National Institute on Drug Abuse

RESEARCH

MONOGRAPH SERIES

Neurobiological

Approaches to

Brain-Behavior

Interaction

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

124
Neurobiological Approaches to

Brain-Behavior Interaction

Editors:

Joseph Frascella, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

Roger M. Brown, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

Research Monograph 124

1992

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, MD 20857
ACKNOWLEDGMENT

This monograph is based on the papers and discussion from a technical review

on “Neurobiological Approaches to Brain-Behavior Interaction,” held on

September 5-6, 1990, in Bethesda, MD. The review meeting was sponsored by

the 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 material in this volume

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

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

Citation of the source is appreciated.

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

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

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

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

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

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

of the studies reported herein.

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

selectively included in the coverage of American Statistics Index, BioSciences

lnformation Service, Chemical Abstracts, Current Contents, Psychological

Abstracts, and Psychopharmacology Abstracts.

DHHS publication number (ADM)92-1846

Printed 1992

ii
Contents

Preface v

Microdialysis in the Study of Psychostimulants and the

Neural Substrate for Reinforcement: Focus on Dopamine

and Serotonin

Bartley G. Hoebel, Luis Hernandez,

Gregory P. Mark, and Emmanuel Pothos

Neurophysiological Approaches to Receptor

Pharmacodynamics

Francis J. White

Psychomotor Stimulant Effects on Single Neurons in

Awake, Behaving Rats

Mark O. West, Laura L. Peoples, Martin

Wolske, and Sfeven I. Dworkin

Brain-Stimulation Reward: A Model for the Study of the

Rewarding Effects of Abused Drugs

Conan Kornefsky and George Bain

New Neuroimaging Techniques for Investigating

Brain-Behavior Relationships

William J. Jagusf and Thomas F.

Budinger

1

35

57

73

95

iii
Studying Psychoactive Drugs With Positron Emission

Tomography: Relationships Between Mood and Metabolic

Rate 117

Harriet de Wit, John Metz, and Malcolm

Cooper

Metabolic Mapping Methods for Identifying the Neural

Substrates of the Effects of Abused Substances 135

Linda J. Porrino

Interactions of Neurotransmitters With Drugs and

Behavior 153

Lewis S. Seiden

of Abused Drugs

Historical Influences Affecting the Behavioral Actions

161

James E. Barrett

Importance of Behavioral Controls in the Analysis

of Ongoing Events 173

Steven I. Dworkin, Linda J. Porrino, and

James E. Smith

Cocaine Self-Administration: Pharmacology and

Behavior 189

William L. Woolvelton

Multivariate and Nonlinear Approaches to Characterizing

Drug Effects on the Locomotor and Investigatory Behavior

of Rats 203

Mark A. Geyer and Martin P. Paulus

Neurobiological Approaches to Brain-Behavior

Interaction: Summary and Overview 237

George F. Koob

iv
Preface

Neurobiological Approaches to Brain-Behavior Interaction is the result of a

NIDA-sponsored technical review meeting held September 5-6, 1989, in

Bethesda, MD. (For an overview of the presentations see “Neurobiological

Approaches to Brain-Behavior Interaction: Summary and Overview.“)

Neuroscientists and behavioral scientists were brought together to share ideas

and techniques for studying brain mechanisms that underlie or modify behavior.

Technically, we have come a long way from the early 1960s when large

inferential leaps were necessary to describe a behavior or function with respect

to neurophysiological, neuropharmacological, and neurochemical events. With

this approach it was necessary, for example, to run drug dose-response or

time-response evaluations on a behaving group of animals in parallel with a

different set of animals that was used for neurochemical determinations; less

precise and specific methods were used to determine whole-brain monoamine

levels and metabolite data to explain synaptic events as well as to provide a

basis for drawing correlations with behavioral effects of a drug.

Recently, however, advances in technology and methodological developments

have been made that allow for direct correlations between brain changes and

behavioral events. Direct measurement of synaptic events occurring in discrete

brain regions now can be assessed during ongoing behavior. This capability

has the potential for immensely magnifying our understanding of relationships

between brain structure and function tremendously enhances our capacity for

dealing with malfunctions at the cellular level, especially with respect to

understanding the direct effects of drugs of abuse on the central nervous

system (CNS), and further, to reveal how these drug-CNS interactions alter

behavior.

The purpose of this technical review was twofold. First, an important aim was to

examine the various techniques for monitoring neural activity, synaptic events,

and biochemical interactions during ongoing behavior by discussing the

strengths and weaknesses of the current methodologies. Toward this end,

discussions are provided in this monograph on the 2-deoxyglucose technique

(“Importance of Behavioral Controls in the Analysis of Ongoing Events); in vivo

v
voltammetry and microdialysis (“Microdialysis in the Study of Psychostimulants

and the Neural Substrate for Reinforcement: Focus on Dopamine and

Serotonin”); computer imaging techniques such as positron emission

tomography, single-photon emission computed tomography, and magnetic

resonance tomography (“Studying Psychoactive Drugs With Positron Emission

Tomography: Relationships Between Mood and Metabolic Rate” and “New

Neuroimaging Techniques for Investigating Brain-Behavior Relationships”); and

neurophysiological recording methods (“Neurophysiological Approaches to

Receptor Pharmacodynamics” and “Psychomotor Stimulant Effects on Single

Neurons in Awake, Behaving Rats”).

As we enter the Decade of the Brain, information is becoming available

concerning the complex response of the brain to drugs of abuse as well as the

underlying events that unfold during the development of addiction. Drug

addiction begins with a relatively simple neuropharmacological action of a drug

acting at a specific site. A resultant cascade of neural and chemical events

takes place to produce an experience, which is usually positive. This positive

experience is highly reinforcing and often leads to the compulsive behavior and

abuse of the drug despite its damaging consequences. With repeated drug

exposure, brain mechanisms most likely change or adapt to the presence of the

drug, often resulting in an alteration in the ability to process information.

Because drug addiction is a behavioral phenomenon resulting from a

modification of neural function, the role of the behavioral sciences in

understanding drug abuse is absolutely essential. The Neuroscience Research

Branch of the National Institute on Drug Abuse strongly encourages interaction

between the neurobiological sciences and the behavioral sciences.

Consequently, the second purpose of this technical review was to emphasize

the equally important perspective that behavioral events influence the brain

and, thus, the drug-brain-behavior interaction is an important consideration in

evaluating neuropharmacological data.

Neurobiologists should be aware that a behavioral action of a psychotropic drug

depends on many factors, of which the interaction between a drug and its

corresponding receptor or receptors is only one. The modification of behavior is

the final common pathway of a drug’s action at a synapse whose influence

might lie on a continuum from being very critical to being far removed from

other circuits that determine behavior. Consequently, the ultimate effect of a

drug on behavior might be modified by changes in activity within the brain’s

complex circuitry. These changes might come from environmental or situational

factors that affect neural events, such as previous history, environmental

vi
stimuli, and situation (“Importance of Behavioral Controls in the Analysis of

Ongoing Events”). Locomotor activity is sometimes thought to be a simple drug

screen, yet even this seemingly simple behavior is made up of complex

components (“Multivariate and Nonlinear Approaches to Charcterizing Drug

Effects on the Locomotor and Investigatory Behavior of Rats”). The use of

behavior to study the neural basis of drug action is a powerful tool

(“Brain-Stimulation Reward: A Model for the Study of the Rewarding Effects of

Abused Drugs” and “Cocaine Self-Administration: Pharmacology and Behavior”).

Assuredly, during the Decade of the Brain and thereafter, multidisciplinary

approaches to behavioral neuroscience will have major influences in dealing

with behavioral problems and diseases of the brain. The overall strength of this

meeting on brain-behavior interactions was that it reviewed some

state-of-the-art neuroscience and neurobehavioral techniques currently in use

and also allowed for direct interaction between neuroscientists studying specific

aspects of brain function with those interested in the precise nature of

behavioral events related to drug abuse.

vii

Microdialysis in the Study of

Psychostimulants and the Neural Substrate

for Reinforcement: Focus on Dopamine and

Serotonin

Bartley G. Hoebel, Luis Hernandez, Gregory P. Mark, and

Emmanuel Pothos

INTRODUCTION

Microdialysis is a useful technique in the study of addiction. It provides a means

of measuring neurochemical changes in localized regions of the brain (Delgado

et al. 1972; Kissinger 1990; Ungerstedt 1984). Neurotransmitter release can be

determined periodically while animals receive drugs or engage in drug-seeking

behavior (Becker et al. 1988; Di Chiara and lmperato 1988; Hernandez et al.

1987; Hurd and Ungerstedt 1989; Hurd et al. 1989; Kuczenski and Segal 1989;

Pettit et al. 1989; Sharp et al. 1987; Ungerstedt 1984; Westerink et al. 1987;

Zetterström et al. 1983). Basal release of a neurotransmitter can also reveal

individual differences between animals and differences due to changes in an

animal’s physiology or environment. This chapter illustrates the use of

microdialysis in the study of changes in extracellular dopamine (DA) and

serotonin (5-HT) due to (1) rewarding brain stimulation, (2) natural ingestive

behaviors, (3) classical conditioning, (4) locally infused addictive drugs,

(5) peptides that influence DA release, (6) anorexia in relation to drugs of

abuse, (7) body weight loss, and (8) chronic treatment with haloperidol (HAL)

and clozapine (CLOZ). The goal is to understand (1) the neurochemical actions

of abused psychostimulants, (2) potentiation of these actions by conditioned

stimuli, peptides, and low body weight, (3) development of addiction and

psychosis, and (4) treatment.

1
THE MICRODIALYSIS TECHNIQUE

Microdialysis has an enormous advantage over earlier neurochemical

techniques that required analyses of brain slices, synaptosomes, or tissue

homogenates (Robinson and Justice 1991; Rollema et al. 1991). For most

experiments it is no longer necessary to extract the brain. In vivo microdialysis,

however, has a major disadvantage relative to in vivo voltammetry; researchers

typically must wait 5 to 30 minutes to obtain a microdialysis sample that

contains enough of the neurochemical to be detected by present analytical

techniques. This sampling rate is frustratingly slow when one is dealing with

rapidly changing physiology or behavior. On the other hand, in vivo voltammetry

lacks the wealth of criteria for identifying a variety of neurochemicals.

Compared with open-tip, push-pull cannulas, microdialysis probes (figure 1) are

easier to use because they do not pressurize the brain or become plugged up

for several days. For molecules such as insulin that are too large to pass

through a microdialysis fiber, however, the push-pull approach is advantageous.

We use semipermeable membranes with a molecular weight cutoff of 6,000 that

excludes all enzymes that would degrade the sample. Membranes with a

molecular weight cutoff of 20,000 are also useful, and a variety of materials are

available.

Microdialysis samples are typically analyzed by high-pressure liquid

chromatography (HPLC) or radioimmunoassay. Most of the known

neurotransmitters have been measured in this way, including catecholamines,

5-HT, excitatory and inhibitory amino acids, acetylcholine, and several peptides.

In addition, the second messenger cyclic adenosine monophosphate can be

measured in the extracellular space and could be a valuable tool in a new

approach to the study of addiction (Egawa et al. 1988; Hoebel et al. 1990).

The microdialysis technique involves passing Ringer’s solution through a probe

constructed of input and output tubes with a section of semipermeable dialysis

membrane between them. Neurochemicals diffuse in through the dialysis

membrane and are carried out to a collection vial or directly to the analyzer. The

input and output tubes can go through the brain horizontally, carrying the

Ringer’s solution in one side of the head and out the other (Hamberger et al.

1983; lmperato and Di Chiara 1984; Ungerstedt 1984), vertically side by side in

the form of a U tube (Hernandez et al. 1983; Kalen 1988; Marsden et al. 1986;

Quan and Blatteis 1989; Ungerstedt 1984) or with one tube inside the other

(Benveniste 1989; Hernandez et al. 1986; Johnson and Justice 1983; Kapoor

and Chalmers 1987; Nakahara et al. 1989; Ungerstedt 1986; Westerink et al.

2
FIGURE 1. A removable microdialysis probe in a guide shaft cemented to the

skull. Ringer’s solution from a remote pump enters through the

small inner tube and exits the larger tube to a vial attached above

the rat’s head. Drugs added to the Ringer’s solution diffuse

outward from the semipermeable tip (0.2 mm outer diameter) to

bathe neural tissue. (From Hernandez et al. 1986.)

1987). The horizontal arrangement causes the least local damage because the

microdialysis tube floats in the brain without any steel tubing inside. The U tube

has the most local surface area and therefore collects the most

neurotransmitter in a given region, but it does the most local damage. Any

probe will cause some brain damage but usually not enough to disrupt operant

behavior such as bar pressing for food, self-stimulation, or drugs. If large

probes were placed bilaterally in a small structure, one would have to worry

about bilateral lesions. The concentric arrangement we chose is the smallest

and allows the probe to be removed and reinserted later via an implanted guide

shaft. Small probes, however, require very sensitive analytical chemistry. We

are developing capillary electrophoresis to improve sensitivity for future

experiments (Guzman et al. 1990).

3
MICRODIALYSIS IN BEHAVING ANIMALS

This laboratory has specialized in the measurement of neurotransmitter release

in freely moving animals (Hoebel 1988; Hoebel et al. 1989). This method avoids

any abnormalities caused by anesthesia, and the animals are able to engage in

operant or classical conditioning. We simply adapted well-known microinjection

techniques to microdialysis by implanting a guide shaft with a concentric probe

design (Hernandez et al. 1986). Permanent guide shafts made from hypodermic

needle tubing are implanted in animals while they are anesthetized. The

animals recover for a week before the microdialysis probe is lowered into the

brain. The probe is constructed from two pieces of concentric tubing, with the

dialysis membrane forming the tip in the shape of a 0.2-mm by 3-mm cylinder

(figure 1). There is a tradeoff between choosing a small size and obtaining

enough neurochemical to be detectable. A cellulose dialysis tip only 150 μm by

2 mm can be used if the neurochemical is abundant, for example, DA in the

striatum (STR). For DA in the nucleus accumbens (NAC), we usually use a

probe 200 μm by 3 mm, and for the prefrontal cortex (PFC), a probe 200 μm by

4 mm; in the lateral hypothalamus, DA is usually undetectable unless a drug

such as amphetamine (AMPH) is administered (Hernandez et al. 1986; Parada

et al. 1988a, 1988b). In all these experiments, we typically collect 20-μl samples

over 20 min and then perform a 20-min assay for DA and its metabolites,

dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA). In this way,

sample collection and assay keep pace. It is also possible to measure DA as

often as every 5 min by using smaller HPLC columns and skipping the

measurement of metabolites (Church and Justice 1987; Phebus et al. 1986).

Frequent sampling is a great advantage in behavioral experiments. For

measurements second by second one must turn to in vivo voltammetry, by

which monoamines are oxidized on the tip of an electrode in the brain; in this

case, however, accurate neurochemical identification can be difficult (Gerhardt

et al. 1984; Marsden 1984; Wightman et al. 1988). Microdialysis is more reliable

and more versatile.

The most difficult aspect of behavioral microdialysis is the neurochemical

assay. It is not difficult to place a microdialysis probe with its tip in the

hypothalamus or NAC in a rat. The animal moves freely in a test cage such as

a Skinner box with flexible tubing leading to and from its head via a watertight

swivel (figure 1). Probes are available commercially from Carnegie Medicin

(Sweden), Biological Analytical Systems (United States), and Europhor

(France), although many laboratories make their own. Probe construction is

difficult but can be mastered by a patient person with good eyesight and manual

4
dexterity. We measure 5-HT with an amperometric electrochemical detector

from EG&G: Princeton Applied Research Corp. and DA with a coulometric

detector from ESA Company, although many other brands are also suitable. If

transmitter is abundant in the sample, the Coulochem Electrode Array System

(ESA Co.) with 16 electrodes can be used to detect all the monoamines at

once, along with their metabolites and precursor amino acids. The aim with

HPLC is to achieve very high sensitivity in the low picogram or femtogram

range by having very little dead space and a very smooth baseline free of

electronic noise or pump pulsations. The neurochemical of interest must come

off the column after the massive front of oxidizable compounds has gone by but

not so late that it becomes a broad, smeared-out peak.

A 4:1 signal-to-noise ratio is desirable but not always obtainable. Reuptake

blockers or enzyme inhibitors can be introduced locally by way of the probe to

boost the neurotransmitter signal, but this method has the disadvantage of

departing from natural conditions. The Ringer’s solution itself can be disruptive

(Westerink et al. 1988). Many early studies used high-calcium Ringer’s solution

that boosted DA recovery, but now most laboratories match the Ringer’s

solution calcium level to extracellular levels (about 1.2 mM). Fortunately,

high-calcium Ringer’s solution did not seem to alter the dopaminergic effects of

drugs of abuse in the early studies, but Westerink reports that certain

DA-acetylcholine interactions are blocked by high-calcium Ringer’s solution

(Electrochemical Detection Conference, Nottingham, 1989). Most researchers

still use Ringer’s solution, not a true artificial cerebrospinal fluid. The absence of

magnesium or glucose may be a concern in some experiments. Microdialysis

also has an inherent problem in that it depletes neurotransmitter in the

extracellular space (Benveniste 1989), but this problem is partly solved by the

use of low perfusion rates (0.5 to 1 μl per minute). Nonetheless, microdialysis

has shown logical and consistent effects with drugs of abuse in several

laboratories.

Last, but not least, it is essential to prove beyond doubt that the analytical

procedure actually measures what one claims. This has been done for DA and

5-HT (Brazell et al. 1985; Carboni and Di Chiara 1989; lmperato et al. 1988;

Westerink et al. 1987; Zetterström et al. 1988). For 5-HT we find it is necessary

to oxidize the samples and standards at more than one potential for positive

identification. We also test with 8-OH-DPAT, which is a 5-HT autoreceptor

agonist that stops 5-HT neurons from firing. When treatment with this

compound causes the putative “serotonin peak” to shrink, then (1) the lost 5-HT

probably had come from 5-HT neurons, not glia or platelets and (2) it probably

5
was real 5-HT, not a different compound with the same oxidation characteristics

(Auerbach et al. 1989; Schwartz et al. 1989c).

THE MESOLIMBIC SYSTEM

The mesolimbic DA system has attracted the most attention in the study of the

neural circuitry underlying drug abuse (figure 2). Its importance is undeniable. If

the mesolimbic system is blocked or almost completely depleted of DA, animals

refuse to work for food, self-stimulation, or self-injection (Fibiger and Phillips

1986; Hoebel and Novin 1982; Koob and Bloom 1988; Roberts and Koob 1983;

Wise and Bozarth 1987), whereas depleting norepinephrine has the opposite

effect by increasing feeding and self-stimulation (Hernandez and Hoebel

1989a). Not only is the DA system necessary, its functions are sufficient to

motivate and reward behavior. Rats will work to stimulate certain DA cell

regions electrically or chemically. For example, they will electrically

self-stimulate the mesolimbic DA cell body region in the ventral tegmental area

(VTA) (Phillips et al. 1989). They will also self-inject morphine (Bozarth and

Wise 1981) or neurotensin directly into the VTA (Glimcher et al. 1987); they

self-inject opioids (Goeders et al. 1984), AMPH (Hoebel et al. 1983), or DA into

the NAC (Dworkin et al. 1985), and cocaine into the PFC (Goeders and Smith

1983). Apparently the mesolimbic system is a substrate for both natural rewards

such as feeding and the drug rewards that lead to addiction (Fibiger and Phillips

1986; Hoebell 1988; Mogenson 1987; Wise 1982). The findings of many

researchers are consistent with this suggestion; however, distinguishing among

DA’s roles in stress, arousal, locomotion, priming, and reinforcement is often

troublesome, and much remains to be learned (Salamone et al. 1989).

Numerous conditioning experiments now show that mesolimbic DA is not just

part of a locomotor system; it also mediates reward processes involved in

positive reinforcement (Ettenberg 1989; Fouriezos and Wise 1976; Gallistel et

al. 1982; Spyraki et al. 1983; White and Franklin 1989). It is clear from in vivo

neurochemistry research that the mesolimbic DA system could play similar

roles in natural appetitive behavior and drug appetite.

Microdialysis has been used to study the release of DA by reinforcing stimuli.

Dopaminergic drugs of abuse given systemically caused large increases, e.g.,

ninefold, in extracellular DA in freely moving rats (Imperato and Di Chiara

1984). We have focused on measurement of NAC DA while presenting the rat

with hypothalamic stimulation, food, or intra-accumbens drugs of abuse.

Currently we are testing conditioned stimuli associated with food or drugs.

6
FIGURE 2. Side view of the rat brain showing the brain regions discussed in

the text with some of their interconnections, Sharp arrows indicate

facilitation of the labeled behavioral output function. Blunt arrows

indicate suppression of the function. For example, palatable

“taste” inputs from the nucleus tractus solitarius (NTS) facilitate

the lateral hypothalamic “feeding and reward” system, which in

turn facilitates the ascending “self-administration” DA paths to the

NAC and limbic PFC. Serotonin probably inhibits this process in

combination with postingestive factors by promoting satiety in the

PVN-MH and suppressing feeding-reward in the LH. Not shown

are 5-HT projections to the NAC, HIPP, and related regions where

5-HT might facilitate behavioral arousal. See text for description of

AMPH effects in each of these brain sites corresponding to

monoamine arrows in the diagram. (From Hoebel 1988.)

7
Dopamine Released by Brain Stimulation

Our first step in finding the natural substrate for drug abuse was to stimulate the

perifornical lateral hypothalamus (PFH) using electrodes that induced feeding

and self-stimulation. With electrodes of this type, self-stimulation rate varied

with the animal’s appetite for food (Hernandez and Hoebel 1978; Hoebel 1985).

The PFH is only one of many medial forebrain bundle (MFB) self-stimulation

sites. Electrodes that elicited copulation in males yielded self-stimulation at a

rate that varied with androgen level (Caggiula and Hoebel 1966). Thus, MFB

self-stimulation can reflect natural appetitive behaviors.

The reinforcing properties of MFB self-stimulation can be enhanced by drugs of

abuse. Self-stimulation is often considered an accelerated model of addictive

behavior (Carden and Coons 1990; Hubner et al. 1988; Trujillo et al. 1989).

(See also “Brain-Stimulation Reward, A Model for the Study of the Rewarding

Effects of Abused Drugs.“) MFB self-stimulation sends action potentials along

axons that descend to the midbrain VTA region (Mogenson 1987; Shizgal et al.

1982) and on to taste-sensory cells in the hindbrain (Murzi et al. 1986) as part

of a circuit that is thought to excite the ascending mesolimbic DA system (Wise

1982, 1983). If so, PFH stimulation should release NAC DA. Depending on

electrode configuration and placement, MFB stimulation can also activate the

ascending DA neurons directly as they pass through the hypothalamus

(Nicolaysen et al. 1988; Shizgal 1989; Yeomans 1989). We used placements

and parameters designed to activate descending systems that excite DA

neurons indirectly by way of their normal inputs to the cell bodies and dendrites

(figure 2).

We find that PFH stimulation that induces feeding does release DA in the NAC.

This finding was true whether the rat ate food or not (Hernandez and Hoebel

1989a) (figure 3). As a control, rats with microdialysis probes in the STR

showed no detectable increase in extracellular DA, suggesting that PFH

electrodes for stimulation-induced feeding release much more DA in the NAC

than in the nearby STR. It also showed that the DA that overflowed out of

synapses in the NAC diffused to the local microdialysis probe in the NAC but

did not diffuse as far as the probe in the STR. This demonstration by electrical

stimulation of anatomical specificity between DA subsystems is crucial in the

study of drug abuse. DA-releasing drugs such as AMPH do not distinguish

between DA terminal regions the way PFH-stimulation-induced feeding can.

AMPH would be expected to release DA in all terminal regions whether

involved in abuse or not. Our result suggests that PFH stimulation could have

8
FIGURE 3. Perifornical lateral hypothalamic (PLH) stimulation releases

dopamine (DA) in the nucleus accumbens with food available

(stimulation-induced feeding, left side). The same electrical

stimulation releases DA even without feeding (right side).

Self-stimulation or deprivation-induced feeding will do the same

thing. Feeding also releases DA in the PFC. Psychostimulants

may generate reward by their action in these DA systems.

(From Hernandez and Hoebel 1988a.)

9
acted by way of the VTA (figure 2). It is unlikely that we stimulated mesolimbic

DA axons directly during stimulation-induced feeding, because release was

localized to the NAC rather than to the STR. We are finding, however, that

self-stimulation of the MFB for an hour (approximately 2,500 bar presses) gives

evidence of DA release at both sites (Hunter and Hoebel, unpublished

manuscript). DA in the STR is undoubtedly involved in arousal, stereotypy, and

other motor activities, some of which are involved in feeding (Church et al.

1987), but present evidence suggests DA in the NAC bears some special

relevance to appetitive behavior and reinforcement.

Dopamine Released by Eating

DA release by natural behavior was a crucial test. Eating released DA in the

NAC in rats tested at 80 percent of normal body weight and in rats that were

deprived of food for just a day (Hernandez and Hoebel 1988a; Radhakishun et

al. 1988). The same was true whether rats ate Purina chow or bar-pressed for

food pellets. DA was also released in the PFC during operant feeding, but there

was no comparable release in the part of the STR we sampled. This finding

suggests that DA in the PFC as well as the NAC is involved in the elicitation or

reinforcement of feeding. Therefore, drugs that release DA in the NAC and PFC

mimic natural processes that accompany operant feeding.

Feeding is not the only behavior that releases DA. We found DA release in the

NAC during angiotensin-induced drinking and diuretic-induced salt intake

(Hoebel et al. 1989). Sexual-approach behavior also releases DA (Mas et al.

1990).

Dopamine Release Can Be Conditioned

How does one know whether DA is related only to arousal or whether it carries

the positive valence implied by terms such as elicitation, reward, incentive,

positive reinforcement, preference, or pleasure? That this is a difficult problem

is attested to by long debate. The clearest answers come from conditioning

studies that test animals’ remembrance of past events by having them make

choices long after the training is completed and experimental drugs are gone

(Wise and Bozarth 1987). Therefore, we measured DA release in the NAC

before and after a conditioned taste aversion. Initially, saccharin squirted into

the mouth through an oral catheter released DA. Saccharin taste was then

paired with lithium chloride malaise. As a result, the same concentration of

saccharin had the opposite effect: it caused a significant decrease in DA below

10
baseline levels. This finding suggests that a good-tasting substance can release

DA, but when it is associated with nausea in the animal’s memory, the effect on

DA release is reversed. Thus two opposite effects on DA were obtained even

though the animals appeared to be aroused in both cases (Hoebel et al. 1989;

Mark et al. 1991 a). To save the arousal hypothesis, one would need to

postulate two kinds of arousal: one that increases DA within the NAC and one

that decreases it. We offer instead the view that DA release in the NAC

facilitates appetitive behavior, providing positive reinforcement; conversely, a

decrease in DA release facilitates avoidance, enhancing escape reflexes and

providing negative reinforcement. Because DA in the NAC is released in

response not only to food intake but also to water intake, salt intake (Hoebel et

al. 1989), and sexual appetite (Mas et al. 1990), we can hypothesize that DA

release is naturally involved in many appetitive behaviors. Similarly, it is

possible that DA decreases are involved in numerous avoidance behaviors, but

so far this effect has only been shown to be related to conditioned taste

aversion.

DA can also be conditioned to increase. A novel taste became the conditioned

stimulus for DA release after it had been paired with nutritious food delivered

via a gastric fistula (Mark et al. 1991b).

Dopamine and Addiction

On the basis of the studies that have been described, there are at least two

ways an animal or a person could rectify a conditioned DA decrease: avoid the

stimulus situation that caused it, or take some other ingestant or a drug that

increases extracellular DA. To achieve conditioned DA release, one merely

needs to mimic the stimulus situation and environment.

Many drugs of abuse given systemically increase extracellular DA (Di Chiara

and lmperato 1988; Kuczenski 1986). Similarly, when AMPH, cocaine,

phencyclidine (PCP), or nicotine was injected locally into the NAC or infused

through the microdialysis probe itself, this local treatment increased levels of

extracellular DA in a manner similar to an intraperitoneal injection (Hernandez

and Hoebel 1988b; Hernandez et al. 1987, Hernandez et al. 1988a, 1991;

Kuczenski and Segal 1989; Mifsud et al. 1989; Robinson et al. 1988; Sharp et

al. 1987). The extracellular concentration of the DA metabolite DOPAC

decreased, indicating depletion of intracellular DA or interference with DA

metabolism. This interference adds to the length of time DA would be expected

to persist in the synapses, helping to explain why drugs of abuse are more

11
potent than normal appetitive stimuli such as food. When rats ate a meal, DA

metabolism increased, but after drugs of abuse were administered, DA

metabolism decreased (figure 4, AMPH; figure 5, PCP).

When cocaine is injected locally there is a danger in that it acts as a local

anesthetic. As a control, equimolar procaine and lidocaine were tested.

Procaine is both a mood elevator and a local anesthetic; lidocaine is just a local

anesthetic. Cocaine released the most DA; procaine released less (Hernandez

FIGURE 4. Amphetamine (AMPH) infused through the nucleus accumbens

(NAC) microdialysis probe for 20 min, caused both extracellular

dopamine and serotonin to increase, and dihydroxyphenylacetic

acid to decrease 1 μL per minute perfusate flowrate; 3.37mM

CaCl2 Ringer; 2 μg/μL AMPH in 20 μL of Ringer perfusate.

Assuming 10 percent outward diffusion comparable to 10 percent

monoamine relative recovery, then 4 μg AMPH reached the NAC

during 20 min. (From Herncndez et al. 1987.)

12
In rats with two microdialysis probes, vehicle (VEH) and

phencyclidine (PCP) were injected into the nucleus accumbens

and striatum by temporarily removing the probes. Dopamine and

serotonin recovery increased; dihydroxyphenylacetic acid

decreased (3.37 mM CaCl2 Ringer, 1 μL per minute perfusate flow

rate, PCP 20 μg in 0.5 μL local injection). Postmortem tissue

assays failed to show the increased turnover that is clearly

revealed by microdialysis. (From Hernandez et al. 1988a.)

13
et al. 1990). Lidocaine released no detectable DA at all, thereby controlling for

local anesthesia (figure 6).

Microdialysis can be used to measure extracellular DA during ongoing

self-administration (Hurd et al. 1989; Pettit et al. 1989). Animals self-injecting

cocaine intravenously maintained a level of extracellular DA that varied with

dose, prior experience, and the microdialysis site within the NAC (data

presented at Society for Neuroscience satellite, In vivo Neurochemistry and

Addiction, 1989: Hurd, Ungerstedt, Weiss and Koob; Pettit, Neill and Justice).

Why are drugs of abuse addicting? Solomon (1977) hypothesized an opponent

process in which euphoric effects engender longer lasting dysphoric effects,

thereby creating a need for more of the euphorigenic drug. Koob and Bloom

(1988) have suggested that opiate euphoria and dysphoria are both dependent

on the NAC. Wise and Bozarth (1987) have proposed that the mesolimbic

system mediates euphoria and the periaqueductal gray region is important for

the physical symptoms of opiate withdrawal. We are exploring the possibility

that during withdrawal, release of DA decreases and ACh increases in the NAC

(Rada et al. 1991 a and 1991b; Pothos et al. 1991). In such ways, microdialysis

can be used to search for opponent processes.

THE UNKNOWN IMPORTANCE OF SEROTONIN

We found that local AMPH can increase extracellular serotonin in the NAC

(Hernandez et al. 1987). The same was true of cocaine (Hernandez and Hoebel

1988b) and PCP (Hernandez et al. 1988a, 1988b), showing that these are all

serotonergic drugs at high, local concentrations. AMPH given systemically also

increases extracellular serotonin (Kuczenski and Segal 1989). As a general

rule, the firing rate of serotonin cells is proportional to arousal level (Jacobs et

al. 1984), although local presynaptic control of release and local interactions

with other neurotransmitters must be crucial to local serotonergic function. On

the basis of serotonin-depletion experiments, Dworkin et al. (1988) suggested

that serotonergic innervation of the NAC is involved in the reinforcing effects of

morphine. Perhaps serotonin also plays a role in psychostimulant

reinforcement, but the nature of the serotonin-DA interaction is not clear.

PRESYNAPTIC CONTROL OF DA RELEASE

Nicotine is not in the same class with the other psychostimulants. It is a

cholinergic drug, and yet it too is a drug of abuse. The reason is clear from

14
FIGURE 6. Dopaminergic effect of equimolar (7.3 mM) cocaine, procaine, and

lidocaine infused locally in the nucleus accumbens (NAC) for 20

min. In vitro measurement of drug efflux by capillary

electrophoresis assay showed approximately 28 percent relative

diffusion (outside the probe relative to inside), which suggests

about 40 nmol of drug reached NAC tissue. Local cocaine

increased extracellular dopamine; procaine was about half as

effective; lidocaine had no effect. (From Hernandez et al. 1991.)

15
microdialysis. Nicotine given systemically increased extracellular DA in the NAC

and STR (Di Chiara and lmperato 1988). To determine whether it acts in the

NAC, we infused nicotine locally. It released DA at the microdialysis site, and

the effect could be blocked by systemic administration of a nicotinic-receptor

antagonist, mecamylamine (Mifsud et al. 1989) (figure 7). This finding suggests

that nicotine can act directly in the NAC to release DA by way of nicotinic

receptors on the DA terminals or somewhere in the immediate region.

What is the role of peptides in local control of DA release? In our pilot tests,

local cholecystokinin injected in the posterior NAC gave negative results

(Schwartz et al. 1988). Ruggeri and colleagues (1987), however, observed an

increase in DA when cholcystokinin was given in the anterior NAC or with

neurotensin in the posterior NAC of anesthetized rats. Route of administration,

doses, anesthesia, and microdialysis site within the NAC are all crucial

FIGURE 7. Nicotine infused into the nucleus accumbens via the microdialysis

Ringer, caused release of dopamine from nearby terminals (first

peak and second peak). Mecamylamine given intraperitoneally to

block nicotinic receptors prevented the DA release. (From Mifsud

et al. 1989.)

16
variables in such experiments (De Witte et al. 1987; Wang and Schoenfeld

1988). Bombesin given locally gave positive results; it increased extracellular

DA (Merali 1989). The result of administration of bombesin is exciting because

the administration was local, the dose was low, and the rats were awake. This

suggests that bombesin may exert presynaptic control of DA release along with

acetylcholine.

We need to understand both presynaptic control of release and postsynaptic

synergy of multiple neurotransmitters. DA cell bodies fire at a fairly constant

rate (Steinfels et al. 1982), and DA depletion or blockade causes rather

nonspecific impairments in locomotor and reinforcement functions. Presynaptic

control of DA release, however, by acetylcholine or bombesin might endow the

local system with circuit specificity and, thereby, behavioral specificity.

Postsynaptic synergy between DA and other transmitters could also help create

a behavior-specific code.

MICRODIALYSIS AND DRUG DISCRIMINATION

In a drug discrimination study that involved training rats at 80 percent body

weight, we noted that high doses of phenylpropanolamine (PPA), i.e.,

d,l-norephedrine, generalized to AMPH, which was the training drug (Lee et al.

1989). This finding, in turn, suggested that PPA might have some effect like

AMPH in the NAC. This was also suggested by the finding that rats trained on

systemic AMPH generalized to the cue properties of AMPH injected locally in

the NAC (Nielson and Scheel-Kruger 1986). Rats apparently sense the effects

of AMPH in the NAC and interpret these sensations as they do those of

systemic treatment. PPA and AMPH shared some stimulus property after the

animals received extensive AMPH training.

When we used microdialysis to compare the dopaminergic effects of PPA and

AMPH, the two drugs bore a resemblance in that both increased extracellular

DA, although PPA was about 20 times weaker. Therefore, the effects of DA in

the NAC could be part of what the rats feel. In another way, however, the two

drugs were different. With PPA, the metabolites DOPAC and HVA increased,

as happens after feeding, whereas AMPH decreased these metabolites.

Therefore, PPA might be nonaddicting in part because the released DA is

metabolized relatively rapidly (Hoebel and Hernandez, 1990). Both drugs had

insignificant effects in a group of naive rats at 80 percent body weight; however,

the extensive AMPH training used in drug discrimination has never been

duplicated in a microdialysis experiment.

17
DISTINGUISHING A DRUG’S ANORECTIC ACTION FROM ABUSE

POTENTIAL

Both PPA and AMPH presumably increase monoamines in various parts of the

hypothalamus as well as the NAC. In the medial and paraventricular

hypothalamus, norepinephrine induces feeding at night and serotonin inhibits it.

Thus fenfluramine and fluoxetine have a serotonergic anorectic action in the

medial regions (Weiss et al. 1986). In the lateral hypothalamic area,

norepinephrine and DA can inhibit feeding (Leibowitz 1980; Leibowitz and

Stanley 1986); AMPH has been shown to cause anorexia when injected there

(Leibowitz 1982; Wellman and Cockroft 1990). We find that local AMPH can

release DA, norepinephrine, and serotonin in the PFH region (Parada et al.

19886). Therefore, we can hypothesize that systemic AMPH acts, in part, in the

PFH to inhibit feeding. At the same time, these drugs mimic food by increasing

extracellular DA in the NAC. Certain doses of AMPH given locally in the NAC

can decrease food and water intake, perhaps by increasing secondary or

distracting reinforcers in the environment (Carr and White 1986). We have

come to suspect that all drugs that increase synaptic DA and decrease its

metabolites are prime candidates for both NAC reward and PFH anorexia. This

view is supported by observations that DA-receptor blockers at moderate doses

can blunt reward (Fibiger and Phillips 1986; Wise and Bozarth 1987) and cause

overeating (Parada et al. 1988a).

Some drugs cause anorexia without addiction. Besides PPA, the classic

examples are serotonergic drugs. Microdialysis shows that d-fenfluramine

increases extracellular serotonin in both the medial and lateral hypothalamus

when given systemically (Schwartz et al. 1989a, 1989c).

Microdialysis also showed us that a meal or the anticipation of one releases

serotonin naturally in these regions (Schwartz et al. 1989b, 1989c). In the

medial nuclei serotonin clearly inhibits feeding (Weiss et al. 1986), but we have

only a hint of what it does in the PFH. It may inhibit reward because

intraperitoneal d-fenfluramine increases synaptic serotonin (Schwartz et al.

1989a) and decreases the rate of self-stimulation (McClelland et al. 1989).

Apparently, serotonin acts somewhere to inhibit aspects of a reward system for

feeding. This does not necessarily mean that serotonin will inhibit rewards of

drug self-administration, because the addictive drugs may act “downstream,” for

example, by releasing DA in the NAC.

18
In summary, according to present evidence, if a drug increases extracellular DA

in the NAC, it probably will be a drug of abuse. If it does so in the PFH, it will

also be an anorectic (for example, AMPH), but if it acts primarily to increase

extracellular 5-HT, it is likely to be an anorectic without abuse potential. AMPH

releases all three—DA, norepinephrine, and serotonin-and interferes with their

metabolism. This helps to explain why (1) AMPH in the PVN/medial

hypothalamus induces noradrenergic feeding (Leibowitz and Stanley 1986); (2)

AMPH in the PFH induces dopaminergic and adrenergic satiety (Leibowitz

1982); (3) AMPH given intraperitoneally after norepinephrine depletion loses its

catecholamine satiety effects, but fenfluramine gains serotonergic satiety

(Ahlskog et al. 1984; Hoebel et al. 1989); (4) AMPH in the NAC supports local

self-injection (Hoebel et al. 1983) and enhances self-stimulation (Broekkamp et

al. 1975), and (5) systemic AMPH can both facilitate and inhibit

stimulation-induced feeding depending on the dose (Colle and Wise 1988).

Extra-cellular DA can be conditioned to increase or decrease (Mark et al. 1991),

and it decreases during opiate withdrawal (Pothos et al. 1991). Also in the NAC,

acetylcholine release apparently increases during withdrawal (Rada et al.

1991 b), and its relation to DA and reinforcement is under study as a potential

new approach to addiction.

EFFECTS OF BODY WEIGHT ON DA AND DRUG SELF-ADMINISTRATION

Body weight loss augments self-administration (Carroll 1985; Carroll et al. 1979;

Carroll and Meisch 1980; Meisch 1987; Papasava and Singer 1985). Having

found that PPA can generalize to AMPH in underweight rats and that both

drugs have dopaminergic effects in the NAC, we next injected PPA (20 mg/kg)

and AMPH (1 mg/kg) during a dialysis session in underweight rats. The result

was puzzling. First, basal DA levels in the underweight rats seemed to be lower

than in the control group. Second, systemic PPA and AMPH injections had no

significant effect in these underweight animals (Hoebel and Hernandez, 1990).

To determine whether the low levels of NAC DA were real, the experiment was

repeated using rats as their own controls. Extracellular DA again decreased

when male rats were diet restricted for a week to 80 percent of their starting

weight. Subsequent ad libitum refeeding for a week failed to reverse the effect

(figure 8).

Why do starving animals want more of the addictive drugs? Carroll et al. (1979)

suggest that weight loss has a cueing effect that enhances drug intake.

Perhaps animals find dopaminergic drugs more rewarding under these

circumstances. A number of neurochemical possibilities exist; for example,

19
FIGURE 8. Decrease of 40 to 50 percent in extracellular dopamine in rats

deprived of food to 80 percent of normal body weight. This

treatment is known to increase drug intake (see text). (From

Pothos et al. 1989.)

(1) self-administration might increase in underweight rats to compensate for

decreased extracellular DA release in the NAC; (2) self-administration might

increase because of receptor supersensitivity when extracellular DA levels are

low; or (3) there may be an augmented reward effect in some other area such

as the PFC. More research is needed before we can understand the interaction

of body weight and drug taking. All we can add now is that low body weight

seems to lower extracellular DA in the NAC.

20
MICRODIALYSIS AND ANTIPSYCHOTIC DRUGS

Abusers of AMPH, cocaine, or PCP can suffer from drug-induced psychosis.

Therefore, a clue to the neural basis of drug abuse and psychosis can be

obtained from drugs used to treat schizophrenia.

Antipsychotic drugs (neuroleptics) are DA-receptor blockers (Carlsson 1988).

HAL is the classic example. It has some antipsychotic effect immediately,

especially at high doses. Lower doses are used in nonemergencies and then

continued for weeks to gain improvement in symptoms. Microdialysis confirms

earlier work showing that an immediate effect of DA receptor block by HAL or

clozapine (CLOZ) is a compensatory release of DA (Hernandez et al. 1990;

Hernandez and Hoebel 1989b; O’Connor et al. 1989; Zhang et al. 1989). This

extra DA would mitigate the receptor blocking effect. When HAL is used to treat

the psychosis produced by psychostimulants, the situation is further

complicated by drug-induced DA release, which necessitates high doses of

HAL to successfully compete for the DA receptors.

