seen here at 2 weeks survival. However, there is consistent sparing of beaded

axon terminals, especially marked along the inner surface of the dentate granule cell layer.

Despite the loss of fine axons terminals, the 5-HT innervation in this area, as compared with

neocortex, appears relatively spared following administration of neurotoxic amphetamine

derivatives. Scale bar = 100 μm. 

forebrain, and much of the brain stem, except for superior colliculus, which

is markedly denervated. The forebrain denervation indicates a pronounced,

but consistently selective, loss of 5-HT axons at 2 weeks after drug

280
treatment, which persists for many months. as found in later studies

(Molliver et al., in press). The persistent loss of axon terminals reflects

lasting denervation of target structures and parallels the reduction in 5-HT

uptake sites. A study of the timecourse of regeneration and the origin of

regenerating axons is currently in progress.

Axon Terminals Are Selectively Damaged

The fme morphologic detail afforded by the use of transmitter immunocytochemistry has made it feasible to identify the specific cytologic compartments that arc affected by these neurotoxic drugs. At the 2-week survival

times that were analyzed, intact portions of the neurons are stained by 5-HT

immunocytochemistry, while processes that have degenerated cannot be

visualized. A consistent finding was that raphe cell bodies remain normal

in density and ICC staining intensity, and that many smooth, straight,

tangentially oriented 5-HT axons remain in deep layers of cortex, in

subcortical white matter, and in basal forebrain and lateral hypothalamus.

The disappearance of fine, highly arborized axons with sparing of the

straight preterminal axons is evidence for selective vulnerability of

serotonergic axon terminals. Intense 5-HT immunoreactivity seen in dilated

axons of passage (especially in basal forebrain, in deep layers of frontal

cortex, and in MFB) is presumably due to damming up of neurotransmitter

and other axonal constituents in axon stumps secondary to ablation of the

axon terminals. The accumulation of 5-HT and other contents in preterminal axons and cell bodies indicates that these cellular compartments remain

functionally intact and that transmitter synthesis and anterograde axonal

transport are not evidently impaired. The selective destruction of axon

terminals is consistent with the large decrease in density of 5-HT uptake

sites reported by Battaglia et al. (1987).

Raphe Cell Bodies Are Spared

In Nissl-stained sections, the cell bodies in the raphe nuclei are indistinguishable from those in control brains. The morphology of cell bodies and

dendrites appears unremarkable, and the cells exhibit normal shape and size

and show no evidence of increased staining nor any loss of cytoplasmic

Nissl substance that would reflect chromatolysis. Moreover, Nissl-stained

sections throughout other brain regions including cortex indicate no evidence

of altered cellular morphology. Inclusion bodies in DR neurons of the

monkey that are described elsewhere (Ricaurte et al. 1988) were not seen in

raphe neurons in the rat. The lack of retrograde cytologic changes in raphe

cell bodies is somewhat surprising considering the extensive loss of fine

axon terminals. However, the sparing of cell bodies and of preterminal

axons suggests that there may be substantial potential for recovery and

regeneration of 5-HT projections. The failure to detect cytologic alterations

in raphe cell bodies may reflect technical limitations in the experimental

preparations. First, subtle cytologic changes in DR cell bodies would not

281
be easily detected because these cells normally have fine, dispersed Nissl

substance and eccentric nuclei. Moreover, the use of frozen sections fixed

for immunocytochemistry does not reveal cytologic features at the highest

resolution, and subtle changes might not be visualized. Therefore, more

sensitive cytochemical methods are needed to determine whether there may

be subtle retrograde changes in the raphe neurons.

AXON DEGENERATION

One of the goals of this study was to obtain evidence that would establish

whether or not serotonergic axons are damaged or degenerate following

exposure to psychotropic drugs such as MDA or MDMA. At short survival

times (24 hours after drug administration), while there is a marked decrease

in the number of stained axons, cytopathologic changes are seen in some of

the remaining immunoreactive processes. The most frequent abnormalities

are markedly dilated axons with irregular diameter, giant varicosities, and

fragmentation of axon segments. Giant, swollen varicosities were found in

all cortical areas of treated rats but were never observed in controls. Their

diameter was at least 4 times that of the largest axonal varicosities found in

the normal brain. Moreover, the giant varicosities differ in their regional

distribution from the normal, beaded class of 5-HT axons and appear to be

newly formed structural abnormalities. Greatly swollen axonal stumps are

especially prominent in the basal forebrain and ventral to the genu of the

corpus callosum. At longer survival times, swollen axons are not found,

and the persistent loss of fibers reflects lasting denervation. Several

examples (figure 5) of swollen, fragmented axons are shown in figure 6 of

the report by O’Hearn et al. 1988.

Specific evidence for axon degeneration, especially for MA terminals, is

difficult to establish defmitively. The criteria for degeneration applied in

this chapter are based on previously documented changes in degenerating

MA axons observed by histofluorescence (Baumgarten et al. 1972;

Baumgarten et al. 1973; Bjorklund et al. 1973; Bjorkhmd and Lindvall

1979; Wiklund and Bjorklund 1980; Jonsson and Nwanze 1982). In this

study, the direct visualization of greatly swollen and fragmented nerve fibers

demonstrates that 5-HT axons are structurally damaged by exposure to

MDA and MDMA. These changes are presented as positive evidence for

acute degeneration of axon terminals. This conclusion is further supported

by the damming up of transmitter in swollen preterminal fibers that appear

after the destruction of axon terminals. The subsequent disappearance of

these damaged fibers and persistent loss of fine axon terminals reflects

lasting degeneration. A limitation of transmitter immunocytochemistry for

studying neurotoxicity is that visualization of axons depends upon retention

of the neurotransmitter. Since axons that are depleted of 5-HT cannot be

detected by this method, the present results, while providing positive

evidence for degeneration, are likely to underestimate the number of axons

that are degenerating at any one time. While not currently available, the

282
FIGURE 5. Acute degeneration of 5-HT axons at 1-day survival following

four doses of MDA

NOTE: These axons exhibit cytopathologic changes, such as large, swollen varicosities, irregular

thickening, and fragmentation of fibers. Dilatations of this type are severalfold larger than

the largest 5-HT axon seen in control sections. These changes show evidence of structural

degeneration in 5-HT axons following treatment with MDA. Scale bar = 10 μm.

SOURCE: O'Hearn et al. 1988, Copyright 1988, Oxford University Press.

283
FIGURE 6. Histogram shows the nunber of retrogradely labeled neurons

in the DR and MR nuclei in PCA-treated animals and in

controls

NOTE: Retrogradely labeled cells were counted after a fluorescent dye was injected in

frontoparietal cortex. This figue shows that in rats severely denervated by PCA (right

bars) there is a 92-percent decrease in the number of labeled cells in the DR nucleus, with

no change in the member of cells labeled in the MR nucleus or B9. DR cells:

cross-hatched bars; MR/B9 cells: black bars; control animal on left; moderately denervated

rat in center, severely denervated rat on right. Treated animals received two doses of PCA

(6 mg/kg).

SOURCE: Mamounas and Molliver 1988, Copyright 1988, Academic Press.

use in future studies of a marker that is not released by the neurotoxic

drugs is likely to provide evidence of more extensive terminal degeneration.

The occurrence of drug-induced structural damage and degeneration of 5-HT

axons is further supported by the complete profile of effects produced by

psychotropic drugs such as MDA, PCA, and fenfluramine. Structural

evidence for axon damage is provided by the presence of enlarged

varicosities and swollen fragmented axons in identified 5-HT-containing

fibers at 1 to 2 days after MDA treatment. The formation of enormous,

swollen axons is even more marked after treatment with a structurally

related amphetamine derivative, fenfluramine (5.0 mg/kg) (Molliver and

284
Molliver 1988; Molliver and Molliver, in press). Additional structural

evidence for axonal degeneration is summarized above and includes

Fink-Heimer-positive axons (Ricaurte et al. 1985), persistent loss of fine

axon terminals lasting many months, enlarged axon stumps with intense

immunoreactivity for 5-HT, and the loss of retrograde axonal transport to

DR cell bodies (described below). This structural evidence is accompanied

by the loss of most biochemical markers for 5-HT axon terminals (as noted

previously), including decreases in 5-HT levels, 5-HIAA, tryptophan

hydroxylase activity, and 5-HT uptake sites. Despite this constellation of

findings indicative of 5-HT axon degeneration, one may still speculate that

(however unlikely) axon terminals may remain present yet lack any

detectable properties.

DIFFERENTIAL VULNERABILITY OF 5-HT AXON TYPES

The two morphologic classes of 5-HT axons described earlier (Kosofsky and

Molliver 1987) are differentially vulnerable to the neurotoxic effects of

MDA, MDMA, and certain other neurotoxic amphetamine derivatives. The

denervation caused by MDA and MDMA is subtotal, and some 5-HT axon

terminals are consistently spared in most regions of cortex; them. is a

characteristic regional pattern of axon sparing, as noted above. The analysis

of ICC sections from MDA-treated rats using high-resolution brightfield

microscopy reveals that there is a selective loss of fine axon terminals,

which are almost completely ablated, nearly all of the spared 5-HT axon

terminals in cortex and elsewhere are of the beaded type with large

varicosities (O’Hearn et al. 1988; Mullen et al. 1987). Further analysis of

additional treated and control material shows that the spared, beaded axons

are identical in morphology and distribution to beaded axons that are found

in control animals (Mamounas et al. 1988). The differential vulnerability of

two axon types has been consistently confirmed in a series of additional

studies. The effects of MDA and MDMA were compared with those of

two other substituted amphetamines that were previously shown to cause

similar decreases in biochemical markers for 5-HT, namely PCA and

fenfluramine (Mamounas et al. 1988; Molliver and Molliver 1988; MolIiver

and Molliver, in press). Both of these compounds produced a loss of 5-HT

axon terminals that was indistinguishable from that produced by MDA or

MDMA. In a comparative study of drug effects, PCA administered as two

subcutaneous doses of 10 mg/kg produced a profound loss of 5-HT axon

terminals throughout the rat forebrain, with a regional distribution identical

to that described for MDA (Mullen et al. 1987; Mamounas et al. 1988;

Mamounas et al., in preparation; Mamounas and Molliver 1988). As with

MDA, treatment with PCA (or with fenfluramine) caused a preferential loss

of fine 5-HT axon terminals, while terminals with large, spherical

varicosities were unaffected by these drugs. The spared, beaded axons are

identical in morphology to those found in control animals, and they have

the same regional and laminar distribution. The beaded axons that are

spared are consistently found in layers II to III of parietal and occipital

285
cortex, in the hippocampus where they are located in the subgranular zone

of the dentate gyrus and in the stratum lacunosum of CA1, in layer III of

lateral entorhinal cortex, in the olfactory glomeruli and in other regions

including amygdala, lateral hypothalamus, and most of brain stem (Mullen

et al. 1987; Mamounas et al. 1988; Mamounas et al., in preparation).

Beaded, relatively coarse 5-HT axons that line the ependymal surface of the

lateral ventricle, third ventricle, and aqueduct form a unique group of 5-HT

axon terminals that are also consistently spared by all of the neurotoxic

amphetamines that have been tested. A similar regional distribution of axon

loss was obtained after giving the anorexic drug fenfluramine. Repeated

doses of (±)fenfluramine at 12-hour intervals (n=4 to 8 doses) administered

subcutaneously in doses of 5, 10, or 20 mg/kg produced a persistent loss of

5-HT axons at 2-week survival times with the identical anatomic distribution

and morphologic features of spared axons seen with MDA and PCA

(Molliver and Molliver 1988; Molliver and Molliver, in press). Using three

doses at the 5-mg/kg level and shorter survival times (36 hours), 5-HT

immunocytochemistry revealed a large number of enormously swollen, fragmented 5-HT axons with giant varicosities that are typically over 10 times

the size of normal beaded axons. Thus, fenfluramine produces the same

pattern of axon degeneration as that seen with MDA and PCA (Molliver

and Molliver, in press). Selective neurotoxic effects of d-fenfIuramine,

similar to those found in the rat, have also been observed in cerebral cortex

of the primate (Ricaurte et al., in press). These results indicate that MDA,

MDMA, PCA, and fenfluramine, when administered in moderately large

doses, have nearly identical neurotoxic effects upon 5-HT axons. Moreover,

these studies distinguish two classes of 5-HT axons that differ in their

morphology, regional distribution, and differential vulnerability to

psychotropic drugs. In all cases, the fine axon terminals show consistent

vulnerability to the effects of these compounds, while the beaded axons

appear to be unaffected even at relatively large doses (e.g., 40 mg/kg of

PCA) (Mamounas et al. 1988; Mamounas et al., in preparation).

While the results of the ICC studies summarized above indicate that two

classes of 5-HT axons are differentially affected by particular neurotoxic

amphetamines, analogous examples of selective vulnerability to degeneration

also occur among dopamine (DA) and norepinephrine (NE) neurons in

response to different drugs. Thus, differential vulnerability of specific

subtypes of MA axons appears to be a common feature of these neuronal

systems. For example, the DA neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), selectively damages nigro-striatal DA projections,

while sparing most DA axons that arise from the ventral tegmental area

(Langston et al. 1984; German et al. 1988). Moreover, in human cases of

Parkinson’s Disease, a selective loss of DA has been reported in the

putamen, sparing DA projections to the caudate nucleus (Kish et al. 1988).

In addition, recent studies of the neurotoxin, DSP-4, indicate that NE axons

arising from the locus coeruleus are more susceptible to this compound than

are those that arise from other NE cell groups (Lyons et al. 1989; Fritschy

286
and Grzanna 1989). The above results suggest that a general principle

applying to alI types of monoaminergic neurons may be proposed for

experimental verification: at least two classes of neurons utilize each MA

transmitter, and these neuron subtypes are differentially vulnerable to

neurotoxic or cytopathologic agents. The mechanisms that determine the

differential vulnerability of particular MA cell types are currently unknown,

but are of considerable importance for further understanding of the causes

of neurotoxicity.

DIFFERENTIAL ORIGIN OF 5-HT AXONS

The differential vulnerability of fine and beaded 5-HT axons, combined with

evidence from anterograde transport that fine and beaded fibers arise from

the DR and MR nuclei. respectively, led to the proposal that axons from the

DR nucleus are selectively vulnerable to the neurotoxic effects of psychotropic amphetamines, while the MR projection is resistant. The prior

anterograde transport study (Kosofsky 1985; Kosofsky and Molliver 1987)

sampled a relatively small number of neurons in the central portions of the

DR and MR nuclei and suggested a predominantly differential origin of the

two axon types. In order to determine directly whether the DR and MR

projections are differentially sensitive to psychotropic amphetamines,

L. Mamounas conducted a retrograde axonal transport study using

fluorescent dyes in animals treated with PCA and in controls. PCA is a

useful model experimental drug for the neurotoxic amphetamines, since,

when administered in a single dose. it produces the same pattern of

degeneration as MDA. Fluorescent dye was injected in the cortex and

retrogradely labeled cell bodies were mapped; only those axons that survive

PCA administration are able to take up the label and retrogradely transport

it to cell bodies of origin. Thus, by comparing the number and locations of

cortically projecting raphe neurons in control and treated animals,

identification of the nuclei of origin of drug-sensitive vs. resistant axon

terminals has been possible. The number of labeled neurons (figure 6) in

the DR nucleus of PCA-treated animals was decreased by 77 to 90 percent;

in contrast, the number of labeled neurons in the MR was unchanged

(Mamounas and Molliver 1987; Mamounas and Molliver 1988). These

results demonstrate that DR and MR projections are differentially vulnerable

to PCA, and they confii that fine axon terminals, which are highly sensitive to the neurotoxic effects, arise from neurons in the DR. Moreover, the

loss of the capability for axonal transport is additional evidence in support

of axonal damage and degeneration. These results lead to the proposal that

there are dual 5-HT projections to cortex that are anatomically and functionally distinct. These projections have different nuclei of origin, axon

morphology, regional distributions, and pharmacologic properties. These

findings lead to the further proposal that psychotropic amphetamines act

preferentially upon serotonergic projections from the DR nucleus and that

DR neurons may therefore be involved in the control of affective state and

perceptual integration. While the selective vulnerability of DR axons is

287
likely to be of general validity, at the present time the direct demonstration

by retrograde transport showing a loss of DR projections is based solely on

studies of frontoparietal cortex after using the drug PCA. The consistent

morphology of spared axons suggests that beaded axons in other regions of

the forebrain, which are resistant to these drugs, also arise from the MR

nucleus. However, further studies combining retrograde axonal transport

from a variety of forebrain areas and treatment with MDA and/or MDMA

would be desirable to establish the generality of these findings.

