National Institute on Drug Abuse

RESEARCH

MONOGRAPH SERIES

Assessing

Neurotoxicity of

Drugs of Abuse

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

136
Assessing Neurotoxicity of

Drugs of Abuse

Editor:

Lynda Erinoff, Ph.D.

NIDA Research Monograph 136

1993

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

National Institutes of Health

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, MD 20857
ACKNOWLEDGMENT

This monograph is based on the papers and discussions from a technical

review on “Assessing Neurotoxicity of Drugs of Abuse” held on May 20-21, 1991,

in Bethesda, MD. The technical review was sponsored by the National Institute

on Drug Abuse (NIDA).

COPYRIGHT STATUS

NIDA 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 U.S. 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.

The chapters by Jensen and colleagues and Boyes were reviewed in

accordance with US. Environmental Protection Agency policy and approved

for publication: mention of trade names or commercial products in these

chapters does not constitute endorsement or recommendation for use.

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

selectively included in the coverage of “American Statistics Index,” “BioSciences

Information Service,” “Chemical Abstracts,” “Current Contents,” “Psychological

Abstracts,” and “Psychopharmacology Abstracts.”

National Institute on Drug Abuse

NIH Publication No. 93-3644

Printed 1993
Contents

Page

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Lynda Erinoff

Neurobehavioral Methods Used in Neurotoxicology. . . . . . . . . . . . . . . . . . 1

Hugh A. Tilson

Behavioral Consequences of Partial Monoamine Depletion in

the CNS After Methamphetamine-Like Drugs: The Conflict

Between Pharmacology and Toxicology. . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Lewis S. Seiden, William L. Woolverton, Stanley A. Lorens,

Joseph E.G. Williams, Rebecca L. Corwin, Norio Hata, Mary Olimski

Strategies for Detecting Subclinical Monoamine Depletions in Humans . . 53

Una D. McCann and George A. Ricaurte

Sensory Evoked Potentials: Measures of Neurotoxicity . . . . . . . . . . . . . 63

William K. Boyes

Silver Staining as a Tool for Neurotoxic Assessment . . . . . . . . . . . . . . . . . 101

Carlos A. Beltramino, José de Olmos, Ferenc Gallyas,

Lennart Heimer, and Lászó Záborszky

Mapping Toxicant-Induced Nervous System Damage

With a Cupric Silver Stain: A Quantitative Analysis of Neural

Degeneration Induced by 3,4-Methylenedioxymethamphetamine . . . . . . . 133

Karl F. Jensen, Jeanene Olin, Najwa Haykal-Coates,

James O’Callaghan, Diane B. Miller, and José S. de Olmos

iii
Reactions of 5-HT Neurons to Drugs of Abuse:

Neurotoxicity and Plasticity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Mary Ann Wilson, Laura A. Mamounas, Kenneth H. Fasman,

Karen J. Axt, and Mark E. Molliver

Quantification of Reactive Gliosis as an Approach to

Neurotoxicity Assessment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

James P. O’Callaghan and Diane B. Miller

Pharmacokinetic Approaches to the Study of

Drug Action and Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Arthur K. Cho, Masayuki Hiramatsu, Yoshito Kumagai,

and Nita Patel

Mechanisms Mediating Biogenic Amine Deficits Induced

by Amphetamine and Its Congeners. . . . . . . . . . . . . . . . . . . . . . . .

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

and Glen R. Hanson

Neurotoxicity and Drugs of Abuse: Cannabinoid Interaction

With Brain Glucocorticoid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . .

Philip W. Landfield and J. Charles Eldridge

155

188

213

226

242

Nitric Oxide as a Mediator of Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . 253

Ted M. Dawson, Valina L. Dawson, and Solomon H. Snyder

List of NIDA Research Monographs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

iv
Preface

This monograph is the result of a technical review meeting that was held

May 20-21, 1991. The National Institute on Drug Abuse (NIDA) supports

neurotoxicity research as part of its mandate to explore the consequences of

drug abuse. This research has provided important data on the neurotoxicity

of several abuse substances, such as amphetamine and its congeners;

tetrahydrocannabinol, the psychoactive component of marijuana; and

phencyclidine. These NIDA-supported studies also have served to further

public health on a broader scale by leading to the development of new

methodologies for studying neurotoxicity and by elucidating general principles

that will guide future research in neurotoxicology.

During the course of the technical review meeting, a consensus was reached

that no one technique could be used to define a neurotoxic effect but, rather,

that confluent data from a variety of tests are needed to establish neurotoxicity.

A variety of different methodologies were presented, although by no means

all those being employed in the field. In general, the methodologies could

be divided into those for screening or hazard identification and those for

characterizing neurotoxicity, including studies of mechanism of action. Screens

for evaluating large numbers of compounds are of great value to regulatory

scientists and are useful in the drug abuse field to evaluate new drugs of abuse.

Several presentations focused on screening methodologies based on behavior,

neurophysiology, neuroanatomy, or neurochemistry. Other presentations

focused on different methodologies designed to study underlying mechanisms

of neurotoxicity. A knowledge of the basic mechanisms of neurotoxicity may

help researchers understand some of the effects of drugs of abuse and may

provide useful models for studying neurodegenerative diseases.

At the meeting, Dr. John Olney presented a paper on MK801 neurotoxicity, but

time constraints prevented him from writing a chapter for this monograph. To

get a sense of the content of Dr. Olney’s talk, I recommend the paper by Olney,

J.W.; LaBruyere, J.; Wang, G.; Wozniak, D.F.; Price, M.T.; and Sesma, M.A.,

“NMDA antagonist neurotoxicity: Mechanism and prevention,” Science, 254,

1515-1518, 1991. In addition to presenting different methodologies, Dr. Olney

and colleagues pointed out the importance of understanding the neural circuitry

mediating neurotoxic effects.

V
A number of neurotoxicologists and neuroscientists who did not present papers

were active participants in this meeting and contributed to the lively discussion

following each talk. Unfortunately, there is no roster of meeting participants, so

I cannot thank each person individually. In transcribing the discussion, where

possible I have identified the questioner. Several participants were identified

by name on the tape, and they are Tom Sobotka, Ph.D., and Nate Appel, Ph.D.,

of the US. Food and Drug Administration; Diane Miller, Ph.D., of the U.S.

Environmental Protection Agency; and Rona Ramsay, Ph.D., of the University

of California, San Francisco. I think the discussions were exceedingly valuable,

and they have been included following each chapter. Because of taping failure,

the discussions following the presentations by Dr. O’Callaghan and Dr. Cho

were lost.

I hope that the readers of this monograph get some sense of the enthusiasm

of the participants and of the spirited discussions that took place on the state

of the science. All the authors have provided superb chapters that should

stimulate readers to think about the methodological issues raised in this

monograph.

AUTHOR

Lynda Erinoff, Ph.D.

Project Officer

Neurosciences Research Branch

Division of Basic Research

National Institute on Drug Abuse

Parklawn Building, Room 1DA-31

5600 Fishers Lane

Rockville, MD 20857

vi
Neurobehavioral Methods Used in

Neurotoxicology

Hugh A. Tilson

INTRODUCTION

Neurotoxicity has been defined as any adverse effect on the structure or

function of the central and/or peripheral nervous systems produced by a

biological, chemical, or physical agent (Tilson 1990a). Historically, toxicity has

been defined in anatomical or structural terms (Weiss 1988). It is now generally

accepted that neurotoxic effects can be identified and characterized at one of

many levels of organization of the nervous system, including the chemical,

anatomical, physiological, and neurobehavioral levels. Recently, there has

been an increased reliance on neurobehavioral indices of neurotoxicity to

detect and characterize neurotoxicity (Weiss and Laties 1975; Zbinden et al.

1983; Annau 1986; Tilson 1987, l990b; Cory-Slechta 1989). It has been

suggested that because behavior is the net result of integrated sensory,

motor, and cognitive function occurring in the nervous system, chemicalinduced changes in behavior may be a relatively sensitive indicator of nervous

system dysfunction (National Academy of Sciences 1975). In some cases,

the dose-effect curve for neurobehavioral measures may lie to the left of

other endpoints of toxicity. As pointed out by Weiss (l988), neurobehavioral

assessments are important because it is often the case that one of the earliest

indications of exposure to neurotoxicants is subtle behavioral impairment such

as paresthesias or short-term memory dysfunction; frequently, such behavioral

effects precede more obvious and frank neurological signs. Furthermore,

neurobehavioral measures are generally noninvasive, facilitating the study

of the onset and duration of neurotoxicity.

One measure of the acceptance of neurobehavioral measures in environmental

toxicology is their increased use in the regulatory context. Since 1975, several

expert panels or committees have recommended that neurobehavioral tests be

used in the assessment of chemicals for potential neurotoxicity (Reiter 1987;

Tilson 1990a). As early as 1975, Great Britain and Japan began requiring

neurobehavioral testing of animals to evaluate the developmental neurotoxicity

of new drugs (Kimmel 1988). In 1983 the European Economic Community

adopted testing protocols similar to those required by Great Britain and Japan

(Kimmel 1988), and in 1984 the World Health Organization published proposed

1
testing guidelines for drugs and other agents (World Health Organization

1984). The Organization for Economic Cooperation and Development is now

considering several neurotoxicity testing protocols, many of which involve

neurobehavioral assessments, In the United States, regulatory agencies

utilize neurobehavioral data in the premarket approval of several different

kinds of chemicals. For example, the Food and Drug Administration requires

a number of clinical observations in the routine testing of drugs (Tilson 1990a).

Neurobehavioral data have been used to set threshold limit values of exposure

to many chemicals in the workplace (Anger 1984). In addition, the Environmental

Protection Agency (EPA) has published several testing guidelines covering the

use of various neurobehavioral endpoints, including a functional observational

battery (FOB), motor activity, and schedule-controlled behavior (Tilson 1990a).

Neurobehavioral endpoints are also included in EPA testing guidelines for

organophosphate-induced delayed neuropathy and developmental neurotoxicity.

DEFINITION AND USE OF NEUROBEHAVIORAL ENDPOINTS IN

TOXICOLOGY

Behavior can be defined as whatever an organism is doing (Cory-Slechta 1989).

Many behavioral neurotoxicologists classify behavior as being either respondent

or operant (Tilson and Harry 1982; Cory-Slechta 1989). Respondent behaviors

generally occur in close temporal contiguity with an eliciting stimulus. Operant

behavior is emitted and occurs within the context of many environmental stimuli

but is not elicited by any one stimulus.

Behaviors also can be learned or conditioned. Most behavioral scientists

recognize at least two types of processes associated with learning: classical

and instrumental conditioning. Classical or respondent conditioning involves

the pairing of two stimuli, one of which elicits a reflex and one of which is

neutral. With repeated pairing of the two stimuli, the previously neutral stimulus

becomes a conditioned stimulus and elicits the response in the absence of the

original eliciting stimulus, Instrumental or operant conditioning involves the

repeated presentation or removal of a stimulus following a behavior to increase

the probability of the behavior (i.e., reinforcement). A reinforcer is a stimulus

that increases the probability of behavior. If the probability of a behavior goes

up following the presentation of some stimulus, then positive reinforcement

has occurred. If the probability of a behavior goes up after the removal of a

stimulus, then negative reinforcement has occurred.

The use of neurobehavioral procedures in environmental toxicology varies

according to their intended use (National Academy of Sciences 1975). Several

expert panels have recommended a tiered approach to the evaluation of

chemicals for neurotoxicity (Reiter 1987; Tilson 1990a). Each stage of

assessment incorporates decision points as to whether available information

is sufficient for determining the neurotoxicity of a chemical. Procedures

2
used in later stages of assessment study more specific questions about

the neurotoxicity of a chemical. Such a strategy typically begins with

simple, rapid, inexpensive methods to detect the presence of neurobehavioral

toxicity. Examples of such tests include simple measures of locomotor activity,

sensorimotor reflexes, and neurological signs. Studies concerning biological

mechanism(s) of action or providing data to determine a no-observable-adverseeffect level or lowest-observed-adverse-effect level involve more sensitive

tests and special equipment. Examples of techniques used in later stages of

assessment include discriminated conditioned response techniques to assess

sensory or motor dysfunction and most procedures to evaluate cognitive

function.

Neurobehavioral tests that are used to assess chemical-induced changes in

neurobiological function in animals are frequently based on human neurotoxicity.

Table 1 summarizes the various types of signs and symptoms reported by

humans exposed to a wide range of neurotoxicants (World Health Organization

1986; Tilson and Mitchell 1984). Chemicals can cause a diverse range of

neurobiological effects in humans, including sensory, motor, autonomic, and

cognitive dysfunction (World Health Organization 1986). Clinicians also

recognize sensorium changes in humans, including irritability, attention

TABLE 1. Examples of signs and symptoms reported by humans exposed to

neurotoxicants

Function Manifestation

Sensory Changes in smell, vision, taste, hearing, balance,

proprioception, feeling, pain

Motor Weakness, decreased strength

Tremor

Incoordination or ataxia

Abnormal movements, including myoclonia,

fasciculations, behavioral seizures

Hypermotor or hypomotor activity

Autonomic

Cognitive

Body temperature changes, “cholinergic crisis”

Learning, memory deficits

Sensorium Hallucinations, delusions, apathy, stupor, coma

SOURCE: Adapted from World Health Organization 1986 and Tilson and

Mitchell 1984

3
difficulties, hallucinations, dementia, depression, stupor, and coma. Table 2

summarizes several neurobehavioral endpoints that have been used to

quantitate neurotoxicity in animals. Many neurobiological deficits described

in humans exposed to neurotoxicants can be quantified objectively in animal

models. In the following sections of this chapter, several procedures used in

animal neurobehavioral toxicology to assess neurotoxicant-induced alterations

in sensory, motor, and cognitive function are described. Selection of the

tests for discussion is not meant to be inclusive but rather to provide examples

of the utility of different kinds of neurobehavioral tests.

EXAMPLES OF ANIMAL NEUROBEHAVIORAL TESTS IN TOXICITY

SCREENING

Functional Observatlonal Batteries

Procedures used to identify the presence of neurotoxicity are usually designed to

test many animals and are not considered to be as sensitive to subtle effects as

TABLE 2. Examples of behavioral endpoints of neurotoxicity in animals

Measurement Effect

Sensory Alterations in visual, auditory, gustatory,

somatosensory, and pain sensations

Sensorimotor reflexes Absence or decreased occurrence,

magnitude, or latency; potentiated reflex

Neuromuscular impairment

Motor activity

Abnormal motor movements

Altered grip strength, hindlimb splay

Increases or decreases

Incoordination, ataxis, abnormal gait,

dystonia, myoclonia, tremor, retropulsion,

behavioral seizures

Autonomic dysfunction Piloerection, salivation, increased urination,

diarrhea, hypothermia

Cognitive deficits Alterations in learning or memory

Schedule-controlled

behavior

Change in rate or temporal patterning of

responding

4
more specialized tests for neurobiological dysfunction. The use of an FOB for

screening is now generally accepted. Several batteries for rodents (Gad 1982;

Moser et al. 1988; Haggerty 1989; O’Donoghue 1989; Moser l988), dogs

(Schaeppi and Fitzgerald 1989), and nonhuman primates (O’Keeffe and Lifshitz

1989) have been developed to assess major overt behavioral and neurological

effects. FOB evaluations in animals are similar to clinical neurological

examinations in humans in that they rate the presence and, in some cases,

severity of specific neurological signs.

An FOB typically assesses several neurobiological domains, including

neuromuscular (i.e., weakness, incoordination, gait, and tremor), sensory (i.e.,

audition, vision, and somatosensory), and autonomic (i.e., pupil response and

salivation) functions (table 3). FOB data may be interval, ordinal, or continuous,

and test results are judged according to the number and type of signs affected,

the dose(s) at which neurotoxic signs are observed, and the persistence of

such effects. FOB assessments are usually performed in a sequential fashion,

proceeding from the least to the most interactive with the animal. An FOB can

provide data concerning dose-response and onset, duration, and persistence

of an effect; can differentiate neurotoxicants from nonneurotoxicants; and can

differentiate neurotoxicants having different mechanism(s) or site(s) of action.

The application of an FOB to assess neurotoxicants generally results in a

relatively large data set. In a recent paper, Moser (1991) described an approach

TABLE 3. Behavioral endpoints used in a functional observational battery

Home Cage and Open Field Manipulative

Posture

Convulsions and tremors

Palpebral closure

Lacrimation

Piloerection

Salivation

Vocalizations

Rearing

Urination and defecation

Gait

Arousal

Mobility

Stereotypy

Bizarre behavior

Ease of removal

Ease of handling

Approach response

Auditory startle

Response to aversive stimulus

Righting reflex

Landing foot splay

Grip strength

Pupil reflex

SOURCE: Adapted from Moser 1989

5
to summarize FOB data from neurotoxicity experiments. Rats were dosed with

acrylamide or 2-hydroxyethyl acrylate (HEA) and assessed for neurotoxicity

after 30, 60, or 90 days of dosing. Moser (1991) normalized all the data from

each rat for the 29 measures in the FOB on a scale of 1 to 4. The normalized

scores were then summed for each animal within measures assigned to one of

six functional domains (i.e., autonomic, central nervous system (CNS) activity,

CNS excitability, neuromuscular, sensorimotor, and physiological). Using this

approach, acrylamide-treated rats showed statistically significant effects only in

the neuromuscular domain, which consisted of six different tests, including gait

score, mobility score, landing foot splay, forelimb and hindlimb grip strength, and

righting reflex. HEA had no significant effects on any of the functional domains.

Figure 1 shows the average neuromuscular scores for male and female rats

exposed to acrylamide or HEA for 90 days. Routine analysis of variance was

used to show a significant treatment by time interaction. Male and female rats

were significantly affected after 90 days of dosing with 12 mg/kg of acrylamide.

Figure 1 also shows that HEA had no effects on neuromuscular function at any

time during dosing. The strategy of normalizing and summarizing FOB data into

functional domains is a promising advance in the routine analysis of screening

data from studies using FOB.

Motor Activity. Motor activity includes a broad class of behaviors reflecting the

net integrated output of the sensory, motor, and associative processes of the

nervous system (Reiter and MacPhail 1982). Motor activity is usually quantified

as the frequency of movements over time. Effects of chemicals on motor activity

are usually expressed as total number of counts or as a percentage of some

control value (i.e., control group or preexposure baseline). The frequency of

motor activity within a test session usually decreases, a phenomenon known

as habituation.

Automated procedures to assess motor activity have been used to examine

the effects of psychopharmacological and neurotoxic chemicals (Reiter and

MacPhail 1982; MacPhail et al. 1989). Although it is generally accepted that

motor activity measurements are sensitive, reliable, and efficient (MacPhail et

al. 1989), it has been argued that motor activity measurements alone lack

specificity and do not easily differentiate the effects of psychoactive and other

chemicals from neurotoxicants (Maurissen and Mattsson 1989). However,

motor activity procedures are frequently used in conjunction with other

screening tests such as the FOB. One major conclusion from motor activity

is that the acute effect of most chemicals is a depression of motor activity.

However, some neurotoxicants (i.e., trimethyltin (TMT), triadimefon, triadimenol,

toluene, and p-xylene) and many psychoactive control agents increase motor

activity. It has been suggested that hyperactivity may be evidence of a specific

effect on the nervous system (Crofton et al. 1988).

6
Neuromuscular Sum

Acrylamide

2-Hydroxyethyl Acrylate

FIGURE 1. Average severity scores ±S.E. for the neuromuscular domain

for male and female rats during exposure to various doses of

acrylamide (top) or 2-HEA (bottom). Data for each animal’s

response on tests making up the neuromuscular domain were

normalized on a 1 to 4 scale, and the cumulative score was divided

by the number of tests (six) making up the neuromuscular domain.

Data are average domain scores +/- for 10 rats per group.

SOURCE: Adapted from Moser 1991

7
A recent interlaboratory comparison (Crofton et al. 1991) found a high degree

of comparability in the data generated by different devices and experimental

protocols, including within laboratory control variability, within laboratory

replicability of control data, between laboratory variability in the effects of

prototypic chemicals, and between laboratory comparison in the control rates

of habituation. In the interlaboratory comparison, effects of several psychoactive

or neurotoxic agents were studied in five different testing apparatuses, including

a figure-eight maze, Woodard photoactometer, Motron photoactometer, square

field with photocells, and rack-mounted cages with photocells, Studies were

performed under the conditions standard for each laboratory using either

Long-Evans or Fischer-344 rats. Figure 2 shows the effects of triadimefon,

a fungicide, on motor activity measured in six different laboratories. All

laboratories reported that triadimefon increased motor activity, with a peak

effect observed at about 100 mg/kg. Doses higher than 100 mg/kg had a

less stimulatory effect on motor activity. Other chemicals studied include

FIGURE 2. Dose-related effects of triadimefon on automated motor activity

measured in five different laboratories. Data are plotted as the

percentage of the vehicle control mean for each laboratory.

Control means ranged from 257 to 4,033 activity counts per

session (25 to 90 minutes).

SOURCE: Adapted from Crofton et al. 1991

8
d-amphetamine, scopolamine, carbaryl, chlorpromazine, endosulfan,

physostigmine, cypermethrin, and methylscopolamine. Similar agentinduced effects on motor activity were observed by the different laboratories.

SPECIALIZED TESTS OF NEUROBEHAVIORAL FUNCTION

Motor Dysfunction

Anger (1984) reviewed the effects of 588 chemicals for which the American

Conference of Governmental Hygienists (ACGH) had recommended threshold

limit values (TLV) and found that 167 (28 percent) had a TLV based totally or

in part on neurological or behavioral effects. Anger also evaluated the types

of neurotoxic effects reported for the 167 chemicals and reported a list of 69

different effects, which were classified under 5 major groups, including motor,

sensory, cognitive, general changes, and affective/personality. Of the 69

different effects, 20 percent (14) were motor deficits. The 14 different types

of motor effects can be grouped into 4 categories, including tremor, convulsions

and spasms, weakness, and incoordination (Newland 1988). Examples of

chemicals known to produce motor dysfunction in humans are acrylamide,

2,5-hexanedione, carbaryl, chlordecone, 1 -methyl-4-phenyl-1,2,3,6-

tetrahydropyridine, and manganese. Table 4 summarizes tests developed

to assess motor dysfunction in animal neurobehavioral pharmacology and

toxicology. Because tremors and weakness in animals have a high frequency

of occurrence, procedures used to measure them will be discussed in greater

detail.

Tremor. Chemical-induced tremor is frequently reported following exposure

to neurotoxicants. Anger and Johnson (1985) reviewed 750 chemicals affecting

the nervous system and identified 120 nervous system-related effects. Anger

(1990) listed the behavioral effects produced by 25 or more of the 750 chemicals

and found tremor/twitching to be associated with 177 agents.

Various techniques and approaches have been devised to measure tremor

in animals, and these have been reviewed critically by Newland (1988). One

procedure used to study tremor in rats is that developed by Gerhart and

colleagues (1982)) who assessed whole body movement by measuring the

displacement of a freely moving platform attached to a load cell-transducer.

A spectral analyzer was used to quantitate chemical-induced alternations in

movement. Figure 3 shows dose- and time-related tremorigenic activity

produced by p,p’-DDT, an organochlorine insecticide (Hudson et al. 1985).

In this study, rats were given 25, 50, 75, or 100 mg p,p’-DDT/kg and tested

12 hours later or given 75 mg/kg and tested 2, 5, 12, or 24 hours later.

p,p’-DDT increased the average power of whole body movements in a dosedependent manner, with the greatest increases occurring in the 7.5 to 15.0 Hz

regions of the spectrum. The peak time of effect for p,p’-DDT was 5 to 12 hours

9
TABLE 4. Examples of behavioral procedures to measure motor dysfunction

in animals

Function Test Reference

Tremor

Convulsions

Weakness

Incoordination and

abnormal movements

or posture

Rating scale

Spectral analysis

Rating scale Racine 1972

Grip strength Pryor et al. 1983a

Suspension from rod Molinengo and Orsetti 1976

Swimming endurance Bhagat and Wheeler 1973

Response force titration Elsner et al. 1988

Nautilus Newland 1988

Rotorod performance

Rope climbing

Inclined screen

Hindlimb splay

Automated hindlimb test

Gait analysis

Negative geotaxis

Hong et al. 1984

Newland 1988

Gerhart et al. 1982

Bogo et al. 1981

Carlini et al. 1967

Graham et al. 1957

Edwards and Parker 1977

Tanger et al. 1984

Lee and Peters 1976

Pryor et al. 1983a

postdosing. The apparatus described by Gerhart and colleagues (1982) has

been used to study the biological substrate underlying tremor produced by

chlordecone, p,p’-DDT, and permethrin (Tilson et al. 1985, 1966).

Weakness. In evaluating the most frequently reported behavioral and

neurological effects produced by 750 chemicals affecting the nervous system,

Anger (1990) found 179 agents produced weakness. One technique used in

many laboratories to measure chemical-induced weakness in rodents is the

forelimb and hindlimb grip strength procedure, which measures the force

required to cause an animal to release its grip from a bar or wire screen (Meyer

et al. 1979; Haggerty 1989; Kulig 1989; Moser 1989; O’Donoghue 1969). Kulig

(1989) dosed rats with 3.6, 7.2, or 14.4 mg acrylamide/kg for 12 weeks and

measured grip strength every 3 weeks during dosing and for up to 6 weeks

after cessation of dosing. Figure 4 shows that the higher dose of acrylamide

significantly decreased grip strength after 9 weeks of dosing. Strength began

to recover following cessation of dosing. Grip strength measurements are

included in many neurotoxicity testing batteries (Haggerty 1989; Kulig 1989;

Moser 1989; O’Donoghue 1989; Tilson 1990b).

10
FIGURE 3. Dose- and time-related effects of p,p'-DDT on whole body

movement of rats. Five hours after oral dosing with 25 to 100 mg/

kg p,p’-DOT, whole body movements were evaluated by spectral

analysis (left). A separate experiment examined the effects of 75

mg/kg p,p'-DDT at 2 to 24 hours after dosing (right). Data are

average values (-dBV)=± S.E. for six rats per group.

SOURCE: Data derived from Hudson et al. 1985

Sensory Dysfunction

Many chemicals are known to affect sensory systems. Of the 69 separate

neurotoxic effects reported in ACGH documentation of TLV determinations for

chemicals used occupationally, 10 (14 percent) concerned effects on sensory

systems (Anger 1984). In his review of the effects produced by 750 agents

having neurotoxicity, Anger (1990) reported visual and equilibrium disorders

were associated with 121 and 135 chemicals, respectively. Tactile, pain, and

auditory disorders were associated with 77, 64, and 37 chemicals, respectively.

Examples of chemicals having effects on sensory systems include toluene

(auditory), methyl mercury (visual), acrylamide (somatosensory), parathion

(pain), and 3-methylindole (olfaction).

11
FIGURE 4. The effects of subchronic exposure to acrylamide on forelimb and

hindlimb grip strength of rats. Animals were dosed 5 days per

week (intraperitoneal) with 3.6, 7.2, or 14.4 mg/kg acrylamide.

Measurements were made prior to dosing; 3, 6, 9, or 12 weeks

during dosing; and 3 and 6 weeks after cessation of dosing. Data

are average scores of eight rats per group.

SOURCE: Reprinted with permission from Kulig, B.M. A neurofunctional

test battery for evaluating the effects on long-term exposure to

chemicals. J Am Coll Toxicol 8(1):71-83, 1989. Copyright 1989

by Mary Ann Liebert, Inc. (New York City).

12
Several tests developed to assess animals for chemical-induced neurotoxicity

can be classified into three categories: classical conditioning, operant behavior,

and reflex modification (Maurissen 1988). Neurobehavioral procedures used

to assess sensory function are based on psychophysical methodology, which is

the study of the relationship between a physical dimension of a stimulus and

the response that is generated (Maurissen 1988). Psychophysical assessments

involve the presentation of a stimulus along some physical dimension (wave

length, brightness) and require the animal to make or withhold a specific

response in the presence or absence of that stimulus. Psychophysical methods

have been recently described by Maurissen (1988) and include the method of

limits, constant stimuli, adjustment, and tracking. Table 5 summarizes various

tests that have been used to assess sensory function in animals. Two

representative procedures that have been used to measure chemical-induced

changes in sensory function are described below.

Multisensory Conditioned Avoidance Response (CAR). Pryor and colleagues

(1983b) used a tone-intensity discrimination task in rats to determine the ototoxic

effects of toluene exposure: they exposed weanling rats to 1,200 or 1,400 ppm

toluene for 14 hours a day, 7 days per week, for 5 weeks, Some animals were

trained on a multisensory CAR task during the last week of exposure or during

the first or third weeks following cessation of exposure. Training consisted of

presentation of one of three stimuli (i.e., light, tone, or nonaversive current on

the grid floor of the test chamber) prior to the presentation of an aversive electric

shock. The rats could avoid presentation of the aversive shock by pulling or

climbing a pole suspended from the ceiling of the test apparatus during the

TABLE 5. Examples of behavioral tests used to assess sensory dysfunction

in animals

Test Type Procedure Reference

Classical conditioning Conditioned avoidance

Operant behavior Conditioned suppression

Tracking procedure

Flicker sensitivity

Constant stimulus

Discrimination response

Duration discrimination

Reflex modification Prepulse inhibition

Pryor et al. 1983b

Fox et al. 1982

Maurissen et al. 1983

Merigan 1979

Stebbins and Moody 1979

Hastings 1990

Daniel and Evans 1985

Eastman et al. 1987

Crofton et al. 1990

Fechter and Carlisle 1990

13
presentation of the warning stimuli. Rats learned to avoid the aversive shock

whenever the nonaversive shock or light warning stimuli were presented,

but not when the 20 kHz auditory stimulus was presented (figure 5). In a

subsequent manipulation beginning 2.5 months after the last exposure, the rats

were given a series of training sessions that varied the frequency (i.e., 4, 8, 12,

16, or 20 kHz) and intensity (20 to 60 dB) of the auditory warning stimuli. The

hearing of the toluene-exposed rats was unimpaired at 4 kHz, slightly impaired

at 8kHz, and markedly impaired at 16 and 20 kHz. Subsequent experiments

(Rebert et al. 1983) found auditory dysfunction in these animals using brain

stem-evoked potential methodology to determine auditory thresholds.

Reflex Modification. In the prepulse inhibition procedure, a weak stimulus

presented before a second eliciting stimulus can suppress a reflex response

to the eliciting stimulus. Because the degree of suppression is dependent on

the intensity of the inhibiting stimulus, sensory functioning can be assessed

by determining the intensity of the inhibiting stimulus required to produce

significant inhibition of the reflex (i.e., a sensory threshold). Crofton and

coworkers (1990) gave rats a single exposure to TMT (3, 5, or 7 mg/kg), and

low (5 kHz) and high (40 kHz) auditory function was assessed 11 weeks later.

TMT produced a dose-dependent decrease in the amplitude of the acoustic

startle reflex (figure 6). However, TMT increased the auditory threshold at 40

kHz only. Morphological examination of the inner ear confirmed a preferential

loss of outer hair cells, which are associated with high-frequency hearing.

Cognitive (Learning and Memory) Dysfunction

Of the 69 signs and symptoms in humans exposed to neurotoxicants identified

by Anger (1984), only about 6 percent were classified as cognitive. Memory

problems were associated with 33 of 750 chemicals having neurotoxic effects;

examples of chemicals that affect memory in humans include carbon disulfide,

styrene, organ ophosphates, lead, and mercury (Anger 1990). The concern

about cognitive dysfunction is greater in the area of developmental

neurotoxicology where there is a frequent association between mental

retardation and congenital anomalies (Illingworth 1959; Smith and Bostian

1964).

The experimental assessment of cognitive functions such as learning and

memory in animals is confounded by the fact that such processes must be

inferred from a change in behavior following exposure. Learning is the process

of adaptation to changed contingencies, whereas memory is a construct used to

describe the influence of previous events on behavior. Furthermore, alterations

in learning and memory must be separated from other changes in performance

that depend on intact sensory and motor functions and the motivational state

of the animal. Toxicant-induced changes in learning and memory should be

demonstrated over a range of doses and neurobehavioral procedures.

14
FIGURE 5. Effect of subacute exposure to toluene on performance of a

conditioned avoidance response to tone as a function of the

frequency and intensity of the stimulus. Testing began

approximately 2.5 months after the last exposure.

SOURCE: Reprinted with permission from Pryor, G.; Dickinson, J.; Howd, R.A.;

and Rebert, C.S. Transient cognitive deficits and high-frequency

hearing loss in weanling rats exposed to toluene. Neurobehav

Toxicol Teratol 5:53-57, 1983b. Copyright 1983 by Pergamon

Press, plc. (Elmsford, NY).

15
FIGURE 6. TMT-induced alterations in auditory thresholds and the acoustic

startle reflex amplitude. Panel A: TMT produced a significant

depression in baseline startle response. Data are group averages

±S.E. of the reflex on blank trials. Panel B: TMT-induced increases

in frequency-dependent auditory thresholds. Data are group

averages ±S.E. of the auditory thresholds for 5- and 40-kHz pure

tones. Asterisks indicate a significant difference from saline control

group (p<0.025).

SOURCE: Crofton et al. 1990. Copyright 1990 by Academic Press (Orlando, FL).

16
Neurobehavioral techniques to assess learning and memory have been reviewed

by Peele and Vincent (1989) and Heise (1984). In general, tests for learning and

memory can be categorized into measures of nonassociative learning, classical

conditioning, and instrumental procedures (Heise 1984). Table 6 summarizes

several techniques that have been used to assess neurotoxicant-induced

changes in learning and memory in animals. Examples of three procedures

that have been used to assess learning and memory in rats are described in

greater detail below.

Long-Delay Flavor Aversions. The conditioned flavor aversion procedure is

a classical conditioned task based on the finding that rats will avoid consuming

solutions with flavors previously paired with the consequences of an aversive

stimulus such as an injection of lithium chloride. If delays are imposed between

the initial intake of the flavor and injection of lithium, the aversion to the flavor is

weaker. Peele and colleagues (1989) exposed rats with TMT and tested them

30 days later for conditioned flavor aversions. Lithium was injected 0.5, 3, or 6

hours after rats were allowed to drink an 0.1 percent w/v sodium saccharin

solution. Two days later the rats received concurrent access to water and

saccharin and the preference for the saccharin solution was determined.

TABLE 6. Behavioral endpoints used to assess learning and memory in

animals

Type of Function Procedure Reference

Habituation

Classical conditioning

Startle reflex

Eye-blink reflex

Flavor aversion

Instrumental conditioning Passive avoidance

Active avoidance

Radial arm maze

Biel water maze

Morris water maze

Repeated acquisition

Delayed alternation

Matched to position

Reversal learning

Overstreet 1977

Yokel 1983

Riley and Tuck 1985;

Peele et al. 1989

Walsh et al. 1982a

Tilson et al. 1987

Walsh et al. 1982b

Vergieva and Zaikov 1981

Tilson et al. 1990

Paule and McMillan 1986

Bushnell 1988; Rice

and Karpinski 1988

Bushnell et al. 1991

Rice 1990

17
Figure 7 shows that rats injected with TMT 0.5 or 3 hours after pairing with

saccharin were not different from controls. However, rats in the 6-hour delay

group showed a significant impairment relative to controls. Subsequent

experiments found similar deficits in TMT-exposed rats in tasks for

somatosensory and gustatory avoidance learning. TMT also produced a

relatively specific destruction of pyramidal cells in the hippocampus, an

area important for short-term memory.

FIGURE 7. Relative saccharin intake (saccharin intake expressed as a

proportion of total fluid intake) for rats receiving lithium chloride

following saccharin consumption during a flavor preference test.

Rats were given either 8 mg/kg TMT (triangles) or saline (circles)

and tested 30 days later. Data are average preference scores

± S.E. of nine rats as a function of the delay separating saccharin

availability and lithium chloride administration.

SOURCE: Adapted from Peele et al. 1989. Copyright 1989 by Springer-Verlag

(New York).

18
Morris Water Maze. Spatial memory tasks are being used with greater

frequency in behavioral toxicology. One such task is the Morris water maze,

which measures the ability of a rat to find a submerged platform located in a vat

of water (Morris et al. 1986). Tilson and coworkers (1990) gave rats a single

dose of tris (2-chloroethyl) phosphate (TRCP) and tested for learning and

memory capabilities 3 weeks later. When rats were trained to swim to a platform

always hidden in the same location in the water maze, a significant attenuation in

the rate of acquisition was observed (figure 8). When TRCP-treated rats were

trained in a task where the position of the maze changed daily, TRCP-exposed

rats showed clear deficits in acquisition, Examination of the brains of the TRCPtreated animals revealed significant destruction of pyramidal cells in the CA3

and CA4 regions and of granule cells in the dentate gyrus of the hippocampus.

Other experiments (Tilson et al. 1990) found that the neurobehavioral deficits

produced by TRCP could be blocked by prior administration of atropine or

chlordiazepoxide, both of which reduced the severity of TRCP-induced seizures.

Discrete-Trial Operant Procedure. Working, short-term memory can be

evaluated by using a delayed response task with a spatial location as the

discriminative stimulus. Bushnell (1988) trained rats to receive food for

pressing one of two retractable levers in the choice phase of a trial if that lever

had been presented in the prior sample phase of that trial. Accuracy of the

choice decreases whenever delays are interposed between the sample trial

and the choice, with the largest effect occurring with the longest delay. Choice

accuracy also decreases more at long delays between sample and choice with

shorter times between trials, indicating that interference from previous trials

impairs memory but not discrimination. TMT (7 mg/kg, intravenous [IV]) was

given to rats trained to perform on the spatial delayed response memory task,

and behavior was tested up to 4 weeks later. TMT significantly altered the

retention gradient 4 weeks after treatment (figure 9), suggesting a disruption

of memory associated with temporal delays. Neurochemical studies performed

at the end of the behavioral experiments found a highly significant correlation

between deficits in spatial delayed responding and levels of glial fibrillary acidic

protein (GFAP) in the hippocampus. GFAP levels in the CNS increase over

basal levels in response to cellular injury caused by several known

neurotoxicants (O’Callaghan 1988).

Schedule-Controlled Behavior

Schedule-controlled behavior refers to a special application of operant behavior,

which can be defined as movement that operates on or alters the environment.

As described in a previous section, operant responses become conditioned or

learned when their frequency is modified by the consequences of the response

through a process known as reinforcement. Schedule-controlled behavior is

based on the principle that reinforcement is usually applied intermittently and

that behavior can be predicted on the basis of the programing or scheduling of

19
FIGURE 8. Effects of TRCP exposure on acquisition of spatial memory task

in rats. (Top) Animals were required to find submerged platform

located in the same place each day (reference memory task) for four

trials per day for 9 days. (Bottom) Location of submerged platform

changed each day (repeated acquisition task). Data are average

latencies to find platform ±S. E. Asterisks indicate significant

difference from control group (p<0.05).

SOURCE: Tilson et al. 1990. Copyright 1990 by Academic Press (Orlando, FL).

20
FIGURE 9. Effect of TMT, 7 mg/kg IV on retention gradient during week 4

after exposure. Values are averages ±S. E. for controls ( n=8)

and TMT-exposed rats ( n=5).

SOURCE: Adapted with permission from Bushnell, P.J. Effects of delay,

inter-trial interval, delay behavior and trimethyltin on spatial delayed

response in rats. Neurotoxicol Teratol 10:237-244, 1988. Copyright

1988 by Pergamon Press, plc. (Elmsford, NY).

reinforcement that follows responding, For example, reinforcement can occur

after a certain number of responses have been emitted, after a specified period

of time, or after a combination of both. A schedule of reinforcement specifies

the relation between a specific response and the delivery of reinforcement. For

example, fixed interval (FI) schedules require that a specific period of time

elapse before a response is reinforced, whereas a fixed ratio schedule requires

that a certain number of responses occur before reinforcement is delivered.

Interval and ratio schedules can also be variable where the time or number of

21
responses varies around some average. A complete description of simple,

compound, complex, and higher order schedules can be found elsewhere

(Ferster and Skinner 1957).