It is believed that HAL given chronically to humans gives therapeutic results by

altering DA circuits in some way. The problem is to determine what changes

take place after chronic treatment. Therefore, rats were prepared with

microdialysis probes in three DA terminal regions: PFC, NAC, and STR. Rats

were given HAL for a month and DA was measured at the beginning and end of

the period. The result at the end of the treatment was a selective decrease in

basal DA in the PFC (figure 9). This finding may help explain the improvement

in cognitive functioning that is gained with long-term treatment in psychotic

patients, because it suggests that endogenous psychosis involves an excess of

PFC DA. It follows that stimulant-induced psychosis with AMPH, cocaine, or

PCP probably also involves DA excess in the PFC. As a chronic treatment, HAL

can have disastrous motor side effects in some patients, presumably due to

actions in the nigrostriatal system and its associated pathways (Ichikawa and

Meltzer 1990). Atypical neuroleptics such as CLOZ, however, offer some hope.

New triple microdialysis studies suggest that CLOZ has effects on DA level and

turnover in the PFC and not in the STR (Hernandez et al. 1990; Hernandez and

Hoebel 1989b).

CONCLUSION

In summary, our studies illustrate the use of microdialysis in freely moving rats

and show that psychostimulants can increase extracellular DA by a local action

21
FIGURE 9. Acute haloperidol (HAL) (1st injection) increasing extracellular

dopamine (DA) in the prefrontal cortex (upper left asterisks). After

a month of daily HAL injections, the basal DA level is significant/y

decreased (upper right graph, daggers), and the 39th injection

was relatively ineffective, showing decreased DA turnover. HAL

had less effect in the nucleus accumbens and striatum of the

same rats, (From Hernandez and Hoebel 1989b, 1990.)

22
in the NAC. Also, electrical stimulation of the PFH feeding-reward system

increases extracellular DA in the NAC. Natural appetitive behavior patterns

such as feeding and drinking do the same thing. The release of DA by a sweet

taste can be counterconditioned by pairing the taste with malaise. When the

taste becomes a conditioned signal for toxicity, it causes DA release to

decrease instead of increase. Drugs of abuse that release DA apparently mimic

natural rewarding stimuli. Psychostimulants such as AMPH, cocaine, and PCP

increase extracellular serotonin as well as DA, but serotonin’s role in the NAC is

not clear. In feeding-control centers such as the hypothalamus, serotonin

probably suppresses appetite and self-stimulation. This serotonergic effect,

along with a dopaminergic satiety effect partially explains the anorectic effects

of psychostimulants. Drugs that selectively increase synaptic serotonin are

anorectic and not abused. PPA, which is an over-the-counter anorectic, was

interesting in that it increased extracellular DA in the NAC and also DA

metabolites, showing a marked departure from the dopaminergic drugs of

abuse that decreased DOPAC and HVA. Nicotine was also a special case,

because it acted via nicotinic cholinergic receptors to release DA

presynaptically without measurably altering DA metabolism. The drugs like

cocaine that interfere with the formation of DOPAC and HVA seem to be those

with a high potential for causing psychosis. Microdialysis after chronic

antipsychotic drug treatment showed that basal DA was selectively lowered in

the PFC. Therefore, we suspect that psychosis is partly a PFC abnormality, and

DA antagonists such as CLOZ that act with some selectivity in the PFC might

be effective in cases of drug-induced psychosis. It is known from a variety of

self-injection studies that dopaminergic drugs are positive reinforcers in both the

NAC and the PFC, so it is hard to say which DA projection system will prove to

be more important in addiction therapy. The observation that DA release can be

controlled presynaptically by neurotransmitters such as acetylcholine and

bombesin offers new avenues to addiction treatment. Clearly, microdialysis will

be a valuable tool in the evaluation of these treatments in the experimental

stages.

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ACKNOWLEDGMENTS

This work was supported by Public Health Service grant DA 03597.

AUTHORS

Bartley G. Hoebel

Luis Hernandez

Gregory P. Mark

Emmanuel Pothos

Department of Psychology

Princeton University

Princeton, NJ 08544-1010

34
Neurophysiological Approaches to Receptor

Pharmacodynamics

Francis J. White

INTRODUCTION

A variety of specific neurophysiological techniques have been used with in vivo

and in vitro preparations to further our understanding of how drugs affect

neurotransmission at both the molecular (ion channel gating, conductance

changes) and cellular (single-cell firing) levels; how such actions are related to

changes at a systems level (alterations in activity of identified neuronal

populations); and how these changes are ultimately translated into behavioral

output. Although it is clear that the most direct means of correlating changes in

neuronal activity with specific behaviors would involve the use of single-cell

recordings in freely moving animals (see “Psychomotor Stimulant Effects on

Single Neurons in Awake, Behaving Rats”), such techniques greatly limit the

extent to which pharmacologic manipulation is possible. From the perspective

of receptor pharmacodynamics, the use of in vivo anesthetized-animal

preparations and in vitro slice or cell preparations allows considerably better

definition of relevant drug effects and underlying mechanisms at the single-cell

level.

As an example of the tremendous potential of single-cell recording procedures

in neurobehavioral research, one need look no further than the pioneering

studies of Aghajanian and colleagues (Aghajanian 1978; Andrade and

Aghajanian 1985). The work of this group typifies the use of single-cell

recording techniques as a means of studying receptor pharmacodynamics and

the extension of such findings to clinical investigation. For example, with the

ability to identify defined populations of monoamine-containing neurons,

Aghajanian and colleagues identified similar inhibitory effects of opioids and

alpha-2 selective agonists on norepinephrine-containing neurons within the rat

locus coeruleus (Aghajanian 1978). In addition, they were able to demonstrate

that these drugs worked via separate receptor populations to alter a common

35
transduction mechanism, i.e., inhibition of adenylate cyclase (Andrade and

Aghajanian 1985). This knowledge and the finding that cyclic adenosine

monophosphate (cAMP) formation is greatly exaggerated during opiate

withdrawal provided clinical investigators with a theoretical framework on which

to base the use of clonidine for opiate withdrawal (Gold et al. 1978).

This chapter reviews recent electrophysiological findings from my laboratory

that have identified behaviorally relevant interactions between D1 and D2

dopamine (DA) receptors within the mesoaccumbens and nigrostriatal DA

systems. I hope to demonstrate that although the physiological and behavioral

results were obtained from separate groups of rats, the close parallels between

the findings make it possible to reach certain important conclusions regarding

the cellular sites and mechanisms of action of dopaminergic drugs as they

relate to specific components of behavior and as they may affect clinical

aspects of dopaminergic transmission.

MULTIPLE DA RECEPTORS: FUNCTIONAL INTERACTIONS

Although D1 and D2 DA receptors have historically been defined on the basis of

their (often opposing) effects on CAMP production (Kebabian and Calne 1979;

Stoof and Kebabian 1981) and have been shown to exert apparently opposing

effects on a variety of other biochemical measures, several behavioral and

electrophysiological studies have indicated that these receptor subtypes can

also interact synergistically to control DA-mediated functions (Clark and White

1987; Waddington and O’Boyle 1987). The first evidence of an apparent

synergistic interaction between D1 and D2 receptors was provided by Gershanik

and colleagues (1983), who demonstrated that the presence of both the D1

selective agonist SKF 38393 and the D2 selective agonist LY 141865 was

required to reverse the akinesia produced by administering reserpine to mice.

We provided the initial electrophysiological evidence for synergistic interactions

between D1 and D2 DA receptors. In these studies (White 1986; White and

Wang 1986), we tested the effects of iontophoretic administration of D1 and D2

agonists on neurons within the rat nucleus accumbens (NAC), the terminal area

for the majority of DA neurons within the ventral tegmental area (A10 DA cells).

Both spontaneously active and quiescent NAC neurons, which were made to

fire by the iontophoretic administration of glutamate, were inhibited during

iontophoretic administration of either SKF 38393 or LY 141865. In addition,

receptor selectivity was apparent, because the D2 antagonist sulpiride blocked

only the effects of LY 141865, whereas the D1 antagonist SCH 23390

36
completely blocked the effects of SKF 38393 while partially attenuating the

effects of LY 141865.

Not only were we able to identify both D1 and D2 receptive NAC neurons, but

we also determined that a subpopulation of neurons was inhibited by both

agonists. More important, on those cells that were responsive to both D1 and D2

receptor agonists, coadministration of SKF 38393 and LY 141865 produced a

supra-additive or synergistic inhibition. These findings indicated that certain

NAC neurons may possess both D1 and D2 receptors and that these receptor

subtypes can interact in a synergistic manner to regulate NAC neuronal activity

(White and Wang 1986).

Accounts of several behavioral results were subsequently published, furthering

the notion of synergistic D1-D2 receptor interactions and providing hints as to

how such synergism might occur. For example, Braun and Chase (1986)

reported that in rats acutely depleted of DA with the tyrosine hydroxylase

inhibitor alphamethylparatyrosine (AMPT), the production of various

stereotyped behaviors (licking, gnawing) required the combined administration

of D1 and D2 selective agonists. Subsequently, this group demonstrated that

SKF 38393 potentiated circling behavior induced by the D2 agonist quinpirole

(QUIN; the active enantiomer of LY 141865) in rats with excitotoxin-induced

lesions of the striatum, a rotational model in which DA receptors remain

normosensitive. Moreover, when AMPT was used to deplete DA in these rats,

the QUIN response was markedly reduced unless the D1 agonist was also

injected (Barone et al. 1986). At nearly the same time, Jackson and Hashizume

(1986) reported that the locomotor-stimulant effect of the D2 agonist

bromocriptine was abolished by acute DA depletion, but reinstated by SKF

38393.

Taken together, these various behavioral studies suggested that D1 receptor

stimulation may be required for D2 agonist-induced responses. Thus, when DA

was acutely depleted, the effects of D2 agonists were abolished unless a D1

agonist was also administered, suggesting that the critical event missing in the

depleted animal was D1 receptor stimulation by endogenous DA. Several

previous studies had reported the apparently paradoxical finding that the D1

antagonist SCH 23390 blocked the ability of D2 agonists to induce locomotion

and stereotypy without directly blocking the D2 receptor (Clark and White 1987).

In hindsight, this earlier finding could also be interpreted as indicating a

necessary role for D1 receptor stimulation in these particular D2 agonist-induced

behavioral responses.

37
THE ENABLING ROLE OF D1

 RECEPTORS: ELECTROPHYSIOLOGICAL

STUDIES

In view of these various behavioral findings indicating that D1 receptor

stimulation was necessary for (or enabled) certain behavioral effects of D2

selective agonists, we continued our electrophysiological studies by

determining whether the synergistic interactions we had previously observed

between D1 and D2 receptors within the NAC might also represent an enabling

effect of D1 receptors. We first observed that SKF 38393 potentiated the

inhibitory effects of quinpirole even on neurons not inhibited by the D1 agonist

alone (White 1986). We then studied the interaction of the two receptors in the

absence of receptor occupation by endogenous DA. When we acutely depleted

DA levels (by 80 percent) within the NAC by pretreating with AMPT (White et al.

1988), the normal inhibitory effects of QUIN on NAC cells were significantly

attenuated (White 1986, 1987). Importantly, SKF 38393 administration (at low

ejection currents, which alone produced little inhibition) “reinstated” the

inhibitory response to QUIN (figure 1). Thus, activation of D1 receptors by SKF

38393 in rats acutely depleted of DA enabled D2 receptor occupation by QUIN

to produce an inhibition of neuronal activity. This finding clearly suggested that

endogenous DA may act at D1 receptors to enable Ds-mediated events in the

intact rat (White 1986, 1987).

We have now confirmed and extended these original observations (Wachtel et

al. 1989). First, we demonstrated that D1 receptor activation is required for the

inhibitory effects of other selective D2 DA receptor agonists, such as RU 24213

(Wachtel et al. 1989) and B-HT 920 (Johansen et al. 1991), on NAC neurons.

We also found that iontophoretic administration of other D1 agonists, SKF

75670 and SKF 81297 (Andersen et al. 1987; Arnt et al. 1988), also enabled

the inhibitory effects of QUIN on NAC neurons in AMPT-pretreated rats

(Johansen et al. 1991). Thus, the requirement of D1 receptor activation for D2

receptor-mediated inhibition of NAC neurons is not specific to the particular

agonists employed but is a general phenomenon of D1-D2 receptor interaction.

Second, we reported that the inhibitory effects of the mixed D1-D2

receptor-agonist apomorphine (APO), which possesses nearly equivalent

affinity for the two receptors (Arnt and Hyttel 1985), were still evident in

AMPT-pretreated rats (figure 2). Thus, acute DA depletion failed to attenuate

the effect of a DA agonist that activates both D1 and D2 receptors (Wachtel et

al. 1989).

38
FIGURE 1. Inhibition of the firing of nucleus accumbens neurons by

iontophoretic administration of the D2 agonist quinpirole (QUIN).

Current-response curves illustrate the attenuation of the inhibitory

effect of QUlN by pretreatment with alphamethylparatyrosine

(AMPT and the reinstatement of the QUIN-induced inhibition on

the same neurons with coiontophoresis of SKF 38393 (4 nA).

Each point represents the mean±SEM (QUIN control, n = 11;

AMPT, n = 12, AMPT + SKF 38393, n = 11). (From Wachtel et al.

1989.)

Next, we extended our findings by showing that D1 receptor stimulation also

enables the inhibitory effects of D2 receptors within the lateral

caudate-putamen. AMPT pretreatment nearly abolished the inhibitory effects of

QUIN and RU 24213 (figure 3), but not of APO, on caudate-putamen neurons,

effects that were readily reversed by coiontophoretic administration of SKF

38393 (Wachtel et al. 1989). Therefore, the enabling role of D1 receptor

stimulation for D2 receptor-mediated inhibition of neuronal activity appears to

exist throughout both the dorsal and ventral striata, although apparently not

within the rat medial prefrontal cortex (Sesack and Bunney 1989).

39
FIGURE 2. Inhibition of nucleus accumbens neurons by iontophoretically

administered apomorphine (APO) and quinpirole (QUIN).

Current-response curves illustrate the inability of pretreatment

with alphamethylparatyrosine (AMPT) to alter the inhibitory effect

of APO, whereas the effect of QUIN was attenuated. Each point

represents the mean±SEM (APO control, n = 10; AMPT + APO,

n = 15; QUlN control, n = 9; AMPT + QUIN, n = 8). (From Wachtel

et al. 1989.)

Paralleling our results from studies of the striatal complex are the findings of

Walters and colleagues (1987), who demonstrated D1-D2 interactions with rat

globus pallidus neurons after intravenous (IV) injections of selective D1- and

D2-receptor agonists (Carlson et al. 1987b). Acute depletion of DA with AMPT

was found to attenuate the partial (as compared with APO) excitatory effects of

IV QUIN but not the marked excitatory effects of APO. Subsequent

administration of SKF 38393 to AMPT-pretreated rats that had received QUIN

produced marked increases in pallidal firing equivalent to those produced by IV

APO or by the combination of SKF 38393 and QUIN in normal rats (Walters et

al. 1987). Recently, this group has also demonstrated that the excitatory effects

of IV APO on pallidal neurons are attenuated by ipsilateral quinolinic acid

lesions of the rostral striatum (Pan et al. 1987; Pan and Walters 1988),

40
suggesting that the excitatory effects of APO on pallidal neurons result from

disinhibition subsequent to inhibition of striatopallidal caudate-putamen

neurons, which are thought to be mostly GABA-ergic in content (Fonnum et al.

1978).

Our findings obtained during recordings of caudate-putamen neurons suggest

that the permissive role of D1 receptor stimulation for D2 agonist-induced

excitation observed on globus pallidus neurons (Walters et al. 1987) may be the

result of direct enabling actions in the caudate-putamen occurring on

FIGURE 3. Inhibition of caudate-putamen neurons by iontophoretic

administration of RU 242 13. Current-response curves illustrate

the attenuation of the RU 24213-induced inhibitory effect on

caudate-putamen neurons in rats pretreated with

alphamethylparatyrosine (AMPT) and the ability of SKF 38393

(4 nA) to restore the inhibition produced by RU 24213. The

attenuation of the RU 24213 effect by AMPT pretreatment is

greater in the caudate-putamen than in the NAC (fig. 2). Points

represent the mean±SEM (n = 10 for each group). (From Wachtel

et al. 1989.)

41
striatopallidal GABA-ergic neurons. Similarly, Yang and Mogenson (1989) have

reported that SKF 38393 enables the ability of QUIN to increase the amount of

firing of neurons within the rat ventral pallidum, which receive an inhibitory input

from NAC GABA-ergic neurons when the D1 agonist is infused directly into the

NAC before the D2 agonist.

THE ENABLING ROLE OF D1 RECEPTORS: BEHAVIORAL STUDIES

In parallel behavioral experiments, we have demonstrated the necessity of

combined stimulation of D1 and D2 receptors for the full expression of oral

stereotyped behaviors (licking and gnawing). As shown in figure 4, combined

administration of SKF 38393 and QUIN produced nearly maximal stereotyped

gnawing comparable to that observed with the mixed D1-D2 agonist APO,

whereas QUIN alone, even at extremely high doses (Walters et al. 1987), fails

to produce oral stereotypies (figure 4). In addition, we have demonstrated that

acute depletion of DA with AMPT completely abolishes the low-level,

stereotyped sniffing typically observed in rats following administration of a D2

selective agonist such as QUIN (White et al. 1988). Administration of SKF

38393 along with QUIN reinstates the behavior and intensifies its quality to oral

stereotypies (figure 4). Other investigators have reported similar findings for

locomotor activity and oral stereotypy (Arnt et al. 1987; Braun and Chase 1986;

Jackson and Hashizume 1986; Longoni et al. 1987b; Meller et al. 1988), D2

agonist-induced yawning (Longoni et al. 1987a; Ushijima et al. 1988), and

circling in rats with quinolinic acid lesions of the striatum (Barone et al. 1986).

As in our electrophysiological experiments, acute DA depletion failed to alter

APO-induced stereotyped behavior (figure 4), presumably because APO

supplies the necessary D1 receptor stimulation (White et al. 1988).

D1-AGONIST-INDUCED FUNCTIONAL EFFECTS DO NOT REQUIRE

D2-RECEPTOR STIMULATION

A remaining unsettled question regarding the interactions between D1 and D2

receptors is the extent to which the enabling relationship is reciprocal. Our

studies indicate that D1 receptor-mediated electrophysiological responses do

not require D2 receptor stimulation (Wachtel et al. 1989), because the inhibitory

effects of SKF 38393 on both NAC (figure 5) and caudate-putamen cells are

unaltered by acute DA depletion. Similarly, the characteristic grooming

responses produced by the D1 agonist SKF 38393 (Molloy and Waddington

1984, 1987a, 19876; Starr and Starr 1986a, 1986b) are still evident in rats

acutely depleted of DA (>99.5 percent reductions in striatal DA) by pretreatment

42
with reserpine plus AMPT (figure 6). Indirect evidence also supports our

position, because blockade of D2 receptors unmasks APO-induced grooming

FIGURE 4. The ability of various drug treatments to induce stereotyped

behaviors in normal rats and rats depleted of dopamine by

alphamethylparatyrosine (AMPT). Both quinpirole (QUIN) and

apomorphine (APO) were administered at a dose of 2.0 mg/kg,

whereas SKF 38393 was administered at 16 mg/kg. Each bar

represents the mean±SEM obtained from eight rats. Stereotypy

ratings for QUIN, APO, AMPT + APO, SKF + QUIN, and AMPT +

SKF + QUIN were all significantly higher than saline (SAL,

p < .01). The SKF + QUIN treatment was not significantly different

from APO, although the APO and QUIN groups were significantly

different from one another (p < . 01). The effects of the QUIN +

SKF combination were significantly greater than QUIN alone

(p < .05). AMPT pretreatment significantly reduced the effects of

QUIN (p < .01); however, the SKF + QUIN combination reinstated

the stereotyped behaviors in the AMPT group. Stereotypy scores

were based on a standard 0-6 rating scale. (From White et al.

1988.)

43
FIGURE 5. Inhibition of nucleus accumbens (NAC) neurons by iontophoretic

administration of the D1 agonist SKF 38393. Current-response

curves illustrating the failure of alphamethylparatyrosine (AMPT)

pretreatment to affect the inhibition of NAC neurons by SKF

38393. Each point represents the mean±SEM (SKF 38393

control, n = 10; AMPT + SKF 38393, n = 11). (From Wachtel et al.

1989.)

responses, suggesting that D1 stimulation produced grooming in the absence of

D2 tone (Molloy and Waddington 1984; Starr and Starr 1986a; Starr et al. 1987).

Although some (not all) D2 selective antagonists have been reported to

attenuate SKF 38393-induced grooming (Molloy and Waddington 1987b), we

believe that such a finding may be due to competing behavioral effects of the

antagonist, e.g., catalepsy.

D2 DA AUTORECEPTORS DO NOT REQUIRE ENABLING

Unlike postsynaptic D2 DA receptors within the striatal complex,

impulse-regulating somatodendritic autoreceptors on midbrain DA neurons are

not responsive to D1 receptor activation. For both A10 (White and Wang 1984)

and A9 nigral (Carlson et al. 1987a) DA neurons, D2, but not D1, agonists inhibit

44
FIGURE 6. Grooming behavior induced by a 16-mg/kg dose of SKF 38393

(n = 8) versus saline (n = 5) in rats pretreated with a combination

of reserpine (5 mg/kg and alphamethylparatyrosine (AMPT,

250 mg/kg) and in nontreated controls (n = 8). In rats that

received reserpine and AMPT, SKF 38393 induced a significant

increase in grooming versus saline (p < .001). Data indicate the

amount of time the rats engaged in grooming behavior during a

30-min observation period. (From White et al, 1988.)

neuronal activity. The lack of effect of SKF 39383 is not due to the partial

agonist characteristic of this drug because the full D1 agonist SKF 81297

(Andersen et al. 1987) produced similar results (Wachtel and White,

unpublished findings). Given that SKF 38393 can potentiate QUIN-induced

inhibition of NAC cells that are not directly inhibited by the D1 agonist (White

1986; 1987), such “silent” effects of D1 receptor stimulation might also occur on

DA neurons to modulate the effects of D2 agonists at impulse-regulating

autoreceptors. SCH 23390, however, fails to alter the inhibitory effects of D2

agonists on either A9 or A10 DA cells (Carlson et al. 1986; Wachtel et al. 1989).

45
In addition, acute DA depletion does not alter the effects of D2

 agonists,

because neither IV nor iontophoretic administration of SKF 38393 alters

QUIN-induced inhibitions of midbrain DA cells (Wachtel et al. 1989). Thus, it is

apparent that impulse-regulating somatodendritic DA autoreceptors are

exclusively of the D2 subtype (White and Wang 1984). Similar manipulations of

nerve-terminal, synthesis-modulating receptors also fail to reveal D1 receptor

involvement in (or enabling of) D2 receptor-mediated inhibition of DA synthesis

(Wachtel et al. 1989).

CHRONIC DA DEPLETION RELIEVES D2 RECEPTORS OF THE

NECESSITY FOR D1 STIMULATION

In contrast to their actions in normal rats, D1 receptor antagonists fail to block

the effects of both mixed D1-D2 agonists and selective D2 agonists in behavioral

models using rats with supersensitive DA receptors (Arnt 1985a, 1985b; Arnt

and Hyttel 1985; Breese and Mueller 1985). Whereas both D1 and D2 agonists

elicit rotation in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the

nigrostriatal pathway, these effects are blocked only by antagonists selective for

those receptors (Arnt and Hyttel 1984, 1985). Similarly, the hyperactivity

produced by D1 or D2 agonists in rats with bilateral 6-OHDA lesions (Arnt

1985b Breese and Mueller 1985) or in rats treated repeatedly with reserpine

(Arnt 1985a) is blocked only by the appropriate receptor-selective antagonist.

These findings suggest that the necessity of D1 receptor stimulation for D2

receptor-mediated functional responses may be relieved after chronic DA

depletion.

Despite this apparent lack of interaction between D1 and D2 receptors in rats

with supersensitive DA receptors, several investigators have reported

synergistic rotational responses to D1 and D2 DA agonists in rats with unilateral

6-OHDA lesions of the nigrostriatal tract (Koller and Herbster 1988; Robertson

and Robertson 1986, 1987; Rouillard and Bedard 1988; Sonsalla et al. 1988). In

addition, synergistic interactions between D1 and D2 selective agonists have

also been reported with respect to the self-mutilating biting observed in rats that

had received 6-OHDA as neonates (Breese et al. 1985). Thus, despite the lack

of an enabling relationship between D1 and D2 receptors, these studies clearly

indicate that D1 and D2 receptors may still work in concert to regulate behavior

in DA-denervated rats.

We have recently investigated these relationships using our single-cell

electrophysiological techniques (Hu et al. 1990). In striking contrast to the

46
effects of acute DA depletion, long-term depletion of DA produced either by

repeated reserpine administration or by 6-OHDA lesions of the nigrostriatal DA

system did not attenuate the inhibitory effects of iontophoretic QUIN on

caudate-putamen neurons. In fact, the inhibitory potency of QUIN was

enhanced (supersensitive) compared with that observed in the intact

caudate-putamen (figure 7). The inhibitory effects of QUIN in the denervated

caudate-putamen could not be attributed to residual DA stimulating

supersensitive D1 receptors (and thereby enabling D2 receptor-mediated

inhibition), because further DA depletion produced by additional acute

administration of AMPT failed to reduce the effects of QUIN (figure 7). In

addition, the lack of D1 receptor-mediated enabling in the 6-OHDA rat cannot be

attributed to the loss of DA terminals and possible cell-cell D1-D2 interaction,

because similar findings were observed with repeated reserpine treatment (Hu

et al. 1990).

These electrophysiological results, indicating that D2 receptor-mediated

functional effects are relieved from the requirement of D1 receptor stimulation,

provide a single-cell correlate of previous behavioral findings indicating that D1

receptor stimulation is not required for D2 agonist-induced behaviors in rats with

chronic DA depletion. Taken together, these behavioral and electrophysiological

findings indicate that D1 and D2 receptors located in the caudate-putamen

become functionally uncoupled after long-term depletion of DA. Whether D1

and/or D2 receptor supersensitivity or both are required for the uncoupling of

these receptor subtypes is a question currently under investigation in our

laboratory. It has been reported that selective upregulation of D1 receptors

produced by repeated administration of the D1 antagonist SCH 23390

potentiates the behavioral effects of D2 agonists (Hess et al. 1986), suggesting

that selective D1 receptor supersensitivity may cause enhanced enabling rather

than functional dissociation of D1 and D2 receptors.

CONCLUSIONS

The findings reviewed in this chapter demonstrate a close parallel between the

effects of D1 and D2 DA receptor agonists at the behavioral and cellular levels.

Although the data were not obtained from the same animals, the similarities

between the interactions of D1 and D2 receptors observed on unconditioned

DA-dependent behaviors and those seen on single neurons within the striatal

complex (caudate-putamen and NAC) clearly suggest that the behavioral

effects may be the reflection of D1-D2 receptor interactions occurring at the

cellular level within the striatum. Not only have these findings generated

47
considerable interest in the underlying mechanisms involved in D1

-D2 receptor

interactions at the molecular level, but they also have many important clinical

implications.

FIGURE 7. Comparison of responses of caudate-putamen neurons to

coiontophoretic administration of D1 and D2 selective agonists in

control rats (n = 10 cells) (A); rats acutely depleted of dopamine

(DA) with alphamethylparatyrosine (AMPT) (n = 15 cells) (B); rats

chronically depleted of DA with 6-hydroxydopamine (6-OHDA)

(n = 23 cells) (C); and rats that received both 6-OHDA and AMPT

(n = 10 cells) (D). (A) In control rats, low (subinhibitory) currents of

SKF-38393 potentiated the inhibitory effects of quinpirole (QUIN)

on the same caudate-putamen neurons (F[1, 18] = 4.65, p < 05).

(B) In rats acutely depleted of DA by AMPT, WIN-induced

inhibition was attenuated without simultaneous stimulation of D1

receptors (F[1, 28] = 47.73, p < .0001). (C) In rats chronically

depleted of DA by 6-OHDA lesions, the requirement of D1

48
One of the most important implications of the enabling relationship between D1

and D2 DA receptors is its potential relevance for the treatment of parkinsonism

(see Clark and White 1987; Waddington and O’Boyle 1987). With the exception

of Ldopa, currently available antiparkinsonian drugs (bromocriptine, pergolide)

are selective D2 agonists. With the discovery that D1 receptor stimulation is

required for certain behavioral effects of D2 agonists, many investigators

suggested that mixed D1-D2 agonists or combinations of D1 and D2 agonists

would be more effective in alleviating parkinsonian symptoms (Clark and White

1987). The more recent finding that D1 receptor stimulation is no longer

necessary for D2-mediated responses in the DA-denervated striatum appears to

argue against the need for simultaneous stimulation of D1 and D2 receptors in

patients with parkinsonism. Synergistic behavioral effects of selective D1 and D2

agonists, however, have been reported in the unilateral 6-OHDA rotational

model (Koller and Herbster 1988; Robertson and Robertson 1986, 1987;

Rouillard and Bedard 1988; Sonsalla et al. 1988) and in neonatal

6-OHDA-treated rats (Breese et al. 1985). In view of these findings, it was

somewhat surprising that, in our electrophysiological study, DA denervation not

only relieved the requirement of D1 receptor stimulation for D2 agonist-induced

inhibition (enabling), also abolished the D1-mediated potentiation of D2

agonist-induced inhibition (a quantitative synergistic effect). This failure to

observe either quantitative or qualitative synergism on caudate-putamen

neurons was not due to a maximal “floor effect” produced by QUIN at

supersensitive D2 receptors, because additional DA depletion produced by

AMPT resulted in greater inhibition of caudate-putamen neurons by QUIN

(figure 7). These findings suggest that the behavioral synergism reported in

previous studies of DA-denervated rats is not due to synergistic actions at

adjacent D1 and D2 DA receptors located in the caudate-putamen.

FIGURE 7. receptor stimulation for QUIN-induced inhibition was abolished, as

(continued) was the potentiating (synergistic) effect of SKF-38393 on

QUIN-induced inhibition (n = 15). (D) Additional depletion of DA

with AMPT in 6-OHDA-lesioned rats not only enhanced the

inhibitory effect induced by QUIN (F[1, 29] = 8.54, p < .01) but

also failed to produce a synergistic inhibition on caudate-putamen

neurons, indicating that (1) the supersensitive response observed

in 6-OHDA-pretreated rats was not maximal, thereby masking

possible potentiation by SKF-38393 and (2) the ability of D2

receptor stimulation to inhibit caudate-putamen neurons in

6-OHDA rats was not due to residual DA stimulating

supersensitive D1 receptors (enabling). (From Hu et al. 1990.)

49
Robertson and Robertson (1986, 1987) have proposed that the synergistic

rotational responses produced by submaximal doses of D1 and D2 selective

agonists may be due to the combined actions at D2 receptors within the

caudate-putamen and D1 receptors located on the terminals of striatonigral

neurons within the substantia nigra pars reticulata. Such systems-level (distant)

D1-D2 receptor interactions could explain the observation of behavioral

synergism in the absence of adjacent D1-D2 interactions at the single-cell level.

Electrophysiological studies have also provided a possible correlate of this

behavioral interaction. Thus, Weick and Walters (1987) found a synergistic

inhibition of substantia nigra pars reticulata neurons produced by combined IV

administration of D1 and D2 selective agonists only in rats with 6-OHDA lesions.

These findings, combined with the previous behavioral and electrophysiological

studies, provide new evidence for mechanisms through which the behavior of

patients with parkinsonism might be influenced. Although it appears that DA

receptor supersensitivity relieves D2 receptors from the requirement of D1

receptor stimulation (i.e., the loss of adjacent D1-D2 receptor interactions on

these neurons), additional distant (perhaps compensatory) functional

interactions between D1 and D2 receptors are evident at a systems level. Thus,

the possible utility of combined D1-D2 stimulation in the treatment of

parkinsonism cannot be ruled out.

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6-hydroxydopamine lesioned rats: D1/D2 coactivation induces potentiated

responses. Brain Res 405:234-246, 1987.

White, F.J. Electrophysiological investigations of the D-1 dopamine receptor.

Clin Neuropharmacol 9:29-31, 1986.

White, F.J. D1 dopamine receptor stimulation enables the inhibition of nucleus

accumbens by a D2 receptor agonist. Eur J Pharmacol 135:101-105, 1987.

White, F.J.; Bednarz, L.M.; Wachtel, S.R.; Hjorth, S.; and Brooderson, R.J. Is

stimulation of both D1 and D2 receptors necessary for the expression of

dopamine-mediated behaviors? Pharmacol Biochem Behav 30:189-193,

1988.

White, F.J., and Wang, R.Y. Pharmacological characterization of dopamine

autoreceptors in the rat ventral tegmental area: Microiontophoretic studies. J

Pharmacol Exp Ther 231:275-280, 1984.

White, F.J., and Wang, R.Y. Electrophysiological evidence for the existence of

D1 and D2 dopamine receptors in the rat nucleus accumbens. J Neurosci

6:274-280, 1986.

Yang, C.R., and Mogenson, G.J. Ventral pallidal neuronal responses to

dopamine receptor stimulation in the nucleus accumbens. Brain Res

489:237-246, 1989.

ACKNOWLEDGMENTS

This research was supported by U.S. Public Health Service grants DA 04093

from the National Institute on Drug Abuse and MH 40832 from the National

54
Institute of Mental Health and by the State of Michigan, Department of Mental

Health.

AUTHOR

Francis J. White, Ph.D.

Department of Psychiatry

Neuropsychopharmacology Laboratory

Wayne State University School of Medicine

Lafayette Clinic

951 East Lafayette

Detroit, Ml 48207

55

Psychomotor Stimulant Effects on Single

Neurons in Awake, Behaving Rats

Mark O. West, Laura L. Peoples, Martin Wolske,

and Steven I. Dworkin

INTRODUCTION

The current quest to understand the neural mechanisms of drug abuse requires

the use of methods that provide instantaneous measures relating the brain to

behavior. Several such methods are reviewed in this volume. This paper

describes the application of an established technique, electrophysiology, in a

relatively uncharted area: drug administration to awake, freely moving animals.

Combining electrophysiology, pharmacology, and behavior in one preparation is

a valuable but challenging endeavor. Neuronal activity varies with behavior, and

drug effects on brain activity and behavior vary as a function of complex, but

quantifiable, behavioral factors (Johansen 1988). (See also “Importance of

Behavioral Controls in the Analysis of Ongoing Events” and “Cocaine

Self-Administration: Pharmacology and Behavior.“) In the preparation

described, such variables are evaluated in terms of conscious neuronal

processing involved in and responding to seeking drugs of abuse. Certain

compromises are necessary, however, when electrophysiology, pharmacology,

and behavior are combined. It is not possible to adhere to the rigorous protocols

that strengthen each individual approach (see “Neurophysiological Approaches

to Receptor Dynamics”). A thorough review of highly relevant theoretical and

methodological issues has appeared elsewhere (Deadwyler 1986).

This chapter focuses on strategies for studying single-unit responses to

psychomotor stimulants in freely moving rats. Of particular interest are the

effects of stimulants on cellular processing related to motor function and

reinforcement. First, with respect to stimulant effects on striatal activity, we

describe a protocol that accounts for a combination of variables, including the

behavioral effects of the drug and the behavioral correlations of recorded units.

57
Second, with the intent of studying the neurobiological mechanisms underlying

the reinforcing properties of cocaine, we describe an extension of the first

protocol to a recently developed method for recording single cell activity during

cocaine self-administration. Each strategy is illustrated with results obtained

from specific experiments. In spite of certain compromises, the strengths of a

combined approach are manifested by demonstrating drug effects on specific

patterns of neuronal activity related to particular behaviors and

neurotransmitters.

MEASURES OF UNIT ACTIVITY APPROPRIATE FOR DRUG STUDIES

In addition to a neuron’s tonic (spontaneous) firing rate, its phasic firing patterns

serve as useful substrates for assessing drug effects on neural circuits. Phasic

patterns are transient neuronal signals related to specific behaviors, sensory

and motor events, electrical stimulation of afferent pathways, agonist-antagonist

interactions, and so on. Phasic neuronal patterns that have been used

effectively in studying dopamine (DA) function include evoked auditory unit

discharges (Rolls et al. 1984), accumbens discharges activated by stimulation

of limbic afferents (Yim and Mogenson 1982; Yang and Mogenson 1984;

DeFrance et al. 1985b; Hakan and Henriksen 1989), and DA receptor

agonist-antagonist interactions in the accumbens (White and Wang 1986). The

better characterized the phasic pattern in terms of the synaptic input or receptor

subtype involved, the more meaningful is the interpretation of drug effects

(Bloom 1974). In turn, drug studies can contribute to the basic understanding of

synaptic events that mediate particular phasic neuronal patterns.

In the rat striatum (caudate-putamen), striking differences can be demonstrated

between the behavioral correlates of neurons in medial subregions and of those

in lateral subregions. Whereas medial units show only general relationships to

movement-e.g., increased firing during whole-body movements such as

locomotion (West et al. 1987)—a substantial percentage (25 percent to

35 percent) of lateral striatal units fire during specific sensorimotor activity of a

particular part of the body (West et al. 1990). Virtually all body parts are

represented in the lateral striatum, including limbs, individual vibrissae, snout,

chin, neck, shoulder, trunk, and so on (Carelli and West 1991). The location of

these units corresponds with the location of termination of projections from

somatic sensorimotor cortex (Wise and Jones 1977; Cospito and Kultas-Ilinsky

1981; Donoghue and Herkenham 1986).

58
These phasic, sensorimotor firing patterns provide excellent substrates for

(1) assessing the response of these cells to drugs and (2) studying basal

ganglia function. An important neurobiological question addressed in this

chapter is, how do DA and psychomotor stimulants modulate the activity of DA

target cells involved in sensorimotor function?

CONTROLLING BEHAVIORAL VARIABLES IN STUDIES OF

PSYCHOMOTOR STIMULANT EFFECTS ON NEURONAL ACTIVITY

Measures of both tonic and phasic neuronal patterns are influenced by

behavioral variables. Therefore, when neural activity is compared between

predrug and postdrug conditions, the animal’s behavior must be comparable

across conditions. The following studies of psychomotor stimulants illustrate

that this comparability can be accomplished by controlling for drug-induced

behaviors. One approach is to produce these behaviors experimentally in the

predrug control period. Drug-induced behaviors also can be avoided either by

administering low doses that have minimal effects on behavior or by applying

drugs microiontophoretically.

Use of Forced Treadmill Locomotion in Conjunction With a

Locomotion-Inducing Dose of Amphetamine

The study of amphetamine’s effects on striatal-unit activity in awake rats has

required the development of a detailed strategy, the description of which is a

useful illustration of some of the issues involved in electrophysiological study of

drug effects in behaving animals. A critical issue concerns the conditions under

which the baseline (control) firing rate is measured. Spontaneous firing rates of

striatal neurons vary widely across conditions. They have been shown to be at

or near zero in numerous studies performed in anesthetized animals. In marked

contrast, we have found that the majority of neurons in the central and medial

portions of the striatum of the awake rat are spontaneously active during resting

behavior and show robust increases in overall firing rate during general,

whole-body movements such as locomotion (West et al. 1987). Therefore, to

measure the effects of a psychomotor stimulant on unit firing, it is inappropriate

to obtain a control firing rate during resting behavior. Control measures must be

taken during motor behaviors comparable to those observed following drug

administration (Trulson and Jacobs 1979).

Control firing rates were obtained during alternating 30-s periods of treadmill

locomotion versus rest. Animals were given a dose of dexamphetamine

59
(1.0 mg/kg intraperitoneally) that is known to produce locomotion in rats.

Neuronal activity was compared between control (saline) and experimental

epochs of treadmill locomotion. Across conditions, no changes were observed

in locomotor pace or posture.

We have studied the effects of amphetamine on both types of striatal unit

described above, i.e., those related to specific body parts and those related to

general body movement. Medial striatal units related to general movement

uniformly showed increases (range, 5 percent to 300 percent) in

locomotor-related firing 5 to 30 min following amphetamine injection (compared

with locomotion following saline injection). Firing recovered after 60 to 120 min;

that is, units fired faster after drug injection than they did during the same

behavior before injection.

Amphetamine also enhanced the timelocked firing of lateral striatal units related

to specific movements (Peoples et al. 1991). A forelimb unit showed a greater

likelihood of firing and a shorter latency to respond to footfall after amphetamine

injection, although no drug-induced changes were observed in the rate or length

of individual strides. The movement-related discharges of two units timelocked

to stereotyped, vertical head movement were increased 10-fold after

amphetamine injections, with no measurable change in the amplitude or

velocity of head movement. The time course of increased unit firing coincided

with an increased frequency of stereotyped head movements.

These data suggest a potential correlation between the level of striatal firing

and the frequency of a stereotyped movement during psychomotor stimulation.

One possible interpretation is that the increased firing represented enhanced

responsiveness of striatal neurons to cortical or thalmic inputs carrying

sensorimotor information. This could involve facilitation by amphetamine of DA

transmission at nigrostriatal synapses, which appears to be a critical substrate

mediating the stimulant effects of amphetamine on the motor system. In any

case, our data indicate that psychomotor stimulant activation of motor output

may be linked to an increased excitability of striatal neurons during the

processing of sensorimotor information.

Use of Low Stimulant Doses To Minimize Confounds Resulting From

Motor-Related Neural Firing

The response of striatal neurons to the psychomotor stimulant cocaine has also

been examined by means of a different strategy to control for possible

60
motor-related neural activity. In these studies, a low dose (1.0 mg/kg

intraperitoneally) of cocaine was used that did not produce any observable

changes in motor behavior. Thus, following drug injection, animals walked

normally during treadmill epochs and rested normally during intervening 30-s

rest periods. The result of these experiments was that drug-related changes in

neuronal firing were observed only during locomotor periods, but not during

rest. In every case, units showed increased firing rates (compared with

locomotion following saline injection) that recovered within 60 to 120 min

(Shimizu et al. 1987).

In view of cocaine’s mechanism of blocking DA reuptake (Heikkila et al. 1975;

Ritz et al. 1987), an interesting interpretation of these findings is that the

increases were selective for locomotion because DA release was greater during

this behavior than during rest. Indeed, there is substantial literature showing

correlations between dopaminergic transmission and movement (Ungerstedt et

al. 1977) (as well as DA antagonism and poverty of movement). It follows that

the present locomotor-related increases in striatal firing rate may have been

mediated, in part, by increased striatal DA release. These data provide further

support for the idea-derived from the amphetamine studies described-that

DA agonists may increase striatal responsiveness to inputs related to

movement. The cocaine study further emphasizes the importance of using a

context that allows motor behavior: cocaine’s effects on striatal firing would not

have been observed if the animal had remained at rest.