EFFECTS OF MDMA IN PRIMATES

Evidence that MA neurons in the primate brain are susceptible to the toxic

effects of amphetamines was first reported following chronic methamphetamine treatment in rhesus monkeys (Seiden et al. 1975) before the toxicity

was characterized in rodents. Most studies of amphetamine neurotoxicity

have been conducted in rats; however, data from rodents do not always

predict the mechanism of drug action or degree of toxicity in primates. In

order to predict the potential neurotoxic effects of MDMA in humans, it is

essential to analyze the drug effects in monkeys, since there is evidence that

the metabolism of amphetamines in primates differs substantially from that

in other species (Caldwell 1976). In order to determine the sensitivity of

5-HT neurons to MDMA, Ricaurte and colleagues administered various

doses of this drug (2.5 to 5.0 mg/kg) to a series of squirrel monkeys using

subcutaneous injections, repeated twice daily for 4 days. Determination of

5-HT levels by HPLC revealed that multiple doses of MDMA produced

large depletions of 5-HT in many parts of the forebrain including neocortex,

caudate nucleus, hippocampus, and hypothalamus (Ricaurte, this volume).

At 2 weeks after the last dose, the neocortex was markedly depleted of

5-HT, with the lowest dose (2.5 mg/kg) producing a 44-percent depletion

and the highest dose (5.0 mg/kg) producing a 90-percent depletion of 5-HT

(Ricaurte et al, 1987; Ricaurte et al. 1988). Immunohistochemical

preparations from treated monkeys revealed a marked reduction in the

number of serotonergic axon terminals throughout cerebral cortex at 2

weeks survival, with the persistence of some structurally damaged

intracortical axons that were abnormally swollen (Ricaurte et al. 1988). In

addition, examination of cell bodies showed the presence of abnormal

cytoplasmic inclusion bodies in the DR nucleus. These inclusions were

periodic acid-Schiff (PAS)-positive and appeared to contain lipofuscin.

Based on the evidence that MDMA is highly toxic to serotonergic neurons

in primates, a detailed neuroanatomic analysis was conducted by

M.A. Wilson to characterize the morphology and regional distribution of

5-HT axons that are affected by MDMA in the macaque monkey. Two

weeks after treatment with MDMA (eight doses, 5 mg/kg, subcutaneous) the

density of 5-HT immunoreactive axon terminals was strikingly reduced

throughout the cerebral cortex (Wilson et al. 1987; Wilson et al. 1989). A

characteristic regional distribution of serotonergic denervation was found in

different cortical areas. For example, in somatosensory cortex, which is

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densely innervated by 5-HT axons in control animals, few 5-HT axon

terminals remain after MDMA treatment, except for beaded axons in layer

I. In other regions, there was a significant denervation, yet a subgroup of

5-HT axons consistently survives, e.g., in visual cortex, hippocampus,

dentate gyrus, and amygdala

As in the rat, the morphology of 5-HT axons in the normal primate is

heterogeneous, with both fine and beaded axon terminals intermixed. The

fine axon terminals are profoundly vulnerable to MDMA, as found in the

rat, while nearly all of the surviving axons are of the beaded type with

large spherical varicosities. The loss of 5-HT axons in monkeys is greater

than that in rats that were given a fourfold higher dose of MDMA and,

therefore, MDMA is far more neurotoxic in the primate than in the rat

(Ricaurte et al. 1988; Wilson et al. 1989). While cell bodies and

preterminal axons are stained, the morphologic changes in some 5-HT axons

and the persistent loss of fine axon terminals provide evidence that MDMA

produces axon terminal damage and degeneration in the primate cortex.

Furthermore, the selective vulnerability of fine axon terminals and the

sparing of beaded axons indicates that multiple classes of 5-HT axons can

be distinguished in primates, as in rodents. Thus, the morphologic

differences between 5-HT axons, their differential vulnerability to psychotropic drugs, and characteristic regional distributions suggests that--in

primates--there may be two parallel, ascending serotonergic projections

subserving different functions. The susceptibility of fine axons to MDMA

supports the hypothesis that these axons are one of the sites of action of

this drug and am involved in the control of affective state.

The effects of MDMA in the primate indicate that the anatomic organization

and pharmacologic properties of ascending 5-HT projections in the primate

are similar to those in the rodent These studies employing relatively large,

repeated, subcutaneous doses of MDMA were not designed to analyze the

toxicity of this drug in humans, but to obtain results indicating the potential

toxicity, site of action, and biological effects of this drug on 5-HT neurons.

It should be noted that the drug administration schedule may not be comparable to typical human use, that humans generally take MDMA via the

oral route, and that the sensitivity of human and subhuman primates to the

effects of MDMA may not be the same. However, in view of the

extensive destruction of 5-HT axon terminals at doses that are approximately twice that commonly used for recreational purposes by humans,

MDMA may have a relatively small margin of safety, and it would be

prudent to consider this drug potentially hazardous for human use.

Therefore, if human administration of MDMA-like compounds is considered

clinically efficacious, further studies are needed to determine whether there

may be a safe dose range or if there may be related compounds with less

potential toxicity and similar beneficial effects. The studies reported here,

and in other papers in this volume, describe several methodological

approaches and well-characterized parameters to study the effects and

289
neurotoxicity of new psychoactive compounds. The effects of such drugs

on pharmacologic and structural properties of 5-HT neurons in rodents are

highly predictive of the action of these drugs in primates. However, it is

clear from the above results that drug potency varies considerably among

species and must be evaluated separately in primates. The results indicate

that MDMA, combined with biochemical and immunohistochemical studies,

provides a useful experimental tool to study the activity of such drugs and

their neurotoxicity.

It would also be important to determine whether 5-HT axons are altered in

clinical dementias, since a preliminary study shows that swollen 5-HT axons

are associated with amyloid-containing plaques in aged monkeys (Kitt

et al. 1989). Since the swollen 5-HT axon terminals in Alzheimer-like

plaques are similar to degenerating axons seen after MDMA treatment, it is

possible that endogeneous or environmental toxins derived from phenethylamines may play a role in the etiology of dementias. Since illicit recreational use of MDMA and related drugs may produce similar structural

damage to 5-HT axons, it is plausible that the long-term effects of such

damage might predispose susceptible individuals to degenerative disorders of

the Alzheimer’s type. Although this possibility is highly speculative,

long-term prospective followup of MDMA users for subtle psychological

changes in memory and cognitive processes are certainly warranted.

MECHANISM OF MDMA ACTION AND TOXICITY

The mechanisms by which MDMA and related drugs produce their pharmacologic actions and neurotoxic effects are not well understood, making it

difficult to predict what structural or metabolic differences may account for

the differential vulnerability of specific 5-HT axon types. However, new

information from several laboratories has provided insight into the

mechanisms of these drugs and indicates the importance of multidisciplinary

approaches in this area of investigation. The commonality of both

morphologic and biochemical effects of the methylenedioxy-substituted

amphetamines with fenfluramine and PCA suggests that all these compounds

may act via the same mechanism. Both in vivo and in vitro preparations

have shown that this class of compounds acts by acutely releasing serotonin

from 5-HT axon terminals in forebrain (Fuller et al. 1975a; Nichols

et al. 1982; Johnson et al. 1986; Sanders-Bush and Martin 1982; Schmidt

1987a; Trulson and Jacobs 1976). Several studies have shown that the

acute release of 5-HT is distinct from the long- term neurotoxic effects

produced by PCA or MDMA and that separate mechanisms may be

involved (Fuller et al. 1975b; Sanders-Bush et al. 1975; Schmidt 1987a);

both effects depend on a carrier-mediated mechanism. In particular, the

long-term degenerative effects can be prevented by administration of

fluoxetine or citalopram, which block the 5-HT uptake carrier (Fuller

et al. 1975b; Schmidt 1987a). The role of a carrier-mediated mechanism is

further supported by high affinity of MDMA for the 5-HT uptake site

290
(Steele et al. 1987; Battaglia et al. 1988). In summary, previous

pharmacologic studies indicate that MDMA and related psychotropic

amphetamines have a multiphasic effect marked by an acute release of

5-HT, which may be reversible, followed by a chronic decrease in 5-HT

markers probably due to axon degeneration.

Morphologic studies from this laboratory provide strong support for the

multiphasic mechanism described above, and also indicate that fine axon

terminals are selectively affected. In a series of acute in vivo experiments,

a single intraperitoneal injection of PCA (10 mg/kg) or MDA (20 mg/kg)

produced a dramatic reduction in the number of 5-HT immunoreactive

axons in cortex and hippocampus of rats at survivals of 30 minutes to 4

hours following treatment (Berger et al. 1987). When administered by

itself, the 5-HT uptake inhibitor fluoxetine had no effect on the staining or

density of 5-HT axons after acute or repeated doses (Berger et al. 1987;

Berger et al., in preparation); however, fluoxetine (10 mg/kg) coadministered

with either MDA or PCA completely prevented the acute and chronic

decrease in 5-HT immunoreactive axons. The results of these and further

studies demonstrate that both MDA and PCA cause acute depeletion of 5-HT

from fine axon terminals; however, the beaded axons stain intensely and

appear unaffected (Berger et al. 1987; Mamounas et al. 1988). It is of

interest that single or repeated doses of two other ring-substituted

psychotropic amphetamines, DOM and 2,5-dimethoxy-4-ethylamphetamine

(DOET), did not produce a reduction in 5-HT levels or the staining of

5-HT axons (Berger et al. 1987), consistent with the evidence that the latter

drugs act at 5-HT2 receptors (Glennon 1985). The contrasting effects of

these several drugs led to the proposal that there are at least two classes of

psychotropic amphetamines with different sites of action: one type

exemplified by DOM or DOET acts postsynaptically at 5-HT2 receptors,

while the other type, such as PCA and MDA, acts presynaptically by a

carrier-mediated mechanism to release 5-HT from axon terminals (Berger

et al. 1987). Since PCA and MDA (but not DOM or DOET) cause degeneration of 5-HT axons, the ability of amphetamine derivatives to cause

massive release of 5-HT appears related to the neurotoxicity of these

compounds. The fact that fluoxetine prevents the neurotoxicity supports the

idea that the neurotoxic amphetamines act at a presynaptic site located on

5-HT axon terminals and bind to the 5-HT uptake carrier. The selective

releasing effect of psychotropic drugs such as MDA and PCA upon fine

axon terminals is relevant to the finding noted above that these terminals

appear selectively associated with 5-HT2 receptors (Blue et al. 1988b). The

release of 5-HT from this set of terminals may selectively activate 5-HT2

receptors at postsynaptic sites that are linked to activation of

phosphoinositide hydrolysis (Conn and Sanders-Bush 1987).

291
IS A NEUROTOXIC METABOLITE FORMED?

Based on observed differences between the in vivo and in vitro effects of

amphetamine derivatives, several laboratories have suggested that the

neurotoxic effects may depend upon the formation of an active drug

metabolite (Sanders-Bush et al. 1972; Hotchkiss and Gibb 1980; Stone

et al. 1987b); others have suggested that a metabolite might be formed from

DA or 5-HT that is released in large quantities (Johnson et al. 1988;

Commins et al. 1987a; Stone et al. 1988). To pursue this issue, this

laboratory has employed several strategies in an effort to determine whether

the parent amphetamine derivative is itself neurotoxic.

INTRACEREBRAL DRUG ADMINISTRATION

In view of the marked neurotoxicity of systemically administered MDA,

E. O’Hearn administered MDA and/or MDMA directly into cerebral cortex

by stereotaxic microinjection (6 μg in 0.5 μl). At both long and short

survival times (3 days to 3 weeks) the 5-HT innervation density at the

injection site could not be distinguished from that in normal animals or

after saline injections (Molliver 1987; Molliver et al. 1986; O’Hearn et al.,

in preparation). These results suggested that large doses of MDA or

MDMA administered directly into the brain are not neurotoxic and that the

formation of a peripheral drug metabolite may be an essential step in

inducing neurotoxicity. However, several caveats to this interpretation are

raised, particularly that neurotoxic effects may require prolonged exposure to

the drug and these lipophilic compounds are likely to diffuse rapidly from

the injection site. To address the duration of exposure issue, U. Berger has

done a series of experiments in which PCA was continuously infused

directly into the cerebral cortex using an Alzet minipump at a rate of 10 μg

per hour for 48 hours. Following a 2-week survival period, ICC

preparations from the injection site revealed a small zone of local tissue

damage due to the implanted cannula, similar in both drug-injected and

saline control animals. However, despite continuous infusion of PCA (or

MDMA) over 2 days, the serotonergic innervation in the surrounding tissue

appeared normal, with no detectable loss of 5-HT immunoreactive staining.

In contrast, a similar injection of the neurotoxin 5,7-dihydroxytryptamine

(5,7-DHT) produced a zone of total 5-HT denervation at least 2 to 3 mm in

diameter. These chronic intracerebral microinjection experiments lend

further support to the view that the parent compound is not itself neurotoxic

(Berger 1989; Berger et al., in preparation; Molliver et al. 1986).

DRUG EFFECTS IN THE HIPPOCAMPAL SLICE PREPARATION

In order to circumvent the difficulties of maintaining known, constant drug

concentrations in the brain in vivo, the hippocampal slice preparation was

adapted to study the acute anatomic effects of psychotropic drugs. This

method was implemented in conjunction with Drs. K. Stratton and

292
J. Baraban, who have experience in maintaining hippocampal slices under

in vitro conditions for electrophysiologic recording. Several experimental

paradigms have been tested with this method, showing that the hippocampal

slice preparation combined with immunocytochemistry is a useful tool for

studying in vivo and in vitro effects of psychotropic drugs. Freshly

prepared hippocampal slices are incubated in oxygenated buffer, with or

without drugs added, and are then immersion fixed and sectioned for ICC

staining. The quality and sensitivity of axonal visualization in slices is

equivalent to that in sections prepared from perfusion-fixed rats. In slices

from control rats, a high density of morphologically intact 5-HT axons is

seen in the hippocampus, with the same distribution as in conventional

sections. In order to verify the 5-HT-depleting effects of in vivo treatment,

hippocampal slices were prepared from rats that were given a single dose of

PCA (10 mg/kg) subcutaneously, and the animals were sacrificed 3 hours

later. A marked decrease of 5-HT immunoreactive axons was observed in

slices that were fixed immediately after sacrifice of PCA-treated rats. Slices

that were maintained in vitro in physiological saline showed progressive

recovery of 5-HT axon staining over 0.5 to 2 hours. However, if the

survival time of the animal after PCA treatment was extended for over 24

hours, then no recovery was seen, and the loss of 5-HT axons was

irreversible. These results provide direct immunochemical and anatomic

support for previous pharmacologic studies that showed a biphasic effect of

PCA and related amphetamine derivatives, characterized by an early phase

of 5-HT depletion that is potentially reversible during the first 24 hours

(Fuller et al. 1975a; Sanders-Bush et al. 1975; Schmidt 1987a). During this

acute phase, the fine axons are depleted of 5-HT but have not degenerated;

the terminals retain the ability to synthesize and store 5-HT if the toxic

compound dissociates, as observed in the slice incubation bath (Molliver

et al. 1988). With longer in vivo survival times (4 to 6 days after PCA)

the lack of subsequent recovery in vitro provides evidence for irreversible

axon degeneration. The timecourse of this biphasic effect closely matches

that reported by Fuller et al. (1975b) based on the use of 5-HT uptake

blockers to displace PCA in vivo.