Schedule-controlled behavior reflects the net integrated sensory, motor,

and cognitive abilities of an organism. The study of neurotoxicants on

human schedule-controlled behavior is limited, although it has been used in

psychopharmacology to study the effects of psychoactive drugs. Schedulecontrolled behavior has been used extensively in animal behavioral toxicology

where it has been shown to be sensitive to a wide range of neurotoxicant agents

(Tilson 1987; Rice 1988). Schedule-controlled behavior is useful in that the

experimental animal frequently serves as its own control. Two examples in

which schedule-controlled behavior has been used to study neurotoxicant

effects on behavior are described below.

Effects of Lead on Intermittent Schedules of Reinforcement. Several studies

have demonstrated that developmental exposure to lead has significant effects

on operant behavior tested at later ages (Rice et al. 1979; Cory-Slechta and

Thompson 1979). Cory-Slechta and colleagues (1985) exposed male weanling

rats repeatedly to 25 ppm lead acetate via the drinking water. The rats were

trained to respond for food on an FI 1 -minute schedule of reinforcement starting

at 50 days of age. Performance was assessed over 90 experimental sessions

or 136 days. Figure 10 shows that lead exposure resulted in an increased

response rate during the first 40 sessions. However, after 40 sessions, response

rates of the lead-exposed group approached those of the control group. These

data are important in that behavioral effects were observed in animals having

blood lead levels ranging from 15 to 20 upg/dL throughout the study. Such blood

levels are within the range observed in many children (Mahaffey et al. 1982).

Effects of Solvents on Schedule-Controlled Behavior. Solvents include

several types of chemicals (i.e., aromatic, aliphatic, and halogenated

hydrocarbons: alcohols: esters; ketones; aliphatic nitrites; anesthetics: and

propellants). These volatile organic solvents are prevalent in the environment,

especially in the air and drinking water. The behavioral effects of the volatile

solvents have been studied by many investigators; however, comparisons

between studies have been confounded due to differences in behavioral

technique, routes of exposure, and lack of adequate dose-response

determinations. Glowa (1985) compared the range of effects of several volatile

organic solvents using schedule-controlled behavior in mice. Response on an

FI 60-second schedule of reinforcement was studied during exposure to the

solvents. Figure 11 shows that with few exceptions (e.g., toluene) solvents

from several different chemical classes produced dose-dependent decreases

in the rate of responding. Glowa (1985) suggested that the behavioral toxicity

of the solvents should be described as a decrease in response rate.

Decrements in responding were more closely associated with higher

22
FIGURE 10. Individual overall response rates (responses per minute) of rats

(n= 12 per group) exposed from weaning to 25 ppm sodium

acetate (controls, top panels) or 25 ppm lead acetate (bottom

panels) over 40 sessions of an Fl 1-minute schedule of

reinforcement. Increased response rates of the lead-exposed

rats were determined using a randomization test using the linear

coefficients resulting from the cubic polynominal fit to each

animal’s curve.

SOURCE: Cory-Slechta et al. 1985. Copyright 1985 by Academic Press

(Orlando, FL).

23
FIGURE 11. Average rates of responding (proportion of control) as a function

of exposure concentration (ppm) for the rate-decreasing effects

of cumulative exposure to acetone (ACE), carbon disulfide

(CS2), ethyl acetate (EAC), n-octane, (OCT), methyl ethyl

ketone (MEK), trichloroethane (TCE- l), halothane (HAL), and

toluene (TOL), for FI 60-second responding in mice.

SOURCE: Glowa, J.R. Behavioral effects of volatile organic solvents, In:

Seiden, L.S., and Balster, R.L., eds. Behavioral Pharmacology:

The Current Status. New York: Alan R. Liss, 1985. pp. 537-552.

Copyright © 1985 by John Wiley & Sons, Inc. Reprinted by

permission of John Wiley & Sons, Inc.

concentrations and were relatively linear. This latter aspect is important

because it permits direct comparison of EC50 values for diverse classes of

chemicals. Linear dose-response functions are also useful in the determination

of benchmark doses or no-observed-adverse-effect or lowest-observed-adverseeffect levels used in quantitative risk assessment.

SUMMARY

The use of neurobehavioral techniques in toxicology has increased dramatically

over the past several years. Several national and international groups have

recommended that neurobehavioral tests be included in the initial stages of

24
hazard identification, and regulatory agencies have responded by preparing

testing guidelines or requiring behavioral test data for premarket approval of

environmental and pharmaceutical chemicals. In addition, neurobehavioral

data have been used to set exposure limits in the workplace.

In the future, neurobehavioral data will be used more frequently in the area

of risk assessment, which has been defined as the “characterization of the

potential adverse effects of human exposure to environmental hazards”

(National Academy of Sciences 1983). Good risk assessment depends on

the ability to determine whether a particular agent is or is not causally linked

to a particular health effect and on the availability of dose-response data for

quantitative risk assessment. Neurobehavioral techniques used in animal

behavioral toxicology measure neurobiological functions similar to those

measured in humans. In addition, neurobehavioral procedures can be used

in longitudinal studies where the onset and duration of effects of chemical

exposure can be measured in the same animal. Neurobehavioral techniques

are also amenable to the study of tolerance and compensation following

repeated exposure or following recovery of function that can occur following

cessation of exposure. Therefore, neurobehavioral procedures provide a

valuable tool for research designed to reduce major uncertainties associated

with the risk assessment process, such as animal to human extrapolation

(homology of animal models) and dosing issues (i.e., high-to-low dose, acute

vs. repeated dosing, and continuous vs. episodic dosing).

Although the use of neurobehavioral procedures has had a significant impact

on neurotoxicology, their use in the risk assessment process and in monitoring

populations for possible subtle changes in neurobiological function will be

limited if additional research is not done to understand the neural substrates

underlying neurobehavioral endpoints. The ability to link chemically induced

behavioral changes to alterations at the neurophysiological, neurochemical,

and neuroanatomical levels will lead to a greater acceptance of the validity and

reliability of neurobehavioral endpoints in defining adverse effects of chemicals

on the nervous system.

REFERENCES

Anger, W.K. Neurobehavioral testing of chemicals: Impact on recommended

standards. Neurobehav Toxicol Terafol 6:147-153, 1984.

Anger, W.K. Worksite behavioral research. Results, sensitive methods, test

batteries and the transition from laboratory data to human health.

Neurotoxicology 11:629-720, 1990.

Anger, W.K., and Johnson, B.L. Chemicals affecting behavior. In: O’Donoghue,

J., ed. Neurotoxicity of Industrial and Commercial Chemicals. Boca Raton,

FL: CRC Press, 1985. pp. 51-148.

25
Annau, Z. Neurobehavioral Toxicology. Baltimore, MD: Johns Hopkins

University Press, 1986.

Bhagat, B., and Wheeler, M. Effect of nicotine on the swimming endurance of

rats. Neuropharmacology 12:1161-1165, 1973.

Bogo, V.; Hill, T.A.; and Young, R.W. Comparison of accelerod and rotarod

sensitivity in detecting ethanol- and acrylamide-induced performance

decrement in rats: Review of experimental considerations of rotating rod

systems. Neurotoxicology 2:765-787, 1981.

Bushnell, P.J. Effects of delay, intertrial interval, delay behavior and trimethyltin

on spatial delayed response in rats. Neurotoxicol Teratol 10:237-244, 1988.

Bushnell, P.J.; Padilla, S.S.; Ward, T.; Pope, C.N.; and Olszyk, V.B. Behavioral

and neurochemical changes in rats dosed repeatedly with diisopropyl

fluorophosphate (DFP). J Pharmacol Exp Ther 256:741-750,199l.

Carlini, E.A.; Silva, M.T.A.; Cesare, L.C.; and Endo, R.M. Effects of chronic

administration of ß-(3,4-dimethoxyphenyl)-ethylamine and ß-(3,4,5-

trimethoxyphenyl)-ethylamine on the climbing rope performance of rats.

Med Pharmacol Exp 17:534-542, 1967.

Cory-Slechta, D.A. Behavioral measures of neurotoxicity. Neurotoxicology

10:271-298, 1989.

Cory-Slechta, D.A., and Thompson, T. Behavioral toxicity of chronic

postweaning lead exposure in the rat. Toxicol Appl Pharmacol 47:151-159,

1979.

Cory-Slechta, D.A.; Weiss, B.; and Cox, C. Performance and exposure indices

of rats exposed to low concentrations of lead. Toxicol Appl Pharmacol

78:291-299, 1985.

Crofton, K.M.; Boncek, V.M.; and Reiter, L.W. Hyperactivity induced by

triadimefon, a triazole fungicide. Fundam Appl Toxicol 10:459-465, 1988.

Crofton, K.M.; Dean, K.F.; Menache, M.G.; and Janssen, R. Tri-methyl effects

on auditory function and cochlear morphology. Toxicol Appl Pharmacol

105:123-132, 1990.

Crofton, K.M.; Howard, J.L.; Moser, V.C.; Gill, M.W.; Reiter, L.W.; Tilson, H.A.;

and MacPhail, R.C. Interlaboratory comparison of motor activity experiments:

Implications for neurotoxicological assessments. Neurotoxicol Teratol 13:599-

609, 1991.

Daniel, D.A., and Evans, H.L. Effects of acrylamide on multiple behavioral

endpoints in the pigeon, Neurobehav Toxicol Teratol 7:267-273, 1985.

Eastman, C.L.; Young, J.S.; and Fechter, L.D. Trimethyl ototoxicity in albino

rats, Neurotoxicol Teratol 9:329-332, 1987.

Edwards, P.M., and Parker, V.H. A simple, sensitive, and objective method for

early assessment of acrylamide neuropathy in rats. Toxicol Appl Pharmacol

40:589-591, 1977.

Elsner, J.; Fellmann, C.; and Zbinden, G. Response force titration for the

assessment of the neuromuscular toxicity of 2,5-hexanedione in rats.

Neurotoxicol Teratol 10:3-13, 1988.

26
Fechter, L.D., and Carlisle, L. Auditory dysfunction and cochlear vascular injury

following trimethyltin exposure in the guinea pig. Toxicol Appl Pharmacol

105:133-143, 1990.

Ferster, C.B., and Skinner, B.F. Schedules of Reinforcement. New York:

Appleton-Century-Crofts, 1957.

Fox, D.A.; Wright, A.A.; and Costa, L.G. Visual acuity deficits following neonatal

lead exposure: Cholinergic interactions. Neurobehav Toxicol Teratol 4:689-

693, 1982.

Gad, S.C. A neuromuscular screen for use in industrial toxicology. J Toxicol

Environ Health 9:691-704, 1982.

Gerhart, J.M.; Hong, J.S.; Uphouse, L.L.; and Tilson, H.A. Chlordecone-induced

tremor: Quantification and pharmacological analysis. Toxicol Appl Pharmacol

66:234-243, 1982.

Glowa, J.R. Behavioral effects of volatile organic solvents. In: Seiden, L.S., and

Balster, R.L., eds. Behavioral Pharmacology: The Current Status. New York:

Alan R. Liss, 1985. pp. 537-552.

Graham, R.C.B.; Lu, F.C.; and Allmark, M.G. Combined effect of tranquilizing

drugs and alcohol on rats. Fed Proc 16:302-318, 1957.

Haggerty, G.C. Development of Tier I neurobehavioral testing capabilities for

incorporation into pivotal rodent safety assessment studies. J Am Coll Toxicol

8:53-70, 1989.

Hastings, L. Sensory neurotoxicology: Use of the olfactory system in the

assessment of toxicity. Neurotoxicol Teratol 12:455-459, 1990.

Heise, G.A. Behavioral methods for measuring effects of drugs on learning and

memory in animals. Med Res Rev 4:535-558, 1984.

Hong, J.S.; Tilson, H.A.; Uphouse, L.L.; Gerhart, J.; and Wilson, W.E. Effects of

chlordecone exposure on brain neurotransmitters: Possible involvement of

the serotonin system in chlordecone-induced tremor. Toxicol Appl Pharmacol

73:336-344, 1984.

Hudson, P.M.; Chen, P.H.; Tilson, H.A.; and Hong, J.S. Effects of p,p’-DDT

on the rat brain concentrations of biogenic amine and amino acid

neurotransmitters and their association with p,p’-DDT-induced tremor

and hyperthermia. J Neurochem 45:1349-1355, 1985.

Illingworth, R.S. Congenital anomalies associated with cerebral palsy and

mental retardation. Arch Dis Child 34:228-238, 1959.

Kimmel, CA. Current status of behavioral teratology: Science and regulation.

Crit Rev Toxicol 19:1-10, 1988.

Kulig, B.M. A neurofunctional test battery for evaluating the effects of long-term

exposure to chemicals. J Am Coll Toxicol 8(1):71-83, 1989.

Lee, C.C., and Peters, P.J. Neurotoxicity and behavioral effects of thiram in

rats. Environ Health Perspect 17:35-43, 1976.

MacPhail, R.C.; Peele, D.B.; and Crofton, K.M. Motor activity and screening for

neurotoxicity. J Am Coll Toxicol 8:117-125, 1989.

27
Mahaffey, K.R.; Annest, J.L.; Roberts, J.; and Murphy, R.S. National estimates

of blood lead levels: United States, 1976-1980. Association with selected

demographic and socioeconomic factors, N Engl J Med 307:573-579, 1982.

Maurissen, J.P. Quantitative sensory assessment in toxicology and

occupational medicine: Applications, theory, and critical appraisal. Toxicol

Left 43:321-343, 1988.

Maurissen, J.P.J., and Mattsson, J.L. Critical assessment of motor activity as

a screen for neurotoxicity. Toxicol Ind Health 5:195-202, 1989.

Maurissen, J.P.J.; Weiss, B.; and Davis, H.T. Somatosensory thresholds in

monkeys exposed to acrylamide. Toxicol Appl Pharmacol 71:266-279,1983.

Merigan, W.H. Effects of toxicants on visual systems. Neurobehav Toxicol

1[Suppl]:15-22, 1979.

Meyer, O.A.; Tilson, H.A.; Byrd, W.C.; and Riley, M.T. A method for the routine

assessment of fore- and hindlimb grip strength of rats and mice. Neurobehav

Toxicol 1:233-236, 1979.

Molinengo, L., and Orsetti, M. Drug action on the “grasping reflex” and

on swimming endurance: An attempt to characterize experimentally

antidepressant drugs. Neuropharmacology 15:257-260, 1976.

Morris, R.G.M.; Anderson, E.; Lynch, G.S.; and Baudry, M. Selective

impairment of learning and blockade of long-term potentiation by a N-methylD-aspartate receptor antagonist AP5. Nature 319:774-776, 1986.

Moser, V.C. Screening approaches to neurotoxicity: A functional observational

battery. J Am Coll Toxicol 8:85-94, 1989.

Moser, V.C. Applications of a neurobehavioral screening battery. J Am Coll

Toxicol 10:661-669, 1991.

Moser, V.C.; McCormick, J.P.; Creason, J.P.; and MacPhail, R.C. Comparison

of chlordimefon and carbaryl using a functional observational battery.

Fundam Appl Toxicol 11:189-208, 1988.

National Academy of Sciences. Principles for Evaluating Chemicals in the

Environment. Washington, DC: National Academy of Sciences, 1975.

National Academy of Sciences. Risk Assessment in the Federal Coverment:

Managing the Process. Washington, DC: National Academy of Sciences,

1983.

Newland, MC. Quantification of motor function in toxicology. Toxicol Left

43:295-319, 1988.

O’Callaghan, J.P. Neurotypic and gliotypic proteins as biochemical markers

of neurotoxicity. Neurotoxicol Teratol 10:445-452, 1988.

O’Donoghue, J.L. Screening for neurotoxicity using a neurologically based

examination and neuropathology. J Am Coll Toxicol 8:97-116, 1989.

O’Keeffe, R.T., and Lifshitz, K. Nonhuman primates in neurotoxicity screening

and neurobehavioral toxicity studies. J Am Coll Toxicol 8:127-140, 1989.

Overstreet, D.H. Pharmacological approaches to habituation of the acoustic

startle response in rats. Physiol Psychol 5:230-238, 1977.

Paule, M.G., and McMillan, D.E. Effects of trimethyltin on incremental repeated

acquisition (learning) in the rat. Neurobehav Toxicol Teratol 8:245-253, 1986.

28
Peele, D.B.; Farmer, J.D.; and Coleman, J.E. Time-dependent deficits in delay

conditioning produced by trimethyltin. Psychopharmacology 97:521-528,

1989.

Peele, D.B., and Vincent, A. Strategies for assessing learning and memory,

1978-1987: A comparison of behavioral toxicology, psychopharmacology,

and neurobiology. Neurosci Biobehav Rev 13:33-38, 1989.

Pryor, G.; Dickinson, J.; Howd, R.A.; and Rebert, C.S. Transient cognitive

deficits and high-frequency hearing loss in weanling rats exposed to toluene.

Neurobehav Toxicol Teratol 5:53-57, 1983b.

Pryor, G.T.; Uyeno, E.T.; Tilson, H.A.; and Mitchell, C.L. Assessment of

chemicals using a battery of neurobehavioral tests: A comparative study.

Neurotoxicol Terarol 5:91-118, 1983a.

Racine, R. Modification of seizure activity by electrical stimulation, II. Motor

seizure. Electroencephalogr Clin Neurophysiol 32:281-294, 1972.

Rebert, C.S.; Sorenson, R.A.; Howd, R.A.; and Pyror, G.T. Toluene-induced

hearing loss in rats evidenced by the brainstem auditory evoked potential

response. Neurobehav Toxicol Teratol 5:59-62, 1983.

Reiter, L.W. Neurotoxicology in regulation and risk assessment. Dev Pharmacol

Ther 10:354-368, 1987.

Reiter, L.W., and MacPhail, R.C. Factors influencing motor activity

measurements in neurotoxicology. In: Mitchell, C.L., ed. Nervous System

Toxicology. New York: Raven Press, 1982. pp. 45-65.

Rice, D.B. Lead-induced behavioral impairment on a spatial discrimination

reversal task in monkeys exposed during different periods of development.

Toxicol Appl Pharmacol 106:327-333, 1990.

Rice, D.B., and Karpinski, K.F. Lifetime low-level lead exposure produces

deficits in delayed alternation in adult monkeys. Neurotoxicol Teratol 10:207-

214, 1988.

Rice, D.C. Quantification of operant behavior. Toxicol Left 43:361-379, 1988.

Rice, D.C.; Gilbert, S.G.; and Willes, R.F. Neonatal low-level exposure in

monkeys: Locomotor activity, schedule-controlled behavior, and the effects

of amphetamine. Toxicol Appl Pharmacol 51:503-513, 1979.

Riley, A.L., and Tuck, D.L. Conditioned taste aversions: A behavioral index of

toxicity. Ann N Y Acad Sci 443:272-292, 1985.

Schaeppi, U., and Fitzgerald, R.E. Practical procedure of testing for

neurotoxicity. J Am Coll Toxicol 8:29-34, 1989.

Smith, D.W., and Bostian, K.E. Congenital anomalies associated with idiopathic

mental retardation. J Pediatr 65:189-199, 1964.

Stebbins, W., and Moody, D.B. Comparative behavioral toxicology. Neurobehav

Toxicol 1[Suppl]:33-44, 1979.

Tanger, H.J.; Vanwersch, R.A.P.; and Wolthius, 0.L. Automated quantitative

analysis of coordinated locomotor behaviour in rats. J Neurosci Methods

10:237-245, 1984.

Tilson, H.A. Behavioral indices of neurotoxicity: What can be measured?

Neurotoxicol Teratol 9:427-443, 1987.

29
Tilson, H.A. Neurotoxicology in the 1990s. Neurotoxicol Teratol 12:293-300,

1990a.

Tilson, H.A. Animal neurobehavioral test battery in NTP assessment. In:

Johnson, B.L., ed. Advances in Neurobehavioral Toxicology: Applications

in Environmental and Occupational Health. Chelsea, Ml: Lewis, 1990b. pp.

403-418.

Tilson, H.A., and Harry, G.J. Behavioral principles for use in toxicology and

pharmacology. In: Mitchell, C.L., ed. Nervous System Toxicology. New York:

Raven Press, 1982. pp. 1-27.

Tilson, H.A.; Hong, J.S.; and MacTutus, C.F. Effects of 5,5-diphenylhydantoin

(phenytoin) on neurobehavioral toxicity of organochlorine insecticides and

permethrin. J Pharmacol Exp Ther 233:285-289, 1985.

Tilson, H.A.; Hudson, P.M.; and Hong, J.S. 5,5-Diphenylhydantoin antagonizes

neurochemical and behavioral effects of p,p’-DDT but not chlordecone.

J Neurochem 47:1870-1878, 1986.

Tilson, H.A., and Mitchell, C.L. Neurobehavioral techniques to assess the

effects of chemicals on the nervous system. Annu Rev Pharmacol Toxicol

24:425-450, 1984.

Tilson, H.A.; Shaw, S.; and McLamb, R.L. The effects of lindane, DDT, and

chlordecone on avoidance responding and seizure activity. Toxicol Appl

Pharmacol 88:57-65, 1987.

Tilson, H.A.; Veronesi, B.; McLamb, R.L.; and Matthews, H.B. Acute exposure

to tris(2-chloroethyl)phosphate produces hippocampal neuronal loss and

impairs learning in rats. Toxicol Appl Pharmacol 106:254-269, 1990.

Vergieva, T., and Zaikov, H.R. Behavioral change in rats with inhalation of

styrene. Hig Zdraveopazvane 24:242-247, 1981.

Walsh, T.; Gallagher, M.; Bostock, E.; and Dyer, R.S. Trimethyltin impairs

retention of a passive avoidance task. Neurobehav Toxicol Teratol 4:163-167,

1982a.

Walsh, T.; Miller, D.B.; and Dyer, R.S. Trimethyltin, a selective limbic system

neurotoxicant, impairs radial-arm maze performance. Neurobehav Toxicol

Teratol 4:177-183, 1982b.

Weiss, B. Quantitative perspectives on behavioral toxicology. Toxicol Left

43:285-293, 1988.

Weiss, B., and Laties, V. Behavioral Toxicology. New York: Plenum, 1975.

World Health Organization. Principles for Evaluating Health Risks to Progeny

Associated With Expouse to Chemicals During Pregnancy. Environmental

Health Criteria. Document 30. Geneva: World Health Organization, 1984.

World Health Organization. Principles and Methods for the Assessment of

NeurotoxicityAssociated With Exposure to Chemicals. Environmental Health

Criteria. Document 60. Geneva: World Health Organization, 1986.

Yokel, R.A. Repeated systemic aluminum exposure effects on classical

conditioning of the rabbit. Neurobehav Toxicol Teratol 5:41-46, 1983.

Zbinden, G.; Cuomo, V.; Racagni, G.; and Weiss, B. Application of Behavioral

Pharmacology in Toxicology. New York: Raven Press, 1983.

30
Discussion

Q: Do you have any simple tests for physical dependence that will give

evidence of neurotoxicity?

A: I think some of the measurements in the functional observational battery

exam might be able to show withdrawal signs due to morphine because many

of these tests are designed to detect chemical-induced changes in nervous

system excitability or arousal. Under the appropriate conditions, schedulecontrolled behavior has been used to determine withdrawal.

Q: It is known that amphetamine addicts eat an awful lot and sleep a long time

after they crash. The thing that we have to show is that they actually eat more

or sleep longer-that would somehow be evidence of physical dependence.

A: There are behavioral tests involving diurnal measures of consummatory

responses (i.e., eating and drinking behaviors) and activity patterns that could

be used to study consequences of withdrawal from physical dependence.

Q (Ricaurte): If you take as a positive control a compound like MPTP (the

dopaminergic neurotoxicant) and you put it through a screen such as the screen

you have described in the rat, what kind of information does the screen give?

A: The rat is not very sensitive to MPTP, so a neurological screen would

probably not detect the presence of neurotoxicity. If a chemical produces motor

dysfunction in a test species, then a neurological screen such as the functional

observational battery should detect it.

Q (Ricaurte): A related question is to what extent is consideration given not

only to rodents but also to using a nonhuman primate to screen.

A: For premarket approval of pesticides and for toxic substances, EPA requires

testing in rodent species. In certain cases, avian species are required for

assessment of organophosphate-induced delayed neuropathy.

Q (Ricaurte): Is there a particular sequence or array of tests that you use to

cover the eventuality that one animal may not be adequate?

A: Our testing guidelines do not specify a strain of rats. In the case of the

developmental neurotoxicity, there is one strain of rats that is discouraged.

Multispecies assessments are important to cover this eventuality.

Q (Sobotka): Just one clarification. It was my understanding that with MPTP

[1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine] you can get effects in rats at

proper doses. It seems that there is a growing misconception that a rat is not

31
responsive to MPTP. It is true that rats do not reproduce the lasting

neuropathological effects seen in primates, dogs, and humans, but then

you’re talking about the FOB identifying the chemical. A rat is able to do

that. Where you may have a problem is when you do the dose-response

characterization stage where you’re looking for a lowest-effect level or

no-effect level. Then you have a problem.

COMMENT (Ricaurte): Is the endpoint that you are using specific? Is it a

measure of, for example, dopamine levels?

A (Sobotka): No, no. I’m talking about an observational battery. I have not

done this myself; this is based on the literature. The only animal species that I

have used MPTP on is the dog. And we can see these types of neurological

problems in the dog. In the rodent relative to the primate, you have to use

higher doses, but according to the literature, the kinds of effects that you will

see here would be picked up by FOB.

COMMENT (Tilson): Jim O’Callaghan may have something of an answer for

this, because he has looked at rats.

A (O’Callaghan): I’m going to defer to my colleague, Diane Miller, for

behavioral expertise.

A (Miller): In the literature, you can pick up catalepsy in both mice and rats if

you go to high enough doses. So with the FOB, you would pick that up. Now,

that is not a long-lasting effect. You have the question of reversibility and

whether an effect is related to any kind of substantia nigra damage, any kind of

dopaminergic long-lasting changes.

A (Tilson): I think that the combined use of a functional observational battery,

motor activity, neuropathological assessment, and GFAP measurements would

pick up most neurotoxicants as we know them today. Lew?

Q (Seiden): The time after the insult has to be an important variable in

assessing the toxicity vs. the pharmacology of these compounds or drugs. I

don’t know of a good benchmark for discriminating between the short effects

and the more long-lasting, perhaps permanent effects of these drugs. Do you

have any, as a regulatory agency?

A (Tilson): Changes lasting more than 24 to 48 hours after dosing cause a

greater level of concern than those that are acutely reversible.

32
AUTHOR

Hugh A. Tilson, Ph.D.

Director

Neurotoxicology Division (MD-74B)

U.S. Environmental Protection Agency

Health Effects Research Laboratory

Research Triangle Park, NC 27711

33
Behavioral Consequences of Partial

Monoamine Depletion in the CNS After

Methamphetamine-Like Drugs: The

Conflict Between Pharmacology and

Toxicology

Lewis S. Seiden, WiIliam L. Woolverton, Stanley A. Lorens,

Joseph E.G. Williams, Rebecca L. Corwin, Norio Hata,

and Mary Olimski

INTRODUCTION

There is considerable evidence that dopamine (DA) and serotonin (5-

hydroxytryptamine [5-HT]), by virtue of their role as tonic neurotransmitters

in the central nervous system (CNS), play an important role in locomotion and

motor control, food intake, mood, sexual behavior, aggression, sleep, and pain

perception (Seiden et al. 1979; Carlsson 1988; lversen et al. 1990; Seiden

and Dykstra 1977). Part of that evidence is derived from pharmacological

studies: Drugs that either directly or indirectly stimulate DA receptors have a

characteristic profile of behavioral effects (Creese and lversen 1973, 1975;

Goodman and Gilman 1990). For example, amphetamine (AMPH) is an indirect

DA agonist. At low-to-moderate doses, AMPH increases locomotion, apparently

involving DA in the limbic forebrain; decreases food and water intake; has ratedependent effects on schedule-controlled behavior; and functions as a positive

reinforcer. At higher doses, AMPH induces species-specific stereotypic

behaviors involving striatal DA (Cooper et al. 1991; Ellinwood 1974; Kelleher

and Morse 1964; Scheel-Kruger and Randrup 1967). In humans, AMPH

increases positive mood, has a high abuse liability, and when used in high

doses, can induce stereotypic and maladaptive behaviors. When AMPH is

used repeatedly, paranoid psychosis develops that may also be related to a

hyperdopaminergic state. AMPH can also prevent sleep and is prescribed in the

treatment of narcolepsy (Goodman and Gilman 1990). Although some of the

effects of AMPH may involve 5-HT and/or the norepinephrine (NE) mechanisms,

DA appears to play a particularly prominent role (Ando et al. 1985; Azzaro et al.

1974; Chiueh and Moore 1975; Heikkila et al. 1975; Raiteri et al. 1979). It also

should be noted in this context that DA antagonists are used as antipsychotic

agents and DA agonists are used to treat Parkinson’s disease.

34
Similarly, pharmacological studies suggest that 5-HT plays an important role

in the behavioral effects of antidepressant, antipsychotic, and antianxiety drugs

(Asberg and Wagner 1986; Garattini and Samanin 1988; Marek et al. 1988;

Peroutka and Snyder 1980; Plic 1987; Seiden and O’Donnell 1985; Sulser

1983 a; Vetulani et al. 1976). Several clinically efficacious antidepressant

drugs block the 5-HT transporter (e.g., chlorimpramine, imipramine, fluoxetine)

(Cowen 1990; Richelson 1990; Sulser 1983b; Willner 1985; Crews et al. 1983;

Marek and Seiden 1988a, 1988b). These compounds alter locomotor activity,

food and water intake, and pain perception. In addition, they increase time

spent in the forced swim test, increase the number of reinforcers obtained on

a differential reinforcement of low rate 72-second (DRL 72-s) schedule, and

have been reported to affect sleep. The above effects are consistent with the

idea that 5-HT plays a role in the maintenance of physiological and behavioral

function (Curzon 1990; Humphrey et al. 1990; Jimerson et al. 1990; Morin

et al. 1990; Richardson 1990; Wauquier and Dugovic 1990).

Data from experiments with neurotoxins also suggest important behavioral

functions for DA and 5-HT. Lesions to DA systems induced by 6-

hydroxydopamine (6-OHDA) have profound behavioral effects shortly after the

toxin is administered. These include severe decreases in food and water intake

and locomotion. In addition, DA lesions alter the behavioral effects of drugs

that act via DA mechanisms (Kostrzewa and Jacobowitz 1974). Depletion of

5-HT by neurotoxins or electrolytic lesions have been shown to have effects on

sleep, reaction to painful stimuli, sexual behavior, and aggression, although

there has been some difficulty in interpreting some of these findings (Seiden

and Dykstra 1977). It is reasonable to suspect, therefore, that chronic depletion

of DA or 5-HT would lead to long-term changes in behavior.

Methamphetamine (MA) and certain related phenethylamines are drugs of

abuse that are toxic to DA- and/or 5-HT-containing neurons in the brain

(Hotchkiss et al. 1979; Koda and Gibb 1973; Seiden et al. 1976; Seiden and

Kleven 1989). When administered repeatedly to animals, these compounds

induce long-lasting monoamine depletions on the order of 50 percent. It seems

likely that these drugs are neurotoxic in humans. Therefore, it is important to

determine whether monoamine depletions of the magnitude produced by these

drugs have significant behavioral consequences. Although these compounds

have been found to have little or no obvious long-lasting effect on behavior,

behavioral testing has not been extensive. Therefore, the present series of

experiments was designed to examine the effects of small-to-moderate

depletions of DA and 5-HT induced by neurotoxins or neurotoxic amphetamines

on a wide range of behaviors that were predicted to be sensitive to small

changes in monoamine concentrations. Those behaviors included locomotion,

feeding, drinking, avoidance, escape, and open-field testing as well as several

different operant tasks that are known to be sensitive to antidepressant,

anxiolytic, and antipsychotic drugs.

35
METHODS

Male Sprague-Dawley rats (3 to 4 months old) were housed individually in a

temperature- and humidity-controlled facility on a 12-hour light-dark cycle (lights

on at 7 a.m.). Rat chow and water were available ad lib, except in experiments

with schedule-controlled behavior (fixed conservative number [FCN], variable

interval [VI], and DRL) and eight-arm radial maze when the rats were maintained

at 80 to 85 percent of their predeprivation body weights.

Neurotoxins

Rats were pretreated with pargyline (20 or 30 mg/kg, intraperitonally [IP] 60

to 75 minutes prior to the neurotoxin injections) and anesthetized with sodium

pentobarbital (30 mg/kg, IP, 40 to 55 minutes prior) supplemented with

methoxyflurane. Desipramine (20 mg/kg, IP) was administered 30 to 45 minutes

prior to neurotoxins. 5,7-Dihydroxtryptamine creatinine sulfate (5,7-DHT) (25,

50, 75, or 100 μg/ventricle, as the base) (Regis Chemical Co., Morton Grove, IL)

or 6-OHDA (25, 37.5 and 50 μg/ventricle, as the base) (Sigma Chemical Co.,

St. Louis, MO) was injected bilaterally intracerebroventricularly (ICV). Ampicillin

(50 mg/kg, intramuscularly 25 to 40 minutes prior) was given to prevent infection.

Postoperatively, the 6-OHDA lesion rats were given access to preferred foods

(vanilla custard with whole milk and vanilla wafers) and gradually returned to

standard chow.

Neurotoxic Drugs

(+)-MA HCI (12.5, 25, and 50 mg/kg/injection) or (±)-3,4-

methylenedioxymethamphetamine (MDMA) HCI (MDMA) (10, 20, and

40 mg/kg/injection) was injected subcutaneously (sC), b.i.d. (morning and

evening, 12 hours apart), for 4 consecutive days. Drugs were dissolved

in 0.9 percent saline, and doses are expressed as the salts.

Parachloroamphetamine (PCA) HCI was purchased from Sigma.

Behavioral Testing

A variety of behavioral tests were conducted. Methods and results from each

test are described below. In most cases, testing began 2 weeks after treatment

and continued for at least 2 weeks. The major exception was food and water

intake for which one group was tested approximately 18 months after treatment.

Analysis of Monoamines

After behavioral testing, rats were sacrificed by decapitation and their brains

were rapidly dissected over ice; the striatum, hippocampus, and frontal cortex

were assayed for 5-HT and DA (Heffner et al. 1980). Concentrations of DA and

36
5-HT were analyzed using high-performance liquid chromatography with

electronic detection, by the method of Kotake (Kotake et al. 1985).

RESULTS

Neurochemical effects of neurotoxins and neurotoxic drugs were dose related.

Generally, only the maximum depletion that was observed is reported.

Behavioral changes are summarized in table 1.

Locomotor Activity

Locomotor activity (Erinoff et al. 1979) was measured in a stabilimeter

apparatus for a period of 24 hours (12 hours light, 12 hours dark) once per

week in rats treated with 6-OHDA or 5,7-DHT and in appropriate control rats.

TABLE 1. Summary of behavioral effects of neurotoxins and neurotoxic drugs

37
TABLE 1. (continued)

KEY: i = increased; 0 = no effect; = decreased/impaired; = not tested.

NOTES:

* See Methods section above for abbreviations, doses, routes, and schedules

of drug administration.

†

 A decrease in center squares entered was seen only in one experiment

(Lorens et al. 1990) and only after the 50 mg, ICV total dose. This finding

was not replicated nor observed at higher doses (100 to 200 mg, ICV).

‡

 Alterations produced by 6-OHDA were not consistent across groups (see

text).

§

 PCA (5 mg/kg x 2d) also was ineffective.

|| Impaired swimming ability was reported by Lorens and colleagues (1990) but

was not replicated.

** Morphine (5.0 mg/kg, SC) analgesia was potentiated by 200 mg (ICV total

dose) but not 50 mg 5,7-DHT. This effect was observed 8 but not 2 weeks

postinjection.

†† This finding is in contrast to the report by Nencini and colleagues 1988.

No changes were observed in the face of an approximately 50-percent

depletion of DA or 50 percent of 5-HT. Rats showed a low level of activity

during the light and a slightly higher level during the dark.

Food and Water Intake

Food (45 mg BioServ dustless pellets) and water intake were measured using

photo-beam detection units (Coulbourn Instruments, Lehigh, PA) and monitored

by a computer in an adjacent room. Food intake was analyzed in terms of total

grams, number of meals, meal duration, meal size, and intermeal interval.

Water intake was analyzed in terms of total mL, number of bouts, bout duration,

bout size, and interbout interval. The room was on a 12-hour light-dark cycle

38
and the last 5 days of stable intake were used for data analysis. After treatment,

one group of rats (short term) was returned to the experimental chambers after

a 2-week recovery period, for at least 2 weeks. A second group (long term) was

singly housed and maintained at stable body weight (approximately 350 g) for

18 months, after which it was returned to the experimental chambers for at least

2 weeks, For both groups, the last 5 days of stable intake were compared

based on drug dose received.

Short-Term Studies. In rats given 6-OHDA, DA was depleted to 44 percent of

control in the caudate, 60 percent of control in the frontal cortex, 73 percent of

control in the hypothalamus, and 63 percent of control in the hippocampus; DA

was unaffected in the spinal cord. Total grams, meal duration, and bout size

increased. Intermeal interval decreased. In rats given 5,7-DHT, 5-HT was

reduced to 30 percent of control in the caudate, 14 percent of control in the

hypothalamus, 7 percent of control in the hippocampus, and 16 percent of

control in the spinal cord. 5-HT was not affected in the frontal cortex. Number

of meals increased, whereas meal size and bout duration decreased.

The MDMA regimen depleted 5-HT to 61 percent of control in the caudate,

37 percent of control in the frontal cortex, and 49 percent of control in the

hippocampus. 5-HT in other regions was unaffected. There were no effects on

food intake. In rats given MA, DA was depleted to 70 percent of control in the

caudate; DA in other regions was unaffected. 5-HT was depleted to 43 percent

of control in the caudate, and 29 percent of control in the frontal cortex; it was

unaffected in other regions. There were no changes in food intake.

Long-Term Studies. In rats given MDMA and tested approximately 18 months

after the last injection, DA in the spinal cord was depleted to 55 percent of

control, whereas DA in other regions was unaffected. 5-HT was not affected

in any region. There were no changes in food intake. In rats given MA and

tested approximately 18 months after the last injection, 5-HT was depleted

to 16 percent of control in the frontal cortex and 26 percent of control in the

hippocampus, increased to 131 percent in the hypothalamus, and was

unaffected in other regions, DA was increased to 221 percent of control in

the spinal cord and unaffected in other regions. There were no changes in

food intake.

Schedule-Controlled Behavior

Three schedules of reinforcement were used to study the effects of neurotoxins

on schedule-controlled behavior: an FCN, a VI 90-second (VI 90-s), and a DRL

72-s. After stable behavior was established under each schedule, neurotoxins

were administered. Retesting began approximately 2 weeks after administration

of neurotoxins.

39
Fixed Consecutive Number

The FCN was conducted in standard two-lever operant chambers (Gerbrands

Corp., Arlington, MA). Five or more consecutive responses on the left lever

followed by one response on the right lever were required for delivery of a food

pellet (45 mg, Noyes). A response on the right lever before five consecutive

responses on the left lever reset the response requirement on the left lever.

This schedule contained a signalled (SD) and an unsignalled (SO) component;

each component consisted of 10 trials. The components (SD-SO) alternated

until a total of 10 components (5 each) were completed. During the SD, the

left lever light was illuminated until five consecutive responses were made; then

the left lever light was extinguished, and the right lever light was illuminated.

During the SO component, only the houselight was illuminated. The probability

of reinforcement during the SD component was adjusted so that the number of

reinforcers for both components was approximately equal. Responding was

analyzed as percent correct for each component.

In rats that received 6-OHDA, DA was depleted to 14 percent of control in

caudate, increased to 172 percent of control in the spinal cord, and was

unaffected in other regions. There was no effect on FCN performance. In rats

that received 5,7-DHT, 5-HT was depleted to 20 percent of control in caudate,

16 percent of control in frontal cortex, 34 percent of control in hypothalamus,

12 percent of control in hippocampus, and 15 percent of control in the spinal

cord. DA was reduced to 76 percent of control in caudate, 51 percent of control

in hypothalamus, and 14 percent of control in spinal cord; it was unaffected in

other regions. NE was depleted to 4 percent of control in the hippocampus

but was unaffected in other regions. FCN performance was unaffected.