Microiontophoretic Application of Drugs Directly Onto Recorded Neurons

A definitive means of preventing drug-induced behavioral changes that might

alter neuronal activity independent of direct drug action is the iontophoretic

application of drugs directly onto recorded neurons (West and Woodward 1984;

West et al. 1986). Preliminary studies using this technique have further

implicated striatal DA release in locomotor-related increases in striatal firing. Of

sixty striatal units studied, direct iontophoretic application of DA excited

50 percent and inhibited 23 percent of the units. Of the units that showed

increased activity during locomotion, 80 percent were also excited by DA.

Furthermore, iontophoretically applied trifluoperazine (DA receptor blocker)

attenuated the increased striatal firing correlated with locomotion (Shimizu et al.

1987). These findings suggest that DA participates in neuronal processing in

the striatum during locomotion and that its effect in freely moving rats can

elevate striatal firing, either through direct or modulatory actions (Woodward et

al. 1979).

61
Moreover, the studies illustrate certain significant features of the iontophoretic

method in awake animals. The method not only eliminates changes associated

with systemic drug administration (e.g., changes in behavior and in multiple

neurotransmitter systems) but also, by means of systematic manipulations of

behavioral variables, makes it possible to use selective agonists and

antagonists to relate a particular transmitter to a particular behavior.

INTERPRETATION OF STRIATAL RESULTS

Despite the variability that is presumably inherent in studying drug effects on

neuronal activity in awake animals, results have shown a reasonable degree of

uniformity (most likely attributable to efforts to control behavioral variables) in

suggesting that striatal firing rates are enhanced by DA and related compounds.

The findings are consistent with the hypothesis that DA agonists may modulate

the responsiveness of striatal neurons to synaptic inputs carrying sensorimotor

information (Freund et al. 1984). The increased excitability of striatal neurons

appears to be linked in some way, perhaps causally, to stimulation of motor

output by psychomotor stimulants.

ACCUMBENS UNIT RESPONSES TO SELF-ADMINISTERED COCAINE

Psychomotor stimulants are potent reinforcers in drug self-administration

paradigms (Aigner and Balster 1978). Stimulants both activate motor behavior

and reinforce drug-seeking behaviors; some combination of these properties

apparently underlies the abuse potential of these drugs (Wise and Bozarth

1987). In order to study the as yet unknown neuronal mechanism or

mechanisms involved in psychomotor stimulant reinforcement, methods for

intravenous (IV) self-administration of cocaine have recently been combined

with chronic electrophysiological techniques. The focus of initial studies has

been on the nucleus accumbens septi (NAS), which is strongly implicated in the

neural circuitry that mediates psychomotor stimulant self-administration

(Dworkin and Smith 1988; Koob and Hubner 1988). The approach used was an

extension of the one used in the above-mentioned studies of stimulant effects

on striatal activity. The activity of accumbens cells in response to cocaine was

interpreted in light of processing involved in producing drug-seeking behavior

versus “nonspecific” motor behaviors. A detailed strategy was used to isolate

movement-related variables that may covary with unit activity in order to

evaluate the extent to which they contribute to the overall variance in neuronal

activity during self-administration.

62
Four animals were prepared with a chronic catheter implanted in the jugular

vein for drug infusion, as well as recording electrodes (microwires 25 to 60 μm

in diameter) in the NAS for recording single-neuron activity during behavior. A

bipolar electrode implanted in the ipsilateral fimbria was used to stimulate

subicular afferents and activate recorded neurons (n = 6) at a monosynaptic

latency (5 to 10 ms) in order to identify them as NAS neurons (DeFrance et al.

1985a). In the home cage, animals were infused with heparinized saline every

2 hr to maintain the patency of the catheter. One week was allowed for recovery

from surgery. Animals were placed in the experimental chamber daily and

connected to a harness that led to a fluid and electronic swivel. Baseline firing

rates were recorded during resting behavior and during treadmill locomotion to

obtain a measure of each unit’s general correlation with movement. The lever

for self-administering cocaine was then inserted into the chamber.

Before and after drug administration, behavior was videotaped by a

computerized process in which each video frame (30 frames per second) as

well as each unit discharge was time stamped. In subsequent analysis,

individual frame numbers corresponding to a repeated movement (e.g. rearing,

turning, nose poking, head bobbing) were entered into the computer as nodes

around which perievent histograms were constructed, depicting neural activity

timelocked to each particular behavior (Carelli and West 1991).

Figure 1 shows a graph of unit firing rates as a function of behavior and initial

cocaine infusions. During the first and second self-administration training

sessions of rat 1, the firing rates of four NAS neurons were recorded during

(1) a 3-to 4-min period in which the subject rested, i.e., sat in place; (2) a 2-min

period of treadmill locomotion; and (3) a 6-to 10-min period in which the rat

roamed the chamber freely and received the first three infusions (0.2 mL of

1.65 mg/mL [0.33 mg] cocaine per infusion). These infusions were administered

as part of the shaping procedure. All units showed small increases in firing

during locomotion (compared with rest) and then further increases in response

to initial infusions of cocaine. These results suggest that the increased firing

following cocaine infusion was not simply related to drug-induced motor activity.

Figure 2 shows the results of the entire second cocaine self-administration

training session (2.5 hr) for the same rat. The following dependent measures

are plotted as a function of 15-min blocks: (1) cumulative number of IV infusions

(top left panel); (2) number of lever presses (top left panel); (3) number of nose

pokes into any of the four corners of the operant chamber (bottom left panel);

(4) number of 180” turns made by the animal (bottom left panel); and (5) mean

63
FIGURE 1. Firing rates (mean Hz + SEM) of four accumbens units during rest

and locomotion and following the first three infusions of cocaine.

See text for details.

64
FIGURE 2. Motor behaviors and accumbens firing rates plotted as a function of 75-min blocks during a 2.5-hr

session of cocaine self-administration. Top left panel: Cumulative infusions, white squares;

number of lever presses, black squares. Bottom left panel: Number of nose pokes, white squares;

number of 180° turns, black squares. Right panels: Mean firing rates (Hz) of two accumbens

neurons recorded simultaneously. Arrows indicate onset of lever presses made by animal. Firing

rates of these neurons during rest (R) and locomotion (L) and following the first three cocaine

infusions (C) are taken from figure 1. Dashed vertical lines indicate time when saline replaced

cocaine in infusion syringe.
firing rate recorded from each of two nucleus accumbens neurons recorded

simultaneously (right panels). Note that during the first six 15-min blocks, each

lever press was followed by an infusion of cocaine; during the last four 15-min

blocks, each lever press was followed by a 0.2-mL infusion of isotonic saline

(extinction).

The shaping procedure ended after three 15-min blocks and 13 infusions. After

that point (arrows), the animal lever-pressed at a steady rate ranging from 0.25

to 0.5 presses per minute. Thus, the cumulative number of infusions increased

linearly to 30. The stimulant effects on behavior were exhibited as increases in

both nose pokes and locomotion back and forth across the chamber (i.e., 180°

turns). Both behaviors, however, exhibited inverted U-shaped curves, reflecting

the fact that the animal became ataxic during the sixth 15-min block (cumulative

dose = 29.7 mg/kg cocaine).

In the right panels of figure 2, unit firing rates from figure 1 are inserted for

reference. During the first 15-min block, firing rates were elevated after the first

three cocaine infusions, relative to rates observed during rest or locomotion.

Firing rates of both units decreased throughout the remainder of the period of

cocaine availability. Note that both units showed transient increases during the

top left block in which the animal lever-pressed (arrows). Within an hour after

saline replaced cocaine in the infusion syringe, both units recovered to firing

rates observed at the beginning of the session.

Using the data from figure 2, Pearson Product Moment Correlations were

calculated between the mean firing rate of each unit and the (1) cumulative

number of cocaine infusions, (2) number of nose pokes, (3) number of 180”

turns, and (4) number of lever presses (table 1).

TABLE 1. Pearson Product Moment Correlations from data in figure 2

Mean firing rate

Measure Unit 1 Unit 2

Cumulative cocaine infusions -.847* -.793*

Nose pokes -.500 -.076

180° turns -.341 -.090

NOTE: Correlations between mean firing rate and cumulative number of cocaine infusions were

calculated across the period of cocaine availability; other correlations were calculated across all

10 blocks of 15 min.

66
Correlational analysis revealed that the firing rate of each accumbens unit

correlated significantly with the cumulative number of cocaine infusions but not

with any recorded motor behavior. That is, relative to elevated firing rates after

the first three cocaine infusions, continued cocaine infusions significantly

reduced the firing rates of both units tested to precocaine levels. Thus,

preliminary findings suggest that cocaine may initially excite accumbens

neurons, whereas subsequent exposure to the drug appears to produce no

further activation but might produce a depression of firing or a return to

precocaine levels. Furthermore, the lack of correlation with any measure of

motor behavior suggests that these results were not due to unit correlations

with drug-induced motor activity. Finally, perievent time histograms depicting

unit activity with respect to lever pressing have thus far shown no patterns

indicative of neuronal firing related to drug-seeking behavior.

CONCLUSION

To characterize the effects of a particular drug on neuronal activity in an awake

animal, it is essential to use appropriate behavioral controls. Doing so involves

both accounting for the behavioral effects of the drug and characterizing each

unit’s behavioral correlates. Although advances have been made in

understanding the functions of striatal neurons during behavior, little is known

about the behavioral correlates of accumbens neurons. Initial studies have

demonstrated emphatically the need for a better understanding in this regard.

Thus far, the most reliable correlation observed is that 90 percent of accumbens

units (n = 224) increase firing rate during general movement, such as

locomotion, (relative to rest). Correlations with more specific behaviors have not

yet been observed. Because cocaine produces motor simulation, accumbens

activity in response to cocaine could potentially be related to motor processing.

In fact, substantial evidence suggests that stimulant activation of motor output is

mediated by accumbens and striatal neurons, neural substrates apparently

directly influenced by cocaine’s effect at dopaminergic synapses (Gershanik et

al. 1983). Therefore, the responses of these cells to cocaine must be

interpreted in light of sensorimotor processing involved in producing

drug-seeking behavior. It is not yet clear to what extent explanations in these

terms will contribute to an understanding of the neural mechanisms mediating

reinforcement. The approach described in this chapter, and the continued

refinement of it, might be suitable for addressing such issues and for making

significant advances in understanding the neurobiological effects of

psychomotor stimulants.

67
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ACKNOWLEDGMENTS

This work was supported in part by U.S. Public Health Service grants DA 04551

and DA 06886 to Mark West and DA 03628 to Steven Dworkin from the

National Institute on Drug Abuse.

AUTHORS

Mark O. West. Ph.D.

Laura L. Peoples, Ph.D.

Martin Wolske

Department of Psychology

70
Rutgers University

New Brunswick, NJ 08903

Steven I. Dworkin, Ph.D.

Department of Physiology and Pharmacology

Bowman Gray School of Medicine

300 South Hawthorne Road

Winston-Salem, NC 27103

Address correspondence to

Mark O. West

Department of Psychology

Rutgers University, Busch Campus

New Brunswick, NJ 08903

71

Brain-Stimulation Reward: A Model for the

Study of the Rewarding Effects of Abused

Drugs

Conan Kornetsky and George Bain

During the past decade, there has been increased acceptance of the

hypothesis that the reinforcing effects of abused substances are related more to

their ability to enhance neuronal activity of the brain reward systems than to

ameliorate withdrawal symptoms or reverse some underlying psychological or

somatic pathology. Much of the change in attitude about bases of the

reinforcing effects of abused substances has been shaped by the experimental

work of investigators who, during the past 18 years, have studied the action of

these drugs on behavior maintained by rewarding intracranial stimulation. Most

abused substances increase the sensitivity of animals to (i.e., increase the

rewarding value of) this intracranial stimulation. This model of drug-induced

euphoria is not only predictive of abuse liability, but it also has the further

advantage of lending itself to the study of the neuronal events that underlie the

reinforcing effects of abused substances. James Olds (1977, p. 4), in

commenting about the discovery of self-stimulation, described it as “a new

window on the brain.”

The phenomenon that animals would work to obtain intracranial stimulation was

first described by Olds and Milner (1954), who stated, “It is clear that electrical

stimulation in certain parts of the brain, particularly the septal area, produced

acquisition and extinction curves which compare favorably with those produced

by conventional primary reward.”

An early report of the effects of pentobarbital and d-amphetamine on

brain-stimulation reward (BSR) (Killam et al. 1957) suggested that the method

might be a useful technique for studying the rewarding effects of abused

substances. With few exceptions, however-notably a series of experiments of

the effects of d-amphetamine on BSR by Stein and associates (Stein and Ray

73
1960; Stein and Seifter 1961; Stein 1962a, 1962b; Stein 1964; Stein and Wise

1969)—BSR was not considered a useful mode for the study of abused drugs.

Among the reasons for this lack of interest was that the effects of drugs on BSR

were not significantly different from those of natural reinforcers such as food.

Starting, however, in the early to mid-1970s approximately 20 years after the

original report by Olds and Milner, there was renewed interest in the procedure

because of the potential it offered for understanding the neurobehavioral bases

of drug-induced euphoria, reward, and subsequent reinforcement.

METHODOLOGY

The experimental paradigms of BSR studies and behavioral studies that use

other reinforcers such as food are similar. Most investigators use an operant

procedure employing a lever manipulandum in one wall of the experimental

chamber. The critical factor in selecting a manipulandum is that the response

required of the animal should not be so great that completing a response is

difficult for the animal or so easy that any random movement would result in the

delivery of a rewarding stimulation.

Electrode Implantation and Stimulus Parameters

Both monopolar and bipolar stereotaxically implanted electrodes are used in

brain-stimulation studies. In the monopolar configuration a single wire is

implanted and a screw anchored to the skull serves as the indifferent electrode

or collector. This monopolar arrangement leads to a greater spread of current

and has been reported to require higher stimulus intensities to obtain response

rates similar to those found with bipolar electrodes (Valenstein and Beer 1961).

These differences in effective intensities were attributed to the alternation of the

cathode and anode during biphasic stimulation between the neural site at the tip

of the electrode and the indifferent surface electrode. The bipolar configuration

consists of two straight wires cemented together or two wires twisted together

and implanted so that their uninsulated tips alternate as cathode and anode

during the stimulation to the neural site.

The parameters of the actual electrical stimulation employed by the several

groups who are conducting brain-stimulation studies vary to some degree.

Some investigators employ brief trains of constant-current, cathodal,

monophasic, rectangular pulses and vary the frequency (e.g., Fouriezos and

Nawiesniak 1987; West and Wise 1988). In many laboratories the current

intensity is varied and trains of either sine waves or rectangular pulses of

74
alternating polarity with each positive or negative rectangular pulse lasting

approximately 0.2 ms are typically used. The train durations generally are

0.5 ss; the fixed frequency of pulse pairs is usually between 60 and 160 Hz.

Current intensities for effective stimulation may vary from approximately 50 to

300 μA; the lower current intensities are characteristic of bipolar electrodes and

the higher intensities are more characteristic of monopolar configurations. The

lower the frequency used for stimulation, the greater the amount of current

needed before for the stimulus reaches a rewarding level; thus the

effectiveness of the stimulus is probably a function of the total charge

(coulombs), which is resultant of frequency, current intensity, and stimulus

duration.

Figure 1 shows an example of the relationship between the probit of the percent

of responding and the current intensity at two different frequencies in the same

animal. In this figure the curve for the lower frequency is to the right of the curve

for the higher frequency, indicating that at the same intensity the lower

FIGURE 1. Relationship between probit of percent response and current

intensity. Two frequencies (160 Hz and 180 Hz) are shown for a

single subject on two different days, respective/y. A. Probit of

percent response versus intensity μA) in log representation. B.

Probit of percent response for the same two frequencies versus

electrical charge (picocoulombs) in log representation.

75
frequency is less rewarding. However, when the same data from the two

experiments are presented as a function of the electrical charge (coulombs), the

curves are almost superimposed. Thus, the effective stimulus is the power

(electrical charge) of the stimulus and not the components of the electrical

charge.

Lever-Pressing-Rate Methods

The most commonly used BSR method has been to use the simple rate of lever

pressing (i.e., rate of response) as the dependent variable. For the most part,

investigators have used continuous reinforcement or low fixed-ratio

reinforcement schedules. When rate of response is used, there is a direct

relationship between the intensity of stimulation and the rate of response. This

increase in response rate will asymptote at some level and increasing the

intensity further will result in a decrease in rate (figure 2). The example shown is

a single animal tested on two successive days for 5 minutes at each indicated

intensity. On day 1, the order of intensities was increasing; on day 2, the animal

was started at the previous day’s ending intensity, and a descending order of

intensities was used.

In the simplest paradigm, a level of intensity is selected that generally allows for

a high rate of responding. An increase in rate of response after a drug is

administered is usually interpreted as the result of the animal becoming more

sensitive to the stimulation (the stimulation is more rewarding) whereas a

decrease in responding is interpreted as the result of the animal becoming less

sensitive to the reinforcing stimulus (the stimulation is less rewarding). The

major problem in using rate of response as the dependent variable in drug or

lesion studies employing BSR is the lack of specificity of the mechanisms

involved. An increase in rate of response may be the result of a general level of

psychomotor stimulation, and a decrease may be the direct result of treatment

on the ability of the animal to press a lever at the high rates usually obtained

when the reinforcer is BSR. In an experiment in which each animal was

prepared with two stimulating electrodes bilaterally implanted and each of two

levers was wired to one of the electrodes, Hodos and Valenstein (1962) found

no relationship between the rate of response and the preference for a

stimulation site.

Despite the problems of interpretation when the dependent variable is rate of

response, it is still extensively used to determine the effects of drugs on the

reinforcing value of the stimulation. Trying to measure changes in the reward

76
FIGURE 2. An example of a rate-intensity function for a single animal. The

solid line reflects rates of response on day 1 when the order of

reinforcement current was ascending. The dashed line reflects

rates of response on day 2 when descending order of

reinforcement current was used. Note that at the higher intensities

the rate of response was less than maximal. (From Payton 7986.)

value of the stimulation more directly, a number of investigators have used a

range of stimulus intensities. Using this paradigm, investigators look for shifts in

the intensity-rate function after administering of drugs.

An example of testing at a full range of intensities (rate-intensity method) was

the method used by Leith and Barrett (1976), who demonstrated tolerance to

the facilitatory effects of d-amphetamine on BSR as well as a poststimulation

depression in response rate (figure 3). The current intensity was adjusted to an

intensity that resulted in maximal response rates for each animal. Then, within

each session, the intensity was attenuated every 5 s by one-twentieth of the

77
FIGURE 3. Effects of d-amphetamine (0.5 mg/kg) on the rate of response for

BSR before and after chronic (2 wk) treatment with high doses

(20 mg/kg per day) of the drug in a single subject. The current

intensity is decreased by one-twentieth of the initial value every

5 s. After the initial 0,5-mg/kg dose of d-amphetamine, a clear

shift of the response-intensity curve to the right of saline is shown

for the subject prior to chronic administration. A similar shift of

lesser magnitude is shown after chronic d-amphetamine

administration of the same dose. (From Leith and Barrett 1976.)

78
starting intensity until 15 levels of stimulation had been tested. At this point, the

intensity was reset to the starting level, and the procedure was repeated

throughout the length of the 60-min session. Figure 3 illustrates the effects of

0.5 mg/kg of damphetamine in a single animal. Facilitation in response rate

after d-amphetamine is clear from the shift of the response intensity curve to the

right of the saline curve. After 2 weeks of daily 20-mg/kg doses of

d-amphetamine, animals were again tested after 0.5 mg/kg of d-amphetamine

or saline. As shown, there was not only evidence of tolerance to

d-amphetamine, but also a depression in the rate of responding even after

saline. This depression is indicated by the shifi of the curve to the left. Other

investigators (e.g., West and Wise 1988) have used multiple intensities in the

study of drug effects. In these studies, rate of response is expressed as a

function of frequency (rate-frequency function). A shift in the rate-frequency

function is called a phase shift; a shift to the right after the drug is administered

is interpreted as a decrease in the reward value; a shift to the left is interpreted

as an increase in reward value. Failure to reach the same asymptotic rate of

response after the drug is obtained following saline administration is interpreted

as evidence that the drug causes a performance deficit. The phase shift method

has been used in which the dependent variable was speed of running combined

with a lever press (Gallistel et al. 1974). Figure 4 illustrates the effects of

various doses of pimozide using this technique (Stellar et al. 1983). In this

experiment the animal was required to negotiate a one meter long alley to a

goal box where a lever was available. A single lever press resulted in the

delivery of the intacranial stimulation.

Because the rate-intensity and rate-frequency methods also use rate of

response or running speed as an integral part of the procedure, the effects of

drugs on the reward value and on performance might still be confounded. And

because threshold and asymptotic values are at the two extremes of the curve

(the asymptotic low and high rate points) there is greater chance for error than

when the threshold is in the mid-range of the rate-frequency or current curve.

Alternative Operant Manipulandum

To avoid the possible confounding effects of drugs on motor performance with

the reinforcement value of the stimulation, many investigators have used an

alternative manipulandum (Liebman 1983). Despite the simplicity of the lever

press, it is not a response that is programmed into the repertoire of the rat.

Schiff (1976) clearly demonstrated that not all lever pressing is equivalent.

79
FIGURE 4. Shift in the baseline running speed (cm/set) (reward summation

functions) for a single subject after 0.125, 0.25, and 0.5 mg/kg of

pimozide. Then a 0.25-mg/kg dose produced a clear shift of the

curve to the right without a significant decrement of asymptotic

running speed (as indicated along the ordinant). The 0.5-mg/kg

dose, however, not only shifted the curve but also decreased the

asymptotic running speed. N = the number of current pulses per

burst. (From Stellar et al. 1983.)

Animals with caudate lesions had greater decreases in rate of response when

the lever was 64 mm versus 10 mm above the chamber floor.

An operant response that is indigenous to the rat and requires little motor effort

is the nose poke. Rats will readily stick their noses into small apertures in the

80
floor or wall of an experimental chamber. By use of a photocell beam in the

aperture, whose interruption results in the delivery of the stimulus, this natural

and easily made response can be exploited. This manipulandum has been used

in both lesion and pharmacologic studies (Gerhardt and Liebman 1981;

Ettenberg et al. 1979; Simon et al. 1979). From these experiments, it became

clear that if the performance demands of a manipulandum become too great,

then changes in response rate might well be a function of the effects of the

lesions or drugs on the motor performance of the animal and of any change in

the reward value of the stimulation.

Rate-Independent Instrumental Response Methods

Despite attempts to circumvent the motor response effects without

circumventing the reward value of the stimulation, Liebman (1983) argued that

using other manipulanda did not solve the problem, because the effects on

reward might be confounded with the operant rate per se. In an attempt to

separate performance effects from rewarding effects, a number of investigators

have employed rate-independent procedures. Valenstein and Meyers (1964)

used a “place preference” procedure; Gallistel and colleagues (1974) used a

procedure that incorporated running speed to a goal box, where a lever press

resulted in delivery of the stimulation (see figure 4). Other investigators have

employed a shuttle box procedure (Levitt et al. 1977; Atrens et al. 1976;

Liebman et al. 1982; Liebman 1983; Wauquier et al. 1983). Many of these

investigators also have criticized these procedures because of the difficulty in

interpreting the specificity of the drug effects and because these methods are

more cumbersome than using operant rate as the dependent variable. A further

problem (rarely mentioned when there is a high density of stimulation, as when

rate is the dependent variable or is an integral part of the procedure) is the

marked oral stereotypy seen in the animals. A great deal of mouthing behavior

is observed; subjects often press the lever not only with a paw but also with the

mouth simultaneously. Similar behavior is seen when animals receive direct

injections of dopamine agonists into the ventral lateral striatum (Kelley 1988).

This indication of marked dopamine nigrostriatal stimulation suggests that the

effects of drugs on BSR that are seen with using rate-dependent procedures

might be combined effects of the action on reward systems and on the

nigrostriatal motor system.

81
Threshold Measures

Very early in the investigations of BSR effects of drugs, the term threshold

change often was used to define the effect of a drug. In these studies, threshold

was defined as the intensity that caused a particular defined rate of responding

or the lowest intensity at which any responding took place. Often, when a range

of intensities are used, the threshold is defined as the intensity at which the

animal responds at 50 percent of the maximum response rate. This definition is

similar to what has been referred to as the “locus of rise” by some investigators

(Edmonds and Gallistel 1977). Despite the usefulness of these procedures,

they are by definition rate dependent. To circumvent this problem some

investigators have developed alternative procedures for determining threshold.

Two-Lever Reset Titration Method. The first attempt at a threshold

procedure that was independent of response rate was the two-lever reset

method (Stein and Ray 1960). This procedure makes use of a chamber with a

two-lever manipulandum. Pressing the left lever, for example, results in the

delivery of a suprathreshold stimulation. Each subsequent response (or a fixed

number of responses) on this lever results not only in the delivery of a

rewarding stimulation but also in the attenuating of the stimulus intensity. A

response on the other lever resets the stimulus level to its original intensity. The

average intensity at which the animal resets the stimulus intensity is defined as

the threshold.

Although this procedure has a number of advantages over a simple rate

procedure, it does have some problems in interpretation. Fouriezos and

Nawiesniak (1987) have pointed out that the higher the starting level, the higher

the reset value. Among other criticisms is that there may be habituation to the

stimulation; that is, the animals may reset at higher levels as a function of

experience with the procedure. Also, the differences observed after drug may

be confounded with the rate of response. A major advantage of the procedure is

the relative ease in the control and delivery of stimulation. Among the

investigators who recently have employed this procedure in the study of drug

effects are Gardner et al. (1988) and Van Wolfswinkel and Van Ree (1985).

Psychophysical Discrete Trial Method. In attempting to determine the

effects of drugs on BSR without the contamination of motor effects and

independent from rate of response, we have developed a psychophysical

discrete trial method for determining the threshold for rewarding intracranial

stimulation (Marcus and Kornetsky 1974; Esposito and Kornetsky 1977;

82
Kornetsky 1985). This procedure consists of discrete trials, each requiring a

single response by the subject to receive the stimulation. Figure 5 shows a

cartoon of an animal in the experimental chamber and a schematic

representation of the experimental procedure.

A trial begins with an experimenter-delivered (noncontingent) stimulation (S1),

which signals the animal that the appropriate response (turning a wheel

manipulandum within 7.5 s) results in a second (contingent) stimulation (S2).

This contingent stimulation is identical to the noncontingent stimulation. If the

animal does not respond within the 7.5-s available response time, the trial is

terminated; the only consequence is that the subject does not receive the

second stimulation. In most of our experiments the inter-trial interval varies

randomly between 7.5 s and 22.5 s (mean, 15 s). To preclude responding

during the intertrial interval, any response during the interval postpones the

onset of the next trial for 15 s. In the final form of the procedure the current

intensities are varied in a stepwise fashion of descending and ascending order,

and the S2 intensity is exactly the same as the S1 intensity.

An example of the methods we have used for collecting the data and

determining the threshold is given in figure 6. The simplest method we have

used to determine the threshold is to average the column thresholds, which are

arbitrarily defined as the midpoint intensity between the two lowest steps in

which there are three contingent responses and fewer than three contingent

responses, respectively. More recently, we have used a more exact estimate of

the threshold, which allows the slope of the response-intensity curve to be

determined. In this method, the percentage of times the animal responded at

each intensity is determined. When plotted against the intensity of stimulation,

this percentage is expressed as a sigmoid curve (figure 7A). When percentage

is converted to probit and plotted against intensity expressed on a log scale

(figure 7B), the data fit a straight line. With this procedure the threshold is the

intensity of stimulation corresponding to probit 5. If the slope of the regression

line not significantly different from zero, the animal is not under stimulus control.

Using the psychophysical discrete trial method, Markou and colleagues (1989)

demonstrated the specificity of this threshold procedure and the independence

of the threshold measure from motor effects. In their experiment, they found

that increasing the force necessary to turn the wheel manipulandum had no

effect on the threshold but did increase the latency of response. Additional

evidence that the discrete trial procedure is independent from motor effects is

the failure of rate of response for rewarding brain stimulation to correlate with

83
FIGURE 5. Schematic of the two possible outcomes during a sing/e trial of the

psychophysical discrete trial method. In example I, the rat is

presented a noncontingent stimulus (S1), affer which if does not

respond during the indicated 7.5-s response interval. Failure to

respond has no scheduled consequences, and a new trial begins

affer a 15-s interval. In example II, the rat is presenfed a

noncontingent stimulus, responds by turning the wheel

manipulandum within 7.5 s, and receives a second stimulus (S2)

of the same intensity. The 7.5- by 75-cm cylindrical

manipulandum is mounted in one wall of a plastic chamber 25- by

25- by 35-cm. A four-cog cam attached to one end of the

manipulandum triggers a microswifch for each quarter-rotation.

84
FIGURE 6. Shown are the number of responses made at each intensify by a

single animal in an experiment in which the step size was 10 μA

and five trials were given at each intensity before moving to the

next intensify. In this experiment, the stimulation started with five

trials at 90 μA, followed by five trials at each of the lower

intensities until the animal made fewer than two responses out of

the five trials at two consecutive intensities (in this case, 50 μA

and 40 μA). These are indicated by a minus sign. The next five

trials were started at one step size lower (30 μA), and the

ascending intensity trials were begun, When three or more

responses out of the five trials were made at two consecutive

intensities, indicated by a plus sign, a descending series was

begun starting at one step size higher. This procedure was

repeated until four columns were completed. Percentage of

responses at each intensity were then computed and converted to

probif. Because a probif of 100 does not exist, the probit for

99 percent is used. These percentage and probif values are used

for computation of the power functions shown in figure 4.

Threshold can also be approximated by faking the midpoint in

each column between the step in which the animal made three or

more and two or fewer responses. The threshold for the session is

the mean of the column thresholds.

85
FIGURE 7. Power functions from data shown in figure 6. a. Percentage

response-intensity function in log representation.

b. Probif-intensity function in log representation.

the threshold obtained in the same group of animals (r= –.24, 31 df)

(Kornetsky 1985; Payton 1986).

EFFECTS OF DRUGS ON BSR

Using the psychophysical discrete trial method, we have found that all drugs

that lower the threshold for rewarding brain stimulation (table 1) are abused,

have the potential for abuse as determined by studies in human subjects, or will

be self-administered by animals. Although we have not been able to

demonstrate that 9-tetrahydrocannabinol (9-THC) or the barbiturates lower the

threshold for BSR, Gardner et al. (1988) have reported that by the

psychophysical discrete trial method 9-THC will lower the threshold when the

two-lever reset method is used, but only in the Lewis rat. Although results are

somewhat mixed, some investigators have found that barbiturates as well as

the benzodiazepines (Liebman 1985) will increase response rates at low doses.

The rate-independent threshold method often gives results similar to those in

the rate-response method. There are, however, some marked differences,

especially with opiate drugs (Kornetsky and Bain 1990). Most investigators

have failed to find clear evidence that BSR will be facilitated the first time the

86
TABLE 1. Effects of various drugs on brain-stimulation reward threshold

Lowers No change Raises

Morphine

6-acetylmorphine

Buprenorphine

Nalbuphinea

Methamphetamine

Amfonelic acid

Tripelennamineb

MDMA

Heroin

Cocaine

Pentazocinea

d-amphetamine

PCP

Bromocriptine

Nicotine

Ethanolc

THC

LSD

Naloxone

Naltrexone

Cyclazocine

EKC

Nisoxetine

Apomorphine

U50,488

Pentobarbital

Procaine

Haloperidol

Pimozide

Chlorpromazine

lmipramine

Atropine

Scopolamine

NOTE: The drugs listed are only those that have been tested in our laboratory using the

rate-independent threshold procedure. Some of those that cause no change, especially

pentobarbital, might lower the threshold under drug self-administration conditions, as observed with

ethanol.

aEspecially in combination with tripelennamine.

bEspecially in combination with pentazocine or nalbuphine.

cOnly under conditions of self-administration (Moolten and Kometsky 1990).

animal is given morphine (Reid 1987). This has not been the case using the

psychophysical discrete trial procedure. Figure 8 shows the threshold-lowering

effects of the first exposure to 4.0 mg/kg of morphine (Izenwasser and

Kornetsky 1987). We have consistently obtained threshold lowering at the first

exposure to morphine (e.g., Marcus and Kornetsky 1974). The major difference

between our finding and those of others in regard to the first exposure to

morphine is probably related to the independence of the rate-independent

threshold method of motor effects. The motor-depressant effects of morphine

are probably paramount the first few times a rat receives morphine; this effect

translates into decreases in rate of response. Changes in rate of response after

drug are, as in most operant procedures, subject to rate dependency. Motor

effects of a drug are often reflected in changes in rate of response in animals

87
FIGURE 8. Mean z scores of first-time administration of 4 mg/kg of morphine

to five rats. The shaded area indicates the 95 percent confidence

limits. (Adapted from lzenwasser and Kornetsky 1987.)

that are working for natural reinforcers such as food, so that increases in

response rate may be due to psychomotor stimulation-a phenomenon often

seen after low doses of sedative-hypnotics-and decreases in response rate

may reflect motor impairment.

The results we have obtained-as well as those of other investigators using

different methods-indicate that BSR is a powerful model for studying of the

rewarding effects of abused substances. Another major use for the technique is

in studying the neurobehavioral bases of the reinforcing effects of such drugs.

Although the model is not homologous with drug use in humans, as the drug

self-administration model is, it has the advantage of being a direct measure of

the action of drugs on the brain-reward system. The understanding of the

88
anatomy and the neurochemistry of this system and of how drugs change this

system tells us much about the bases for the rewarding effects of the abused

substances. For a review of the neurochemical bases of the reward system and

the effect of drugs on this system see Phillips and Fibiger (1989).

SUMMARY

The robustness of the findings of the BSR effects of abused substances

indicates that almost any technique used to measure these effects will be

useful. The simplest procedure, however, of selecting a single intensity of

stimulation and determining the effects of drugs on the rate of response for that

selected intensity is fraught with difficulty in interpretation. More than 25 years

ago the interpretation of changes in response rate as a reflection of changes in

the reward value of the stimulation was challenged (Hodos and Valenstein

1962). Today it is rare to see a single stimulation intensity used in a published

manuscript. However, most studies still use procedures of which rate of

response is an integral part. Because animals will press a lever more than

80 times per minute and-on a continuous reinforcement schedule-receive as

many as 80 stimulations a minute, conclusions about the specificity of the

effects of drugs are difficult.

The results of BSR experiments on mechanisms of action of abused

substances clearly indicate that the reinforcing effect of most, if not all, such

substances is probably the result of activation of a reward system that

originates in the cell bodies of the ventral tegmental area and courses rostrally

to the limbic and frontal projection sites of the mesocortical system. Thus, we

believe, the technique has clearly fulfilled its promise as a “window on the brain”

(Olds 1977).

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ACKNOWLEDGMENTS

This work was supported in part by grant DA02326 from the National Institute

on Drug Abuse and Research Scientist Award DA00099 from the National

Institute on Drug Abuse to Conan Kornetsky.

AUTHORS

Conan Kornetsky, Ph.D.

George Bain, Ph.D.

Division of Psychiatry

Boston University School of Medicine

80 East Concord Street

Boston, MA 02118

93

New Neuroimaging Techniques for

Investigating of Brain-Behavior Relationships

William J. Jagust and Thomas F. Budinger

INTRODUCTION

Neuroscientists using brain-imaging techniques to study human behavior have

characteristically relied on methods that demonstrate anatomic structure to

understand the neural substrates of cognitive function. Inferences about the

relationship between structure and function are drawn by correlating of lesion

locations with their effects on behavior. Characteristic methods for

demonstrating lesion location have included neurological examination,

neuropathological study at autopsy, and anatomic imaging techniques such as

pneumoencephalography, angiography, and x ray computed tomography (CT).

Studies of this sort have formed the basis of much of our present-day

understanding of human cortical function and have provided important insights

into the cerebral organization of language, memory, attention, and emotional

processes in humans.

Within the past decade, new techniques for the imaging of brain function have

been developed. These techniques, positron emission tomography (PET) and

single photon emission computed tomography (SPECT), use radiation-detecting

instruments (tomographs) to provide three-dimensional images of the

distribution of injected radiotracers. By labeling a variety of molecules with

radionuclides, researchers can study a variety of physiological processes.

This chapter reviews the recent developments in brain imaging. In particular,

the use of functional imaging techniques to study a specific illness, Alzheimer’s

disease (AD), is discussed to demonstrate potential applications of these

methods. The use of magnetic resonance imaging (MRI) for studying the

relationship of brain structure to function is also discussed.

95
BASIC PRINCIPLES OF EMISSION TOMOGRAPHY

All emission tomographic techniques use an injected radioactive tracer that is

detected by a tomograph. The tracer consists of a molecule of interest, for

example, a metabolic substrate such as deoxyglucose, which is labeled with a

radioactive isotope, such as 11C or 18F, that can be detected by the tomograph.

The tomograph may use different principles to map accurately the distribution of

the tracer (depending to a great extent on the nature of the emitted radiation),

but the final result is a series of tomographic brain “slices” that provide

three-dimensional information about the distribution of the tracer. The ultimate

goal of these techniques is not simply the production of the image, but rather

the quantitation of physiological processes. Models that rely on a priori

information about the behavior of the tracer in vivo (such as the transport and

metabolism of the molecule), in conjunction with time-varying measurements of

the tracer in the circulation and the brain, are necessary for measuring these

physiologic processes quantitatively. Once such information is available, the

mathematics of the modeling is relatively straightforward, although assumptions

about the behavior of the tracer must be carefully evaluated in each

experimental situation.

The essential difference between PET and SPECT is in the types of radioactive

isotopes used to label the molecules of interest. PET uses isotopes in which the

decay process results in two high-energy photons that are always oriented 180”

opposite each other. SPECT uses isotopes that decay by emitting single

photons in random directions. Most of the PET isotopes have shorter half-lives

than the SPECT isotopes. These differences have profound consequences for

the radiochemical synthesis methods as well as for the nature of the

tomographs and measurements that can be made.

PET tomographs consist of a radial array of crystal detectors that are

electronically coupled to detect the simultaneous excitation of two opposing

crystals by the simultaneously emitted high-energy photons. Thus, the positron

emission can be localized to a line, and the reconstruction of an image from

these multiple projection lines results in a map of the distribution of the tracer.

The intrinsic property of electronic collimation leads to high sensitivity (generally

defined as counts per millicurie of activity per second) for detecting of

radionuclides. The consequence is better statistics describing tracer location

than SPECT provides, because PET resolution is several millimeters higher

than SPECT resolution. Also, the high sensitivity of PET translates into the

96
ability to acquire many counts in a short time and thus allows dynamic,

time-varying measurements to be made quickly.

In SPECT imaging, the tracer map is ascertained by using collimation to

localize the line along which a decay event occurred. Collimation results in

restricting of the field of view of each detector element. Consequently, the

decrease in sensitivity is greater with SPECT than with PET, which collimates

electronically. The decrease in sensitivity of SPECT scanners is greater as the

resolution is improved; the practical limitation is at resolutions less than about

10 mm. In addition, the problems of radiation scatter and attenuation by brain

and skull are more difficult to solve with SPECT imaging than with PET,

resulting in somewhat less accurate measurements of brain radioactivity. A

more detailed analysis of the physics and instrumentation of these two

techniques has been published by Budinger (1987).

The isotopes used in SPECT and PET obviously differ as well. These isotopes,

and some of the compounds of interest that have been labeled with them, are

shown in tables 1 and 2. The commonly used PET isotopes, particularly carbon

and oxygen, are natural constituents of organic molecules. Therefore, use of

these isotopes in synthesizing tracers frequently, preserves the biological

behavior of the molecule of interest. The use of 18F is also advantageous,

because its relatively longer half-life makes it easier to work with. The breadth

of possibilities for synthesizing tracers chemically is not as great for SPECT as

for PET, because the radionuclides used in are not generally natural

constituents of the biological molecules, e.g., iodine and technetium versus

carbon and oxygen. The result is that SPECT tracers for human brain studies

have been widely used only for measuring regional cerebral blood flow (rCBF).

Many potential SPECT tracers for studying the dopamine, serotonin, and

adrenergic systems are in various stages of development, however. New

tracers for both PET and SPECT are being developed in many laboratories; the

enterprise is important but time-consuming, because the in vivo behavior of a

tracer must be understood entirely before mathematical modeling and

quantitating of the physiological process of interest can be done.

PET and SPECT differ considerably in their applicability to neuroscience

research questions. The short half-life of many PET isotopes makes using them

simpler for repeated measurements and permits sequential studies to be made

in the same patient. Because the variability of PET data appears to increase

with the test-retest time period, studying the same subject at one sitting

minimizes the problem of differences in the subject’s physiological states at

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TABLE 1. Some PET tracers that have been used to study the human brain,

the radionuclides that have been used to label them, and the

physiological processes that they quantitate

Isotope Tracer Physiological process

18F

15O

11C

FDG (fluorodeoxyglucose)

Fluormethane

N-methyl spiperone

Fluoro-DOPA

Haloperidol

O2

H2O

CO2

CO2

Deoxyglucose

DMO

N-methyl spiperone

Carfentanil

Diprenorphine

68Ga

Leucine, methionine

Methyl-bromo-LSD

N-methyl nicotine

EDTA

Glucose metabolism

Blood flow

D-2 dopamine receptors

Dopamine uptake and metabolism

D-2 dopamine receptors

Oxygen metabolism

Blood flow

Blood flow

Blood volume

Glucose metabolism

Tissue pH

D-2 dopamine receptors

Mu-opiate receptors

Mu-, delta-, and kappa-opiate

receptors

Protein synthesis

S-2 serotonin receptors

Nicotinic receptors

Blood-brain barrier permeability

TABLE 2. Some SPECT tracers that have been used to study the human

brain, the radionuclides that have been used to label them, and

the physiological processes that they quantitate

Isotope Tracer Physiological process

123I

99mTc

IMP

HIPDM

QNB

IBZM

HM-PA0

ECD

Blood flow

Blood flow

Muscarinic cholinergic receptors

D2 dopamine receptors

Blood flow

Blood flow

98
different test times and decreases errors in repositioning the patient in the

scanner. The longer half-life of SPECT isotopes generally requires that

test-retest paradigms be performed on different days. Although many PET

studies can be done using the longer lived tracer 18F, which can be generated

by a regional cyclotron for delivery to several local PET centers, using the

shorter lived PET tracers such as 15O and 11C necessitates locating a cyclotron

very near the PET scanner; additional expense and technical support are

therefore involved. Improvements in the technology of compact cyclotrons

specifically designed to produce radionuclides of interest for PET studies will

lessen the technical support and space requirements for PET in the future.