In order to test the cytotoxic potential of PCA alone, hippocampal slices

from untreated control animals were incubated in buffer containing PCA,

over a wide range of concentrations (typically 50 μM) for 2 to 3 hours.

The incubation of slices directly in the parent compound (PCA) did not

induce 5-HT depletion, and the 5-HT innervation in these slices was indistinguishable from that in control animals. Moreover, incubation of slices

from PCA-treated animals in PCA-containing buffer did not prevent the

recovery of 5-HT immunoreactive axons. The absence of 5-HT depletion

after immersion of hippocampal slices in PCA strongly supports the proposition that PCA and related drugs are not directly neurotoxic. Thus, in vivo

systemic administration of the drug appears necessary for the formation of a

neurotoxic compound, such as a metabolite of the drug or of 5-HT, which

is released.

293
THE PROTECTIVE EFFECT OF 5-HT DEPLETION

To determine whether the release of endogeneous 5-HT mediates the neurotoxic effects of PCA in the brain, several pharmacologic regimens were

employed to deplete animals of 5-HT prior to treatment with PCA. In a

series of studies conducted by U. Berger, rats were depleted of 5-HT by

prior treatment with reserpine (2.5 mg/kg), the 5-HT synthesis inhibitor

parachlorophenylalanine (PCPA) (250 mg/kg), or a combination of both

drugs. These drugs initially produce depletion of 5-HT in brain and other

tissues (over several days) followed by recovery to normal levels over

2 weeks; after that time, normal 5-HT ICC axon staining is obtained, and

no evidence of axonal swelling or degeneration was observed. Animals

depleted of 5-HT by different regimens were subsequently treated with PCA

(10 mg/kg) and tested after 2 weeks for 5-HT neurotoxicity using both

HPLC and immunocytochemistry. This study revealed a marked protective

effect on 5-HT neurons after combined treatment with PCPA plus reserpine.

After the extensive 5-HT depletion produced by combined treatment with

both drugs, PCA produced only a small reduction in brain 5-HT levels, and

nearly all 5-HT axons in forebrain were spared. Reserpine pretreatment

alone, although producing substantially reduced brain MA levels, did not

afford significant protection against the effects of PCA. This result

indicates that depletion of 5-HT (and other biogenic amines) from vesicular

storage sites in the brain does not provide significant protection against the

neurotoxic effects of PCA, whereas more extensive depletion from brain,

platelets, and intestine by inhibition of 5-HT synthesis does prevent the

toxicity of PCA (Berger, in preparation). Since a primary pharmacological

effect of PCA is the release of 5-HT from nerve terminals and platelets,

these results suggest that the neurotoxicity of PCA is dependent upon the

presence of a releasable pool of 5-HT and is not mediated directly by the

drug or one of its metabolites. The site of the PCA-induced 5-HT release

essential for neurotoxicity is not known, although depletion of vesicular

5-HT is not itself sufficient for protection. The data suggest that an

extensive depletion of 5-HT pools is required to block the PCA-induced

toxicity. These results lead to the suggestion that the 5-HT stores in

platelets (or mast cells), the main 5-HT storage sites in the periphery, may

play a central role in the neurotoxic mechanism (Berger et al. 1989).

Therefore, it is postulated that a neurotoxic metabolite is formed from 5-HT

released by the action of PCA on platelets or on other 5-HT storage sites.

PCA is also a strong inhibitor of MA oxidase (Fuller 1966) and may

therefore facilitate the formation of an unusual neurotoxic indolamine

metabolite from peripherally released 5-HT. This metabolite, not yet

identified, may enter the brain and cause selective destruction of 5-HT

axons. This proposal is consistent with the findings from Seiden’s

laboratory that the neurotoxin 5,6-dihydroxytryptamine can be detected in

the brain following PCA administration (Commins 1987a). Moreover, since

reserpine depletes other biogenic amines, a role for catecholamines in the

toxicity of amphetamine derivatives should be considered, as proposed

294
earlier (Johnson et al. 1988; Stone et al. 1988). While further investigation

is needed to determine the origin and identity of the neurotoxic compound,

the present studies indicate that exposure to the parent compound itself, e.g.,

PCA or MDA, is not sufficient to produce a lasting neurotoxic effect, nor

does it produce acute 5-HT depletion.

CONCLUSION

The present studies demonstrate the value of combining morphologic with

biochemical methods to study the neurotoxicity of psychotropic drugs upon

central 5-HT neurons and to identify the specific neurons and neuronal

compartments that are affected. There are two distinct serotonergic projections to forebrain that arise from the DR and MR nuclei, respectively, and

have different patterns of termination in cortex, morphologically distinct

axon terminals, and dissimilar pharmacologic properties. Substituted amphetamine derivatives PCA, MDA, MDMA, and fenfluramine have similar

profiles of neurotoxicity in the brain and all act selectively upon the fine

axon terminals that arise from the DR nucleus. Direct anatomic evidence

for structural damage to 5-HT axon terminals has been obtained after treatment with MDA and with fenfluramine. These cytopathologic changes in

axons combined with the pharmacological profile of effects, which include

persistent decreases in 5-HT levels, turnover, synthesis, and uptake sites,

provide convincing evidence that these psychotropic amphetamines can

produce axon terminal degeneration. The exact mechanism of neurotoxicity

has not yet been elucidated, nor has the specific neurotoxin been identified.

Present evidence indicates that neither the parent compound alone nor a

drug metabolite produces 5-HT depletion or degeneration. Preliminary

evidence that depletion of central and peripheral 5-HT affords protection

against the effects of RCA leads to the hypothesis that a metabolite of 5-HT

released in the periphery, possibly from platelets, is essential for the

expression of amphetamine-induced neurotoxicity. The selective toxic effect

of these compounds upon one class of 5-HT axon terminals with sparing of

other 5-HT axons and of raphe cell bodies provides a setting in which

regenerative sprouting is likely to occur, a subject of ongoing investigation.

The augmented neurotoxicity of MDMA in primates raises concern about

the possible neurotoxic effects of this drug in humans. Further studies are

needed to determine whether there may be a safe range of doses for human

use of compounds in this class and whether clinically efficacious drugs

similar to MDMA but without toxic effects can be designed. Current data

also suggest that it may prove useful to explore experimentally the use of

5-HT uptake blockers such as citalopram, paroxetine, or fluoxetine to

protect against the cytotoxic effects of drug overdose. The use of selective

neurotoxic drugs in experimental studies should continue to enhance our

understanding of the complex functional organization of 5-HT projections in

the brain and the multiplicity of effects that have been ascribed to this

neurotransmitter.

295
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ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service Research grants

DA 04431, NS21011, NS15199, and HD19920.

AUTHORS

Mark E. Molliver, M.D.

Professor

Department of Neuroscience and Neurology

Laura A. Mamounas, Ph.D.

Fellow

Department of Neuroscience

Mary Ann Wilson, B.A.

Fellow

Biochemistry, Cellular, and Molecular Biology Program

Department of Neuroscience

Johns Hopkins University School of Medicine

725 North Wolfe Street

Baltimore, MD 21205

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Studies of MDMA-Induced

Neurotoxicity in Nonhuman

Primates: A Basis for Evaluating

Long-Term Effects in Humans

George A. Ricaurte

INTRODUCTION

Studies of (±)3,4-methylenedioxymethamphetamine (MDMA) neurotoxicity

in nonhuman primates are potentially of great importance to both basic

science and public health. Scientifically, such studies could shed light on

the functional role of serotonin in the primate central nervous system

(CNS). In this regard, it is pertinent to recall that it was not until the

effects of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP) were explored in the monkey that its profound dopaminergic neurotoxic effects were noted (Chiueh et al. 1983) and then utilized to develop

the first complete animal model of Parkinson’s disease (Burns et al. 1983).

Given this precedent, it seems not unreasonable to speculate that studies of

MDMA in the primate could similarly enhance our understanding of seroto

nergic function in higher animals. From a public health perspective,

MDMA studies in monkeys are of value because they will help to define

the risk that MDMA poses to humans. Additionally, these studies could

help identify functional consequences of MDMA neurotoxicity in primates,

and thus guide the clinical assessment of MDMA-exposed individuals.

This chapter will review some recently completed studies on the long-term

effects of MDMA in nonhuman primates. The goals of these studies were

to (1) determine if the neurotoxic effects of MDMA, which have been well

documented in the rodent (see below), generalize to the primate; (2)

compare the relative sensitivity of primates and rodents to the neurotoxic

effects of MDMA; (3) ascertain if the toxic effects of MDMA in the

monkey are restricted to nerve fibers (as they are in the rat). or if they

involve cell bodies as well; (4) evaluate how closely toxic doses of MDMA

in the monkey approximate those used by humans; and (5) examine whether

5-hydroxyindoleacetic acid (5-HIAA) in the cerebrospinal fluid (CSF) can

be used to detect MDMA-induced serotonergic damage in the CNS of primates. Before presenting the results of these studies, previous results in the

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rodent will be briefly summarized, so as to put findings in the primate in

proper perspective.

PRIOR FINDINGS IN RODENTS

Bats given MDMA show prolonged reductions in the concentration of brain

serotonin (Schmidt 1987; Commins et al. 1987; Stone et al. 1986; Battaglia

et al. 1987; Mokler et al. 1987; Ricaurte et al. 1987), the number of

serotonin uptake sites (Schmidt 1987; Battaglia et al. 1987; Commins

et al. 1987), the level of 5-HIAA (Mokler et al. 1987; Stone et al. 1986),

and the activity of tryptophan hydroxylase (TPH) (Stone et al. 1986).

Correlative anatomical studies indicate that these neurochemical changes are

due to damage of serotonergic axons (O’Hearn et al. 1988). Cell bodies in

the brainstem of the rodent do not appear to be damaged by MDMA.

Serotonin-containing perikarya in the raphe nuclei of rats have a normal

cytological appearance and show no obvious reduction in number (Molliver

1987; O’Hearn et al. 1988). In guinea pigs, MDMA produces long-term

neurochemical effects similar to those in rats (Commins et al. 1987). This

is noteworthy because guinea pigs (like humans) metabolize amphetamine

primarily by side-chain deamination, whereas rats do so mainly by ring

hydroxylation (Caldwell et al. 1976). In contrast to guinea pigs and rats,

mice do not develop long-term depletions of serotonin after MDMA, even

after high doses (Stone et al. 1987). This provocative finding raises the

important question of whether other animals might not also be resistant to

MDMA’s neurotoxic effects. In this regard, monkeys are of special interest

because of their close phylogenetic relationship to humans and because they

metabolize amphetamine in a manner similar to humans (Caldwell

et al. 1976). For these reasons. as well as for those previously mentioned,

studies were undertaken to evaluate the neurotoxic potential of MDMA in

nonhuman primates.

OBSERVATIONS IN PRIMATES

Studies were performed in the squirrel monkey (Saimiri sciureus). This

primate species was selected because of its size, availability, and prior use

in neurotoxicity studies (Langston et al. 1984). Initial dose-response

determinations were carried out using the following doses of MDMA:

2.50, 3.75, and 5.00 mg/kg. Each dose of MDMA was administered

subcutaneously twice daily (at approximateIy 0800 and 1700 hours each

day) for 4 consecutive days. This particular regimen of drug administration

was employed because its prior extensive use in the rat (Commins

et al. 1987; Battaglia et al. 1987; Ricaurte et al. 1987) would permit

comparison of results in the monkey with those in the rat. Two weeks

after drug treatment, the animals were killed, the brains were removed,

dissected, and then analyzed for their regional content of serotonin,

doparnine, and noradrenaline using the method of Kilpatrick et al. (1986), as

previously described (Ricaurte et al. 1988a).

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MDMA produced a dose-related depletion of serotonin without altering the

concentration of either dopamine or norepinephrine in the monkey brain

(table 1). Even the lowest dose of MDMA produced a substantial depletion

TABLE 1. Selective dose-related depletion of serotonin in cerebral cortex

of monkeys administered MDMA 2 weeks previously

Treatment 5-HT DA NE

Saline 0.167 ± 0.015 10.4 ± 0.5 0.39 ± 0.03

MDMA - 2.50 mg/kg 0.093 ± 0.010* NT** NT

(-44%)

MDMA - 3.75 mg/kg 0.037 ± 0.013* NT NT

(-78%)

MDMA - 5.00 mg/kg 0.017 ± 0.003* 9.7 ± 0.8 0.41 ± 0.02

(-90%) (ns) (ns)

*p<0.05, determined by individual comparison to control after one-way analysis of variance showed

F value p<0.05

**NT=not tested because higher dose was without effect.

NOTE: Values in μg/g represent the mean ± SEM (n=3).

of serotonin in the cerebral cortex of the monkey (-44 percent). MDMA

also reduced the concentration of cortical 5-HIAA (table 2). Reduced levels

of serotonin and 5-HIAA were evident not only in the cerebral cortex, but

also in the caudate nucleus (-86 percent), hippocampus (-77 percent), hypothalamus (-77 percent), thalamus (-84 percent), and putamen (-90 percent)

(table 3). Anatomical studies were subsequently carried out in collaboration

with Dr. Mark Molliver and Marianne Wilson of the Johns Hopkins University School of Medicine to determine if there was a structural basis for the

serotonin and 5-HIAA depletions induced by MDMA. These morphological

studies showed that there was a marked reduction of serotonin-immunoreactive axons in the monkey forebrain (figure 1). and that, at high power,

some of the remaining axons appeared swollen and misshapen (Wilson

et al. 1988). Coupled with the biochemical observations, these morphological findings suggest that MDMA produces neurochemical deficits by

damaging serotonergic axons. Further, they demonstrate that the long-term

effects of MDMA originally documented in the rodent generalize to the

primate.

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TABLE 2. Decreased concentration of 5-HIAA in the monkey brain 2 weeks

after MDMA (5 mg/kg)

N Neocortex Caudate Hippocampus Hypothalamus

Control 3 0.250 ± 0.010 0.232 ± 0.005 0.245 ± 0.006 0.897 ± 0.042

MDMA 3 0.040 ± 0.006* 0.056 ± 0.002 0.060 ± 0.001* 0.543 ± 0.084*

*p<0.05, two-tailed student's t-test.

NOTE: Values represent the mean ± standard error of the mean.

TABLE 3. Regional concentrations of serotonin in the monkey brain

2 weeks after MDMA (5 mg/kg)

Somatosensory

Cortex Caudate Putamen Hippocampus Hypothalmus Thalamus

CONTROL (n=3)

0.14 ± 0.01 0.21 ± 0.03 0.28 ± 0.02 0.13 ± 0.03 0.90 ± 0.06 0.73 ± 0.01

MDMA (n=3)

0.02 ± 0.01* 0.03 ± 0.01* 0.03 ± 0.01* 0.03 ± 0.01* 0.21 ± 0.01* 0.12 ± 0.01*

*p<0.05, two-tailed student's t-test. 

NOTE: Values represent the mean ± standard error of the mean. 

RELATIVE SENSITIVITY: PRIMATES VS. RODENTS

Next, the relative sensitivity of monkeys and rats to the serotonin-depleting

effects of MDMA was evaluated. This was done by comparing doseresponse data in these two experimental animals. In the monkey, a

2.5 mg/kg dose regimen of MDMA produced a 44 percent depletion of

serotonin (figure 2). By contrast, in the rat, a 10 to 20 mg/kg dose

regimen of MDMA was required to produce a comparable effect. Thus,

monkeys are 4 to 8 times more sensitive than rats to the serotonin-depleting

effects of MDMA. Inspection of the data in figure 2 also showed that the

dose-effect curve of MDMA in the monkey is much steeper than in the rat.