VI 90-s responding (Levine et al. 1980a, 1980b) was unchanged following

approximately 50-percent depletions of either DA or 5-HT. The rates of both

responding and reinforcement were the same between the treated and the

sham-treated rats.

Responding under the DRL 72-s was also unchanged (response rate and

reinforcement rate did not differ) as a result of approximately 50-percent

depletions of either DA or 5-HT.

Behavioral Test Battery

An additional battery of behavioral tests, designed to assess various aspects

of behavioral function, was used to examine the effects of neurotoxins and

neurotoxic drugs. The test battery included open-field behavior, one-way

avoidance, discriminated two-way avoidance, and the forced swim test (Lorens

et al. 1990). The effects of both neurotoxins and neurotoxic drugs were

examined using this test battery. The effects of 5,7-DHT and MDMA on

40
eight-arm radial maze performance and morphine-induced analgesia also were

determined. In addition, the effects of 6-OHDA on a home-cage-intrusion test

were examined. Groups of rats were treated with neurotoxins and neurotoxic

drugs and evaluated in multiple tests.

In rats that received 5,7-DHT, 5-HT was depleted to about 20 to 40 percent

of control. MDMA also produced dose-dependent and selective reductions

in regional CNS 5-HT concentrations that were virtually equivalent (18 to 34

percent reductions) in all CNS regions assayed (catecholamine concentrations

were not altered). The lowest dose (50 μg) of 5,7-DHT significantly decreased

open-field behavior and impaired swimming ability. MDMA did not alter openfield behavior or swimming ability. There was no effect of 5,7-DHT or MDMA

on one-way (spatial, unsignalled) or visually discriminated two-way (nonspatial,

signaled) avoidance conditioning. Additionally, neither treatment affected

acquisition of one- or two-way avoidance or performance in the eight-arm

radial maze.

Although dose-dependent effects of 6-OHDA were obtained, the changes in

regional DA concentrations were highly variable. Thus, the 6-OHDA-treated

rats were grouped according to their caudate-putamen DA levels: 6-OHDA-1,

83 percent of control: 6-OHDA-2, 66 percent of control; and 6-OHDA-3, 34

percent of control. In the frontal cortex, DA reductions reached 56 percent, but

the variability precluded statistical significance. Decreases in hypothalamic DA

were more modest but statistically significant. No systematic alterations were

detected in hippocampal or spinal cord monoamines and their metabolites.

Some significant behavioral changes were observed that varied as a function

of lesion group and test. In the open field, the 6-OHDA-2 group exhibited a

reduction in center squares entered. The 6-OHDA-3 group made more errors

during one-way conditioned avoidance response (CAR) training, whereas the

6-OHDA-1 group returned to the shock compartment (errors of commission)

more often during two-way CAR acquisition. There was no effect in the swim

test, and there were no changes in aggressive or social behavior in the homecage-intrusion test.

In rats treated with MA, DA levels were reduced only in the neostriatum (by

34 percent). Additionally, MA reduced 5-HT in the frontal cortex (by 61

percent), neostriatum (by 57 percent), hippocampus (by 81 percent), and spinal

cord (by 29 percent) but not in the hypothalamus. NE concentrations were not

altered in any CNS region. There was no effect on open-field exploration, the

acquisition of a one-way or visually discriminated two-way CAR, swimming

ability, or morphine analesia (5 mg/kg, SC).

4 1
DISCUSSION

An overview of the behavioral results obtained in the present series of studies

shows that although neurotoxins had some long-lasting behavioral effects

(e.g., food intake, VI and DRL performance), a wide variety of behaviors were

unaffected by partial denervation of the DA or 5-HT systems by MDMA or MA.

In addition, some of the changes that were observed with neurotoxins were

generally unsystematic with regard to dose and, therefore, may have been the

result of random variation. One might draw the conclusion from these results

that DA and/or 5-HT do not play an important role in mediating these behaviors.

However, these results should be interpreted with caution because the effects

of drugs on 5-HT and DA systems strongly suggest that these systems play a

role in behavior.

An account of the results presented here must, at this point, remain largely

speculative. It may be that the functional reserve capacity of the CNS

readily compensated for neurotransmitter changes. Within a particular

neurotransmitter system, the amount of release that is compromised by druginduced toxicity may be too small to be reflected in behavior. There is some

evidence to support this view in that larger depletions of DA or 5-HT cause

more persistent changes in behavior. VI 90-s responding has been found to

increase with time as a result of 6-OHDA lesions that depleted both DA and

NE by greater than 70 percent. Similarly, large depletions of DA cause

response rate increases and reinforcement rate decreases in DRL 72-s

behavior, and 5,7-DHT lesions that produce a large depletion of 5-HT increase

the response rate and decrease the rate of reinforcement on the DRL 72-s

schedule.

Alternatively, it may be that the behaviors studied, rather than relying on the

function of any particular neurotransmitter system, rely on several synergistic

transmitter systems. A partial reduction in the presynaptic terminal field of one

neurotransmitter may be readily compensated for by one of the synergistic

systems.

Other possibilities involve functional compensation in CNS neurotransmission.

It may be that transmission in DA and 5-HT neurons was normal or at least

adapted to maintain normal behavioral function. Although the concentrations

of monoamines in the brain were decreased, this measure may not reflect

the amount that is released. If synthesis and release were increased as a

compensatory mechanism, then the same number of receptors would be

stimulated and function would not change. Although it is difficult to understand

how postsynaptic tone would be maintained if one assumes that the DA and

5-HT systems comprise tight chemical junctions, the assumption of volume

transmission makes the results more reasonable. In volume transmission, the

42
released transmitter can diffuse many millimeters from the release site and

thereby stimulate receptors not located within microns of the presynaptic

terminal. Thus, one could readily see why function would not be compromised

even in the face of significant decreases in the levels of DA and/or 5-HT. It is

also possible that the number of receptors changed in such a way as to give a

normal response in the face of only partial agonism. These events do occur in

the face of denervation, but there have not been adequate studies to determine

how these changes translate into effects on brain function and behavior.

Regardless of the precise mechanisms involved, one is left with the somewhat

surprising conclusion that drug-induced CNS monoamine neurotoxicity had few,

if any, long-lasting behavioral consequences in these studies. However, this

should not be construed as meaning that CNS neurotoxicity is inconsequential.

It is possible that the behavioral tasks used were not sensitive to functional

changes. Moreover, behavioral consequences of neurotoxicity may be more

apparent under different conditions. For example, depletions of DA in neonatal

rats have been shown to cause hyperactivity that persists into adulthood

(Erinoff et al. 1979). It may also be the case that the appearance of agerelated deficits is hastened by drug-induced neurotoxicity. It remains for

future research to examine these possibilities.

REFERENCES

Ando, K.; Johanson, C.E.; Seiden, L.S.; and Schuster, C.R. Sensitivity changes

to dopaminergic agents in fine motor control of rhesus monkeys after

repeated methamphetamine administration. Pharmacol Biochem Behav

22(5):737-743, 1985.

Asberg, M., and Wagner, A. Biochemical effects of antidepressant treatment—

Studies of monoamine metabolites in cerebrospinal fluid and platelet

[3H]imipramine binding. Ciba Found Symp 123:57-83, 1986.

Azzaro, A.J.; Ziance, R.J.; and Rutledge, C.O. The importance of neuronal

uptake of amines for amphetamine-induced release of 3H-norepinephrine

from isolated brain tissue. J Pharmacol Exp Ther 189(1):110-118, 1974.

Carlsson, A. The current status of the dopamine hypothesis of schizophrenia.

Neuropsychopharmacolgy 1(3):179-186, 1988.

Chiueh, C.C., and Moore, K.E. D-amphetamine-induced release of “newly

synthesized” and “stored” dopamine from the caudate nucleus in vivo.

J Pharmacol Exp Ther 192(3):642-653, 1975.

Cooper, J.R.; Bloom, F.E.; and Roth, R.H. The Biochemical Basis of

Neuropharmacology. New York: Oxford University Press, 1991.

Cowen, P.J. Antidepressants: Current concepts of mode of action. Int Clin

Psychopharmacol 3:45-55, 1990.

Creese, I., and lversen, S.D. Blockage of amphetamine induced motor

stimulation and stereotypy in the adult following neonatal treatment with

6-hydroxydopamine. Brain Res 55:369-382, 1973.

43
Creese, I., and Iversen, SD. The pharmacological and anatomical substrates

of the amphetamine response in the rat. Brain Res 83:419-436, 1975.

Crews, F.T.; Scott, J.A.; and Shorstein, N.H. Rapid down-regulation of

serotonin, receptor binding during combined administration of tricyclic

antidepressant drugs and alpha2 antagonists, Neuropharmacology

22(10):1203-1209, 1983.

Curzon, G. Serotonin and appetite. In: Whitaker-Azmitia, P.M., and Peroutka,

S., eds. The Neuropharmacology of Serotonin. Ann N Y Acad Sci 600:521-

531, 1990.

Ellinwood, E.H. Behavioral and EEG changes in the amphetamine model of

psychosis. Adv Biochem Psychopharmacol 12:281-297,1974.

Erinoff, L.; MacPhail, R.C.; Heller, A.; and Seiden, L.S. Age-dependent effects

of 6-hydroxydopamine on locomotor activity in the rat. Brain Res 164:195-

205,1979.

Garattini, S., and Samanin, R. Biochemical hypotheses on antidepressant

drugs: A guide for clinicians or a toy for pharmacologists? Psychol Med

18(2):287-304, 1988.

Goodman, L., and Gilman, A. The Pharmacological Basis of Therapeutics. New

York: Pergamon Press, 1990.

Heffner, T.G.; Hartman, J.A.; and Seiden, L.S. A rapid method for the regional

dissection of the rat brain. Pharmacol Biochem Behav 13(3):453-456, 1980.

Heikkila, R.E.; Orlansky, H.; Mytilineou, C.; and Cohen, G. Amphetamine:

Evaluation of d- and l-isomers as releasing agents and uptake inhibitors for

3H-dopamine and 3H-norepinephrine in slices of rat neostriatum and cerebral

cortex. J Pharmacol Exp Ther 194(1):47-56, 1975.

Hotchkiss, A.J.; Morgan, M.E.; and Gibb, J.W. The long-term effects of multiple

doses of methamphetamine on neostriatal tryptophan hydroxylase, tyrosine

hydroxylase, choline acetyltransferase and glutamate decarboxylase

activities. Life Sci 25:1373-1378, 1979.

Humphrey, P.A.; Fenium, W.; Perren, M.J.; Beresford, J.M.; Skingle, M.; and

Whalley, E.T. Serotonin and migraine. In: Whitaker-Azmitia, P.M., and

Peroutka, S., eds. The Neuropharmacology of Serotonin. Ann N Y Acad Sci

600:587-600, 1990.

Iversen, L.L.; Iversen, S.D.; and Snyder, S.H. Handbook of

Psychopharmacology. New York: Plenum, 1990.

Jimerson, D.C.; Lesem, M.D.; Hegg, A.P.; and Brewerton, T.D. Serotonin in

human eating disorders. In: Whitaker-Azmitia, P.M., and Peroutka, S., eds.

The Neuropharmacology of Serotonin. Ann N Y Acad Sci 600:532-544, 1990.

Kelleher, R.T., and Morse, W.H. Escape behavior and punished behavior.

Federations Proc 23:808-817, 1964.

Koda, L.Y., and Gibb, J.W. Adrenal and striatal tyrosine hydroxylase activity

after methamphetamine. J Pharmacol Exp Ther 185:42-50, 1973.

Kostrzewa, R.M., and Jacobowitz, D.M. Pharmacological actions of 6-

hydroxydopamine. Pharmacol Rev 26:199-288, 1974.

44
Kotake, C.; Heffner, T.; Vosmer, G.; and Seiden, L. Determination of dopamine,

norepinephrine, serotonin and their major metabolic products in rat brain by

reverse-phase ion-pair high performance liquid chromatography with

electrochemical detection, Pharmacol Biochem Behav 22:85-89, 1985.

Levine, T.E.; Erinoff, L.; Dregits, D.P.; and Seiden, L.S. Effects of neonatal and

adult 6-hydroxydopamine treatment on random-interval behavior. Pharmacol

Biochem Behav 12(2):281-285, 1980a.

Levine, T.E.; McGuire, P.S.: Heffner, T.G.; and Seiden, L.S. DRL performance

in 6-hydroxydopamine-treated rats. Pharmacol Biochem Behav 12(2):287-

291, 1980b.

Lorens, S.A.; Hata, N.; Cabrera, T.; and Hamilton, M.E. Behavioral effects of

5,7-DHT and MDMA in rats. In: Paoletti, R.; Van Houtte, P.M.; Brunello, N.;

and Maggi, F.M. Serotonin From Cell Biology to Pharmacology and

Therapeutics. Dordrecht, The Netherlands: Kiuwer Academic Publishers,

1990. pp. 615-623.

Marek, G.J.; Li, A.: and Seiden, L.S. Antidepressant-like effects of (+)-

oxaprotiline on a behavioral screen. Eur J Pharmacol 157(2-3):183-188, 1988.

Marek, G.J., and Seiden, L.S. Effects of selective 5-hydroxytryptamine-2

and nonselective 5-hydroxytryptamine antagonists on the differentialreinforcement-of-low-rate 72-second schedule. J Pharmacol Exp Ther

244(2):650-658, 1988a.

Marek, G.J., and Seiden, L.S. Selective inhibition of MAO-A, not MAO-B, results

in antidepressant-like effects on DRL 72-s behavior. Psychopharmacology

(Berlin) 96(2):153-160, 1988b.

Morin, L.P.; Michels, K.M.; Smale, L.; and Moore, R.Y. Serotonin regulation of

circadian rhythmicity. In: Whitaker-Azmitia, P.M., and Peroutka, S., eds. The

Neuropharmacology of Serotonin. Ann N Y Acad Sci 600:418-426, 1990.

Nencini, P.; Woolverton, W.L.; and Seiden, LB. Enhancement of

morphine-induced analgesia after repeated injections of

methylenedioxymethamphetamine. Brain Res 457(1):136-142, 1988.

Peroutka, S.J., and Snyder, S.H. Long-term antidepressant treatment decreases

spiroperidol-labeled serotonin receptor binding. Science 210(4465):88-90,

1980.

Pilc, A. The role of alpha 2-adrenoceptors in the mechanism of action of

antidepressant drugs. POl J Pharmacol Pharm 39(6):691-713, 1987.

Raiteri, M.; Cerrito, F.; Cervoni, A.M.; and Levi, G. Dopamine can be released

by two mechanisms differentially affected by the dopamine transport inhibitor

nomifensine. J Pharmacol Exp Ther 208(2):195-202, 1979.

Richardson, B.P. Serotonin and nociception. In: Whitaker-Azmitia, P.M., and

Peroutka, S., eds. The Neuropharmacology of Serotonin. Ann N Y Acad Sci

600:511-520, 1990.

Richelson, E. Antidepressants and brain neurochemistry. Mayo Clin Proc

65(9):1227-1236, 1990.

45
Scheel-Kruger, J., and Randrup, A. Stereotype hyperactive behavior produced

by dopamine in the absence of noradrenaline. Life Sci 6:1389-1398, 1967.

Seiden, L.S.; Andresen, J.; and MacPhail, R.C. Methylphenidate and damphetamine: Effects and interactions with alphamethyityrosine and

tetrabenazine on DRL performance in rats. Pharmacol Biochem Behav

10(4):577-584, 1979.

Seiden, L.S., and Dykstra, L.A. Psychopharmacology: A Biochemical and

Behavioral Approach. New York: Van Nostrand, 1977.

Seiden, L.S.; Fischman, M.W.; and Schuster, C.R. Long-term

methamphetamine induced changes in brain catecholamines in tolerant

rhesus monkeys. Drug Alcohol Depend 1(3):215-219, 1976.

Seiden, L.S., and Kleven, MS. Methamphetamine and related drugs: Toxicity

and resulting behavioral changes in response to pharmacological probes,

In: Asghar, K., and De Souza, E., eds. Pharmacology and Toxicology of

Amphetamine and Related Designer Drugs. National Institute on Drug Abuse

Research Monograph 94. DHHS Pub. No. (ADM)89-1640. Washington, DC:

Supt. of Docs., US. Govt. Print. Off., 1989. pp. 146-160.

Seiden, L.S., and O’Donnell, J.M. Effects of antidepressant drugs on DRL

behavior. In: Seiden, L.S., and Balster, R.I., eds. Behavioral Pharmacology:

The Current Status. New York: Alan R. Liss, 1985. pp. 323-338.

Sulser, F. Mode of action of antidepressant drugs. J Clin Psychiatry 44:14-20,

1983a.

Sulser, F. Deamplification of noradrenergic signal transfer by antidepressants:

A unified catecholamine-serotonin hypothesis of affective disorders.

Psychopharmacol Bull 19(3):300-304, 1983b.

Vetulani, J.; Stawarz, R.J.; Dingeli, J.V.; and Sulser, F. A possible common

mechanism of action of antidepressant treatments: Reduction in the

sensitivity of the noradrenergic cyclic AMP generating system in the rat limbic

forebrain, Naunyn Schmiedebergs Arch Pharmacol 293(2):109-114, 1976.

Wauquier, A., and Dugovic, C. Serotonin and sleep-wakefulness. In: WhitakerAzmitia, P.M., and Peroutka, S., eds. The Neuropharmacology of Serotonin.

Ann N Y Acad Sci 600:447-459,1990.

Willner, P. Antidepressants and serotonergic neurotransmission: An integrative

review. Psychopharmacology (Berlin) 85(4):367-404, 1985.

DISCUSSION

Q: I was wondering if you saw any long-term changes in body weight?

A: Not when they are compared with their own rate of growth. In other words,

you would get some initial losses, particularly with methamphetamine [meth], in

the body weight, but then if you compared them with controls in a relatively freefeeding situation, they will gain weight at about the same rate as the controls.

So it does lag a little bit.

46
Q: Second question: On one of your slides you said something about the

recovery of the serotonin, and I was wondering, are those changes that you

were talking about relatively permanent or were you looking at them in some

point in the recovery phase?

A: Now, that is a good question. It depends upon which drug you are talking

about. For the meth, we’ve looked for long times after we have treated these

animals, and they are down in serotonin, and they are down in dopamine. But

for fenfluramine, we do see a certain amount of recovery in certain regions in

the brain as we do for MDMA; furthermore, it depends on what brain region

you are talking about and at what aspect of the system you are talking about.

Mainly, we look at levels, and the levels will tend to come back toward normal

in certain brain regions and stay flat in other brain regions. Dr. Molliver will talk

about this in more detail: there seems to be some recovery in levels. Nate Appel

has seen some recovery in the uptake sites. It is a matter of controversy as to

how to interpret some of these data, and maybe we can get some of that worked

out here.

Q (Gibb): Lew, having spent my life looking at changes in neurochemistry, I find

your results discouraging. On the other hand, the fact is that we do know that

fluoxetine is effective in the treatment of depression. Also, as I understand it,

it is very effective in the treatment of obesity. If my information is correct, the

primary focus of that drug’s effect is on the serotonergic system. So I would

conclude that 5-HT is important, even though at this point none of the data

that we have available from animal studies would suggest that. That would

be the skeptic’s view. It seems to me that the question that really needs to be

addressed now (and I think you have said it) is whether the tests that we are

using are sophisticated enough to pick up those kinds of changes. That is the

important issue here. You would think that the feeding behavior would address

the issue. Obviously, I am not a behaviorist, and I wondered if you care to

comment on that?

A: The project took a period of 3 years, We honestly thought that the tests that

we were using would be sensitive enough to pick up changes as they occurred.

Q: Have you looked at sex at all? Because as I understand it, I don’t know. . .

A: Lorens looked at sexual behavior, but he had a very limited trial. And there

were no changes.

Q (Heimer): Lew, I was wondering about the definition of the term

“neurotoxicity” in relation to the techniques you have covered here. I

suppose that you take for granted, as does everybody who uses the so-called

“suppressive” silver methods, that when you see heavily argyrophilic particles

or a strongly argyrophilic neuron, this neuron is degenerating and will eventually

47
die. When you do see black “measles” representing degenerating terminals,

you take for granted that they will die and that the related neuronal cell bodies

will also die. Although many people have been obsessed by silver methods,

there are only a few who have been experimenting with more and more refined

techniques. Maybe you saw the material at the Anatomy Meeting that Dr. de

Olmos and his colleagues from Argentina produced. They kept modifying their

silver technique to the point where, in quinolinic acid experiments, they used the

same dose that people have used to make “restricted” lesions in the striatum.

De Olmos and his colleagues detected degenerating neurons not only in

striatum but in cerebral cortex, thalamus, and various other places even 15

minutes after the injection. I was just wondering if the silver methods have

become so sensitive that they might even pick up changes that are reversible.

I am sure that there are many who know this area better, so they might even

have thought about these things, but as I remember from the time I used

silver techniques, there are few people studying the ultrastructure of these

argyrophiiic particles and neurons. I wonder what you think about this problem?

A: George Ricaurte spent a lot of time working at the level of the electron

microscope and did most of the silver staining procedures along with Ray

Guillery. I thought that the changes using the electron microscope were not

reversible. I must say that, since that time, Phil Groves has obtained evidence

showing very nicely that you can see a silver-positive neuron in a cell that has

been stained immunologically for tyrosine hydroxylase, indicating that it is the

same cell that takes up silver. Now, if you combine that with the idea that the

uptake site is down and that the levels are down, I think the conclusion

becomes inescapable. But I would agree with you that any single benchmark

in and of itsetf may not be a hallmark for neurotoxicity. We have converging

evidence from chemistry, from function (if you take uptake as a function of the

ceil), and the argyrophilic nature of the fiber that degeneration can occur.

But taken singly, I think that any one of these things could lead us astray.

Q (Molliver): About 10 years ago, S. Ögren and colleagues published a series

of reports on animals treated with PCA [parachloroamphetamine] that have a

selective serotonin deficiency. They found profound behavioral changes: a

tenfold decrease in conditioned avoidance learning. Would you comment on

these data?

A: We have never looked at it using PCA. We have looked at conditioned

avoidance acquisition using other 5-HT toxins, but we have not been able to

replicate these data; but we have never tried it with PCA.

Q (Molliver): That was a profound effect. Have others been able to replicate

these data?

A: I don’t think so. I think they have tried.

48
Q (Gibb): Lew, one other thing that I think needs to be taken into consideration

here is (I am sure George will tell us about this) that the rat is relatively less

sensitive to these compounds than primates. And I wonder, therefore, if there

is any possibility that there is impairment of behavior in primates subsequent,

down the line. What I think your data show is that there is a tremendous

amount of plasticity in the system. And we know that if you use 6-

hydroxydopamine, you have to deplete more than 80 percent or so of the

dopamine to show any parkinsonian symptoms. I wonder if the same thing

applies here, and if so, is it worthwhile conducting tests. I guess you couldn’t

do it in rats, maybe, but to see whether with age there is a deficit that develops.

I think the concern that all of us have with regard to the abuse of these

compounds is what is going to happen to these individuals in their sixties or

earlier, possibly, as they abuse these amphetamine compounds; will they be

early parkinsonian patients? Is there anything that we can do as far as tests

are concerned?

COMMENT (Schuster): We did look extensively at the behavior of the

primate after serotonin and dopamine depletions induced by high levels of

methamphetamine. And I must say that we were as completely disappointed

in those investigations as Lew had been with these more recent rat studies.

Our studies used very sophisticated behavioral methods such as force-lever

techniques in which you are able to measure hand tremor, and despite the fact

that the monkeys had 60 to 70 percent depletion of dopamine, we did not see

any changes in their performance.

COMMENT (Ricaurte): It is a nice idea. However, I actually favor the other

possibility you mentioned, and that is that there are functional deficits but

that you need the right task and a sufficient degree of damage within a given

neurotransmitter system to detect it. So, for example, in the case of the

force-lever paradigm, I would maintain that the animals didn’t have as much

dopamine depleted from the basal ganglia that one would have liked; I also

would maintain that perhaps the task, although it is a motor task, may not be

the appropriate task to get at extrapyramidal motor dysfunction. Before going

on to other systems, I would favor the notion of stressing a given system and

selecting the appropriate task to see whether it is possible to detect subclinical

deficits.

A (Seiden): These monkeys were impaired when they were challenged with

physostigmine, as I recall.

Q (Molliver): I have a question about Dr. Gibb's comment regarding fluoxetine.

You must continue to give fluoxetine to see its effects on the depression, so this

is a pharmacological action as opposed to a permanent alteration induced by

the given compound. If you give MDMA every day, you will see behavioral

49
changes. However, if you give a course of it, wait for 3 weeks, and then look

at the system, you don’t see any permanent changes in behavior.

A (Seiden): That takes you back to the dilemma again. If you are modifying

the presynaptic level pharmacologically and you get a change, and then you

deprive the system of the presynaptic transmitter and you don’t get a change,

I have trouble interpreting those data.

Q (Landfield): I think this issue you raised on effects in animals is really very

Important, and it is going to be a recurring issue because we see the same

thing in our THC [tetrahydrocannabinol] studies-the same issues. There are

two factors that make me think that one shouldn’t be too discouraged about

the discrepancies. We have to remember that human behavior has a lot

of redundancy built in, a lot of subtlety. Even in the case of Aizheimer’s

disease, there has to be massive neuronal degeneration before a patient is

institutionalized. Human behavior is much more complex; at least, we like to

think so. We can see subtle differences in human behavior that we might not

see in the rat behavior. A few cocktails can change people’s speech: we can

notice changes. Whereas to observe an alcohol effect on behavior in a rat,

you might need to induce a level of alcohol toxicity that would have a human

completely knocked out. We have to remember that with human behavior, we

can detect very subtle changes. The ratio between toxicity and observable

behavioral effect may be very different in different species. Now, on the other

side of the coin, some conditions allow us to detect a larger effect in a rat. In

rats, one can damage the nervous system and see much more degeneration

from certain toxic drugs because of experimental design: for example, dosage

can be controlled to obtain a much higher rate of delivery. As you said earlier,

if you don’t see that effect in humans, it can be disappointing or seem less

Interesting. However, that may not be so, because toxicity in animals can at

least provide clues to which brain systems are being changed by the drug or

what is happening to membranes. These toxic effects in animals can provide

clues to mechanisms, even though they are not seen in humans. You might

have to treat for 20 years in humans to see the same effect. We also have to

remember that the lifespan of the rat is 2 years, so everything is telescoped.

What takes 2 months in a rat may take 10 years in human beings. I don’t think

we should be disappointed about discrepancies-just recognize the large

differences in the experiments with different species. The discrepancies are

providing important clues, and we shouldn’t expect to see the same things in

rats and humans exactly. I think the points that were made here are very

important. You need different types of tests, more subtle tests, to see subtle

changes in rat behavior.

A (Seiden): I couldn’t agree more. I think that is exactly what I wanted to do

here. I think that I’ve stimulated our discussion. Tom?

50
Q (Sobotka): Could I suggest looking at more local areas of the brain for

depletion? I was working in the area of norepinephrine and noticing that

Dr. Molliver and his colleagues have shown us that there is quite a bit of

differentiation in where norepinephrine was located in the brain. I tried to

locally deplete norepinephrine in rabbits. At least with conditioned avoidance

tests, many people have looked for behavioral effects of depletion of

norepinephrine, and there really isn’t a lot of consensus in that area. I thought

that since you tried a global depletion, there may be some kind of global

compensation. Norepinephrine and serotonin are interacting with many other

things locally in the brain, so what if we depleted norepinephrine locally and

found significant behavioral changes in our task?

AUTHORS

Lewis S. Seiden, Ph.D.

Professor

William L. Woolverton, Ph.D.

Associate Professor

Department of Pharmacological and Physiological Sciences

University of Chicago

947 East 58th Street

Chicago, IL 60637

Stanley A. Lorens, Ph.D.

Professor

Department of Pharmacology

Loyola University Medical Center

2160 First Avenue

Maywood, IL 60153

Joseph E.G. Williams, Ph.D.

Assistant Professor

Queen Mary and Westfield College

Mile End Road

London El 4NS

ENGLAND

51
Rebecca L. Corwin, Ph.D.

Research Associate

Behavioral Neuropharmacology Unit

Experimental Therapeutics Branch

National Institute of Mental Health

National Institutes of Health

Building 10, Room 4N 214

Bethesda, MD 20892

Norio Hata, Ph.D.

Instructor

Department of Psychiatry

Shinshu University School of Medicine

3-1-1 Asahi

Matsumato 390

JAPAN

Mary Olimski

Research Technician

4629 North Winchester

Chicago, IL 60640

52
Strategies for Detecting Subclinical

Monoamine Depletions in Humans

Una D. McCann and George A. Ricaurte

INTRODUCTION

Certain lesions of the central nervous system (CNS) have obvious manifestations

that are readily detected by an experienced clinician. For example, a righthanded individual who develops sudden right-sided weakness and language

problems is likely to have pathology involving the left cerebral hemisphere.

A careful neurological examination might yield more specific information

regarding the anatomical distribution of the lesion, which often can be confirmed

radiologically using imaging techniques such as computerized tomography or

magnetic resonance imaging (MRI). Unfortunately, other lesions of the CNS

may remain undetected for long periods, both because they do not have obvious

clinical manifestations under ordinary conditions and because behavioral

functions sewed by the involved brain areas may be subtle and difficult to

test and measure.

Parkinson’s disease, a neurodegenerative disease involving brain dopamine

neurons, provides an example of a naturally occurring CNS lesion that can

exist in a preclinical or asymptomatic form and that can go undetected for years

(Koller 1992). Patients with late-stage Parkinson’s disease are readily diagnosed

by a well-defined symptom complex that includes slow movement, rigidity,

resting tremor, and postural instability. Often, there is also a loss of facial

expression and some degree of cognitive impairment. However, none of

these impairments becomes apparent until 60 to 70 percent of dopamine

neurons have degenerated and brain dopamine concentrations are depleted

by 80 to 90 percent (Hornykiewicz 1975; Forno and Alford 1987; Koller 1992).

The absence of symptoms in the face of such large dopamine depletions

underscores the problem of preclinical diagnosis. This problem is magnified

severalfold when brain structures with more subtle functions (e.g., serotonin

or mesocortical dopamine systems) are involved.

Several recreationally used amphetamine analogs, including

methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA),

and 3,4-methylenedioxyethylamphetamine (MDEA), have the potential to

53
damage CNS dopamine and serotonin neurotoxins in a variety of animal

species (Seiden and Ricaurte 1987). Whereas the toxic effects of MA involve

dopamine and serotonergic neurons, the toxic effects of MDMA appear to be

specific for serotonergic neurons (Stone et al. 1986; Battaglia et al. 1987;

Ricaurte et al. 1988a). Moreover, it appears that nonhuman primates are at

an increased risk for developing MDMA neurotoxicity (Ricaurte et al. 1988b;

Ricaurte and McCann 1992), and there is increasing concern that humans who

use amphetamines recreationally may also incur damage to brain dopamine

and serotonin neurons. Because the recreational use of MDMA and related

drugs may be on the rise (Noticeboard 1992), it is important to determine their

neurotoxic potential in humans and develop reliable methods for detecting

preclinical monoamine neurotoxicity, if it exists. This chapter reviews the

strategies available for detecting partial depletions of CNS dopamine or

serotonin in humans. Strengths and weaknesses of each strategy are

discussed, and potential tools for enhancing current methodologies are

proposed.

CEREBROSPINAL FLUID ANALYSIS

Cerebrospinal fluid (CSF) bathes the brain and spinal cord. Because of the

CSF’s close proximity to brain structures and its isolation from other major

organs, changes in the neurochemistry of CSF are thought to reflect changes

specific to the CNS (Wood 1980). Furthermore, because the total volume of

CSF (100 to 150 mL) undergoes complete replacement approximately six

times per day (approximately 600 to 700 mL per day), CSF measures can be

used to detect relatively short-term dynamic changes in the CNS milieu.

Although plasma and urine measurements also have been utilized as indices

of CNS monoamine neurotransmitter function, these are generally considered

to be less specific because multiple organ systems (including the adrenal

medulla) contribute considerably to their components (Kagedal and Goldstein

1988).

CSF measures not only are used routinely for the determination of

neuropathological processes in clinical populations but also have been

used as a strategy for detecting suspected neurotransmitter alterations in

neuropsychiatric diseases. More specifically, CSF collected by lumbar

puncture has been used to measure concentrations of relevant neurotransmitter

metabolites (Moir et al. 1970). With regard to amphetamine neurotoxicity,

measurements of lumbar CSF homovanillic acid can be used as an indirect

measure of brain dopamine activity (Kagedal and Goldstein 1988), whereas

5-hydroxyindoleacetic acid (5-HIAA) can be used to detect changes in brain

serotonin (Ricaurte et al. 1988a; Banki and Molnar 1981). Decreases in either

54
of these metabolites in individuals who had been exposed to amphetamines

provides support for the occurrence of amphetamine neurotoxicity in humans.

The advantages of using CSF measures to detect subclinical neurotoxicity

include easy accessibility of CSF, reproducibility of the assay (Menachem et al.

1989) and that it is a dynamic measure. Drawbacks of this strategy include

that CSF composition provides an indirect reflection of brain neurochemical

activity, that it is nonspecific (numerous factors are known to influence the

composition of CSF) (Moir et al. 1970; Post and Goodwin 1977; Wood 1980),

that it is not particularly sensitive (i.e., concentration changes in the cervical

spine underestimate changes in the forebrain) (Sourkes 1973; Sjostrom et al.

1975; Ricaurte et al. 1988a), and that the lumbar puncture procedure can be

unpleasant and can have complications (e.g., “lumbar puncture headache”).

NEUROENDOCRINE STUDIES

A second strategy for detecting subclinical damage to dopamine and

serotonin systems takes advantage of the important roles of these systems

in the hypothalamic-pituitary-adrenal (HPA) axis. Both dopamine and

serotonin are involved in the normal regulation of prolactin and growth

hormone secretion (for reviews, see Kato et al. 1985; Arimura and Culler

1985) and damage to either monoaminergic system can sometimes be

detected using neuroendocrine measures. For example, dopamine is known

to tonically inhibit prolactin release at the level of the pituitary (Weiner and

Bethea 1981). When dopamine levels are severely depleted, or postsynaptic

dopamine stimulation is decreased, abnormally high baseline prolactin levels

result. In instances where depletion of dopamine is not extensive, it may be

necessary to pharmacologically challenge the dopamine system before

endocrine disturbances are noted. For example, in a normal individual, an

oral dose of L-dihydroxyphenylalanine (L-dopa), the precursor to dopamine,

leads to a rise in growth hormone levels. In an individual with damaged

dopamine neurons, there might be a blunted growth hormone response to

L-dopa because fewer dopamine neurons would lead to reduced conversion

of L-dopa to dopamine. By contrast, the same individual given a postsynaptic

dopamine receptor agonist (e.g., bromocriptine) might be expected to have an

abnormally robust rise in growth hormone, because of postsynaptic dopamine

receptor supersensitivity.

In the case of serotonin, similar neuroendocrine strategies have been utilized

to measure neurotransmitter dysfunction. Because serotonin plays a role in

regulating normal secretion of prolactin, growth hormone, and cortisol, damage

to serotonin pathways in the HPA axis can be detected through disturbances in

the homeostatic mechanisms of these hormones (Heninger et al. 1984). For

55
example, both direct and indirect serotonin agonists (e.g., mchlorophenylpiperazine and L-tryptophan) cause acute elevations in

serum prolactin concentrations (Meites and Sontag 1981; Charig et al. 1986),

possibly via a prolactin-releasing agent at the level of the hypothalamus

(Garthwaite and Hagen 1979). In an individual with severe serotonin damage

in the hypothalamus, the rise in prolactin following administration of Ltryptophan might be expected to be diminished (assuming that postsynaptic

supersensitivity is not sufficient to mask presynaptic dysfunction). If the

damage is partial, or if a postsynaptic 5-hydroxytryptamine agonist is

administered, a supraphysiologic response might be anticipated. Because

of the possibility of compensatory mechanisms over time (e.g., postsynaptic

receptor supersensitivity or decreased inhibitory feedback), timing of the

neuroendocrine challenge relative to the time of the suspected neurotoxic

insult may be crucial for proper interpretation of the neuroendocrine challenge.

The previous examples of neuroendocrine methods for detecting preclinical

monoaminergic damage are not exhaustive and are intended to illustrate the

general neuroendocrine strategy. The advantages to neuroendocrine studies

include their sensitivity, low cost, relatively noninvasive nature, and the ease

with which procedures can be performed. However, like CSF measures,

neuroendocrine measures are indirect and nonspecific. Furthermore, because

of the possibility of compensatory mechanisms, results of the neuroendocrine

challenge may not be consistent over time. Therefore, although potentially

lending support for the diagnosis of neurotoxicity, neuroendocrine disturbances

are not definitive.

NEUROIMAGING TECHNIQUES

Recent advances in imaging technology and the development of

neurotransmitter-specific ligands hold promise for the detection of subclinical

neurotoxic injury. As further advances are made, imaging techniques that

can be used to this end will include single photon emission computerized

tomography, positron emission tomography (PET), and possibly MRI (at least

in cases where the extent of neuronal volume loss is sufficient). However, to

date only PET has been used successfully in humans for the detection of

neurotoxic damage, and only in the dopamine system (Calne et al. 1985).

The reasons for this have been largely caused by the limited number of suitable

ligands available rather than because of limitations of imaging techniques.

However, in the case of serotonin, which is present in the brain at much lower

concentrations than dopamine, sensitivity of imaging techniques also may prove

to be problematic in detecting subtle neurotoxic lesions.

56
Because it is known that neurotoxic amphetamines damage the presynaptic

element, the ideal ligand for detecting amphetamine neurotoxicity would be

a presynaptic marker. Postsynaptic receptor markers, although possibly

demonstrating receptor down- or up-regulation, would not provide compelling

evidence of neurotoxicity. Of the various possible presynaptic markers, ligands

for the uptake site may prove to be superior for the detection of neurotoxic

injury because they label a “structural” macromolecular element of the nerve

terminal (as opposed to neurotransmitter precursors such as 6-fluorodopa

that can be altered by metabolic events). In the case of the dopamine

system, several such ligands exist and include GBR-12909, nomifensine,

and WIN-35428. Neurotoxic damage in humans caused by the potent

neurotoxin 1 -methyl-4-phenyl-1,2,3,8-tetrahyropyridine has been detected

using 6-fluorodopa, and this ligand also should be useful in detecting

dopaminergic damage caused by amphetamines such as methamphetamine,

which damages dopamine nerve terminals.

Development of presynaptic markers for serotonin neurons has been more

difficult, although there are several promising candidates in the development

phase. In particular, a variety of ligands that label the serotonin transporter

complex (e.g., paroxetine derivatives) may prove to be ideal for studies in

individuals exposed to potential serotonin neurotoxins such as MDMA or

MDEA.

Advantages to PET and other neuroimaging techniques are that they

provide evidence of neuronal alteration, they can be anatomically and

pharmacologically specific, and they are relatively noninvasive (intravenous

catheters are required). Disadvantages to neuroimaging studies are their

potential lack of sensitivity, their expense, the large number of personnel

required for completion of the study, and the inability to perform studies

without special equipment and personnel.

PHARMACOLOGICAL CHALLENGE

A specific pharmacological challenge to a compromised neurotransmitter

system has not been widely used in the detection of subclinical monoaminergic

neurotoxicity but has potential utility. This method would take into account

that the neurotransmitter system in question, although not eradicated, has

limited reserves. In essence, under normal conditions, a compromised

neurotransmitter system might be able to function, but if it is stressed,

abnormalities might become apparent. An analogy can be drawn with the

use of the cardiac stress test in instances where coronary artery insufficiency

is suspected but unconfirmed. In an individual with “silent” coronary artery

disease, cardiac abnormalities are inapparent under ordinary physical

57
conditions. Abnormalities such as irregular heart rhythms or angina pectoris

may become manifest when the Individual is subjected to physical stress such

as the treadmill test (Astrand 1978; Kilpatrick 1986).