Because of the nature of the quantitative studies usually carried out by PET

centers, only small numbers of subjects can practically be studied in a particular

investigative protocol. SPECT is less technically demanding and consequently

enables larger patient series to be studied. Tissue radioactivity can be

measured more accurately with PET than with SPECT, mainly because the

actual radioactivity-concentration measurement in brain structures requires an

instrument resolution smaller than the structures of interest. PET resolution is

close to the size of most small cerebral structures. A second advantage of PET

is the ease with which dynamic studies can be performed because of its high

sensitivity.

PET IN STUDIES OF ALZHEIMER’S DISEASE

Dementia is a progressive loss of cognitive abilities that interferes with function.

It is most commonly caused by AD, which accounts for between 50 percent and

70 percent of all cases of adult dementia in numerous series (Tomlinson et al.

1970). Multiple cerebral infarctions (multi-infarct dementia, or MID) accounts for

the second most common cause of dementia. Besides being a medical problem

of enormous social and economic importance (Katzman 1976), AD presents a

clinical problem as well, because no direct laboratory marker can confirm its

presence during life. Thus, AD is a diagnosis of exclusion, which can be arrived

at only after ruling out numerous other causes of dementia (some of which are

curable) and can be confirmed only at autopsy. Although some aspects of AD

have been reproduced in a variety of laboratory animals, there is no complete

animal model of the illness; therefore, studies of the pathophysiology of the

disease must be performed in humans.

Anatomic imaging studies have not generally been helpful in diagnosing AD;

results are nonspecific. CT has confirmed pathological findings that gross

atrophy is seen in dementia, with smaller brain size and enlarging sulci and

99
ventricles (Zatz et al. 1982). This atrophy is not specific for AD, because it

occurs in other dementias as well as in normal aging. Although studies that use

volumetric CT measurements generally show better separation of AD and

control groups than linear measurements do, studies that use volumetric CT

measurements usually find overlap among groups on these measures and have

not been useful diagnostic criteria for individual cases (McGeer 1986). As a

research tool, CT has demonstrated correlations between neuropsychological

measures of dementia severity and brain atrophy (Chawluk et al. 1987) and has

shown differences in the rate of progression of atrophy between AD and

age-matched control subjects (Luxenberg et al. 1987). At present, however, CT

scanning has been used clinically to exclude other dementia-causing illnesses

rather than to diagnose AD directly.

The application of PET to the study of AD has delineated changes in regional

cortical physiology and thus expanded our understanding of this disease.

Frackowiak and colleagues (1981), using the isotope 15O as elemental O2 to

study oxygen metabolism and as H20 to study CBF, found diminished flow and

metabolism in temporal and parietal cortex in AD. Concurrent measurement of

flow and metabolism permits the cerebral oxygen extraction ratio (OER) to be

evaluated. Frackowiak and colleagues found OER to be normal-evidence that

the diminished flow is a consequence, and not a cause, of the metabolic deficit.

Other laboratories have used glucose metabolic tracers and also found

generally diminished glucose metabolism in temporal and parietal cortex

(Benson et al. 1983; Duara et al. 1986).

In our laboratory we have used the glucose metabolic tracer

fluorodeoxyglucose (FDG) in conjunction with the PET-280, a single-slice

tomograph with 8-mm resolution (full width at half maximum) and a 10-mm slice

thickness. More detailed characteristics of this instrument have been published

previously (Derenzo et al. 1981). Initial studies of ten AD patients (Friedland et

al. 1983) showed significant decreases in relative FDG uptake in

temporoparietal cortex. Further investigation of this phenomenon in our

laboratory was designed to quantitate these differences and attempt to

understand their mechanisms.

Glucose metabolic rates may be estimated by two methods. The first, an

autoradiographic method developed by Sokoloff and colleagues (1977) for use

in animals, uses an operational equation subsequently modified by Phelps and

colleagues (1979) for use with PET in humans. The technique permits the

quantitation of regional cerebral metabolic rate for glucose (rCMRglu), with the

100
measurement of brain radioactivity at only one time point approximately 45 min

following tracer injection. In addition, the time course of the tracer in plasma

must be known and is generally obtained by multiple sampling from an artery or

arterialized vein. Assumed values of rate constants (which may be measured

separately or obtained from the literature) are then used in a correction term to

account for the small amount of the tracer in the brain that remains

unmetabolized. This technique has the advantages that it requires only one

PET measurement to estimate rCMRglu and is relatively insensitive to small

errors in the rate constants. Two shortcomings of this technique are that large

errors in the rate constants may cause errors in the calculation of rCMRglu

(Hawkins et al. 1981) and that the pathophysiology of the diminished glucose

metabolism remains unknown when the operational equation is used, because

diminished glucose metabolism may result from either diminished transport or

phosphorylation.

The second method for quantitating rCMRglu uses dynamic PET data, that is,

sequential PET data beginning immediately following injection of the tracer and

continuing for 40 min or more. The simultaneous acquisition of a

time-versus-activity curve describing the tracer time course in blood permits

iterative least-squares fitting for the individual parameters of the Sokoloff model:

k1 and k2 for forward and reverse transport of glucose, respectively, and k3 for

phosphorylation by the hexokinase enzyme. Estimating of these individual

parameters might further elucidate of the mechanism of decreased glucose

utilization.

We performed PET studies in 16 patients who met current research criteria for

AD (McKhann et al. 1984) and in 7 age-matched controls. Subjects in both

groups were free of significant medical illnesses and were taking no medication.

The AD group averaged 65.5 years of age (SD = 6.6) and the control group

averaged 63.0 years (SD = 3.0). The AD patients were mildly to moderately

demented, and disease onset had occurred before age 65 in 12 of them. A

subset of these patients (11 with AD and 6 controls) underwent dynamic PET

scanning. A more detailed report of this study can be found in a recent

publication (Friedland et al. 1989).

The dynamic PET experiments were designed to acquire time-activity curves for

the PET data and the time course of the activity in blood. Immediately after the

tracer was injected, blood was withdrawn from a heated hand vein, at short

intervals at first, then at progressively longer intervals. Simultaneously, PET

data were acquired, initially every 2.5 s or 5 s, then at increasingly longer

101
aintervals. Regions of interest were then drawn in PET images, and the

time-activity curves for each brain region were calculated. These data were

then fit with the blood time-activity curves by use of an iterative least-squares

fitting technique to derive the rate constants for glucose transport and

phosphorylation. Figure 1 shows sample PET data from a single region in one

patient, in conjunction with blood data and the fit. Values for each rate constant

were averaged in each patient group and then applied with the operational

equation to determine metabolic rates for the patients who did not have

dynamic studies and to determine metabolism in tomographic levels at which

dynamic data were not obtained.

Although dynamic studies did not reveal differences between controls and AD

patients for any of the model parameters K1 through K3, the variability of the

data was large; coefficients of variation ranged from 18 percent to 45 percent.

When this variability was reduced by normalizing the data with ratios of the

parameter in a given brain region to the value in the entire cortex, we found that

AD patients showed relatively reduced K3 in the temporal cortex, the brain

region that has been consistently most abnormal in AD.

Results using the operational equation also confirmed our earlier results, and

those of others, that glucose metabolism is significantly decreased in temporal

and parietal cortex. We also reduced the variability of these data by normalizing

regional values for glucose metabolism by dividing the rCMRglu for a given

region by the glucose metabolism in relatively normal brain regions, the

thalamus and striatum. When this was done, relative reductions in glucose

metabolism were apparent in all neocortical regions (frontal, parietal, temporal,

and occipital cortex) but not in cerebellum or white matter.

These PET studies demonstrate a disturbance in neocortical glucose

metabolism that, while involving all neocortical regions, is most prominent in

temporal and parietal cortex and appears to be due to abnormalities of the

hexokinase step of glucose metabolism and not to glucose transport.

Nevertheless, it remains possible that the disturbed glucose transport is a result

of loss of neuronal number in the image volume rather than a primary

disturbance of glucose metabolism. Friedland and colleagues (1985a) have

noted that the regions showing diminished glucose metabolism are also the

sites of the most severe neuropathological involvement (Brun and Englund

1981).

102
FIGURE 1. Blood and brain time-activity curves from a dynamic positron

emission tomography (PET) study of fluorodeoxyglucose (FDG)

uptake. The (*) symbols represent the brain data obtained from

sequential PET scans, the (O) symbols represent the time course

of the blood radioactivity, and the solid line is the result of the

model fit.

Although the pathophysiology of glucose hypometabolism in AD is still not

completely resolved, the technique has utility both for diagnosing AD and for

understanding the production of disease symptoms. Several laboratories have

shown that the neocortical glucose hypometabolism occurs early in the illness

103
when memory loss is the only significant abnormality (Haxby et al. 1986; Kuhl

et al. 1987; Cutler et al. 1985) raising the possibility that the pattern of

hypometabolism can predict which amnesiac patients will subsequently develop

AD. In addition, Pick’s disease, a dementia marked by frontal and temporal lobe

neuropathology, shows a metabolic pattern different from that of AD (Kamo et

al. 1987), with prominent frontal lobe hypometabolism. Whereas other

dementias, including MID (Kuhl et al. 1985) and normal-pressure

hydrocephalus (Jagust et al. 1985), have not generally shown the same

metabolic pattern as AD, the pattern is clearly not entirely specific; it also has

been seen in Parkinson’s disease (Kuhl et al. 1984) and in one patient with

Creutzfeldt-Jakob disease (Friedland et al. 1984).

The metabolic abnormalities in AD have been found to correlate with

neuropsychological functioning and are thus clearly related to the disease

symptoms. Patients frequently demonstrate asymmetries of glucose

metabolism, and the nature of neuropsychological impairment is related to the

hemisphere that is more severely hypometabolic (Friedland et al. 1985b; Haxby

et al. 1985). AD patients with prominent disturbances in language, visuospatial

relations, or other cognitive functions also demonstrate glucose metabolic

abnormalities in brain regions known to correspond to the localization of these

functions. Evidence suggests that early metabolic abnormalities may predict

later neuropsychological deficits that will develop as the disease progresses

(Haxby et al. 1987). It appears that as the disease progresses the frontal lobes

eventually become affected and asymmetries tend to diminish (Jagust et al.

1988).

PET has also been used to investigate the blood-brain barrier in AD.

Neuropathological evidence showing the frequent deposition of amyloid in

leptomeningeal vessels suggests that abnormalities of the blood-brain barrier

may play a role in the development of AD. PET studies using 68Ga-labeled

ethylenediamine tetra-acetic acid (EDTA), a large molecule normally excluded

by the blood-brain barrier, have shown no transport of the tracer into brain

tissue from the vascular compartment (Friedland et al. 1986; Schlageter et al.

1987).

Higher resolution instruments will be used in future PET work on AD. Our new

tomograph, the Donner PET-600, has an in-plane resolution of 2.6 mm and an

axial thickness of 5 mm (Derenzo et al. 1987). Images from a control and an AD

subject are shown in figure 2. Improved resolution provides the ability to

quantitate physiologic processes in small brain regions, particularly the mesial

104
PET 600

Control AD

FIGURE 2. Results of PET-FDG studies using the new PET-600 tomograph

with 2.6-mm resolution. The subject with Alzheimer’s disease

shows bilateral decrements in tracer uptake in temporal cortex.

temporal lobe structures, which we suspect are the earliest to be involved in

AD. The potential use of PET with tracers to study human brain neurochemistry

in AD will also provide a model for examining the effects of neurochemical

lesions on behavior.

SPECT IMAGING IN AD

Initial findings using PET with FDG suggested that the abnormal pattern of

glucose metabolism in AD could have diagnostic utility. The technical factors

that have been described, however, largely limit the availability of PET to

tertiary care university centers. SPECT imaging technology is immediately

accessible to most community hospital nuclear medicine departments, and

tracers for studying rCBF are now commercially available. Findings that rCBF

appears normally coupled to regional metabolism in AD have supported the use

of this technique in investigations of demented patients.

105
Studies of rCBF in AD patients have used both 123I-labeIed

N-isopropyl-piodoamphetamine (IMP) and 99mTc-labeled hexamethyl

propyleneamine oxime (HM-PAO) with similar results (Johnson et al. 1987;

Sharp et al. 1986; Cohen et al. 1986; Gemmell et al. 1987). Prominent temporal

and parietal hypoperfusion is seen when both tracers are used. Methods of data

analysis have included visual inspection and within-patient ratios of regional

radioactivity distribution and generally show some overlap between AD and

control subjects. In addition, the frequent asymmetry of the temporoparietal

lesions has resulted in the misclassification of AD patients as having MID, a

problem that can be avoided by using an anatomic imaging technique in

conjunction.

In our initial series (Jagust et al. 1987) we studied nine AD patients, five

controls, and two patients with MID using the tracer IMP. Because of the

technical problems limiting accurate measurements of tracer uptake, we did not

model and quantitate rCBF in these patients but rather used a ratio of the

radioactivity in a region of interest to the activity in the entire tomographic slice.

Initial results showed that the temporoparietal-to-whole-slice ratio completely

discriminated the AD from the control and MID subjects. In addition, this ratio

strongly correlated with dementia severity as measured using the Mini-Mental

State Examination (MMSE) (Folstein et al. 1975).

Other groups also have noted relationships between tracer uptake and

dementia severity (Johnson et al. 1988). Studies of patients with non-Alzheimer

types of dementia, particularly those that clinically appear to affect the frontal

lobes, have shown abnormalities of frontal lobe structures rather than the

temporoparietal abnormalities of AD (Jagust et al. 1989).

SPECT series of AD patients also show relationships between regions of

diminished tracer uptake and cognitive performance (Perani et al. 1988). These

results are quite similar to results obtained by PET and demonstrate the

pathophysiological basis for the disease’s heterogeneity. Indeed, a

considerable advantage of SPECT over PET is that its relative low cost and

ease of use allow larger subject groups to be studied. This point is important in

illnesses like AD, in which biological differences between individuals may be

large.

The study of larger patient groups has also indicated that the technique is

neither completely sensitive nor specific for the diagnosis of AD. We recruited

patients in the earliest stages of AD to evaluate the sensitivity of SPECT and

106
IMP for diagnosing the cause of dementia (Reed et al. 1989). We studied 21

patients, all of whom had SPECT scans and neuropsychological testing. Nine

were mildly demented, with scores on the MMSE in the normal range

(mean = 26.6, SD = 1.7), although all complained of memory loss and were felt

to have AD; 12 were moderately demented (MMSE mean score = 15.0,

SD = 5.4). Thirty-six healthy, age-matched control subjects were studied; 14

received SPECT scans and 22 underwent neuropsychological testing, but no

control subject received both. We quantitated SPECT data by calculating an

rCBF ratio defined as the ratio of regional radioactivity normalized to occipital

cortical radioactivity. We studied four cortical rCBF ratios-orbitofrontal and

dorsolateral frontal cortex-and temporal and parietal cortexand found

considerable overlap among patients (both mild and moderately demented) and

control subjects in all brain regions. Furthermore, when SPECT and

neuropsychological abnormalities were defined as scores outside the

95 percent confidence intervals for the control-group means, all AD patients

showed memory abnormalities, whereas only 55 percent of the mildly demented

patients and 83 percent of the moderately demented patients showed temporal

or parietal perfusion deficits. Temporal or parietal SPECT abnormalities,

however, were seen in only 7 percent of control subjects, suggesting that the

presence of abnormalities might indicate dementia more reliably than does the

presence of abnormal memory function, which was seen in 27 percent of

control subjects. Although other methods of SPECT data analysis might be

more sensitive for diagnosing AD (such as visual inspection by a trained

observer), it is clear that a normal SPECT scan does not rule out the presence

of AD.

These preliminary results with SPECT provide general guidelines for both the

clinical and research uses of the technique. As a diagnostic tool in dementia,

SPECT is clearly not a definitive tool. It may be useful, however, in difficult

diagnostic cases, such as patients with early symptoms, because findings of

temporal and parietal hypoperfusion support the diagnosis of AD. SPECT may

also be useful in diagnosing patients with unusual dementia symptoms, such as

the apathy and depression seen in frontal lobe dementia syndromes. As a

research tool, SPECT is useful for studying clinical problems in which biological

heterogeneity limits PET studies of small patient groups.

MRI IN DEMENTIA

A primary advantage of MRI over traditional methods of anatomic brain imaging

is its inherent contrast sensitivity. The use of different pulse sequences to

107
image the brain allows the distinct visualization of grey and white matter and

the delineation of both from cerebrospinal fluid (CSF). The sensitivity of the

technique to small changes in water content is also useful in detecting small

lesions in grey or white matter.

An important application of MRI to the study of dementia has been its use to

define the extent of cortical atrophy, because sulcal detail is not obscured by

the beam-hardening effects of CT. When MRI measurements are compared

with CT measurements, they show similar estimates of ventricular size but

different estimates of sulcal size; MRI provides a better measure of cortical

atrophy (Chawluk et al. 1987).

MRI has also been used to evaluate changes in the white matter in dementia.

Several studies have demonstrated that white-matter lesions (WMLs) occur in

both the nondemented and demented elderly; similarities exist between groups

in both the configuration and extent of the lesions except for the presence of

large, confluent WMLs in MID (Hershey et al. 1987). These lesions have not

correlated with dementia severity, and some AD patients have not had any

WMLs at all. Although finding these lesions on MRI frequently guides the

physician toward the diagnosis of MID or Binswanger’s disease, these

abnormalities may be misleading, because their pathological significance is not

well understood.

Recent work from our laboratory has made use of the greater contrast

sensitivity of MRI to evaluate specifically the brain region that is most implicated

in AD-the hippocampus (Seab et al. 1988). We used the IBM-MIT-LBL 0.5 T

imager and a phase-corrected inversion-recovery sequence. By taking scans

5 mm thick at 8-mm intervals (figure 3), we were able to localize the slice

containing the hippocampus and model its size as an elongated ellipse. The

comparison of AD patients and controls demonstrated that hippocampal size

(normalized to the size of the lenticular nucleus) completely differentiated all ten

AD subjects from seven controls. Although this is a preliminary study of a small

patient group, it demonstrates that anatomical imaging may be useful in

studying of a specific brain structure in AD.

NEUROIMAGING STUDIES OF BRAIN AND BEHAVIOR

The imaging studies reviewed here infer relationships between brain physiology

and behavior through correlations, such as the relationship between

metabolism in a given brain region and performance on a neuropsychological

108
NMR

Normal Alzheimer’s Disease

Pattern of hippocampal atrophy: Solid area represents atrophic hippocampus.

Dashed line is outline of normal hippocampus. H=hippocampus. WM=white

matter. V=ventricle.

FIGURE 3. Nuclear magnetic resonance imaging of a patient with Alzheimer’s

disease compared with that of a control subject, demonstrating

hippocampal atrophy. Studies used a phase-corrected

inversion-recovery pulse sequence (TR = 200, TI = 500,

TE = 33 ms) with 5mm-thick sections spaced at 8-mm intervals.

109
test in a patient group. Such methods have obvious drawbacks, including the

difficulty in concluding a causal mechanism because of the possible eff acts of

an unknown and unmeasured variable. Studies that use behavioral tasks to

activate specific brain regions during the performance of a PET scan have

many advantages for studying the relationships between brain and behavior.

These techniques are demanding, for they generally involve the use of

short-lived tracers and performance of multiple cognitive tasks at a single

sitting. Several reviews of these experimental procedures and their results have

been published (Petersen et al. 1988; Posner et al. 1988), demonstrating the

utility of the technique for uncovering functional consequences of cognitive

activity in humans.

PET tracers designed to study regional changes in neurochemistry also have

wide potential application to behavioral research. Changes in neurotransmitter

receptor number may be important in the production of disease symptoms in

schizophrenia (Seeman 1987) and depression (Mayberg et al. 1988). The use

of ligands to evaluate opiate receptors and dopamine receptors in the brain also

has obvious potential applications to the study of drug abuse. Similar ligands

will undoubtedly also become available for SPECT imaging.

The advantages of MRI in localizing and quantitating the size of different brain

structures will enhance our ability to relate structure to function in normal

subject populations and in illnesses such as AD, in which no discrete brain

lesion is present. Thus, high-resolution MRI studies of patients with amnesia of

unknown cause have demonstrated bilateral structural changes in the

hippocampus (Press et al. 1989). Gazzaniga (1989) has noted that magnetic

resonance images of the corpus callosum in monozygotic twins provide strong

evidence of genetic factors influencing brain structure.

The study of behavior by means of these new imaging techniques has

important implications for neuroscientists. It is now possible to image human

metabolism and blood flow in three dimensions and to quantitate such

physiologic processes accurately. Measurements of neurotransmitter receptor

number and affinity are also possible. The ability to make these measurements

noninvasively and safely allows them to be repeated over time, so that the

effects of mental activation, drugs, treatments, or disease can be evaluated.

The coupling of such measurements with the use of MRI to image anatomy in

detail holds great promise for understanding the neural mechanisms underlying

human behavior.

110
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ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants AGO7793 and

AGO5890 from the National Institute on Aging and by the Director, Office of

Energy Research, Office of Health and Environmental Research of the U.S.

Department of Energy under contract DE-AC03-76SF00098.

AUTHORS

William J. Jagust, M.D.

Thomas F. Budinger, M.D., Ph.D.

Research Medicine, 55-121

Lawrence Berkeley Laboratory

1 Cyclotron Road

Berkeley, CA 94720

115

Studying Psychoactive Drugs With Positron

Emission Tomography: Relationships

Between Mood and Metabolic Rate

Harriet de Wit, John Metz, and Malcolm Cooper

INTRODUCTION

The study of neural mechanisms underlying the effects of abused drugs has, for

practical and ethical reasons, been limited largely to studies using laboratory

animals (Snyder 1984; Koob and Bloom 1988). Most current techniques in

neuroscience require invasive procedures that cannot be conducted with

human subjects. The use of animals as subjects, however, requires inferences

across species, and it is sometimes difficult to determine which of a drug’s

multiple pharmacologic effects are related to its potential for abuse in humans.

Recently, techniques that permit us to study the effects of psychoactive drugs

on neural activity in normal, awake human subjects have become available.

These techniques eliminate the need for cross-species inferences and allow us

to study neural events in relation to other drug effects that are known to be

related to drug abuse, such as the drugs’ subjective effects. The subjective, or

mood-altering, effects of drugs (such as euphoria) are considered to be central

to the initiation and maintenance of drug-seeking behavior; therefore, a better

understanding of the relationship between the subjective experiences and

corresponding neural events will greatly advance our knowledge of the

biological basis of drug abuse.

In the present study, positron emission tomography (PET) was used with a

radioactively labeled glucose analog-18-F-2-deoxy-2-fluoro-D-glucose (FDG)

as the tracer. This technique provides a measure of regional cerebral glucose

uptake, which is thought to be an indicator of neuronal activity. Two

placebo-controlled studies were conducted in normal, awake volunteers,

examining the effects of (1) ethanol (ETOH) and (2) diazepam (DZP). We

117
studied the metabolic changes induced by the drugs and the relationship of

these metabolic effects to the drugs’ effects on mood and subjective state.

An important feature of these studies was that the subjective effects of the

drugs were monitored at the same time the metabolic data were obtained. It is

known that many effects of drugs, including subjective effects, vary both across

individuals and across settings (Galizio and Maisto 1985). Even drugs that are

usually considered to be reliable euphorigenic agents, such as amphetamine

and alcohol, produce dysphoric effects in some individuals (de Wit et al. 1986,

1987, 1989), and the environmental settings in which drugs are administered

are known to alter profoundly their mood-altering effects (McCarty 1985).

Therefore, in studying the neural substrates of subjective drug effects, it is

important to verify that the drugs are actually producing their expected

subjective effects.

The studies described here were initiated to explore the biological basis of

individual differences in responses to ETOH in normal volunteers. It had been

observed in previous studies (de Wit et al. 1987, 1989) that normal social

drinkers varied widely in their subjective responses to a low dose of ETOH:

some subjects experienced stimulantlike effects, whereas others experienced

sedation. These differences in subjective effects corresponded to differences in

ETOH preference in a choice test: the subjects who experienced ETOH as

stimulantlike chose it more often than a placebo, whereas those who

experienced ETOH as sedativelike chose the placebo more often than ETOH.

One goal of the present studies with PET was to explore possible physiologic

bases of individual differences such as these. The more immediate goals were

simply to assess the effects of ETOH and DZP on regional cerebral metabolic

rate for glucose (CMRglu) and to determine the relationship between the

metabolic and the mood-altering effects of the drug.

STUDY 1: ETHANOL

Study 1 consisted of two phases: a behavioral phase and a PET phase. The

behavioral phase was a seven-session preference experiment conducted under

seminatural conditions to assess the subjective and reinforcing effects of ETOH

in a recreational setting. After completing the behavioral phase, subjects were

tested in the PET phase, which consisted of three brain scans-one with

placebo, one with 0.5 g/kg of ETOH, and one with 0.8 g/kg of ETOH. The

behavioral phase provided a “natural” referent against which to assess the

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generality of the subjective responses to ETOH that were obtained in the highly

unnatural conditions of the PET setting.

Procedure

Subjects. Eight normal, healthy males participated in study 1. All were

students aged between 21 and 29 years, and they were light social drinkers

(mean, 6.0 drinks per week; range, 1-20 drinks per week). Most subjects

reported having tried marijuana at least once but had little experience with other

drugs. They were screened for physical and psychiatric health before

participating, and they had read and signed a consent form that outlined the

procedures to be used.

Behavioral Phase. Subjects participated in seven sessions, which were

conducted one or two evenings a week from 7 p.m. to 11 p.m. The sessions

were conducted in a recreational environment consisting of two comfortably

furnished rooms with television, radio, movies, and a variety of games. Subjects

were tested in groups of four individuals to create a natural social situation.

During the sessions when no other events were scheduled, subjects engaged in

leisure activities of their choice but were not allowed to work or study. The first

four sessions were sampling sessions, during which either ETOH (0.5 g/kg) or

placebo was administered in color-coded cups. The last three sessions were

choice sessions, during which subjects chose whichever beverage they

preferred. Subjects were told that they might receive a tranquilizer, a stimulant,

alcohol, or a placebo, but that the same substance would always be in a cup of

the same color. During sampling sessions, ETOH and placebo were

administered during alternating sessions in counterbalanced order across

subjects. The beverages were administered in divided unit doses of 0.1 g/kg,

ingested at 15-min intervals. During the sampling sessions, five unit doses were

administered between 7 p.m. and 8 p.m. During choice sessions, subjects could

take from 1 to 11 doses of their chosen substance-i.e., a total dose of 1.1 g/kg

of ETOH if they chose the ETOH beverage—between 7 p.m. and 10 p.m.

ETOH beverages consisted of 10 percent ETOH by volume mixed with tonic

and lime (total volume, 450 mL for a 70-kg subject), and placebo beverages

consisted of mix with 1 percent ETOH as taste mask. The number of choice

sessions during which ETOH was selected over placebo was the measure of

drug preference, and the number of unit doses taken was the measure of dose

preference.

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During each session, subjects completed mood and drug effects questionnaires

before ingesting the beverages and at regular intervals throughout the sessions.

They completed the Profile of Mood States (POMS) (McNair et al. 1971), a

72-item adjective checklist consisting of eight primary scales (anxiety,

depression, anger, vigor, fatigue, friendliness, confusion, and elation) and two

composite scales (arousal and positive mood). At regular intervals, subjects

also completed drug-liking and identification questionnaires and tests of

psychomotor performance. Several of the questionnaires were also used in the

PET phase that followed, to facilitate comparisons of the subjective effects of

ETOH across settings.

PET Phase. After the behavioral phase was completed, subjects participated

in the PET phase, which consisted of three weekly sessions. A PET scan was

conducted during each of these sessions, preceded by administration of a low

or moderate dose of ETOH (0.5 g/kg or 0.8 g/kg) or a placebo beverage.

For 12 hours before each session, subjects fasted and abstained from

beverages containing sugar, caffeine, and alcohol. They reported to the PET

laboratory between 9 and 11 a.m., at which time a transmission scan was

conducted for attenuation correction. Intravenous catheters were inserted in

each arm, one for injecting the tracer and one for blood sampling. After the

catheters had been inserted, subjects completed a predrug POMS and

practiced a visual monitoring task (VMT). The VMT consisted of bright and dim

light flashes presented at 4-s to 7-s intervals. Subjects were instructed to press

a handheld button after each dim flash but not after the bright flashes, and their

reaction times and accuracy were recorded. Subjects performed this task

throughout the scanning period, except for a brief pause to complete the first

postdrug POMS. The function of the VMT was to stabilize cognitive activity

across sessions and across individuals; in the absence of such a task there is

wide variability in the level of cognitive activity (e.g., wakefulness) during scans,

which could obscure the effects of drug treatments. After practicing the VMT

and completing the presession POMS, subjects ingested (double-blind) a

beverage containing 0.5 g/kg ETOH, 0.8 g/kg ETOH, or placebo. The

beverages, which were similar to those administered in the behavioral phase

but smaller in volume, were administered in five equal portions at 1-min

intervals. Immediately after ingesting the beverage, a subject was positioned in

the scanner (this took about 5 min), began the VMT, and was then injected with

the FDG. The subject remained in the scanner for the next 40 min, continuously

performing the VMT except for a brief pause after 20 min to complete

subjective-effects questionnaires. The POMS and a drug-liking questionnaire

120
were presented on a Macintosh computer placed in front of the subject while he

remained positioned in the scanner. At the end of the scanning period, the

subject completed a final POMS and drug-liking questionnaire.

PET studies were performed in “dynamic” mode (Huang et al. 1980), although

the data reported here were reconstructed using the autoradiographic method

(Phelps et al. 1979; Reivich et al. 1979). Data were obtained by use of a

three-ring PETT VI scanner (Ter-Pogossian et al. 1982) which provides five

transaxial image slices with an interslice spatial resolution of 8 mm (full width at

half maximum) and an interslice distance of 14 mm. The PET images integrated

metabolic activity occurring during the period from 10 to 50 min after the ETOH

was ingested. Subjects were injected intravenously with 5 to 10 mCi of FDG

within 5 min of ingesting the ETOH beverage. Periodically throughout the

scanning period 1-cm3 blood samples were taken for use in converting

radioactive counts to metabolic rates as well as to verify the stability of glucose

levels after ETOH administration and to determine blood ETOH level.

CMRglu was assessed bilaterally in seven regions of interest: four cortical

regions (temporal, parietal, occipital, and frontal), basal ganglia, thalamus, and

cerebellum. These regions were identified on each reconstructed image by a

technician who was blind to the experimental conditions. The regions of interest

were relatively large so that they remained well within the limits of resolution of

the scanner (8 mm). A measure of global CMRglu was obtained in each PET

session by averaging all 14 regions for each subject. To compare across

conditions, relative CMRglu in each region of the brain was calculated as a ratio

of the average (whole-brain) rate.

Results

Behavioral Phase. Seven of the eight subjects chose the ETOH-containing

beverage in at least two of the three choice sessions and reported liking the

ETOH beverage more than the placebo. The remaining subject chose the

placebo during all three sessions and reported liking ETOH less than the

placebo. Most of the subjects correctly identified ETOH during sampling

sessions, except for the subject who chose the placebo and labeled ETOH a

“tranquilizer.” The average dose of ETOH ingested during choice sessions was

0.62 g/kg. In sampling sessions, blood ETOH level reached an average peak

level of 0.028 g/dL at approximately 17 min after the last dose. In choice

sessions, blood ETOH level varied according to the self-administered dose. The

POMS data obtained on the sampling sessions indicated that most subjects

121
experienced typical, ETOH-like subjective effects from the drug, including

increases on the vigor, friendliness, and elation scales and decreases on the

anxiety and fatigue scales (figure 1). Subjects’ ratings of drug liking during the

sampling sessions were positively correlated with the number of doses they

subsequently ingested during the choice sessions (R = .73). Thus most, but not

all, subjects showed positive mood responses to ETOH in the naturalistic

situation, and their subjective responses to ETOH were consistent with their

choice of the drug.

PET Phase. On average, ETOH produced subjective effects in the PET

setting similar to those produced in the naturalistic setting, e.g., increases in

friendliness and elation (figure 1). There was considerable variability in

responses across individuals, however, and the POMS scores of individual

subjects after ETOH were not significantly correlated across the two settings.

Ratings of drug liking for individual subjects, however, were moderately

correlated across the two settings, indicating some stability of responses within

individuals. The one subject who did not choose the ETOH in the behavioral

phase was also the only subject to rate his liking of ETOH lower than his liking

of placebo in the PET phase, and to misidentify both doses of ETOH (he

labeled it “tranquilizer” at the low dose and “stimulant” at the higher dose).

Blood ETOH level reached a mean peak level of 0.044 g/dL at 46 min following

the 0.5-g/kg dose of ETOH and 0.062 g/dL at 58 min following the 0.8-g/kg

dose. The fact that blood ETOH level was higher in the PET phase after the

0.5-g/kg dose than after the same total dose in the behavioral phase can be

accounted for by differences in rate of drug administration (5 min vs. 1 hr) and

possibly by the fact that subjects fasted before drug ingestion in the PET phase

but not in the behavioral phase. ETOH did not affect either reaction times or

accuracy on the VMT, nor did it change plasma glucose levels.

Average global metabolic rates were 8.88 mg/100 g per minute during the

placebo sessions, 8.55 mg/100 g per minute after the low dose of ETOH, and

8.35 mg/100 g per minute after the moderate dose of ETOH. Only the

difference between the higher dose and placebo approached statistical

significance (p < .06). At the 0.5-g/kg dose, global CMRglu decreased in five of

the eight subjects relative to placebo, and at the 0.8-g/kg dose, CMRglu

decreased in seven of the eight subjects. In one subject (the same individual

who showed an idiosyncratic response in the behavioral phase) average

CMRglu increased after each dose of ETOH.

122
FIGURE 1. Mean scores on four Profile of Mood States scales after

administration of placebo and ethanol during the behavioral phase

and the position emission tomography (PET) phase of study 1.

Behavioral phase data are based on the mean scores from the

two sampling sessions; the PET phase data are based on the

mean of the three scores (1) before beverage administration,

(2) 20, and (3) 40 min into the scanning period.

The effects of ETOH on CMRglu (figure 2) appeared to be fairly uniform across

all the regions of interest, although at both doses the thalamus was relatively

less affected than other parts of the brain. During all sessions, regardless of

drug administration, CMRglu was higher in the left hemisphere than in the

right-a difference that has been observed previously (Wagner et al.

1986)—but relative CMRglu in different regions of the brain remained the same.

Neither global nor regional changes in CMRglu were related to blood ETOH

123
FIGURE 2. Mean (and SEM) cerebral metabolic rate for glucose in each

region of interest after administration of placebo, and 0.5 g/kg of

ethanol (ETOH) and 0.8 g/kg of ETOH in eight healthy males.

Whole-brain CMRglu was calculated by averaging the values for

regions of interest for each subject.

level, reaction times, or plasma glucose level. Regional changes in CMRglu

after ETOH were examined by calculating ratio scores of particular brain

regions relative to the rest of the brain. The ratio of regional CMRglu to

whole-brain CMRglu during placebo sessions was subtracted from the ratio of

regional CMRglu to whole-brain CMRglu during ETOH sessions. These

regional-change scores were then correlated with mood changes after ETOH.

Mood changes for each POMS scale were calculated by subtracting predrug

scores from the first postdrug score (20 min into the scan) and then subtracting

the placebo-difference score from the ETOH-difference score. The effects of

ETOH on regional CMRglu were then correlated with its effects on mood. The

results of this exploratory correlational analysis are presented in tables 1 and 2,

which show correlations that exceeded an arbitrarily selected cutoff value of

r = .70 (.71 is the level that would be significant at the p = .05 level if only two

variables were being compared). In view of the small number of subjects and

the large number of dependent variables, these correlations can only be

124
TABLE 1. Summary of correlations between changes in mood and CMRglu

after administration of low dose of ethanol (0.5 g/ks)

Region of

interest

Negative moodsa

Co Dp El

Positive moodsa

PM Vg Fr

Left parietal

Left basal ganglia

Right occipital

— -.71 .82 .85 .83 .89

-.86 — — — — —

.71 .71 — — — —

NOTE: Co = confusion; Dp = depression; El = elation; PM = positive mood; Vg = vigor;

Fr = friendliness; — = correlation < .70.

aPOMS scales.

TABLE 2. Summary of correlations between changes in mood and CMR

after administration of moderate dose of ethanol (0.8 g/kg)

Region of

interest

Negative moodsa

Co Fa Ag

Positive Moodsa

El PM Ar

Left temporal -.77 -.94 — — — .86

Left thalamus — .79 — — — —

Left cerebellum -.78 — — — — —

Right parietal — — .80 — — —

Right occipital — — — -.74 -.72 —

NOTE: Co = confusion; Fa = fatigue; Ag = anger; El = elation; PM = positive mood; Ar = arousal;

— = correlation < .70.

aPOMS scales.

considered exploratory. The lack of consistency in the patterns of correlations

between CMRglu and mood across the two doses suggests, however, that no

systematic relationships exist between the mood-altering effects of ETOH and

its effects on regional CMRgIu.

125
STUDY 2: DIAZEPAM

In study 2, a separate group of eight subjects participated in three PET

sessions—one with placebo, one with 0.07 mg/kg of DZP, and one with

0.14 mg/kg of DZP. (These doses of DZP are equivalent to about 5 mg and

10 mg for a 70 kg person.) (Although six of the eight subjects also participated

in a natural-choice experiment with DZP before the PET scans, data from that

study are incomplete and are not reported here.)

Procedure

Eight normal, healthy males similar to those described in study 1 participated.

Their average age was 24.5 years (range, 21-29 years), most were students,

and they drank on average 6.25 alcoholic drinks per week (range, 2-20). Use of

other drugs was minimal. The subjects were screened for physical and

psychiatric health.

The three PET sessions were conducted according to the same procedures

described for ETOH, except that capsules containing DZP or placebo were

administered 30 min before the FDG was injected. To further blind the drugs, a

placebo beverage similar to that used in the ETOH experiment was also

administered immediately before the FDG was injected; subjects were told that

they might receive a stimulant, a tranquilizer, alcohol, or a placebo.

Subjective-effects questionnaires were administered before subjects ingested

the capsules and 20 and 40 min after scanning had begun (i.e., 50 and 70 min

after subjects had ingested the capsules). All other procedures were as

described in study 1.

Results

Relative to placebo, average global CMRglu decreased significantly (t tests,

p < .04) after both doses of DZP. The means were as follows: (1) placebo,

8.25 mg/100 per minute; (2) 0.07 mg/kg DZP, 7.36 mg/100 per minute; and

(3) 0.14 mg/kg DZP, 7.42 /100 per minute.) In seven of the eight subjects,

global CMRglu decreased after both doses of DZP, whereas in one subject,

CMRglu increased after both doses. CMRglu decreased to about the same

level in all regions of the brain after DZP (figure 3), except in the thalamus and

basal ganglia, which were relatively less affected by the drug, particularly at the

higher dose. Although DZP decreased accuracy in the VMT task (96 percent

correct after placebo and 86 percent correct after both doses of DZP), it did not

126
change reaction times. Subjects most often thought they had received a

tranquilizer, even when they had received a placebo. Accordingly, POMS

arousal scores decreased over the course of all sessions, i.e., from predrug to

20 and 40 min after FDG injection, regardless of whether subjects received

DZP or placebo (figure 4). The only measure that clearly differentiated DZP

from placebo was the POMS anxiety scale; scores decreased after the higher

dose of DZP. Liking ratings were higher after DZP was ingested at both the low

dose and the moderate dose than after placebo was ingested. The one subject

whose metabolic rate increased after ingesting DZP was also the only subject

to label the higher dose of the drug “placebo,” and he was the only subject who

rated his liking of DZP lower than placebo.

Correlations between regional CMRglu and mood were conducted using the

same formula described in study 1 (tables 3 and 4). The consistently high

correlations between CMRglu and measures of sedation (fatigue and arousal)

FIGURE 3. Mean cerebral metabolic rate for glucose (CMRglu) in each region

of interest after administration of placebo, and 0.07 mg/kg of

diazepam (DZP) and 0.14 mg/kg of DZP in eight healthy males.

Whole-brain CMRglu was calculated by averaging the values for

regions of interest for each subject.

127
indicate that subjects who experienced the strongest sedative effects also

showed the largest decreases in CMRglu. The correlations between measures

of sedation and left temporal cortex responses were also observed in the

previously described ETOH experiment. Neither global nor regional CMRglu

was correlated with other measures obtained in the experiment (i.e., subjects’

demographic characteristics or drug-use histories, VMT performance, or blood

levels of DZP).

FIGURE 4. Mean scores on four Profile of Mood States scales: after placebo,

0.07 mq/kg of diazepam (DZP), and 0.14 mg/kg of DZP; before

drug administration; and 20 and 40 min into the scanning period

(50 and 70 min after drug administration).

128
TABLE 3. Summary of correlations between changes in mood and CMRglu

after administration of a low dose of diazepam (0.07 mg/kg)

Region of

Interest Depressiona Arousal b

Left temporal

Left basal ganglia

— .76

.86 —

aPOMS scales, negative moods.

bPOMS scales, positive moods.

TABLE 4. Summary of correlations between changes in mood and CMRglu

after administration of moderate dose of diazepam (0.14 mg/kg)

Region of

interest

Negative moodsa

Co Fa

Positive moodsa

Ar Vg

Left frontal

Left temporal

Left occipital

Left cerebellum

Right frontal

Right occipital

Right thalamus

Right cerebellum

Left mean

Right mean

Global mean

Global s.d.

— —

— -.79

— —

— -.76

— —

— .79

-.71 —

— -.72

— -.73

— -.72

— -.72

-.73 —

— .74

.71 —

.74 —

.72 —

.70 —

.75 —

— —

— —

NOTE: Co = confusion; Fa = fatigue; Ar = arousal; Vg = Vigor.

aPOMS scales.

DISCUSSION

These studies demonstrate the feasibility of using PET to study drug-induced

changes in cerebral metabolic rate and of exploring the relationship between

these metabolic changes and the drugs’ mood-altering effects. The studies

129
illustrate both strengths and limitations of the PET methodology in the study of

brain mechanisms underlying drug actions. This discussion focuses on these

strengths and limitations, using the above studies as examples.