Consequently, small increments in dose cause large increases in serotonin

depletion in the monkey but not in the rat. Clearly, this could have serious

implications for humans experimenting with higher doses of MDMA, as it

suggests that the margin of safety of MDMA in primates is narrow.

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MDMA CONTROL

FIGURE 1. Marked reduction of serotonin-immunoreactive axons in the somatosensory cortex of

MDMA-treated monkey
FIGURE 2. Dose-response data in rats and monkeys administered MDMA

2 weeks previously

INVOLVEMENT OF NERVE CELL BODIES

The severity of the axonal damage caused by MDMA in the forebrain of

the monkey raised the question of whether cell bodies in this experimental

animal might also be damaged by MDMA. As noted above, cell body

damage does not occur in the rat (O’Hearn et al. 1988). However, it is to

be recalled that in the case of MPTP, it was not until it was tested in the

monkey that MPTP’s toxic effects on cell bodies were appreciated (Chiueh

et al. 1983; Langston et al. 1984). To determine if this was also the case

for MDMA, the brainstem of monkeys treated with the high dose (5 mg/kg)

regimen of the drug 2 weeks previously was examined histologically.

MDMA-treated monkeys showed no obvious cell loss in either the dorsal or

median raphe nuclei. However, there were clear cytopathologic changes in

nerve cells of the dorsal (but not median) raphe nucleus. Specifically,

nerve cells in the dorsal raphe nucleus appeared shrunken and contained

brownish-red intracytoplasmic spherical inclusions, which were acid fast in

Ziehl-Nielsen stain for lipofuscin, granular in LFB-PAS-stained sections, and

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vividly PAS positive. While the significance of these inclusions remains

unclear, their staining characteristics suggested the presence of an increased

amount of lipofuscin. More recent studies investigating the fate of these

inclusion-bearing nerve cells have suggested that they do not die, and that

their survival is associated with partial recovery of serotonin in the monkey

forebrain (Ricaurte et al. 1988c). Similar recovery of serotonin has been

noted in MDMA-treated rats (Battaglia et al. 1988). It remains to be

determined if recovery of serotonin is related to regeneration of serotonergic

axons, and if the new axons innervate their original targets or form aberrant

synaptic contacts.

RELEVANCE TO HUMANS

Clearly, much of the impetus for investigating the neurotoxic effects of

MDMA in animals has come from concern that MDMA may produce

similar toxic effects in humans. However, the extent to which findings in

animals can be extrapolated to humans has been unclear, largely for three

reasons: First, in most animal studies, MDMA has been administered

subcutaneously or intraperitoneally (Schmidt 1987; Commins et al. 1987;

Battaglia et al. 1987; Mokler et al. 1987), even though humans invariably

take the drug orally (Seymour 1986). Second, most animal studies have

used multiple doses of MDMA, and these have been given over relatively

short periods of time (Commins et al. 1987; Battaglia et al. 1987; Mokler

et al. 1987). By contrast, humans typically take single doses of MDMA,

usually weeks apart (Seymour 1986). Third, doses of MDMA tested in

animals have often far exceeded those used by humans.

In an effort to bridge the gap between studies of MDMA in animals and

human MDMA use patterns, the following studies were performed. These

studies tested the importance of dose, route, and schedule of drug

administration as determinants of MDMA neurotoxicity. In the frost

experiment, one group of monkeys received MDMA (5 mg/kg twice daily

for 4 days) subcutaneously; another group received an identical dosage

regimen of the drug orally. The animals were killed 2 weeks later, and

regional brain serotonin concentrations were determined. Monkeys given

oral MDMA showed depletions of serotonin that, depending on brain region,

ranged from one-third to two-thirds of those found in monkeys given the

drug subcutaneously (table 4). Recently, similar results have been obtained

in rhesus monkeys (Kleven et al. 1989). Taken together, the results of

these studies indicate that the oral route of administration does not afford

significant protection against the long-term effects of MDMA on serotonin

neurons.

A second study compared the effects of single versus multiple doses. One

group of monkeys received a single 5 mg/kg dose of MDMA orally;

another group received the same dose by the same route, but on a twice

daily basis for 4 days. As before, the multiple dose regimen produced a

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large depletion of serotonin in all forebrain regions examined (table 5). By

contrast, the single dose produced a depletion of serotonin only in the

thalamus and hypothalamus. In both brain regions, the depletion was

smaller than that produced by the multiple dose regimen, but achieved

statistical significance. The long-term effects of even single doses of

MDMA further attest to the high sensitivity of the primate to the serotonindepleting effects of MDMA. Further, they raise the question of whether

humans might be similarly affected, particularly since they take doses that

are only 2 to 3 times lower than the dose that produce an effect in the

monkey (1.7 to 2.7 vs. 5.0 mg/kg).

STUDIES OF CSF 5-HIAA

Detecting a depletion of serotonin in the brain of a living human poses a

major challenge. To date, only two methods have been attempted. The

first involve measurement of 5-HIAA in the CSF (Garelis et al. 1974; Moir

et al. 1970); the second calls for neuroendocrine challenge with various

serotonergic agents (Cowen and Anderson 1976; Heninger et al. 1984).

Unfortunately, both of these methods are indirect, and neither has been fully

validated. Accordingly, it was necessary to test the usefulness of CSF

5-HIAA as a marker of MDMA neurotoxicity in the monkey before

attempting to use it on humans.

To validate the CSF 5-HIAA method in the monkey, three monkeys were

given MDMA at a dose that produces extensive serotonergic damage

(5 mg/kg twice daily for 4 days, SC); three other age- and sex-matched

animals were given saline and served as controls. Two weeks later, all of

the animals were lightly anesthetized with ether, and 200 to 300 μL of CSF

were removed by cervical puncture. Later that same day, all animals were

killed for determination of regional CNS and CSF serotonin and 5-HIAA

levels. These measurements showed that MDMA lowered the concentration

of 5-HIAA in the CSF but not that of homovanillic acid (HVA) or

3-methoxy-4-hydroxyphenylene-glycol (MHPG) (table 6). The reduction of

CSF 5-HIAA was associated with a marked depletion of serotonin in the

CNS (table 7). The decrease in 5-HIAA in cervical CSF was smaller than

the depletion of serotonin in the forebrain (59 percent vs. 90 percent), but

greater than the depletion of serotonin in the cervical spinal cord

(45 percent vs. 59 percent) (Ricaurte et al. 1988b). Hence, cervical CSF

5-HIAA underestimates serotonin depletion in the forebrain, but overestimates serotonin depletion in the cervical spinal cord. These results

indicated that while 5-HIAA in CSF does not fully reflect the depletion of

serotonin in the forebrain, it can serve as a partial indicator of serotonergic

damage induced by MDMA in the forebrain of primates.

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TABLE 4. Effect of Oral vs subcutaneous MDMA on regional brain serotonin in the primate 2 weeks later

TABLE 5. Effect of single vs multiple doses of MDMA on regional brain serotonin in the primate 2 weeks later
TABLE 6. Selective reduction in 5-HIAA in CSF of monkeys administered

MDMA 2 weeks previously

Treatment N 5-HIAA HVA MHPG

Saline 3 101 ± 7 255±44 30 ± 4

MDMA 3 41 ± 7* 264 ± 20 40 ± 11

*p<0.05, two-tailed student's t-test.

NOTE: Values represent the mean ± SEM (expressed in μg/mg of tissue).

TABLE 7. Selective reduction in serotonin in the caudate nucleus of

monkeys administered MDMA 2 weeks previously

Treatment N

Saline 3

Serotonin

0.218 ± 0.023

Dopamine

11.6 ± 0.9

Norepinephrine

2.59 ± 0.18

MDMA 3 0.021 ± 0.005* 9.7 ± 0.2 2.84 ± 0.91

*p<0.005, two-tailed student's t -test.

NOTE: Values represent the mean ± SEM (expressed in μg/mg of tissue).

CSF 5-HIAA STUDIES IN HUMANS

In light of this, studies of CSF 5-HIAA have been initiated in a cohort of

human volunteers with a history of extensive MDMA use. Most participants in the study are individuals who have recently learned of the

neurotoxic properties of MDMA and have asked to be evaluated for

possible serotonergic damage. To qualify for the study, subjects must (1)

have used MDMA on at least 20 to 25 occasions, (2) be drug-free for at

least 2 weeks prior to participating in the study, and (3) not have a history

of neuropsychiatric illness thought to involve alterations in serotonin

metabolism. To date, 34 individuals have participated in the study. The

study is now in progress, and completion is anticipated by 1991. At this

time, it would be premature to comment on the results.

NEUROENDOCRINE STUDIES

As noted earlier, the only other method presently available for detecting

serotonergic dysfunction in living humans involves neuroendocrine challenge

with serotonin-active drugs (Cowen and Anderson 1976). One such

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neuroendocrine test is the L-tryptophan challenge test (Heninger

et al. 1984). Briefly, this test calls for intravenous administration of

L-tryptophan to human subjects with subsequent measurement of serum

prolactin concentration. A rise in serum prolactin is taken as a measure of

central serotonergic activity. Using the L-tryptophan challenge tests,

serotonergic function was recently evaluated in nine MDMA subjects in

collaboration with Drs. Price and Heninger of the Yale University School of

Medicine. L-tryptophan induced a robust rise in serum prolactin in controls

but not in MDMA subjects (figure 3). The peak change in serum prolactin

concentration and the area under the prolactin response curve were

diminished in MDMA subjects, but the difference was not statistically

signifiit (Price et al. 1989). Additional studies are now in progress to

assess the significance of these findings.

FIGURE 3. Prolactin response to IV L-tryptophan in control and

MDMA subjects (n=9)

SUMMARY AND CONCLUSION

The results of the studies reviewed here show that the neurotoxic effects of

MDMA generalize to the primate. Further, they indicate that monkeys are

considerably more sensitive than rats to the serotonin-depleting effects of

MDMA, and that the dose-response curve of MDMA in the monkey is

much steeper than in the rat. Perhaps as a consequence of this, the toxic

effects of MDMA in the monkey involve serotonergic nerve fibers as well

as cell bodies, whereas in the rat, only nerve fibers are affected. The

present studies also show that the toxic dose of MDMA in the monkey

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(5 mg/kg) closely approaches the dose typically used by humans (1.7 to

2.7 mg/kg). This finding heightens concern that MDMA may be neurotoxic

in humans, particularly since the steepness of the dose-response curve of

MDMA in the primate suggests a narrow margin of safety. Finally,

preclinical studies in monkeys have shown that CSF 5-HIAA can be used to

detect MDMA-induced serotonergic damage in the primate CNS. Studies

now underway in MDMA-exposed humans should help determine if MDMA

exerts long-term toxic effects on serotonergic neurons in the human brain.

DISCUSSION

QUESTION: How long after the administration of the MDMA in your

human subjects did you measure your parameter?

ANSWER: The request we made to the subjects was that they remain

entirely drug-free for 2 weeks. We were trying to simulate the situation

that we had in animals.

QUESTION: How long after the administration of the drug did you

measure the 5-HIAA?

ANSWER: We did not administer MDMA. We were dealing with humans

who had been previously exposed to MDMA. The request we made was

that they not take the drug for at least 2 weeks. Some subjects had not

taken the drug for over a year. Some had taken it as recently as 19 days.

QUESTION: Is there a decrease in serotonin metabolites, and does it show

any relationship to age, cumulative dose, and so forth? Did you see

anything there? You have that one person who had 42 grams. Was he any

more or any less affected?

ANSWER: We are in the midst of analyzing the data. You will remember

that the mean 5-HIAA level in control subjects is approximately 19 to 20.

In that one individual, 5-HIAA level turned out to be 14 or 13. You might

predict that he would have been the lowest one on the scale. I subsequently learned the importance of body height, and he happens to be a very

short, stocky man. So we are now in the process of reanalyzing the data,

trying to take in the appropriate variables into account. And, again, these

arc studies that have to be replicated in our own hands and extended by

others.

QUESTION: Do you see a progressive decline as a function of age, and if

you have someone who has been damaged, is there any age relationship in

this depletion?

ANSWER: No. Only one individual was 70 or 71 years old. By and

large, the individuals are younger than 40. The other side of the coin, of

317
course, is does 5-HIAA really change with age in the control population?

That was one of the variables that I listed. I would emphasize that the data

for that are actually very weak. My guess is that there are no age-related

changes in 5-HIAA, at least out to 50 to 60 years of age. But that is

something we are going to have to contend with as well.

QUESTION: You showed CSF levels in monkeys after the 4-day treatment

regimen, but you also showed neurochemical data after a single oral

administration. Did you have the opportunity to look at the CSF levels

after a single oral administration?

ANSWER: No. That study was actually completed before the CSF studies.

We did not get CSF data on the animals that received the single dose

because that study was done before the CSF studies were undertaken.

COMMENT: With regard to the CSF levels, I want to emphasize that the

decreases that you get are probably a gross underestimate, as was pointed

out, Another reason is that the ventricular plexus of serotonin fibers is

completely unaffected, and that is probably a very large source. I do not

know to what extent that contributes to CSF levels of S-HT and S-HIAA,

but those fibers are in the CSF and bathing in it, and they appear to be

quite active. So they must be biasing against your seeing an effect. The

fact that you are getting such a sizable effect must mean that there is a

very profound depletion in the forebrain.

QUESTION: Does anything suggest that people who have taken these

drugs or MDMA for a period of time are subject to episodes of depressive

disorders or affective disorders?

ANSWER: Frankly, at this point, we have only anecdotal evidence. And

as Dr. Schuster mentioned yesterday, people’s responses can be very

misleading. I could cite three individuals who attribute some mood

disturbances to their prior MDMA use, but one wonders how much their

reports are based on what you want to hear.

One individual was prescient enough to realize that his depression coincided

with loss of his job so he did not know if his depression was related to

losing his job or to MDMA ingestion. I think these people are going to

need to be looked at by people who know what they are doing in terms of

analyzing depression, and that has not been done.

COMMENT: It is interesting in that letter that George Greer wrote to me

informally, off the record, that he had seen 10 patients in psychotherapy

who had been treated extensively with fenfluramine for dieting. And, after

several weeks on fenfluramine, they became very depressed, and two of

them committed suicide. So that is a very serious consideration.

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RESPONSE: Another interesting consideration is that a number of the subjects have participated with the intent of helping the rest of the country see

that this is not such a harmful drug. A number of the proponents of

MDMA use the paucity of behavioral abnormalities in MDMA users to

point to the fact that literally thousands of subjects have used the drug, and

that they are not walking around like zombies; they do not appear to be

harmed.

My answer always is that no one has yet done a detailed neurobehavioral

study of these individuals and the deficit that they may have. It may be

very subtle in nature, and I am not sure that we have the methods available

to detect and quantify those deficits. The fact that these people are not

walking in with overt behavioral disturbances as the people with MPTP did.

I think, is related to the fact that, one, they may not have the kind of

neurotoxicity we are suspecting, and two, if they do, the kind of functional

consequences that you may get from serotonergic dysfunction may be much

more subtle than the kind of functional consequences you get with

dopamine dysfunction, where it is very easy to recognize the parkinsonian

patient

QUESTION: Do you have any plans to study whether it would be possible

to eliminate the large variation in dosage level and frequency and duration

since the last dose by studying fenfluramine, where patients are receiving

prescribed doses every day for finite periods of time? Perhaps one could

set up a study where you sample CSF before and after the therapy so that

you would avoid any concern about whether you had selected a group of

patients who had low 5-HIAA levels.

ANSWER: Yes, that is one of the groups that we would hope to

incorporate.

COMMENT: I am not an advocate of this view, but my colleague

Efrain Azmitia from NYU has suggested that perhaps rather than seeing

deficits, that pruning our serotonin projections now and then might be a

very advantageous and beneficial thing to do.

RESPONSE: I am not going to try to follow up on that one.

QUESTION: We are used to seeing a lot of those big beaded axons which

we, alter methamphetamine. have interpreted as damage. Do you think it is

what is left over rather than big axons that are damaged?