A pharmacologic neuronal stress test might be useful in the evaluation of

patients with suspected subclinical neurotoxicity. For example, in an individual

with a history of significant methamphetamine use, diminished dopaminergic

neurotransmission might be expected but, under ordinary conditions, might

be clinically inapparent because it was not sufficiently severe. If that

individual were given a drug that specifically interfered with dopaminergic

neurotransmission, it might be anticipated that the subject would demonstrate

exquisite sensitivity to its effects. Drugs that could be used to challenge the

dopaminergic system include -methyl-para-tyrosine, a synthesis inhibitor, or

haloperidol, a postsynaptic dopamine receptor blocker. Endpoints that could

be used to measure drug effects include parkinsonian symptoms and

elevations in serum prolactin levels, as mentioned above.

Specific challenges of the serotonin system might be accomplished using the

serotonin synthesis inhibitor parachlorophenylalanine. Unlike challenges to the

dopamine system, challenges to the serotonin system have no obvious and

easily measured functional endpoint. However, because serotonin has been

implicated in the regulation of sleep, mood, anxiety, impulsivity, and pain

sensitivity, disturbances in these behavioral spheres might be expected.

SUMMARY

Given the reported increase in the recreational use of controlled substance

analogs such as MDMA and related drugs, it is important to determine whether

these drugs produce neurotoxic effects in humans. Several strategies available

for detecting preclinical neurotoxicity to dopamine and serotonin neurons have

been discussed, and their strengths and limitations have been listed. In

addition, some promising strategies that are still in the development stage (e.g.,

PET) have been mentioned. None of the available methods for detecting

neurotoxicity is conclusive: therefore, converging lines of evidence will be

essential to provide convincing indication of subclinical neurotoxicity in humans.

Such studies will help define the public health risk of recreationally used drugs.

Furthermore, documentation and determination of drug-induced neurotoxic

changes may shed light on the pathophysiology of idiopathic neurodegenerative

diseases involving monoaminergic neurons in humans and could be useful in

the development of new treatment strategies. Finally, detailed neuropsychiatric

evaluation of individuals with confirmed subclinical serotonergic neurotoxicity

may enhance knowledge regarding the functional role of brain serotonin

neurons in health and disease.

58
REFERENCES

Arimura, A., and Culler, M.D. Regulation of growth hormone secretion. In:

Imura, H., ed. The Pituitary Gland. New York: Raven Press, 1985. pp, 221-

259.

Astrano, P.O. Evaluation and quantification of exercise capability and of

physical capacity in man. Prog Cardiovasc Dis 19(1):51 -67, 1976.

Banki, C.M., and Molnar, G. CSF 5-HIAA as an index of serotonin processes.

Psychiatry Res 5:23-32, 1981.

Battaglia, G.; Yeh, S.Y.; O’Hearn, E.; Molliver, M.E.; Kuhar, M.J.; and De

Souza, E.B. 3,4-Methylenedioxymethamphetamine and 3,4-

methylenedioxyamphetamine destroy serotonin terminals in rat brain:

Quantification of neurodegeneration by measurement of [3H]paroxetinelabelled serotonin uptake sites. J Pharmacol Exp Ther 242:911-916, 1987.

Calne, D.B.; Langston, J.W.; Martin, W.R.; Stoessl, A.J.; Ruth, T.J.; Adam, M.J.;

Pate, B.D.; and Shulzer, M. Positron emission tomography after MPTP:

Observations relating to the cause of Parkinson’s disease. Nature 317:246-

248, 1985.

Charig, E.M.; Anderson, I.M.; Robinson, J.M.; Nutt, D.J.; and Cowen, P.J.

L-tryptophan and prolactin release: Evidence for interaction between 5HT1

and 5HT2 receptors. Hum Psychopharmacol 1:93-105, 1986.

Forno, L.S., and Alford, E.C. Pathology of Parkinson’s disease. In: Keller, W.C.,

ed. Handbook of Parkinson’s Disease. New York: Marcel-Dekker, 1987.

pp. 209-236.

Garthwaite, T.L., and Hagen, T.C. Evidence that serotonin stimulates a

prolactin-releasing factor in the rat. Neuroendocrinology 29:215-223, 1979.

Heninger, G.; Charney, D.S.; and Sternberg, D.E. Serotonergic function in

depression: Prolactin response to intravenous tryptophan in depressed

patients and healthy subjects. Arch Gen Psychiatry 41:398-402, 1984.

Hornykiewicz, O. Parkinson’s disease and its chemotherapy. Biochem

Pharmacol 24:1061-1065, 1975.

Kagedal, B., and Goldstein, D.S. Catecholamines and their metabolites.

J Chromatogr 429:177-233, 1988.

Kato, Y.; Matsushita, N.; Ohta, H.; Tojo, K.; Shimatsu, A.: and Imura, H.

Regulation of prolactin secretion, In: Imura, H., ed. The Pituitary Gland.

New York: Raven Press, 1985. pp. 261-278.

Kilpatrick, D. Exercise vectocardiography in the diagnosis of ischaemic heart

disease. Lancet 14;2(7981):332-334, 1986.

Koller, W.C. When does Parkinson’s disease begin? Neurology 42(Suppl 4):27-

31,1992.

Meites, J., and Sontag, W.E. Hypothalamic hypophysiotropic hormones and

neurotransmitter regulation: Current views. Annu Rev Pharmacol Toxicol

21:295-321, 1981.

59
Menachem, E.B.; Persson, L.; Schecter, J.; Haegele, D.; Huebert, N.; and

Hardenberg, J. Cerebrospinal fluid parameters in healthy volunteers during

serial lumbar punctures. J Neurochem 52:632-635, 1989.

Moir, A.T.; Ashcroft, G.W.; Crawford, T.B.; Eccleston, D.; and Guldbert, H.C.

Cerebral metabolites in cerebrospinal fluid as a biochemical approach to the

brain. Brain 93:357-368, 1970.

Noticeboard: Drug culture. Lancer 339:117, 1992.

Post, R., and Goodwin, F.K. Approaches to brain amines in psychiatric patients:

A reevaluation of cerebrospinal fluid studies. In: Iversen, S.D., and Snyder,

S.H., eds. Handbook of Psychopharmacology. Vol. 8. New York, London:

Plenum Press, 1977. pp. 147-l 85.

Ricaurte, G.A.; DeLanney, L.E.; Wiener, S.G.; Irwin, I.; and Langston, J.W. 5-

Hydroxyindoleacetic acid in cerebrospinal fluid reflects serotonergic damage

induced by 3,4-methylenedioxymethamphetamine in CNS of non-human

primates. Brain Res 474:359-363, 1988a.

Ricaurte, G.A.; Forno, L.S.; Wilson, M.A.; DeLanney, L.E.; Irwin, I.; Molliver, M.;

and Langston, J.W. (±) 3,4-Methylenedioxymethamphetamine selectively

damages central serotonergic neurons in nonhuman primates. JAMA

260(1):51-55, 1988b.

Ricaurte, G.A., and McCann, U.D. Neurotoxic amphetamine analogs: Effects in

monkeys and implications for humans. Ann N Y Acad Sci 648:371-382,1992.

Seiden, L.S., and Ricaurte, G.A. Neurotoxicity of methamphetamine and related

drugs. In: Meltzer, H.Y., ed. Psychopharmacology-A Generation of

Progress. New York: Raven Press, 1987. pp. 359-366.

Sjostrom, R.; Ekstedt, J.; and Amggard, E. Concentration gradients of

monoamine metabolites in human cerebrospinal fluid. J Neuro Neurosurg

Psychiatry 38:668-668, 1975.

Sourkes, T.L. On the origin of homovanillic acid (HVA) in the cerebrospinal fluid.

J Neural Transm 34:153-157, 1973.

Stone, D.M.; Stahl, D.S.; Hanson, G.L.; and Gibb, J.W. The effects of 3,4-

methylenedioxymethamphetamine (MDMA) and 3,4-

methylenedioxyamphetamine on monoaminergic systems in the rat brain.

Eur J Pharmacol 28:41-48, 1986.

Weiner, R.I., and Bethea, CL. Hypothalamic control of prolactin secretion, In:

Jaffe, R.B., ed. Prolactin. New Holland, NY: Elsevier Press, 1981. pp. 19-55.

Wood, J.H. Neurochemical analysis of cerebrospinal fluid. Neurology 30:645-

651, 1980.

DISCUSSION

Q: George, do you think that we will ever be able to get a clear answer by

comparing people who use MDMA with control groups? Are we going to need

to use people as their own controls?

60
A: If we compare results of an MDMA cohort with those of a control group,

the comparison is compromised for a number of reasons, including the fact

that invariably people have used other drugs as well. Also, there may be

differences in educational background as well as a host of other differences

that make intergroup comparisons difficult. One strategy that might be used

to get around such problems would be an epidemiologic one, where numbers

are such that you can begin to factor out some of the variables; but still the

evidence is going to be indirect. Between-group comparisons are flawed, but

I am afraid for now it is really the only viable option given the fact that to try to

do before-and-after comparisons using subjects as their own controls is difficult

unless (1) you have the authorization to do such a study and (2) you are willing

to do such a study because (as you know) there are risks associated with

testing the effects of MDMA on humans.

Q: How far do you think you have to go without doing that?

A: If you can develop a consistent line of argument with numerous lines of

evidence where various results all point to the same conclusion, I think you

can go reasonably far. I doubt that we will ever reach the point where we

can definitely state that there is such a percent depletion of serotonin in a

given individual’s brain. However, using the strategies I have discussed in

combination, looking for converging lines of evidence, I think you can probably

develop a reasonable case for the presence or absence of neurotoxicity in a

human cohort. In that regard, the human situation is not terribly different from

the preclinical situation, where converging lines of evidence also are necessary

to reach the conclusion that you are in fact dealing with neurotoxicity. None of

the measures is going to make a compelling case by itself. But if different

measures are considered together, a picture begins to emerge.

Q (Miller): Has anyone tried to look at the same issues with the fenfluramine

populations in Europe?

A: Not that I know of. There is one disturbing result in the literature.

Fenfluramine is often used instead of L-tryptophan as a challenge agent to

study serotonin systems in humans. The disturbing observation is that when

Coccaro and colleagues’ used fenfluramine as the challenge drug, they found

a shift in the baseline of their “control group,” so that when a control group was

rechallenged with fenfluramine 1 week later, the prolactin response appeared

blunted, suggesting a change in serotonin system as a function of fenfluramine

exposure. Aside from that one study, fenfluramine-exposed individuals have

1 Coccaro, E.F.; Siever, L.J.; Klahr, H., et al. Diminished prolactin responses to repeated

fenfluramine challenge in man. Psychiatry Res 22(3):257-259, 1987.

61
not been studied either with CSF or neuroendocrine challenge strategies, to

my knowledge.

COMMENT (Seiden): There was one study done in Europe that was published

in Lancet, I think last year-a fairly large study of people who were getting

fenfluramine for a whole year. They were testing various neurological and

psychological outcomes in these patients who had taken fenfluramine for

weight reduction. There was nothing marked in that study. But the only way

a factor would come up in that study was if the patient reported to the

physician that he was experiencing x, y, or z effect. Patients weren’t given

questionnaires. So it is a little biased toward nonreporting.

AUTHORS

Una D. McCann, M.D.

Chief, Sleep Laboratory

Section on Anxiety and Affective Disorders

Biological Psychiatry Branch

National Institute of Mental Health

National Institutes of Health

Building 10, Room 3S-237

9000 Rockville Pike

Bethesda, MD 20892

George A. Ricaurte, M.D., Ph.D.

Assistant Professor

Department of Neurology

Francis Scott Key Medical Center

Johns Hopkins University School of Medicine

Baltimore, MD 21224

62
Sensory-Evoked Potentials:

Measures of Neurotoxicity

William K. Boyes

NEED FOR TESTS OF SENSORY FUNCTION

Only a portion of approximately 65,000 chemicals used in commerce have an

adequate toxicity database (Office of Technology Assessment 1990). Although

no hard figures are available, it has been estimated that approximately 25

percent of the compounds tested produce significant neurotoxicity (Office

of Technology Assessment 1990). Of those compounds reported to have

neurotoxic effects, one analysis found that approximately 44 percent produced

sensory system effects, with visual effects being most common (Crofton and

Sheets 1989). Combining these values would yield an estimate, admittedly

crude, that approximately 11 percent of all compounds are potential sensory

toxicants. In comparing the types of visual system effects reported in humans

with those observed in laboratory animals, the human reports are more likely to

involve loss of visual perception, such as blurred or double vision, whereas the

animal reports are more likely to be easily observable lesions such as cataracts

(Boyes 1992). Rarely are deficits of sensory function reported in laboratory

animal toxicology studies. The reasons for this are fairly obvious, because

animals do not readily reveal what they see, hear, or feel. Furthermore, animals

have not been “asked” these questions with technology capable of providing

some of the answers. Given the likelihood that many chemicals are toxic to

sensory systems, it is necessary to develop and implement procedures that

will enable the detection and characterization of sensory toxicity in routine

animal toxicology studies. At the same time, it would be advantageous to

obtain information about mechanisms of action, perception, and the

comparability of responses in laboratory animals and humans.

SENSORY-EVOKED POTENTIALS

The analysis of sensory-evoked potentials (SEPs) has much to offer in filling

such needs. These responses are averaged electrophysiological events

recorded from appropriately positioned electrodes following repeated

presentation of sensory stimuli. Typical recording situations for measuring

63
visual-evoked potentials from human and rat subjects are depicted in figure 1.

A sensory stimulus, in this case a changing visual pattern, is presented to the

subject. Electrical activities recorded from electrodes positioned over the

primary visual projection areas are differentially amplified with respect to a

reference site, and several responses are averaged together by computer in

synchrony with stimulus presentation. Thus, activity that is unrelated to

stimulation averages out, whereas activity that occurs repeatedly in a timelocked fashion following stimulus presentation is enhanced. The resulting

Pattern-Evoked Potential Recording Procedure

Pattern Onset-Evoked Potentials

FIGURE 1. Typical evoked potential recording arrangements for humans and

rats, along wifh pattern onset-evokedpotentials recorded from

eachspecies

64
averaged evoked potential reflects summed neural responses involved in

processing sensory input. A variety of sensory modalities can be studied,

including vision, audition, and somatosensation, which yield visual-, auditory-,

or somatosensory-evoked potentials, respectively. Variations in the stimulus

parameters, and in the recording procedures, can be utilized to obtain

information about the operational characteristics of the neural systems that

process stimulus information. Evoked potentials can be used in conjunction

with behavioral, neurochemical, or histopathologlcal techniques. A great deal

has been written about SEPs and related procedures, and excellent sources

exist (Cracco and Bodis-Wollner 1986; Regan 1989).

SEPs AND CLINICAL DISORDERS

The practical utility of SEPs in clinical medicine has long been recognized.

Halliday and coworkers first demonstrated the usefulness of pattern reversal

visual-evoked potentials (VEPs) in detecting clinical disorders, including optic

neuritis (Halliday et al. 1972) and multiple sclerosis (Halliday et al. 1973).

During episodes of visual impairment, VEPs recorded following stimulation

of the affected eye were smaller in amplitude and prolonged in latency in

comparison with periods of remission. Comparisons of VEP latencies recorded

from normal subjects and patients with multiple sclerosis reveal clear differences

(Bodis-Wollner et al. 1979; Halliday 1981). These and other successes led to

widespread clinical use of SEPs. Some of the clinical conditions in which

evoked potentials have been reported to be altered are presented in table 1.

In studying clinical disorders, evoked potentials have been found to provide

objective confirmation of other reports of sensory dysfunction, to be sensitive to

both mild and severe sensory impairments, to aid in localization of lesions, to

help in charting the course of diseases and treatments, and to be invaluable in

studying patients with poor ability to communicate, such as infants and young

children. Evoked potentials are used widely to monitor the status of sensory

tracts during the course of neurosurgery (Jones 1986). These versatile tools

have much to offer the neurotoxicologist.

SEPs AND NEUROTOXIC DISORDERS

Properties that make SEPs clinically useful also make them attractive for

studying neurotoxicity. Over the past 15 years, a number of review articles

have been written that serve to chronicle the development of the technology as

well as the growing application to toxicological problems (Woolley 1977; Fox et

al. 1982; Dyer 1982,1983,1985,1986,1987a, 1987b; Rebert 1983; Dyer and

Boyes 1983; Arezzo et al. 1985; Otto et al. 1985, 1988; Otto 1986; Seppalainen

1988; Mattsson and Albee 1988; Mattsson et al. 1989a, 1990a, 1992; Boyes

1992). What began as a relatively limited and experimental application of the

65
TABLE 1. Examples of neurological conditions in which SEPs are altered

Condition Reporting Source

Aging

Albinism

Alzheimer’s disease

Amblyopia

Amyotrophic lateral

sclerosis

Aphasia

Asphyxia

Cochlea/auditory

nerve damage

Blood-brain barrier

disruption

Cerebral lesions

Color blindness

Coma

Dementia

Diabetes

Down’s syndrome

Epilepsy

Glaucoma

Guillain-Barre

Head trauma

Celesia and Daly 1977; Morrison and Reilly 1989;

Simpson and Erwin 1983

Creel et al. 1981; Apkarian and Spekreijse 1986

Coben et al. 1983; Smith et al. 1990

Levi and Manny 1986

Cosi et al. 1984; Dasheiff et al. 1985; Bosch et

al. 1985

Rothenberger et al. 1982

Hrbek et al. 1977; Hecox and Cone 1981

Don 1986

Warnke et al. 1989

Giblin 1964; Bodis-Wollner 1976; Nakanishi et

al. 1978; Obeso et al. 1980; Chu 1986

Regan and Spekreijse 1974

Starr and Achor 1975; Starr 1976; de la Torre et al.

1978; Hume et al. 1979; de la Torre 1981; Karnaze

et al. 1982; Stohr et al. 1987; Facco et al. 1988

Abbruzzese et al. 1984; Goodin et al. 1978;

Michalewski et al. 1986

Trick 1991

Bigum et al. 1970

Ratliff and Zemon 1984

Atkin et al. 1983; Bodis-Wollner 1989

Brown and Feasby 1984

Greenberg et al. 1977; Shaw 1986; Jabbari et al.

1987; Mahapatra and Bhatia 1989

66
TABLE 1. (continued)

Condition Reporting Source

Human

immunodeficiency

virus

Huntington’s disease

Hydrocephalus

Hyperactivity

lschemia

Learning disability

Leukodystrophy

Migraine

Minamata disease

Multiple sclerosis

Parkinson’s disease

Phenylketonuria

Optic neuritis

Optic tract lesions

Retinopathy

Reye syndrome

Spinal cord injury

Syringomyelia

Tumors

Vascular lesions

Koralnik et al. 1990

Noth et al. 1984; Josiassen et al. 1982

McSherry et al. 1982; Onofrj et al, 1981

Halliday et al. 1984

Mizrahi and Crawford 1984

Duffy 1986; 0llo and Squires 1986

Markand et al. 1982; Garg et al. 1983

Nyrke et al. 1989

Mukuno et al. 1984

Halliday et al. 1973; Cant et al. 1978; Dorfman et al.

1978; Nuwer and Namerow 1981; Barajas 1982;

Bottcher and Trojaborg 1982; Matthews and

Wattam-Bell 1982; Matthews and Small 1983; Oishi

et al. 1985; Toyonaga 1988; Sartucci et al. 1989;

Ghilardi et al. 1991

Mintz et al. 1981; Bodis-Wollner et al. 1981;

Hansch et al. 1982; Bodis-Wollner et al. 1986

Giovannini et al. 1988

Halliday et al. 1972

Onofrj et al. 19826; Sherman 1986

Papakostopoulos et al. 1984

Goff et al. 1983

Perot 1973

Anderson et al. 1986

Takayasu et al. 1987

Noel and Desmedt 1975

67
techniques grew steadily, utilizing more sophisticated methodology and providing

more frequent and broader applications. Now it is possible to incorporate

evoked potential testing into toxicological testing protocols (Mattsson et al.

1992). Guidelines have been drafted for conducting visual-, auditory-, and

somatosensory-evoked potential procedures in testing the potential toxicity of

compounds regulated by the U.S. Environmental Protection Agency under the

Toxic Substances Control Act (1976) or the Federal Insecticide, Fungicide,

and Rodenticide Act (1988 amendment) (Boyes 1990). If such guidelines are

adopted, the routine use of SEPs in studying compounds suspected of damaging

sensory systems is likely to continue growing.

There are many interesting examples of toxic damage produced to sensory

systems that have been studied using evoked potentials. For example,

ototoxicity is produced by exposure to several different solvents, including

toluene, xylene, carbon disulfide, methylene chloride, and styrene in addition

to trichloroethylene (Pryor et al. 1983, 1984a, 1984b, 1987; Rebert et al.

1983,1989 a, 1989b, 1991; Rebert and Becker 1986). Rats exposed to

trichloroethylene for 12 weeks showed dose-related amplitude deficits in brain

stem auditory-evoked responses (BAER) that were more severe at 16 kHz than

at 8 or 4 kHz (figure 2). These permanent, frequency-dependent changes are

indicative of hearing loss and are thought to result from damage to cochlear hair

cells. The auditory deficit is clearly evident in the amplitude/intensity functions

presented in figure 2. Clinically, it is probably more common to use BAER

latency/intensity functions in studying auditory dysfunction because amplitudes

are somewhat more variable across subjects. BAER latency/intensity functions

were shown to be sensitive to alterations produced by developmental exposure

to alcohol (Church 1987), and both latency and amplitude/intensity functions

were altered by prenatal cocaine exposure (Church and Overbeck 1990).

Finally, the systematic reduction of BAER stimulus levels, such as would be

required to generate amplitude or latency/intensity functions, can be used

to estimate auditory thresholds. These procedures have been shown to

correlate well with behaviorally determined changes in auditory thresholds

and histologically measured loss of cochlear hair cells produced by the

ototoxicant trimethyltin (Crofton et al. 1990).

Somatosensory deficits induced by neurotoxic compounds have also been

studied using SEPs (Dyer 1987b). For example, acrylamide-induced changes

in somatosensory-evoked potentials have been reported in rats (Boyes and

Cooper 1981) (figure 3) and monkeys (Arezzo et al. 1981) (figure 4). Acrylamide

produced progressive latency increases and amplitude reductions of

somatosensory-evoked potentials in both species. The evoked potential

changes in monkeys preceded both behavioral and neuropathological signs

of intoxication. Thus, somatosensory-evoked potentials can be sensitive

68
FIGURE 2. Trichloroethylene-induced reductions in amplitude of tone-elicited

BAERs

SOURCE: Reprinted with permission from Neurobehavioral Toxicology and

Teratology 13(1), Rebert, C.S.; Day, V.L.; Matteucci, M.J.; and

Pryor, G.T. Sensory-evoked potentials in rats chronically exposed

to trichloroethylene: Predominant auditory dysfunction. Copyright

1991, Pergamon Press plc. (Elmsford, NY).

indicators of central/peripheral distal axonopathy. Somatosensory-evoked

potentials are typically elicited with electrical shock stimuli applied to a selected

peripheral nerve through either skin or needle electrodes. Such stimuli bypass

the sensory receptors that normally transduce stimulus properties such as

pressure, vibration, or temperature into sensory neural activity. Evoked potentials

recorded under these conditions provide somewhat limited information regarding

sensory perception. It is likely that techniques under development involving more

natural skin stimulation such as brushing or vibration (Pratt and Starr 1986) will

correlate better with perception and also find useful toxicological applications.

VEPs have been used to study many known or potential neurotoxic compounds.

Boyes (1992) lists some of the compounds studied following acute exposures,

Virtually every compound tested that was previously thought to be neurotoxic

was found to produce changes in VEPs at some dose level. These studies

suggest that VEPs might be useful in the detection phase of neurotoxicity

investigations. However, because of practical constraints involved in screening

many compounds to detect neurotoxicity, VEPs will likely be more useful in

69
FIGURE 3. Acrylamide-induced reduction in amplitude of far-field

somatosensory-evoked potentials in rats

SOURCE: Reprinted with permission from Neurobehavioral Toxicology and

Teratology, 3, Boyes, W.K., and Cooper, G.P. Acrylamide

neurotoxicity: Effects on far-field somatosensory evoked potentials

in rats. Copyright 1981, Pergamon Press plc. (Elmsford, NY).

70
FIGURE 4. Acrylamide-induced changes in amplitude and latency of far-field

somatosensory-evoked potentials in monkeys. SLSEP-short

latency SEP.

SOURCE: Reprinted with permission from Neurobehavioral Toxicology and

Teratology, 7(4), Arezzo, J.C.; Simson, R.; and Brennan, N.E.

Evoked potentials in the assessment of neurotoxicology in humans.

Copyright 1985, Pergamon Press plc. (Elmsford, NY).

71
characterization of changes in the visual system once abnormalities are

discovered. Effects on evoked potentials with complementary data from other

procedures can reveal much regarding the nature of the visual dysfunction

produced. For example, reports that developmental lead exposure produced

scotopic visual deficits in behavioral studies (Bushnell et al. 1977) prompted a

series of investigations into the nature of lead-induced visual deficits by Fox

and coworkers. VEP deficits (Fox et al. 1977, 1979; Fox 1984), in conjunction

with other data, including electroretinograms (ERGS), prompted the discovery of

selective action of lead on rod, but not cone, photoreceptors (Fox and Sillman

1979; Fox and Farber 1988; Fox and Chu 1988). In another example, VEP

deficits, again along with changes in ERG, helped demonstrate the development

of visual toxicity in metabolically altered rats treated with methanol (Lee 1987;

Eells 1991). Finally, increased latencies and decreased peak-to-peak amplitudes

of VEPs recorded from both visual cortex and the optic tract following exposure

to trimethyltin indicated that this well-known limbic-system toxicant also produced

retinal damage (Dyer et al. 1982), a supposition subsequently confirmed

histologically (Chang and Dyer 1983).

SEPs AND MORE SOPHISTICATED QUESTIONS

Given that SEPs aid in the detection and characterization of neurotoxicity, what

do changes in evoked potentials mean? The answer to this question takes

several directions, two of which are considered here: (1) What is the cellular

locus or pharmacological nature of the action of the compound, and (2) what

are the perceptual correlates of the effects (i.e., how well can the subject hear,

see, or feel)? The first answer leads to investigations of the mechanism of

action of intoxication. To provide the second answer, it is necessary to study

the relationship between evoked potentials and sensory psychophysics.

SEPs and Mechanisms of Action

Evoked potentials, as neuronal population responses, provide a broad level

of analysis and therefore are not generally considered for use in mechanism

of action studies. However, there is nothing to prevent the design of studies

using evoked potentials that can help elucidate possible mechanisms of action.

When used in conjunction with other techniques, they can be powerful.

Several authors have shown that acute and chronic exposure to toluene can

produce a variety of effects on SEPs in rodents and humans (Dyer et al. 1984,

1988; Rebert et al. 1989c; Urban and Lukäs 1990) in addition to the ototoxicity

discussed previously (Pryor et al. 1983, 1984a, 1984b; Rebert et al. 1983,

1989a). Mattsson and colleagues (1989b) demonstrated that acute inhalation

of high concentrations of toluene vapors, as might occur during human toluene

72
abuse, produced an unusual episode of high frequency oscillations overriding

normal rat SEPs (figure 5). EEG power spectra were also altered in a fashion

that, along with the SEP changes, suggested the excitatory phase of the druginduced users’ “high.” The spectra showed that this set of changes also could

be produced by the toluene metabolite o-cresol, but not by other major toluene

metaboliteS. These results suggested that the o-cresol metabolic pathway

of toluene could be of importance in sustaining drug abuse behavior. It is

unknown whether the neurotoxicity that follows repeated exposure to high

concentrations of toluene, and that in a simulated abuse paradigm produced

changes in flash-, auditory-, and somatosensory-evoked potentials and caudal

nerve action potentials, is likewise attributable to o-cresol (Mattsson et al. 1990b).

Another example illustrating the use of evoked potential technology to address

more mechanistic scientific questions is a set of studies regarding the effects

of chlordimeform, a member of the formamidine class of insecticides and

acaricides. Chlordimeform has an unusually selective effect on rat VEPs.

Maximal SEP Excitation

FIGURE 5. Toluene and o-cresol-induced osillations in rat somatosensoryevoked potentials

SOURCE: Reprinted with permission from Neurotoxicology and Teratology,

11(1), Mattsson, J.L.; Albee, R.R.; and Gorzinski, S.J., “Similarities

of toluene and o-cresol neuroexcitation in rats.” Copyright 1989,

Pergamon Press plc. (Elmsford, NY).

73
When reversing black-and-white patterns were used as stimuli to elicit pattern

reversal-evoked potentials (PREPS), the response amplitude was increased

many times by acute treatment with chlordimeform (Boyes and Dyer 1984).

However, the amplitude of flash-evoked potentials (FEPs) recorded from the

same rats was unchanged. Amitraz, another formamidine pesticide, had similar

acute effects on VEPs, but the duration of the effects was more prolonged

(Boyes and Moser 1987). In contrast to the selective effects on amplitudes,

peak latencies of both PREPS and FEPs responses were increased. In

subsequent studies it was shown that the latency increases were a secondary

consequence of chiordimeform-induced hypothermia. However, the selective

amplitude effects could not be attributed to body temperature changes (Boyes

et al. 1985c), nor could they be attributed to monoamine oxidase inhibition

(Boyes et al. 1985a), at one time a leading candidate for the mechanism of

pesticidal properties of the formamidines (Aziz and Knowles 1973; Beeman

and Matsumura 1973).

This left two leading hypotheses regarding the possible mechanism of action of

chlordimeform in altering rat VEPs. One was that the effects could have been

attributed to local anesthetic-like actions (Chinn et al. 1977; Lund et al. 1978;

Pfister et al. 1978). Recently, the formamidines have been shown to enhance

susceptibility to amygdaloid kindling through local anesthetic-like properties

(Gilbert and Mack 1989). However, the author and colleagues were unable to

mimic the VEP effects of chlordimeform across a range of doses of lidocaine

(unpublished observation), so this mechanism of action did not appear to be

relevant to the visual effects of the formamidines.

An alternative hypothesis was suggested from reports that chlordimeform,

and more potently its demethyl and di-demethyl metabolites, were agonists

of octopamine, a neurotransmitter in firefly light organs (Hollingworth and

Murdock 1980) and locust neuromuscular junctions (Evans and Gee 1980).

Insect octopamine receptors are pharmacologically similar to mammalian

-adrenoceptors, suggesting that chlordimeform might act as a mammalian

-agonist (Hollingworth and Lund 1982). Yohimbine, an -antagonist,

attenuates other toxic manifestations of formamidines (Moser and MacPhail

1985), and some actions of the formamidines had been demonstrated on

mammalian -adrenergic systems, largely in the peripheral nervous system

(Hsu and Kakuk 1984; Hsu and Lu 1984; Hsu and McNeel 1985; Hsu and

Hopper 1986; Hsu et al. 1986). In VEP studies, rats treated with clonidine, an

-agonist, showed PREP amplitude augmentations that were very similar to

those produced by chlordimeform (Boyes and Moser 1988). Pretreatment with

yohimbine, which was not effective given alone, was able to block the effects of

subsequent treatment with either clonidine or chlordimeform (figure 6). These

results suggested that the actions of chlordimeform in the rat visual system may

74
FIGURE 6. Chlordimeform-induced increases in amplitude of PREPS in rats,

which are blocked by pretreatment with yohimbine

SOURCE: Boyes and Moser 1988

involve agonist-like properties at -adrenoceptors, Interestingly, the actions of

amitraz on dentate gyrus field potentials following perforant path stimulation, to

increase hippocampal excitability and reduce tonic inhibition, also are mimicked

by clonidine (Gilbert and Dyer 1988). Costa and coworkers have demonstrated

in a series of studies that metabolites of chlordimeform, as well as amitraz, bind

potently to -adrenoceptors in the mammalian central nervous system (CNS)

(Costa and Murphy 1987; Costa et al. 1988, 1989a,1989b). Thus, the VEP

studies in conjunction with other reports helped demonstrate a mechanism

of action of the formamidine insecticides in the mammalian CNS.

SEPs and Sensory Function

Answering the question “What do changes in evoked potentials mean?” also

involves the relationship between changes in SEPs and changes in sensory

function. When there are changes in evoked potentials, does that mean that

the ability to see, hear, or feel is altered, and if so, in what fashion? In general,

when there are changes in evoked potentials, changes in sensory capacities

are expected, but the fidelity of the relationship between evoked potential

changes and sensation depends on a number of factors.

75
Perhaps the most important of these factors is the sensory stimuli used to study

sensory function and elicit evoked potentials. One basic concept in sensory

physiology is that within sensory modalities there exist parallel processing

channels, sometimes involving anatomically distinct cells or regions of the brain

(Campbell and Robson 1968; Sachs et al. 1971; Campbell 1980; Lennie 1980;

Regan 1982; Stone and Dreher 1982; Livingstone and Hubel 1988; Lennie et al.

1990). Each of these channels is responsible for encoding distinct stimulus

parameters, such as visual color or motion, sensation of vibration or pain, or

hearing high as opposed to low pitch. Thus, it is reasonable to expect that

selective deficits within a sensory modality can be produced by exposure to

neurotoxic compounds.

If evoked potentials are to be compared with other measures of sensory function,

it is important to use the same stimulus conditions. Most commonly, sensory

function is examined using behavioral procedures referred to as sensory

psychophysics (Evans 1982; Moody and Stebbins 1982; Maurissen 1988).

When evoked potentials and sensory psychophysics have been studied in the

same subjects and using the same sensory stimuli, the two procedures have

shown remarkably similar results. Campbell and Maffei (1970) and Campbell

and Kulikowski (1972) measured VEPs to stimulus patterns for which the

stimulus contrast (the difference in luminance between the bright and dark parts

of the pattern, adjusted for the mean luminance) varied. They showed that

the amplitude of the evoked potentials was a linear function of log contrast, and

when the perceptual contrast thresholds were measured, they corresponded

very closely with the contrast levels that failed to elicit evoked potentials.

Although there are some exceptions (Regan 1989), the general conclusion of

studies carefully comparing evoked potentials and sensory perception is that

there is a close correspondence between the measures (Campbell and Maffai

1970; Campbell and Kulikowski 1972; Bodis-Wollner et al. 1972: Cannon

1983; Bobak et al. 1984, 1988). In laboratory animal studies, and in some

human cases, SEP measures can be more readily performed than sensory

psychophysics, because in part of the extensive time required to train animals

to perform stimulus discrimination tasks. In addition, in human studies the need

to communicate with and elicit the cooperation of subjects can be problematic

in certain situations. For these reasons, SEPs have become an accepted tool

for both the experimental study of sensory psychophysics and clinical tests of

sensory function. An indication of the wide applicability and acceptance of

evoked potential measures of sensory function is that a variety of measures

of visual resolution, including visual acuity and visual contrast sensitivity, have

been estimated using evoked potentials in many species, including goldfish

(Regan et al. 1975) rats (Meyer and Salinsky 1977; Onofrj et al. 1982a; Boyes

and Dyer 1983; Fox 1984; Harnois et al. 1984; Silveira et al. 1987), opossum

(Silveira et al. 1982; Picanco-Diniz et al. 1983), rabbits (Kulikowski 1978; Pak

76
1984) ground squirrels (Jacobs et al. 1980), cats (Berkley and Watkins 1971,

1973; Campbell et al. 1973; Freeman and Marg 1975; Harris 1978), dogs

(Odom et al. 1983), and nonhuman primates (Padmos et al. 1973; Previc 1986;

Bonds et al. 1987). In humans, infants (Pirchio et al. 1978; Harris et al. 1976;

Spekreijse 1978; Atkinson et al. 1979; Fiorentini et al. 1980; Sokol et al. 1983),

healthy adults (Campbell and Maffei 1970; Campbell and Kulikowski 1972), and

clinical patients (Steele et al. 1989) also have been studied.

The close correspondence between evoked potential and psychophysical

results means that evoked potential measurements can be used to provide an

indication of the nature of the deficits in sensory perception. For example,

electrophysiologically determined audiograms have been used to study the

toxicity of adult exposure to trimethyltin (Crofton et al. 1990; Fechter and

Carlisle 1990) and developmental exposure to monosodium glutamate

(Janssen et al. 1991).

Another example involves a return to the study of chlordimeform discussed

previously, in which chlordimeform increased the amplitude of PREPS, but not

FEPs (Boyes and Dyer 1984) through agonist-like actions at -adrenoceptors

(Boyes and Moser 1988). However, this mechanism of action tells us nothing

regarding the nature of the visual changes following chlordimeform exposure

or why PREPS were affected but not FEPs. To help answer these questions,

Boyes and colleagues (1985c) investigated a number of stimulus manipulations

in conjunction with treatment with chlordimeform, including stimulus luminance,

pattern size, orientation, and contrast. The effects of chlordimeform were

clearly dependent on stimulus contrast, appearing only for high-contrast stimuli.

This explained the lack of effects on FEP amplitudes, because the FEP-eliciting

stimuli, bright flashes, were devoid of contrast. This result implies that

adrenergic receptors are involved somehow in regulating the neural encoding

of stimulus contrast. Saying what this means for visual perception is still a

difficult proposition, but one would expect differences in the perception of

high-contrast patterns.

The issue of evoked potential studies of sensory function should not be left

without noting that evoked potentials provide information regarding behavioral

states or cognitive processes, in addition to sensory information. For example,

a negative wave in the FEP of rats, occurring at about 160 ms (N160), is

influenced by a number of factors that are independent of the sensory stimulus

per se, such as the number of days tested (Dyer 1989; Herr et al. 1991a).

This peak, which is actually the first portion of a rhythmic flash evoked after

discharge, appears to reflect behavioral sensitization and habituation-like

processes. These extrasensory correlates of evoked potentials also can be

exploited profitably in neurotoxicity studies, such as those showing (N160 to

77
be reduced in amplitude following exposure to solvents, including toluene and

xylene (Dyer et al. 1988), carbon disulfide (Herr et al. 1992a), and others

(Herr et al. 1991b, 1992b).

ANIMAL-TO-HUMAN EXTRAPOLATION

The procedures for recording SEPs from laboratory animals and from human

subjects can be superficially quite similar (figure 1). The stimuli may be either

identical or scaled for the relative sensory capacities of the species being

studied. The electrodes, although affixed differently, should record electrical

activity from analogous portions of the sensory systems, to the extent that

the systems of different species have analogous components. Creel and

colleagues (1974) and Mattsson and coworkers (1989a) have presented

evoked potentials that are generally similar in a number of species. The

extrapolation of toxicological data from one species to another is a major

source of uncertainty in assessing exposure risks (National Research Council

1983). Therefore, if SEPs are to be recorded from laboratory animals and

used to make decisions regarding the potential for human neurotoxicity, then

a systematic comparison of the evoked potential responses among species

is warranted.

A strategy for comparing the responses among different species is needed.

Perhaps the strongest comparison would be to show that a variety of neurotoxic

compounds produced corresponding changes in laboratory animals and humans

and with similar dose-response relationships. Making this comparison is not

possible because of the ethical problems in deliberately delivering known

neurotoxic compounds to human subjects. Occasionally, human cases of

inadvertent overexposure to neurotoxic compounds are discovered, but the

sporadic nature of the occurrence of the cases, the unknown dose levels, and

the presence of confounding variables make systematic comparisons with

animal studies impractical, if not impossible. A more available approach

consists of parametric stimulus manipulations, drug treatments, and studying

cases of human neurodegenerative diseases.

The first part of the strategy involves recording evoked potentials from laboratory

animals and healthy human volunteers and comparing changes in responses to

manipulations of stimulus variables such as pattern size and rate of stimulus

presentation (Hudnell et al. 1990a; Hudnell and Boyes 1991). The rationale for

these comparisons is that if the sensory systems of the different species operate

similarly (i.e., if they show comparable changes in response to the stimulus

manipulations), then it follows that they may respond similarly to toxic compounds

that reach the target tissue.