One of the primary advantages of PET is that it can be used with normal,

awake human subjects. It is safe and relatively noninvasive and yet provides a

direct measure of brain activity in both cortical and deeper, subcortical

structures of the brain. There are several advantages to using humans as

subjects: (1) Because a primary goal of many of the studies is to improve our

understanding of human drug use, the use of human rather than nonhuman

subjects eliminates the need for inferences across species. (2) Human

subjects, unlike laboratory animals, can describe the quality and magnitude of

their subjective responses to the drugs (e.g., euphoria), which are thought to be

closely related to the drugs’ likelihood of being abused (Martin et al. 1971). (3)

Although this study used only normal, healthy volunteers, the technique is well

suited to studying individual differences in drug effects among different

subgroups of individuals, such as those with presumed biological

predispositions (e.g., family history of alcoholism), extensive drug-use histories

(e.g., ex-addicts), or other presumed risk factors (e.g., antisocial personality

disorder). Even within a relatively homogeneous population of subjects such as

those used here, the correlations between metabolic changes and subjective

effects may be informative. For example, in each group of eight subjects in the

ETOH and DZP studies, one individual exhibited unusual metabolic changes

after drug administration relative to the rest of the group, and in both cases

those individuals also showed unusual subjective responses to the drugs.

Although little can be concluded from these isolated instances, it is possible that

individual variations in metabolic responses to drugs are meaningfully related to

variability in their mood-altering effects.

In these studies, the subjective effects of ETOH and DZP were measured at the

same time and in the same setting as the metabolic effects. In addition, in the

ETOH study the subjective and reinforcing effects were also assessed in the

same subjects in a more natural setting. Because it cannot be assumed that

drugs produce the same subjective (or other pharmacologic) effects in different

environmental settings and in all individuals, concurrent assessment of the

metabolic effects and the mood effects was a critical feature of this study.

Indeed, it was found that some subjects did not exhibit the expected subjective

effects of either ETOH or DZP in the PET studies. A related question is whether

the subjective drug effects reported by subjects in the relatively unnatural PET

setting are representative of the effects they might experience in a more

130
natural, recreational setting. The data from the ETOH study indicate that

although drug-liking scores of individual subjects were correlated across the two

settings, the quality of their mood responses as measured by subjective-effects

questionnaires varied across the two settings. These findings suggest that

generalizations from laboratory drug studies to the drugs’ presumed effects

outside the laboratory should be made cautiously.

Another important feature of our methodology was the use of the VMT to

stabilize the subjects’ cognitive activity. If cognitive activity is left uncontrolled

(resting state), wide variability is observed both across and within subjects.

Duara and colleagues (1987) have shown that this variability can be reduced

greatly by engaging subjects in a uniform cognitive task during the scanning

period. The vigilance task used in the present studies was used in a previous

study by Wagner and colleagues (1986), who obtained highly stable metabolic

data from the same subjects across two repeat scans when they performed the

VMT. Despite the improved control achieved by such a behavioral “clamp” on

mental activity, however, the possibility exists that performance of the task may

mask or inhibit other potential drug effects. In our studies, for example, either

the drugs’ effects on metabolism or their effects on mood might be altered by

performance of the task. This possibility will be explored in future studies.

Among the limitations of the current FDG-PET technology is the relatively poor

spatial and temporal resolution. The regions of interest in these studies were

defined to be relatively large so that they would be well within the limit of

resolution of the PETT VI scanner (8 mm). The inability to study smaller brain

regions is clearly a limitation of the current technique and may account for the

absence of regional changes in the present studies. Improvements in PET

technology, including an improved scanner and improved image-correlation

techniques (Pelizzari et al. 1989) and improved methods of analyzing existing

data, have been and are being developed to improve the spatial resolution of

PET. Similarly, the relatively long period required for FDG uptake (40 min) may

obscure changes of shorter duration, such as those expected to occur as

short-lived effects of abused drugs. Alternative PET techniques, such as the

use of oxygen-15 to measure oxidative metabolism or blood flow, will provide

greater temporal sensitivity.

Another limitation of studies such as those described here is that they are

correlational. Without convergence of data from other sources, it is not possible

to determine whether systematic relationships between the two measures (i.e.,

metabolic rate and mood) reflect causal relations (and, if so, the direction of the

131
causality) or coincidental relationships. Changes in regional CMRglu may

reflect the initial sites of drugs’ actions, or they may reflect neural events at

some point beyond the initial site of action. Correlations between CMRglu and

mood might either reflect effects of the drugs directly or the consequences of

the mood-altering (or other) effects of the drugs. These possibilities can be

explored in future studies-including, for example, investigations of other

(similar and dissimilar) drugs and manipulations that affect mood without drug

administration.

The assumptions underlying the PET methodology are highly complex (Phelps

et al. 1979), and the models for reconstructing images have been controversial.

Although the methods used in our studies are well accepted (Nelson et al.

1987), the complexity of the models increases the risk of error in data handling

and interpretation. In particular, when drugs are administered as an

experimental treatment, the possibility exists that the drugs may alter brain

chemistry or neural function in a way that invalidates the model. For example,

alcohol is known to have widespread effects on the characteristics of cell

membranes (Tabakoff and Hoffman 1981; Goldstein et al. 1982), which might

invalidate the rate constants used in reconstructing the images. Fortunately,

data are available to rule out some of these possibilities (rate constants, for

example, can be calculated from data collected in the dynamic mode), but

caution is clearly necessary in interpreting drug-induced changes in CMRglu.

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C.-T. Accurate three-dimensional registration of CT, PET and MR images of

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Phelps, M.E.; Huang, S.C.; Hoffman, E.J.; Selin, C.J.; Sokoloff, L.; and Kuhl,

D.E. Tomographic measurement of local cerebral glucose metabolic rate in

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133
Metabolic Mapping Methods for ldentifiying

the Neural Substrates of the Effects of

Abused Substances

Linda J. Porrino

The administration of an abused substance to an organism, whether

self-administration or passive administration, can result in a variety of

pharmacologic actions that may include both central and peripheral effects.

These physiological and behavioral responses are more likely to be the product

of multiple processes at a number of anatomic sites than the result of a single

action in a single location. To determine the neural substrates of the actions of

drugs of abuse, it is necessary, therefore, to identify neural events in circuits

and pathways throughout the brain, not just at one location. Doing so requires

either that we investigate small portions of the brain one at a time (with

electrophysiological methods, for example, or that we use methods capable of

surveying the entire brain simultaneously. The 2-[14C]deoxyglucose (2-DG)

method developed by Sokoloff and colleagues (1977) has provided such a

capability. The 2-DG method is a means by which neuroscientists can

investigate the functional events in the brain related to various physiological,

pharmacologic, and behavioral states. It has been used extensively in

neuropharmacology to identify the neural circuits that mediate the effects of a

wide variety of pharmacologic agents (for reviews see McCulloch 1982; Porrino

and Pontieri, in press). In this chapter, the application of the 2-DG method will

be described with particular attention to the advantages and disadvantages of

its use in pharmacology.

The 2-DG method measures rates of local cerebral glucose utilization

throughout all portions of the central nervous system (CNS). Although the 2-DG

method may appear to be essentially an anatomic technique on the basis of

autoradiographic images that are produced, it is in fact a biochemical method

that measures a biological process: glucose utilization, or the rate at which

energy is consumed in neuroanatomically defined regions in the CNS of

135
conscious animals. The value of measuring rates of glucose utilization stems

from the fact that in the brain, as in other tissues that do physicochemical work,

the amount of energy used is correlated with the amount of work done in that

tissue. Glucose utilization in the brain is a measure of energy use under normal

physiological conditions, because glucose is virtually the exclusive substrate for

energy metabolism. The basic rationale of the 2-DG method is that functional

activity in any given brain region is directly related to energy metabolism in that

region. It is possible, therefore, by measuring changes in rates of glucose

utilization, to identify brain regions in which functional activity is altered during

various experimental manipulations.

There are a number of energy-requiring processes in brain that contribute to

basal rates of glucose utilization; for example, transmitter synthesis, release

and reuptake, and synthesis of lipids and proteins. The largest proportion of

energy in the CNS, however, is used to maintain and restore ionic gradients. In

fact, it has been estimated that 80 percent of the energy generated in brain is

used for this purpose (Kurumaji et al., in press). It is important to appreciate that

the changes in rates of glucose utilization that are evoked by an experimental

manipulation are thought to result mainly from increases or decreases in

electrical activity or synaptic activity in the CNS. These changes in electrical

activity, in turn, produce corresponding increases or decreases in the activity of

Na+, K+-ATPase, the energy-consuming enzyme involved in the restoration of

neuronal ionic gradients to their resting state. Experiments with deoxyglucose

have shown that the coupling of glucose utilization to functional activity is

dependent on Na+,K+-ATPase activity, in that ouabain, an ATPase inhibitor, can

block the increases in glucose utilization that accompany electrical stimulation

(Mata et al. 1980).

One significant issue in the interpretation of changes in glucose utilization is the

site of these changes. A number of studies (Schwartz et al. 1979; Kadekaro et

al. 1985) have clearly demonstrated that functional activation of glucose

utilization occurs mainly in nerve terminals rather than in cell bodies. Terminals

undoubtedly make a larger contribution than cell bodies because of the greater

ratios of surface area to volume in neuronal terminals (Schwartz et al. 1979).

Alterations in energy metabolism in a specific brain area are due to changes in

terminals of the afferent inputs to that structure and not in the cell bodies

contained within the structure. This is the reason why data from

electrophysiological recording studies and 2-DG studies frequently do not agree

(Palombo et al. 1990). Different sets of events are being measured by the two

met hods.

136
The measurement of rates of glucose utilization follows the general principles

for measuring of rates of any reaction with radioactive tracers (figure 1). The

amount of product formed over an interval of time is determined, related to the

integrated specific activity (which is the ratio of the labeled precursor to the total

precursor pool integrated over the time of measurement), and corrected for

kinetic differences between the labeled and unlabeled compounds. To measure

rates of local cerebral glucose utilization, a radioactively labelled analog of

glucose, 2-DG, is used. Like glucose, 2-DG is transported into cerebral tissue

by the same carrier and phosphorylated by hexokinase, but, unlike glucose, it is

not metabolized further and is, therefore, trapped within cells. It is this trapping

that allows quantitative autoradiography to be used to measure actual rates of

glucose utilization in individual brain regions, Glucose, in contrast, is

metabolized further via the glycolytic and tricarboxylic acid pathways or the

pentose phosphate pathway eventually to carbon dioxide, which is cleared

rapidly from cerebral tissue. The use of [14C]glucose as a tracer for the

measuring of cerebral glucose utilization can be inaccurate because it is difficult

to correctly estimate the loss of labeled product from tissue. The amount of

label in tissue as measured autoradiographically with [14C]glucose is not a true

reflection of the amount of glucose used in that tissue. Direct comparisons of

the two tracers have shown that rates of glucose utilization in animals in which

the visual system has been stimulated are underestimated when [14C]glucose is

used as the tracer (Collins et al. 1987).

The calculations of rates of glucose utilization with the 2-DG method (figure 1)

are made from two measurements. The first is the concentration of radioactivity

in the tissue measured autoradiographically and is a measure of the product

formed. The concentration of radioactivity in the tissue, however, is composed

of both the product formed (in this case, 2-[14C]deoxyglucose-6-phosphate) and

unmetabolized precursor (in this case, 2-DG)’ The two cannot be distinguished

autoradiographically. The percentage of unmetabolized precursor must be

subtracted from the total to measure the rate of the reaction. The second

measurement is the levels of glucose and 2-DG in plasma during the

experimental period, which are required to determine the integrated specific

activity of the precursor in tissue. Levels in plasma are used because it is

impossible to make direct tissue measurements. Corrections are made for the

lag in tissue equilibration with plasma. The rate of glucose utilization is

described mathematically by the operational equation of the method (figure 1).

The details of the mathematical derivation of the 2-DG method and an

extensive discussion of its theoretical basis are beyond the scope of this

chapter (see Sokoloff et al. 1977; Sokoloff 1982; Sokoloff and Porrino 1986). In

137
FIGURE 1. Operational equation of the radioactive deoxyglucose method and

its functional anatomy. T represents the time at the termination of

the experimental period; C* represents the total 14C concentration

in brain tissue; Cp* and Cp represent the concentrations of

[

14C]deoxyglucose and glucose in the arterial plasma. The

constants k1*, k2*, and k3* represent the rate constants for

carrier-mediated transport of [14C]deoxyglucose from plasma to

tissue, for carrier-mediated transport back from tissue to plasma,

and for phosphotylation by hexokinase, respectively. equals the

ratio of the distribution space of deoxyglucose in the tissue to that

of glucose; 0 equals the fraction of glucose, which, once

phosphorylated, continues down the glycolytic path; and Km* and

Vm* and Km and Vm represent the Michaelis-Menten kinetic

constants of hexokinase for deoxyglucose and glucose,

respectively. (From Sokoloff 1983 with permission.)

138
addition, the techniques used for the application of the 2-DG method have been

described in detail elsewhere (Porrino and Crane 1990).

ADVANTAGES

A number of the characteristics of the 2-DG method are advantageous for its

use in neuropharmacologic as well as in other physiological investigations. One

of the primary advantages of the 2-DG method is that it uses autoradiography,

which allows the visualization not only of individual structures within the brain,

but also of small subnuclei and anatomical subdivisions within a given structure.

The resolution of the 2-DG method is approximately 100 microns. The limiting

factor is the diffusion and migration of the labeled compound in the tissue

during freezing and cutting of the brain. Even though cellular resolution is not

possible, autoradiography still provides a considerable degree of anatomic

resolution. This point is apparent in 2-DG studies of the visual system of

primates, in which a pattern of light and dark striations in the visual cortex can

be seen following the occlusion of one eye (Kennedy et al. 1978). These

columns correspond to ocular dominance columns identified

electrophysiologically and anatomically (figure 2). Even in the rat, it is possible

to distinguish subdivisions of a structure as small as the lateral habenula.

Glucose utilization in the lateral portion of the lateral habenula is decreased

significantly after dopaminergic agonists are administered (McCulloch et al.

1980; Wechsler et al. 1979; Porrino et al. 19846, 1988).

Another advantage is the ability to examine the entire CNS. Autoradiography

allows a broad survey of possible effects at all levels of the brain, not limiting

investigations to loci in which effects are expected. Table 1 shows

representative values for local cerebral glucose utilization in normal conscious

rats and primates in a variety of structures. If the whole brain is examined,

effects of an experimental manipulation may be seen that would otherwise have

gone unnoticed. In a primate study on the effects of lesions of the substantia

nigra induced by the infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,

alterations in cerebral glucose utilization rates were evident not just within the

basal ganglia, as expected, but also in circuits that mediate eye movements

(Porrino et al. 1987). By using this kind of an approach it is possible to survey

effects in a number of systems without using different sets of animals.

Although originally experiments with the 2-DG method were first conducted in

restrained or partially restrained animals, methods have been developed

recently that permit the 2-DG method to be used in freely moving, conscious

139
FIGURE 2. Autoradiograms of corona/ brain sections at the level of the striate

cortex. A: Animal with normal binocular vision. Note the laminar

distribution of the density; the dark band corresponds to layer 4.

B: Animal with bilateral visual deprivation. Note the reduced

density and the disappearance of the dark band corresponding to

layer 4. C: Animals with right eye occluded. Note the alternate

dark and light striations. These columns are most apparent in

layer 4 but extend through the entire thickness of the cortex. The

arrows point to regions of bilateral asymmetry where the ocular

dominance columns are absent. These are presumably areas ‘with

only monocular inputs. (From Kennedy et al. 1978 with

permission.)

140
TABLE 1. Representative Values for Local Cerebral Glucose Utilization in

Normal Conscious Rat and Monkey. (μmol/100 g per minute)

Structure

Prefrontal cortex

Motor cortex

Auditory cortex

Visual cortex

Caudate

Nucleus accumbens

Globus pallidus

Lateral hypothalamus

Ventral thalamus

Medial geniculate

Lateral geniculate

Substantia nigra compacta

Inferior colliculus

Superior colliculus

Locus coeruleus

Cerebellum

Rata Monkeyb

(n=4) (n=4)

66±3 57±2

94±1 47±2

154±3 73±2

102±2 69±1

107±2 64±4

87±2 37±2

55±1 25 ± 1

55±1 28±1

95 ±1 43±2

113±4 61±2

92±2 39±2

72±2 46±1

167±2 133±6

91 ± 1 52±1

71 ± 1 36±3

58±1 37±2

aValues from Porrino et al. 1988.

bValues from Porrino et al. 1987.

animals (Crane and Porrino 1989). This approach allows the method to be

applied to the study of behavior. Animals that were lever-pressing for electrical

brain stimulation have been studied (Porrino et al. 1984a, 1990), as have the

effects of the administration of psychostimulants on locomotor activity (Porrino

et al. 1988; Pontieri et al. 1990). One potential problem with this modification is

that the catheters are necessarily rather long. Using catethers requires that a

relatively greater amount of blood be drawn to clear the dead space before

sampling. Clearing the dead space prevents the sampled blood from mixing

with the blood that may be present in the catheter before sampling and assures

that the sampled blood actually reflects the internal state of the animal. To

eliminate the problems of a large volume of dead space in experiments, an

amount of blood equivalent to approximately twice the volume of the catheter is

removed and discarded before sampling of blood (Crane and Porrino 1989). In

this way, rates of glucose utilization can still be measured accurately in

behaving animals without compromising their physiological state.

141
One problem with the 2-DG method is that the uptake of 2-deoxyglucose is

dependent on variables such as blood pressure, cardiac output, and plasma

glucose levels. Overall differences in uptake based on these variables could

obscure differences among groups that result from changes in functional

activity, particularly if non- or semiquantitative methods are used. Because the

calculation of absolute rates of glucose utilization with the 2-DG method takes

these variables into account, however, valid comparisons can be made among

groups; the brain areas with altered functional activity can be identified and the

magnitude of the changes can be measured. These capabilities are clearly an

advantage in pharmacologic studies in which the physiological status of the

animal is frequently altered by drug administration.

DISADVANTAGES

Despite its advantages, the 2-DG method has a number of limitations that

should be considered. First, the method is unable to differentiate between direct

and indirect effects of a given stimulus. An entire pathway or circuit may be

metabolically activated even though the direct action of the stimulus may occur

only at the origin of or at some point along the pathway. The 2-DG method,

therefore, identifies the sites in the brain that are activated when a drug initiates

its actions. Apomorphine (a dopaminergic receptor agonist), for example, has

widespread effects on rates of cerebral glucose utilization (figure 3). In a study

by McCulloch et al. (1982), the areas in which glucose utilization was altered

included brain regions devoid of dopaminergic receptors. In addition,

metabolism was unchanged in regions in which high concentrations of

dopaminergic receptors are found, e.g., the nucleus accumbens. Although this

lack of specificity may be a limitation in some instances, as in the case of

determining the primary site of action of a drug, it is an advantage when the

goal is to identify the neural circuits or pathways that mediate a specific

behavioral response.

Another disadvantage is that it is not possible to identify the specific

neurotransmitters, neuromodulators, or receptor types that are responsible for

changes in brain electrical activity. This point is illustrated by a study in which

both rates of glucose utilization and turnover rates of dopamine,

norepinephrine, and serotonin were measured simultaneously in animals

receiving electrical brain stimulation to the ventral tegmental area (Smith et al.

1990). The increased rates of glucose utilization observed in the nucleus

accumbens of self-stimulating animals were accompanied by increased

dopamine turnover but decreased serotonin turnover. In contrast, in the

142
FIGURE 3. Schematic representations of three coronal sections of rat brain at

the levels of the nucleus accumbens (top), caudate-putamen

(middle), and substantia nigra (bottom). Areas rich in dopamine

receptors are shown on the left of each section; regions in which

alterations in cerebral metabolism were observed following

apomorphine administration on the right of each section. (Data

from McCulloch et al. 1982.)

143
amygdala-where increased rates of glucose utilization were also

found-norepinephrine turnover was altered. It is not possible to predict the

nature of neurotransmitter changes on the basis of alterations in glucose

utilization.

Another limitation is the inability to distinguish between inhibitory and excitatory

processes on the basis of increases or decreases in 2-DG uptake. Both

excitation and inhibition involve similar metabolic processes at the neuronal

level, in that maintenance and restoration of ionic gradients have similar energy

requirements regardless of whether excitatory or inhibitory neurotransmitters

are secreted at synaptic terminals. Ackermann and colleagues (1984)

demonstrated that when either the fimbria-fornix or perforant paths were

stimulated electrically, the resulting increases in 2-DG uptake within the

hippocampus were a consequence of the long-lasting recurrent inhibitory

processes rather than of the brief excitatory potentials that also accompany the

stimulation. The increased 2-DG uptake in this study was the consequence of

the increased activity of afferents to the hippocampus. Neither excitation nor

inhibition was the critical determinant of the direction of changes in rates of

glucose utilization.

Because constant conditions are considered necessary for measuring rates of

glucose utilization (Sokoloff et al. 1977) the long experimental time period can

be a significant limitation in drug studies. Physiological and behavioral

responses to drug administration can vary significantly during the 45 minutes

required for applying the 2-DG method. Although the total experimental time is

45 minutes, the uptake and phosphorylation of 2-DG in brain reflect

predominantly the metabolism of 2-DG in the first 5 to 10 minutes. It is so

because the tissue concentrations of free 2-DG available for metabolism are

highest during the first 5 to 10 minutes after it is injected (Sokoloff et al. 1977).

Choosing the optimal time for drug administration in relation to the time of

injecting the 2-DG is important to ensure maximal sensitivity of the method as

well as to ensure that data will be comparable across treatments. Drug

administration should be timed so that the maximum drug response coincides

with the time of maximum incorporation of 2-DG.

A final disadvantage that should be considered is that repeated measurements

are not possible in the same animal. Groups of separate animals must be used

to determine the effects of each experimental condition. The use of separate

animals increases variability as well as the number of animals necessary.

144
FACTORS INFLUENCING INTERPRETATION

One of the most important considerations in the interpreting alterations in rates

of glucose utilization is the choice of measure. Because it can sometimes be

difficult to design experimental procedures that allow for blood sampling of

freely moving, behaving animals, the method has been modified to eliminate the

necessity for catheterization and blood collection. These modifications have led

to the use of alternatives to rates of glucose utilization as measures of

functional activity. Some of these modifications, however, compromise the

validity of the method and, in effect, produce erroneous results (for additional

discussion see Sokoloff et al. 1983). In many of these modifications (Meibach et

al. 1980; Gallistel et al. 1982) the radioactive tracer is injected intraperitoneally.

This route of administration results in a high proportion of unphosphorylated

deoxyglucose as a percentage of the total radioactivity in tissue at the end of

the experiment-as much as 20 percent in gray matter and 40 percent in white

matter (Kelly and McCulloch 1983a). Following an intravenous pulse, in

contrast, most of the unmetabolized 2-DG is cleared from the tissue, leaving as

little as 5 percent in gray matter and 10 percent to 12 percent in white matter

(Kelly and McCulloch 1983a). With intraperitoneal tracer injections, the higher

proportion of unmetabolized tracer can mask true changes in metabolism.

Three other factors besides route of administration can affect the clearance of

unmetabolized 2-DG from brain tissue: plasma glucose concentration, cardiac

output, and the rate of glucose consumption in other tissues. These factors may

vary significantly with experimental conditions, particularly in pharmacologic

experiments, leading to errors. Optical densities, or even tracer concentrations,

of given brain regions cannot be compared accurately from animal to animal

without taking into account the proportion of unmetabolized tracer.

To circumvent these problems, some investigators (Collins 1978; Sharp et al.

1983) have used relative rates of glucose utilization, or the ratio of optical

densities, or tracer concentrations of a given structure to that of white matter.

The validity of gray-white matter ratios as a measure of glucose utilization

depends on the assumption that optical densities or tracer content in white

matter are constant across conditions. This assumption may be valid at times,

but many pharmacologic treatments can cause changes that invalidate it. The

choice of the particular white matter measured may also be critical; the fornix or

the internal capsule, for example, may be more sensitive to drug-induced

changes than the corpus callosum or the optic tract. In addition, the relationship

between tracer concentration in tissue and optical density is not a linear one,

145
and it varies with exposure time (Kelly and McCulloch 1983b). The exposure

times chosen must be on the linear portion of the curve for the particular film

type.

Other investigators have used a measure in which the average optical density

of a given structure is related to the average optical density of the entire section

in which the structure is analyzed (Gallistel et al. 1982). The relative optical

density (ROD) measure and the gray-white ratios suffer from the same

problems: (1) lack of constancy of the referent; (2) nonlinearity of the

relationship of optical density to tracer concentration; (3) failure to take into

account differences in the percentage of unmetabolized tracer; and (4) the use

of intraperitoneal injections. There is, however, an additional problem with this

measure. Because increased or decreased 2-DG uptake may occur in a

number of structures sampled in the same section (i.e., the average rate in the

entire section is likely to be increased or decreased), it may not be possible with

the ROD method to detect changes in some structures if those changes are

proportional to the changes in the entire section. If a particular structure

happens to be sampled in sections in which tracer uptake has not been altered,

changes in that structure are more likely to be detected than if sampling

occurred in sections in which many changes occurred.

RODS and local rates of cerebral glucose utilization were measured in

autoradiograms of two groups of animals treated either with methamphetamine

(2.5 mg/kg, intravenously) or saline vehicle. Identical brain regions on each

section were analyzed simultaneously for each measure. Of the structures

analyzed, rates of glucose utilization were increased in the caudate-putamen

(+62 percent), nucleus accumbens (+39 percent), olfactory tubercle

(+42 percent), and anterior cingulate cortex (+18 percent). In contrast, RODS

measured simultaneously in the same autoradiograms were increased in the

caudate-putamen (+15 percent) and olfactory tubercle (+23 percent),

unchanged in the nucleus accumbens (+4 percent), and decreased in the

anterior cingulate (-12 percent). The ROD measure not only underestimated

the increases in glucose utilization in some structures but also failed to detect

changes in others and yielded spurious results regarding the direction of

change in still others (figure 4). Clearly, caution must be used in interpreting the

results of any study in which this measure is employed.

146
FIGURE 4. Comparison of changes in rates of glucose utilization and relative

optical densities (RODs) in the posterior caudate (top) and the

anterior cingulate cortex (bottom) in rats treated with

methamphetamine (METH; 2.5 mg/kg, intravenously) and saline

vehicle (CONT). Scales have been adjusted so that measures of

glucose utilization and ROD of control are equivalent on each

scale. Scales of glucose utilization and ROD have been adjusted

so that the values of the methamphetamine-treated group

illustrate the percentage of change from control for each measure.

147
CONCLUSIONS

The 2-DG method provides a number of unique advantages for

neuropharmacologic investigations. First, the effects of a drug can be assessed

comprehensively because the entire brain can be examined at one time.

Second, studies can be carried out in conscious animals that are not restricted.

Third, the 2-DG method is a powerful means to relate structure to function, in

that a dynamic neuronal process can be measured with the resolution of

autoradiography. Finally, since there is a unique pattern of alteration in

functional activity associated with any given drug, the effects of a variety of

drugs of the same and different pharmacologic classes can be compared and

contrasted in a novel and highly informative way.

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AUTHOR

Linda J. Porrino

Department of Physiology and Pharmacology

Bowman Gray School of Medicine

Medical Center Boulevard

Winston-Salem, NC 27157-1083

151

Interactions of Neurotransmitters With Drugs

and Behavior

Lewis S. Seiden

The behavior of organisms can be considered either a dependent variable or an

independent variable. Since the brain affects behavior, behavior can be

considered dependent on neural activity. On the other hand, the brain’s status

(e.g., how it synthesizes neurotransmitters) depends on the amount of ongoing

behavior (an independent variable). Consequently, there is interaction among

behavioral events, both dependent and independent. In this chapter I review

evidence that leads to a concept of brain-behavior interactions and then

propose a framework for explaining a way in which drugs, behavior, and

neurochemistry interact with one another.

In 1867, Claude Bernard proposed that life forces are geared toward

maintaining a constancy of the internal milieu and that the behavior and

physiology of the organism maintain this balance; this postulate implied that a

balance is restored after chemicals are utilized. For example, food deprivation

elicits a feeding response to restore nutrient balance. A few years later Bernard

put forward the concept of homeostasis to designate the maintenance of the

internal milieu (Bernard 1927). The concept of homeostasis had profound

implications for nervous system function as well as for basic biochemical and

cellular processes. Bernard went on to consider stress. A stressor was

conceptualized as a massive sensory stimulation that emanates most often

from the external environment and alters the physiology of the organism. After a

stressor occurs, physiological responses take place that mitigate against the

stress-induced response to achieve homeostasis. In humans, it is possible to

identify internal stimuli such as anxiety or pain, which could also lead to a

physiological stress response. Thus, Bernard, with his notion of the internal

milieu, set the conceptual framework for much of the research into central

nervous system function that has taken place in the past century.

153
Later, W.B. Cannon, a major figure in physiological psychology, observed that

stress results in an increase in circulating corticosterone (Cannon 1922). This

observation became the operational definition of a stress response, and the

stimuli eliciting the increase were called stressors. Furthermore, Cannon noted

that the stress response is accompanied by an increase in sympathetic tone

and a decrease in parasympathetic tone, and so he suggested that stress is

mediated by sympathetic nervous system activity. Von Euler later couched this

idea in chemical terms by showing that animals with hypertension (which

occurs with stress) had alterations in adrenergic tone (von Euler and Sjostrand

1943). Cannon also noted that behavioral changes are part of the response to

stress, and this point is consistent with Bernard’s ideas of homeostasis. An

example of a behavioral concomitant to stress is the increase in an animal’s

aggressive posturing in response to threat. Avoidance might also occur as a

response to threat. Thus, Cannon proposed the famous flight-or-fight response

(Cannon 1922).

A few years later, Hans Selye became interested in stress and coined the term

general adaptation syndrome for a group of stereotypic responses to stressors

(Selye 1950). Examples of stimuli eliciting the syndrome are extreme heat or

cold, infection, muscular fatigue, and large doses of drugs.

A combined definition of stress can be derived from the writings of Selye and

Cannon: stress is a group of responses to a specific pattern of stimuli, and the

responses include an increase in adrenal cortisol secretions, an increase in

circulating epinephrine and norepinephrine (NE), and an increase in the rate of

specific behaviors. Responses may be short-term (acute) or long-term

(chronic). Over a long period of exposure to stressors, either hypersensitive or

tolerant responses may occur. The short-term response is a flight-or-fight

response, in contrast to the long-term general adaptation syndrome, which

includes habituation or tolerance and in which the flight-or-fight response is

often extinguished.

Dopamine (DA) and NE were discovered in the brain in the 1950s.

Considerable data supported the notion that the effects of certain psychotropic

drugs, as well as the stress response, are mediated by centrally located NE and

DA (Gordon et al. 1966). Several investigators reported that NE levels as well

as turnover rates were changed by stressful stimulation. Early attempts to

determine central nervous system events as a simple function of stress, such

as continuous electrical shock, produced variable results (Bliss et al. 1966;

Gordon et al. 1966; Modigh 1974). Levels of NE and DA were found to be lower

154
in brains of individually housed mice than in brains of group-housed mice.

Turnover was also increased in isolated animals.

Other work showed altered NE and DA turnover in brains of animals exposed to

shock or shock avoidance (Hurwitz et al. 1971). Even without shock in the

experiments, once the rat had been trained to avoid shock, there was a change

in NE turnover as a result of training on the avoidance schedule. Bliss et al.

(1966) noted a change in levels of central NE between self-stimulating and

shocked rats; Thierry et al. (1968) noted long-term changes in NE metabolism

in response to electroconvulsive shock (ECS) in rats and found that the effects

of ECS persisted after animals had received it. This discovery was important

not only because the changes were acute but also because the changes could

persist. The persistent nature of the changes implies that the history of the

animal is an important determinant of the turnover rate of the amines, which, in

turn, may become an important determinant of behavior.

Looking at a paradigm that did not involve shock, Draskoczy and Lyman (1967)

measured the turnover rate of NE in hibernating and awake ground squirrels.

The ground squirrel responds to the lowering of its core temperature by

hibernating. During hibernation, NE metabolism decreases; the rate of NE

metabolism in the brain decreases to zero, but the rate in the adrenal medulla

and in adipose tissue, while very low, is still measurable. When core

temperature is increased, the squirrels come out of hibernation and the rate of

NE metabolism returns to normal; this rate is comparable to that of other

mammals that are awake. The rate of NE metabolism in ground squirrels can,

therefore, be manipulated by changing the environmental circumstances.

Changes in catecholamine (CA) turnover in brain can also be observed in an

operant response (Schoenfeld and Seiden 1969). Tyrosine hydroxylase

inhibition by alpha-methyltyrosine (AMT) engendered a decrease in

fixed-interval (FI) and fixed-ratio (FR) responding, and when tyrosine

hydroxylase was inhibited, the rates of responding under the FR schedules

showed a decrease proportional to the FR requirement. This change in

behavior was a function of the ratio size and suggested that ongoing behavior

itself was an important mediator of the drug effect. We concluded from these

data that the initial rate is an important factor in determining the depletion of

CAs resulting from tyrosine hydroxylase inhibition because the rates were

higher in FR schedules, but there were only small differences in rates among

the various FI schedules. The effect of tyrosine hydroxylase inhibition was

related to the pre-drug rate of responding (Schoenfeld and Seiden 1969). It is

155
also important to point out that the rate of responding decreased over time in

rats that were lever-pressing on the FR 10 schedule. At the beginning of the

session, the rate of lever pressing in the treated rats started close to

saline-treated rats, but the rate of lever pressing among the AMT-treated rats

rapidly decreased after about 15 minutes of the session had elapsed. DA and

NE in brain were partially depleted by the AMT treatment at the beginning of the

session. The rate of lever pressing decreased during the session, and it was

shown that this rate decrease over time could be prevented with L-dopa, which

replenished DA and NE. The decrease in responding with time and the

restoration with L-dopa suggested that responding itself depleted DA or NE or

both.

Lever pressing also had a direct effect on brain DA and NE in rats trained on a

variable-interval (VI) schedule of reinforcement. Two groups were trained on the

VI schedule, but one group was given tyrosine hydroxylase inhibitor while they

were in their home cage, and the other group was given the drug while they

were in the operant chamber. There was a significantly greater depletion of DA

and NE depletion in the group in the operant chamber. This observation

strongly suggested that lever pressing in the face of tyrosine hydroxylase

inhibition could further deplete DA and NE. Another experiment confirmed that

lever pressing influences NE metabolism in brain (Lewy and Seiden 1972).

Tritiated NE was introduced into the lateral ventricle of rats pressing a lever on

a VI schedule and the rats were sacrificed at the end of the session. Both the

total activity of NE in brain and the specific activity of NE in brain were reduced

as a result of lever pressing, suggesting that the behavior or some

environmental aspect of the operant procedure was fed back to the brain and

increased the release and synthesis of NE.

By using a multiple schedule, it was shown that amphetamine (AMPH)

increases low response rates and decreases high response rates. This

phenomenon is known as rate dependency (Kelleher and Morse 1968; Dews

and Wenger 1977; Dews 1958). On the basis of the data that have been

presented, which demonstrate that behavior can alter turnover of DA and NE

and that drug effects depend on ongoing behavior, we could begin to

conceptualize the mutual interactions among drugs, brain chemistry, and

behavior (figure 1). Drugs influence brain chemistry, which can influence

behavior. Behavior can alter the environment, and a specific alteration in the

environment can reinforce behavior. In addition, behavior and the environment

can also alter brain chemistry. Therefore, brain chemistry, behavior, and the

environment are highly interactive. Drugs effects on behavior are not

156
DRUGS BRAIN

CHEMISTRY

BEHAVIOR

ENVIRONMENT 

FIGURE 1. Interactions among brain chemistry behavior, and environment.

predictable entirely by the way in which drugs alter brain chemistry, because

other mechanisms operate on brain chemistry, and these must be taken into

account before one can determine the resultant behavior. A number of variables

may account for the effects of operant behavior on brain chemistry: the

consummatory response, deprivation, the nature of the operant, the amount of

locomotion, the effects of the contingent relationship between the response and

the reinforcement, and the history of the organism.

Feeding behavior affects the release of DA in several regions of the brain. The

ratio between the DA metabolite dihydroxyphenylacetic acid (DOPAC) and DA

was measured as a function of food deprivation; access to food and food

consumption followed. There are regions of the brain where the levels of NE

and DA do not change. In one study, food deprivation did not increase the

turnover of DA but feeding for an hour engendered an increase in rate of

turnover of DA in the hypothalamus and in some mesolimbic areas. Alterations

in DA turnover as a function of feeding reveals specificity for certain areas of

the brain. In the caudate-putamen and some mesolimbic areas, no changes

occurred; changes in DA metabolism resulting from continuous access to food

after food deprivation revealed that the nucleus accumbens, the amygdala, and

157
the hypothalamus showed an increase in DA turnover. Ingestion of food in a

normal pattern, nutrition, and sensory signals engendered by food intake may

account for the changes; these factors play a role in the food-deprived feeding rat.

For this reason, tube-fed rats were used as controls in some experiments. These

studies showed that all three factors-food, nutrition, and stimulation of the

gut-increased DA turnover in the amygdala. In the hypothalamus and

accumbens, only food and oral intake increased DA turnover (Heffner et al. 1980).

In another experiment NE turnover was measured as a function of water

consumption. Rats were presented with 0.02 mL of water as a reinforcer on an

operant schedule. The frequency of presentation and quantity of water were varied,

and, in some experiments, the water was presented without a lever-pressing

contingency. The results of these experiments show that the number of water

presentations is an important determinant of the NE turnover rate. The number of

presentations was proportional to the rate of turnover even when the total amount

of water presented was held constant. The total amount of water was not related to

the turnover rate in a linear fashion (Heff ner et al. 1984; Heff ner et al. 1981;

Luttinger and Seiden 1981; Emmett-Oglesby et al. 1975; Albert et al. 1977).

In the caudate-putamen the turnover was directly related to the response rate. An

experiment used lever pressing on an FR 5 schedule of water reinforcement. Rates

of turnover differed depending on the area, but no direct relationship to the

response rate existed in the areas of the brain examined (Heffner et al. 1984).

In the last experiment, we demonstrated that the effects of AMPH and lever

pressing on DA turnover were additive and may account for the rate-decreasing

effects engendered by AMPH because of the mutual interactions of AMPH and

lever pressing on DA turnover. AMPH caused an increased rate of DA turnover

in rats that were not pressing; saline-treated animals that were lever-pressing

on a VI 30-s schedule also showed an increase in the rate of DA turnover. If

AMPH and operant behavior were combined, the rate of DA turnover doubled.

In this case, the response went down because rats that were lever-pressing on

a VI 30-s schedule have an intermediate response rate. The drug effect on DA

added to the behavioral effect and resulted in an altered behavioral effect

(Seiden and Heff ner 1984). These results are consistent with the idea that the

chemistry of the brain is determined by a multiplicity of factors and that these

factors can result in the final state that influences behavior.

The data presented in this paper are quite consistent with the basic ideas

expressed by Claude Bernard some 125 years ago. Bernard maintained that

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stimulation of the organism that alters physiology will lead to further alterations

in physiology or behavior or both, which will tend to correct the original

alteration. Viewed in these terms, the rat, pressing the lever and depleting its

CAs faster than they can be replenished, stops pressing the lever; this

alteration in behavior then slows down the rate of amine utilization. Two

important factors then are reciprocally interacting with each other: the brain

chemistry (which is a necessary antecedent for the occurrence of the behavior)

and the change in that biochemistry (which is a consequence of the behavior).

By considering the dynamic interaction of the two with regard to Cannon’s

homeostasis principle, one should be able to predict behavior accurately over

time, provided one understands the effects of the environment and behavior

itself on brain chemistry as well as the role of that chemistry in behavior.

REFERENCES

Albert, L.H.; Emmett-Oglesby, M.W.; and Seiden, L.S. Effects of schedules of

reinforcement on brain catecholamine metabolism in the rat. Pharmacol

Biochem Behav 6:481-486, 1977.

Bernard, C. Quoted by Nitzescu and Runceanu. Compt. Rend. Soc. de Biol

97:1109, 1927.

Bliss, E.L.; Wilson, V.B.; and Zwanziger, J. Changes in brain norepinephrine in

self-stimulating and “aversive” animals. J Psychiatr Res 4:59-63, 1966.

Cannon, W.B. New evidence for sympathetic control of some internal

secretions. Am J Psychiatry 2:15, 1922.

Dews, P.B. Studies on behavior. IV. Stimulant actions of methamphetamine.

J Pharmacol Exp Ther 122:137-147, 1958.

Dews, P.B., and Wenger, G.R. Rate-dependency of the behavioral effects of

amphetamine. In: Thompson, T. and Hughes, P.B., eds. Advances in

Behavioral Pharmacology. Vol. 1. New York: Academic Press, 1977.

pp. 167-227.

Draskoczy, P.R., and Lyman, C.P. Turnover of catecholamines in active and

hibernating ground squirrels. J Pharmacol Exp Ther 155:101-111, 1967.

Emmett-Oglesby, M.W.; Lewy, A.J.; Albert, L.H.; and Seiden, L.S. Role of lever

responding and water presentation in altering at brain catecholamine

metabolism. J Pharmacol Exp Ther 204:406-415, 1975.

Gordon, R.; Spector, S.; Sjoerdsma, A.; and Udenfriend, S. Increased synthesis

of norepinephrine and epinephrine in the intact rat during exercise and

exposure to cold. J Pharmacol Exp Ther 153:440-447, 1966.

Heffner, T.G.; Hartman, J.A.; and Seiden, L.S. Feeding increases dopamine

metabolism in the rat brain. Science 208:1168-1170, 1980.

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Heffner, T.G.; Luttinger, D.; Hartman, J.A.; and Seiden, L.S. Regional changes in

brain catecholamine turnover in the rat during performance on fixed ratio and

variable interval schedules of reinforcement. Brain Res 214:215-218, 1981.

Heffner, T.G.; Vosmer, G.; and Seiden, L.S. Time-dependent changes in

hypothalamic dopamine metabolism during feeding in the rat. Pharmacol

Biochem Behav 20:947-949, 1984.

Hurwitz, D.A.; Robinson, S.M.; and Barofsky, I. The influence of training and

avoidance performance on disulfiram-induced changes in brain

catecholamines. Neuropharmacology 10:447-452, 1971.

Kelleher, R.T., and Morse, W.H. Determinants of the specificity of behavioral

effects of drugs. Ergebn Physiol Biol Chem Exp Pharmakol 60:1-56, 1968.

Lewy, A.J., and Seiden, L.S. Operant behavior changes in norepinephrine

metabolism in rat brain. Science 175:454-456, 1972.

Luttinger, D., and Seiden, L.S. Increased hypothalamic norepinephrine

metabolism after water deprivation in the rat. Brain Res 208:147-165, 1981.

Modigh, K. Effects of social stress on the turnover of brain catecholamines and

5-hydroxytryptamine in mice. Acta Pharmacol Toxicol 34:97-105, 1974.