ANSWER: Yes. We have very carefully evaluated that question and

Dr. O’Hearn is a great skeptic who is forcing us to look at it very closely.

We are finding that the large varicosity fibers that are left are identical in

distribution, morphology, and density to those present in the normal fibers.

The damaged fibers are of a completely different nature; they are 10 times

319
as big, fragmented, and very, very damaged. They can be easily

distinguished.

QUESTION: So you get both?

ANSWER: Yes.

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ACKNOWLEDGMENTS

This research was supported in part by National Institute on Drug Abuse

grant number DA 05707-01.

AUTHOR

George A. Ricaurte, M.D., Ph.D.

Department of Neurology

John Hopkins University School of Medicine

Francis Scott Key Medical Center

4940 Eastern Avenue

Baltimore, MD 21224

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Dose- and Time-Dependent Effects

of Stimulants

Everett H. Ellinwood, Jr., and Tong H. Lee

INTRODUCTION

Considerable confusion abounds in discussion of stimulant-induced toxicity,

pathology, psychopathology, and the mechanisms underlying these changes.

Although there is laboratory and clinical evidence for histochemical and

structural pathologies induced by chronic and high doses of stimulants, the

specific relationship to behavioral pathology has not been clearly

demonstrated. Clinically, the confusion originates in part from several

sources including lack of clear distinctions (1) between phases of stimulant

syndrome; (2) between the types of dosing, routes of administration, and

differential pharmacokinetic parameters for different utilization styles;

(3) between outcomes or other dependent variables; and (4) between

proposed mechanisms mediating the outcome. In basic research, the confusion often results from description of a singular effect or even multiple

effects of chronic stimulant treatment without clearly delimiting the timeframe, dosing schedule, or mutual exclusiveness of competing behavioral

effects.

Our whole task from a clinical perspective includes (1) delineation of the

patterns of behavioral pathology induced both during the active stimulant

abuse phase and the phases of withdrawal; (2) description of the sequential

profile of underlying structural and functional pathology at each of the

clinical phases; and (3) an attempt to elucidate the relationship between

(1) and (2). In addition, an understanding of the pharmacological and other

parameters sufficient and necessary for inducing components of the

stimulant syndrome is clearly needed and can be obtained only from basic

laboratory studies.

In figure 1 we have outlined phases of stimulant abuse and withdrawal;

this pattern does not depict the larger spectrum of patterns seen in stimulant

abusers. Instead, we have emphasized what is called a high-dose transition

pattern (Gawin and Ellinwood 1988), which leads to the greatest behavioral

pathology. In focusing on the high-dose transitional form, the periodic

dosing over months to years is deemphasized, as is considerable basic

research literature dealing with once or twice-a-day dosing schedules.

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Animal studies have shown that the periodic dosing regimen often leads to

behavioral augmentation or “sensitization” in animals (Post 1981).

Stimulant Use Withdrawal Phases

FIGURE 1. Phases of stimulant abuse and withdrawal seen with

high-transition pattern

PHENOMENOLOGY OF STIMULANT ABUSE

High-Transition Pattern

Initial phases in high-transition pattern are similar to those in other abuse

patterns. Typically, individuals are initially exposed to single doses of

stimulants for therapeutic (e.g., weight reduction) or other purposes

(Ellinwood 1973). Euphoria produced by single doses of stimulants before

development of tolerance are proportional to plasma levels (Fischman et al.

1976; Javaid et al. 1978). Higher levels of euphoria are achieved with

intravenous (IV) route secondary to rapid rise to a peak concentration

(figure 2, A). During the “single bolus” phase, conditioning to euphoriant

“rush” of stimulants is especially profound in individuals using a rapid route

of administration (e.g., IV or smoking). The single bolus phase is followed

by increasing doses and frequency (“dose frequency escalation” phase)

mainly secondary to a development of tolerance to euphoriant effect of

stimulants.

The high-dose transition is defined as a transition phase in which the

individual suddenly increases the doses of stimulants or switches to smoking

(e.g., cocaine “crack”) or IV route of administration (Gawin and Ellinwood

1988). This change leads to a rapid escalation of plasma levels and intense

euphoria (i.e.. rush) often with subsequent increase in dosing frequency. ln

its most severe form, the high-dose pattern is characterized by binges of

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FIGURE 2. Dose frequency escalation patterns, cocaine and amphetamine

stimulant use, in which the individual repeatedly administers high doses of

stimulant in an attempt to “chase” the euphoric state against the background

of rapidly developing acute tolerance. Each bingeing episode can last from

a few hours to days and is usually terminated by extreme physical

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exhaustion and/or exhaustion of drug supply (Gawin and Ellinwood 1988).

It is the high, sustained plasma levels that appear to lead to the greatest

pathology in both stimulant abusers and laboratory animals (Ellinwood and

Kilbey 1977). Figure 2, B through D, illustrates drug plasma levels during

the high-dose transition and bingeing phase. A severe compulsive pattern

of repeated cocaine administration is necessary to maintain sustained drug

levels during cocaine binges, whereas, because of its longer plasma half-life,

amphetamine bingeing is usually characterized by longer intervals between

injections. During this compulsive phase, severely addicted individuals

report stereotyped patterns of behavior and thiig with near exclusion of

other concerns. Possible mechanisms responsible for different phases of

high-transition pattern are summarized in figure 3 under abuse dependence.

At present. there is only speculation on these mechanisms.

During the high-dose transition, the intense euphoria associated with the

rush leads to profound conditioning of associated abuse behaviors; not only

are the injection behaviors highly conditioned to the circumstances

surrounding the injections, but the behaviors leading up to the procurement

of the drug and the preparation prior to injection are also conditioned

(Ellinwood 1973).

Aspects of Withdrawal

The clinical withdrawal period may be considered as a sequence of phases

beginning with “crash” and ending with long-term withdrawal that can only

be presented descriptively. Long-term residual effects may be noted weeks

or months afterwards. Consideration for each different phase of withdrawal

is critical in understanding and treating the evolving stages of withdrawal.

The treatment consideration in the intermediate withdrawal phase is quite

different from that in the long-term withdrawal phase (Gawin and Ellinwood

1988).

Crash, the initial phase of stimulant withdrawal, immediately follows a

bingeing episode. Initially marked depressive dysphoria, anxiety, and

agitation are noted, followed by craving for sleep over the next few hours

(Ellinwood 1973; Gawin and Ellinwood 1988). Often the individual uses a

wide variety of sedatives or anxiolytics, such as alcohol, to initiate a

sustained hypersomnia. Prolonged sleep, often lasting 24 to 36 hours, is not

unusual during this phase. Notably, addicts report minimal desire for the

abused drug during this immediate phase of withdrawal (Gawin and Kleber

1986).

As the individual recovers from the crash phase, a period of anhedonia,

dysphoria, and decreased mental and physical energy ensues (intermediate

withdrawal phase). This phase can last from several days to weeks.

Emerging from the mood and energy dysfunction, craving or high urge for

the stimulant returns, frequently leading to recidivism (Ellinwood 1973).

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FIGURE 3. Toxicity and development of dependence under single-dose,

escalated, and binge conditions

The stimulant urge-impulses are sensitive to environmental cues such as

returning home and associated multiple situational stimuli (e.g., paraphernalia, friends, etc.). With continued drug availability, it is not unusual to

observe repetitious cycles of bingeing with intervening crash and

intermediate withdrawal phases over a period of months. It is noted that

conditioned withdrawal responses are less pronounced than with opiates; yet,

withdrawal appears to have a phase-specific relation to the reemergence of

cue-sensitive responses that deserves further research.

With continued abstinence through the intermediate withdrawal phase, a

more natural baseline affective state returns (long-term withdrawal phase).

Although decreased in frequency and intensity, however, urges to return to

stimulant use can recur after months to years of abstinence, again frequently

triggered by environmental cues (Ellinwood 1973; Gawin and Kleber 1986).

Moreover, the individual can exhibit a “grease slide” return to previously

conditioned behavioral responses with a single “taste” of stimulants. Full

expression of the drug-induced paranoid, stereotyped thinking pattern within

minutes to hours of return to stimulant use is a well-documented long-term

sequela of high-dose stimulant abuse (Ellinwood 1973; Bell 1973). This

full expression of behavioral pathology may have originally taken weeks or

months of chronic stimulant use to evolve. These examples highlight the

latent propensity to recidivism even after long-term withdrawal from

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stimulants. A question that arises from the residual behavioral pathologies

is whether such changes are related to toxicity associated with stimulant

use. Thus, careful consideration of the acute and chronic toxicity is

warranted.

TOXICITIES ASSOCIATED WITH SINGLE DOSES

OF STIMULANTS

Peripheral Toxicities

Several types of toxicities are responsible for stimulant-related morbidity

and mortality (figure 3). Reported cardiovascular toxicities include acute

myocardial infarction, “stunned” myocardium syndrome, arrhythmias, myocarditis, and ruptured aorta (Cregler and Mark 1986). Significantly, a

history of underlying disease appears not to be a prerequisite. For example,

acute myocardial infarction following administration of cocaine has been

documented in patients without fixed or spastic coronary diseases or history

of cardiac symptoms (Isner et al. 1986). Interestingly, cocaine appears to

be more frequently associated with the above cardiac complications than are

amphetamines. The exact reason for this preponderance of cocaineassociated toxicity is not clear; the significant local anesthetic effect of

cocaine may contribute to its cardiac toxicity. Alternatively, because of its

ultrashort half-life, cocaine may be more liable to overdosing with attempts

to maintain effective plasma levels.

The relative contribution of different mechanisms to stimulant-induced

cardiac toxicities is not known. Currently, sympathetic overstimulation is

thought to mediate many of these effects (Cregler and Mark 1986).

Increased oxygen demand secondary to increased heart rates and blood pressure has been hypothesized to lead to myocardial infarction (especially in

patients with fixed coronary disease) and/or ventricular arrhythmias. In

patients with no history of cardiac disease, cocaine is thought to induce

acute ischemic complications via vasospasm of the coronaries (Ascher et al.

1988). In addition, Virmani et al. (1988) have reported a 20 percent

incidence of myocarditis thought to be secondary to accumulated

microvascular injuries.

One critical factor that has been neglected in considering mechanisms of

cardiac fatalities is the timeframe for various types of toxicities. For

example, a majority of cocaine-related fatalities and near fatalities reported

from emergency rooms are attributed to one or more types of cardiac

ischemic or hypertensive episodes (Isner et al. 1986). Thus, these studies

may discount the cocaine-induced arrhythmias and conduction defects as

important direct causes of fatalities. Yet, if coroner reports are used as data

sources (Virmani et al. 1988; Wetli and Wright 1979; Mittleman and Wetli

1984), there are great numbers of deaths in which pulmonary effusion and

lack of evidence for coronary occlusion, acute myocardial infarction, or

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other events tend to discount the preponderance of coronary mechanisms as

the key factor leading to death

The above discrepancy may be partly explained by studies by Kabas et al.

(1988) demonstrating that, during the first 1 to 5 minutes of large IV

cocaine dose, the His bundle-ventricular conduction time is markedly

prolonged in dogs. These results indicate that an intense but transient

conduction defect occurs almost immediately after escalation of plasma

cocaine level. Local anesthetics impair cardiac conduction by interacting

with the sodium ion channel (Starmer et al. 1984). A cardiac arrhythmia

may develop rapidly secondary to combination of the conduction defect and

cardiac irritability (due to massive cardiac stimulation by catecholamine

potentiation). Furthermore, since the local anesthetic effect is potentiated by

reduced extracellular pH (Moorman et al. 1986), acidosis due to increasing

myocardial ischemia and/or seizure activity may potentiate the arrhythmogenic effect, ultimately leading to a fatal cardiac arrhythmia. Precipitous

cardiac deaths, both with and without preceding seizure activities, have been

documented following IV administration of cocaine (Wetli and Wright

1979). It should be mentioned that seizures are not necessary for the

cardiac effect, and seizure threshold is above that necessary for cardiac

conduction prolongation (Kabas et al. 1988). In conclusion, many

cocaine-related sudden deaths coming directly to coroners’ attention may be

precipitated by a brief conduction defect leading to a terminal ventricular

arrhythmia.

Central Toxicity

Although the incidence of cerebrovascular accidents from stimulant usage is

low, case reports following acute intake of cocaine or amphetamines have

appeared (Cregler and Mark 1986). Persons with subclinical cerebrovascular

abnormalities such as arteriovenous malformation or cerebral aneurysm

appear to be particularly susceptible. In addition to preexisting structural

abnormalities, stimulants themselves, when abused chronically, may induce

cerebral microarteriolar pathology predisposing individuals to stroke

(Rumbaugh 1977). A sudden surge in blood pressure induced by the drug

with the background of various types of vascular abnormalities is likely to

mediate the cerebrovascular accidents. Intracranial hemorrhage should be

included in differential diagnoses for patients complaining of headaches after

stimulant use.

High doses of stimulants lead to progressive hyperthermia; death from a

gradual overdose of stimulants (e.g., those occurring in “body packers”) are

often associated with hyperpyrexia, convulsions, and cardiovascular shock

(Ellinwood 1973; Wetli and Wright 1979). Hyperpyrexia is more frequently

noted with amphetamine, perhaps due to the longer half-life of this agent,

Life-threatening hyperpyrexia usually ensues an hour or more following

large doses of stimulants and is more prevalent in relatively naive

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nontolerant users. In animals, amphetamines produce hyperthermia and

death in a dose-dependent manner (Zalis et al. 1967). Stimulants increase

body temperature by affecting both the central and peripheral temperatureregulating mechanisms, as well as by stimulating motor activity (Ellinwood

1973).

Not only do stimulants induce hyperthermia, but elevated ambient or body

temperature itself may augment various effects of stimulants (Weihe 1973).

For example, elevated environmental temperature has been associated with

fatalities among amphetamine abusers taking their usual doses of the drug,

and exercise potentiates the toxicity, as demonstrated by fatalities among

athletes during the sixties when use of amphetamine to enhance performance

was prevalent (Ellinwood 1973). The behavioral stereotypy induced by

amphetamine is also potentiated by increased ambient temperature (Horita

and Quock 1974), as is depletion of dopamine (DA) following chronic

methamphetamine (METH) administration (Seiden and Ricaurte 1987). A

recent study shows that acute hyperthermia may attenuate adaptive

compensatory mechanisms of dopamine pathways (e.g., regulation of DA

impulse-flow) in response to methylphenidate (Lee et al. 1988). This

finding, then, suggests that the increased toxicity of stimulants under

hyperthermic conditions may be due not only to the increased temperature

per se but also to a direct impairment of the body’s ability to compensate

for stimulant toxicity.

Another modality of stimulant-induced toxicity is the induction of

generalized seizures and associated anoxia (Ellinwood 1973; Jonsson et al.

1983). As noted above, the seizure during the hyperthermic condition is

frequently associated with more gradual overdosing of stimulants, and,

indeed, status epilepticus may ensue. The complication may also result

from direct lowering of threshold by stimulants. For example, cocaine, via

its local anesthetic properties, can alter amygdala electrical activity and

produce seizures (Post et al. 1987); seizures due to local anesthetic effects,

in contrast to hyperthermia-associated seizures, appear immediately after

dosing. It should be mentioned that periods of nonfatal anoxia need to be

considered in the accumulative neuropathology associated with chronic

stimulant administration.

TOXICITIES ASSOCIATED WITH CHRONIC STIMULANT

ADMINISTRATION

Peripheral Toxicities

During the escalation and bingeing phases of stimulant abuse, higher doses

and frequency, as well as propensity for more rapid route of administration,

may lead to increased susceptibility to various medical complications. On

the other hand, development of either tolerance or sensitization to different

stimulant effects is well known (Ellinwood 1973; Post 1981) and should be

330
properly assessed along with other variables already mentioned. For

example, one recent report (Avakian and Manneh 1987) demonstrated that

chronic cocaine pretreatment reduced susceptibility to epinephrine-induced

arrhythmia in rabbits, suggesting chronic abusers may become tolerant to the

arrhythmogenic effect of stimulants. It is not known whether this tolerance

indeed develops in clinical settings and, if so, whether it shows time-, abuse

pattern-, or dose-dependencies.