78
In parametric comparisons of human and rat VEPs, the stimuli used were light

and dark vertical bars in which the luminance varied in a sinusoidal pattern

across the face of the stimulus screen. These so called sine-wave gratings are

commonly used in visual research, and the pattern size, that is, the width of the

bars, is expressed as spatial frequency or the number of cycles of the stimulus

pattern per degree of visual angle. Spatial frequency is to be distinguished from

temporal frequency, which refers to the rate at which the stimulus is modulated

over time, expressed in Hz. When the temporal rate is less than approximately

3 Hz, the individual “transient” evoked potentials are visible, and data analysis

typically involves measuring the amplitude and latency of individual components

in the response. With faster stimulus rates, the response takes on “steady-state,”

sinusoidal appearance in which the primary temporal frequencies of the response

are driven by the stimulus rate. Steady-state responses are typically analyzed

using Fourier-transformation-based spectral analyses, and data are expressed

as phase and either spectral power or spectral amplitude of response

components at various frequencies, Of particular interest is the response

amplitude at the stimulus rate and harmonics. The temporal modulation can

take several different forms, including pattern reversal, in which the light and

dark portions of the pattern alternate positions, and pattern appearance/

disappearance, in which the stimulus pattern alternates with a nonpatterned,

blank screen of the same mean luminance as the stimulus pattern. Note that

in either pattern reversal or pattern appearance/disappearance, there is no

overall change in stimulus luminance, so the recorded potentials reflect

aspects of the neural encoding of the change in stimulus pattern.

Pattern-evoked potentials recorded using either transient or steady-state

temporal modulation have a number of similar properties in humans and rats.

For example, the transient pattern-evoked potential peak labeled N110 in

figure 1 showed a selective reduction of amplitude after pattern adaptation in

both species (Hudnell et al. 1990a, 1990b). The spectral amplitudes of steadystate pattern appearance/disappearance VEPs recorded under comparable

conditions from rats and humans are presented in figure 7. The pattern sizes

were smaller in humans (higher stimulus spatial frequency), which reflects the

ability of humans to resolve finer visual details than rats, but otherwise the

stimuli were similar. The responses were recorded from extradural skull

electrodes chronically implanted in rats, but necessarily from the scalp of

humans. The closer proximity of the recording electrodes to the cortical tissue

generating the potentials accounts for the higher voltage responses recorded

from rats than from humans. In response to manipulations of pattern size

(spatial frequency), the 1F component (the response activity occurring at the

stimulus rate) in both rats and humans showed a “bandpass” spatial frequency

profile in which amplitudes were greatest at intermediate spatial frequencies. In

both species, the peak and overall shape of the 1F amplitude curves resembled

79
FIGURE 7. Rat and human spectral amplitudes following on-off pattern

modulation at the stimulus frequency (1F) and first harmonic (2F),

as a function of pattern size (spatial frequency) and stimulation

fate (temporal frequency [TF])

SOURCE: Hudnell and Boyes 1991

the peak and shape of the respective contrast sensitivity function curves of the

two species (Campbell and Robson 1968; Birch and Jacobs 1979). However,

the 1F curves of the human and rat subjects did appear to differ in response to

temporal frequency manipulations. The response profiles of the two species

were rather similar at low temporal frequencies, but differed as the stimulus

rate increased, with humans better able to follow high stimulus rates. In

contrast, the 2F curves of both species were similar in both spatial and temporal

domains. However, the 2F spatial profiles differed from the 1F profiles in that the

responses tended to be largest at low spatial frequencies. Also, in contrast to the

1F data that differed in temporal response across species, as temporal frequency

increased, the 2F responses of both rats and humans declined at a similar rate.

Thus, stimulus manipulations revealed both similarities and differences between

the rat and human response profiles. It follows that some aspects of human

spatial vision, as reflected in the pattern VEPs, may be adequately modeled in

laboratory rats to be predictive of human neurotoxic susceptibilities, but others,

such as the response to high temporal stimulation rates, may not be predictive.

80
In the second phase of this strategy, the responses to neuroactive drugs of

laboratory animals and human volunteers are compared. If the responses to

drugs are similar, then perhaps the responses to other, less benign, exogenous

compounds also will be similar. Human volunteers and rats were given

diazepam, an agonist at the benzodiazepine-binding site on the gammaaminobutyric acid (GABA)-receptor complex, and pattern appearance/

disappearance VEPs were recorded. Both species showed an amplitude

reduction of 2F at intermediate spatial frequencies but no significant changes

in 1F amplitude (Hudnell and Boyes 1991). These results suggest a similar

role for GABA-ergic modulation of the pattern-elicited responses in the rat and

human visual systems and imply that other compounds altering the homeostasis

in this system may act similarly in the two species. Although the larger

implications of findings such as these to other neurotoxicants acting on other

neurotransmitter systems or at nontransmitter sites are unknown, the more that

two systems in different species can be shown to operate on similar principles,

the more likely it is that neurotoxicants acting on unknown mechanisms would

produce similar results in the two species.

The third part of the experimental strategy, still under way, involves comparing

the responses to known neurodegenerative diseases in humans with animal

models of the human conditions. If a disease such as generalized epilepsy

results in SEP changes (Ratliff and Zemon 1984) and similar effects can

be quantified in animal models of epilepsy, then greater confidence is warranted

that degenerative conditions caused by neurotoxicants also may be expressed

similarly in evoked potentials of laboratory animals and humans,

The research program on recording human and rat evoked potential data using

comparable procedures has also led to cross-species mapping, which involves

the development of a mathematical equation to predict the value of a dependent

variable in one species from data derived in another species (Benignus et al.

1991). This process was applied to some of the rat and human VEP data

presented in figure 7, namely the 1F amplitude values across spatial frequency,

recorded at a temporal frequency of 3 Hz. First, a mathematical function relating

amplitude to spatial frequency was fitted to the data from each species, and

95-percent confidence limits were calculated (figure 8). Next an equation was

derived, the cross-species map, that describes the relationship between the

functions for each species. In this case, the cross-species map was a linear

function (figure 9), actually two linear functions, reflecting the prediction of the

rising and falling portions of the human curve from the corresponding and

noncorresponding limbs of the rat function. The fact that both curves are

linear is a consequence of fitting both rat and human raw data using a single,

symmetrical function, differing only in the parameters of fit. Perhaps functions

81
FIGURE 8. Tuning curves and 95-percent confidence bounds fit to the rat

and human data at a 3-HZ stimulation rate from figure 7.

SOURCE: Benignus et al. 1991

such as these will someday be useful in attempting to predict potential human

neurotoxicity from laboratory animal data.

In summary, these approaches to cross-species comparisons give a measure

of confidence that most neurotoxic changes found in rats will be predictive of

similar changes in humans. It would be prudent to expect that sole reliance

on a species such as the rat will open the possibility of missing some relevant

sensory toxicity because rats do not possess all the sensory capacity, such as

color vision, present in humans. The rate of false negatives, perhaps the most

important category of errors in toxicity testing and risk assessment, cannot be

assessed from these types of investigations. In addition, these comparisons

do not take into account factors such as metabolic differences, which may

produce cross-species differences in toxicity, but focus only on comparability

of the measured endpoints. However, for both economic and ethical reasons,

rodents likely will be the mainstay of routine toxicity testing for some time.

These types of cross-species comparisons do address the relevance of the rat

data for estimating human risk. Our results to date suggest a good degree of

comparability between pattern-evoked potentials in rats and humans, suggesting

that neurotoxicity data collected in rats using these procedures may be predictive,

82
FIGURE 9. Equations for mapping rat spatial frequencies as equivalent

human spatial frequencies

SOURCE: Benignus et al. 1991

with some exceptions, of similar changes in humans. In other words, the falsepositive rate may be relatively low. The comparability of evoked potential

measures across species in combination with the good relationship between

evoked potential changes and changes in sensory function, discussed above,

lead to the conclusion that changes in SEPs produced in laboratory animals

following exposure to potential neurotoxic compounds may be predictive of

neurotoxicant-induced deficits in sensory function in humans.

CONCLUSIONS

There is a need to incorporate tests of sensory function in laboratory animal

toxicity testing. It is clear that sensory dysfunction may frequently occur, but go

undetected, in standard animal toxicological testing protocols. SEP technology

can be employed to address this need, can provide information regarding

perceptual deficits arising from toxic insults, and can yield information regarding

possible mechanisms of action. In addition, SEP changes in laboratory animals

can be predictive of sensory changes in humans. Further research is needed

to better define the neural substrates of evoked potentials and their perceptual

correlates so that the implications of evoked potential changes for the cellular

83
targets, mechanisms of actions, and perceptual correlates of evoked potential

changes can be better understood. More extensive use of these tools in animal

toxicology is needed to better understand the ability of this methodology to

detect sensory changes and to predict human toxicity from animal data.

REFERENCES

Abbruzzese, G.; Reni, L.; Cocito, L.; Ratto, S.; Abbruzzese, M.; and Favale, E.

Short-latency somatosensory evoked potentials in degenerative and vascular

dementia. J Neurol Neurosurg Psychiatry 47:1034-1037, 1984.

Anderson, N.E.; Frith, R.W.; and Synek, V.M. Somatosensory evoked potentials

in syringomyelia. J Neurol Neurosurg Psychiatry 49:1407-1410, 1986.

Apkarian, P., and Spekreijse, H. The VEP and misrouted pathways in human

albinism. In: Cracco, R.Q., and Bodis-Wollner, I., eds. Evoked Potentials.

New York: Alan R. Liss, 1986. pp. 211-226.

Arezzo, J.C.; Schaumburg, H.H.; Vaughan, H.G.; Spencer, S.P.; and Barna,

J. Hind limb somatosensory evoked potentials in the monkey: The effects of

distal axonopathy. Ann Neurol 12:24-32, 1981,

Arezzo, J.C.; Simson, R.; and Brennan, N.E. Evoked potentials in the

assessment of neurotoxicology in humans, Neurobehav Toxicol Teratol

74:299-304, 1985.

Atkin, A.; Bodis-Wollner, I.; Podos, SM.; Wolkstein, M.; Mylin, L.; and Nitzberg,

S. Flicker threshold and pattern VEP latency in ocular hypertension and

glaucoma. Invest Ophthalmol Vis Sci 24:1524-1528, 1983.

Atkinson, J.; Braddick, O.; and French, J. Contrast sensitivity of the human

neonate measured by the visual evoked potential. Invest Ophthalmol Vis Sci

18:210-213, 1979.

Aziz, S.A., and Knowles, C. Inhibition of monoamine oxidase by the pesticide

chlordimeform and related compounds. Nature 242:417-418, 1973.

Barajas, J.J. Evaluation of ipsilateral and contralateral brainstem auditory

evoked potentials in multiple sclerosis patients. J Neurol Sci 54:69-78, 1982.

Beeman, R.W., and Matsumura, F. Chlordimeform: A pesticide acting upon

amine regulatory mechanisms, Nature 242:273-274, 1973.

Benignus, V.A.; Boyes, W.K.; and Hudnell, H.K. Quantitative methods in

animal-to-human extrapolations, Neurosci Biobehav Rev 15:165-171, 1991.

Berkley, M.A., and Watkins, D.W. Visual acuity of the cat estimated from

evoked cerebral potentials. Nature 234:91-92, 1971.

Berkley, M.A., and Watkins, D.W. Grating resolution and refraction in the cat

estimated from evoked cerebral potentials. Vision Res 13:403-415, 1973.

Bigum, H.B.; Dustman R.E.; and Beck, E.C. Visual and somato-sensory evoked

responses from mongoloid and normal children. Electroencephalogr Clin

Neurophysiol 28:576-585, 1970.

84
Birch, D., and Jacobs, G.H. Spatial contrast sensitivity in albino and pigmented

rats. Vision Res 19:933-937, 1979.

Bobak, P.; Bodis-Wollner, I.; Harnois, C.; and Thornton, J. VEPs in humans

reveal high and low spatial contrast mechanisms. Invest Ophthal Vis Sci

25:980-983, 1984.

Bobak, P.; Bodis-Wollner, I.; and Marx, M.S, Cortical contrast gain control in

human spatial vision. J Physiol 405:421-437, 1988.

Bodis-Wollner, I. Vulnerability of spatial frequency channels in cerebral lesions.

Nature 261:309-311, 1976.

Bodis-Wollner, I. Electrophysiological and psychophysical testing of vision in

glaucoma. Surv Ophthalmol 33:301-307, 1989.

Bodis-Wollner, I.; Hendley, CD.; and Kulikowski, J.J. Electrophysiological and

psychophysical responses to modulation of contrast of a grating pattern.

Perception 1:341-349, 1972.

Bodis-Wollner, I.; Hendley, C.D.; Mylin, L.H.; and Thornton, J. Visual evoked

potentials and the visuogram in multiple sclerosis. Ann Neurol 5:40-47, 1979.

Bodis-Wollner, I.; Onofrj, M.; Marx, M.S.; and Mylin, L.H. Visual evoked

potentials in Parkinson’s disease: Spatial frequency, temporal rate, contrast,

and the effects of dopaminergic drugs, In: Cracco, R.Q., and Bodis-Wollner,

I., eds. Evoked Potentials. New York: Alan R. Liss, 1986. pp. 307-319.

Bodis-Wollner, I.; Yahr, M.D.; Mylin, L.; and Thornton, J. Dopaminergic

deficiency and delayed visual evoked potentials in humans. Ann Neurol

11:478-490, 1981.

Bonds, A.B.; Casagrande, V.A.; Norton, T.T.; and DeBruyn, E.J. Visual

resolution and sensitivity in a nocturnal primate (Galago) measured with

visual evoked potentials. Vision Res 27:845-857, 1987.

Bosch, E.P.; Yamada, T.; and Kimura, J. Somatosensory evoked potentials in

motor neuron disease. Muscle Nerve 8:556-562, 1985.

Bottcher, J., and Trojaborg, W. Follow-up of patients with suspected multiple

sclerosis: A clinical and electrophysiological study. J Neurol Neurosurg

Psychiatry 45:809-814, 1982.

Boyes, W.K. "Proposed Test Guidelines for Using Sensory Evoked Potentials

as Measures of Neurotoxicity.” Office of Research and Development, U.S.

Environmental Protection Agency. Deliverable 2555. Document number

600x-90166. 1990.

Boyes, W.K. Testing visual system toxicity using evoked potential technology.

In: Isaacson, R.L., and Jensen, K.F., eds. The Vulnerable Brain and

Environmental Risks: Volume 1. Malnutrition and Hazard Assessment. New

York: Plenum, 1992. pp. 193-222.

Boyes, W.K., and Cooper, G.P. Acrylamide neurotoxicity: Effects on far-field

somatosensory evoked potentials in rats. Neurobehav Toxicol Teratol 3:487-

490, 1981.

85
Boyes, W.K., and Dyer, R.S. Pattern reversal visual evoked potentials in awake

rats. Brain Res Bull 10:817-823, 1983.

Boyes, W.K., and Dyer, R.S. Chlordimeform produces profound, selective and

transient changes in visual evoked potentials of hooded rats. Exp Neurol

86:434-447, 1984.

Boyes, W.K.; Jenkins, D.E.; and Dyer, R.S. Chlordimeform produces contrastdependent changes in visual evoked potentials of hooded rats. Exp Neurol

89:434-449, 1985c.

Boyes, W.K., and Moser, V.C. Investigations of amitraz neurotoxicity in rats. II.

Effects on visual evoked potentials, Fund Appl Toxicol 9:140-153, 1987.

Boyes, W.K., and Moser, V.C. An -adrenergic mode of action of

chlordimeform on rat visual function. Toxicol Appl Pharmacol 92:402-418,

1988.

Boyes, W.K.; Moser, V.C.; MacPhail, R.C.; and Dyer, R.S. Monoamine oxidase

inhibition cannot account for visual evoked potential changes produced by

chlordimeform. Neuropharmacology 24:853-860, 1985b.

Boyes, W.K.; Padilla, S.; and Dyer, R.S. Body temperature-dependent and

independent actions of chlordimeform on rat visual evoked potentials and

optic nerve axonal transport. Neuropharmacology 24:743-749, 1985a.

Brown, W.F., and Feasby, T.E. Sensory evoked potentials in Guillain-Barre

polyneuropathy. J Neurol Neurosurg Psychiatry 47:288-291, 1984.

Bushnell, P.J.; Bowman, R.E.; Allen, J.R.; and Marlar, R.J. Scotopic vision

deficits in young monkeys exposed to lead. Science 196:333-335, 1977.

Campbell, F.W. The physics of visual perception. Philos Trans R Soc Lond Biol

290:5-9, 1980.

Campbell, F.W., and Kulikowski, J.J. The visual evoked potential as a function

of contrast of a grating. J Physiol 222:345-356, 1972.

Campbell, F.W., and Maffei, L. Electrophysiological evidence for the existence

of orientation and size detectors in the human visual system. J Physiol

207:635-652, 1970.

Campbell, F.W.; Maffei, L.; and Puccolino, M. The contrast sensitivity of the cat.

J Physiol 229:719-731, 1973.

Campbell, F., and Robson, J. Application of Fourier analysis to the visibility of

gratings. J Physiol 197:551-566, 1968.

Cannon, M.W. Contrast sensitivity: Psychophysical and evoked potential

methods compared. Vision Res 23:87-95, 1983.

Cant, B.R.; Hume, A.L.; and Shaw, N.A. Effects of luminance on the pattern

visual evoked potential in multiple sclerosis. Electroencephalogr Clin

Neurophysiol 45:496-504, 1978.

Celesia, G.G., and Daly, R.F. Effects of aging on visual evoked responses.

Arch Neurol 34:403-405, 1977.

Chang, L.W., and Dyer, R.S. Trimethyltin induced pathology in sensory

neurons. Neurobehav Toxicol Teratol 5:337-350. 1983.

86
Chinn, C.; Lund, A,E.; and Yim, G.K.W. The central actions of lidocaine and a

pesticide, chlordimeform. Neuropharmacology 16:867-871, 1977.

Chu, N.-S. Median and tibial somatosensory evoked potentials: Changes in

short and long-latency components in patients with lesions of the thalamus

and thalamo-cortical radiations. J Neurol Sci 76:199-219, 1986.

Church, M.W. Chronic in utero alcohol exposure affects auditory function in rats

and in humans. Alcohol 4:231-239, 1987.

Church, M.W,, and Overbeck, G.W. Prenatal cocaine exposure in the LongEvans rat. III. Developmental effects on the brainstem auditory-evoked

potential. Neurotoxicol Teratol 12:345-351, 1990.

Coben, L.A.; Danziger, W.L.; and Hughes, C.P. Visual evoked potentials in mild

senile dementia of Alzheimer type. Nectroencephalogr Clin Neurophysiol

55:121-130, 1983.

Cosi, V.; Poloni, M.; Mazzini, L.; and Callieco, R. Somatosensory evoked

potentials in amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry

47:857-861, 1984.

Costa, L.G., and Murphy, SD. Interaction of the pesticide chlordimeform with

adrenergic receptors in mouse brain: An in vitro study. Arch Toxicol 59:323-

327,1987.

Costa, L.G.; Olibet, G.; and Murphy, S.D. Alpha2-adrenoceptors as a target for

formamidine pesticides: In vitro and in vivo studies in mice. Toxicol Appl

Pharmacol 93:319-328, 1988.

Costa, L.G.; Olibet, G.; Wu, D.S.; and Murphy, SD. Acute and chronic effects

of the pesticide amitraz on alpha2

-adrenoceptors in the mouse brain. Toxicol

Lett 47:135-143, 1989a.

Costa, L.G.; Wu, D.S.; Olibet, G.; and Murphy, S.D. Formamidine pesticides

and alpha2

-adrenoceptors: Studies with amitraz and chlordimeform in rats

and development of a radioreceptor binding assay. Neurotoxicol Teratol

11:405-411, 1989b.

Cracco, R.Q., and Bodis-Wollner, I., eds. Evoked Potentials. New York: Alan R.

Liss, 1986.

Creel, D.; Dustman, R.E.; and Beck, E.C. Intensity of flash illumination and the

visually evoked potential of rats, guinea pigs and cats. Vision Res 14:725-

729,1974.

Creel, D.; Spekreijse, H.; and Reits, D. Evoked potentials in albinos: Efficacy

of pattern stimuli in detecting misrouted optic fibers. Nectroencephalogr Clin

Neurophysiol 52:595-603, 1981.

Crofton, K.M., and Sheets, L.P. Evaluation of sensory system function using

reflex modification of the startle response. J Am Coll Toxicol 8:199-211, 1989.

Crofton, K.M.; Dean, K.F.; Menache, M.G.; and Janssen, R. Trimethyltin effects

on auditory function and cochlear morphology. Toxicol Appl Pharmacol

105:123-132, 1990.

87
Dasheiff, R.M.; Drake, M.E.; Brendle, A.; and Erwin, C.W. Abnormal

somatosensory evoked potentials in amyotrophic lateral sclerosis.

Electroencephalogr Clin Neurophysiol 60:306-311, 1985.

de la Torre, J.C. Evaluation of brain death using somatosensory evoked

potentials. Biol Psychiatry 16:931-935, 1981.

de la Torre, J.C.; Trimble, J.L.; Beard, R.T.; Hanlon, K.; and Surgeon, J.W.

Somatosensory evoked potentials for the prognosis of coma in humans.

Exp Neurol 60:304-317, 1978.

Don, M. Value of ABR and cochlear potentials in differentiating cochlear from

auditory nerve pathology. In: Cracco, R.Q., and Bodis-Wollner, I., eds.

Evoked Potentials. New York: Alan R. Liss, 1986. pp. 366-372.

Dorfman, L.J.; Bosley, T.M.; and Cummins, K.L. Electrophysiological

localization of central somatosensory lesions in patients with multiple

sclerosis. Electroencephalogr Clin Neurophysiol 44:742-753, 1978.

Duffy, F.H. Topographic mapping of evoked potentials in learning-disabled

children. In: Cracco, R.Q., and Bodis-Wollner, I., eds. Evoked Potentials.

New York: Alan R. Liss, 1986. pp. 485-496.

Dyer, R.S. Physiological methods for assessment of trimethyltin exposure.

Neurobehav Toxicol Teratol 4:659-664, 1982.

Dyer, R.S. In vitro approaches to neurotoxicity-a physiologist’s perspective. In:

Kolber, R.; Wong, T.K.; Grant, L.D.; DeWoskin, R.S.; and Hughes, T.J., eds.

In Vitro Toxicity Testing of Environmental Agents, Part A. New York: Plenum,

1983. pp. 487-497.

Dyer, R.S. The use of sensory evoked potentials in toxicology. Fundam Appl

Toxicol 5:24-40, 1985.

Dyer, R.S. Interactions of behavior and neurophysiology. In: Annau, Z., ed.

Neurobehavioral Toxicology. Baltimore, MD: Johns Hopkins University Press,

1986. pp. 193-213.

Dyer, R.S. Macrophysiological assessment of organometal neurotoxicity. In:

Tilson, H.A., and Sparber, S.B., eds. Neurotoxicants and Neurobiological

Function: Effects of Organoheavy Metals. New York: Wiley, 1987a.

pp. 137-184.

Dyer, R.S. Somatosensory evoked potentials. In: Lowndes, H.E., ed.

Electrophysiology in Neurotoxicology, Vol. II. Boca Raton, FL: CRC Press,

1987b. pp. 1-69.

Dyer, R.S. Peak N160 of rat flash evoked potential: Does it reflect habituation

of sensitization? Physiol Behav 45:355-362, 1989.

Dyer, R.S.; Bercegeay, M.S.; and Mayo, L.M. Acute exposures to p-xylene and

toluene alter visual information processing. Neurotoxicol Teratol 10:147-153,

1988.

Dyer, R.S., and Boyes, W.K. Use of neurophysiological challenges for the

detection of toxicity. Fed Proc 42:3201-3206, 1983.

88
Dyer, R.S.; Howell, W.E.; and Wonderlin, W.F. Visual system dysfunction

following acute trimethyltin exposure in rats. Neurobehav Toxicol Teratol

4:191-195, 1982.

Dyer, R.S.; Muller, K.E.; Janssen, R.J.; Barton, C.N.; Boyes, W.K.; and

Benignus, V.A. Neurophysiological effects of 30 day chronic exposure to

toluene in rats. Neurobehav Toxicol Teratol 6:363-368, 1984.

Eells, J.T. Methanol-induced visual toxicity in the rat. J Pharmacol Exp Ther

257:56-63, 1991.

Evans, H.L. Assessment of vision in behavioral toxicology. In: Mitchell, C.L., ed.

Nervous System Toxicology. New York: Raven Press, 1982. pp. 81-107.

Evans, P.D., and Gee, J.D. Action of formamidine pesticides on octopamine

receptors. Nature 287:60-62, 1980.

Facto, E.; Caputo, P.; Liviero, M.C.; Munari, M.; Toffoletto, F.; Fabiani, F.; and

Giron, G. Auditory and somatosensory evoked potentials in brain-dead

patients. Riv Neurol 58:140-145, 1988.

Fechter, L.D., and Carlisle, L. Auditory dysfunction and cochlear vascular injury

following trimethyltin exposure in the guinea pig. Toxicol Appl Pharmacol

105:133-143, 1990.

Fiorentini, A.; Pirchio, M.; and Spinelli, D. Scotopic contrast sensitivity in infants

evaluated by evoked potentials, Invest Ophthalmol Vis Sci 19:950-955, 1980.

Fox, D.A. Psychophysiologically and electrophysiologically determined spatial

deficits in developmentally lead-exposed rats have a cholinergic component.

In: Narahashi, T., ed. Cellular and Molecular Neurotoxicology. New York:

Raven Press, 1984. pp. 123-140.

Fox, D.A., and Chu, L.W.F. Rods are selectively altered by lead. II. Ultrastructure

and quantitative histology. Exp Eye Res 46:613-625, 1988.

Fox, D.A., and Farber, D.B. Rods are selectively altered by lead. I.

Electrophysiology and biochemistry. Exp Eye Res 46:597-611, 1988.

Fox, D.A., and Sillman, A.J. Heavy metals affect rod, but not cone,

photoreceptors. Science 206:78-80, 1979.

Fox, D.A.; Lewkowski, J.P.; and Cooper, G.P. Acute and chronic effects of

neonatal lead exposure on the development of the visual evoked response

in rats. Toxicol Appl Pharmacol 40:449-461, 1977.

Fox, D.A.; Lewkowski, J.P.; and Cooper, G.P. Persistent visual cortex

excitability alterations produced by neonatal lead exposure. Neurobehav

Toxicol 1:101-106, 1979.

Fox, D.A.; Lowndes, H.E.; and Bierkamper, G.G. Electrophysiological techniques

in neurotoxicology. In: Mitchell, C.L., ed. Nervous System Toxicology. New

York: Raven Press, 1982. pp. 299-335.

Freeman, D.N., and Marg, E. Visual acuity development coincides with the

sensitive period in kittens. Nature 254:614-615, 1975.

89
Garg, B.P.; Markland, O.N.; DeMyer, W.E.; and Warren, C. Evoked response

studies in patients with adrenoleukodystrophy and heterozygous relatives.

Arch Neurol 40:356-359, 1983.

Ghilardi, M.F.; Sartucci, F.; Brannan, J.R.; Onofrj, M.C.; Bodis-Wollner, I.; Mylin,

L.; and Stroch, R. N70 and P100 can be independently affected in multiple

sclerosis. Electroencephalogr Clin Neurophysiol 80:1-7, 1991.

Giblin, D.R. Somatosensory evoked potentials in healthy subjects and in patients

with lesions of the nervous system. Ann N Y Acad Sci 112:93-142, 1964.

Gilbert, M.E., and Dyer, R.S. Increased hippocampal excitability produced

by amitraz. Neurotoxicol Teratol 10:229-235, 1988.

Gilbert, M.E., and Mack, C.M. Enhanced susceptibility to kindling by

chlordimeform may be mediated by a local anesthetic action.

Psychopharmacology 99:163-167, 1989.

Giovannini, M.; Valsasina, R.; Villani, R.; Ducati, A.; Riva, E.; Landi, A.; and

Longhi, R. Pattern reversal visual evoked potentials in phenylketonuria.

J Inherited Metab Dis 11:416-421, 1988.

Goff, W.R.; Shaywitz, B.A.; Goff, G.D.; Reisenauer, M.A.; Jasiorkowski, J.G.;

Venes, J.L.; and Rothstein, P.T. Somatic evoked potential evaluation of

cerebral status in Reye syndrome. Electroencephalogr Clin Neurophysiol

55:388-398, 1983.

Goodin, D.S.; Squires, K.C.; and Starr, A. Long latency event-related components

of the auditory evoked potential in dementia. Brain 101:635-648, 1978.

Greenberg, R.P.; Mayer, D.J.; Becker, D.P.; and Miller, J.D. Evaluation of brain

function in severe human head trauma with multimodality evoked potentials.

J Neurosurg 47:150-177, 1977.

Halliday, A.M. Visual evoked potentials in demyelinating disease. In: Waxman,

S.G., and Ritchie, J.M., eds. Demyelinating Disease: Basic and Clinical

Electrophysiology. New York: Raven Press, 1981. pp. 201-215.

Halliday, A.M.; McDonald, W.J.; and Muskin, J. Delayed visual evoked response

in optic neuritis. Lancet 1:982-985, 1972.

Halliday, A.M.; McDonald, W.J.; and Muskin, J. Visual evoked response in

diagnosis of multiple sclerosis. Br Med J 4:661-664, 1973.

Halliday, R.; Callaway, E.; and Rosenthal, J.H. The visual ERP predicts clinical

response to methylphenidate in hyperactive children. Psychophysiology

21:114-121, 1984.

Hansch, E.C.; Syndulko, K.; Cohen, S.N.; Goldberg, Z.I.; Potvin, A.R.; and

Tourtellotte, W.W. Cognition in Parkinson disease: An event-related potential

perspective. Ann Neurol 11:599-607, 1982.

Harnois, C.; Bodis-Wollner, I.; and Onofrj, M. The effect of contrast and spatial

frequency on the visual evoked potential of the hooded rat. Exp Brain Res

57:1-8, 1984.

Harris, L.; Atkinson, J.; and Braddick, O. Visual contrast sensitivity of a 6-monthold infant measured by the evoked potential. Nature 264:570-571, 1976.

90
Harris, L.R. Contrast sensitivity and acuity of a conscious cat measured by the

occipital evoked potential. Vision Res 18:175-178, 1978.

Hecox, K.E., and Cone, B. Prognostic importance of brainstem auditory evoked

responses after asphyxia. Neurology 31:1429-1433, 1981.

Herr, D.W.; Bercegeay, M.S.; and Boyes, W.K. A comparison of the effects of

dichloromethane and 1,2-dichlorobenzene on peak N160 amplitude of flash

evoked potentials in rats. Toxicologist 11:390, 1991b.

Herr, D.W.; Bercegeay, M.S.; and Boyes, W.K. Alterations in flash evoked

potentials produced by 1,3-dichloropropane: Further evidence that lipid

soluability alone does not predict neuroactive potency. Toxicologist 12:277,

1992b.

Herr, D.W.; Boyes, W.K.; and Dyer, R.S. Rat flash-evoked potential peak N160

amplitude: Modulation by relative flash intensity. Physiol Behav 49:355-365,

1991a.

Herr, D.W.; Boyes, W.K.; and Dyer, R.S. Alterations in rat flash and pattern

reversal evoked potentials after acute or repeated administration of carbon

disulfide. Fundam Appl Toxicol 18:328-342, 1992a.

Hollingworth, R.M., and Lund, A.E. Biological and neurotoxic effects of amidine

pesticides, In: Coates, J.R., ed. Insecticide Mode of Action. New York:

Academic Press, 1982. pp. 189-227.

Hollingworth, R.M., and Murdock, L.L. Formamidine pesticides: Octopamine-like

actions in a firefly. Science 208:74-76, 1980.

Hrbek, A.; Karlberg, P.; Kjellmer, I.; Olsson, T.; and Riha, M. Clinical application

of evoked electroencephalographic responses in newborn infants. I. Perinatal

asphyxia. Dev Med Child Neurol 19:34-44, 1977.

Hsu, W.H., and Hopper, D.L. Effect of yohimbine on amitraz-induced CNS

depression and bradycardia in dogs. J Toxicol Environ Health 18:423-429,

1986.

Hsu, W.H., and Kakuk, T.J. Effects of amitraz and chlordimeform on heart rate

and pupil diameter in rats: Mediated by -adrenoreceptors. Toxicol Appl

Pharmacol 73:411-41 5, 1984.

Hsu, W.H., and Lu, Z.-X. Amitraz-induced delay of gastrointestinal transit in

mice: Mediated by -adrenergic receptors. Drug Dev Res 4:655-660, 1984.

Hsu, W.H.; Lu, Z.-X.; and Hembrough, F.B. Effect of amitraz on heart rate and

aortic blood pressure in conscious dogs: Influence of atropine, prazosin,

tolazoline, and yohimbine. Toxicol Appl Pharmacol 84:418-422, 1986.

Hsu, W.H., and McNeel, S.V. Amitraz-induced prolongation of gastrointestinal

transit and bradycardia in dogs and their antagonism by yohimbine:

Preliminary study. Drug Chem Toxicol 8:239-253, 1985.

Hudnell, H.K., and Boyes, W.K. The comparability of rat and human visualevoked potentials. Neurosci Biobehav Rev 15:159-164, 1991.

91
Hudnell, H.K.; Boyes, W.K.; and Otto, D.A. Stationary pattern adaptation and

the early components in human visual evoked potentials. Electroencephalogr

Clin Neurophysiol 77:190-198, 1990b.

Hudnell, H.K.; Boyes, W.K.; and Otto, D.A. Rat and human visual-evoked

potentials recorded under comparable conditions: A preliminary analysis to

address the issue of predicting human neurotoxic effects from rat data.

Neurotoxicol Teratol 12:391-398, 1990a.

Hume, A.L.; Cant, B.R.; and Shaw, N.A. Central somatosensory conduction

time in comatose patients. Ann Neurol 5:379-384, 1979.

Jabbari, B.; Vance, S.C.; Harper, M.G.; Salazar, A.M.: Smutok, M.A.; and Amin,

D. Clinical and radiological correlates of somatosensory evoked potentials in

the late phase of head injury: A study of 500 Vietnam veterans.

Electroencephalogr Clin Neurophysiol 67:289-297, 1987.

Jacobs, G.H.; Blakeslee, B.; McCourt, M.E.; and Tootell, R.B.H. Visual

sensitivity of ground squirrels to spatial and temporal luminance variations.

J Comp Physiol 136:291-299, 1980.

Janssen, R.; Schweitzer, L.; and Jensen, K.F. Glutamate neurotoxicity in the

developing rat cochlea: Physiological and morphological approaches. Brain

Res 552:255-264, 1991.

Jones, M.A. The value of evoked potentials in surgical monitoring. In: Cracco,

R.Q., and Bodis-Wollner, I., eds. Evoked Potentials. New York: Alan R. Liss,

1986. pp. 421-427.

Josiassen, R.C.; Shagass, C.; Mancall, E.L.; and Roemer, R.A. Somatosensory

evoked potentials in Huntington’s disease. Electroencephalogr Clin

Neurophysiol 54:483-493, 1982.

Karnaze, D.S.; Marshall, L.F.; McCarthy, C.S.; Klauber, M.R.; and Bickford,

R.G. Localizing and prognostic value of auditory evoked responses in coma

after closed head injury. Neurology 32:299-302, 1982.

Koralnik, I.J.; Beaumanoir, A.; Hausler, R.; Kohler, A.; Safran, A.B.; Delacoux,

R.; Vibert, D.; Mayer, E.; Burkhard, P.; Nahory, A.; Magistris, M.R.; Sanches,

J.; Myers, P.; Paccolat, F.; Quoex, F.; Gabriel, V.; Perrin, L.; Mermillod, B.;

Gauthier, G.; Waldvogel, F.A.; and Hirschel, B. A controlled study of early

neurologic abnormalities in men with asymptomatic human immunodeficiency

virus infection. N Engl J Med 323:864-870, 1990.

Kulikowski, J.J. Pattern and movement detection in man and rabbit: Separation

and comparison of occipital potentials. Vision Res 18:183-189, 1978.

Lee, E.W. A rat model for the study of methanol visual neurotoxicity.

Toxicologist 7:97, 1987.

Lennie, P. Parallel visual pathways: A review. Vision Res 20:561-594, 1980.

Lennie, P.; Trevarthen, C.; Van Essen, D.; and Wassle, H. Parallel processing

of visual information. In: Spillman, L., and Werner, J.S., eds. Visual

Perception: The Neurophysiological Foundations. San Diego, CA: Academic

Press, 1990. pp. 103-128.

92
Levi, D.M., and Manny, R.E. The VEP in the diagnostic evaluation of amblyopia.

In: Cracco, R.Q., and Bodis-Wollner, I., eds. Evoked Potentials. New York:

Alan R. Liss, 1986. pp. 437-446.

Livingstone, M., and Hubel, D. Segregation of form, color movement and depth:

Anatomy, physiology, and perception. Science 240:740-749, 1988.

Lund, A.E.; Shankland, D.L.; Chinn, C.; and Yim, G.K.W. Similar cardiovascular

toxicity of the pesticide chlordimeform and lidocaine. Toxicol Appl Pharmacol

44:357-365, 1978.

Mahapatra, A.K., and Bhatia, R. Predictive value of visual evoked potentials in

unilateral optic nerve injury. Surg Neurol 31:339-342, 1989.

Markand, O.N.; Garg, B.P.; DeMyer, W.E.; Warren, C.; and Worth, R.M.

Brain stem auditory, visual and somatosensory evoked potentials in

leukodystrophies. Electroencephalogr Clin Neurophysiol 54:39-48, 1982.

Matthews, W.B., and Small, M. Prolonged follow-up of abnormal visual evoked

potentials in multiple sclerosis: Evidence for delayed recovery. J Neurol

Neurosurg Psychiatry 46:639-642, 1983.

Matthews, W.B., and Wattam-Bell, J.R.B. Evoked potentials in the diagnosis of

multiple sclerosis: A follow up study. J Neurol Neurosurg Psychiatry 45:303-

307, 1982.

Mattsson, J.L., and Albee, R.R. Sensory evoked potentials in neurotoxicology.

Neurotoxicol Teratol 10:435-443, 1988.

Mattsson, J.L.; Albee, R.R.; and Eisenbrandt, D.L. Neurological approach to

neurotoxicological evaluation in laboratory animals. J Am Col Toxicol 8:271-

286, 1989a.

Mattsson, J.L.; Albee, R.R.; and Gorzinski, S.J. Similarities of toluene and

o-cresol neuroexcitation in rats. Neurotoxicol Teratol 111:71-75, 1989b.

Mattsson, J.L.; Boyes, W.K.; and Ross, J.F. Incorporating evoked potentials into

neurotoxicity test schemes. In: Tilson, H.A., and Mitchell, CL., eds. Target

Organ Toxicology Series: Neurotoxicology. New York: Raven Press, 1992.

pp. 125-145.

Mattsson, J.L.; Eisenbrandt, D.L.; and Albee, R.R. Screening for neurotoxicity:

Complementarity of functional and morphological techniques. Toxicol Pathol

18:115-127, 1990a.

Mattsson, J.L.; Gorzinski, S.J.; Albee, R.R.; and Zimmer, M.A. Evoked potential

changes from 13 weeks of simulated toluene abuse in rats. Pharmacol

Biochem Behav 36:683-689, 1990b.

Maurissen, J.P.J. Quantitative sensory assessment in toxicology and

occupational medicine: Applications, theory, and critical practice. Toxicol Lett

43:321-343, 1988.

McSherry, J.W.; Walters, CL.; and Horbar, J.D. Acute visual evoked potential

changes in hydrocephalus. Nectroencephalogr Clin Neurophysiol 53:331-

333, 1982.

93
Meyer, G.E., and Salinsky, M.C. Refraction of the rat: Estimation by pattern

evoked visual cortical potentials. Vision Res 17:883-885, 1977.

Michalewski, H.J.; Rosenberg, C.; and Starr, A. Event-related potentials in

dementia. in: Cracco, R.Q., and Bodis-Wollner, I., eds. Evoked Potentials.

New York: Alan R. Liss, 1986. pp. 521-528.

Mintz, M.; Tomer, R.; Radwan, H.; and Myslobodsky, MS. Visual evoked

potentials in hemiparkinsonism. Electroencephalogr Clin Neurophysiol

52:611-616, 1981.