Schoenfeld, R.I., and Seiden, L.S. Effect of alpha-methyltyrosine on operant

behavior and brain catecholamine levels. J Pharmacol Exp Ther

167:319-327, 1969.

Seiden, L.S., and Heffner, T.G. Alteration of brain catecholamine metabolism by

environmental and behavioral events: An explanation of drug-behavior

interactions. In: Catecholamines, Neuropharmacology and the Central

Nervous System: Theoretical Aspects. New York: Alan R. Liss, Inc., 1984.

pp. 275-284.

Selye, H. The Physiology and Pathology of Exposure to Stress. 1st ed.

Montreal, Canada: Acta, Inc., 1950.

Thierry, A.; Javoy, F.; Glowinski, J.; and Kety, S.S. Effects of stress on the

metabolism of norepinephrine, dopamine and serotonin in the central

nervous system of the rat. I. Modifications of norepinephrine turnover. J

Pharmacol Exp Ther 163:163-171,1968.

von Euler, S., and Sjostrand, T. Pressor substances in urine and plasma from

normal and hypertensive subjects. Nature 151:168, 1943.

AUTHOR

Lewis S. Seiden, Ph.D.

Department of Pharmacological and Physiological Sciences

University of Chicago

Chicago, IL 60637

160
Historical Influences Affecting the Behavioral

Actions of Abused Drugs

James E. Barrett

INTRODUCTION

Many attempts to describe factors contributing to the etiology of drug abuse

have concentrated solely on the inherent characteristics of the drug and on the

neurobiological substrates at which those drugs potentially exert their complex

effects. An overriding emphasis on the pharmacology of the drug and on its

interaction with multiple neuronal systems, while of unquestionable importance,

potentially neglects other variables that can contribute significantly to the effects

of a drug and, perhaps, to the likelihood that it will be abused. Experiments

summarized in this chapter focus on what appears to be a behaviorally

determined malleability in the effects of drugs of abuse. These studies indicate

that the behavioral actions of abused drugs can be determined by conditions

that have occurred in the past, even in the absence of overt features of

behavior that normally predict drug activity. Importantly, the altered effects of

these drugs persist long after those initiating conditions have terminated,

leaving residual traces of previous experience that become apparent only after

an abused drug is administered. These findings suggest that prior behavioral

experience may actually predispose an individual to a particular drug effect by

altering the initial drug experience, which may then influence the likelihood of

subsequent drug use. This outcome could represent an initial step toward

eventual drug abuse. Conversely, it is possible that other behavioral

experiences could block particular drug effects, serving as “immunizations”

against subsequent drug use. Together, these outcomes could account for

some of the individual differences in drug effects and in the behavioral

disposition to abuse drugs. Conditions that render an individual more or less

vulnerable to the effects of abused drugs warrant careful evaluation. The

experimental analysis of those conditions, with a corresponding emphasis on

arriving at a better understanding of the involvement of correlated

neurobiological processes, should yield considerable information about

161
individual vulnerability to the effects of abused drugs. This information should

then permit more effective behavioral and pharmacologic strategies to be

developed, which could be of use in treating and preventing drug abuse.

BEHAVIORAL HISTORY

The behavioral effects of many drugs often can be predicted on the basis of the

characteristics of behavior occurring at the time the drug is administered. An

example of this principle is frequently referred to as rate dependency, in which

the effects of psychomotor stimulants are related inversely to the rate of

responding in the absence of the drug (Kelleher and Morse 1968). The

dependence of a drug effect on the existing rate or pattern of behavior is an

instance of control by relatively proximal variables that exert more immediate

influences on the effects that a drug has on behavior. There are, however,

several examples in which the effects of a drug appear to be related to

conditions that have occurred in the past but are not present in the immediate

environment. For example, early experiments conducted by Steinberg and

colleagues using exploratory behavior of rats found that an

amylobarbitone-amphetamine mixture produced large increases in exploratory

behavior in rats that had no experience in the maze, whereas with experienced

subjects, these drugs had little effect (Steinberg et al. 1961). Subsequent

experiments by these investigators evaluating both behavioral and

pharmacologic history led to the general conclusion that “behavior under the

influence of psychoactive drugs depends on striking a flexible balance between

the drugs themselves, the precise circumstances under which they are

administered, and the state of the recipient at the time of administration. The

state of the recipient, in turn, depends on its previous experiences” (Rushton et

al. 1963, p. 888).

One aspect of these studies involving exploratory or locomotor behavior

warrants attention. In every instance in which experience was shown to alter

the effects of a drug or drug combination in these studies, the experience itself

also directly changed the behavior under study. For example, in the Steinberg

et al. (1961) study, after repeated exposure to the maze, the experienced rats

showed activity levels almost two times higher than those of the inexperienced

rats. In view of the important contribution of baseline levels of activity in

determining the effects of drugs (i.e., rate dependency), these findings must be

interpreted somewhat cautiously. Any experience that modifies behavior before

a drug is administered and leaves an enduring measurable effect on overt

behavior would demonstrate an “experiential” effect. It would be the case, for

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example, if animals trained under one schedule of reinforcement were provided

with experience under a different schedule and then returned to the original

procedure at which time the response rates differed from those obtained

initially. If the behavioral effects of a drug differ before and after the intervening

schedule history, it is impossible to separate the historical influences from those

that directly affect behavior (Urbain et al. 1977). The isolation of a “pure” effect

of experience on behavior should, therefore, be observed in the absence of a

lasting overt influence on the behavior being studied, given that no other

intervention such as the administration of a drug has occurred. Otherwise, it

would be difficult to differentiate between effects that change behavior directly

and then modify subsequent drug action and affects that do not appear to

modify overt behavior but that, nevertheless, produce a change in the typical

effects of a drug.

More recent experiments using squirrel monkeys, rats, and pigeons have

supported and extended this conclusion about the influence of behavioral

history on drug activity. For example, in one experiment with squirrel monkeys,

d-amphetamine decreased food-maintained lever pressing that was punished

by the delivery of electric shock (Barrett 1977). This outcome is typical for this

drug under these conditions and has been reported by several investigators and

in several species (e.g., Geller and Seifter 1960; Hanson et al. 1967; Spealman

1979). However, when monkeys trained initially under the punishment

procedure were exposed subsequently to a shock-postponement schedule and

then returned to the punishment schedule, d-amphetamine produced large

increases in punished responding. These increases were not dependent on

changes in response rate since these behavioral measures were comparable

before and after the interpolated training under the avoidance schedule. In

addition, the altered rate-increasing effects of amphetamine persisted unless

other interventions occurred (Barrett 1985). Effects of different drugs, such as

chlorpromazine, were not altered by the sequence of conditions that modified

the effects of d-amphetamine (Bacotti and McKearney 1979), suggesting that

only the effects of certain compounds may be changed by these conditions. It

may be that only drugs of abuse, for reasons yet unknown, share this

malleability to engender different effects that depend on nonpharmacologic as

well as pharmacologic influences.

Additional experiments have shown that previous experience can reverse the

effects of other abused drugs such as cocaine, morphine, and chlordiazepoxide

(Barrett and Stanley 1983; Barrett and Witkin 1986; Barrett et al. 1989) as well

as influence the effects of methadone and the development of tolerance to

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methadone (Egli and Thompson 1989; Nader and Thompson 1987, 1989). One

very striking feature of many of these studies is that drug effects were different

after exposure to a particular condition even though no changes in ongoing

behavior were manifested after that condition and even though those conditions

were no longer part of the current environment. The administration of a drug

can reveal characteristics of behavior and its control by previous environmental

conditions that are otherwise not evident in ongoing behavior.

Despite the several experiments showing the importance of behavioral history

in altering the effects of drugs, there are few studies directly addressing the

specific variables that might contribute to those effects. It is known that a history

of avoidance is necessary to reverse the effects of d-amphetamine on punished

behavior, because monkeys in a yoked control condition that received

response-independent shock did not show any changes in the effects of

d-amphetamine (Barrett and Witkin 1986). Thus, it is not shock exposure per se

that reverses the effects of d-amphetamine but a specific avoidance history. A

history of exposure to certain schedules can modify drug actions, but it is not

clear what aspect of that schedule history is important (Barrett 1986).

Several possible approaches might clarify how behavioral variables alter the

effects of a drug. For example, in many of the experiments in which drug effects

were modified by behavioral experience, the same event was used at different

times to control behavior in different ways. The effects of d-amphetamine on

responding suppressed by the presentation of shock were reversed by

interpolated exposure to a shock postponement schedule (Barrett 1977). Thus,

the same shock stimulus was used at different times either to maintain or

suppress responding. As a result of these complex behavioral histories, stimuli

may be established that have multiple behavioral functions, which are then

revealed by the administration of a drug. How important is it that the stimulus be

the same in order to obtain these effects? Is only an avoidance history

important, regardless of the nature of the event, or must the punishment and

avoidance stimuli be identical? These questions could be addressed by using

different types of response consequences to control behavior. Studies of this

type may also provide fundamental information about the functional

consequences of establishing complex environmental stimuli (Sidman 1986)

that may be of general significance to our understanding of how complex

histories and stimulus control contribute to the behavioral effects of drugs.

It is also not clear at present whether changes in the effects of a drug by

behavioral history also modify that drug’s ability to function as a reinforcer. A

164
recent study that demonstrated individual vulnerability in response to the effects

of amphetamine (AMPH) seems particularly appropriate to this issue. Piazza et

al. (1989) reported that individual rats could be identified for “addiction liability”

on the basis of their individual response to either environmental or

pharmacologic challenges. Rats that displayed high levels of locomotor activity

in a novel environment responded with even higher activity levels after AMPH

was administered than did rats that showed lower activity levels in the absence

of the drug. Moreover, the high-activity rats rapidly acquired intravenous AMPH

self-administration, whereas rats from the other group did not. These findings

suggest that there are individual differences in the effects of AMPH and in the

predisposition to self-administer this drug and that such differences may be

related to observable behavior. Although the study suggests that differences

between rats in the behavioral and reinforcing effects of AMPH are genetically

determined, it also raises the possibility, in view of the results that have been

summarized, that differences in these effects may also be induced by

behavioral manipulations.

NEUROBIOLOGICAL CORRELATES OF BEHAVIORAL HISTORY

Although the molecular properties of the drug do not seem solely responsible

for its behavioral effects and abuse potential, it is clear that these compounds

interact with neuroanatomically and neuropharmacologically distinct substrates

in the central nervous system (CNS). How these substrates are modified by

factors such as past behavioral history is not currently understood, but it is likely

to be a fertile avenue for future research. In many respects, the factors that

contribute to the qualitative and quantitative changes in drug effects may be

related to more general principles involved in learning. Historical influences

suggest that plasticity of the CNS continues well beyond the initial

developmental period during which such changes normally occur. Techniques

are now available for monitoring neurochemical correlates of behavioral

processes. The combined approach to many of these issues, including the

development of molecular biological procedures, should yield considerable

insight into the transient and more enduring changes in CNS activity that are

related to behavioral interventions (Barrett 1991; Barrett and Nader 1990;

Dworkin and Smith 1989).

PHARMACOLOGIC HISTORY

Experiments demonstrating that the effects of abused drugs can be modified by

a particular behavioral history are paralleled by experiments demonstrating that

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pharmacologic history can also modify a drug’s behavioral and discriminative

stimulus effects. For example, the effects of pentobarbital on punished

responding of squirrel monkeys maintained under a stimulus-shock schedule

depended on whether those monkeys were previously administered

d-amphetamine or morphine (Glowa and Barrett 1983). In monkeys that had

received morphine, pentobarbital did not increase punished responding,

whereas in those that had received AMPH, pentobarbital produced increases in

punished behavior. These effects occurred long after pharmacologic factors

related to the administration of either morphine or AMPH could have played a

role in the effects of pentobarbital.

A similar result was obtained in studies examining the discriminative stimulus

effects of novel antianxiety drugs. Using a drug discrimination procedure in

which the mixed dopamine (DA) antagonist and serotonin (5-HT) agonist

spiroxatrine was established as a discriminative stimulus, it was found that

either the DA or the 5-HT elements of the compound stimulus could be salient,

depending on the animal’s drug history (Barrett and Olmstead 1989). If the

progression of drug substitution testing was initially biased toward DA, the

animals responded to buspirone (which is similar to spiroxatrine in its mixed

5-HT1A and DA activity) but did not generalize to more specific 5-HT1A agonists

such as 8-OH-DPAT, which shares buspirone’s 5-HT1A actions. If the

progression of drug substitution testing was initially biased toward 5-HT1A

compounds, then compounds such as 8-OH-DPAT and several other 5-HT1A

ligands occasioned spiroxatrine-key responding. Thus, the relative

neurochemical balance of these drugs, as measured by their discriminative

stimulus effects, could be altered by the pharmacologic history.

These results indicate that pharmacologic histories, much like behavioral

histories, may either enhance or diminish a particular effect of a drug. The

results with the drug discrimination procedure could be especially important for

future analysis. It is commonly accepted that the discriminative stimulus effects

of drugs are related to their subjective effects and that such effects may

correlate with abuse potential. In the experiments with spiroxatrine as a

discriminative stimulus, either the DA or the 5-HT component of spiroxatrine

could be made dominant by varying the pharmacologic history. If this history

can “direct” the discriminative stimulus effects of drugs with multiple

pharmacologic actions by enhancing the relative balance of controlling stimulus

influences, similar factors may operate in modifying the behavioral actions of

drugs. Perhaps one mechanism that accounts for the modification of effects of a

complex drug such as cocaine is that some components of the drug’s

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neuropharmacologic actions become more prominent while others are muted. If

these factors are uniquely correlated with abuse, then they may begin a

cascade of events culminating in enhanced abuse potential.

SELF-ADMINISTRATION IN PHARMACOLOGIC HISTORY

Results similar to those that have been described have been obtained using

drug self-administration studies. For example, Schlichting et al. (1970) found

that monkeys with a codeine self-administration history showed lower and more

variable levels of d-amphetamine self-administration than monkeys with prior

histories of either pentobarbital or cocaine self-administration. Young et al.

(1981) reported that the ability of dextrorphan to maintain responding was much

higher in animals with a history of ketamine self-administration than in animals

with no such history or with a history of codeine self-administration. Similarly, it

has been demonstrated recently that the self-administration of the

noncompetitive N-methyl-D-aspartate antagonist MK-801 can depend on drug

reinforcement history (Beardsley et al. 1990). In monkeys with a history of

cocaine self-administration, MK-801 was not self-administered. However, in

monkeys with a history of phencyclidine self-administration, MK-801 maintained

self-administration behavior. The abuse potential of a particular drug, as

measured by its ability to maintain drug self-administration behavior, may be

critically related to the pharmacologic history or experience.

Recent work by Falk and colleagues also has shown that different drug histories

can affect schedule-induced drug intake and preferences (Falk and Tang

1989a, b; Tang and Falk 1986, 1988). Thus, the ability of certain drugs to

maintain responding can depend on a subject’s self-administration history and

the type of drugs with which the subject has had experience. It is significant that

many of these same variables appear to play a role in determining the

subjective effects of a variety of drugs in humans, as well as in the likelihood of

drug abuse (Haertzen et al. 1983).

DRUG-BEHAVIOR INTERACTION HISTORY

Experimental studies also have demonstrated that the subsequent effects of a

drug can be modified by the behavioral consequences that occurred at the time

the drug was administered. A clear example of the importance of a

drug-behavior interaction history was reported by Smith and McKearney (1977).

Key pecking by pigeons was maintained under a procedure in which food was

produced only if at least 30 s elapsed between pecks. Increases in responding

167
produced by an initial injection of d-amphetamine decreased the frequency of

food presentation because the time between responses after drug typically was

less than the 30-s schedule requirement. However, subsequent drug

administrations, spaced days apart, resulted in progressively smaller increases

in responding, eventually resulting in the failure of d-amphetamine to have any

effect. When the contingency was removed during drug administration, these

drugs continued to produce increases in responding. In many respects, these

results resemble tolerancelike actions but actually derive from the dynamic

interactions between behavior and the environment that are induced by the

drug.

Another example of drug-behavior interaction history is provided by an

experiment in which morphine, which does not typically increase punished

behavior, did so after a drug effect occurred under a particular set of

experimental conditions (Brady and Barrett 1986). The effects of morphine were

studied under a multiple fixed-interval schedule in which responding in each of

two components terminated a stimulus correlated with electric shock delivery.

Morphine was first studied when responding of monkeys during one component

was suppressed by arranging that a shock also followed every 30th response

during the interval; this procedure reduced responding during that interval, but

responding remained at a high rate during the alternate unpunished component.

Morphine unexpectedly increased punished responding, an effect not previously

reported for this drug; these increases also occurred when the punishment

schedule was studied alone under a single-component schedule condition.

Extensive analyses of the conditions responsible for this atypical effect resulted

in the conclusion that the context in which behavior occurs when a drug is

administered can interact with the effects that drug will have on behavior.

Behavioral and pharmacologic histories can both be important, alone and in

combination. What is remarkable about these results is that the conditions

under which a particular effect is induced by the environment become less

important once the effect has been obtained. The modified actions of the drug

are then sustained in the absence of the inducing condition.

SUMMARY AND CONCLUSIONS

The studies summarized briefly in this chapter provide several examples of

experiments in which a behavioral or pharmacologic history produced profound

changes in the effects of an abused drug. In many instances these changes

were qualitative, that is, the direction of the effects that drug usually has on

behavior was modified dramatically. AMPH and cocaine-both of which may

168
normally decrease a particular behavior—will, after a suitable history, produce

large increases in the behavior under study. If the behavioral effects of a drug

are related to the abuse potential of that drug, then any condition that alters the

behavioral effects of that drug could be an important component underlying

vulnerability to drug abuse.

A noteworthy aspect of the studies that have been described is the lasting

influence that prior experience can have on behavior and the behavioral effects

of drugs in the absence of overt changes in behavior itself. The traces of past

experience may not be apparent in ongoing behavior until a drug is

administered. This feature makes drugs an important tool for studying

behavioral processes and raises several related questions pertaining to the

mechanisms responsible for these effects (Barrett 1986). Although at present

there is little information available to answer the several questions raised by

these experiments, newer techniques available in the neurosciences promise to

help reveal the neurobiological correlates of these changes that should be of

general importance in understanding both behavioral and pharmacologic

processes.

REFERENCES

Bacotti, A., and McKearney, J.W. Prior and ongoing experience as

determinants of the effects of d-amphetamine and chlorpromazine on

punished behavior. J Pharmacol Exp Ther 211:80-85, 1979.

Barrett, J.E. Behavioral history as a determinant of the effects of

d-amphetamine on punished behavior. Science 198: 67-69, 1977.

Barrett, J.E. Modification of the behavioral effects of drugs by environmental

variables. In: Seiden, L.S., and Balster, R.L., eds., Behavioral Pharmacology:

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ACKNOWLEDGMENTS

Grant support is acknowledged from the National Institute on Drug Abuse, DA

02873 and DA 06828.

AUTHOR

James E. Barrett, Ph.D.

Lilly Research Laboratories

Eli Lilly and Co.

Lilly Corporate Center

Indianapolis, Indiana 46285

172
Importance of Behavioral Controls in the

Analysis of Ongoing Events

Steven I. Dworkin, Linda J. Porrino, and James E. Smith

INTRODUCTION

One of the most compelling factors that have increased interest in the

neurobiological mechanisms of substance abuse has been concern about the

deleterious effects of the direct and indirect actions of behaviorally active

compounds. For example, cocaine was considered a relatively benign

compound until recent findings indicated the drug severely disrupts social and

occupational functioning and is associated with a high degree of morbidity and

mortality (Fischman 1987). Additional concerns resulting in increased efforts to

understand and treat drug abuse include the deleterious effects of these drugs

on the immune system and the increased risk of acquired immunodeficiency

syndrome associated with the intravenous (IV) use of these compounds.

A second factor that may account for the growing interest in delineating the

neurobiology of substance abuse is the rapid rise in technology over the past

several years in pharmacology and the neurosciences. New or refined

procedures have been developed for investigating the processes that occur in

the central nervous system directly related to the behavior of an organism

(Dworkin and Smith 1987, 1989). It is now reasonable to believe that a

neurobiological approach to substance abuse not only will lead to a greater

understanding of the factors responsible for drug abuse but will also provide

some reasonable indications and directions for the improved treatment of this

problem (Koob and Bloom 1988).

Most of the techniques and procedures for directly investigating the

neurobiologic components of substance abuse are discussed in this

monograph, ranging from those that determine the effects of pharmacologic

agents with known mechanisms of action on measures of drug abuse to those

that provide direct assessments of neuronal activity associated with drug

173
administration. Between these extremes are procedures that (1) provide

correlational analysis of receptor binding with behavioral effects (Ritz et al.

1987; Spealman et al. 1989; Smith et al. 1984b); (2) determine the effects of

specific neurotoxin lesions on the reinforcing and other behavioral effects of

abused drugs (Dworkin and Smith 1988; Koob and Goeders 1989); (3) provide

direct measures of the content, release, and utilization of neurotransmitters

correlated with ongoing behavior (Miyauchi et al. 1988, 1989; Smith et al. 1981,

1984a); and (4) provide measures of functional neuronal activity (Pettit and

Justice 1989; Porrino et al. 1984).

These neurobiological procedures must be coupled with appropriate and well

designed behavioral procedures in order to provide an unambiguous analysis of

the behavioral significance of any observed neurobiological changes. This

paper deals with some of the issues related to the selection of appropriate

behavioral baselines for neurobiological investigations.

Research in experimental analysis of behavior and behavioral pharmacology

has provided several insights into some of the important variables that should

be considered in any research effort to determine the behavioral effects of

abused drugs. These variables include nonpharmacologic as well as

pharmacologic factors (Barrett 1987). These same variables may also

determine the behavioral effects of neurobiological manipulations. The

nonpharmacologic variables that should be considered in any

behavioral-neurobiological investigation include (1) the control rate of

responding or activity, (2) the type of reinforcer or punisher, (3) the

environmental context, and (4) the behavioral history of the organism. The list of

pharmacologic factors is also of considerable importance: (1) the class and

dose of the drug, (2) the route of administration, and (3) the pharmacologic

history of the organism.

Analyzing the neurobiological aspects of drug abuse is further complicated by

the interactions that can take place between the nonpharmacologic and the

pharmacologic factors. For example, the effects of d-amphetamine on behavior

are influenced by the previously mentioned factors. Amphetamine typically

results in an inverted-U-shaped dose-effect curve on schedule-controlled

behavior. Low doses of the drug have little or no effect on responding, and

moderate doses increase operant responding, whereas larger doses decrease

response rates; moreover, the effects of amphetamine are also related to the

control rate of responding (Dews and Wenger 1977). Low to moderate rates of

responding are increased by doses of the drug that have no effect on, or

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decrease high rates of, responding in the same animal even in the same

experimental session. Thus, both the dose and the control rate of responding

can be important determinants of the behavioral effects of amphetamine. In

addition, the behavioral effects of amphetamine are influenced by both the

behavioral history of the organism and the current environmental context

(Barrett and Witkin 1986). Two studies have shown that the typical effects of

amphetamine on punished behavior can be reversed by behavioral variables in

the absence of any apparent changes in the baseline performance.

Amphetamine does not typically increase low rates of punished responding

(Houser 1978). Significant increases in punished responding, however, were

observed when d-amphetamine was given to monkeys with a history of

responding maintained by electric shock (Barrett 1977) or when responding was

maintained in alternating shock avoidance and shock punishment components

(McKearney and Barrett 1975). These effects and their determinants influence

the behavioral effects of other drugs of abuse as well as amphetamines (Barrett

and Witkin 1986).

In addition to their rate-dependent effects, abused substances have both

reinforcing and discriminative-stimulus functions. Their reinforcing effects can

be directly measured by self-administration studies, and the stimulus functions

are typically determined by means of drug discrimination procedures. These

behavioral effects are of importance to any neurobiological investigation of drug

abuse. Although the same or similar neurobiological techniques can be used to

evaluate these effects, it is unlikely that similar behavioral designs can be used

to measure all three effects. This chapter focuses on a few behavioral

procedures that are used to evaluate the neurobiological components of the

reinforcing effects of drugs and presents some of the behavioral controls that

are necessary for these investigations. In particular, the influences of behavioral

context and response contingency are presented.

BEHAVIORAL CONTEXT OF NEUROTOXIN LESIONS

Besides altering the effects of drugs on schedule-controlled behavior,

environmental context can also influence the effects of 6-hydroxydopamine

(6-OHDA) lesions of the nucleus accumbens on morphine self-administration

(Dworkin et al. 1988). Neurotoxin-lesion procedures are used to assess the

involvement of specific neurotransmitter systems in the neurobiological

components of drug reinforcement. A number of studies have reported that the

selective destruction of dopaminergic pathways from the ventral tegmental area

to the nucleus accumbens results in a decrease in the reinforcing efficacy of

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stimulants (Roberts et al. 1980; Roberts and Koob 1982). Several different

behavioral schedules have been used to evaluate the effects of dopaminergic

lesions on cocaine self-administration, including fixed-ratio (FR) and

progressive-ratio schedules and a concurrent chained schedule of food, water,

and drug self-administration (Koob and Goeders 1989). All of these studies

reported a decrease in cocaine self-administration, suggesting that this lesion

causes a selective reduction in the reinforcing effects of cocaine and thus

eliminating two other potential interpretations of the behavioral effects of this

lesion, i.e., nonselective response decrements and changes in rate-dependent

effects of the drug.

Similar attempts to elucidate the role of dopaminergic innervations of the

nucleus accumbens in the reinforcing effects of opiates have not resulted in

consistent effects. Lesions of the nucleus accumbens induced by 6-OHDA have

resulted in increased morphine self-administration in opiate-dependent rats

(Smith et al. 1985). It has been suggested that the increased rate of

self-administration resulted from a decrease in the reinforcing efficacy of the

drug. A similar lesion did not alter food, water, or morphine self-administration in

opiate-dependent rats that were studied on a concurrent chained schedule

(Dworkin et al. 1988). Furthermore, a more extensive dopamine lesion of the

nucleus accumbens resulted in a 70 percent decrease in cocaine

self-administration and only a 24 percent decrease in heroin self-administration

in rats trained to self-administer both compounds on alternate days (Pettit et al.

1984). Because several reports indicate that nucleus accumbens dopaminergic

neurons are involved in the behavioral effects of opiates, the results reported in

the latter two studies may have been a function of the environmental context;

i.e., in both of these studies, responding was maintained by other reinforcers in

addition to the opiate. Moreover, the two lesion studies on morphine

self-administration (Dworkin et al. 1988; Smith et al. 1985) were conducted in

the same laboratory using similar procedures; the obvious difference was the

food and water contingency. Thus, the results of these two studies clearly

demonstrate that environmental context can influence the effect of this

dopaminergic lesion on morphine self-administration in opiate-dependent rats.

RESPONSE CONTINGENCY

The term reinforcementas used in behavioral research has a fairly well

accepted definition: an increase in the probability or frequency of some

observable behavior when the presentation of an environmental event is

contingent on that behavior (Skinner 1938). Behavior can be changed by the

176
presentation or removal of the event; the term positive reinforcer is used to

describe the event in the first case, and negative reinforcer in the second. This

definition stresses the contingent relationship between some measurable

behavior and the delivery of the environmental event. The precise relationships

between behavior and the environmental event are collectively referred to as

schedules of reinforcement. A traditional view of reinforcement asserted that

there were three mutually exclusive categories of contingent events: reinforcers,

punishers, and neutral stimuli. An additional implication was that every

environmental event is an incontrovertible member of one of these three

categories, and thus it was assumed that the effects of all environments were

trans-situational. After 30 years of research in experimental analysis of behavior

and behavioral pharmacology, it is common knowledge among researchers that

environmental events such as administration of food and electric shock can

have both reinforcing and punishing effects (Morse and Kelleher 1977).

Furthermore, abused drugs such as cocaine can function as both positive and

negative reinforcers (Spealman 1979). The hedonic relevance of environmental

events depends on several factors, including the behavioral and pharmacologic

history of the organism and the current condition or environmental context in

which the organism is placed. Most of the research in these areas has focused

on the contingent delivery of environmental events. If the defining feature of

reinforcement is the contingent delivery of these events, however, then what is

the hedonic relevance of noncontingent presentations of putative reinforcers or

punishers? The remainder of this chapter presents some of the behavioral and

neurobiological differences observed between the contingent and

noncontingent delivery of environmental events.

NEUROBIOLOGY OF DRUG REINFORCEMENT

The yoked-box procedure (figure 1) is the behavioral paradigm that has been

used in several studies investigating the neurobiology of reinforcement. This

procedure involves the use of triads to assess relative differences related to the

contingent versus noncontingent presentation of environmental events. All

subjects are randomly assigned to one of three conditions. Responding by one

of the subjects results in the contingent delivery of a reinforcer and the

simultaneous delivery of the same environmental event to a second member of

the triad. The third subject is typically used as a control for nonspecific factors

(i.e., handling, food deprivation, exposure to the experimental chamber) and is

not exposed to the environmental event under investigation. As shown at the

bottom of figure 1, if the contingent delivery of the environmental event results

in a specific behavioral or neurobiological effect in the subject that is receiving

177
FIGURE 1. Diagram of the yoked-box procedure.

contingent deliveries of the reinforcer, then the effect is considered to be a

function of reinforcement. If similar changes are observed in both subjects that

are exposed to the environmental event under investigation, then the effect is

considered to be a nonspecific activation produced by the delivery of the event.

The third situation, in which all three subjects show the same effect, indicates

that the event has no significant behavioral or neurobiological relevance under

these conditions.

The yoked-box procedure for evaluating the neurobiology of reinforcement has

several advantages. Many behaviorally relevant variables can be controlled

simultaneously, and the potential influences of historical or contextual variables

can be eliminated. The procedure is also relatively easy to establish.

Disadvantages include various sources of bias and random error, such as

individual differences in the effectiveness of the event-which would result in

178
misleading data-and potential fluctuations in attention, which might favor the

contingent condition (Church 1964; Gardner and Gardner 1988). Selecting

environmental events with a high degree of saliency (i.e., presentation of food

to meal-deprived subjects, electric foot shock, or intravenous drug delivery) will

most likely eliminate most of the disadvantages inherent in this behavioral

design.

The yoked-box procedure was used by Smith and Dworkin (1986) to investigate

the neurobiological correlates of the reinforcing effects of morphine. A triad of

littermates, male F-344 rats, was used for these studies. Two of the rats in each

triad were made physically dependent on morphine and then randomly

assigned to one of two conditions. One rat was allowed to self-administer the

drug, while the other received noncontingent injections yoked to the delivery

schedule of the self-administering rat. A third subject was never exposed to

morphine and received yoked saline infusions. It was reasoned that any

differences observed between the rats receiving yoked-morphine and those

receiving yoked-vehicle injections would be the result of the pharmacologic

actions of the drug, whereas any additional differences between the

self-administration condition and the yoked-morphine condition would be related

to the reinforcing effects of the drug. A small FR schedule (FR 10) was used for

the self-administration condition to minimize the influence of responding on the

neurochemical measures. Neurotransmitter turnover rates (Smith et al. 1981,

1984a) and receptor-binding studies (Smith et al. 19846) indicated that the

contingent administration of the drug produced more extensive and larger

neurobiological changes than did the noncontingent administration. As a result

of these studies, it was suggested that two potential neuronal circuits are

involved in the reinforcing effects of opiates. Moreover, the proposed systems

are consistent with systems identified by 2-deoxyglucose (2-DG)

autoradiography in rats exposed to contingent versus noncontingent electrical

stimulation of the ventral tegmental area (Porrino et al. 1984).

In our attempts to evaluate the neurobiological aspects of the reinforcing effects

of cocaine using the yoked-box procedure, we have observed a more profound

and obvious difference in contingent versus noncontingent administration of

cocaine. Littermate triads of male F-344 rats were surgically prepared with

intravenous (IV) catheters. The rats were placed in three individual operant

conditioning chambers in single sound-attenuating chambers and allowed to

recover from the surgery. When food and water intake returned to baseline

values, each rat was assigned to one of three conditions. One was trained to

self-administer cocaine under an FR 1 schedule that was increased to FR 2

179
when stable rates of cocaine intake were observed. The other two littermates

received noncontingent injections of either cocaine or saline yoked to the intake

of the self-administering subject. The number of food pellets available each day

for the yoked subjects was also yoked to the food consumed by the rat that was

exposed to the contingent administration of cocaine. All subjects were

continuously housed in the operant chambers under a reversed cycle of 12 hr

light and 12 hr dark. Each drug and vehicle infusion (0.2 mL) was delivered over

approximately a 5-s period and was paired with a 20-s light and tone

presentation. Rats were exposed to one of two doses (0.33 or 0.67 mg per

infusion) under one of three different access conditions: 2-hr drug availability,

6-hr drug availability, or termination of access after the 80th injection during a

24-hr period (80 infusions per day). For the latter condition, the drug was

available from 5 a.m. until the 80th infusion was delivered. The rats were fed

twice a day, with a maximum of 20 1-mg food pellets. The data obtained from

the 0.33-mg-per-infusion, 80-infusion-per-day condition, and the 2-hr,

0.67-mg-per-infusion condition are shown in figure 2.

Three of the rats (14 percent) exposed to 0.33 mg per infusion for 80 infusions

per day died within 30 days of contingent drug administration, whereas 12 of

the rats (55 percent) that received yoked infusions of cocaine died before the

30th session. None of the rats exposed to yoked-vehicle injections died. The

major behavioral difference observed between rats exposed to contingent

administration of cocaine and those exposed to noncontingent administration of

cocaine was the lack of, or slower development of, tolerance to the anorectic

effects of cocaine. Figure 2, which shows the number of infusions and the

number of food pellets consumed for three representative triads, also shows the

development of tolerance to the anorectic effects of the drug. If tolerance to the

effect of the drug on food intake did develop in the rats that were exposed to

noncontingent infusions of cocaine, it took several more days to occur in them

versus the self-administering littermate. These findings indicate that an

enhanced morbidity and mortality are associated with noncontingent

administration of cocaine compared with contingent administration of the drug

and emphasize the importance of contingent relationships as defining features

of reinforcement mechanisms.

Eight additional triads were prepared with indwelling IV catheters for the 2-DG

studies. Cocaine infusions (0.67 mg per infusion) were available for a 2-hr

period each day Monday through Friday. The animals were placed into operant

conditioning chambers for the cocaine exposure period; when not in the operant

chambers, they were placed in a home cage and given bihourly infusions of

180
FIGURE 2. Number of infusions (solid line) and food consumption for the rat

receiving contingent (SA, dotted line) and noncontingent (YC,

dashed line) over consecutive sessions. Panels shown contain

data from three representative trials in which the yoked animal

died.

saline. The self-administering rats were trained under an FR 2 schedule. Three

of the yoked-cocaine littermates died within 14 days of exposure to cocaine.

Four of the triads were pulse labeled with 2-DG during the 15 days of drug

exposure. All rats exposed to cocaine engaged in excessive conditioned

stereotypy by the second day of exposure. The contingent administration of

cocaine resulted in an increase in local cerebral glucose utilization in several

regions: prefrontal cortex, ventral pallidum, globus pallidus, and nucleus

181
accumbens. It has been suggested that these regions are involved in mediating

the reinforcing effects of cocaine. Furthermore, decreases in glucose utilization

were observed in the lateral septum, lateral hypothalamus, and amygdala. The

effects in these regions may indicate the potential aversive effects of

noncontingent cocaine (figure 3).

NEUROCHEMICAL CORRELATES OF PUNISHMENT

The yoked-box procedure has also been used by Dworkin and colleagues to

determine the neurobiological components of punishment (Dworkin and Smith

1989; Miyauchi et al. 1988, 1989; lzenwasser et al. 1989). Littermate triads

were studied by means of an operant procedure that maintained similar

patterns and rates of responding in punished and unpunished situations

(Dworkin et al. 1989). A third rat received noncontingent food and electric shock

presentations. Specifically, a random-ratio schedule of food reinforcement and

electric foot-shock punishment was used to maintain low rates of responding by

the punished subject, and a yoked-interval schedule maintained similar rates

and patterns of responding by the unpunished subject. Pentobarbital (Dworkin

et al. 1989) and chlordiazepoxide (Izenwasser et al. 1989) produced selective

increases in punished responding and only dose-related decreases in

unpunished responding, whereas cocaine increased only unpunished

responding (Dworkin et al. 1989). A third subject that received noncontingent

food and shock presentations yoked to the punished subject was added for the

neurochemical studies. Initial studies indicated that punishment resulted in a

selective increase in serotonin turnover in the frontal cortex and increased

GABA utilization in the cingulate cortex and dentate gyrus. The turnover of

serotonin and dopamine was shown to decrease in the hypothalamus and

frontal cortex, respectively. Punishment-specific decreases in benzodiazepine

receptor binding in the cerebellum were observed in an additional group of rats

(Izenwasser et al. 1989). A replication of the neurotransmitter turnover studies

has recently been completed using of 12 additional triads and more sensitive

neurochemical procedures. The data shown in figure 4 indicate some of the

selective punishment effects observed in that study.

CONCLUSION

The studies reviewed in this paper strongly indicate that behavioral variables

can profoundly influence the neurobiological effects of manipulations of central

nervous systems. The observation that environmental context can alter the

behavioral effects of neurotoxin lesions suggests that behavioral factors that

182
FIGURE 3. Local cerebral glucose utilization of nine brain regions in groups of rats exposed to contingent

(self-administering, SA bars) infusions of cocaine or noncontingent infusions of cocaine (yoked

cocaine, YC bars) and saline (yoked saline, YS bars). Vertical bars indicate ±1 SEM.
FIGURE 4. Neurotransmitter utilization rates for dopamine (DA, top panels),

serotonin (5 HT, middle panels) and norepinephrine (NE, bottom

panels) for rats exposed to the different conditions of the

punishment procedure. Vertical bars indicate ±1 SEM: Significant

differences (p < .05) between the punished condition and the yoke

condition (*) and between the punished and unpunished

conditions (**) were determined using Dunnett’s t test for multiple

comparison.

184
have been demonstrated to alter the behavioral effects of pharmacologic agents

may also alter the effects of other neurobiological manipulations. The

importance of behavioral factors is further indicated by the general findings that

the contingent administration of several environmental events results in

significant differences in numerous neurobiological measures compared with

the noncontingent delivery of the same environmental event. The effects of

these behavioral factors must be evaluated and controlled if a complete

neurobiological analysis of behavior is to emerge. Thus, a complete

understanding of the neurobiological aspects of behavior requires a

sophisticated behavioral as well as neurobiological approach. The behavioral

procedures reviewed here are certainly not the only procedures that can or

should be used, but they have provided results that stress the importance of

behavioral variables in investigations of the neurobiology of behavioral

processes. At best, only a limited neurobiological account of behavior will come

from investigations that do not include an analysis of behavioral factors; at

worst, without such an account our conclusions will be wrong.

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ACKNOWLEDGMENTS

This work was supported in part by U.S. Public Health Service grants DA 01999

and DA 03628, and by National Institute on Drug Abuse contract 271-87-8118.

AUTHORS

Steven I. Dworkin, Ph.D.

Linda J. Porrino. Ph.D.

James E. Smith, Ph.D.

Department of Physiology and Pharmacology

Bowman Gray School of Medicine

Wake Forest University

Winston-Salem, NC 27103

188
Cocaine Self-Administration: Pharmacology

and Behavior

William L. Woolverton

INTRODUCTION

As the title implies, the purpose of this chapter is to consider cocaine

self-administration from the point of view of two categories of independent

variables that are determinants of the behavioral effects of drugs:

pharmacologic and behavioral variables. By definition, pharmacologic variables

are those related to the drug, such as neurotransmitters and receptors, events

that are usually initiated inside the organism. Behavioral variables are those

relating to the behavior and environment, generally events initiated outside the

organism. I emphasize cocaine self-administration for two related reasons:

because interest in cocaine, among both the public and the scientific

community, is waxing at this time and because my primary research interest for

the past 15 years has been cocaine.

The basic message-that drug abuse is an interaction among an organism, a

drug, and the environment-is not new. It is, however, a message that bears

reiterating lest it be forgotten in the euphoria generated by our newfound ability,

well characterized in this meeting, to measure neurotransmitter changes and

the like taking place inside living organisms. The new technologies clearly offer

the opportunity for developing a more comprehensive understanding of the role

of the brain in drug abuse. At the same time, it is essential to emphasize that

behavior, including drug-seeking behavior, is powerfully determined by

variables that originate outside the organism as well. Any comprehensive

understanding of drug abuse necessarily includes a complete understanding of

each of these categories and, ultimately, of their interaction. What follows, then,

is a brief review of a series of laboratory self-administration experiments, some

published and some not, that address the roles of behavioral and

pharmacologic variables in cocaine self-administration.

189
PHARMACOLOGY

The first series of studies involves evaluating the reinforcing effects of a series

of compounds in an intravenous (IV) self-administration paradigm that has

proved to be highly predictive of abuse potential (Johanson and Balster 1978).

The assumption of these studies is that if a compound shares a pharmacologic

action with cocaine, and both the compound and cocaine function as positive

reinforcers under the same conditions, there is then reason to suspect that the

common pharmacologic action plays a role in the reinforcing effect of cocaine.

Obviously, this result does not demonstrate that the common mechanism

determines the reinforcing effects of both compounds, but it does imply that the

mechanism merits further investigation. Because cocaine has several neuronal

actions-among which are blockade of the reuptake of the monoamines

dopamine (DA), norepinephrine (NE), and serotonin (5-HT) and local anesthetic

effects-we evaluated the reinforcing effects of drugs that are more or less

selective in each of these actions. In the present context, the question is which,

or which combination, of these actions is involved in the reinforcing effect of

cocaine. A second assumption is that, because the brain is the organ that

controls behavior, we are talking about central nervous system (CNS)

pharmacology.

The preparation used in these studies has been described in detail elsewhere

(Woolverton et al. 1984). Briefly, rhesus monkeys were prepared with chronic

IV catheters and allowed to self-administer a baseline drug (cocaine or

d-amphetamine) for 2 to 3 hr per day by pressing a lever under a fixed-ratio

10 (FR 10) schedule of reinforcement. When behavior maintained by the

baseline drug was stable, the drug vehicle (usually saline) was substituted for

the baseline drug until responding declined to low levels (usually less than

10 sessions). Subsequently, responding maintained by the baseline drug was

reestablished, and when it was again stable, a dose of the test drug was made

available for the same number of sessions as had been required for responding

to decline to low levels when the drug vehicle was available. In this way, rates

of self-administration of several doses of the test drug were determined with a

return to baseline conditions between doses. A dose of a test drug was

considered to function as a positive reinforcer if the mean rate of

self-administration over the last three sessions of availability was higher than

the rate maintained by the vehicle and the ranges did not overlap.