Central Toxicities

Effects in Laboratory Animals. As highlighted in other chapters, the

central toxicities during and after repeated stimulant bingeing may be related

to neuronal or terminal destruction and/or depletion of neurotransmitter in

the brain. In monkeys and cats, the report by Duarte-Escalante and

Ellinwood (1970) of neuronal chromatolysis associated with decreased

catecholamine histofluorescence following chronic METH intoxication has

been followed by extensive neurochemical demonstrations of damage to the

monoamine pathways by chronic stimulants (Seiden and Ricaurte 1987).

The most consistent changes have been observed in the DA systems with

more variable effects on norepinephrine (NE) and serotonergic neurons.

Given current attempts in clinical neuroscience to relate monoamine changes

to a variety of mental and movement disorders (including mood disorders

and schizophrenia), reported changes in NE and serotonin levels following

chronic stimulant administration deserve careful consideration, despite

variabilities in findings. The earlier studies by Seiden et al. (1977) are

interesting in that, in contrast to their later study using rats (Wagner et al.

1980), they demonstrated, in monkeys, 40 to 60 percent depletion of NE in

the pons-medulla, midbrain, and frontal cortex regions, both shortly and

3 to 6 months after chronic METH treatment. Molliver et al. (this volume)

also describe extensive loss of finely beaded serotonin terminal areas, yet no

loss or even an increase in serotonin in the medial and posterior raphe. In

an earlier study Duarte-Escalante and Ellinwood 1970) in cats and

monkeys, we also found increase in serotonin histofluorescence in and

around the medial raphe neurons. These findings are in sharp contrast to

more frequently reported effects of METH on brain serotonin levels, i.e., a

decrease (Seiden and Ricaurte 1987).

In addition to changes in monoamines, those in other modulators or transmitters may alter the functional responsiveness following chronic stimulant

administration. For example, the marked increase in acetylcholinesterase

noted in the mesencephalon and brain stem (especially in areas containing

major catecholamine cell bodies) after chronic METH (Duarte-Escalante and

Ellinwood 1970) takes an added significance in light of recent findings that

this enzyme is, like DA, released from dendrites of DA cells in the

substantia nigra (Greenfield 1984). Functionally, the released enzyme can

inhibit DA cells firing in the compacta region. The effect of chronic

331
stimulants on other substances colocalized in DA neurons, such as

cholecystokinin and cytochrome P450 reductase, has not been well studied.

The latter enzyme has been proposed to participate in a possible endogenous

formation of the neurotoxin 6-hydroxydopamine (Sasame et al. 1977); could

the same enzyme be involved in stimulant-induced neurotoxicity via a

similar mechanism as has been proposed by Seiden’s group (Seiden and

Ricaurte 1987)? Changes need to be assessed carefully in a wider spectrum

of modulators, transmitters, and their possible functional consequences.

When determining effects of chronic stimulant administration, it is essential

to distinguish specific drugs (e.g., d-amphetamine vs. METH), doses and

regimens of administration, and differential sensitivities among species as

well as the time at which measurements are made. For example, METH

causes more serotonin depletion than does d-amphetamine (Seiden and

Ricaurte 1987). Cocaine may not induce monoamine depletions (Kleven

et al. 1988), although Hitori et al. (1989) have reported a selectively

decreased binding to DA uptake sites in the prefrontal cortex. This issue

awaits further evaluation. Evidence also indicates that monoamine damage

induced by stimulants is more marked after continuous exposure (Lee and

Ellinwood 1989) or higher doses of stimulants (Seiden and Ricaurte 1987),

and this effect is perhaps more pronounced in higher animals such as the

cat and monkey (Wagner et al. 1980; Owen et al. 1981; Trulson and Crisp

1985).

One of the most critical factors determining specific changes is the time of

determination after chronic dosing. Yet this variable has not been carefully

controlled in many basic and clinical studies. For example, too frequently

in studies of neuronal damage (e.g., chromatolysis), deaths following chronic

stimulant administration is the time variable neglected. Clear demonstration

of the importance of time is provided by recent findings that the sensitivity

of DA autoreceptors undergoes a rapid change (from sub- to supersensitivity) during the first week of withdrawal (Ellinwood and Lee 1983; Lee and

Ellinwood 1989). The autoreceptor supersensitivity will be discussed

further.

Effects In Humans. Neither postmortem nor functional cerebrospinal fluid

(CSF) studies in humans provide firm evidence for similar, long-term

damages or alterations to monoaminergic neurons in chronic stimulant

abusers. In part, the lack of demonstrable neurochemical changes may well

be due to the obvious preclusion of well-controlled prospective experimentation in humans, as well as to variability in critical variables (e.g., individual

sensitivity or pattern of abuse) encountered in clinical research. Possible

relationship of the various complications of stimulant abuse including hyperpyrexia, seizure, anoxia, and metabolic exhaustion to neuronal chromatolysis,

terminal destruction. and monoamine and enzymatic depletion have not been

systematically explored in human autopsy cases. It should be also noted

that, under nonperturbed conditions, overt behavioral deficits are rare in

332
animals depleted of monoamines with chronic stimulants (Lee and

Ellinwood 1989; Kokkinidis 1984). We need to evaluate carefully a

possible relationship between the fatigue, neurasthenia, and mood

dysfunction reported in the protracted stimulant withdrawal in humans and

an underlying neurochemical or anatomical state.

MORE ISSUES IN CHRONIC STIMULANT RESEARCH

In chronic stimulant abusers, one observes interactions among direct longterm toxic consequences and various compensatory behavioral and physiological mechanisms; consequently, it is necessary to evaluate multiple effects

over different phases of stimulant abuse, to sort out the contributions of

each of these mechanisms. Lack of attention to the complex interaction has

contributed to the confusion in stimulant research. Often, in basic research,

a singular mechanism for effects of chronic stimulant treatment (e.g., those

for stereotypy sensitization vs. tolerance) has been examined without consideration of other concomitant changes. For example, only a few investigators have attempted to sort out the conditioned effects in assessment of

sensitization and tolerance (Post et al. 1987; Ellinwood et al. 1973). One

goal of future research should be formulation of a clear concept of how the

changes induced by chronic stimulants integrate over time and which

mechanisms are “rate limiting” in induction of different functional changes.

In addition to interaction among different mechanisms, we need to consider

that there is a competitive economy of behaviors in the animal’s repertoire,

as these behaviors undergo time-dependent changes during chronic

administration. If a single behavior, such as stimulant stereotypy, comes to

the foreground, then other behaviors, such as locomotion or grooming, have

to recede into the background, thus leading to constriction of behavioral

repertoire. The response competition of species-specific behaviors

(Ellinwood and Kilbey 1979) is rarely considered, but it may be a major

contributor to the simultaneous appearance of tolerance and sensitization

reported in many of the basic laboratory studies.

This constriction of behavioral repertoire occurs in the clinical setting.

Examples include not only the compulsive profile of drug-seeking behaviors

(with exclusion of other types of behaviors) but also compulsive ritualistic

(1) “paranoid” thinking patterns, (2) sexual behavior, and (3) cleaning,

sorting, collecting, and grooming behaviors. These are the same behaviors

that rapidly reemerge shortly after readministration of drug following a long

period of abstinence. Unfortunately, we have no clear perspective on

whether or how central toxicity is involved in the initiation, maintenance, or

reemergence of these psychopathologic changes.

Although the sequential periods of withdrawal from chronic stimulants are

an integral part of an abuse pattern, detailed studies are lacking. In this

respect, we have recently demonstrated that DA autoreceptor sensitivity

333
undergoes timedependent changes during withdrawal. Thus, 7-day infusion

of amphetamine induces marked subsensitivity of both terminal and somadendritic DA autoreceptors immediately following the 7-day infusion; more

important, these receptors become supersensitive over the next 7 days

(Ellinwood and Lee 1983; Lee and Ellinwood 1989). This supersensitivity

is manifested by enhanced effects of apomorphine in inhibiting cell firing

and/or DA synthesis in the nigrostriatal and mesolimbic DA pathways. We

have questioned whether the increased autoregulation may in part underlie

the characteristic lethargy and loss of mental energy observed in human

stimulant abusers during the intermediate withdrawal phase (Gawin and

Ellinwood 1988). Enhanced autoregulation may lead to a decreased ability

to “turn on” the DA transmission-regulating behavioral arousal systems.

These functional changes due to changes in autoreceptor sensitivity or other

variables could prove to be an important factor in pathogenesis and rational

treatment of chronic stimulant syndrome.

CONCLUSION

Time is an important variable in the study of the neuropathological and

psychopathological changes noted in chronic stimulant syndromes. It is

important in (1) frequency, timing, and chronicity of dosing, (2) the

evolution of neuropathology and behavioral changes over time; and

(3) evaluation of reversible and residual stages of withdrawal. Careful

delineation of the changes at each stage of the ontogeny and withdrawal of

the stimulant syndrome is warranted. As is summarized in other chapters,

there are many residual pathological changes following chronic amphetamine

stimulant dosing. The relation of neuropathology to psychopathology in the

stimulant abuse syndrome and withdrawal is tantalizing, yet essentially

unknown. This lack of understanding of the relationship certainly applies to

functional changes such as autoreceptor alterations. Whether and how the

chronic waxing and waning atypical depression seen after withdrawal is

related to the stimulant-induced central toxicities demonstrated in laboratory

studies need to be determined. Is it related to the neuronal destruction

and/or monoamine depletion in the brain, is a chronic functional state (e.g.,

DA autoreceptor supersensitivity) sufficient to facilitate this behavioral state,

or is terminal depletion and some other change a necessary covariable?

More important, can we develop a rational approach that allows the

clinician to manipulate the mechanisms to prevent relapse? The marked

variability of therapeutic agents tried for the stimulant withdrawal period

(e.g., tricyclic antidepressants, monoamine oxidase inhibitors, DA agonists,

and uptake inhibitors) attests to our lack of understanding of the ratelimiting mechanisms involved. Understanding of the relationship between

the neuropathological and functional changes noted with the stimulant of

these syndromes may lead to a more fundamental understanding of the

development of psychopathology in the psychoses and addictions in general.

334
DISCUSSION

QUESTION: What is your view of the role of the supersensitive autoreceptor after 7 days? Can you precipitate or replicate a psychosis?

ANSWER: No, I am not relating it to psychosis.

COMMENT/QUESTlON: I was not relating it to psychosis either. I am

trying to put it in a functional context. Have you speculated about the role

of the supersensitive autoreceptor at that point? You could speculate early

on that the subsensitivity autoreceptor favors the potentiation of the

behavioral effect. But what might happen when it becomes supersensitive?

RESPONSE: We have primarily related it to the withdrawal phase of

fatigue and lethargy. We have a system that is set to turn itself off as

rapidly as possible. What are the treatments that reverse autoreceptor

supersensitivity? Thinking ideologically, to be hit with this huge dose of

amphetamine over and over again means doing whatever must be done for

the brain to turn off that response. If you take out the more sensitive

regulation, because these receptors are now supersensitive, you immediately

turn off the impulse coupled with the release of dopamine. If you give

even a small dose of amphetamine, you now have, if you are looking at

this neuron in isolation, a terminal that is not being regulated by impulsecoupled mechanisms. I don’t know how important that is.

COMMENT: I would favor the view that lethargy and fatigue of postamphetamine withdrawal during the withdrawal phase would be consistent

with the shutting off of the dopamine neuron. Still, it is hard to imagine

how that would be. First, the amphetamine-induced release is not regulated

by the autoreceptor. And, as you say, if it would be impulse related,

however weak. it would be regulated. But we do know that after a period

of amphetamine intoxication, an individual is supersensitive behaviorally.

QUESTION: Are you talking about augmentation?

ANSWER: Yes. I am talking about the influence of a subsequent dose on

an individual who has had a repetitive binge of amphetamine. At that time,

he or she is withdrawn. Then he or she comes back and you can give a

relatively low dose that will reinstitute the endstage symptoms, as was being

discussed earlier.

COMMENT: That particular phenomenon, maximum sensitization of

augmentation, is best elicited by single daily doses. We were attempting to

mimic the high-dose continuous binge phenomenon where you sustain

plasma levels (in this case, for 7 days) with an Alzet pump. You don’t see

it going the same augmentation route in that regime. In these Alzet pump

animals, at the end of 7 days, even though they are getting about a

335
5 mg/kg/day dose, there is very little stereotypy. There is massive

tolerance.

I am not relating primarily the amphetamine response or the subsequent

amphetamine response to this autoreceptor phenomenon. I have tried to say

that, in the beginning, when you give a substantial dose of amphetamine,

the autoreceptors are out of the picture. I don’t think they play a part. If

you give a substantial enough dose, it wipes out the autoreceptor response.

I don’t think that we are dealing with that part of the phenomenon. I hope

we are dealing with the beginning model for this loss of mental energy, the

incapacity of the normal responses during the intermediate withdrawal

phase.

QUESTION: Have you looked at the behavioral consequence of the low

dose of apolmorphine in these particular animals?

ANSWER: Yes. In very low doses it turns off the animals. We are

talking about 50, 75 μg, IV. So they are more sensitive to turning down

locomotion, which would fit in with the hypothesis that they would turn

themselves off before they turn on.

There is no way to explain sensitization tolerance using autoreceptors.

QUESTION: Could these changes in the autoreceptors account for the

cravings for cocaine or amphetamine? If you are shutting down dopamine

activity, that may lead to the desire to return to cocaine.

ANSWER: Yes. We think that it is a neat hypothesis. In the absence of

natural reinforcement forces, craving for cocaine becomes more intense. I

think one of the things that would be important is to figure out some way

of testing it.

QUESTION: A possibility that comes to mind is from reading

Dr. Larry Stein’s work. His theory of a reward system suggests that the

cerebral cortex has basically inhibitory behavioral characteristics. And that

the reward system, when it is activated, inhibits the cerebral cortex so that

there is an inhibition of an inhibitory mechanism, thus releasing behavior.

If that is a valid concept, could that have anything to say about the

consequence of this supersensitization having behaviorally inhibitory effects?

ANSWER: I think at this point that even Larry Stein would agree that the

norepinephrine is probably not the major mediator of the reward systems. I

think that we have enough evidence to indicate that is not the case.

QUESTION: If the reward system is not being activated, for whatever

reason, is dopamine considered to be more of a neurotransmitter of the

reward system?

336
ANSWER: I think there is fairly good evidence that dopamine is

substantially involved.

QUESTION: If anything was preventing a reward system from being

operative, you would, perhaps, tend to see this inhibitory effect behaviorally.

With supersensitization, do you have this kind of a consequence to beat the

reward system not being activated?

ANSWER: Well, that certainly would be one of the things we would like

to find some way of testing specifically.

COMMENT: The main point that I wanted to make is that it is very important to attempt to develop models where one is looking at least at some

sort of in vivo integrated preparation. We look at serotonin depletion. We

look at dopamine depletion. We have a variety of different mechanisms.

Again we really do not know what, at this point in time, the serotonin

depletion is doing. I think I know what it means if you deplete dopamine

beyond a certain level. But even there, it is difficult to put an exact degree

of impairment on the levels of dopamine depletion that we see in most of

these models.

I would really like to see development of models that are explant or in vivo

models, where we can see the animal in a more integrated role and look at

the corresponding in vitro events.

I don’t think we know what the rate-limiting mechanisms are for most of

the behaviors that we think we are concerned with.

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AUTHORS

Everett H. Ellinwood, Jr., M.D.

Tong H. Lee, M.D., Ph.D.