Mizrahi, E.M., and Crawford, E.S. Somatosensory evoked potentials during

reversible spinal cord ischemia in man. Electroencephalogr Clin Neurophysiol

58:120-126, 1984.

Moody, D.D., and Stebbins, W.C. Detection of the effects of toxic substances

on the auditory system by behavioral methods. In: Mitchell, CL., ed. Nervous

System Toxicology. New York: Raven Press, 1982. pp. 109-131.

Morrison, J.D., and Reilly, J. The pattern visual evoked cortical response in

human ageing. Q J Exp Physiol 74:311-328, 1989.

Moser, V.C., and MacPhail, R.C. Yohimbine attenuates the delayed lethality

induced in mice by amitraz, a formamidine pesticide. Toxicol Lett 28:99-104,

1985.

Mukuno, K.; Ishikawa, S.; and Okamura, R. Grating test of contrast sensitivity in

patients with Minamata disease. Br J Ophthalmol 65:284-290, 1984.

Nakanishi, T.; Shimada, Y.; Sakuta, M.; and Toyokura, Y. The initial positive

component of the scalp-recorded somatosensory evoked potential in normal

subjects and in patients with neurological disorders. Nectroencephalogr Clin

Neurophysiol 45:26-34, 1978.

National Research Council. Risk Assessment in the Federal Government:

Managing the Process. Washington, DC: National Academy Press, 1983.

Noel, P., and Desmedt, J.E. Somatosensory cerebral evoked potentials after

vascular lesions of the brain-stem and diencephalon. Brain 98:113-128, 1975.

Noth, J.; Engel, L.; Friedemann, H.-H.; and Lange, H.W. Evoked potentials in

patients with Huntington’s disease and their offspring. I. Somatosensory

evoked potentials. Electroencephalogr Clin Neurophysiol 59:134-141, 1984.

Nuwer, M.R., and Namerow, N.S. Somatosensory evoked potential testing in

multiple sclerosis, In: Waxman, S.G., and Ritchie, J.M., eds. Demyelinating

Disease: Basic and Clinical Electrophysiology. New York: Raven Press, 1981.

pp. 183-199.

Nyrke, T.; Kangasniemi, P.; and Lang, A.H. Difference of steady-state visual

evoked potentials in classic and common migraine. Electroencephalogr Clin

Neurophysiol 73:285-294, 1989.

Obeso, J.A.; Marti-Masso, J.F.; and Carrera, N. Somatosensory evoked

potentials: Abnormalities with focal brain lesions remote from the primary

sensorimotor area. Electroencephalogr Clin Neurophysiol 49:59-65, 1980.

94
Odom, J.V.; Bromberg, N.M.; and Dawson, W.W. Canine visual acuity: Retinal

and cortical field potentials evoked by pattern stimulation. Am J Physiol

245(5 Pt. 1):R637-R641, 1983.

Office of Technology Assessment, US. Congress. Neurotoxicity: identifying and

Controlling Poisons of the Nervous System. OTA-BA-436. Washington, DC:

Supt. of Docs., U.S. Govt. Print. Off., 1990.

Oishi, M.; Yamada, T.; Dickins, S.; and Kimura, J. Visual evoked potentials by

different check sizes in patients with multiple sclerosis. Neurology 35:1461-

1465, 1985.

Ollo, C., and Squires, N. Event-related potentials in learning disabilities. In:

Cracco, R.Q., and Bodis-Wollner, I., eds. Evoked Potentials. New York: Alan

R. Liss, 1986. pp. 497-512.

Onofrj, M.; Bodis-Wollner, I.; and Bobak, P. Pattern visual evoked potentials in

the rat. Physiol Behav 28:227-230, 1982a.

Onofrj, M.; Bodis-Wollner, I.; and Mylin, L. Visual evoked potential latencies in

papilledema and hydrocephalus. Neurophthalmology 2:85-92, 1981.

Onofrj, M.; Bodis-Wollner, I.; and Mylin, L. Visual evoked potential diagnosis of

field defects in patients with chiasmatic and retrochiasmatic lesions. J Neurol

Neurosurg Psychiatry 45:294-302, 1982b.

Otto, D. The use of sensory evoked potentials in neurotoxicity testing of

workers. Seminar Occup Med 1:175-183, 1986.

Otto, D.; Baumann, S.; and Robinson, G. Application of a portable

microprocessor-based system for electrophysiological field testing of

neurotoxicity. Neurobehav Toxicol Teratol 7:409-414, 1985.

Otto, D.; Hudnell, D.; Boyes, W.; Janssen, R.; and Dyer, R. Electrophysiological

measures of visual and auditory function as indices of neurotoxicity.

Toxicology 49:205-218, 1988.

Padmos, P.; Haaijamn, J.J.; and Spekreijse, H. Visually evoked potentials to

patterned stimuli in monkey and man. Electroencephalogr Clin Neurophysiol

35:153-163, 1973.

Pak, M.A. Ocular refraction and visual contrast sensitivity of the rabbit

determined by the VECP. Vision Res 24:341-345, 1984.

Papakostopoulos, D.; Dean Hart, C.; Cooper, R.; and Natsikos, V. Combined

electrophysiological assessment of the visual system in central serous

retinopathy. Electroencephalogr Clin Neurophysiol 59:77-80, 1984.

Perot, P.L. The clinical use of somatosensory evoked potentials in spinal cord

injury. Clin Neurosurg 20:367-381, 1973.

Pfister, W.R.; Chinn, C.; Noland, V.; and Yim, G.K.W. Similar pharmacological

actions of chlordimeform and local anesthetics. Pestic Biochem Physiol

9:148-156, 1978.

Picanco-Diniz, C.W.; Silveira, L.C.L.; and Oswaldo-Cruz, E.

Electrophysiological determination of the refractive state of the eye of the

opossum. Vision Res 23:867-872, 1983.

95
Pirchio, M.; Spinelli, D.; Fiorentini, A.; and Maffei, L. Infant contrast sensitivity

evaluated by evoked potentials. Brain Res 141:179-184, 1978.

Pratt, H., and Starr, A. Somatosensory evoked potentials to natural forms of

stimulation, In: Cracco, R.Q., and Bodis-Wollner, I., eds. Evoked Potentials.

New York: Alan R. Liss, 1986. pp. 28-34.

Previc, F.H. Visual evoked potentials to luminance and chromatic contrast in

rhesus monkeys. Vision Res 26:1897-1907, 1986.

Pryor, G.T.; Dickerson, J.; Feeney, E.; and Rebert, C.S. Hearing loss in rats first

exposed to toluene as weanlings or as young adults. Neurobehav Toxicol

Teratol 6:111-119, 1984a.

Pryor, G.T.; Dickinson, J.; Howd, R.A.; and Rebert, C.S. Transient cognitive

deficits and high-frequency hearing loss in rats exposed to toluene.

Neurobehav Toxicol Teratol 5:53-57, 1983.

Pryor, G.T.; Rebert, C.S.; Dickinson, J.; and Feeney, E. Factors affecting

toluene-induced ototoxicity in rats. Neurobehav Toxicol Teratol 6:223-238,

1984b.

Pryor, G.T.; Rebert, C.S.; and Howd, R.A. Hearing loss in rats caused by

inhalation of mixed xylenes and styrene. J Appl Toxicol 7:55-61, 1987.

Ratliff, F., and Zemon, V. Visual evoked potentials elicited in normal subjects

and in epileptic patients by windmill-dartboard stimuli. In: Nodar, R.H., and

Barber, C., eds. Evoked Potentials II. Stoneham, MA: Butterworth Publishers,

1984. pp. 251-259.

Rebert, C.S. Multisensory evoked potentials in experimental and applied

neurotoxicology. Neurobehav Toxicol Teratol 5:659-671, 1983.

Rebert, C.S., and Becker, E. Effects of inhaled carbon disulfide on sensoryevoked potentials of Long Evans rats. Neurobehav Toxicol Teratol 8:533-541,

1986.

Rebert, C.S.; Day, V.L.; Matteucci, M.J.; and Pryor, G.T. Sensory-evoked

potentials in rats chronically exposed to trichloroethylene: Predominant

auditory dysfunction. Neurotoxicol Teratol 13(1):83-90, 1991.

Rebert, C.S.; Matteucci, M.J.; and Pryor, G.T. Multimodal effects of acute

exposure to toluene evidenced by sensory-evoked potentials from Fischer344 rats. Pharmacol Biochem Behav 32:757-768, 1989a.

Rebert, C.S.; Matteucci, M.J.; and Pryor, G.T. Acute effects of inhaled toluene

on adult male Long-Evans rats. Pharmacol Biochem Behav 33:157-165,

1989c.

Rebert, C.S.; Matteucci, M.J.; and Pryor, G.T. Acute effects of inhaled

dichloromethane on the EEG and sensory-evoked potentials of Fischer-344

rats. Pharmacol Biochem Behav 34:619-629, 1989b

Rebert, C.S.; Sorenson, S.S.; Howd, R.A.; and Pryor, G.T. Toluene-induced

hearing loss in rats evidenced by the brainstem auditory-evoked response.

Neurobehav Toxicol Teratol 5:59-62, 1983.

96
Regan, D. Visual information channeling in normal and disordered vision.

Psychol Rev 89:407-444, 1982.

Regan, D. Human Brain Electrophysiology: Evoked Potentials and Evoked

Magnetic Fields in Science and Medicine. New York: Elsevier, 1989.

Regan, D.; Schellart, N.A.M.; Spekreijse, H.; and van den Berg, T.J.T.P.

Photometry in goldfish by electrophysiological recording: Comparison of

criterion response method with heterochromatic flicker photometry. Vision

Res 15:799-807, 1975.

Regan, D., and Spekreijse, H. Evoked potential in indications of colour

blindness. Vision Res 14:89-95, 1974.

Rothenberger, A.; Szirtes, J.; and Jurgens, R. Auditory evoked potentials to

verbal stimuli in healthy, aphasic, and right hemisphere damaged subjects.

Arch Psychiatr Nurs 231:155-170, 1982.

Sachs, M.B.; Nachmias, J.; and Robson, J.G. Spatial-frequency channels in

human vision. J Opt Sot Am 61:1176-1186, 1971,

Sartucci, F.; Scardigli, V.; and Murri, L. Visual evoked potentials and magnetic

resonance imaging in the evaluation of pre- and retrochiasmatic lesions in

multiple sclerosis. Clin Vision Sci 4:229-238, 1989.

Seppalainen, A.M. Neurophysiological approaches to the detection of early

neurotoxicity in humans. Crit Rev Toxicol 18:245-298, 1988.

Shaw, N.A. Somatosensory evoked potentials after head injury: The

significance of the double peak. J Neurol Sci 73:145-153, 1986.

Sherman, J. ERG and VEP as supplemental aids in the differential diagnosis of

retinal versus optic nerve disease. In: Cracco, R.Q., and Bodis-Wollner, I.,

eds. Evoked Potentials. New York: Alan R. Liss, 1986. pp. 343-353.

Silveira, L.C.L.; Heywood, CA.; and Cowey, A. Contrast sensitivity and visual

acuity of the pigmented rat determined electrophysiologically. Vision Res

27:1719-1731, 1987.

Silveira, L.C.L.; Picanco-Diniz, C.W.; and Oswaldo-Cruz, E. Contrast sensitivity

function and visual acuity of the opossum. Vision Res 22:1371-1377, 1982.

Simpson, D.M., and Erwin, C.W. Evoked potential latency change with age

suggests differential aging of primary somatosensory cortex. Neurobiol Aging

4:59-63, 1983.

Smith, A.T.; Early, F.; and Jones, G.H. Comparison of the effects of Alzheimer’s

disease, normal aging and scopolamine on human transient visual evoked

potentials. Psychopharmacology 102:535-543, 1990.

Sokol, S.; Moskowitz, A.; and Paul, A. Evoked potential estimates of visual

accommodation in infants. Vision Res 23:851-860, 1983.

Spekreijse, H. Maturation of contrast EPs and development of visual resolution,

Arch ltal Biol 116:358-369, 1978.

Starr, A. Auditory brain-stem responses in brain death. Brain 99:543-554, 1976.

Starr, A., and Achor, L.J. Auditory brain stem responses in neurological

disease. Arch Neurol 32:761-768. 1975.

97
Steele, M.; Seiple, W.H.; Carr, R.E.; and Klug, R. The clinical utility of visualevoked potential acuity testing. Am J Ophthalmol 108:572-577, 1989.

Stohr, M.; Riffel, B.; Trost, E.; and Ullrich, A. Short-latency somatosensory

evoked potentials in brain death. J Neurol 234:211-214, 1987.

Stone, J., and Dreher, B. Parallel processing of information in the visual

pathways: A general principle of sensory coding? Trends Neurol Sci 5:441-

446, 1982.

Takayasu, M.; Shibuya, M.; Suzuki, Y.; Harada, T.; Kanamori, M.; and

Kageyama, N. Trigeminal sensory evoked potentials in patients with

trigeminal neurinoma: Report of two cases. Neurosurgery 20:453-456, 1987.

Toyonaga, N.; Adachi-Usami, E.; and Kakisu, Y. Visually evoked cortical

potentials to pattern disappearance stimuli in multiple sclerosis. Neuroophthalmology 8:81-86, 1988.

Trick, G.L. Pattern evoked retinal and cortical potentials in diabetic patients.

Clin Vision Sci 6:209-217, 1991.

Urban, P., and Lukas, E. Visual evoked potentials in rotogravure printers

exposed to toluene. Br J Ind Med 47:819-823, 1990.

Warnke, PC.; Phillips, A.; Bernstein, L.P.; and Groothuis, D.R. The

somatosensory evoked potential as a noninvasive method to determine flow

rates for hyperosmotic disruption of the blood-brain barrier. Neurosurgery

25:405-411, 1989.

Woolley, D.E. Electrophysiological techniques in toxicology. In: Zenick, H., and

Reiter, L.W., eds. Behavioral Toxicology: An Emerging Discipline. EPA-600/9-

77-042, 1977.

DISCUSSION

Q: Do you expect these neurophysiological or neuroanatomical changes?

A: Yes, they occurred in the monkeys. In the monkeys there was a

degeneration of the retinal ganglion cells, and I would expect that is what we

would see in the rats as well.

Q (Landfield): I would imagine one of the major control problems that you have

is the arousal state of the animal. Arousal can have dramatic effects on the

size of the evoked potential. It can cut it in half.

A: That is an issue that we have to deal with, particularly when some of the

neuroactive compounds can have effects on arousal level, stress, etc. It comes

out in a variety of ways: pupil size and body temperature. Body temperature,

particularly in rats, can be strongly affected by exposure to toxic compounds.

And the latency to EPs [evoked potentials] can be a linear relationship to body

temperature. So you have to model that. We have been looking at the later

waves of the EPs. The ones that I have talked about today are pretty early,

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and they are thought to be closely tied to the sensory phenomenon itself. But

there are later latency potentials which are thought to be more closely related

to arousal, habituation, and sensitization-like phenomena. Those potentials

actually can be very sensitive to neurotoxic compounds, particularly the

solvents. We have a peak occurring at about 160 milliseconds after a flashevoked potential which has been altered in a variety of solvent exposures, like

toluene, xylene, carbon disulfide-a whole series of those. We are in the

middle of a research program right now that tries to tie those changes to what

we know about the behavioral effects. It is something that we need to consider,

but it is also something that we can use.

Q (Landfield): I was just thinking that you could be lesioning the reticular of the

arousal system, thinking you are affecting the visual system. Even the initial

primary sensory-evoked potential is affected by arousal.

A: Yes, you are right. In our tests, our animals are harnessed (and we test

them without anesthetic because that may interfere with the neurotoxic

compound that we are testing), and the restraint itself can be stressful; and

I think that may equate them all at a high level of arousal. It is not a good

answer. The other aspect to your question-having lesions elsewhere and

detecting the effects in the visual system-is for us a mixed blessing because,

when we are testing unknown compounds, we want to know if they are

effective. That is the first question, And if you see effects on evoked potentials,

you can subsequently do a research program to trace that down. But at least

we have detected it. The fact that they may be dependent and interdependent

on other systems can be seen as a mixed blessing.

Q (Tilson): When you are testing evoked potentials in a screen situation, you

wouldn’t be looking at just one, you would probably be looking at several

endpoints.

A: Yes, you would be looking at visual, auditory, and somatosensory combined

together. And if you see effects on all of those, then you may suspect that it is

a more general phenomenon. That is a good point.

ACKNOWLEDGMENTS

D.W. Herr, Ph.D.; C.S. Rebert, Ph.D.; and L. Erinoff, Ph.D., reviewed an earlier

version of the manuscript.

99
AUTHOR

William K. Boyes, Ph.D.

Chief

Neurophysiological Toxicology Branch

Neurotoxicology Division

Health Effects Research Laboratory

U.S. Environmental Protection Agency

Research Triangle Park, NC 27711

100
Silver Staining as a Tool for Neurotoxic

Assessment

Carlos A. Beltramino, José S. de Olmos, Ferenc Gallyas,

Lennart Heimer, and Lázló Záborszky

INTRODUCTION

Neurotoxicology and neurotoxicity have become matters of great public concern,

and efforts have intensified to find reliable and convenient methods to identify

those chemicals and substances that are potentially harmful to health because

of their toxic effect on the central nervous system (CNS). A reliable answer is

needed for the following question: Have the basic units of the CNS (i.e., the

neurons) suffered irreversible damage as a result of the toxic effect? In other

words, the task is to find a handy method to detect signs of degeneration in

neurons and to determine the location and extent of such neuronal degeneration

in the CNS. The reduced silver methods’ have in the past been the most effective

procedures for mapping experimentally induced neuronal degenerations, and the

results demonstrated by modern silver methods indicate that they also have the

qualities necessary to make them a powerful method for screening neuronal

damage caused by various neurotoxic substances including drugs of abuse.

Since the early part of of this century, silver methods have been used with great

success to stain both normal and pathologic components of nervous tissue (von

Fajersztajn 1901; Bielschowsky 1904; Ram n y Cajal 1904). The ease with which

various tissue components attract silver and the high-contrast microscopic images

provided by metallic silver deposits are two of the most attractive features of silver

impregnation.

Because the molecular basis for silver staining is still largely unknown, silver

methods have generally been improved and refined by the empirical method

and then modified by individual investigators to serve the requirements of a

special line of research. As a result, many silver staining protocols have been

developed through the years for various purposes, and the choice of method is

rarely a foregone conclusion. This makes it especially difficult for researchers or

histologists just beginning to use these procedures. Different methods and their

various modifications often must be tried to obtain optimal results. Because

tinkering with methods is time consuming, the tinker-and-toil process is often

neglected, with the result that silver procedures are frequently inconsistent or

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inadequate. Therefore, silver methods have the reputation of being capricious, or

in the words of Wolman (1955), “their practical application represents more often

an art than a science.”

But the art of silver staining has continued to attract the attention of experimental

neuroanatomists and even an occasional neurochemist, and some procedures

have been refined to the point where they can demonstrate certain components

of the tissue with a high degree of specificity and consistency. To appreciate the

unique advantages that the silver methods can bring to the morphologic screening

of neurotoxicity, it is useful to review some of the accomplishments made possible

with the aid of silver methods.

THE NAUTA PERIOD: THE SILVER AGE OF NEURONAL TRACT TRACING

When the reduced silver methods were introduced at the turn of the century,

they were used primarily for the study of normal axons and for visualizing various

pathological conditions in the human brain. Several years elapsed before the

first reports on the use of silver methods for the tracing of experimentally induced

axon degeneration appeared in the literature (e.g., Rasdolsky 1923, 1925; Hoff

1932a, 1932b; Schimert2 1938, 1939; Szentágothai-Schimert 1941). However,

the real breakthroughs in the use of silver methods for the tracing of Wallerian

degeneration3 (Waller 1850) came with the discovery of the Bielschowsky

modifications by Glees (1946), Nauta (1950), and Nauta and Gygax (1951)

for frozen sections, followed by the popular ‘suppressive” Nauta-Gygax method

(Nauta and Gygax 1954). In the 1954 method, Nauta and Gygax introduced

a pretreatment of the sections in phosphomolybdic acid and potassium

permanganate before the treatment in an aqueous silver nitrate solution followed

by an ammoniacal silver solution and development in a formaldehyde solution

acidified with citric acid (Nauta reducer). The pretreatment was important because

it allowed the suppression of the staining of normal axons, which in turn greatly

facilitated the selective visualization of degenerating axons. It was later shown

that selective demonstration of degenerating axons and terminals could be

achieved by simply treating the sections for a short time in ammoniacal silver

solution followed by a reducer (Eager and Barnett 1964, 1966), particularly if

the tissue had been stored in fixative for a long time (Loewy 1969). Nonetheless,

the pretreatment introduced by Nauta and Gygax became the method of choice

for suppressing the staining of normal fibers, and a number of laboratories around

the world made use of various modifications of the Nauta-Gygax method,

especially after having realized that the demonstration of the finest degenerating

axon arborizations and boutons is often optimal with rather short survival times,

usually 2 to 3 days following the experimental lesion.

The experimental tracing of pathways by the aid of the Nauta-Gygax method

and many other silver methods (e.g., Fink and Heimer 1967; de Olmos 1969;

Ebbesson and Rubinson 1969; Wiitanen 1969; Eager 1970; Kalaha-Brunst et al.

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1974; Desclin and Escubi 1975) was in large part responsible for the dramatic

expansion of neuroanatomical knowledge in the sixties and seventies. One in

every four or five papers published in the Journal of Comparative Neurology or

Brain Research in the early 1970s was based on silver staining of degenerating

axons. Several examples could be mentioned in which the increased precision

made possible by silver methods produced results that decisively changed the

way the brain is perceived. One example from a key topic may be sufficient to

illustrate this point. The first anatomical demonstration of the familiar oculardominance columns in the monkey striate cortex was made possible by the

Fink-Heimer modification of the Nauta method in 1968, and the results were

published in Nature the following year (Hubel and Wiesel 1969). It is also

worth mentioning that the 1960s renaissance in comparative neuroanatomy of

nonmammalian species was in large part fueled by students of the Nauta school

(e.g., Ebbesson 1970; Lohman and Mentink 1972; Karten et al. 1973) and many

others, who mastered and adapted the silver methods for the tracing of pathways

in nonmammalian vertebrates.

The heyday of the silver staining epoch lasted from the early 1950s into

the 1970s. During this time, silver staining was the only practical method

available for the tracing of long pathways in the brain. The classic text titled

“Contemporary Research Methods in Neuroanatomy” (Nauta and Ebbesson

1970) reflects the heavy reliance on silver methods at the time; more than onethird of the chapters pertain directly to silver methods used in the experimental

tracing of degenerating pathways. These chapters did not introduce new silver

methods; there was, rather, considerable discussion about how to interpret

the results obtained by the experimental silver methods. Although it had

been convincingly shown that the silver methods commonly used for the

demonstration of so-called “terminal degeneration” were sensitive enough to

identify a large number of degenerating boutons (Heimer and Peters 1968),

one lingering problem concerned the question of sensitivity; another problem

was related to the unavoidable damage of fibers passing through areas lesioned

to induce degeneration. This made it difficult sometimes to identify the cells of

origin for the pathway under study.

In regard to the problem of sensitivity, and with the benefit of hindsight, the

introduction of the pretreatment of the sections in phosphomolybdic acidpotassium permanganate was a mixed blessing. The pretreatment allowed the

suppression of normal fiber staining but also introduced the risk of suppressing

argyrophilia in the most delicate degenerating fibers; too extensive pretreatment

had the effect of suppressing all staining. The degree of pretreatment, although

crucially important, was a subjective and somewhat arbitrary decision.

As for the unavoidable damage of fibers passing through the lesioned area,

this shortcoming on the part of the experimental silver methods was probably

one of the most important factors for the rapidly increasing popularity of the

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autoradiographic tracing method (Lasek et al. 1968; Cowan et al. 1972; Edwards

1972). Many structures, especially in the deeper parts of the forebrain and brain

stem, could be more easily analyzed with the autoradiographic technique than

with methods based on Wallerian degeneration, and the number of publications

using experimental silver methods declined precipitously in the late 1970s and

early 1980s. However, the dwindling popularity of the silver methods was

caused not only by the arrival of the autoradiographic method but also by the

introduction of many other powerful tracer methods, including the horseradish

peroxidase method (Kristensson et al. 1971; LaVail and LaVail 1972), the

fluorescent tracer method (Kuypers et al. 1977; Van der Kooy et al. 1978), and

the Phaseolus vulgaris leuco-agglutinin technique (Gerfen and Sawchenko

1984). Because of the successes of these newer tracer methods, the silver

method was increasingly viewed as old fashioned in the context of experimental

tracing of pathways, and in recent textbooks on tract-tracing methods (e.g.,

Heimer and Záborszky 1989; Björklund and Hökfelt 1990), not a single chapter

was devoted specifically to experimental silver methods. However, as discussed

in some detail below, the silver methods did not disappear from the field of

experimental neuroanatomy. Silver methods still offer some unique advantages

in particular tract-tracing experiments (e.g., Carlsen et al. 1982; Záborszky et al.

1985; Heimer et al. 1987); but more important in the context of this review is a

growing awareness of the advantages that reduced silver methods can offer in

other important areas of neuroscientific endeavor, including neurotoxicology and

ischemia research.

As the conclusion of this short history of silver staining, it should be emphasized

that the tracing of pathways by means of silver staining of degenerating axons

and terminals during the third quarter of this century provided a firm anatomical

foundation for the modern era of neurobiology. It is an ironic coincidence that as

the close of the century nears and the “decade of the brain” is celebrated, the

reduced silver methods seem poised to provide a further legacy to neuroscience.

APPLICATION OF SILVER METHODS FOR SELECTIVE DEMONSTRATION

OF DEGENERATION REMAINS A LARGELY UNEXPLOITED OPPORTUNITY

IN NEUROPATHOLOGY

Although reduced silver methods lost much of their attraction in experimental

neuroanatomy when new tracer methods based on specific neuronal uptake and

axoplasmic transport were introduced in the 1970s and 1980s some silver

staining procedures remained the methods of choice for specific neuropathologic

phenomena including neurofibrillary tangles and senile plaques. Reflecting the

recent surge of interest in Alzheimer’s disease, several new protocols for the

staining of neurofibrillary changes have been published during the last few years

(e.g., Sevier and Munger 1965; Yamamoto and Hirano 1986; Campbell et al.

1987; Braak et al. 1988; Hedreen et al. 1988; Yamaguchi et al. 1990). These

methods are modifications of the classic Bielschowsky method (e.g., Sevier and

104
Munger 1965; Yamamoto and Hirano 1986) or incorporate principles that were

developed in the field of experimental neuroanatomy in more recent years (e.g.,

the method by Campbell et al. 1987; Braak et al. 1988; Hedreen et al. 1988).

For the neuropathologist, the usefulness of silver methods is by no means limited

to the detection of neurofibrillary changes. As already indicated, practically every

tissue component is potentially argyrophilic, and specific silver methods have

been developed for the study of various nervous tissue components, One of the

best-known examples of such specialized methods is the silver carbonate

technique for microglia developed by Del Rio-Hortega (1919, 1932).

With the possible exception of the many silver procedures developed by Del

Rio-Hortega for the study of glia cells and the various modifications of the

Bielschowsky silver technique for the study of axons and neurofibrillary

changes in Alzheimer’s disease and related disorders, metallic silver techniques

never have become part of the technical arsenal in laboratories of general

neuropathology. This may be explained in part by silver procedures’ reputation

for being difficult and capricious and by the rather natural tendency to avoid

the unfamiliar. Nevertheless, this perception is unfortunate considering the

advantages that the reduced silver methods can offer to those interested in

degeneration of neurons and their dendritic and axonal processes (e.g., Grant

1970; Leonard 1979).

A striking experimental example of the superiority of the silver degenerating

methods vis-a-vis regular Nissl stains for detecting degenerating neurons can be

seen in the olfactory cortex following olfactory bulb removal. Reports of chronic

atrophic changes in olfactory cortex several weeks or months after lesions in the

olfactory bulb or olfactory peduncle had occasionally appeared in the literature

(e.g., Winkler 1918; Uyematsu 1921; Allison 1953). However, in spite of many

earlier studies with conventional Nissl methods, transsynaptic induction of

degeneration in olfactory cortex neurons as a result of an ipsilateral bulbectomy

was detected only when sensitive silver methods were employed (Heimer 1968;

Price 1976; Heimer and Kalil 1978; Carlsen et al. 1982). This especially striking

illustration of the power of the silver method for screening neuronal degeneration

is illustrated in a recent paper by Switzer (1991) who argues persuasively for

the general utility of silver methods in assessing neurotoxicity.

If certain steps in the basic silver protocols are modified, silver ions can be

induced to form metallic silver preferentially on mitochondria, boutons, myelin,

or glia or on mesodermal elements in blood vessels. In spite of this versatility

and, in some cases, exclusive sensitivity (e.g., degenerating boutons cannot

in general be detected on the light microscopic level with any technique

other than the silver method), it is apparent that a number of more specific

immunohistochemical techniques (e.g., for glial fibrillary acidic protein) are

more attractive to the neuropathologists than the reduced silver methods. The

105
reading of well-known reviews of neuropathological methodology (e.g., Duchen

1984; Okazaki 1989) confirms that the advantages of modern reduced silver

methods have rarely been exploited in general neuropathology. Nonetheless,

the identification of damaged pathways in the human brain must certainly be

considered a desirable goal of a postmortem examination, and the reduced

silver methods offer some unique advantages in this regard (e.g., Voogd et al.

1990). Contrary to common belief, some products of axonal degeneration can

remain for several years after damage to the human brain (Grafe and Leonard

1981). Therefore, even if people survive for a considerable time following

lesions in the CNS, postmortem silver staining of such brains could provide

important signs for the existence of such lesions as well as answers regarding

the effect on specific pathways. This approach could become a sensitive

complement to the more traditional methods of histological evaluation in

neuropathology.

SILVER IMPREGNATION IN THE 1980s: NEW METHODOLOGICAL

DEVELOPMENTS

Although the silver methods have been used only occasionally in experimental

neuroanatomy during the past decade, several developments have nevertheless

taken place in regard to the procedures of silver impregnation. For instance,

important and selective methods have been introduced by Gallyas and

colleagues, and the cupric-silver method for degenerating neuronal elements

(de Olmos 1969) has appeared in several new and increasingly more powerful

versions during the past decade.4

The basic principle of silver staining was outlined many years ago by Liesegang

(191 I), who suggested that reducing groups in the tissue transform ionic silver

to ultramicroscopic “nuclei” of metallic silver, which are then enlarged by the

deposition of additional silver to the point where they become visible in the light

microscope (photographic development). In other words, the final result

depends on the initial distribution of binding sites for silver ions. Several

investigators have attempted to clarify the mechanisms involved and the

conditions that determine the success and the specificity of the staining (e.g.,

Nauta and Gygax 1951; Samuel 1953a, 1953b: Peters 1955a, 1955c, 1955c;

Wolman 1955; Evans and Hamlyn 1956; White 1960; Eager and Barnett 1964,

1966; Giolli 1965; Loewy 1969; Giolli and Karamanlidis 1978). As already

indicated, the use of a suitable pretreatment (Nauta and Gygax 1954) to

accentuate the difference between degenerating and normal fibers was an

important step in modern silver methods including the many modifications of

the Nauta-Gygax method, the various methods developed by Gallyas and

collaborators, and the cupric-silver method by de Olmos.

During the 1970s and early 1980s, Gallyas (1982a, 1982b) conducted a series

of experiments on the mechanisms of silver staining and identified some of the

106
factors that determine the formation of metallic silver. Besides consolidating

the Liesegang theory, Gallyas and coworkers (1980a, 1980b, 1980c) identified

conditions that favored the staining of either degenerating axons or degenerating

terminals. The success of the method for demonstrating degenerating axons

(Gallyas et al. 1980b) is due in large part to the use of a physical developer

(a reducer containing silver ions) rather than the commonly used Nauta reducer

(formaldehyde acidified with citric acid). The method of staining degenerating

terminals (Gallyas et al. 1980c), on the other hand, resembles the Eager

(1970) method in its simplicity: following an alkaline pretreatment and a short

immersion in an ammoniacal silver solution, the development takes place in

a Nauta reducer. Characterized by low background, high contrast, and good

reproducibility, the Gallyas methods are also well suited for dark field illumination

at low magnification as well as for quantitative evaluations (e.g., Wolff et al.

1981; Záborszky and Wolff 1982).

The ability to induce a Golgi-like staining of neurons in many parts of the brain

was described by Switzer (1976), who used injections of puromycin, a metabolic

cytotoxin, to traumatize neurons before staining them with the cupric-silver

method. In more recent publications, Gallyas and collaborators have described

a method for the Golgi-like demonstration of “dark” neurons consequent to

various pathological conditions or artificially produced by postmortem

traumatization (Gallyas et al. 1990; van den Pol and Gallyas 1990). The

success of this method depends on the use of an esterification procedure,

which in combination with the physical development produces a selective and

easily controllable demonstration of traumatized neurons. The most remarkable

feature of the method described by Gallyas and colleagues is how quickly

(a few minutes) after trauma the affected neurons can be revealed.

The cupric-silver method for the tracing of degenerating pathways was

developed several decades ago (de Olmos 1966), but the excellent quality of

the results was overshadowed to some extent by a rather involved procedure,

and the method was not widely applied in tract-tracing studies. Although a

cupric-silver solution was used by Richardson in 1960 for the demonstration

of autonomic nerves, de Olmos discovered by accident that cupric ions could

be helpful in suppressing the staining of normal fibers in the CNS. This result

is amply supported by more recent examination of metal ions for suppressing

normal tissue argyrophilia (Gallyas and Wolff 1986). In contrast to the

suppressive step in the Nauta-Gygax method, which has a tendency to interfere

with the argyrophilia of both normal and degenerating fibers, the cupric-sulfate

treatment seems to be less prone to suppress the argyrophilia of degenerating

axons and terminals, The original cupric-silver method (de Olmos 1969) was

later modified, first to improve the demonstration of terminal degeneration (de

Olmos and Ingram 1972) and then for the demonstration of traumatized neurons

(retrograde and transneuronal degeneration) by Carlsen and de Olmos (1981).

107
In contrast to the various Gallyas versions of the silver techniques, which

demonstrate particular subsets of neuronal degeneration (i.e., axonal, terminal,

or soma-dendritic degeneration), the cupric-silver method provides an overall

view of these changes occurring in traumatized neurons.

It appears at first that the Gallyas methods and the cupric-silver method might

represent two approaches to demonstrate the same phenomena. However,

some significant difference may exist with regard to the morphological

molecular substrate responsible for the result in the Gallyas methods and the

cupric-silver method. One possible reflection of this difference is the ability to

reliably impregnate specific parts of normal forebrain and brain stem when

using the cupric-silver procedure (de Olmos 1969).

USE OF SILVER METHODS IN NEUROTOXIC RISK ASSESSMENT

With the growing realization that functions of the CNS can be irreversibly

compromised by a rapidly increasing number of drugs and chemical

substances, neurotoxic risk assessment has become a matter of great urgency.

A review of the literature suggests that neurotoxicologists are rediscovering the

silver methods as a means of finding answers to questions of neuronal damage.

Reduced silver methods have been used to great advantage for demonstrating

neuronal degeneration in response to a number of neurotoxins, including

6-hydroxydopamine (Hedreen and Chalmers 1972; Maler et al. 1973),

methamphetamine and methylenedioxymethylamphetamine (Ricaurte et al.

1982; Commins and Seiden 1986; Commins et al. 1987; Jensen et al. 1990,

1991), 3-acetylpyridine (Balaban 1985), diazepam (Balaban et al. 1988a),

trimethyltin (Balaban et al. 1988b; Whittington et al. 1989), organophosphorus

(Tanaka and Bursian 1989), and glutamate (Janssen et al. 1991; Schweitzer

et al. 1991). The neurotoxic effect of tetanus toxin (Illis and Mitchell 1970) and

capsaicin (Jancsó and Király 1980; Ritter and Dinh 1988, 1991) as well as the

neuronal degeneration caused by excitotoxic amino acids including kainic acid

(Jarrard 1983; Ben Ari et al. 1981), ibotenic acid (Jarrard 1989), and quinolinic

acid (de Olmos and Beltramino 1991) also have been successfully

demonstrated by the aid of silver methods.

To demonstrate the high degree of sensitivity available through the use of

modern silver methods, the authors prepared material from animals injected

with trimethyltin (figure 1) and quinolinic acid (figures 2 and 3). In the case

demonstrated in figure 1 (prepared in collaboration with Karl Jensen at the

Health Effects Research Laboratory at the Environmental Protection Agency

at Triangle Park, NC), rats were injected intraperitoneally with trimethyltin in a

dose of 8 mg/kg body weight; the rats survived for 48 hours (figure 1, panels A

and B) or 24 hours (figure 1, panel C) following the injection. Pronounced

neuronal degeneration is evident, not only in hippocampus (figure 1, panels A

and B) and proisocortex (figure 1) panel C), but also in many other structures

108
FIGURE 1. A. Degeneration in the rat hippocampus following an

intraperitoneal trimethyltin injection (8 mg/kg body weight) 48

hours before sacrifice of the animal. Note the large number of

degenerating neurons, especially in CA3 and CA4, and the

massive degeneration of terminals as reflected in the pitch-black

areas in most of the hippocampus. B. Detail of some of the

degenerating neurons in CA3. C. Degenerating neurons in rat

insular cortex following a trimethyltin injection 24 hours before

sacrifice of the animal (de Olmos’ cupric-silver method).

109
FIGURE 2. Degeneration in parietal cortex of a rat in which quinolinic acid

(60 nmol in 0.5 μL) was injected into the striarum (A, B, and D) or

thalamus (C) 24 hours before sacrifice of the animal. The section

in A was stained with Gallyas' methods for “dark” neurons,

whereas those in B, C, and D were stained with de Olmos’ cupricsilver method. Note the corkscrew appearance of the apical

dendrite of the pyramidal cell in A and in many of the pyramidal

cells in B (black arrows). The section in B was counterstained

with neutral red. Like the neurons in A and B, the densely spined

neuron in D shows signs of degeneration including an irregular

cell body and beaded axon. The insert in D demonstrates the

detailed rendition of dendritic spines from the dendrite indicated

by the arrow. The heavy band of terminal degeneration in C is

located in layers 3 and 4.

110
FIGURE 3. Direct print of a sagittal section of a rat brain with a striatal

injection of quinolinic acid (60 nmol/0.5 μL) 24 hours before

sacrifice of the animal. The figure illustrates the high-contrast

panoramic view of massive degeneration in the striatum,

substantia nigra, and related pathways made possible by the

silver method, in this case, the cupric-silver method.

KEY: CPu=caudate-putamen; SN=substantia nigra; Tu=olfactory tubercle

throughout the forebrain and brain stem as described by Balaban and colleagues

(1988b).

Figures 2 and 3 demonstrate degenerative neuronal changes in the rat brain

following striatal or thalamic injections of quinolinic acid. The dose of quinolinic

acid injected into striatum of the rats illustrated in these figures was 60 nmol in

0.5 μL. This is considerably less than the dose generally used in comparable

experiments (e.g., Foster et al. 1983; Björklund et al. 1986; El-Defrawy et al.

1986; Foster and Schwarcz 1989; Beal et al. 1988; Boegman and Parent 1988;

Calderon et al. 1988). Nonetheless, significant degeneration is manifest, not

only in striatum, but in many distant areas, including the cerebral cortex (figure 2),

endopiriform nucleus, claustrum, globus pallidus, septum, bed nucleus of stria

terminalis, thalamus, substantia nigra, and several other brain-stem areas.

111
Although the low-magnification picture in figure 3 does not allow full

appreciation of the neuronal degeneration in all these areas, it does give

a synoptic view of the dramatic degeneration in dorsal and ventral striatum

and closely related nigrostriatal and striatonigral fiber systems.

The advantages of the silver techniques may also be exploited in cases where

brain tissue is damaged by hypoxic events (figure 4, panels A and B) or other

forms of trauma (Crain et al. 1988; Franck and Roberts 1990; Freund et al.