The compound GBR 12909 is a highly selective DA reuptake blocker (Heikkila

and Manzino 1984) that has high affinity for cocaine binding sites (Madras et al.

190
1989). When GBR 12909 was made available for self-administration,

responding was maintained above vehicle levels in all three of the monkeys

tested to date (figure 1) (Bergman et al. 1989; Kleven et al. 1988). In the

monkey in which a complete dose-response function was determined (8618),

the dose-response function was a typical inverted U, with doses that were too

low to maintain behavior, a dose that maintained the peak rate of responding,

and higher doses at which rates of self-administration decreased. The potency

of GBR 12909 in this preparation was approximately equal to that of cocaine. In

contrast, neither the NE reuptake blocker nisoxetine (Woolverton 1987) nor the

5-HT reuptake blocker fluoxetine (figure 2) functioned as a positive reinforcer. I

should emphasize that the study with fluoxetine is now under way in my

laboratory, and the data are not complete. Nevertheless, there was no evidence

of fluoxetine self-administration at the doses that have been tested. Although

the rate of responding for saline was unusually high in monkey 8623, I have

included the data to make an important point: that responding maintained by

FIGURE 1. Intravenous self-administration of the dopamine reuptake blocker

GBR 72909 by rhesus monkeys. The number of injections was

limited to 20 at this dose because the monkey became

preconvulsive. The number of injections per 2-hr session is on the

ordinate, and the dose of GBR 12909 is on the abscissa. Each

point represents the mean of the last three sessions of availability

of a dose, and the vertical lines represent the range.

191
FIGURE 2. Intravenous self-administration of the 5-HT reuptake blocker

fluoxetine by rhesus monkeys. The number of injections was

limited to 20 at this dose because the monkey became

preconvulsive. The number of injections per 2-hr session is on the

ordinate and dose of fluoxetine is on the abscissa. Each point

represents the mean of the last three sessions of availability of a

dose, and the vertical lines represent the range.

fluoxetine occurred at a lower rate than did responding for saline is evidence

that the doses of fluoxetine we have tested were behaviorally active.

In a study designed to examine the role of local anesthetic actions in the

reinforcing effects of cocaine, we evaluated the reinforcing effects of a series of

local anesthetics: procaine, lidocaine, tetracaine, and procainamide

(Woolverton and Balster 1979). In fact, it is well known that some local

anesthetics can function as positive reinforcers (Ford and Balster 1977;

Hammerbeck and Mitchell 1978; Johanson 1980). This effect, however, seems

to be limited to local anesthetics that are ester-linked compounds (Woolverton

and Balster 1979). The amide-linked local anesthetics have not been found to

function as positive reinforcers. Because not all local anesthetics function as

positive reinforcers under these conditions, it seems unlikely that local

anesthetic actions are responsible for the reinforcing effects of cocaine. Rather,

the data suggest that cocaine and local anesthetics that can function as positive

192
reinforcers share some other action in common (blockage of DA reuptake?) that

mediates their reinforcing effects.

If CNS DA is involved in the reinforcing effects of cocaine, then it is reasonable

to ask which DA receptor subtype is involved. Woolverton and colleagues

(1984) tested a number of direct DA agonists in this self-administration

paradigm. All the compounds that stimulate D2 receptors (apomorphine,

propylbutyldopamine, bromocriptine, and piribedil) functioned as positive

reinforcers in at least half the monkeys. The compound most selective for D2

receptors (piribedil) was the most consistent positive reinforcer; four of four

monkeys tested self-administered piribedil. In fact, we have used piribedil since

then as a baseline drug in antagonist studies (Woolverton 1986). On the other

hand, the D1 agonist SKF 38393 was not self-administered by any monkey up

to high doses, i.e., to doses that have been found to decrease the rate of lever

pressing maintained by food in rhesus monkeys (Kamien and Woolverton 1989).

Taken together, these data support the not altogether surprising conclusion that

pharmacology is important in drug self-administration. Across a constant set of

behavioral conditions, drugs that act primarily as agonists of DA systems in the

brain, either indirectly or directly, consistently functioned as positive reinforcers,

whereas drugs that act primarily to block uptake of other monoamines or are

local anesthetics did not. These findings support the belief, which has been held

for some time (Wise and Bozarth 1981), that DA-reuptake blockage is the

operative mechanism in cocaine pharmacology, mediating its reinforcing effect,

and that an action of DA at D2 receptors plays a necessary role in this effect.

Pharmacology, however, is not sufficient to explain or characterize

drug-maintained behavior.

BEHAVIOR

Drug-maintained behavior is not limited to the final drug self-administration

response. It may be, particularly in humans, a long and elaborate sequence of

complex behaviors that evolves over time and is ultimately maintained by the

self-administration of a drug. Furthermore, alternatives to drug

self-administration are usually available. The next series of studies involves

evaluating the reinforcing effects of cocaine under several different behavioral

conditions. The assumption (perhaps it is a question) of these studies is that the

pharmacology of cocaine alluded to previously is constant across behavioral

conditions. Ultimately, this assumption can and should be tested directly.

Despite CNS pharmacology, however, rates and patterns of drug-maintained

193
behavior (indeed, whether a drug functions as a positive reinforcer at all) vary

widely with the environmental circumstances under which a drug is made

available.

The first environmental variable to emphasize, and the one for which there are

the most data, is the schedule of reinforcement. It is well established that

cocaine can function as a positive reinforcer under a wide variety of schedules

of reinforcement (Spealman and Goldberg 1978; Johanson and Fischman

1989). In a study evaluating potential antagonists of the reinforcing effects of

cocaine, we trained monkeys under a multiple schedule of food or cocaine

availability in which food was available in the first and third component of the

multiple schedule and cocaine was available in the middle component. The

schedule of reinforcement was an FR 30, and there was a 2-min timeout (TO)

after delivery of each reinforcer. As can be seen in figure 3, high rates and

patterns of responding typical of FR schedules were maintained by cocaine

injections. In contrast, when the same dose of cocaine was made available

under a fixed-interval (FI) schedule of reinforcement, responding occurred at

low rates early in the interval and gradually accelerated to high rates at the end

of it (figure 4) (Johanson 1982); that is, simply manipulating the conditions of

cocaine availability dramatically alters the rate and pattern of drug-maintained

behavior. It seems unlikely that schedule of reinforcement has changed the

CNS pharmacology of cocaine.

An experiment by Spealman (1979) makes this point in an even more dramatic

way. In that study, squirrel monkeys were prepared with IV catheters and

trained to press one lever (right lever) under a variable-interval (VI) 3-min

schedule of reinforcement for injections of cocaine. After VI behavior was stable

(approximately 2 wk), a second lever was added to the chamber. Responding

on the second (left) lever terminated cocaine availability under an FI 3-min

schedule of reinforcement, i.e., the chamber went dark and responding had no

consequence for 1 min. Responding was maintained on both levers under these

conditions, with rates and patterns typical of each schedule of reinforcement

(figure 5); that is, behavior was simultaneously maintained by cocaine injection

and by the termination of cocaine availability. When conditions were modified

so that responding on the left lever produced cocaine injections under the VI

schedule and responding on the right lever was maintained under the FI

schedule of cocaine termination, rates and patterns of responding changed

appropriately to the new schedule conditions. When saline was substituted for

cocaine, responding declined on both levers, indicating that cocaine injections

were necessary to maintain behavior on both levers. When the amount of

194
FIGURE 3. Cumulative response records for a rhesus monkey responding

under a multiple schedule of food (Fd) or cocaine (Coc)

availability. Food was available in the first and third components,

noted by the upward deflection of the lower pen. Cocaine (0.025

mg/kg per injection) was available in the middle component. In all

three components, the schedule of reinforcement was FR 30, with

a 2-min time out (TO) after delivery of each reinforcer. In addition,

there was a 15-min TO between components of the multiple

schedule. The recorder did not run during TO periods.

195
FIGURE 4. Cumulative response records for a rhesus monkey responding

under a fixed-interval 5-minute schedule of cocaine availability.

Doses of cocaine are indicated in the upper left of each panel.

Each record is the first 30 min of a 3-hr session. (From Johanson

1982.)

cocaine that the monkey normally self-administered in a session was infused

noncontingently, immediately before the session, responding was not seen on

either lever, indicating that cocaine was not nonspecifically increasing lever

pressing, i.e., to maintain behavior on both levers, cocaine had to be contingent

on responding. When responding on the FI lever no longer terminated cocaine

availability, responding on that lever decreased, whereas responding on the VI

lever to receive cocaine continued to be maintained. This is convincing

196
FIGURE 5. Cumulative response records for squirrel monkeys responding

under schedules of cocaine injection and termination of cocaine

availability. Responses on the right lever (upper record in each

panel) produced intravenous injections of cocaine under a

variable-interval (VI) schedule. Responses on the left lever (lower

record in each panel) terminated the schedule of cocaine injection

for 1 min under a fixed-interval (FI) schedule. Pens were reset at

the beginning of time out periods, during which recorders did not

run. Doses of cocaine are indicated in the upper left of each

panel. Each record is the first 30 min of a 3-hr session. (From

Spealman 1979, copyright 1979, American Association for the

Advancement of Science.)

197
evidence that cocaine was functioning simultaneously as a positive reinforcer

and as a negative reinforcer in the same monkey. Again, the question to ask is,

How can we explain cocaine functioning simultaneously in the same organism

as both a positive and a negative reinforcer on the basis of the pharmacology of

cocaine?

The third study was conducted in my laboratory by Michael Nader (Nader et al.

1989). In this study, monkeys were trained in a discrete-trials choice paradigm

and allowed to choose between an injection of cocaine and food pellets. The

procedure has been described in detail elsewhere (Woolverton and Johanson

1984). Briefly, when a session begins, white lights are illuminated above the left

lever, and red or green lights are illuminated above the right lever. Red and

green are each associated with one of the reinforcers, cocaine or food.

Responding on the left lever changes the light color above the right lever from

red to green and from green to red. After a minimum of three color switches, a

monkey can choose a reinforcer by pressing the right lever when the light

associated with that reinforcer is illuminated. After a choice is completed, all the

lights in the chamber go off and there is a 10-min TO between trials. After that

TO, a new trial is initiated as before. Sessions consisted of 15 trials and were

conducted each day.

When the number of food pellets available as the alternative to cocaine was

held constant and the dose of cocaine was increased, the frequency of cocaine

choice increased (figure 6). On the other hand, when the dose of cocaine was

held constant and the number of food pellets available as the alternative to

cocaine was increased, the frequency of drug choice decreased. Put simply,

the reinforcing effect of cocaine varied with dose and with the magnitude of an

alternative reinforcer that was simultaneously available.

SUMMARY

I would like to stress several major points. The first is that CNS pharmacology is

important in drug self-administration. In the case of cocaine, the necessary

effect seems to be blockade of reuptake of DA rather than blockade of reuptake

of NE or 5-HT or local anesthetic effects. An action of that DA on the D2

subtype of DA receptors appears to play an important role.

The second point is that increased DA in synapses in the CNS is not sufficient

to explain drug-maintained behavior. The schedule of reinforcement critically

determines rates and patterns of drug-maintained behavior. Barrett (this

198
FIGURE 6. Frequency of cocaine choice in monkeys given a choice between

cocaine and food. Dose of cocaine is on the abscissa. Different

bars represent different numbers of food pellets available as the

alternative to cocaine. Vertical lines are the range of at /east two

evaluations of each choice condition.

volume) has presented convincing evidence that behavioral history, i.e., the

sequential exposure to alternative behavioral conditions, can dramatically alter

drug effects. What I hope to have made clear is that the simultaneous

opportunity to engage in other behaviors can dramatically alter the reinforcing

effects of drugs. Indeed, under appropriate conditions, a drug can function as

both a positive and a negative reinforcer simultaneously in the same animal.

The drug-choice studies I have described emphasize the dynamic nature of the

interaction between behavioral and pharmacologic variables. The frequency of

choosing to self-administer cocaine varied with the magnitude of the dose of

cocaine (a pharmacologic variable) and the magnitude of an alternative positive

reinforcer that was available (a behavioral variable). Increasing the magnitude

of the alternative reinforcer decreased cocaine choice, but increasing the

magnitude of the cocaine dose reestablished drug preference. In a sense, this

is not a surprising result. The rhetoric of the times, however, would have it that

199
drug self-administration, particularly cocaine self-administration, is somehow a

different class of behavior that is not subject to the usual laws governing the

behavior of organisms. As our understanding of drug self-administration

evolves, however, that position will become increasingly untenable.

These experimental findings challenge simple neurobiological assumptions

about the determinants of drug self-administration. Clearly, a reinforcing effect,

like any other behavioral effect of a drug, is not simply an immutable effect of a

pharmacologic property of the drug. Given the pivotal role of reinforcing effects

in drug abuse, this realization is critical to a comprehensive understanding of

drug abuse. Any complete neurobiological model of drug abuse will have to

account for these effects. Apparently, increasing the concentration of DA in

synapses in the brain is not sufficient to account for drug-maintained behavior.

As has been noted by Fibiger, neuroleptics increase the concentration of DA in

synapses in the CNS, but there is little reason for concern about neuroleptic

abuse. Alternatively, the conditions of drug availability may alter the

neurobiology of cocaine. This is, indeed, a possibility and one that will ultimately

need to be evaluated. Given the enormous number of environmental conditions

that are known to modify drug effects, this is, at least for me, a sobering

thought. The up side, however, is that there is enough to do to keep us all busy

for a long time to come.

Finally, B.F. Skinner (1989, p. 18) eloquently generalizes the point that I have

attempted to make:

There are two unavoidable gaps in any behavioral account. One

between the stimulating action of the environment and the response

of the organism and one between the consequences and the

resulting change in behavior. Only brain science can fill those gaps.

In doing so it completes the account. It does not give a different

account of the same thing. Human behavior will eventually be

explained (as it can only be explained) by the cooperative action of

ethology, brain science and behavior analysis.

A complete understanding of the behavior of drug abuse will require just such a

collaborative approach.

200
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ACKNOWLEDGMENT

This work was supported by grant DA 00250 from the National Institute on Drug

Abuse.

AUTHOR

William L. Woolverton, Ph.D.

Department of Pharmacological and Physiological Sciences

University of Chicago

947 East 58th Street

Chicago, IL 60637

202
Multivariate and Nonlinear Approaches to

Characterizing Drug Effects on the Locomotor

and Investigatory Behavior of Rats

Mark A. Geyer and Martin P. Paulus

INTRODUCTION

The measurement of motor activity in rodents is one of the most fundamental

behavioral tests used in the study of drugs of abuse. In many cases, the

behavior is referred to as unconditioned or spontaneous motor activity because

it has not been explicitly conditioned by the experimenter. The related term

genera/ motor activity is commonly used when monitoring systems are

employed that do not distinguish between different aspects of locomotor

behavior; while the term locomotor activity is typically applied when ambulatory

movements are monitored more specifically. Various instrumentally and

operationally defined measures of motor activity have been used to assess the

behavioral effects of drugs or other manipulations, either as strict measures of

locomotor activity or as measures of more global and context-dependent

constructs such as arousal, curiosity, emotionality, and exploration. In

psychopharmacology, activity measures are often used as bioassays of drug

effects or to establish macroscopic characteristics for drug classes. For

example, psychoactive drugs are defined as stimulants or depressants largely

on the basis of their effects on gross measures of the motor activity of rodents.

MEASUREMENT CONSIDERATIONS

Motor Behavior Versus Locomotor Behavior

The definitions of terms that have been mentioned rest on the assumption that

motor activity is a category of behavior. However, so-called spontaneous or

general motor activity is not a unitary class of behavior. Rather, depending on

the nature of the recording technique, many different behavioral actions may be

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included within the measured behavior. Each of these behavioral actions could

be defined as a response that can be measured and studied in its own right

(Skinner 1933). Such an approach recognizes that macroscopic behavior

consists of assemblies of microscopic responses. Hence, any attempt to

indiscriminately detect any movement of the animal and combine all these

responses into one category called motor activity is an oversimplification of the

behavioral observations. Although such approaches have demonstrated some

utility insofar as they are able to detect the global effects of drugs, these coarse

measures of motor activity provide little or no information about the behavior of

the animal or the nature of a drug-induced change in behavior.

Such indications have led most researchers to rely on measures of locomotor

behavior rather than motor behavior. Locomotor activity can be defined

operationally as movement from place to place and is virtually always one of

the behavioral responses that provides a major contribution to any measure of

general motor activity. It is, however, a much more specific measure because

the monitored behavior is limited to units that specifically reflect the animal’s

movements of some minimal distance or from one place to another. Such units,

which are often called crossovers or crossings, require ambulation by the

animal. By design, such measures are insensitive to movements related to

sniffing, grooming, eating, drinking, tremor, or breathing. As the field has

advanced, many investigators have begun to measure locomotor responses

concurrently with other behaviors, such as rearings, object contacts, hole

pokes, or patterns of behavior (e.g., circling) to enhance their ability to

characterize drug effects and interpret their results.

Automated Monitoring Systems

Only a limited number of different measurement techniques have been applied

to detecting locomotor activity in rodents. The primary approaches have relied

on photobeams, wheels, touch plates, field detectors based on ultrasonic or

capacitance circuits, and mechanical transducers such as jiggle cages, tilt

cages, and force platforms. More recently, video imaging techniques were

introduced to monitor locomotor activity. Most of the devices, which were

reviewed by Reiter and MacPhail (1979), incorporate mechanical transducers,

infrared motion detectors, or field detectors and are limited by the difficulty of

differentiating locomotor from nonlocomotor movements (e.g., grooming,

sniffing); dependence on the size, weight, or temperature of the animal; and

complications associated with standardization and calibration. Different

measurement techniques that are purported to measure horizontal locomotion

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may yield different results depending on the particular sensitivities of the

recording device. For instance, Ljungberg (1978) combined two standard

activity-monitoring devices, a commercial capacitance-based activity meter and

a photobeam box, for assessing simultaneously the effects of several

catecholaminergic drugs in rats. He found that the two devices reflected

behavioral changes differently and that their results were not correlated.

To date, the most successful and reliable measures of locomotor activity have

come from devices that use infrared photobeams. Photobeams are relatively

trouble free in operation and can be calibrated and standardized easily. Infrared

beams can function independent of the ambient illumination and provide no

feedback to the animal even during the nocturnal portion of the animal’s

sleep-wake cycle. Photobeams can also be used to detect rearings by placing

an array of beams at an appropriate height above the animal; the same

technology can be used to monitor hole pokes as an explicit measure of

exploratory behavior. An important consideration is that repeated interruptions

of the same photobeam should not be counted in a measure of locomotor

behavior, because they may reflect movements on very small scales. Although

the total number of photobeam interruptions has been used widely as a

measure of activity, a measure based on successive interruptions of separate

beams is more specific to locomotor movements and is therefore generally

preferable. There is strong support for the recommendation of Reiter and

MacPhail (1979) that all automated activity devices should include a measure

that is selectively sensitive to locomotor movements to distinguish them from

fine movements associated with grooming, sniffing, tremor, etc. Such an

approach will make possible comparisons with other reports in the literature and

thereby increase our understanding of at least this major component of motor

activity.

Length of Test Sessions

In much of the early work using the traditional open field systems and visual

observations of behavior, test sessions were commonly as brief as 3 min to

5 min. Even in the recent literature, some procedures limit the test session to

only 10 min. Such short test sessions may be suitable for assessing drug

effects on emotionality or exploration, but they are seldom adequate for

characterizing treatment effects on the levels or patterns of locomotor activity.

Short test sessions maximize the influences of factors such as handling, the

familiarity of the animal with the test chamber, and the contribution of behaviors

related to attempts to escape. Most such factors are as likely as locomotor

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activity to be influenced by the drug or other experimental manipulation. The

guidelines for motor activity testing established by the U.S. Environmental

Protection Agency (EPA 1986, p. 17, 891) provide that “the test session shall

be long enough for motor activity to approach asymptotic levels by the last 20

percent of the test session for most treatments and animals’ activity counts

shall be collected in equal time periods of no greater than 10 minutes duration.”

In practice, these considerations suggest making test sessions at least 30 min

long and preferably 60 min or more.

Nature of the Test Chamber

Several factors must be considered in selecting the size and shape of the test

chamber (Geyer 1990). If locomotion is the object of study, the chamber must

be large enough to elicit this behavior. Further, there are advantages to a

chamber that is large enough to permit a meaningful division between central

and peripheral areas. For example, a study on the influences of a depletion of

central norepinephrine on the hyperactivity induced by amphetamine revealed

that although the depletion has no effect on the amount of locomotor

hyperactivity, it resulted in a significant increase in the time spent in the center

and a significant decrease in time spent in the corners of the chamber (Geyer et

al. 1986a). Conversely, a neurotoxin-induced depletion of brain serotonin had

the diametrically opposite effect on the locomotor response to amphetamine,

again without altering the amount of activity (Gately et al. 1986).

The nature of the test chamber also has important consequences with regard to

the sensitivity of the measures to both increases and decreases in the

locomotor activity. Beyond general principles of measurement, the advisability

of using an experimental design in which one can detect bidirectional

changes-i.e., to avoid “ceiling” and “floor” effects-in the measured behavior is

clear. Indeed, EPA (1986, p. 17, 891) includes the requirement in its guidelines

for measuring motor activity that “the device used must be capable of detecting

both increases and decreases in activity, i.e., baseline activity as measured by

the device must not be so low as to preclude decreases nor so high as to

preclude increases.” (Federal Register 1986, p. 17,891).

One reasonable criterion that has been suggested and could be applied more

widely is that the test be capable of detecting the activating effects of

amphetamine and the inhibiting effects of chlorpromazine. To satisfy such

requirements, the test chamber should be sufficiently novel for the animal so

that an adequate level of exploratory locomotor activity is elicited. Potentially

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misleading floor effects may also be avoided by testing animals during the

waking part of their sleep-wake cycle. Unfortunately, the use of paradigms that

have limited ability to detect drug-induced decreases in activity is one of the

most common and unfortunate weaknesses in the behavioral literature on the

effects of psychoactive drugs.

The animal’s degree of familiarity with the test environment can have a

profound influence on the observed effect of a drug. For example, the

reductions in locomotion and investigatory hole poking produced by low doses

of hallucinogens in rats tested in a novel test chamber are not seen if the rats

are already familiar with the chamber (Geyer and Light 1979; Adams and Geyer

1985a). Such effects are comparable to the profound and complex influences of

the animal’s experiential history on the effects of drugs on scheduled behavior.

Importance of the Center of the Chamber (Thigmotaxis)

One of the most noticeable aspects of the locomotor behavior of a rat in an

enclosed chamber is the tendency to remain close to the walls, referred to as

thigmotaxis. In traditional open field devices, entries into the central

portion-usually defined as being more than half a body length away from and

therefore not in contact with any wall-often have been scored separately from

movements around the periphery. Measures of center entries have proved to be

very sensitive to the effects of drugs. For example, hallucinogens decrease

entries into the center of a chamber even when they have no effect on the level

of activity as measured by either peripheral movements or total number of

photobeam interruptions (Adams and Geyer 1985a). Such findings can be

interpreted as a hallucinogen-induced increase in agoraphobia (fear of open

spaces), that is, a specific potentiation of the animals’ normal avoidance of the

presumably threatening central area. The demonstrable significance to the

animal of the central portion of an activity chamber underscores the advantages

of using a chamber that is large enough to elicit thigmotaxis and to enable the

separate detection of central and peripheral movements to be detected

separately.

ADVANTAGES OF MULTIVARIATE ASSESSMENTS

Value of Multiple Concurrent Measures

The fundamental conclusion of virtually all critical reviews of activity measures

is that it is highly advantageous to assess multiple aspects of exploratory and

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locomotor activity concurrently (Geyer 1990; Lat 1965; Reiter and MacPhail

1979; Robbins 1977). The multivariate assessment of unconditioned behavior

allows the investigator to assess the validity of hypothetical constructs, make

more confident comparisons with other results in the literature, examine the

generality and specificity of an observed effect, identify the contribution of

response competition, and detect artifacts. For example, many investigators

have used locomotor activity measures as indicators of constructs such as

arousal and exploration. The challenge has been to distinguish between activity

related to an animal’s internal level of arousal and activity elicited by external

stimuli. Most theorists have concluded that the amount of exploratory behavior

is directly related to the novelty and complexity of stimuli in the environment

and, in reciprocity with the process of habituation, inversely proportional to the

organism’s prior experience with those stimuli (Berlyne 1960; Kumar 1968;

McReynolds 1962; Montgomery 1955).

Because standard activity measures are influenced by many factors, behavioral

scientists have begun to use hole boards to provide specific measures of

investigatory responding. A hole board is simply a test chamber with holes into

which burrowing animals such as rats frequently poke their noses. Thus, the

holes serve as specific stimuli that elicit easily measured inspective responses.

Many hole board chambers rely solely on holes in the floor, a design that

maximizes the likelihood that inadvertent missteps by the animal could lead to

erroneous counts being generated. Such artifactual responses could easily be

related systematically to the drug treatment and therefore could be very

misleading. However, holes in the walls can be used with equal success and

somewhat more reliability (Flicker and Geyer 1982). Just as measures of

locomotion in the absence of specific measures of investigatory behavior are

difficult to interpret in terms of exploratory behavior, inferences based solely on

measures of hole poking or head dipping are very questionable unless

treatment effects on general levels of activity are assessed simultaneously. For

example, although both amphetamine and apomorphine increase locomotor

activity, amphetamine increases the frequency of hole pokes and apomorphine

decreases it (Geyer et al. 1979; Ljungberg and Ungerstedt 1976). Such

observations indicate that measures of hole poking should not be used without

concurrent measures of locomotor activity.

An Example: The Behavioral Pattern Monitor

In the context of characterizing the behavioral effects of psychoactive drugs by

themselves and especially in their interactions with other drugs or experimental

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manipulations, the availability of multiple measures enables the behavioral

profiles to be developed and used. The Behavioral Pattern Monitor (BPM) was

designed to combine the features of activity and hole board chambers and to

measure individual response frequencies and durations (Flicker and Geyer

1982; Geyer 1982). Each chamber consists of a large (30.5- by 60-cm) black

Plexiglas box containing three floor holes and seven wall holes equipped with

infrared beams and a wall touch plate to detect rearings. The chamber is

crisscrossed with infrared beams, which are sampled by a microcomputer. The

computer records the sequences of hole pokes and rearings and the current

position with a temporal resolution of 0.1 s and a spatial resolution of 3.8 cm.

Various descriptors are obtained from the raw data, including the total number

of photobeam breaks and crossovers from one 15-cm square to another, and

more qualitative descriptors such as entries into and time spent in the center

and the corners. Because the record of all movements is permanent, it may be

used for computer reconstructions of the pattern of movements on paper or on

a video display or for calculating of descriptive statistics reflective of

treatment-induced differences in these patterns. Further, objects or lights may

be placed in specific holes to manipulate their novelty or complexity and elicit

discrete inspective and novelty responses (Flicker and Geyer 1982).

Characterizing the Specificity of Drug Action

A major and fundamental issue in the characterizing of drug effects on behavior

is specificity. In the present context, an example is provided by the category of

drugs labeled stimulants. Many, but not all, of these drugs are also drugs of

abuse. In sharp contrast to the advances in precision and specificity so

apparent in most areas of neurosciences and pharmacology, much of the

behavioral psychopharmacology literature continues to rely on measures of

behavioral activity that cannot differentiate between drugs such as

amphetamine and caffeine or between apomorphine and cocaine. That is, if one

collects only a single measure of locomotor activity, drugs as different as

amphetamine, apomorphine, scopolamine, caffeine, methylenedioxymethamphetamine (MDMA), and nicotine are often indistinguishable. Hence, to

assess the value of the multivariate behavioral assessment provided by the

BPM system, the effects of various stimulant drugs have been compared in

systematic dose-response studies (Geyer 1982; Geyer et al. 1986b; Gold et al.

1988). The goal was to determine whether developing profiles of locomotor and

investigatory behaviors would enable distinctions to be made that cannot be

made with standard measures of the amount of activity.

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In the studies summarized here, naive male rats were tested only once during

their initial exposure to the BPM chambers, Each experiment involving a

stimulant drug included four or five groups of 8 to 12 animals each. Test

sessions were conducted during the dark phase of the animals’ light-dark cycle

and lasted 60 min. Subcutaneous injections of saline or one of several doses of

the test drug were given 10 min before the animal was introduced to the

chamber. The following doses (in milligrams per kilogram) of each drug were

tested: amphetamine, 0.25, 0.5, 1.0, and 2.0; scopolamine, 0.125, 0.25, 0.5,

and 2.0; caffeine, 2.5, 5.0, 10.0, 15.0, and 20.0; apomorphine 0.1, 0.5, 1.0, and

2.0; nicotine, 0.0625, 0.125, 0.25, and 0.5; MDMA, 1.25, 2.5, 5.0, and 10.0; and

lisuride, 0.005, 0.015, 0.03, and 0.06. Details of these experiments may be

found in the original reports (Adams and Geyer 1985b; Geyer et al. 1986b; Gold

et al. 1988). For each experiment, repeated-measure and mixed-design

analyses of variance were performed for selected variables. Dunnett’s tests

were used to compare each treatment group with the control group. All

statistical comparisons reported here were derived from comparisons of the

particular dose group with the corresponding control group, although some

figures depict only the results from the most typical control group.

All the drugs elicited dose-related increases in either total photobeam breaks or

crossovers, which are defined as movements from one 15-cm square to

another. The effects of a representative dose of each drug on crossovers during

the first and second halves of the hour-long test session are shown in figure 1.

Clearly, all the drugs can be classified as stimulants on the basis of their shared

ability to increase such measures of locomotor activity. Even the simplest

multivariate assessment, however, began to differentiate these drugs from one

another. Many drugs have differential effects on entries into a central area that

are unrelated to differences in overall levels of activity. Scopolamine,

apomorphine, and MDMA shared a common effect in that animals tended not to

enter the center of the chamber (figure 2).

These three drugs produce characteristic patterns of hyperactivity in which the

animals rarely move away from the walls. On the other hand, the increase in

center entries produced by caffeine is in direct proportion to the effects of the

drug on total movements. Such observations demonstrate that the separate

measurement of central versus peripheral movements is of considerable value

both in detecting the behavioral effects of drugs and in differentiating the effects

of seemingly similar drugs. The dopamine agonist lisuride, which is a structural

relative of lysergic acid diethylamide (LSD) and has important effects on

serotonergic systems (Adams and Geyer 1985b), also increased entries into the

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center to about the same extent that it increased crossovers. Thus, its effects

contrast with those of LSD, which markedly decreases center entries (Adams

and Geyer 1985a). In contrast, amphetamine was the only drug that produced a

preferential increase in center entries.

FIGURE 1. Effects of stimulants on crossovers. The effects of the selected

doses of the various stimulant drugs on crossovers are shown as

group (N = 10-12) means ± SEM for successive halves of the

hour-long test sessions. At these doses (shown in milligrams per

kilogram), each drug significantly increased locomotor activity

during the last ha/f of the test session. The control values shown

are the median values from the separate control groups used for

each stimulant study. Statistical comparisons were based on each

particular control group. SAL = saline; AMP = d-amphetamine;

APO = apomorphine; CAF = caffeine; LIS = lisuride; MDMA =

methylenedioxy-methamphetamine; N/C = nicotine; SCOP =

scopolamine. * = significantly different from corresponding control,

p < 0.05.

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FIGURE 2. Effects of stimulants on center entries. Group means for center

entries are shown as in figure 1. The center region is illustrated in

figure 6. * = significantly different from corresponding control,

p < 0.05.

The BPM also provides explicit measures of exploratory or investigatory

behavior, namely hole pokes and rearings. In all cases, each drug had similar

effects on both these behaviors, as shown for hole pokes in figure 3.

Scopolamine markedly increased both hole pokes and rearings, whereas

apomorphine virtually abolished both behaviors and MDMA reduced them.

Lisuride also significantly decreased both hole pokes and rearings despite

increasing crossovers and center entries. Although caffeine and nicotine

produced similar increases in crossovers and at least tended to increase center

entries, caffeine increased and nicotine decreased both hole pokes and

rearings. Thus, although at the doses selected for comparison all these drugs

produce roughly comparable increases in the amount of locomotor activity, they

are readily discriminable by means of a rather simple form of multivariate or

profile analysis. Just as these drugs may be differentiated biochemically on the

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FIGURE 3. Effects of stimulants on hole pokes. Group means for ho/e pokes

are shown as in figure 1. Each chamber contains three floor holes

and seven wall holes.* = significantly different from corresponding

control, p < 0.05.

basis of binding or other neurochemical effects, or pharmacologically by virtue

of their differential sensitivities to receptor antagonists or synthesis inhibitors, so

may they be differentiated at a behavioral level even by means of a single test

paradigm.

UTILITY OF ANALYSES OF SPATIAL AND TEMPORAL PATTERNS OF

ACTIVITY

Patterns of Locomotor Activity

A new and sensitive approach to the study of drug effects on locomotor and

exploratory behavior is based on examining the patterns of locomotion by

plotting the sequence of movements as the animal explores the chamber. The

BPM system permanently stores all the movement patterns of the animal

together with the duration of each investigatory response or pause in a

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particular x-y position. One of the most instructive uses of these data with

regard to understanding the structured manner in which a rat explores its

environment has been the computer generation of reconstructed visual images

of the sequences of hole pokes, rearings, and locomotor movements. As

expected, virtually all untreated animals tend consistently to avoid the center

region and to stay near a corner of the chamber. The structure of the movement

patterns themselves was most easily identified by observing the video displays

of the movements when animals were tested in a free-exploration paradigm in

which they could move freely between a home cage and the larger BPM

chamber. The animal’s location in the home cage serves as an organizing

focus; excursions to various parts of the larger chamber and back follow

progressively more fixed routes over time. Typically, the outbound part of an

excursion is more frequently interrupted by investigatory hole poking and

rearing than the return. When tested in the more typical forced-exploration

situation, the behavior is similar except that each animal selects a particular

corner as its home area. Each rat, however, clearly develops its own particular

spatial pattern of movements, and this pattern is predictable across time within

a session and between sessions.

Drug Effects on Spatial Patterns of Locomotion

The consistency in the locomotor patterns of untreated rats has led to the study

of drug-induced changes in these patterns per se. For example, the stimulant

drugs that have been discussed above in the context of multivariate

assessments have also been examined in terms of their influences on spatial

patterns of locomotion (Geyer et al. 1986b; Gold et al. 1988). Even at doses

that produce comparable increases in the amount of locomotor activity, some of

the stimulant drugs are readily distinguishable by virtue of qualitative changes

in the animals’ locomotor patterns. Some drugs, such as low doses of

amphetamine, disrupt the normal structure by producing highly varied patterns

of directional changes (figure 4). At higher doses, amphetamine can produce

perseverative patterns (Lat 1965; Schiorring 1979). Other stimulant drugs

essentially replace the normal patterns of locomotion with new, even more

highly structured patterns. For example, both apomorphine and scopolamine

induce movement patterns that are very predictable and seemingly

characteristic of each drug. Most animals treated with apomorphine run around

the perimeter of the chamber consistently in one direction for most of the

session (figure 5). Although scopolamine-treated animals also move around the

perimeter of the chamber, they frequently change directions and pause enroute

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FIGURE 4. The locomotor pattern induced by amphetamine. Shown here are

the movement patterns exhibited by a representative animal given

amphetamine and tested for 1 hr in the Behavioral Pattern

Monitor. To avoid exact retracings of the same lines, ± 40 percent

variability has been added to successive x - y positions.

FIGURE 5. The locomotor pattern induced by apomorphine. Shown here are

the movement patterns exhibited by a representative animal given

apomorphine and tested for 1 hr in the Behavioral Pattern

Monitor. To avoid exact retracings of the same lines, ± 40 percent

variability has been added to successive x - y positions.

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to investigate the holes and rear against the wall, responses only rarely seen

with apomorphine-treated rats (Geyer 1982).

Intermediate combinations have also been noted. For example, doses of 5 to

10 mg/kg of the serotonin-releasing compound MDMA cause animals to move

around the perimeter of the chamber, changing directions occasionally as with

scopolamine, but without hole poking or rearing as they do with apomorphine. It

also appears that stimulants such as caffeine and nicotine do not disrupt the

normal structure of the animals’ spatial patterns of locomotion. With these drug

treatments, it is evident that each animal adopts a preference for a particular

home area and establishes preferred excursion routes that are as predictable

as those of controls. That is, the structure of their locomotor patterns is largely

similar to those exhibited by untreated or saline-treated control animals. These

drugs increase activity primarily by reducing the duration of each visit in the

home corner. Accordingly, the strongest differences between caffeine or

nicotine versus saline controls occur after 20 to 30 min, when the control

animals begin to pause longer in their self-selected home corner.

Measures of Perseverative Patterns of Locomotion

Despite the recent advent of automated devices that can record such patterns,

the statistical description and analysis of the resulting data has posed a difficult

problem. We have had some success with a measure called the spatial

coefficient of variation (CV) (Geyer 1982; Geyer et al. 19866). In our first

attempts to use the metric to statistically describe and evaluate the sequences

of position changes, the data were reduced into transitions among five areas:

the two ends, the center, and the two long walls (figure 6). Subsequent

applications of this approach have involved calculating transitions among nine

areas (Gold et al. 19866; Geyer et al. 1988). In either case, transitions among

the five or nine areas can be displayed in a matrix with 16 or 40 permissible

cells (see figure 6). Relative transition frequencies are then calculated as

percent of total, and the spatial CV is derived from this set of numbers. To the

extent that an animal preferentially repeated certain transitions, the spatial CV

increases. A more uniform or random distribution of spatial transitions produces

a low spatial CV.

Figure 7 illustrates the differential effects of the various stimulant drugs on the

CV statistic, a measure of the degree of structure or predictability in the spatial

pattern of locomotion. As appropriate to the pattern differences described

above, the effects of the drugs on the CV were independent of their effects on

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FIGURE 6. The spatial coefficient of variation (CV) statistic. The diagram at

the top represents the five regions into which the chamber is

divided by the analysis program for assessing the CV and defining

center entries. To calculate the CV, transitions of the rat from one

region to another are cumulated as illustrated in the matrix This

particular matrix comes from an animal treated with scopolamine,

which makes animals run around the periphery of the chamber in

both directions. Hence, the numbers in this matrix include several

large numbers and many small numbers because this animal

exhibited the same movement pattern consistent/y. According/y,

the standard deviation or coefficient of variation-CV-of this set

of numbers is relatively high, i. e., 0.890. An animal that distributed

its movements more random/y or uniform/y throughout the

chamber would produce a matrix filled with more similar numbers

and therefore a low CV. Thus, the CV statistic increases to the

extent that an animal repeats certain spatial movement patterns to

the exclusion of others.

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FIGURE 7. Effects of stimulants on the spatial coefficient of variation. Group

means for CV, or the coefficient of variation, are shown as in

figure 1. The spatial CV is defined in figure 6. An increase in the

spatial CV reflects a more repetitive pattern of movements. * =

significant/y different from corresponding control, p < 0.05.

the amount of activity. The drugs that were observed to produce repetitive or

stereotyped patterns of locomotor hyperactivity—scopolamine, MDMA, and

especially apomorphine—all significantly increased the CV. Conversely,

amphetamine, which increased the frequency of directional changes and

produced an unpredictable pattern of locomotion at the doses tested,

significantly decreased the CV. Nicotine and caffeine had minimal effects on it.

Although lisuride was observed to produce repetitive rotational movements,

these predictable patterns were limited to the early part of the test session and

tended to form oval patterns centered on the diagonal of the chamber (Adams

and Geyer 1985b). Because this particular pattern did not conform to the

arbitrary Cartesian coordinate system used in calculating of the CV, the

statistical measure was inconsistent with the observed behavior. Such an

example reflects the constant need to compare the results of abstracted

descriptive statistics with the object being measured-in this case, locomotor

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behavior. Discussed below are some newer measures that do not suffer from

the low-resolution weaknesses of the CV measure and therefore more

accurately describe the behavioral changes induced by lisuride. Despite such

occasional limitations of the spatial CV measures, the results depicted in

figure 7 reveal that they provide additional information that is not evident in any

other measure and that is independent of drug-induced changes in amount of

activity.

Although descriptive statistics such as the spatial CV have proved useful, much

more work in this area is warranted. The combination of multivariate

assessments and pattern analyses provided by systems such as the BPM

promises to enhance the study of drug effects on locomotor activity in much the

same way the switch from whole-brain to regional analyses of brain chemistry

revolutionized our understanding of drug effects on neurotransmitters. The

advent of new technologies such as video tracking systems and the associated

computer-based pattern analyses should have considerable impact in this area.

Further, the application of methods used for analyzing nonlinear dynamical

systems is certain to supply a variety of additional measures that have

enormous potential to quantify and characterize the effects of drugs on such a

complex, dynamic, and metastable behavioral system as spontaneous

locomotor activity (e.g., Geyer et al. 1986b; Paulus et al. 1990).

SCALING APPROACHES IN BEHAVIORAL PHARMACOLOGY

Problem

The data acquisition systems that are currently being used in behavioral

experiments are far more sophisticated than the concepts that are being used

to deal with the amount and variety of information. The preceding discussions

emphasize that more elaborate techniques are necessary for dealing with the

information obtained from such experiments.

Approach

In the study of physical systems that are near phase transitions (Stanley 1971),

an important concept emerged that is now widely used to categorize different

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systems in so-called universality classes. The basic idea is that an important

quantity that describes the system (such as its free energy) has the following

property:

In words, replacing the argument of this quantity by an argument that is scaled

by a factor of , the quantity can be obtained by multiplying the quantity by the

factor to some power a. Functions that obey this relationship are said to be

scaling. Central to the treatment of statistical mechanical systems near phase

transitions (i.e., complex systems that exhibit large fluctuations) are equations

that yield a function rather than a number as their solution. Transferring these

results to behavioral systems leads to the basic assumption that a single

measure will not describe the results adequately. Rather, a function that relates

the quantity of interest to its variation depending on intervening variables may

provide a more complete description. In other words, insights about the system

may be gained by observing a quantity of interest (e.g., locomotor activity) on

different levels of observational perspective. Scaling concepts have been

extensively used for studying fractal objects in mathematics and physical

experiments. A fractal may be described simply as “a shape made of parts

similar to the whole in some way” (Mandelbrot 1977). A concrete example of a

scaling relationship is that the mass of an object scales with the measuring

resolution with which the object is studied. For a regular “geometric” fractal, this

scaling relationship holds as a strict equality, whereas for a “statistical” fractal, it

may be described as follows:

[mass] — lengthscaleexponent

In addition, the range of this scaling ansatz may be specified.