Behavioral Neuropharmacology Section

Department of Psychiatry

Duke University Medical Center

P.O. Box 3870

Durham, NC 27710

340
Recommendations for Future

Research on Amphetamines and

Related Designer Drugs

Ray W. Fuller

INTRODUCTION

This volume has focused on several amphetamine analogs in addition to

amphetamine and methamphetamine, especially 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA).

Among the pharmacologic actions of these drugs, their behavioral effects in

humans and in laboratory animals have been discussed, with some attention

to electrophysiologic and electroencephalographic effects. Other functional

effects, such as effects on neuroendocrine regulation, sleep, thermoregulation. and appetite and body weight have not been discussed. Consideration

of toxic effects mainly focused on neurotoxic actions that the drugs can

have on specific brain monoaminergic neurons. In relation to this action,

two other amphetamine analogs, p-chloroamphetamine and fenfluramine,

have been compared because of their similar neurotoxic actions in rats.

SOCIAL IMPLICATIONS

General concerns about abuse of amphetaminerelated drugs are similar to

concerns about other illicit or addictive drugs. Dr. G. Nahas wrote an

editorial for the Wall Street Journal arguing that “a strongly expressed

sentiment of societal disapproval” of illicit drugs is necessary for prohibitive

measures to be effective (Wall Street Journal, July 11, 1988, p. 16). He

cited examples from history to support his contention that when illicit

addictive drugs are socially accepted and easily available, they have a very

damaging effect on individuals and on a society. His examples included the

use of cannabis in the Islamic-dominated world several centuries ago, the

chewing of coca leaf in Peru, the use of opium in China at the beginning

of this century, the epidemic of amphetamine abuse in Japan in the 1950s,

and others. In some cases, there was widespread social acceptance.

Although there is acceptance of MDMA and amphetamines in only limited

segments of our society, Nahas argues there is not forceful enough

disapproval of illicit drugs in general in our society today.

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The behavioral and dependence-producing effects of some of these drugs

can be damaging to individuals, but neurotoxic damage to particular brain

neurons can result when these drugs are given to animals, including

nonhuman primates. There continue to be inadequate data about whether

such damage occurs in humans.

Patterns of Abuse of Amphetamine Analogs

The need for more accurate and precise information about human use of

MDMA and MDA was aptly stated by Dr. Gawin (this volume) and others.

There is a general perception that these drugs are widely used, especially on

college campuses, but there are relatively few hard data on the geographic

distribution of use, on the pattern(s) and frequency of use, the doses used,

and so on. For many reasons, such information is needed.

Behavioral Effects of Amphetamines: How Useful, What Mechanisms?

The behavioral effects of amphetamine, methamphetamine, MDMA, MDA,

p-chloroamphetamine, and fenfluramine are not identical. Except for the

last drug, all can cause some degree of behavioral stimulation, but exact

behavioral effects differ markedly. More complete definition of their

behavioral differences is a prerequisite to a better understanding of the

mechanism(s) of these drugs.

Apparently there are psychiatrist and nonpsychiatrist clinicians whose

experience convinces them that MDMA can have therapeutic uses, mainly

as an adjunct to psychotherapy. Despite these convictions, there appear to

be no published data to support these claims. There is an urgent need for

objective data from well-controlled, blinded clinical studies, if these claims

of therapeutic usefulness are to be taken seriously. If a bona fide use is

evident, then it may be possible to produce other drugs with the same

desirable action, lacking the toxicity inherent in MDMA.

NEUROCHEMICAL MECHANISMS

Aside from the therapeutic usefulness of MDMA, there is scientific

importance to elucidating further the mechanism(s) involved in the

seemingly unique behavioral effects of MDMA and MDA. Apparently, a

major action of these drugs is the release of serotonin and dopamine from

brain neurons, leading to enhanced serotonergic and dopaminergic input to

those neuronal systems with which they make synaptic contact. In addition,

MDMA has been shown to interact in vitro with sites including 5HT2,

5HT1A, and adrenergic receptors, among others (Battaglia, this volume).

Do those interactions occur in vivo, and does MDMA interact as an agonist

or as an antagonist at these sites? If the interactions occur in vivo, how do

they contribute to the profile of behavioral effects of MDMA? These

questions can and should be approached experimentally. Further unraveling

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of the effects of MDMA and MDA on serotonergic and dopaminergic

function is also needed. Serotonin neurons and dopamine neurons are

known to interact in many brain regions (Bosler et al. 1984; Benkirane

et al. 1987; Herve et al. 1987), so the release of dopamine may influence

serotonergic function, just as the release of serotonin may influence

dopaminergic function.

Neurotoxicity of Amphetamines

The recognition that amphetamines can be neurotoxic in brain can be traced

back to p-chloroamphetamine studies. In the middle 1960s, p-chloroamphetamine and p-chlorometbamphetamine were found to cause selective depletion of brain serotonin (Pletscher et al. 1963; Fuller et al. 1965). The long

duration of this depletion was not appreciated until later (Sanders-Bush

et al. 1972). and it was subsequently established that the loss of serotonin

was accompanied by changes in other parameters specifically associated

with serotonin neurons, e.g., a loss in tryptophan hydroxylase, a loss in

serotonin uptake capacity, and a reduction in serotonin turnover, as well as

by histologic evidence of neurotoxicity (Puller and Snoddy 1974; Harvey

et al. 1975; Sekerke et al. 1975; Massari et al, 1978). Fenfluramine was

recognized to have similar effects on brain serotonin neuron parameters

(Harvey and McMaster 1975; Clineschmidt et al. 1978), although there has

been controversy about histologic changes (Sotelo and Zamora 1978).

During the 1970s, evidence accumulated that amphetamine and methamphetamine could also be neurotoxic (Ellison et al. 1978; Hotchkiss and Gibb

1980; Wagner et al. 1980). The effects of amphetamine seem mostly

limited to dopamine neurons, whereas methamphetamine affects dopamine

and serotonin neurons (Warren et al. 1984). Most recently, MDMA and

MDA have been shown to produce neurotoxicity toward brain serotonin

neurons much like that of the halogenated amphetamines (Ricaurte

et al. 1985; Stone et al. 1986).

Role of Uptake Carriers

The neurotoxic effects of all these compounds are antagonized by inhibitors

of monoamine uptake (table 1), implicating the membrane uptake carrier on

serotonin and dopamine neurons in the mechanism of neurotoxicity. In this

regard, these amphetamines are like a drug somewhat related in structure,

namely 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a

Parkinsonism-causing neurotoxic drug that has been studied intensely since

1983 (Langston and Irwin 1986). In the case of MPTP, the mechanism by

which inhibitors of the dopamine uptake carrier block the neurotoxicity

toward dopamine neurons (mainly nigrostriatal dopamine neurons) seems

clear. A metabolite of MPTP, l-methyl-4-phenylpyridinium (MPP+), has

been shown to be a substrate for the dopamine uptake carrier (Javitch

et al. 1985). Thus accumulation of MPP+, formed metabolically from

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MPTP, into dopamine neurons seems to be essential, and blockade of that

accumulation prevents the neurotoxicity. MPP+ also can be transported into

norepinephrine neurons (Javitch et al. 1985), leading to neurotoxicity toward

cortical norepinephrine neurons, an effect blocked by inhibitors of the

norepinephrine uptake carrier (Sundstrom and Jonsson 1985).

TABLE 1. Characteristics of monoaminergic neurotoxicity induced by

amphetamine and related compounds in laboratory animals

Membrane Uptake Carrier Drug Metabolite

Drug Involved in Neurotoxicity Involved in Neurotoxicity

Amphetamine Yes

Methamphetamine Yes

MDMA, MDA Yes

p-Chloroamphetamine Yes

Fenfluramine Yes

MPTP Yes Yes

The mechanism by which uptake inhibitors block the neurotoxic effects of

amphetamine, methamphetamine, MDMA, MDA, p-chloroamphetamine, and

fenfluramine is not so clear. A simple explanation (analogous to that with

MPTP) might be that these drugs are accumulated into serotonin or

dopamine neurons via the membrane uptake carrier, and that uptake

inhibitors prevent the neurotoxicity by preventing that accumulation.

However, none of these compounds have been shown to be a substrate for

dopamine or serotonin uptake carriers. Their lipophilicity leads one to

believe they would enter neurons readily without requiring active transport.

There may be, however, both entry and accumulation of these drugs. It is

conceivable that the amphetamines do enter brain monoaminergic neurons

and other cells by passive diffusion. They may in addition be accumulated

by brain monoaminergic neurons, if amphetamines are substrates for the

membrane uptake carriers. For example, p-chloroamphetamine may enter all

cells, but may be selectively concentrated in serotonin neurons, due to its

accumulation via the membrane uptake carrier. That concentration may be

required for the short-term and long-term depletion of brain serotonin, so

that inhibition of the uptake carrier blocks the depletion. No direct

evidence to support this possibility is available. Uptake of radioactive

p-chloroamphet-amine by the serotonin uptake carrier has not been shown in

vitro, although p-chloroamphetamine does have high affinity for that carrier

(Wong et al. 1973).

There has been some evidence that p-chloroamphetamine is preferentially

localized in synaptasomal fractions of brain homogenates (Wong

et al. 1972), and recently Ask and Ross (1987) have published evidence

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consistent with an accumulation of pchloroamphetamine in serotonergic

synaptosomes in vitro. They evaluated the ability of reversible inhibitors of

monoamine oxidase to be accumulated in serotonin nerve endings by the

membrane uptake carrier by comparing two conditions of serotonin

deamination: first, when the radioactive serotonin was being deaminated,

mainly inside serotonergic synaptosomes, after it was accumulated via the

uptake carrier; and second, when serotonin was being deaminated by other

synaptosomes, because its transport via the serotonin uptake carrier was

blocked. In this way, they evaluated the ability of certain reversible

inhibitors of monoamine oxidase to be themselves concentrated in serotonergic synaptosomes due to their transport via the membrane uptake carrier.

Study of p-chloroamphetamine, which is a reversible inhibitor of monoamine

oxidase (Fuller 1978), did indicate accumulation within serotonergic

synaptosomes.

Further investigation of the possibility that inhibitors of the serotonin uptake

carrier protect against serotonin depletion by p-chloroamphetamine, fenfluramine, MDMA, MDA, and methamphetamine because they prevent the

accumulation of those drugs within serotonin nerve terminals is warranted,

but at present compelling evidence for this mechanism does not exist.

Possible Role of Dopamine Release

A second possible mechanism, supported by some existing data on methamphetamine and MDMA, is that these drugs release dopamine, which is then

taken up into serotonin neurons via the membrane uptake carrier, leading to

neurotoxic effects on the serotonin neurons. Inhibitors of dopamine

synthesis or of the dopamine uptake carrier, e.g., -methyltyrosine and GBR

12909, have been reported to prevent the depletion of serotonin by methamphetamine and by MDMA (Schmidt et al. 1985; Gibb et al., this volume).

MDMA does release dopamine both in vitro and in vivo (Yamamoto and

Spanos 1988). Dopamine can be transported into serotonergic synaptosomes

(Schmidt and Lovenberg 1985). Further investigation is needed, especially

to see if the involvement of dopamine is a general phenomenon in the

neurotoxic effects of amphetamines. We have found that potent inhibitors

of dopamine uptake, including mazindol and nomifensin, block depletion of

striatal dopamine by MPTP in mice, but do not block depletion of brain

serotonin by p-chloroamphetamine in mice.

Possible Role of an Active Metabolite of the Drug in the Neurotoxicity

of Amphetamine Analogs

The possibility that an active metabolite is involved in the neurotoxic effects

of amphetamine analogs receives limited discussion in this chapter and has

been considered previously, especially with p-chloroamphetamine (Miller

et al. 1986.) Partly because the chemical structures of these amphetamines

do not suggest ways in which they would be toxic to neurons, the

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possibility that conversion to a more reactive metabolite accounts for the

neurotoxicity has been attractive. The study of numerous analogs of

p-chloroamphetamine and other neurotoxic amphetamines has not yielded a

strong candidate for such a neurotoxic metabolite. Most potential

metabolites of p-chloroamphetamine caused less depletion of serotonin

(Fuller 1978). Although N-hydroxy-p -chloroamphetamine did deplete

serotonin, it was metabolized rapidly and almost quantitatively to

p-chloroamphetamine (Fuller et al. 1974). Inhibitors and inducers of drug

metabolism have generally failed to influence neurotoxicity of amphetamines. MDA is metabolixed to a-methyldopamine (Marquardt et al. 1978;

Midha et al. 1978), a metabolite that should be considered as a possible

mediator of neurotoxicity, especially in view of the properties of dopamine

discussed below.

Involvement of a Metabolite of the Neurotransmitter in the

Neurotoxicity of Amphetamines

Also a possibility is that a product formed from one of the neurotransmitters affected mediates the neurotoxic effects of amphetamines. This

possibility was suggested by Seiden and Vosmer (1984), who reported the

presence of 6-hydroxydopamine in the rat caudate nucleus after a single

injection of a high, neurotoxic dose of methamphetamine. They suggested

that 6-hydroxydopamine was formed from endogenous dopamine released by

methamphetamine and that the 6-hydroxydopamine was responsible for the

neurotoxicity to dopaminergic terminals. Other investigators have not found

6-hydroxydopamine to be present in rat striatum after amphetamine or methamphetamine administration (Rollema et al. 1986).

Commins et al. (1987) have also reported the formation of 5,6-dihydroxytryptamine in rat hippocampus after a single, high doses of methamphetamine. They suggested that the formation of 5,6-dihydroxytryptamine, a

known neurotoxic substance, may mediate the neurotoxic effects of

methamphetamine toward serotonergic nerve terminals.

Molliver (this volume) made the provocative suggestion that a metabolite of

serotonin released from blood platelets by p-chloroamphetamine may

mediate the neurotoxic effects of p-chloroamphetamine on cortical serotonergic neurons in the rat. Such a possibility would be compatible with the

observations of Molliver and his colleagues (this volume) that p-chloroamphetamine is not effective when pumped directly into the brain or when

added to brain slices in vitro. Their demonstration that a combination of

p-chlorophenylalanine and reserpine prevented the neurotoxic effects of

p-chloroamphetamine led them to suggest that platelet serotonin stores were

involved in the neurotoxic mechanism (Berger et al., submitted for publication). This interesting idea deserves testing in various ways. Since it

would not cross the blood-brain barrier, 5,6-dihydroxytryptamine would not

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seem to be a candidate for their hypothesized metabolite, unless the

integrity of that barrier had been lost due to the drug treatment.

Role of Dopamine Involvement in the Neurotoxicity of Amphetamines

Some data suggest that dopamine itself is involved in certain of the

neurotoxic effects. It is worth asking if dopamine might account for the

neurotoxicity of all the amphetamine analogs toward both dopaminergic and

serotonergic neurons. At least three ways in which dopamine might lead to

cytotoxicity have been suggested, First, dopamine might be converted to

6-hydroxydopamine, a known neurotoxin, as discussed above. Second,

dopamine metabolism by monoamine oxidase is known to produce hydrogen

peroxide, and excess hydrogen peroxide formation from this source might,

under some conditions, have deleterious effects on the cell (Cohen and

Mytilineou 1985). Third, dopamine itself is known to undergo autooxidation analogous to, but slower than, that of 6-hydroxydopamine

(Graham et al. 1978; Graham 1984). Persistently increased intraneuronal

but extragranular concentrations of dopamine due to amphetamine-induced

release of granular stores of dopamine and protection against dopamine

oxidation by monoamine oxidase type A have been suggested as possibly

mediating the neurotoxic effects of amphetamine (Fuller and HemrickLuecke 1982). Uptake of dopamine into serotonergic terminals (Schmidt

and Lovenberg 1985) might lead to destruction of serotonergic terminals

after treatment with drugs like methamphetamine and MDMA. It is not

clear why such effects should be less with amphetamine than with methamphetamine, yet amphetamine seems to affect dopaminergic neurons

primarily, whereas methamphetamine is neurotoxic toward serotonin neurons

as well as dopamine neurons (Hotchkiss and Gibb 1980; Ricaurte

et al. 1984). Investigation of the possible involvement of dopamine in the

different neurotoxic process is needed.