1990a, 1990b; Lin et al. 1990; van den Pol and Gallyas 1990; Schmidt-Kastner

and Freund 1991; de Olmos et al., in press). Figure 4, panel C, illustrates

degenerating cell bodies in striatum following injection of the suicide transport

agent volkensin (Wiley and Stirpe 1988) into the substantia nigra. Figure 4,

panel D, demonstrates a Golgi-like staining of neurons traumatized by

hypoglycemia. Degenerative changes can be demonstrated by the aid of the

silver method as early as a few minutes following the insult (de Olmos et al.,

in press: van den Pol and Gallyas 1990).

One of the most attractive features of the silver method is its high degree

of sensitivity. The reason for this resides in the capacity to initiate in situ

silver deposits in response to favorable changes in the chemical groups of

macromolecules.5

 lmmunohistochemical techniques are also sensitive and

able to detect early changes (Yanagihara 1990). In addition, these techniques

are quite specific, but herein lies not only their strength but also their

weakness as potential general screening methods. Furthermore, some of

the immunoprocedures that are now being used in ischemia research (e.g., for

microtubule-associated proteins and a-tubulin) are based on the disappearance

of the immunoreaction in degenerating neurons and for that reason are less

suited as screening methods in cases where a subpopulation of neurons is

affected or in situations where degenerating neurons are isolated and scattered.

On the other hand, if one knows where to look, a slight but detectible loss of

immunoreaction for microtubule-associated protein can be appreciated in

hippocampal dendrites after only 3 minutes of ischemia (Kitagawa et al. 1989).

Other immunohistochemical procedures can reveal specific proteins (e.g.,

heat-shock proteins) (Lindquist 1986) produced by the neurons in response

to stress and trauma. Like the silver method, the immunoreaction of the heatshock proteins can provide a positive Golgi-like image of traumatized neurons

(Gonzalez et al. 1989). The affected neurons can be detected a few hours after

the occurrence of the trauma, but the time that elapses between the synthesis

of these de novo proteins and their disappearance is usually short and varies

from one population of cells to another. Furthermore, it is generally presumed

that only some immunoreactive cells die, whereas others may survive. The

same comments are applicable in general to the recently discovered c-Fos

protein and related immediate early gene products (Sagar et al. 1988; Morgan

112
FIGURE 4. A and B. Pictures from part of hippocampus in a gerbil subjected

to 10 minutes bilateral occlusion of internal carotid arteries 6 hours

before sacrifice of the animal. Whereas it is difficult to pinpoint the

degenerating neurons in the Nissl stained section in A, the adjacent

cupric-silver stained section shown in B leaves little doubt about the

massive neuronal degeneration. C. Degenerating cell bodies in

striatum of the rat following injection of volkensin (0.1 μL contains

3 ng) into substantia nigra 10 days before sacrifice of the animal

(Gallyas’ method for terminal degeneration). D. Degenerating

neurons in the cerebral cortex of a hypoglycemic rat sacrificed

3 hours following intraperitoneal administration of insulin (25 IU/kg)

(Gallyas’ method for “dark” neurons).

113
and Curran 1989), which have been used to localize the site of drug action

(Robertson and Robertson 1989; Young et al. 1989; Graybiel et al. 1990).

It is interesting to note that some of the silver methods used in ongoing

experimental studies of ischemia and other kinds of trauma (de Olmos et al., in

press; van den Pol and Gallyas 1990) can produce positive evidence of neuronal

injury at an earlier time following trauma than the heat-shock and the c-Fos

proteins. Nonetheless, there are a number of immunohistochemical techniques

that can now be applied to the study of neuronal activation and/or damage; the

field is wide open and at an exciting stage, where much progress will be made

by exploiting new interdisciplinary applications of various techniques.

From the experience gained so far, the silver techniques represent tools as

sensitive as immunohistochemical methods for assessing neurotoxicity. The

silver methods cannot indicate the neurochemical characteristics of the affected

neurons, but they can provide a quick, sensitive, and comprehensive survey of

neurodegenerative changes and direct subsequent searches for more specific

pathometabolic changes. Furthermore, with the aid of silver methods, the extent

of irreversible damage can be clearly assessed by direct observation of the

degenerating neurons and their processes. Also, although silver is a precious

metal, it is cheaper to buy the materials needed for silver staining than to

purchase the often expensive antibodies needed for the immunohistochemical

techniques. This additional advantage of using silver methods is a consideration

during times of financial restraints or when screening neurotoxins on a large

scale. However, in the final analysis, the reduced silver methods and the

immunohistochemical methods, although comparable in sensitivity, are very

different in many ways: both approaches have unique qualities that would

seem to ensure their independent and complementary use in various aspects

of neurotoxicology.

The advantage of using the silver method in the screening of neurotoxicity rests

not only on its sensitivity and the high-contrast images it provides but also on

the fact that it seems to be able to register all types of degeneration, and it does

so in a pattern that demonstrates the morphology of the degenerating tissue

elements. At present, it also may be unique in the ability to detect degenerating

axons and boutons, especially when they are the principal site of action for a

neurotoxin and when the parent cell body itself could survive the trauma. It is

not generally appreciated that the silver technique may be adjustable for different

conditions of fixation (e.g., both freshly and thoroughly fixed material). Although

silver methods usually have been applied to frozen free-floating sections, the

methods also have been modified for use on mounted cryostat sections of both

fresh and fixed material (Hamlyn 1957; Hjorth-Simonsen 1970; Loots et al. 1977;

Wheeler and Ritter 1984). Further developments of such modifications could

facilitate the processing of a large amount of material in a routine fashion

114
and include the possibility of using adjacent unfixed sections with other

neurohistological procedures.

As with most histotechnical methods, silver staining does present some initial

hurdles for the newcomer. However, Switzer (1991) reminds researchers that,

although small variations in one of the procedural steps in the silver staining can

ruin the results, “. . . if the same diligence necessary for the success of other

sensitive procedures, such as in biochemistry and immunocytochemistry, is

applied to the silver methods, consistent results will be obtained.” Interpreting

the pattern of argyrophilia also may present an obstacle for a beginner, but this

kind of uncertainty on the part of the inexperienced observer is not unique to the

silver method; it is common to all histological stains. With regard to the silver

methods, the problems of interpretation have been dealt with systematically by

several authors (Heimer 1970; Giolli and Karamanlidis 1978; de Olmos et al.

1981). Furthermore, as in every other field of human endeavor, it is useful for

the novice to profit from the guidance of more experienced individuals.

SUMMARY

There is no denying that the silver methods lost their dominant role as tracttracing methods in the past 10 to 15 years. But it seems equally clear that the

silver technique is headed for a dramatic revival in many fields of neuroscience,

where the scope and localization of neuronal degeneration are a central issue.

Together with the immunostaining of proteins formed or altered in traumatized

neurons, the modern silver techniques provide neurotoxicologists and

neuropathologists with unparalleled opportunities to detect and study injured

and dying neurons. Characterized by great sensitivity and distinct rendition

of the morphology of degenerating neurons and their processes, the reduced

silver methods constitute the ideal tool for screening irreversible neuronal

damage caused by neurotoxic substances including drugs of abuse.

Those interested in the rapidly expanding fields of “excitotoxicity” and

neurodegenerative disorders (Taylor 1991) are also likely to find increasing

use for the silver methods. The pattern of degeneration in so-called “system

degenerations” may be predetermined by the neuronal connections (Saper et

al. 1987), and as the disease progresses from the destruction of the originally

affected neuron population, closely related systems and pathways may be

recruited into the pathophysiologic cascade. Any type of trauma to the CNS

has the potential to produce this type of “domino effect” of degeneration, through

which additional systems are progressively recruited into a degenerative chain

reaction of transneuronal degeneration. In other words, even longstanding

disorders may exhibit signs of more recent degeneration, and the proper use

of silver methods at autopsy may give some important clues regarding the

etiology of disease: it may also provide new insights about the anatomy of

the human brain.

115
Little can be said at present about the chemical basis of argyrophilia in

degenerating and “reactive” neurons, but there is every reason to pay more

attention to this subject. One can expect that a continuing and concerted

effort will result in a rational understanding of the molecular biological and

physicochemical events that fortuitously provide the basis for the selective

impregnation of degenerating neuronal elements, This knowledge can be the

basis for the development of even more reliable and simple, yet sensitive,

silver methods suited for neurotoxic risk assessment on a large scale.

NOTES

1.

2.

3.

4.

5.

If reduction of the silver ions into metallic silver is accomplished by the aid

of a special reducing solution, the silver method is commonly referred to

as a “reduced” silver method. Another main group of silver methods is

represented by the various Golgi methods in which the reduction of the

silver ions takes place without the help of a special reducing solution.

Until 1940 Dr. Szentgothai used his original family name, Schimert.

In 1850 Augustus Waller demonstrated that a nerve fiber that is separated

from its cell body undergoes a gradual disintegration referred to as

Wallerian degeneration. This important phenomenon forms the basis not

only for the experimental silver methods but also for the famous Marchi

method (Marchi and Algeri 1885) for the staining of degenerating myelin.

The cupric-silver method also has been introduced for the detection of

proteins and peptides in gel electrophoresis (Switzer et al. 1979), and this

principle is widely used by biochemists because of the high sensitivity in

detecting amounts of protein in the nanogram to femtogram range.

Silver staining is based on a chemical reaction, the first phase of which is

catalyzed by certain points of the tissue structure, whereas in the second

phase the products of the first phase (submicroscopic metallic silver grains)

serve as catalyst. For this reason, a favorable change in the composition of

one single chemical group in a macromolecule, if it occurs within a potential

catalytic point, is capable of initiating the in situ formation of a silver grain

containing 103

-108 atoms. This is why silver methods are sensitive enough

to visualize disturbances undetectable by other kinds of histological

methods, excluding the immunohistological methods, in case of which a

change in one single chemical group generally results in the binding or

deposition of only one molecule of the coloring agent.

REFERENCES

Allison, A.C. The secondary olfactory areas in the human brain, J Anat 88:481-

488, 1953.

116
Balaban, C.D. Central neurotoxic effects of intraperitoneally administered 3-

acetylpyridine, harmaline and niacinamide in Sprague-Dawley and LongEvans rats: A critical review of central 3-acetylpyridine neurotoxicity. Brain

Res Rev 9:21-42, 1985.

Balaban, C.D.; O’Callaghan, J.P.; and Billingsley, M.L. Trimethyltin-induced

neuronal damage in the rat brain: Comparative studies using silver

degeneration stains, immunocytochemistry and immunoassay for

neuronotypic and gliotypic proteins. Neuroscience 26(1):337-361, 1988b.

Balaban, CD.; Roskams, A.-J.; and Severs, W.B. Diazepam attenuation of

somatostatin-induced motor disturbances and neurotoxicity. Brain Res

458:91-96, 1988a.

Beal, M.F.; Kowall, N.W.; Swartz, K.J.; Ferrante, R.J.; and Martin, J.B. Systemic

approaches to modifying quinolinic acid striatal lesions in rats. J Neurosci

8(10):3901-3908, 1988.

Ben-Ari, Y.; Tremblay, E.; Riche, D.; Ghilini, G.; and Naquet, R. Electrographic,

clinical and pathological alterations following systemic administration of kainic

acid, bicuculline or pentetrazole: Metabolic mapping using the deoxygiucose

method with special reference to the pathology of epilepsy. Neuroscience

6:1361-1391, 1981.

Bielschowsky, M. Die Silberimpregnation der Neurofibrillen. J Psychol Neurol

(Leipzig) 3:169-188, 1904.

Björklund, A., and Hökfelt, T. Analysis of neuronal microcircuits and synaptic

interactions. In: Björklund, A.; Hökfelt, T.; Wouterlood, F.G.; and van den Pol,

A.N., eds. Handbook of Chemical Neuroanatomy. Vol. 8. Amsterdam, The

Netherlands: Elsevier Science Publishers, 1990.

Björklund, H.; Olson, L.; Dahl, D.; and Schwarcz, R. Short- and long-term

consequences of intracranial injections of the excitotoxin, quinoiinic acid, as

evidenced by GFA immunohistochemistry of astrocytes. Brain Res 371:267-

277, 1986.

Boegman, R.J., and Parent, A. Differential sensitivity of neuropeptide Y,

somatostatin and NADPH-diaphorase containing neurons in rat cortex and

striatum to quinolinic acid. Brain Res 445:358-362, 1988.

Braak, H.; Braak, E.; Ohm, T.; and Bohl, J. Silver impregnation of Alzheimer’s

neurofibrillary changes counterstained for basophilic material and lipofuscin

pigment. Stain Technol C63(4):197-200, 1988.

Calderon, SF.; Sanberg, P.R.; and Norman, A.B. Quinolinic acid lesions of rat

striatum abolish D1

- and D2-dopamine receptor-mediated catalepsy. Brain

Res 450:403-407, 1988

Campbell, S.K.; Switzer, R.C. III; and Martin, T.L. Alzheimer’s plaques and

tangles: Control of silver staining through physical development. Neurosci

Abstr 13:678, 1987.

Carlsen, J., and de Olmos, J.S. Silver impregnation of degenerating neurons

and their processes. A modified cupric-silver technique. Brain Res 208:426-

431, 1981.

117
Carlsen, J.; de Olmos, J.; and Heimer, L. Tracing of two-neuron pathway in the

olfactory system by the aid of transneuronal degeneration: Projections to the

amygdaloid body and hippocampal formation. J Comp Neurol 208:196-208,

1982.

Commins, D.L., and Seiden, L.S. -Methyltyrosine blocks methylamphetamineinduced degeneration in the rat somatosensory cortex. Brain Res 365:15-20,

1986.

Commins, D.L.; Vosmer, G.; Virus, R.M.; Wooiverton, W.L.; Schuister, CR.,

and Seiden, L.S. Biochemical and histological evidence that

methylenedioxymethylamphetamine (MDMA) is toxic to neurons in the rat

brain. J Pharmacol Exp Ther 241(1):338-345, 1987.

Cowan, W.M.; Gottlieb, D.I.; Hendrickson, A.E.; Price, J.L.; and Woolsey, T.A.

The autoradiographic demonstration of axonal connections in the central

nervous system. Brain Res 37:21-51, 1972.

Crain, B.J.; Westerkam, W.D.; Harrison, A.H.; and Nadler, J.V. Selective

neuronal death after transient forebrain ischemia in the mongolian gerbil:

A silver impregnation study. Neuroscience 27:387-402, 1988.

Del Rio-Hortega, P. El “tercer elemento” de los centros nerviosos. 1. La

microglia enestado normal. 2. lntervención de la microglia en los procesos

patologicos. 3. Naturaleza probable de la microglia. Bol Sot Esp de Biol 9:69-

120, 1919.

Del Rio-Hortega, P. Microglia. In: Penfield, W., ed. Cytology and Cellular

Pathology of the Nervous System. Vol. 2. New York: Paul B. Hoeber, 1932.

pp. 483-534.

de Olmos, J.S. El sistema granular argirofilico en el cerebro del sapo y de la

tortuga. Acta Physiol Latinoam 16:41-42, 1966.

de Olmos, J.S. A cupric-silver method for impregnation of terminal axon

degeneration and its further use in staining granular argyrophilic neurons,

Brain Behav Evol 2:213-237, 1969.

de Olmos, J.S., and Beltramino, C.A. Early detection of neuronal degeneration

with a cupric-silver procedure after intrastriatal injections of quinolinic acid.

Anat Rec 229(4):21A, 1991.

de Olmos, J.S.; Beltramino, C.A.; Shaffrey, M.E.; Keomahathai, N.S.; and Jane,

J.A. Very early detection of ischemic neuronal degeneration in the CNS by an

amino-cupric-silver technique. Anat Rec Abstr, in press.

de Olmos, J.S.; Ebbesson, S.O.E.; and Heimer, L. Silver methods for the

impregnation of degenerating axopiasm. In: Heimer, L., and Robards,

M.J. Neuroanatomical Tract-Tracing Methods. New York: Plenum, 1981,

pp. 117-170.

de Olmos, J.S., and Ingram, W.R. An improved cupric-silver method for

impregnation of axonal and terminal degeneration. Brain Res 33:523-529,

1972.

Desclin, J.C., and Escubi, J. An additional silver impregnation method for

demonstration of degenerating nerve cells and processes in the central

nervous system. Brain Res 93:25-39, 1975.

118
Duchen, L.W. General pathology of neurons and neuroglia. In: Adams, J.H.;

Corsellis, J.A.N.; and Duchen, L.W., eds. Greenfield’s Neuropathology. 4th

ed. New York: Wiley-Medical, 1984. pp. 1-52.

Eager, R.P. Selective staining of degenerating axons in the central nervous

system by a simplified silver method. Spinal cord projections to external

cuneate and inferior olivary nuclei in the cat. Brain Res 22:137-141, 1970.

Eager, R.P., and Barnett, R.J. Studies on the chemical basis of the Nauta

technique. Anat Ret 148:368, 1964.

Eager, R.P., and Barnett, R.J. Morphological and chemical studies of Nautastained degenerating cerebeliar and hypothalamic fibers. J Comp Neurol

126:487-510, 1966.

Ebbesson, S.O.E. The selective silver-impregnation of degenerating axons and

their synaptic endings in nonmammalian species. In: Nauta, W.J.H., and

Ebbesson, S.O.E., eds. Contemporary Research Methods in Neuroanatomy.

Berlin: Springer-Verlag, 1970. pp. 132-162.

Ebbesson, S.O.E., and Rubinson, K. A simplified Nauta procedure. Physiol

Behav 4:281-282, 1969.

Edwards, S.B. The ascending and descending projections of the red nucleus in

the cat: An experimental study using an autoradiographic tracing method.

Brain Res 48:45-63, 1972.

El-Defrawy, S.R.; Boegman, R.J.; Jhamandas, K.; Beninger, R.J.; and Shipton,

L. Lack of recovery of cortical cholinergic function following quinolinic or

ibotenic acid injections into the nucleus basalis magnocellularis in rats. Exp

Neurol 91:628-633, 1986.

Evans, D.H.L., and Hamlyn, L.H. A study of silver impregnation methods in the

central nervous system. J Anat (London) 90:193-202, 1956.

Fink, R.P., and Heimer, L. Two methods for selective silver impregnation of

degenerating axons and their synaptic endings in the central nervous system.

Brain Res 4:369-374, 1967.

Foster, A.C.; Collins, J.F.; and Schwartz, R. On the excitotoxic properties of

quinolinic acid, 2,3-piperidine dicarboxylic acids and structurally related

compounds. Neuropharmacology 22(12A):1331-1342, 1983.

Foster, A.C., and Schwartz, R. Neurotoxic effects of quinolinic acid in the

mammalian central nervous system. In: Stone, T.W., ed. Quinolinic Acid and

the Kynurenines. Boca Raton, FL: CRC Press, 1989. pp. 173-192

Franck, J.E., and Roberts, D.L. Combined kainate and ischemia produces

“mesial temporal sclerosis.” Neurosci Lett 118:159-163, 1990.

Freund, T.F.; Buzsàki, G.; Leon, A.; Baimbridge, K.G.; and Somogyi, P.

Relationship of neuronal vulnerability and calcium-binding protein

immunoreactivity in ischemia. Exp Brain Res 83:55-66, 1990a.

Freund, T.F.; Buzsàki, G.; Leon, A.; Baimbridge, K.G.; and Somogyi, P.

Hippocampal cell death following ischemia: Effects of brain temperature

and anaesthesia. Exp Neurol 108:251-260, 1990b.

Gallyas, F. Physico-chemical mechanism of the argyrophil III reaction.

Histochemistry 74:409-421, 1982a.

119
Gallyas, F. Equation of the mass-time relationship of the argyrophil I and

argyrophil III reactions. Histochemistry 74:423-433, 1982b.

Gallyas, F.; Güldner, F.H.; Zoltay, G.; and Wolff, J.R. Golgi-like demonstration

of “dark” neurons with an argyrophil II method for experimental

neuropathology. Acta Neuropathol 79:520-628, 1990.

Gallyas, F., and Wolff, J.R. Metal-catalyzed oxidation renders silver

intensification selective: Applications for the histochemistry of

diaminobenzidine and neurofibrillary changes. J Histochem Cytochem

34(12):1667-1672, 1986.

Gallyas, F.; Wolff, J.R.; Böttcher, H.; and Záborszky, L. Selective demonstration

of axonal degeneration. Stain Technol 55:291-297, 1980b.

Gallyas, F.; Wolff, J.R.; Böttcher, H.; and Záborszky, L. A reliable and sensitive

method to locate terminal degeneration and lysosomes in the CNS. Stain

Technol 55:299-306, 1980c.

Gallyas, F.; Záborszky, L.; and Wolff, J.R. Experiments on the mechanism of

impregnation methods demonstrating axonal and terminal degeneration,

Stain Technol 55:281-290, 1980a.

Gerfen, C.R., and Sawchenko, P.E. An anterograde neuronatomical tracing

method that shows the detailed morphology of neurons, their axons and

terminals: lmmunohistochemical localization of an axonally transported plant

lectin. Phaseolus vulgaris-leucoagglutinin (PHA-L). Brain Res 290:219-238,

1984.

Giolli, R.A. A note on the chemical mechanism of the Nauta-Gygax technique.

J Histochem Cytochem 13:206-210, 1965.

Giolli, R.A., and Karamanlidis, A.N. The study of degenerating nerve fibers

using silver-impregnation methods. In: Robertson, R.T., ed. Methods in

Physiological Psychology: Neuroanatomical Research Techniques. Vol II.

New York: Academic Press, 1978. pp. 21 1-240.

Glees, P. Terminal degeneration within the central nervous system as studied

by a new silver method. J Neuropathol Exp Neurol 5:54-59, 1946.

Gonzalez, M.F.; Shiraishi, K.; Hisanaga, K.; Sagar, S.M.; Mandabach, M.; and

Sharp, F.R. Heat shock proteins as markers of neural injury. Mol Brain Res

6:93-100, 1989.

Grafe, M.R., and Leonard, C.M. Successful silver impregnation of degenerating

axons after long survivals in the human brain. J Neuropathol Exp Neurol

39(5):555-574, 1981.

Grant, G. Neuronal changes central to the site of axon transection. A method

for the identification of retrograde changes in perikarya, dendrites and axons

by silver impregnation. In: Nauta, W.J.H., and Ebbesson, S.O.E., eds.

Contemporary Research Methods in Neuroanatomy. Berlin: Springer-Verlag,

1970. pp. 173-185.

Graybiel, A.M.; Moratalla, R.; and Robertson, H.A. Amphetamine and cocaine

induce drug-specific activation of the c-fos gene in striosome-matrix

compartments and limbic subdivisions of the striatum. Proc Natl Acad Sci

U S A 87:6912-6916, 1990.

120
Hamlyn, L.H. Application of the Nauta-Gygax technic for degenerating axons to

mounted sections. Stain Technol 32:123-126, 1957.

Hedreen, J.C., and Chalmers, J.P. Neuronal degeneration in rat brain induced

by 6-hydroxydopamine: A histological and biochemical study. Brain Res

47:1-36, 1972.

Hedreen, J.C.; Raskin, L.S.; Struble, R.G.; and Price, D.L. Selective silver

impregnation of senile plaques: A method useful for computer imaging.

J Neurosci Methods 25:151-158, 1988.

Heimer, L. Synaptic distribution of centripetal and centrifugal nerve fibers in the

olfactory system of the rat. An experimental anatomical study. J Anat

103:413-432, 1968.

Heimer, L. Selective silver-impregnation of degenerating axoplasm. In: Nauta,

W.J.H., and Ebbesson, S.O.E., eds. Contemporary Research Methods in

Neuroanatomy. Berlin: Springer-Verlag, 1970. pp. 106-131.

Heimer, L., and Kalil, R. Rapid transneuronal degeneration and death of cortical

neurons following removal of the olfactory bulb in adult rats. J Comp Neurol

178(3):559-609, 1978.

Heimer, L., and Peters, A. An electron microscope study of a silver stain for

degenerating boutons. Brain Res 8:337-346, 1968.

Heimer, L., and Záborszky, L., eds. Neuroanatomical Tract-Tracing Methods 2:

Recent Progress. New York: Plenum Press, 1989.

Heimer, L.; Záborszky, L.; Zahm, D.S.; and Alheid, G.H. The ventral

striatopallidothalmic projection. I. The striatopallidal link originating in striatal

parts of the olfactory tubercle. J Comp Neurol 255:571-591, 1987,

Hjorth-Simonsen, A. Fink-Heimer silver impregnation of degenerating axons

and terminals in mounted cryostat sections of fresh and fixed brains. Stain

Technol 45:199-204, 1970.

Hoff, E.C. Central nerve terminals in the mammalian spinal cord and their

examination by experimental degeneration. Proc R Soc Lond [Biol] 111:175-

188, 1932a.

Hoff, E.C. The distribution of spinal terminals (boutons) of the pyramidal tract

determined by experimental degeneration. Proc R Soc Lond [Biol] 111:226-

237, 1932b.

Hubel, D.H., and Wiesel, T.N. Anatomical demonstration of columns in the

monkey striate cortex. Nature 221:747-750, 1969.

Illis, L.S., and Mitchell, J. The effect of tetanus toxin on boutons terminaux.

Brain Res 18:283-295, 1970.

Jancs , G., and Király, E. Distribution of chemosensitive primary sensory

afferents in the central nervous system of the rat. J Comp Neurol 190:781-

792, 1980.

Janssen, R.; Schweitzer, L.; and Jensen, K.F. Glutamate neurotoxicity in the

developing rat cochlea: Physiological and morphological approaches. Brain

Res 552:255-264, 1991.

Jarrard, L.E. Selective hippocampal lesions and behaviour: Effects of kainic

acid lesions on performance of place and cue tasks. Behav Neurosci 97:873-

889, 1983.

121
Jarrard, L.E. On the use of ibotenic acid to lesion selectively different

components of the hippocampal formation. J Neurosci Methods 29:251-259,

1989.

Jensen, K.F.; Miller, D.B.; Olin, J.K.; Haykai-Coates, N.; and O’Callaghan, J.P.

Characterization of methylenedioxymethamphetamine (MDMA) neurotoxicity

using the de Olmos cupric-silver stain and GRAP immunohistochemistry:

Changes in specific neocortical regions implicate serotonergic and nonserotonergic targets. Soc Neurosci Abstr 17(2):1429, 1991.

Jensen, K.F.; Miller, D.B.; Olin, J.K.; and O’Callaghan, J.P. Evidence for the

neurotoxicity of methylenedioxymethamphetamine (MDMA) using a cupricsilver stain for neuronal degeneration. Soc Neurosci 16(1):256, 1990.

Kalaha-Brunst, C.; Giolli, R.A.; and Creel, D.J. An improved silver impregnation

method for tracing degenerating nerve fibers and their terminals in frozen

sections. Brain Res 82:279-283, 1974.

Karten, H.J.; Hodos, W.; Nauta, W.J.H.; and Revzin, A.M. Neural connections

of the “visual wulst” of the avian telencephalon. Experimental studies in the

pigeon (Columba livia) and owl (Speotyto cunicularia). J Comp Neurol

150(3):253-277, 1973.

Kitagawa, K.; Matsumoto, M.; Ninobe, M.; Miroshiba, K.; Hata, R.; Ueda, H.;

Handa, H.; Fukunaga, R.; Isaka, Y.; Kimura, K.; and Kamada, T. Microtubuleassociated protein 2 as a sensitive marker for cerebral ischemic damageimmunohistochemical investigation of dendritic damage. Neuroscience

31(2):401-411, 1989.

Kristensson, K.; Olsson, Y.; and Sjöstrand, J. Axonal uptake and retrograde

transport of exogenous proteins in the hypoglossal nerve. Brain Res 32:399-

406, 1971.

Kuypers, H.G.J.M.; Catsman-Berrevoets, C.E.; and Padt, R.E. Retrograde

axonal transport of fluorescent substances in the rat’s forebrain. Neurosci Lett

6:127-135, 1977.

Lasek, R.; Joseph, B.S.; and Whitlock, D.G. Evaluation of a radioautographic

neuroanatomical tracing method. Brain Res 8:319-336, 1968.

LaVail, J.H., and LaVail, M.M. Retrograde axonal transport in the central

nervous system. Science 176:1416-1417, 1972.

Leonard, C.M. Degeneration methods in neurobiology. Trends Neurosci 2:156-

159, 1979.

Liesegang, R.E. Die Kolloid-Chemie der histologischen Silberfärbung. Kolloid

Beitr 3:1-46, 1911.

Lin, C.-S.; Polsky, K.; Nadler, J.V.; and Crain, B.J. Selective neocortical and

thalamic cell death in the gerbil after transient ischemia. Neuroscience

35(2):289-299, 1990.

Lindquist, S. The heat-shock response. Annu Rev Biochem 55:1151-1191,

1986.

Loewy, A.D. Ammoniacal silver staining of degenerating axons. Acta

Neuropathol 14:226-236, 1969.

122
Lohman, A.H.M., and Mentink, G.M. Some cortical connections of the Tegu

Lizard “Tupinammbias teguixin.” Brain Res 45:325-344, 1972.

Loots, J.M.; Loots, G.P.; and Joubert, W.S. A silver impregnation method for

nervous tissue suitable for routine use with mounted sections. Stain Technol

52:85-87, 1977.

Maler, L.; Fibiger, H.C.; and McGeer, P.L. Demonstration of the nigrostriatal

projection by silver staining after nigral injections of 6-hydroxydopamine.

Exp Neurol 40:505-515, 1973.

Marchi, V., and Algeri, G. Sulle degenerazioni discendenti consecutive a lesioni

sperimentale in diverse zone della corteccia cerebrale. Riv Sper Freniatr Med

Leg 11:492-494, 1885.

Morgan, J.I., and Curran, T. Stimulus-transcription coupling in neurons: Role of

cellular immediate-early genes. Trends Neurosci 12(11):459-462, 1989.

Nauta, W.J.H. Über die sogenannte terminale Degeneration im

Zentralnervensystem und ihre Darstellung durch Silberimpragnation. Schweiz

Arch Neurol Psychiatr 66:343-376, 1950.

Nauta, W.J.H., and Ebbesson, S.O.E., eds. Contemporary Research Methods

in Neuroanatomy. Berlin: Springer-Verlag, 1970.

Nauta, W.J.H., and Gygax, P.A. Silver impregnation of degenerating axon

terminals in the central nervous system (1) technic (2) chemical notes. Stain

Technol 26:5-11, 1951.

Nauta, W.J.H., and Gygax, P.A. Silver impregnation of degenerating axons in

the central nervous system: A modified technic, Stain Technol 29:91-93,

1954.

Okazaki, H., ed. Fundamentals of Neuropathology: Morphologic Basis of

Neurologic Disorders. 2d ed. Tokyo: Igaku-Shoin Medical Publishers, 1989.

Peters, A. Experiments on the mechanism of the silver staining. Part I.

Impregnation. J Microsc Sci 96:84-102, 1955a.

Peters, A. Experiments on the mechanism of the silver staining. Part II.

Development. J Microsc Sci 96:103-115, 1955b.

Peters, A. Experiments on the mechanism of the silver staining. Part III.

Quantitative studies. J Microsc Sci 96:301-316, 1955c.

Price, J.L. Rapid transneuronal degeneration in the piriform cortex of the rat.

Soc Neurosci Abstr 2:161, 1976.

Ramón y Cajal, S. Algunos metodos de coloratión de los cilindros-ejes,

neurofibrillas y vidos nerviosos. Trab Lab Invest Biol 3:1, 1904.

Rasdolksky, J. Über die Endigung der extraspinalen Bewegungssysteme in

Rückenmark. Z Gesamte Neurol Psychiatr 86:361-374, 1923.

Rasdolsky, J. Beiträge zur Architektur der grauen Substanz des Rückenmarks

(Unter Benutzung einer neuen Methode der Färbung der

Nervenfasernkollateralen). Virchows Arch (Pathol. Anat.) 257:356-363, 1925.

Ricaurte, G.A.; Guillery, R.W.; Seiden, L.S.; Schuster, C.R.; and Moore, R.Y.

Dopamine nerve terminal degeneration produced by high doses of

methamphetamine in the rat brain. Brain Res 235:93-103, 1982.

123
Richardson, K.C. Studies on the structure of autonomic nerves in the small

intestine, correlating the silver-impregnated image in light microscopy with the

permanganate-fixed ultrastructure in electron-microscopy. J Anat 94:457-472,

1960.

Ritter, S., and Dinh, T.T. Capsaicin-induced neuronal degeneration: Silver

impregnation of cell bodies, axons, and terminals in the central nervous

system of the adult rat. J Comp Neurol 271:79-90, 1988.

Ritter, S., and Dinh, T.T. Prior optic nerve transection reduces capsaicininduced degeneration in rat subcortical visual structures. J Comp Neurol

308:79-90, 1991.

Robertson, H.A., and Robertson, G.S. L-dopa and D1-selective agonists

activate C-fos expression in the striatum of 6-hydroxydopamine-lesioned rats.

Soc Neurosci Abstr 15(1):782, 1989.

Sagar, S.M.; Sharp, F.R.; and Curran, T. Expression of c-fos protein in brain:

Metabolic mapping at the cellular level. Science 240:1328-1331, 1988.

Samuel, E.P. Impregnation and development in silver staining. J Anat 87:268-

277, 1953a.

Samuel, E.P. The mechanism of silver staining. J Anat 87:278-287, 1953b.

Saper, C.B.; Wainer, B.H.; and German, D.C. Axonal and transneuronal

transport in the transmission of neurological disease: Potential role in system

degenerations, including Alzheimer’s disease. Neuroscience 23(2):389-398,

1987.

Schimert, J. Die Endigungsweise des Tractus vestibulospinalis. Z Anat

Entwicklungsgesch 108:761-767, 1938.

Schimert, J. Das Verhalten der Hinterwurzelkollateralen im Rückenmark.

Z Anat Entwicklungsgesch 109:665-687, 1939.

Schmidt-Kastner, R., and Freund, T.F. Selective vulnerability of the

hippocampus in brain ischemia. Neuroscience 41(3):599-636, 1991.

Schweitzer, L.; Jensen, K.F.; and Janssen, R. Glutamate neurotoxicity in rat

auditory system: Cochlear nuclear complex. Neurotoxicol Teratol 13:189-193,

1991.

Sevier, A.C., and Munger, B.L. A silver method for paraffin sections of neural

tissue. J Neuropathol Exp Neurol 24:130-135, 1965.

Switzer, R.C. III. Neural argyrophilia induced by puromycin: A directed Golgilike method. Neurosci Lett 2:301-305, 1976.

Switzer, R.C. III. Strategies for assessing neurotoxicity. Neurosci Biobehav Rev

15:89-93, 1991.

Switzer, R.C. III; Merril, C.R.; and Shjifrin, S. A highly sensitive silver stain for

detecting proteins and peptides in polyacrylamide gels. Anal Biochem 98:231-

237, 1979.

Szentágothai-Schimert, J. Die Endigungsweise der absteigenden

Riickenmarksbahnen. Z Anat Entwicklungsgesch 111:322-330, 1941.

Tanaka, D., Jr., and Bursian, S.J. Degeneration patterns in the chicken central

nervous system induced by ingestion of the organophosphorus delayed

neurotoxin tri-ortho-tolyl phosphate. A silver impregnation study. Brain Res

484:240-256, 1989.

124
Taylor, R. A lot of “excitement” about neurodegeneration. Science 252:1380-

1381, 1991.

Uyematsu, S. A study of the cortical olfactory apparatus. Arch Neurol Psychiatry

6:146-156, 1921.

van den Pol, A.N., and Gallyas, F. Trauma-induced Golgi-like staining of

neurons: A new approach to neuronal organization and response to injury.

J Comp Neurol 296:654-673, 1990.

Van der Kooy, D.; Kuypers, H.G.J.M.; and Catsman-Berrevoets, C.E. Single

mammillary body cells with divergent axon collaterals. Demonstration by a

simple, fluorescent retrograde double labeling technique in the rat. Brain Res

158:189-196, 1978.

von Fajersztajn, J. Ein neues Silberimprägnations-vetfahren als Mittel zur

Fäbung der Achencylinder. Neurol Centralbl 20:98-106, 1901.

Voogd, J.; Feirabend, H.K.P.; and Schoen, J.H.R. Cerebellum and

precerebellar nuclei. In: Paxinos, G., ed. The Human Nervous System.

London: Academic Press, 1990. pp. 321-386.

Waller, A.V. Experiments on the section of the glossopharyngeal and

hypoglossal nerves of the frog, and observations of the alterations produced

thereby in the structure of their primitive fibers. Phil Trans R Soc Lond[Biol]

140:423-469, 1850.

Wheeler, D.A., and Ritter, S. A modification of the Carlsen-de Olmos cupricsilver impregnation method for use on mounted cryostat sections. Soc

Neurosci Abstr 10:424, 1984.

White, L.E., Jr. Enhanced reliability in silver impregnation of terminal axonal

degeneration-original Nauta method. Stain Technol 35:5-9, 1960.

Whittington, D.L.; Woodruff, M.L.; and Baisden, R.H. The time-course of

trimethyltin-induced fiber and terminal degeneration in hippocampus.

Neurotoxicol Teratol 11:21-33, 1989.

Wiitanen, J.T. Selective silver impregnation of degenerating axons and axon

terminals in the central nervous system of the monkey (Macaca mulatta).

Brain Res 14:546-548, 1969.

Wiley, R.G., and Stirpe, F. Modecin and volkensin but not abrin are effective

suicide transport agents in rat CNS. Brain Res 438:145-154, 1988.

Winkler, C.A. The olfactory tract in the rabbit. Opera Omnia 5:397-413, 1918.

Wolff, J.R.; Eins, S.; Holzgraefe, M.; and Záborszky, L. The temporo-spatial

course of degeneration after cutting cortico-cortical connections in adult rats.

Cell Tiss Res 214:303-321, 1981.

Wolman, M. Studies of the impregnation of nervous tissue elements, Q J

Microsc Sci 103:297-310, 1955.

Yamaguchi, H.; Haga, C.; Hirai, S.; Nakazato, Y.; and Kosaka, K. Distinctive,

rapid, and easy labeling of diffuse plaques in the Alzheimer brains by a new

methenamine silver stain. Acta Neuropathol 79:569-572, 1990.

Yamamoto, T., and Hirano, A. A comparative study of modified Bielschowsky,

Bodian and thioflavin S stains on Alzheimer’s neurofibrillary tangles.

Neuropathol Appl Neurobiol 12:3-9, 1986.

125
Yanagihara, T. lmmunohistochemical assessment of cerebral ischemia. In:

Schurr, A., and Rigor, B.M., eds. Cerebral lschemia and Resuscitation. Boca

Raton, FL: CRC Press, 1990. pp. 389-412.

Young, ST.; Porrino, L.J.; and ladarola, M.J. Induction of C-fos by direct and

indirect dopamine agonists. Soc Neurosci Abstr 15(2):1091, 1989.

Záborszky, L.; Alheid, G.F.; and Heimer, L. Mapping of transmitter-specific

connections: Simultaneous demonstration of anterograde degeneration and

changes in the immunostaining pattern induced by lesions, J Neurosci Meth

14:255-266, 1985.

Záborszky, L., and Wolff, J.R. Distribution patterns and individual variations of

callosal connections in the albino rat. Anat Embryol 165:213-232, 1982.

DISCUSSION

Q (Ricaurte): Have you come across the situation where a toxin can produce

an insult and induce silver positivity as visualized by one of these methods, only

to find later that it was a reversible change-that, in fact, a particular terminal or

fiber or cell body did not degenerate, but there was just a reversible change in

the cytoplasm?

A: I have not: I haven’t really looked that much into neurotoxicology, until

preparing for this talk.

A (O’Callaghan): I think George Kreutzberg at Max Planck spent several years

studying a model of the facial nerve and looked at the cell-body reaction in

facial nucleus, at microglial reaction, and at silver reactions in that cell body.

The cell body does not degenerate. It gives you the classic cell-body reactionsilver degeneration pattern around the cell body-and you get a gliosis around

the cell body. All that is reversible.

Q (Ricaurte): Is it a silver reaction of the cell body of origin?

A (Jensen): I think it is. I am not sure.