System

When the influence of drugs on unconditioned locomotion is studied, counts of

activity based on photobeam breaks provide a quantity that may be a candidate

for a scaling approach. The information provided by photobeams depends on

the particular apparatus and is not related in a simple fashion to explicit

behavioral responses. The locomotor activity of rats in the BPM may be

conceptualized as movements that occur on different scales. The movements of

an animal may involve small local movements characterized by consecutive

photobeam breaks within the same region of the chamber. Such patterns of

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beam breaks may reflect small head movements, movements that occur during

grooming or investigatory behaviors such as rearing or hole poking, or repetitive

focal perseverative behaviors such as biting or gnawing. In addition, the animal

will exhibit some long, distance-covering movements, which appear as straight

paths without interruption by local movements and often reflect the frequently

observed rotating pattern of locomotion.

A normally behaving animal will exhibit both local and distance-covering

movements, as well as intermediates that are combinations of these

movements, during the test session. Although this chapter focuses on the

spatial domain, a similar distribution of the durations of events provides a

description of the frequencies of movements in the temporal domain.

Descriptors based on response durations may indicate changes in rate of

activity. Thus, to describe the behavior exhibited in the BPM, a plane can be

constructed that consists of two axes; the first can be used to describe changes

in the spatial composition of local and distance-covering movements, and the

second displays the overall level of activity. Effects of psychoactive substances

in the BPM can therefore be discriminated further to include qualitative aspects

of locomotor behavior indicated by changes in the composition of movements.

The locomotor activity observed in this chamber shown in figure 8 has already

been expressed as a function of the distance between photobeam breaks on

several discrete levels (Geyer et al. 1986b). It was suggested that the total

count of all events serves as a measure of small movement activity, the

recordings of crossovers serves as measures of ambulations, and movements

between regions such as those in figure 6 serve as indicators of larger

movements within the BPM. This idea can be extended using the scaling

approach to introduce the following basic relationship,

where L is the average length of consecutive response sequences, i.e.,

positions of the rat in the BPM chamber, measured with resolution I. The value

d is often identified with the fractal dimension (Mandelbrot 1977) of an object.

We will refer to it here as the spatial scaling exponent and have deliberately

avoided calling it dimension, because new developments in the application of

ergodic theory to experimental data sets have resulted in a variety of dimension

measures that may be obtained by embedding the data into higher dimensional

spaces (for an overview, see Farmer et al. 1983). The average distance of

consecutive responses can be measured by introducing a metric on a space

221
FIGURE 8. Calculation of the spatial scaling exponent d. On the left, two

different patterns of four consecutive movements within the

Behavioral Pattern Monitor are displayed and connected by lines.

By computing the average distance between these responses

using different inter-response distances, the spatial scaling

exponent describes the change in length with the change in the

inter-response distance. The lower part displays an example with

consecutive responses whose distance measures are more

strongly affected by increasing the inter-response measuring unit

leading to shorter distances compared to the upper part with the

same measuring resolution. The scaling exponent is obtained by

finding the slope between the measuring resolution and the

average distance computed.

222
constructed from the recorded data sets. The city block metric was chosen as a

suitable distance metric because it seems to be particularly sensitive to the

correspondence between changes in the photobeams and changes in the (x-y)

position of the animal in the BPM. It is computed as

The average distance of response sequences, L, starting at the mth position in

the data set, reads

where C is a normalization factor ensuring that end effects are not influencing

the distance calculation. The scaling exponent dis computed by fitting the slope

of the line of log(L) vs. log(I) with a least-square procedure. The physical

interpretation of the exponent d can be obtained by considering an example of a

path observed in the BPM chamber, which is schematically given in figure 8. In

the first case, successive observations result in locations of the rat in the box

that are connected by straight lines. The function L(I) results in a linear

dependence on I, thus giving the simplest result, namely, that the scaling

exponent for a straight line is 1. In the second case, consider a path that is very

irregular and covers only a moderate distance after many successive

observations. In this case, the average distance measured is smaller with a

low-resolution instrument than with a high-resolution instrument, resulting in a

stronger decay of the average distance than with the straight path and therefore

in a higher d exponent. The spatial scaling exponent d consequently

characterizes how path lengths depend on the resolution of the instrument and

allows one to quantify different qualitative geometrical features of the locomotor

behavior.

So far, the response was defined to be a movement within a given

measurement resolution without considering how long it takes to observe one

response. A second scaling approach can be formulated by considering a

measuring instrument that is recording the position of the animal in the box with

different time resolutions. Thus, the following relationship may be assumed:

which states that the number of observations within a given time resolution

depends on the time and a characteristic time scaling exponent, alpha or 

223
More simply stated, the number of responses having a certain length (t, t)in

the BPM chamber decays with a power law behavior. The interpretation of

may be facilitated by an example. An animal treated with a stimulant drug like

amphetamine exhibits many fast locomotor responses because of the increased

amount of activity. Conversely, slow responses are correspondingly more

infrequent than saline control animals and faster responses dominate the

behavior. Such an effect results in a rapid decay of the number of responses

observed with increasing response time; thus a larger time scaling exponent

is obtained. A rat that shows less locomotor activity in the BPM than controls

exhibits relatively more slow responses, thereby slowing the decay of the

number of the responses across increasing response times and yielding a

smaller

The Goal

The eventual goals of this line of investigation are (1) to develop metrics that

can facilitate comparisons among the effects of substances and other

manipulations and then (2) to identify a small number of parameters that

describe the macroscopic properties of the complex system. The main concept

of synergetics (Haken 1983) is that interacting systems self-organize and

exhibit global properties that are not dependent on the microscopic details of

the system. These properties are described by order parameters, which consist

of macroscopic collective variables and characterize the self-organized

behavior. The characterization of the effects of substances on different

behavioral paradigms is undoubtedly influenced by many factors, some of

which were presented in papers at this meeting (“Importance of Behavioral

Controls in the Analysis of Ongoing Events” and “Cocaine Self-Administration:

Pharmacology and Behavior”). Substances may influence behavior through the

interaction of several neurotransmitters and neuropeptides in different locations

in the brain, as pointed out by Koob (“Neurobiological Approaches to

Brain-Behavior Interaction: Summary and Overview”). Therefore, with

unconditioned locomotion as the dependent variable, one has to assume that

the effects of different substances reflect combinations of many effects on

different neuronal systems. To capture the differential effects of psychoactive

substances, the spatial scaling exponent dand the temporal scaling exponent

were taken to be order parameters that describe the macroscopic organization

of locomotor behavior in the BPM. The first objective was to identify categories

of substance effects in the d versus plane.

224
Implementation

The implementation and application of scaling approaches to this behavioral

data set follow a scheme that is broadly applicable to a wide variety of data

sets. There are, however, important subtleties that may have strong influences

on the identification of the order parameter or parameters. The implementation

consists of a four-stage process.

1.

2.

3.

4.

A set of behavioral observations is considered for evaluating the effect of

the independent variable in the experiment. In the case of locomotor

activity experiments, these observations depend strongly on the specifics

of the apparatus used; e.g., for the BPM, the sets recorded consist of a

location in the BPM chamber, whether rearing or hole poking has occurred,

and the time since the last response.

The second stage, an important step in determining the order parameters,

involves a transformation of the data set in a suitable space. The choice of

the space depends strongly on the theoretical construct used to assess

changes induced by drugs. In our case, to calculate the spatial scaling

exponent d, we transformed the data set into Euclidean space equipped

with the city block metric.

A measure is defined, i.e., a function that converts elements of the

previously defined space into a number (M:S R). We chose the average

length of the paths traveled in the BPM chamber and the number of

responses with a certain response time as the measures of interest to

describe the effects of the drugs.

It is determined whether a scaling relationship exists between the measure

and a variable that influences the measure. If a scaling relationship is

observed, then this variable may serve as an order parameter, because it

combines the information of the measurement on different levels of

observation. For the paths of the rats in the BPM, the average length

served as the measure and the length scale was used as an order

parameter leading to the calculation of d. Similarly, the number of observed

responses having a response time between t and (t + t) served as

another measure that showed a scaling relationship with the response time

as an order parameter resulting in the calculation of

225
Results

Figure 9 displays the results of the evaluation of amphetamine-treated animals

in the d-versus plane. The results are expressed in terms of deviations in

units of standard deviations from the saline controls, i.e., a z-transform

FIGURE 9. Effects of amphetamine on scaling exponents. The results from

amphetamine-treated animals are shown in z-transformed

coordinates in the d-versus plane. The origin corresponds to the

saline control group and the boxes indicate ± 1 standard deviation

from the mean in the d and directions. Increasing doses are

connected with lines to signify the dose-response curve.

Amphetamine results in a strong activating effect, indicated by a

decrease in a with no strong changes in d.

226
was carried out to obtain these data points. Consecutive doses of drug are

connected with lines to indicate the direction of the dose-response curve. At the

origin and corresponding to the location of the saline control group, a square

with a side length of 2 units indicates the 66 percent confidence interval (±1

standard deviation) of the transformed data. The points in the plane correspond

to the transformed group mean and the boxes around the points are the

transformed standard deviations for d and , respectively.

The animals treated with d-amphetamine did not exhibit significant differences

in the spatial scaling exponent d over the 60-min test session (F[4, 37] = 0.61).

The temporal scaling exponent a yielding information about the distribution of

different “pausing times” during the test session gives the expected result of an

increase in “fast” time bins because of the increased activity of the

amphetamine-treated animals ( : F [4, 37] = 26.99). This hyperactivity leads to

an increased negative slope of the logarithmic decay of number of observed

times versus the response time. The effect of amphetamine on this measure

corresponds to a rotation of the distribution. Contributions from slower time

slices are redistributed into faster bins. The higher doses of amphetamine

(1.0 and 2.0 mg/kg) were significantly different not only from the saline controls

but also from the lowest dose amphetamine group (0.25 mg/kg). In the

d- plane, the dose-response curve is pointed horizontally (no change in d) to

the left toward more negative values.

The rats treated with apomorphine showed more complicated group differences

(d: F[4, 421 = 6.10) (figure 10). The lowest dose (0.1 mg/kg) resulted in an

increased spatial scaling exponent, d, and higher doses (0.5 and 1.0 mg/kg)

reduced this scaling exponent to a level that was somewhat lower (for

2.0 mg/kg) than that of the saline control group. Thus, the paths of the low-dose

apomorphine animals can be characterized as more jagged, covering less

distance with increasing number of consecutive responses. The higher dose

animals exhibit straightened paths that are characterized by a lower scaling

exponent resulting in longer distances observed with increasing response

length. The changes in the temporal scaling exponent which differed

significantly among the different dose groups ( : F[4, 42] = 29.97) documented

an increased decay of time occurrences for the higher doses of apomorphine

(0.5 to 2.0 mg/kg) as a result of the increased activity that was displayed by the

animals. As with amphetamine, the distribution changes correspond to a

rotation of the response time distribution. This response is portrayed in the d-a

plane indicating the biphasic relationship with increasing dose, the lower dose

227
FIGURE 10. Effects of apomorphine on scaling exponents. The z-transformed

results for apomorphine are displayed as in figure 9. The

increasing dose results in an initial small increase of and d

followed by a decrease in d and .

being displaced to the upper right, whereas increasing doses shift the groups to

the lower left.

The dose-response curve of lisuride is displayed in figure 11. Significant

changes among the different dose groups (F[4,38] = 21.93) reflect the

straightened paths characteristic of animals treated with higher doses (30 and

60 μg/kg) and paths that are unchanged with respect to the saline control group

for the lower doses (5.0 and 15.0 μg/kg). The temporal scaling exponent a

showed significant differences (F[4,38] = 28.36), indicating that for the three

lower doses a reduction of locomotor activity occurred, which agrees with other

measures taken from these same animals and reported by Adams and Geyer

(1985b). The distribution of response times is also indicative of a stimulant

effect producing more populated fast time bins for the highest dose (60 μg/kg).

The effect of lisuride can therefore be characterized as a low-dose slowing and

228
FIGURE 11. Effects of lisuride on scaling exponents. The z-transformed results

for lisuride are shown as in figure 9. Lisuride results in a similar

dose-response curve in the d direction but shows less stimulant

properties when compared to apomorphine; see text.

a high-dose stimulating effect. In the d- plane, the biphasic relationship of the

lisuride dose to both the temporal and spatial scaling exponents results in a

slightly different curve than the apomorphine curve. Lisuride suppresses the

activity for most doses tested, and a reduction in d is observed even with lower

levels of activity. The structure of the paths in the BPM chamber is therefore

relatively independent of the amount of activity observed; in this particular case,

long distance-covering moves predominate the behavior even with a reduction

in the overall activity. Furthermore, this pattern of results is more representative

of the observed behavior of the animals than was the spatial CV statistic shown

in figure 7. Although the CV was insensitive to the long distance-covering

movements because they were not oriented in a suitable Cartesian frame, the d

measure captured this effect of the drug more adequately.

229
Finally, the significant changes (F[4,34] = 4.60) between the different doses of

MDMA are characterized by a progressive decrease in d with increasing dose.

Therefore, animals treated with MDMA travel farther in the BPM chamber and

follow straight paths. That both results corroborate previous findings (Gold et al.

1988), indicates a strong circling behavior, particularly for higher doses. The

stimulant effect is substantiated by the distribution of times characterized by a,

which is significantly changed (F[3,34] = 9.50) for the two highest doses versus

the saline control and versus the 1.25- and 2.5-mg/kg groups. This result points

to a temporal restructuring of the responses toward faster movements with less

time being spent at each successive position in the BPM chamber. Across all

the drugs compared here, the sensitivity of a has proven to be much greater

than either crossovers or total beam breaks as a measure of changes in the

amount of activity. Such results indicate that a is an important indicator that

enables us to distinguish even small changes in activity.

Several patterns seem to emerge from this evaluation. First, amphetamine

proved to be the most potent stimulant of the drugs compared; it displaced the

a exponent farthest from the origin. Next were MDMA, apomorphine, and

lisuride, in that order. Second, apomorphine and lisuride show a low-dose

increase in a with a small increase in d, suggesting that in both cases the paths

become more clustered as the temporal behavior slows down. Third, MDMA

and lisuride lead to the strongest decrease in d, followed by apomorphine,

indicating a stimulating action that leads to both a speeding of responses and a

decreased clustering of consecutive responses. Last, it can be summarized that

the change in a is not uniformly associated with a similar change in d for the

different substances evaluated here; thus is emphasized the distinctness of the

information that can be obtained from these measures.

Outlook

The application of scaling concepts to behavioral data points toward several

interesting directions. First, an enormous reduction and compression of

information may be obtained by finding a suitable variable that shows scaling of

the quantities of interest, e.g., measures of locomotor activity. Second, distinct

subgroups of behavioral reactions could be identified based on the basis of

different changes of the temporal and spatial scaling exponents. These

subgroups can be distinguished from one another by a measure of distance

within the plane defined by both exponents d and . Thus, a quantitative

descriptor of similarity may be defined, either as a vector quantity or as a

measure describing the distance between different drug reactions and the

230
directions of the different effects. Finally, to predict drug effects, to distinguish

between different phases of drug effects, or to study rapidly occurring

fluctuations induced by drugs, a quantitative model could be based on the order

parameter concept. In such a model, a set of independent variables-which

may be based on any of the behavioral, biochemical, or neurophysiological

measures described in this volume (e.g., “Psychomotor Stimulant Effects on

Single Neurons in Awake, Behaving Rats,” “Microdialysis in the Study of

Psychostimulants and the Neural Substrate for Reinforcement: Focus on

Dopamine and Serotonin,” and “Neurophysiological Approaches to Receptor

Pharmacodynamics”)—define a function describing the quantitative change of

the order parameter, e.g.,

d(x1, x2)

The change in x1 and x2 may correspond to effects on different receptor types,

which eventually should result in the development of suitable combinations of

substances affecting the different receptor systems in order to obtain a

macroscopic change in behavior compatible with the change in the order

parameters.

CONCLUSIONS

This review has discussed some of the important considerations in assessing

the effects of drugs or other treatments on the locomotor activity of rodents. The

need to clearly define the behavior being studied has led to most researchers,

and this review, focusing on measures of locomotor activity versus more

inclusive measures of general motor activity. For a variety of reasons, the most

reliable measures of rodent activity are those that are selective for locomotor

movements from one place to another. Additional considerations, including the

importance of using a test system that enables the detection of either increases

or decreases in activity, indicate that the test chambers to be used should be of

sufficient novelty or complexity for the control subjects so that they exhibit an

adequate level of activity. For most practical purposes, therefore, the minimal

system suitable for most investigators is one in which the test chamber is (1)

large enough to engender in the animals locomotion that is qualitatively different

from their locomotion in the residential caging and (2) equipped for automated

measures selective to locomotor movements. The further ability to use the

system in the nocturnal portion of the animal’s sleep-wake cycle without

providing feedback to the animal is advantageous from the point of view of

flexibility and sensitivity. Such criteria are readily satisfied by systems based on

231
infrared photobeams, which are clearly the most common and generally

satisfactory detectors. The additional advantage of photobeam systems is that

they can easily provide a secondary measure of general or fine motor activity,

which, while having limited behavioral meaning, can serve as an assay of drug

effects and indicate the need for further studies.

A number of more complex issues are relevant to more thorough and

behaviorally meaningful characterizations of drug effects on locomotor activity.

The foremost concern involves the desirability of multiple concurrent measures,

preferably of explicit investigatory responses as well as locomotor movements.

Maximizing the sensitivity of measures of locomotor activity requires some

recognition of the qualitative features and structure of the spatiotemporal

patterns of locomotion exhibited by rodents in the given test environment. From

a behavioral point of view, the nature of the test chamber has profound effects

on the kinds of questions the investigator may address. The example described

here, the BPM system, demonstrates some of the ways that detailed analyses

of patterns of locomotion may increase the sensitivity and interpretability of

one’s measures. The behavioral profiles provided by such multivariate

assessment techniques greatly enhance the specificity with which drug effects

can be characterized. With the advent of new detector systems such as video

imaging methods and the further development of new metrics, such as those

evolving from the study of nonlinear dynamic systems, future state-of-the-art

studies of locomotor activity are likely to provide ever more informative and

powerful approaches to understanding the behavioral effects of drugs.

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ACKNOWLEDGMENTS

This work was supported by grants from the National Institute on Drug Abuse

(DA02925 and DA6325). M.A. Geyer was supported by a Research Scientist

Development Award from the National Institute of Mental Health (MH00188).

AUTHORS

Mark A. Geyer, Ph.D.

Professor of Psychiatry

Department of Psychiatry, T-0804

School of Medicine

University of California, San Diego

La Jolla, CA 92093

Martin P. Paulus, M.D.

Assistant Research Psychobiologist

234
Laboratory of Biological Dynamics and Theoretical Medicine

Department of Psychiatry, M-0603

School of Medicine

University of California, San Diego

La Jolla, CA 92093

235

Neurobiological Approaches to

Brain-Behavior Interaction:

Summary and Overview

George F. Koob

The technical review developed in such a way that one could easily rearrange

the presentations to reflect three major themes. First, there were dependent

variables from neurobiology ranging from positron emission tomography (PET),

single photon emission computed tomography (SPECT), and magnetic

resonance imaging (MRI) techniques to in vivo voltammetry and dependent

variables from behavior involving measures of brain reward, drug

self-administration, and motor activity. Independent variables that critically

influence the dependent variables included behavioral history that can affect

subsequent behavior and behavioral history that can affect neurochemical

measures. Finally, the important role of contingency control was discussed.

The presentation of PET, SPECT, and MRI techniques by William Jagust

focused on introducing these techniques and their relative resolution and

practicality. It was clear that the PET technique currently provides higher

resolution and more versatility but is more expensive than SPECT. PET also

has the potential advantage that tracers for specific neurotransmitter receptors

may be measured. SPECT is inexpensive but has low sensitivity and poor

temporal resolution and is used largely as a measure of blood flow. MRI

produces a much greater contrast sensitivity between white and gray matter yet

is limited by having no isotopes and thus no tracers by which to measure the

activity of specific receptors.

Using PET, Jagust showed that patients with Alzheimer’s disease had

decreases in glucose metabolism in the temporal and parietal cortex with little

change in subcortical areas. In a preliminary study on drug abusers, labeled

spiroperidol binding was decreased in the caudate nucleus of a nondetoxified

cocaine user.

237
Harriet de Wit then showed the power of the PET technique when combined

with innovative experimental design and correlational analysis. Using a

within-subjects design, de Wit was able to administer drug and take behavioral

measures in a natural setting and then repeat many of these behavioral

measures after drug in the unnatural setting of the PET measurement. Studies

following ethanol ingestion showed increases in measures of positive mood,

elation, friendliness, etc., at blood alcohol levels of 40 to 60 mg percent.

Identical amounts of ethanol produced a decrease in global metabolic rate as

measured by PET in all regions examined, including all the cortices, basal

ganglia, and cerebellum. No major correlations were evident between individual

differences in mood changes and differences in regional metabolism. Similar

results were obtained with diazepam except that the behavioral measures

reflected more decreases in anger, anxiety, and arousal. The experimental

designs used by de Wit were very powerful; these designs combined with better

PET resolution and new methods of PET analysis (see “Importance of

Behavioral Controls in the Analysis of Ongoing Events”) should provide an

exciting means by which to examine the neural basis of human mood states

after drugs.

Linda Porrino discussed the use of the 2-deoxyglucose (2-DG) technique for

evaluating brain activity changes in animals associated with various

independent variables such as electrical brain-stimulation reward, drug

self-administration, and brain pathophysiology. In the context of this thorough

exploration of the 2-DG technique, Porrino emphasized highly critical issues

regarding what was being measured and how it was analyzed.

First, it appears clear that the 2-DG technique measures brain synaptic activity

and changes in the afferent input to neurons but not changes in cell body

activity. The 2-DG measures do not correlate well with electrophysiologic

measures of cell firing, but in support of the hypothesis regarding synaptic

activity, blockade of ion gradients with ouabain blocks glucose metabolism.

(See “Metabolic Mapping Methods for Identifying the Neural Substrates of the

Effects of Abused Substances.“)

The advantage of the 2-DG technique is that it provides a visualization of

activity of the entire central nervous system at the resolution of autoradiography

in conscious, freely moving animals. The disadvantages include inability to

distinguish direct from indirect effects and excitation from inhibition or

neurochemical specificity. In addition, the 2-DG method currently has poor time

resolution.

238
Perhaps the most critical issue, however, centers on the problems of data

analysis. Porrino argued very convincingly that one must show in 2-DG studies

that the reference measure (referent) is not changing with the independent

variable. She showed that the relative optical density measure used by others

can give misleading results and may explain much of the controversy in the

literature. Using measures of local cerebral glucose utilization (nontransformed

rates of glucose utilization), Porrino showed that regions of the ventral striatum

such as the nucleus accumbens (NAC) and olfactory tubercle as well as their

efferent projections had increased 2-DG activity following intracranial

self-stimulation and following administration of drugs that activated the

dopamine (DA) system, such as cocaine and amphetamine.

Mark West demonstrated the utility and versatility of chronic unit recording in

the freely moving rat for identifying the actions of drugs at the cellular/systems

level of analysis. Rats were prepared with multiple microelectrodes using

microwires with which up to 10 electrodes could be recorded at the same time.

This technique can measure spontaneous tonic firing rates as well as phasic

firing patterns that can be correlated with behavior. Specific regional cerebral

stimulation or specific neurotransmitter activation using a four-barreled

micropipette can be combined with electrical recordings in awake animals. The

recordings appear to be stable over days and weeks, indeed, up to 60 days.

Using this technique, West showed that there was a topographical

representation of striatal responses to body stimulation and that other striatal

cells respond to active movement such as a rat moving on a treadmill. The cells

in which firing correlated with treadmill locomotion showed enhanced

responsiveness following administration of amphetamine but decreased

excitation following a dopamine receptor antagonist. Sixty percent of the cells

that fired during locomotion were excited by administration of DA. Recordings of

neural activity from the NAC showed increased firing during the licking of a

spout for chocolate milk but not during licking of a dry spout, suggesting that

increases in spontaneous activity were more related to rewarding stimuli. These

results emphasize the power of this technique in correlating unit activity with

behavioral responses to drugs at the system level. Such a technique provides a

critical bridge between global measures such as the PET and 2-DG techniques

and single-unit responding in anesthetized animals (see “Neurophysiological

Approaches to Receptor Pharmacodynamics”).

Francis White provided an indepth analysis of the power of neurophysiology to

explain receptor pharmacodynamics by exploration of the interaction of the D1

239
and D2

 DA receptor subtypes. Using an anesthetized preparation, sophisticated

electrophysiological recordings allowed specific neuronal systems and the

effects of drugs on these systems to be identified as well as the exploration of

the mechanism and its relevance to behavior. The power of such a technique is

the ability to use classic electrophysiological techniques such as orthodromic

and antidromic stimulation for identifying projection neurons and afferent inputs.

A potential significant weakness of such an approach is the anesthesia.

Although not necessarily a problem with recording from DA neurons,

anesthesia apparently diminishes stimulatory effects particularly in

acetylcholine neurons (comment of H. Chris Fibiger). Consistent with this

observation, the studies discussed by White of recordings from DA neurons in

anesthetized rats show a predominance of DA inhibition whereas in awake

freely moving rats there was a predominance of dopamine excitation (see

“Psychomotor Stimulant Effects on Single Neurons in Awake, Behaving Rats”).

In single-unit recordings from the NAC accumbens in anesthetized rats, White

showed that intact neurons responded to both D1 and D2 agonists with an

inhibition of firing. However, acute depletion of DA from the NAC with

alphamethylparatyrosine (AMPT) produced an abolishment of the decrease in

firing produced by the D2 agonist LY 141865 (quinpirole). This responsiveness

of the DA receptor could be reinstated with a combination of the D2 agonist and

the D1 agonist SKF 38393, suggesting that D1 receptors play a critical enabling

role in the activity of D2 agonists. Similar results were obtained at the behavioral

level, where the stereotyped behavior produced by DA D2 agonists was also

eliminated by pretreatment with AMPT but could be reinstated by simultaneous

administration of the D1 agonist SKF 38393 with the D2 agonist. The converse

requirement of D2 activation for D1 effects did not appear to be true. These

results suggest that the behavioral interactions produced by activation of brain

DA receptors result from an enabling action of the D1 receptor subtype; these

results also show the power of the electrophysiological approach in predicting

pharmacologic interactions at the behavioral level.

Bartley Hoebel’s presentation explored the power of the technique of brain

microdialysis in the context of measuring DA and dihydroxyphenylacetic acid

(DOPAC) from the NAC in freely moving rats. In this technique a chronic

cannula is implanted in the brain of the rat through which a microdialysis probe

can be inserted at any time. Through this probe, brain neurotransmitters that

form an equilibrium with the extracellular space can be measured.

240
The advantage of this technique is that the liberation of neurotransmitters from

synaptic terminals can be correlated with ongoing behavior. Specific

neurotransmitters can be measured that presumably reflect the activity of

synaptic terminals and, because of the size of the dialysate, membrane

enzymes are excluded. Other potential uses for this technique are that it can be

used for drug or transmitter stimulation of specific neuronal receptors by

injecting through the dialysis probe. Disadvantages include a long time

resolution (5 to 20 min), lack of sensitivity, and a limitation in what transmitters

can be measured as well as possible interactions with other simultaneously

released neurotransmitters. Many of these disadvantages will probably be

resolved with refinement of the technique as well as application of more

sophisticated assays and assay techniques.

Using microdialysis, Hoebel has extensively explored the conditions under

which DA release in the NAC can be modified. Most drugs of abuse such as

cocaine, phencyclidine, and nicotine provoked a release of DA when infused

directly into the NAC in freely moving, awake animals. More drug-reinforcing

stimuli such as food and rewarding brain stimulation also caused an increase in

nucleus accumbens DA release. In an elegant experiment, Hoebel showed that

saccharin infused on the tongue of a naive rat increased NAC dopamine

release but actually decreased DA release in a rat for which saccharin had

been the conditioned stimulus in a conditioned taste aversion experiment.

These results demonstrated the power and sensitivity of this technique for

brain-behavior interactions and add impetus to hypotheses suggesting a role for

DA in the NAC in reward/incentive motivation.

Behavior-dependent variables are equally important in brain-behavior

interactions and also have limitations specific to each technique. They also

require important control measures not unlike those for

neurochemical-dependent variables. In addition, behavioral measures can lend

themselves to equally sophisticated analyses, allowing a greater potential for

correlation to discrete electrophysiological and neurochemical events.

The hypothesis that electrical brain self-stimulation provides a direct measure of

the activity of brain reward systems is the premise that guides Conan

Kornetsky’s pursuit of the use of brain-stimulation reward (BSR) as a

behavior-dependent variable. Kornetsky has developed a powerful threshold

measure of BSR that has the major advantage of minimizing the response rate

confounds of other conventional BSR techniques. The technique involves a

discrete trials procedure in which the rat receives a free BSR stimulus train with

241
the option of responding by rotating a wheel manipulandum to obtain a second

stimulation train. Using this procedure Kornetsky can readily obtain reliable

threshold measures that can be subject to probit analysis for statistical testing.

When this procedure was used, most drugs that have abuse potential produced

a lowering of reward threshold. These include drugs such as amphetamine,

cocaine, nicotine, opiates, and phencyclidine. DA receptor antagonists such as

haloperidol and pimozide raised BSR threshold. The selectivity of these effects

has been demonstrated by Kornetsky, who showed that those same drugs and

doses of drugs failed to alter or change in a different direction the threshold for

BSR detection. Here the rat performs exactly the same behavioral task, but the

BSR is presented at an intensity lower than threshold and the rat is asked to

respond to the discriminability of that stimulus.

Finally, BSR has the potential to link drug studies directly to brain function. Two

deoxyglucose measures taken after BSR show that ventral forebrain structures

such as the NAC, olfactory tubercle, and prefrontal cortex show a significant

activation (see “Metabolic Mapping Methods for Identifying the Neural

Substrates of the Effects of Abused Substances”). Drugs of abuse combined

with BSR can potentiate some of these effects, but preliminary results suggest

that some of these drug effects may be common to drugs of abuse and others

may be different for each class of drug.

Drug self-administration as a dependent variable provides one of the most

direct measures of the reinforcing properties of a drug. William Woolverton

described how the behavioral contingencies under which self-administration

was maintained could emphasize either the pharmacology or the history of

reinforcement. A substitution procedure was used in primates to assess the

different potential of drugs to maintain intravenous self-administration. Animals

established a baseline of cocaine self-administration, were subjected to an

extinction probe, and then after several baseline days were subjected to a new

drug probe at a given dose. With this approach, drugs such as the DA agonists,

apomorphine, bromocriptine, and piribedil were readily self-administered,

whereas fluoxetine, a serotonin reuptake inhibitor, was not. Thus, the

pharmacology of the drugs is clearly important for self-administration.

However, the schedule of reinforcement that maintains self-administration can

significantly alter the pattern of responding. Indeed, in earlier work Spealman

showed that cocaine could act as both a positive and negative reinforcer. This

point was demonstrated in monkeys working on a concurrent schedule in which

242
a variable-interval schedule produced cocaine infusions and a fixed-interval

schedule terminated the schedule of cocaine infusion. In addition, choice

procedures can be used to manipulate the relative choice of responding for a

drug. In monkeys trained in a discrete trials procedure for food and cocaine,

increasing the dose of cocaine increased the frequency of cocaine choices;

however, increasing the number of food pellets delivered decreased cocaine

choices. Such a choice procedure may be very useful for brain-behavior

interaction studies, particularly to control for nonspecificity associated with

responding.

Spontaneous locomotor activity has been classically used as a means of

measuring the arousal level of animals and the stimulant and sedative

properties of drugs. However, simple measures of motor activity have

significant limitations in discriminating differential drug effects and even

dose-effect functions. Mark Geyer and Martin Paulus described a new approach

to locomotor activity measures that involves both quantitative and qualitative

measures. A Behavioral Pattern Monitor (BPM) system developed by Geyer not

only incorporates measures of crossovers and investigatory behaviors but also

provides a description of the activity pattern of the animal. Qualitatively, this

pattern can be described by video or actual line drawing. Quantitatively, a

coefficient of variation can be calculated that basically describes the distribution

or uniformity of the locomotor transitions. A high coefficient of variation indicates

a stereotyped pattern of activity. These measures have allowed a clear

discrimination to be made between stimulant drugs that were previously difficult

to separate on motor activity measures. Paulus described a novel approach to

measuring the complex patterns of locomotor activity described by the BPM.

Borrowing from physics and nonlinear dynamics, Paulus used a scaling

transformation to describe the complexity of the locomotor path structure. For

example, the measure d, a spatial scaling component, increased with the

complexity of the path structure but decreased with a preservative path

structure. Thus, apomorphine, which produced stereotyped locomotion and

thigmotaxis (a high coefficient of variation), produced a decrease in d. However,

amphetamine produced nonstereotyped locomotion, a low coefficient of

variation, and no major change in d. These sophisticated mathematical models

provide an innovative way to simplify complex events. They may have great

potential for linking locomotor activation to neurobiological systems and for

identifying and characterizing the actions of stimulant drugs.

Three presentations focused on independent variables that transcend a given

dependent variable or experimental design in experiments designed to

243
elucidate interactions between brain and behavior. These issues affect both the

behavior to be measured and the neurochemistry; not only are they critical for

interpretation of results but they also provide intriguing questions for

brain-behavior interactions.

The role of an organism’s history in altering the behavioral response to drugs

was discussed by James Barrett. Barrett provided several examples in which

the behavioral response to drugs changed dramatically depending on the

previous training of the animal. For example, animals with no history of

avoidance showed a decrease in punished responding following amphetamine

treatment. However, animals with a history of avoidance showed an increase in

punished responding following amphetamine treatment.

Similarly, morphine decreased avoidance responding in animals before an

experience with shock presentation but increased responding on avoidance

after a history of shock presentation. Thus, previous experience left a residual

effect that altered subsequent drug responses. This type of behavioral analysis

appears to be critical for studies of brain-behavior interactions. Unknown at this

time is how such historical effects could alter the reinforcing properties of drugs

or drug dependence. Finally, it should be noted that the residual behavioral

effect produced by a different behavioral history presumably has a

neurochemical correlate.

This thesis was clearly elaborated by Lewis Seiden, who showed that a variety

of behavioral factors can influence catecholamine metabolism; these factors

can differentially affect catecholamine metabolism in different brain regions. For

example, food-deprived rats that were given food showed increases in DOPAC

levels (reflecting increases in DA metabolism) in the hypothalamus, amygdala,

and NAC. However, tube-fed animals given food showed no change in DOPAC

in the NAC and posterior hypothalamus but still showed the increase in DOPAC

in the amygdala. Thus, an increase in DA metabolism in the NAC was

associated with the motor activity of feeding (see “Microdialysis in the Study of

Psychostimulants and the Neural Substrate for Reinforcement: Focus on

Dopamine and Serotonin”; “Psychomotor Stimulant Effects on Single Neurons

in Awake, Behaving Rats”; and “Brain-Stimulation Reward: A Model for the

Study of the Rewarding Effects of Abused Drugs”). Seiden emphasized that the

brain-behavior interface with drugs reflected a dynamic interaction in which both

behavior and the environment could alter brain chemistry with or without drug,

supporting the hypothesis that the residual effects observed with different

behavioral histories can reflect significant changes in brain neurobiology.

244
The importance of behavioral controls in the analysis of brain behavior

interactions was explored by Steven Dworkin. In addition to pharmacologic

factors and behavioral history, the need for subject control over contingencies

was emphasized. To control for nonspecific effects associated with drug effects

when examining brain-behavior interactions, a yoked-control procedure has

been developed. Here, the neurobiological response of the contingent subject is

thought to measure reinforcement mechanisms, whereas the neurobiological

response of the noncontingent subject is thought to reflect nonspecific

activation. The control subject receives no treatment and thus its

neurobiological response presumably reflects no event.

Using this approach, Dworkin and colleagues showed significant differences in

limbic brain regions associated with morphine self-administration between

contingent and yoked subjects. Most notably the amino acids, GABA and

glutamate, and the monoamines, DA, norepinephrine, and serotonin, were

altered. Results from this work have been the driving force in developing

hypotheses about the neurochemical basis for drug reinforcement and

dependence.

This need for appropriate behavioral controls was extended by Dworkin to other

behavioral situations including punishment. DA metabolism was decreased and

serotonin metabolism was increased in the frontal cortex of the punished

subjects versus yoked animals. A behavioral confirmation of the power of the

contingent situation versus the noncontingent situation was manifested by the

significant increase in toxicity associated with noncontingent administration of

cocaine. Thus, control of events can significantly affect brain neurobiology and

the experimental designs used for establishing contingent effects could be

easily used with other neurochemical-dependent variables.

Finally, George F. Koob addressed the question of drug dependence and

different measures of drug dependence. Most of the techniques described in

the technical review focused on behavioral measures of the motivational effects

of drugs and the interface of neurobiological measures in acute drug situations.

Largely lacking were studies in dependent animals.

Previous work exploring the neurobiological substrates for opiate withdrawal

have focused on “physical” signs such as wet dog shakes and ptosis and have

identified a diffuse localization in the amygdala, thalamus, and

mesencephalic-diencephalic function of sites sensitive for opiate antagonist

precipitation of withdrawal. Using more motivational tasks for measuring

245
withdrawal such as response suppression and place aversion, Koob and

colleagues have shown that the region of the nucleus accumbens is a very

sensitive substrate for precipitating the aversive effects of opiate withdrawal

with opiate antagonists. Because the NAC appears to be particularly sensitive

to the effects of opiate antagonists in blocking the reinforcing effects of opiates,

these results suggest that the same sites in the brain important for the acute

motivational effects of drugs may be involved in the plasticity associated with

the motivational aspects of drug dependence.

Several important conclusions developed from this technical review, particularly

in regard to the dependent variables that are used to explore brain-behavior

interactions. Attempts should probably be made to standardize behavioral tests

from rats to primates to humans. Some of the experimental designs elaborated

in the human work attest to this as a possibility. Technical guidelines for the

proper use of voltammetry, microdialysis, neurophysiology, and 2-DG would be

helpful and useful. Clearly, all techniques have some limitations, as amply

elaborated by the presentations. Nevertheless, a standardization of control

procedures and methodology would save unnecessary frustration and lost effort.

Another point evident from the review is that data analysis is continually in need

of improvement and sophistication. New means of analyzing neurochemistry,

motor activity, and glucose metabolism were presented. Such sophisticated

analysis promises an ultimately finer resolution of brain-behavior interactions.

Although the focus on the brain monoamine systems demonstrates the breadth

of knowledge available in this area, studies of brain-behavior interaction

ultimately must demand a broader perspective. New techniques must be

developed for measuring and manipulating neuropeptides and amino acids.

Finally, this technical review focused largely on dependent variables and our

advances therein. However, the presentations on independent variables raised

these questions: What hypothesis is one testing? and What knowledge will be

gained from testing that hypothesis? Crosstalk between the disciplines is still

somewhat limited, and the courageous efforts of participants to transcend

discipline chauvinism and to interact across levels of analysis should continue

and be strongly encouraged.

246
AUTHOR

George F. Koob, Ph.D.

Member (Professor)

Department of Neuropharmacology

The Scripps Research Institute

10666 North Torrey Pines Road

La Jolla, CA 92037

Adjunct Professor

Departments of Psychology and Psychiatry

University of California, San Diego

La Jolla, CA 92037

247
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71 OPIATE RECEPTOR SUBTYPES AND BRAIN FUNCTION. Roger M.

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86 COMPULSORY TREATMENT OF DRUG ABUSE: RESEARCH AND

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107 DRUG ABUSE PREVENTION INTERVENTION RESEARCH:

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111 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH:

VOLUME I. Theresa N.H. Lee, Ph.D., ed.

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112 EMERGING TECHNOLOGIES AND NEW DIRECTIONS IN DRUG

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113 ECONOMIC COSTS, COST EFFECTIVENESS, FINANCING, AND

COMMUNITY-BASED DRUG TREATMENT. William S. Cartwright, Ph.D.,

and James M. Kaple, Ph.D., eds.

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114 METHODOLOGICAL ISSUES IN CONTROLLED STUDIES ON

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Kilbey, Ph.D., and Khursheed Asghar, Ph.D., eds.

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115 METHAMPHETAMINE ABUSE: EPIDEMIOLOGIC ISSUES AND

IMPLICATIONS. Marissa A. Miller, D.V.M., M.P.H., and Nicholas J. Kozel,

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GPO Stock #/017-024-01460-5 $4.00

116 DRUG DISCRIMINATION: APPLICATIONS TO DRUG ABUSE

RESEARCH. R.A. Glennon, Ph.D., T.U.C. Jarbe, Ph.D., and J. Frankenheim,

Ph.D., eds.

GPO Stock #017-024-01470-2 $13.00

117 METHODOLOGICAL ISSUES IN EPIDEMIOLOGY, PREVENTION,

AND TREATMENT RESEARCH ON DRUG-EXPOSED WOMEN AND

THEIR CHILDREN. M. M. Kilbey, Ph.D. and K. Asghar, Ph.D., eds.

GPO Stock #017-024-01472-9 $12.00

118 DRUG ABUSE TREATMENT IN PRISONS AND JAILS. C.G. Leukefeld,

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119 PROBLEMS OF DRUG DEPENDENCE 1991: 53rd ANNUAL

SCIENTIFIC MEETING. The Committee on Problems of Drug Dependence,

Inc. Louis Harris, Ph.D., ed.

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120 BIOAVAILABILITY OF DRUGS TO THE BRAIN AND THE BLOODBRAIN BARRIER. Jerry Frankenheim, Ph.D., and Roger M. Brown, Ph.D., eds.

121 BUPRENORPHINE: AN ALTERNATIVE TREATMENT FOR OPIOID

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122 RESEARCH METHODS IN WORKPLACE SETTINGS. Steven W. Gust,

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123 ACUTE COCAINE INTOXICATION: CURRENT METHODS OF

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ACKNOWLEDGMENTS

This research was supported by grants from the Alcoholic Beverage Medical

Research Foundation, the National Institute on Drug Abuse (DA 02812), and

the Department of Energy (DE-FG02-86ER60438). Use of the Clinical

Research Center in the behavioral phase of study 1 was supported by National

Institutes of Health grant M02 RR00055. The authors acknowledge the

assistance of Wendy Li, Betsy Bodker, and the entire PET Center staff.

AUTHORS

Harriet de Wit, Ph.D.

Department of Psychiatry

John Metz, Ph.D.

Illinois State Psychiatric Institute and Department of Psychiatry

Malcolm Cooper, M.D.

Department of Radiology

Pritzker School of Medicine

University of Chicago

Chicago, IL 60637

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