IMPLICATIONS OF NEUROTOXICITY

Functional Deficits Resulting from Amphetamine Neurotoxicity

Rats that have lost dopamine and/or serotonin terminals following treatment

with amphetamine, methamphetamine, MDMA, MDA, p-chloroamphetamine,

or fenfluramine show little in the way of overt changes in appearance or

behavior. Dr. Ricaurte (this volume) emphasized the need for more studies

in primates, since MPTP-treated mice also show little in the way of

observable functional changes, whereas MPTP-treated monkeys show marked

neurologic deficits. It may be necessary to do more detailed analysis of

specific behaviors and other functional outputs that are influenced by

dopamine and/or serotonin neurons, to detect functional deficits induced by

some neurotoxic drugs. For instance, specific behaviors such as appetitecontrolled behavior (Leibowitz and Shor-Posner 1986), muricidal behavior

(Katz 1980), and sexual behavior (Tucker and File 1983) elicited by drugs

347
or environmental conditions ate known to be influenced by serotonergic

input. Careful analysis of these behaviors in rats that have received

neurotoxic doses of p-chloroamphetamine, MDMA, MDA, fenfluramine, or

methamphetamine may reveal functional deficits. Electroencephalographic

patterns, nociception, sleep, thermoregulation, and endocrine regulation are

other brain-controlled functions that are influenced by serotonergic pathways.

Careful studies of these functions, especially measuring responses elicited by

serotonergic drugs or by environmental stimuli whose actions are mediated

by serotonergic systems (insofar as that is known) may reveal functional

deficits associated with loss of serotonergic terminals. For example, we

have found that the acute increase of serum corticosterone in rats given

p-chloroamphetamine, an increase that appears to be mediated by release of

serotonin from central neurons making input to cells that release corticotropin-releasing factor in the hypothalamus, is blunted in rats pretreated with

a neurotoxic dose of p-chloroamphetamine. There are few examples of

studies of this sort, in which a functional correlate of the loss in serotonin

content has been sought in rats that have received neurotoxic doses of any

of the amphetamine analogs in question. It seems important for such

studies to be done for several masons, including the goal of learning more

about physiologic functions of the serotonin and dopamine pathways that are

affected, and to suggest ways in which possible neurotoxicity in humans

might be investigated.

Does Neurotoxicity Occur in Humans

All the neurotoxic drugs discussed have been taken by human subjects.

Amphetamine and methamphetamine have a long history of therapeutic use

along with illicit misuse. To a limited extent, p-chloroamphetamine has

been used in humans as an investigational drug (Van Praag et al. 1971).

MDMA and MDA have no approved medical uses, but they appear to be

rather widely abused drugs at present. Fenfluramine continues to be

marketed as an appetite suppresant A key question, to which there is no

current answer, is whether any of these drugs produce, in humans, neurotoxic effects on dopamine and/or serotonin neurons in brain analogous to

those produced in rodents and in nonhuman primates (table 2). It is

remarkable that no data exist on this issue, given that the neurotoxic effects

of some of these drugs in animals have been know for more than a

decade.

There are several ways in which possible neurotoxic effects might be

studied. First, measurement of cerebrospinal fluid concentrations of

dopamine or serotonin metabolites would be a straightforward way of

assessing neurotoxicity. There are pitfalls in this approach (as outlined by

Dr. Ricaurte (this volume), such as the facts that lumbar cerebrospinal fluid

might reflect spinal cord neurochemistry more than it reflected brain

neurochemistry, and drugs like p-chloroamphetamine affect serotonin

neurons in spinal cord less than they do those in brain (Sanders-Bush

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et al. 1975). Nonetheless, fenfluramine has been shown to produce marked

decreases in 5-hydroxyindoleacetic acid concentration in the cerebrospinal

fluid during treatment (Shoulson and Chase 1975), and it would be

important to know if those concentrations return to control levels when

fenfluramine is discontinued.

TABLE 2. Nature of neurotoxic damage to brain monoaminergic neurons

Drug

Brain Monoaminergic Neurons Neurotoxicity Occurring

Showing Neurotoxic Damage in Humans

Amphetamine Dopamine

Methamphetamine Dopamine, Serotonin

MDMA, MDA Serotonin

p-Chloroamphetamine Serotonin

Fenfluramine Serotonin

MPTP Dopamine, Norepinephrine Yes

A second approach might be to measure dopamine and serotonin along with

their metabolites and other specific neuronal constituents such as tyrosine

hydroxylase and tryptophan hydroxylase or uptake carrier sites in brain

tissue obtained at autopsy. Accumulating data in this way might be a slow

and tedious process, and drug dosing history might be uncertain and

variable; nonetheless, the approach deserves consideration.

A third approach would be to measure some indicator of functional output

of dopamine and/or serotonin neurons. As mentioned previously, studies in

laboratory animals can be invaluable in defining parameters that change in

correlation with directly measurable neurotoxic effects in the brain.

Changes in serum hormones elicited by a drug whose effects are mediated

by dopamine or serotonin neurons are especially attractive possibilities, since

these changes are already being used as a means of assessing the functional

state of brain serotonergic pathways (Siever et al. 1984). In this regard, it

is intriguing that Coccaro et al. (1987) have observed recently a blunted

elevation in serum prolactin concentration elicited by fenfluramine in

psychiatric patients who had received a previous dose of fenfluramine

within a 12-day period. While there may be numerous possible explanations, one could be that the first dose of fenfluramine had damaged or

destroyed a traction of the serotonin neurons from which release of

serotonin is the mechanism of prolactin elevation by fenfluramine.

A fourth approach to evaluating the intactness of dopamine and/or serotonin

neurons in human subjects who have taken one of the amphetamine analogs

might be to use a probe for labeling a constituent of those neurons in

position emission tomography scanning studies. A label for the serotonin or

dopamine uptake carrier, or a label for tryptophan hydroxylase or tyrosine

349
hydroxylase, would be an ideal agent for use in studies of this sort. Such

methods are not currently available, but the possibilities for development of

methods like this seem excellent.

DISCUSSION

COMMENT: I would like to know why you thought the amphetamine

model of dopamine neurotoxicity might be more suitable or more revealing

for the study of Parkinson’s disease than the MPTP model.

RESPONSE: We do not understand all there is to know about the

mechanisms of MPTP neurotoxicity. but it seems to involve MPP+, which

is potentially cytotoxic to all cells but that attains toxic concentrations after

MPTP administration only in cells that concentrate MPP+. Dopamine

apparently is not involved in the neurotoxic effects of MPTP. I am

attracted to the idea that dopamine itself may be involved in the etiology of

Parkinson’s disease, that dopamine neurons may be at risk because of the

nature of their neurotransmitter.

If there is anything to that line of thought then I am suggesting that the

exact mechanisms involved in MPTP may not be like the mechanisms that

are involved in the &generation of those dopamine neurons in Parkinson’s

disease. And that something like amphetamine neurotoxicity might have

closer parallels to the degeneration in Parkinson’s disease. That

presupposes the way the story is going to end and obviously I don’t know

that any more than anybody else does.

I have argued in the past that looking further at amphetamine toxicity in

terms of understanding the mechanism by which those neurons die, might

be more revealing. That is not to belittle the importance of MPTP as a

model of Parkinson’s disease. Certainly in terms of effects in the MPTPtreated monkeys, these animals are of unquestioned value. But in terms of

the mechanism by which the neurons die, that was the point that I was

questioning, whether the MPTP model would mimic as well as the

amphetamine model.

QUESTION: You mentioned the N-hydroxy parachloroamphetamine. Is it

less or more toxic than PCA?

ANSWER: The same. And the reason is that it is converted very rapidly

to PCA itself almost quantitatively.

QUESTION: If the oxidation of dopamine is proving to be toxic, are there

any natural endogenous substances or nutrients that can help prevent that?

Is it possible that perhaps ascorbic acid would keep the dopamine from

being metabolized or oxidized?

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ANSWER: I think that is an interesting possibility, since cells presumably

have some kind of cytoprotective mechanism. It is possible that in patients

predisposed to Parkinson’s disease there is some breakdown of the patients’

protective mechanisms in those neurons. That might be a reason why they

develop the disease. Fortunately, all of us don’t. I think we simply don’t

know. We should consider all possibiiities.

COMMENT: I would like to follow up with the point that Dr. Rebec,

from the University of Indiana, made here at the NIH in January. The

topic of his talk was ascorbic acid and dopamine in schizophrenia. In

experimenting with amphetamine, he was finding with individual neuron

investigation that amphetamine, in both low and high doses, was inhibitory

in some neurons. In other neurons it was inhibitory in low doses, and in

high doses it became excitatory. But in this investigation he claimed that

he was finding some substance in the brain that was counteracting the effect

of the amphetamine and, by analysis, he said it was determined to be

ascorbic acid.

Now the use of molecular psychiatry of ascorbic acid in schizophrenia by

Linus Pauling and others, where there seems to be some relationship to

dopamine neurons, and fmding that dopamine-dopaminergic neurons or

receptors are present in twice the normal amount, makes this an intriguing

area of investigation.

Dr. Rebec also said that the brains on post mortem studies of

schizophrenics tended to be mushy and to have very low levels of ascorbic

acid in their constituent tissue.

RESPONSE [FROM AUDIENCE]: We tried an opposite strategy where we

made guinea pigs scorbutic. We deprived them of their scorbic acid

contents, then exposed them to amphetamine. In those studies we found

that the ascorbutic animals were protected from some of the neurotoxicities

of the amphetamines. It is a very complex issue. It is not just a matter of

adding vitamin C or ascorbic acid and getting protection; it can work as a

double-edged sword. It can work for or against you.

QUESTION: Has anyone given an uptake blocker in the chronic state?

You showed that in the acute stage that you could get some reversal

effects. Has anyone administered an uptake blocker after chronic use of

amphetamines?

ANSWER: Even after one dose of p-chloroamphetamine, the depletion of

brain serotonin cannot be reversed at later times. You lose the reversibility

after several hours.

QUESTION: Totally lose it?

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ANSWER: Yes. So I feel very sure that in the chronic state there will

come a time when this is not reversible.

QUESTION: We have all these theories about serotonin being involved in

depression. Do you have any explanation for why depression is not seen in

humans if serotonin neurons have been damaged by these drugs?

ANSWER: I think that clearly it is possible that there is no neurotoxicity

in humans. I think all of us would like for this to be the case. And

maybe it is the case. We have talked about this a lot with fenfluramine,

and we have done studies with parachloroamphetamine in which we have

given it orally to rats at relatively low doses, but still anorectic doses, over

90 days. We have seen depletion of serotonin, but that was fully reversible

depletion. It came back when the drug was stopped. I think there may be

no neurotoxicity at the oral doses used in humans. I think that would be

great if that is the case. That would explain why there is no depression or

other kinds of symptoms. But I don’t feel comfortable about relying on the

lack of reporting of depression as real evidence that there is no

neurotoxicity. We simply need to have better data on that.

COMMENT: I think another matter to take into account is that, at least

from the experience of dopamine systems, in order to get overt behavioral

dysfunction you really need a pretty whopping lesion. In the primate, to

get the kind of Parkinsonism that people talk about in animal models, that

animal model actually turns out to be very difficult to produce in chronic

Parkinsonism. The problem is developing an animal that has 90 to

95 percent depletion of dopamine on a chronic basis. As you know, it is a

very narrow window, and it is very difficult to produce that kind of animal

preparation. So I think you have to consider the possibility that lack of

symptoms after serotonergic lesions could, perhaps, be related to the fact

that we are dealing with preparations where there is a 50, 60, 70 percent

depletion where we don’t have enough of a lesion to produce an overt

behavioral disturbance.

RESPONSE: Perhaps I can bridge the dispute by suggesting it is probably

going to vary among neuronal systems. There may be systems in which

you must have a lot of depletion to see a functional change, and there may

be others where it doesn’t take very much.

COMMENT: I would dispute it within the dopamine system itself. And I

would dispute it about Parkinson’s disease. I think that if you did a proper

neuropsychological exam that you would pick up even smaller depletion

effects. I think if you are looking for an overt complete terminal Parkinson

situation, yes, you need a 99 percent depletion. But part of the problem is

that the Parkinson situation involves not only the nigrostriatal system but

also the mesolimbic dopamine system. There are plenty of studies in rats,

and it is very easy to produce a Parkinsonian rat with a very discrete

352
injection of 6-hydroxydopamine in the right place. It doesn’t require a lot

of work. I can take 2 micrograms of 6-hydroxydopamine and put it in

exactly the right location in the ventral tegmental area, and I can produce a

Parkinsonian rat that will die,

So I think you can debate that issue about 99 or 95 percent depletion. I

think that if you probe those animals with the proper pharmacological

agents and proper environmental situation, you will pick up deficits. I think

the lack of knowledge about what the serotonin systems do is the basis of

the problem here. We don’t know what the behavioral consequences of the

serotonin depletion are.

How are we going to probe a person’s gestalt? I think that was brought

out earlier. If we had proper probes we might see the effects. If we have

proper probes for exaggerating serotonergic function or proper probes for

exaggerating deficits associated with serotonergic function we would easily

pick up things. Whether that is important or not, you know it might be

good to trim our serotonin neurons slightly. Maybe we would be better off.

Maybe we would all be somewhat anxiolysed. That is another question.

But I think we have the tools in behavioral pharmacology to conduct tests

in rats that will be sensitive to serotonin depletion. And I assume that

those can be extrapolated to primates.

COMMENT: One of the problems that we have not addressed is the issue

of potential recovery and regeneration.

One of the striking aspects of this toxicity of compounds is selective

destruction terminals and the cell bodies that are left intact. Dr. De Souza

has recently reported some biochemical evidence for recovery of serotonin.

We have now found anatomic evidence for reinnervation of depleted areas

by serotonin neurons. But it is going to be a while before we figure out

whether their reinnervation is appropriate or perhaps aberrant. Do they end

up with complete recovery, do they end up with a better system than they

started with or one that malfunctions? I think that is an important area for

future study.

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AUTHOR

Ray W. Fuller, Ph.D.

Research Advisor

Lilly Research Labortories

Eli Lilly and Company

Lilly Corporate Center

Indianapolis, IN 46285

357
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80 NEEDLE SHARING AMONG INTRAVENOUS DRUG ABUSERS:

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82 OPIOIDS IN THE HIPPOCAMPUS. Jacqueline F. McGinty, Ph.D., and

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83 HEALTH HAZARDS OF NITRITE INHALANTS. Harry W. Haverkos,

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87 OPIOID PEPTIDES: AN UPDATE. Rao S. Rapaka, Ph.D., and Bhola

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88 MECHANISMS OF COCAINE ABUSE AND TOXICITY. Doris H.

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90 PROBLEMS OF DRUG DEPENDENCE, 1988. PROCEEDINGS OF

THE 50TH ANNUAL SCIENTIFIC MEETING. THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris. Ph.D., ed.

GPO Stock #017-024-01362-5 $17

IN PRESS

91 DRUGS IN THE WORKPLACE: RESEARCH AND EVALUATION

DATA. Steven W. Gost, Ph.D., and J. Michael Walsh, Ph.D., eds.

92 TESTING FOR ABUSE LIABILITY OF DRUGS IN HUMANS.

Marian W. Fischman, Ph.D., and Nancy K. Mello, Ph.D., eds.

93 AIDS AND INTRAVENOUS DRUG USE: FUTURE DIRECTIONS

FOR COMMUNITY-BASED PREVENTION RESEARCH. C.G. Leukefeld,

D.S.W.; R.J. Battjes, D.S.W.; and Z. Amsel, Ph.D., eds.

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U.S. GOVERNMENT PRINTING OFFICE: 1989 – 249 - 659 00948
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Alcohol, Drug Abuse, and Mental Health Administration

Printed 1989