COMMENT (Ricaurte): At an early stage, degenerating cell bodies are highly

silver positive. They are very easy to see. However, if someone comes along

4 weeks later using staining techniques and looks in the same area, they might

conclude that there was no cell loss, when in fact, there is. This is difficult to

detect. To show that argyrophilic is a reversible change, someone would need

to go back and do a quantitative study and show that the same cell body that

was silver positive was still present weeks later. Clearly, this would be a difficult

and painstaking task. I always introduce the Fink-Heimer method as one of the

few, if not only, anatomical techniques (at the light microscopic level) that can

show fiber or terminal degeneration.

126
COMMENT (Heimer): I always thought that way.

Q (Seiden): Is the chemical basis for silver reaction in the dying nerve

understood at all?

A: I do not think so. I am not a chemist, but I have read the many papers that

have been written on the mechanism of silver staining. There was a series of

papers published by Alan Peters, many years ago, and he suggested that there

might be a protein, that is, an enzyme, that is argyrophilic. Maybe one way to

think of it is that whatever the argyrophilic substance may be, much more of it

becomes available to react with silver during the process of degeneration. In

the various Nauta methods, potassium permanganate treatment is a key factor

in the suppressive effect of argyrophilic. The argyrophilic-suppressing effect of

permanganate may depend on oxidation of the argyrophilic protein. If you apply

a little “suppression,” you suppress the few available sites for protein-silver

reaction in normal fibers; if you continue the suppression, you also eliminate the

many sites that have become available in degenerating fibers, and with too long

suppression, everything disappears.

Q (Cho): Are these cells permeable with silver? The silver really permeates

these cells?

A: Yes.

Q (Cho): It distributes like water?

A: Yes. Everything is basically argyrophilic in the tissue. You can get silver

into lysosomes and mitochondria.

Q: In the intact cell with no cell damage?

A: Absolutely.

Q (Molliver): A few years ago, Ray Lasek at Case Western reported that silver

stained intermediate filaments. Is there any commonality with silver

degeneration stains? Sternberger has several antibodies against

phosphorylated intermediate filament proteins, and these accumulate in the

damaged cell bodies. Have you looked at structures that stain with silver?

A: No, I haven’t.

COMMENT (Molliver): The neurofilaments are synthesized in the cell body,

transported into the axon, and are phosphorylated; but when axons are

damaged, they accumulate in the cell body.

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Q (Tilson): I have a couple of quick practical questions. As a neurotoxicologist,

you have these changes in silver staining. How comfortable would you be in

calling changes in silver staining following some treatment an adverse effect?

Would you accept an increase in silver stain intensity as evidence of an adverse

effect on the CNS?

A: I am not sure. I think we have to have more studies in this field, and I think

that what is really missing are systematic studies of these processes at the

electromicroscopic level. That might give us some answers in regard to what

happens to these cells that take up silver so quickly. I think there are still many

questions in need of answers.

Q (Tilson): If you saw no changes in silver staining, how comfortable would

you feel in saying that that treatment is not neurotoxic?

A: No, I wouldn’t be comfortable at all, because if there is one thing we

neuroanatomists have learned the hard way, it is that you should never trust

negative results. In short, that answers the question.

Q (Tilson): I have been told that traditional neuroanatomists believe that

20 percent of the cells in an area need to be affected before you can detect a

change at the anatomical level. Do you agree with that?

A: Twenty percent of the cell population ? No, I wouldn’t say so. I think that

you can see neurotoxicity even if only a few cells are affected, provided you

have a good silver stain.

C (Tilson): I meant with the Nissl stain.

A: I agree with you. I think that half the cells in a given area can be affected,

and you still can’t see it.

Q (Tilson): Our testing guidelines for using hematoxylin and eosin and other

stains would seem to be sensitive only to 20- to 50-percent changes. But with

the silver staining, you say that the proportion of cells that would have to die to

see a detectable change would be considerably less?

A: Oh, yes.

Q: What do you think the cupric method is adding to silver to enhance the

sensitivity?

A: The addition of cupric salts improves the staining, probably by having a

suppressive effect on normal tissue argyrophilic, thereby making the silver

impregnation more manageable, as Dr. Gallyas and his coworkers have

128
suggested. Interestingly, Dr. de Olmos discovered the utility of cupric salts

serendipitously; he dropped a beaker containing sections in a modified

Bielschowsky silver solution on a warm copper plate. Eager to save the

material, he collected the sections and the contaminated silver solution floating

on the copper plate. Later he inspected the sections in the microscope, and, to

his amazement, he discovered that the intensity and quality of the silver stain

were dramatically improved. Dr. de Olmos quickly deduced that the contact of

the silver solution with the copper plate was the key factor, and then it was only

a matter of choosing the right cupric solution to repeat this result. The situation

recalls Pasteur’s famous line: “Where observation is concerned, chance favors

only the prepared mind.”

Q: After quinolinate, you can show the degeneration in the cortex after 15

minutes with cupric silver. Have you tried this with another stain?

A: I have not, but I believe Jose de Olmos has tried without much success.

Q (Landfield): Of course, the silver techniques are very valuable for detecting

degeneration. But so many things in neurotoxicology and neuropathology

require quantitative analysis, and I wonder if you would comment on what your

feeling is about the application of these for a quantitative method or whether

there is a potential for it.

A: Yes, that was why I showed the Gallyas technique. I think that with one or

the other of Gallyas’ techniques, one can direct to a certain point the silver to

specific components of the neuron, for example, to degenerating axons or to

the degenerating terminals. It can be done in a systematic fashion with great

reproducibility, so that one can make a computer analysis of, for example, the

density of degenerating boutons. One can follow it over time; it reaches a peak,

and then it goes down. Although I don’t have any experience myself, I think

there is at least some potential with a technique like that.

Q: How about comparing animal to animal? I mean, that was within the same

animal?

A: Yes, the pictures I showed were in the same animal. Well, again, we have

not done it. I have to take their word for it. I think Gallyas and his collaborators

mention this in their papers-even between animals.

COMMENT (Jensen): De Olmos’ most recent modifications are extremely

reproducible from animal to animal.

A (Heimer): By the way, de Olmos’ recent modification is not published yet.

129
Q: You see silver degeneration in 15 minutes if you inject quinolinic acid in the

striatum and look in the temporal cortex? What kind of mechanism would kill a

cell that fast?

A: Good thought. I don’t know-maybe from transneuronal excitotoxicity?

Q: I mean, axonal transport presumably couldn’t get the compound back there

that quickly. Do the cell bodies in the striatum following quinolinic acid

injection? Do they take up the silver? In 15 minutes?

A: Yes, you have a central area which is relatively resistant to argyrophilic; but

in the surrounding area, you do have argyrophilic within the striatum.

COMMENT (Olney): We studied kainic acid toxicity with the de Olmos method

back around 1980 with Joe Price and Jim Schroll. In that case, I think we were

studying seizure-mediated pathology, and so I hesitate to relate it directly to

quinolinate toxicity, which is for the most part, I think, not seizure related.

But we saw the same thing and in many brain regions following subcutaneous

treatment with kainic acid. We saw neurons taking up the silver, showing their

entire arborization, and looking beautiful-but rather sick, probably, some of it.

But we had the same questions. Are these neurons whose cell bodies are

taking up the silver, not just the axons? And if the cell bodies are taking it up,

are they taking it up directly, or is the degenerative process starting at the cell

body or at the axon; and is there a retrograde process here? We have no

answer, but it was seizure-mediated pathology through many seizure circuits

that we were studying.

Q (for Olney from Seiden): What do you think?

A (Olney): I don’t know. I have looked at these kinds of lesions with electron

microscopy and other methods as well as the silver. With striatal lesions, there

is a degenerative reaction that you can see in the cortex, the deep layers of the

cortex, primarily.

COMMENT (Heimer): Excuse me for interrupting. This is interesting because

the degeneration we see is in the superficial layers.

COMMENT (Olney): It is a later degeneration that I am talking about in the

deep layers. But, in addition, if you are talking about degeneration that occurs

in the frontal cortex, for example, I think you mentioned; and if it is ipsilateral to

the striatal lesion-that is, tissue that you have just been poking your needle

through-there is reflux of the quinolinate up around the needle track and it

gets into the cortex and spreads around up in the cortex, causing some direct

excitotoxic damage in the cortex. That is something that I think could happen

130
in 15 minutes and could explain some degeneration in the cortex. But I don’t

know about the temporal cortex.

COMMENT (Heimer): Well, that is an important comment. These are the kinds

of things that would be really important to study.

COMMENT (Olney): And quinolinate is also a peculiar excitotoxin. It has

some characteristics of kainic acid and some of NMDA [N-methyl-D-aspartate].

The NMDA lesion tends to be very circumscribed to the area of injection.

Kainic acid is spread all over the brain because it causes seizure pathology.

All the pathways that interconnect the injection site with the rest of the brain

transneuronally throughout the whole limbic system, in fact, all those systems

get turned on with the propagating excitotoxic mechanism, in which glutamate is

being released at many points throughout the brain. And quinolinate has some

of that property, but not anywhere near as much as kainic acid.

Q (Seiden): In talking with Dr. de Olmos, I found that he could partially block

some of the degeneration with, not MK801, but a different antagonist.

COMMENT (Olney): MK801 would do that. He didn’t try it? He didn’t try

MK801?

COMMENT (Seiden): No, he didn’t, he used one of the other. . .

COMMENT (Olney): MK801 would do that. And tomorrow I am going to show

you that MK801 will also produce its own toxicity in the cortex, and there is a

heat shock protein response to that which is very interesting in relation to the

question about Fos proteins.

COMMENT (Jensen): He used ketamine.

COMMENT (Olney): Yes, ketamine and MK801 have the same mechanism.

ACKNOWLEDGMENTS

The work described in this chapter was supported by U.S. Public Health Service

grants NS-17743 (LH) and NS-23945 (LZ), Fogarty Fellowship TW-04465 (CB),

Consejo National de lnvestigaciónes Cientificas y Tecnicas de Argentina (J de

0), and by OTKA-218 (FG).

131
AUTHORS

Carlos A. Beltramino, M.D., Ph.D.

Forgarty Fellow

Department of Otolaryngology-Head and Neck Surgery

Lennart Heimer, M.D.

Professor

Department of Otolaryngology-Head and Neck Surgery

Department of Neurological Surgery

Lázló Záborszky, M.D., Ph.D.

Associate Professor

Department of Otolaryngology-Head and Neck Surgery

University of Virginia Health Sciences Center

Box 430

Charlottesville. VA 22908

José S. de Olmos, Ph.D.

Research Scientist

lnstituto de Investigación Médica

Casilla de Correo 389

500 Cordoba

ARGENTINA

Ferenc Gallyas, Ph.D.

Professor

Department of Neurosurgery

University of Pécs

Rét utca 2

7623 Pécs

HUNGARY

132
Mapping Toxicant-Induced Nervous

System Damage With a Cupric Silver

Stain: A Quantitative Analysis of

Neural Degeneration Induced by 3,4-

Methylenedioxymethamphetamine

Karl F. Jensen, Jeanene Olin, Najwa Haykal-Coates,

James O’Callaghan, Diane B. Miller, and José S. de Olmos

INTRODUCTION

Evidence continues to accumulate implicating toxic substances in the etiology

of a variety of neurological diseases. Consequently, the process of identifying,

understanding, and regulating neurotoxic substances remains a pressing

challenge. This challenge is complex because toxicants can injure the nervous

system in a variety of ways. In addition, knowledge of the structure and

function of the nervous system is not sufficient to entrust a single endpoint with

predicting the neurotoxic potential of a substance. The purpose of structural

assessments is to provide a convincing picture of the location and extent of

damage to the nervous system. Silver stains that selectively reveal neural

degeneration hold particular promise in this regard. Evidence that such

histological procedures can selectively stain degenerating neurons has

emerged from more than 25 years of anatomical tract tracing experimentation

(Beltramino et al., this volume). In this chapter, the authors describe

preliminary results using a recently developed cupric silver stain (de Olmos

et al., in press) to delineate areas of the brain damaged by administration of

3,4-methylenedioxymethamphetamine (MDMA).

MDMA causes dramatic and long-lasting neurochemical alterations in the brain

(Fuller 1989; Commins et al. 1987a; McKenna and Peroutka 1990; Schmidt

1987; Ricaurte et al. 1988; Scallet et al. 1988; Bittner et al. 1981; Schmidt and

Taylor 1987; Stone et al. 1987; Davis et al. 1987; Schmidt et al. 1986). The

extent to which MDMA-induced neurochemical alterations reflect neural

degeneration remains controversial. To address this issue, the authors have

mapped regions of the brain stained with the cupric silver method following

administration of MDMA. Although a better understanding of the chemical

133
basis of the stain’s selectivity for degenerating neurons is needed before the

observed staining can be uniquely associated with particular degenerative

events, the present results demonstrate that the cupric silver stain is a useful

tool in determining the location and extent of structural alterations resulting from

exposure to a toxicant.

ANALYSIS

MDMA (0, 25, 50, 150 mg/kg as salt) was administered to Long-Evans rats four

times, with a 12-hour interdosing interval. There were four animals per dose

group. The animals were housed individually in wire-bottom hanging cages (to

prevent hyperthermia-induced mortality) and allowed food and water ad libitum.

At various times after the last dose, animals were anesthetized with pentobarbital,

perfused intracardially with saline followed by 4 percent paraformaldehyde in

0.1 M cacodylate buffer (pH 7.4). After perfusion, the brains were removed and a

4-mm sagittal block of the cerebrum was frozen and sectioned at 40 microns. All

sections were collected and allocated to one of six sets. Sets of sections were

stained with either cresyl violet or the cupric silver stain. Regions of silver staining

unique to the treated animals were traced from four homologous sections from a

0.72 mm sample of the sagittal block and reconstructed using a computerized

serial section reconstruction system. A survival time of 48 hours was employed

for the dose-response analysis. For the time-course analysis, MDMA (100 mg/kg)

was administered four times, with a 12-hour interdosing interval, except for

animals in the group labeled “2x,” in which MDMA (100 mg/kg) was administered

only twice. Four animals were sacrificed at each survival time (18 hours, 48

hours, 60 hours, 7 days, and 14 days). An additional four animals were treated

with fluoxetine (5 mg/kg) or MK-801 (1 mg/kg) 30 minutes prior to each of four

doses of MDMA (100 mg/kg). Control groups included either fluoxetine or

MK-801 or MDMA alone. Neurochemical evaluations of animals from

corresponding cohorts are reported by O’Callaghan and Miller (this volume).

RESULTS

Evidence of cupric silver staining could be observed in several regions in control

animals. Consistent and intense staining was apparent in the olfactory bulb.

Diffuse and very light terminal-like staining was occasionally observed in the

hippocampus. Scattered stained neurons occasionally could be seen in the

forebrain. With the exception of the olfactory bulb and hippocampus, staining

in control animals was sufficiently minimal to allow for a systematic comparison

of different brain regions following administration of MDMA.

In animals treated with MDMA and sacrificed 48 hours later, the intensity of

staining in the neocortex is dose related (figures 1 and 2). The staining is most

prominent in the frontoparietal region of the neocortex. The majority of stained

elements within the neocortex are axons and terminals. At lower doses (4x25 to

134
FIGURE 1. Silver staining in sagittal sections of frontoparietal cortex in

animals sacrificed 48 hours following various doses of MDMA.

High magnification of upper neocorticel layers. Panels A through

D: scale bar=100 mm. Lower magnification of frontoparietal

cortex. Panels E through H: scale bar=1 mm. Control (panels A

and E). four doses of 25 mg/kg given 12 hours apart over 2 days

(panels B and F).

4x50 mg/kg), staining is primarily in the upper layers, whereas at higher doses

(4x75 to 4x150 mg/kg), it reaches deeper layers. Perikaryal staining is more

pronounced at the higher doses.

Silver staining is not confined to the frontoparietal neocortex. In some cases,

pronounced staining occurs in other brain regions, including the striatum and

thalamus (figures 3 and 4). Such staining does not occur in all animals in any

one dose group. For example, one animal receiving 4x75 mg/kg MDMA

exhibited extensive staining within the striatum and thalamus (figure 3, panels

A and D, and figure 4), whereas another animal receiving the same dose and

exhibiting the same degree of damage within the neocortex did not exhibit

staining in the striatum (figure 3, panels C and D). The predominant type of

staining differed in the various brain regions. Although terminal, axonal, and

perikaryal staining all were present in the neocortex, striatal staining appeared

to be almost exclusively of terminals, and the thalamus exhibited staining of

both axons and terminals.

135
FIGURE 2. Volumetric reconstructions of regions of intense silver staining

(shaded areas) in the frontoparietal neocortex from four Sagittal

sections after various doses in mg/kg of MDMA.

Silver staining was apparent after the lowest dose (4x25 mg/kg), and the

volume of staining in the neocortex and striatum was dose related (figure 5).

The volume of degeneration was evaluated at various times after repeated

doses of 100 mg/kg (figure 6). Substantial staining was evident as early as

18 hours after only two doses of 100 mg/kg. The extent of staining was only

slightly smaller than that observed 16 hours after four doses of 100 mg/kg.

Staining was maximal at 18 hours and declined thereafter but was still

detectable at 14 days.

The effects of fluoxetine (5 mg/kg) and MK-801 (1 mg/kg) on MDMA-induced

(4x100 mg/kg) silver staining in the frontoparietal cortex also were examined

(figure 7). Neither fluoxetine nor MK-801 induced intense silver staining when

administered alone Fluoxetine reduced by approximately half the volume of

tissue stained and dramatically reduced the intensity of staining throughout the

136
FIGURE 3. Silver staining in the frontoparietal cortex and striatum 48 hours

following the last dose of MDMA (4x75 mg/kg.) Panels A and B:

sagittal section from an animal exhibiting damage to striatum.

Panels C and D: sagittal section from an animal not exhibiting

any silver staining in striatum. Panels A and C: high power of

neocortex from sections shown in panels B and D. Note that the

extent of silver staining in the neocortex is about the Same in both

animals. Thus, it is unlikely that either variation in dosing or

technical considerations pertaining to the histological method

account for the differences. Scale bar=0.2 mm for panels A and

C. Scale bar=1 mm for panels B and D.

137
FIGURE 4. Thalamic and striatal silver staining 48 hours after the fourth dose

of MDMA (75 mg/kg). Panels A and B: section through thalamus

showing both axonal and terminal degeneration. Panels C and D:

section through striatum. From the same animal as in figure 3,

panels A and B. Scale bar=0.2 mm for panels A and C. Scale

bar=0.1 mm for panels B and D.

affected regions. The reduction in intensity of staining was not associated with

a preferential reduction in the staining of terminals, axons, and perikarya. In

contrast to fluoxetine, MK-801 virtually eliminated evidence of MDMA-induced

silver staining.

CONSIDERATIONS FOR THE INTERPRETATION OF SILVER STAINING

The authors’ observations confirm a previous report (Commins et al. 1987a) that

MDMA induces terminal, axonal, and perikaryal staining in the frontoparietal

neocortex. We further assessed the volume of this staining within the neocortex

138
FIGURE 5. Dose dependence of the volume of MDMA-induced neocortical

and striatal silver staining. Each group included four animals. All

animals received each dose of MDMA twice a day for 2 days (a

total of four times). All animals were sacrificed 48 hours after the

last dose. Standard errors indicated by vertical bars.

and demonstrated its dose dependence. Despite the dramatic nature of this

MDMA-induced silver staining, there are several considerations that need to be

borne in mind when interpreting the present findings.

First, all doses of MDMA used in the present study produced intense silver

staining. We therefore do not know a dose level at which MDMA produces no

evidence of silver staining. The time-course data indicate that optimal staining

occurs less than 48 hours following MDMA administration, and significant

staining could be induced by only two doses of 100 mg/kg MDMA. Thus, when

attempting to determine the highest dose of MDMA that does not induce silver

staining, a survival time shorter than 48 hours may be more appropriate. In

addition, a single rather than a multiple, dose also may provide a better estimate

of the highest dose that does not induce silver staining.

Second, the chemical basis for the stain is not currently known. Consequently,

silver staining cannot be specifically associated with particular degenerative

139
FIGURE 6. Time course of the changes in the volume of MDMA-induced

neocortical and striatal silver staining. All animals recieved 4x 100

mg/kg MDMA and were sacrificed at various times after the last

dose except those in the group marked “2x,” which received

only 2x100 mg/kg MDMA and were sacrificed 18 hours after

the second dose. Each group included four animals. Standard

errors indicated by vertical bars.

events. The extent to which the stain may recognize reversible as well as

irreversible alterations also is not known. Longitudinal experiments that include

estimates of the total number of neurons in specific brain regions will be

important in addressing such questions.

Third, we have not measured the intensity of staining; rather, we have relied

on estimates of the volume of intense staining. Consequently, there remains

a subjective component to the volume determination. However, the authors’

impression is that intensity and volume are highly correlated and the subjective

component has only a minor, if any, influence on estimates of the volume of

degeneration. We are currently developing an automated process for objectively

determining the intensity and volume of staining.

140
FIGURE 7. Effect of fluoxetine (fluox) (5 mg/kg) and MK-801 (1 mg/kg) given

30 minutes prior to each of four doses of MDMA (100 mg/kg) on

MDMA-induced silver staining. All groups included four animals.

Animals received either saline or MDMA and were pretreated

with either MK-801 or fluoxetine 30 minutes prior to MDMA. All

animals were sacrificed 48 hours after the last dose. Note that

fluoxetine pretreatment partially blocked MDMA-induced silver

staining; MK-801 eliminated MDMA-induced silver staining.

Standard errors indicated by vertical bars.

Finally, this analysis was not exhaustive because the survey was restricted to

selected sections through a segment of the cerebrum in which unambiguous

staining was present. More extensive studies addressing the extent and nature

of staining in a more precise manner are needed to fully characterize MDMAinduced silver staining.

CONCLUSIONS

Nonetheless, our current results show that maps of silver staining can

demonstrate dose-response relationships. These maps of silver staining

render a profile of MDMA-induced damage that differs from that engendered

by evidence from neurochemical and immunohistochemical studies with respect

141
to two important features. First, MDMA-induced injury as revealed by silver

staining is not restricted to serotonergic neurons. Second, the spatial pattern

of injury as revealed by silver staining does not correspond to the spatial pattern

of MDMA-induced alteration in serotonergic innervation. These two features of

MDMA-induced injury highlight the potential involvement of nonaminergic

mechanisms in MDMA neurotoxicity.

MDMA-Induced injury as Revealed by Silver Staining Is Not Restricted to

Serotonerglc Neurons

Consistent with a previous report (Commins et al. 1987a), we have observed

that MDMA induces silver staining of cell bodies, axons, and terminals in the

neocortex. Because immunohistochemical studies have not revealed

serotonergic perikarya within the neocortex (Lidov et al. 1980; Steinbush

1981) the staining of perikarya constitutes evidence of MDMA-induced injury

to nonserotonergic neurons.

Additional evidence that nonserotonergic processes are also injured by MDMA

comes from the observation that fluoxetine blocked part, but not all, of the

MDMA-induced staining. Fluoxetine pretreatment reduces the volume of

intensely stained tissue by approximately half and also reduces the intensity of

staining throughout the stained region. Staining of terminals, axons, and cell

bodies was still observed after fluoxetine pretreatment. Although we have not

established a dose-response relationship for fluoxetine blockage of MDMAinduced silver staining, the dose of fluoxetine used, 5 mg/kg, was sufficient to

prevent the serotonin depletion produced by the dose, 100 mg/kg of MDMA

(J.P. O’Callaghan, D.B. Miller, and K.F. Jensen, unpublished observations).

These observations support the conclusion that a substantial proportion of the

degenerating terminals, axons, and cell bodies are nonserotonergic.

The Spatial Pattern of MDMA-Induced Injury as Revealed by Silver Staining

Does Not Correspond to the Spatlal Pattern of Alterations In Serotonin

Innervation

The regional pattern of reduction in serotonin is consistent with the idea that

MDMA’s effect on serotonergic neurons is selective for their axons and terminals

(Wilson et al. 1989; Mamounas and Molliver 1988; O’Hearn et al. 1988; Ricaurte

et al. 1988; Molliver et al. 1990). The MDMA-induced loss of uptake binding sites

is largely in agreement with this idea (De Souza et al. 1990; Battaglia et al. 1987,

1991; Battaglia and De Souza 1989; De Souza and Battaglia 1989; lnsel et al.

1989). Molliver and coworkers (Wilson et al. 1989; O’Hearn and Molliver 1984;

Mamounas et al. 1991) have proposed that MDMA is even more selective,

damaging primarily a subpopulation of serotonergic axons. They report that fine

serotonergic axons originating in the dorsal raphe are preferentially vulnerable to

MDMA, whereas coarse serotonergic axons from median raphe are resistant to

142
MDMA. Differences in the pattern of innervation between the fine and coarse

fibers are most striking in the olfactory bulb, hippocampus, and entorhinal cortex

where immunohistochemical studies have demonstrated a preferential loss of

staining of these fine fibers following para-chloroamphetamine (PCA) or

methylenedioxyamphetamine (MDA) (Mamounas et al. 1991).

The regional distribution of MDMA-induced silver staining does not correspond

to the regional distribution of the vulnerable fine fibers. MDMA-induced silver

staining is limited primarily to the frontoparietal cortex. It sometimes involves

regions of posterior neocortex and striatum and is rarely observed in thalamus.

Because terminal-like staining occurs frequently in the olfactory bulb and

sometimes in the hippocampus of control animals, the possibility of MDMAinduced injury in these regions cannot be excluded. However, the alterations in

serotonergic innervation revealed by neurochemical and immunohistochemical

studies are far more widespread than the restricted damage to the frontoparietal

cortex revealed by silver staining.

Within the neocortex, the vulnerable fine fibers can be seen throughout the

cortical lamina but are particularly dense in layer IV (Blue et al. 1988; Kosofsky

and Molliver 1987). MDMA, MDA, and PCA appear to selectively eliminate the

staining of these vulnerable fine fibers (Steinbush 1981; O’Hearn and Molliver

1984; Mamounas et al. 1991). In contrast, the MDMA-induced silver staining is

most pronounced in the upper layers, whereas at higher doses it becomes robust

in layers III to V. Thus, in contrast to its more restricted regional pattern, silver

staining appears to have a more extensive involvement within the frontoparietal

cortex than alterations in the immunohistochemical labeling of serotonergic axons.

Consistent with these findings, corresponding differences between neurochemical

markers of dopamine and silver staining have been described for d-amphetamine

(Ryan et al. 1990).

There are several interpretations that could account for the differences in the

patterns of alterations revealed by serotonin immunohistochemistry and silver

staining. One is that a reduction in the density of immunohistochemically labeled

serotonergic processes can in part be accounted for by a loss of transmitter from

intact axons, rather than solely by degeneration. A similar argument has been

proposed for p-chloro-N-methylamphetamine (Lorez et al. 1976). Another

consideration that may account partially for the results obtained with the two

methods is that silver staining reveals damage to both serotonergic and

nonserotonergic neurons. The contribution of nonserotonergic elements would

not be detected by assays selective for assessing serotonin innervation.

There are other interpretations of these differences. One is that fine serotonergic

degenerating axons do not take up silver stains. This assertion, although

possible, has not been substantiated. However, it does highlight technical

limitations of the anatomical methods used to assess MDMA-induced neural

143
degeneration. There are important limitations to both silver stains and

immunohistochemistry. Silver stains do not distinguish between serotonergic

and nonserotonergic degenerating axons. Serotonin immunohistochemistry

cannot label intact axons that have lost their serotonin as a result of the

pharmacological treatments. Thus, neither silver stains nor immunohistochemistry

can by themselves discriminate the relative vulnerability of serotonergic and

nonserotonergic elements to MDMA. Estimating the relative involvement of

different populations of neurons awaits the development of techniques that can

assess neural damage comprehensively while simultaneously providing the

neurochemical identity of degenerating elements.

Although the precise identity and full complement of neurons vulnerable to

MDMA require further characterization, several hypotheses have been put

forward to account for the neurotoxicity of MDMA as well as other substituted

amphetamines (for reviews, see Fuller 1989; Stone et al. 1991). The majority of

these hypotheses are based on the idea that amphetamines are toxic to particular

neurochemically defined classes of neurons, possibly through the formation of

neurotoxins such as 6-hydroxydopamine and 5,6-dihydroxytryptamine (Commins

et al. 1987b, 1987c; Axt et al. 1990). Our current observation that MK-801, an

N-methyl-D-asparate (NMDA) receptor antagonist, blocks MDMA-induced silver

staining suggests that there may be several alternate, nonserotonin-specific

mechanisms by which substituted amphetamines can cause neural injury. The

most obvious possibility is through an excitotoxic mechanism (Sonsalla et al.

1989; Olney et al. 1987). Interestingly, Carlsson has proposed that the

neurotoxicity of the amphetamines may result from “uncontrolled positive

feedback” from the striatum to the cortex through the thalamus. Such feedback

could involve dopamine (cortical projections to the nigra or as presynaptic

terminations on dopaminergic nigrostriatal afferents) or serotonin (cortical

projections to raphe or as presynaptic terminations on serotonergic afferents to

striatum) with the corticofugal projections being glutaminergic. Thus, one possible

action of MK-801 could be to restabilize positive feedback, preventing a cascade

of overstimulation. This hypothesis is consistent with the observation that MK-801

attenuates amphetamine-induced dopamine release (Weihmuller et al. 1991). It

is also consistent with the currently observed incidence of silver staining in cortex,

striatum, and thalamus following MDMA administration.

Degeneration blocked by MK-801 could also involve mechanisms other than

excitotoxicity. MDMA-induced alteration in thermoregulation may be involved

(Miller 1992; Gordon and Rezvani, in press). MK-801 can induce hypothermia

that can protect against insults such as hyperthermia (Buchan and Pulsinelli

1990). Interestingly, MDMA-induced hyperthermia is also blocked by 5-HT2

receptor antagonists, and at high doses this effect can be differentiated from

long-term reductions in serotonin (Schmidt et al. 1990).

144
Hyperthermia is not the only peripheral influence that could alter the extent of

damage to the brain. Cerebral blood flow is modulated by serotonin (Gilman et

al. 1990) and influenced by MK-801 (Roussel et al. 1992). Stress can alter brain

tryptophan hydroxylase activity (Singh et al. 1990a, 19906; Boadle-Biber et al.

1989) and neocortical dopamine metabolism (Claustre et al. 1986). Because

dopamine is involved in the action of substituted amphetamines on serotonin

innervation (Stone et al. 1988; Schmidt et al. 1985, 1991), stress may play a

role in the observed cortical damage. Indeed, individual variability in stress

response (Miller 1992), particularly to multiple toxicant injections, also may play

a role in the variability reported for several parameters altered by substituted

amphetamines (Seiden 1991; Axt and Molliver 1991; Ryan et al. 1990; O’Dell et

al. 1991), including that of silver staining in the striatum observed in the present

study.

Finally, it is likely that several of MDMA’s actions act in concert to produce silver

staining as well as alterations in serotonergic innervation. Hypotheses that

focus solely on the unique vulnerability of a particular neurochemically defined

class of neurons are not likely to reveal a more complete understanding of such

a complex phenomenon. Conversely, neural degeneration may not be the only

means by which substituted amphetamines produce long-term alterations in

neurotransmitter levels in the brain.

In summary, we have made significant steps toward quantifying a historically

subjective phenomenon, the staining of degenerating neurons with silver. We

have shown how maps of silver staining can be valuable in assessing the dosedependent nature of toxicant-induced brain damage. With refinement, such

maps can help identify components of the brain’s complex circuitry that are

vulnerable to damage by exposure to toxicants.

REFERENCES

Axt, K.J.; Commins, D.L.; Vosmer, G.; and Seiden, L.S. Alpha-methyl-p-tyrosine

pretreatment partially prevents methamphetamine-induced endogenous

neurotoxin formation. Brain Res 515:269.276, 1990.

Axt, K.J., and Molliver, M.E. lmmunocytochemical evidence for

methamphetamine-induced serotonergic axon loss in the rat brain. Synapse

9:302-313, 1991.

Battaglia, G., and De Souza, E.B. Pharmacologic profile of amphetamine

derivatives at various brain recognition sites: Selective effects on serotonergic

systems. In: Asghar, K., and De Souza, E., eds. Pharmacology and

Toxicology of Amphetamine and Related Designer Drugs. National Institute

on Drug Abuse Research Monograph 94. DHHS Pub. No. (ADM)89-1640.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1989. pp. 240-258.

145
Battaglia, G.; Sharkey, J.; Kuhar, M.J.; and De Souta, E.B. Neuroanatomic

specificity and time course of alterations in rat brain serotonergic pathways

Induced by MDMA (3,4-methylenedioxymethamphetamine): Assessment

using quantitative autoradiography. Synapse 8:249-260, 1991.

Battaglia, G.; Yeh, S.Y.; O’Hearn, E.; Molliver, M.E.; Kuhar, M.J.; and

De Souza, E.B. 3,4-Methylenedioxymethamphetamine and 3,4-

methylenedioxyamphetamine destroy serotonin terminals in rat brain:

Quantification of neurodegeneration by measurement of [

3H]paroxetinelabeled serotonin uptake sites. J Pharmacol Exp Ther 242:911-916, 1987.

Bittner, S.E.; Wagner, G.C.; Aigner, T.G.; and Seiden, L.S. Effects of a highdose treatment of methamphetamine on caudate dopamine and anorexia in

rats. Pharmacol Biochem Behav 14:481-486, 1981.

Blue, M.E.; Yagaloff, K.A.; Mamounas, L.A.; Hartig, P.R.; and Molliver, M.E.

Correspondence between 5-HT2 receptors and serotonergic axons in rat

neocortex. Brain Res 453:315-328, 1988.

Boadle-Biber, M.C.; Corley, K.C.; Graves, L.; Phan, T.H.; and Rosecrans, J.

Increase in the activity of tryptophan hydroxylase from cortex and midbrain of

male Fischer 344 rats in response to acute or repeated sound stress. Brain

Res 482:306-316, 1989.

Buchan, A., and Pulsinelli, W.A. Hypothermia but not the N-methyl-D-aspartate

antagonist, MK-801, attentuates neuronal damage in gerbils subjected to

transient global ischemia. J Neurosci 10:311-316, 1990.

Claustre, Y.; Rivy, J.; Dennis, T.; and Scatton, B. Pharmacological studies on

stress induced increase in frontal cortical dopamine metabolism in the rat.

J Pharmacol Exp Ther 238:693-700, 1986.

Commins, D.L.; Axt, K.J.; Vosmer, G.; and Seiden, L.S. Endogenously

produced 5,6-dihydroxytryptamine may mediate the neurotoxic effects of

para-chloroamphetamine. Brain Res 419:253-261, 1987b.

Commins, D.L.; Axt, K.J.; Vosmer, G.; and Seiden, L.S. 5,6-

Dihydroxytryptamine, a serotonergic neurotoxin, is formed endogenously

in the rat brain. Brain Res 403:7-14, 1987c.

Commins, D.L.; Vosmer, G.; Virus, R.M.; Woolverton, W.L.; Schuster, C.R.;

and Seiden, L.S. Biochemical and histological evidence that

methylenedioxymethylamphetamine (MDMA) is toxic to neurons in

the rat brain. J Pharmacol Exp Ther 241:338-345, 1987a.

Davis, W.M.; Hatoum, H.T.; and Waters, I.W. Toxicity of MDA (3,4-

methylenedioxyamphetamine) considered for relevance to hazards of MDMA

(ecstasy) abuse. Alcohol Drug Res 7:123-134, 1987.

de Olmos, J.S.; de Olmos de Lorenzo, S.; Beltramino, C.A.; and Scapellato, D.

An aminoacidic-cupric-silver method for the early detection of degenerative

and regressive changes in neuronal perikarya, dendrites, stem-axons and

axon terminals caused by neurotoxicants and hypoxia. Ada Histochem, in

press.

146
De Souza, E.B., and Battaglia, G. Effects of MDMA and MDA on brain serotonin

neurons: Evidence from neurochemical and autoradiographic studies. In:

Asghar, K., and De Souza, E., eds. Pharmacology and Toxicology of

Amphetamine and Related Designer Drugs. National Institute on Drug Abuse

Research Monograph 94. DHHS Pub. No. (ADM)89-1640. Washington, DC:

Supt. of Docs., U.S. Govt. Print. Off., 1989. pp. 196-222.

De Souza, E.B.; Battaglia, G.; and Insel, T.R. Neurotoxic effects of MDMA on

brain serotonin neurons: Evidence from neurochemical and radioligand binding

studies. Ann N Y Acad Sci 600:882-698, 1990.

Fuller, R.W. Recommendations for future research on amphetamines and related

designer drugs. In: Asghar, K., and De Souza, E., eds. Pharmacology and

Toxicology of Amphetamine and Related Designer Drugs. National Institute

on Drug Abuse Research Monograph 94. DHHS Pub. No. (ADM)89-1640.

Washington, DC: Supt. of Docs., US. Govt. Print. Off., 1989. pp. 341-357.

Gilman, A.G.; Rall, T.W.; Nies, A.S.; and Taylor, P. The Pharmacological Basis

of Therapeutics. 8th ed. New York: Pergamon, 1990.

Gordon, C.J., and Rezvani, A.H. Temperature. In: Boyes, W.; Chang, L.; and

Dyer, R., eds. Handbook of Neurotoxicity. Volume 2: Effects and Mechanisms.

New York: Marcel Dekker, in press.

Insel, T.R.; Battaglia, G.; Johannessen, J.N.; Marra, S.; and De Souza, E.B. 3,4-

Methylenedioxymethamphetamine (“ecstasy”) selectively destroys brain

serotonin terminals in rhesus monkeys. J Pharmacol Exp Ther 249:713-720,

1989.

Kosofsky, B.E., and Molliver, M.E. The serotoninergic innervation of cerebral

cortex: Different classes of axon terminals arise from dorsal and median raphe

nuclei. Synapse 1:153-168, 1987.

Lidov, H.G.; Grzanna, R.; and Molliver, M.E. The serotonin innervation of the

cerebral cortex in the rat-an immunohistochemical analysis. Neuroscience

5:207-227, 1980.

Lorez, H.; Saner, A.: Richards, J.G.; and Da Prada, M. Accumulation of 5-HT in

non-terminal axons after p-chloro-N-methylamphetamine without degeneration

of identified 5-HT nerve terminals. Eur J Pharmacol 38:79-88, 1976.

Mamounas, L.A., and Molliver, M.E. Evidence for dual serotonergic projections

to neocortex: Axons from the dorsal and median raphe nuclei are differentially

vulnerable to the neurotoxin p-chloroamphetamine (PCA). Exp Neurol 102:23-

36, 1988.

Mamounas, L.A.; Mullen, C.A.; O’Hearn, E.; and Molliver, M.E. Dual

serotoninergic projections to forebrain in the rat: Morphologically distinct 5-HT

axon terminals exhibit differential vulnerability to neurotoxic amphetamine

derivatives. J Comp Neurol 314:558-586, 1991.

McKenna, D.J., and Peroutka, S.J. Neurochemistry and neurotoxicity of 3,4-

methylenedioxymethamphetamine (MDMA, “ecstasy”). J Neurochem 54:14-22,

1990.

147
Miller, D.B. Caveats in hazard assessment: Stress and neurotoxicity. In: Isaacson,

R.L., and Jensen, K.F., eds. The Vulnerable Brain and Evironmental Risks.

Volume I. Malnutrition and Hazard Assesssment. New York: Plenum, 1992.

Molliver, M.E.; Berger, U.V.; Mamounas, L.A.; Molliver, D.C.; O’Hearn, E.; and

Wilson, M.A. Neurotoxicity of MDMA and related compounds: Anatomic studies.

Ann N Y Acad Sci 600:649-661, 1990.

O’Dell, S.J.; Weihmuller, F.B.; and Marshall, J.F. Multiple methamphetamine

injections induce marked increase in extracellular striatal dopamine which

correlate with subsequent neurotoxicity. Brain Res 564:256-260, 1991.

O’Hearn, E.; Battaglia, G.; De Souza, E.B.; Kuhar, M.J.; and Molliver, M.E.

Methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine

(MDMA) cause selective ablation of serotonergic axon terminals in forebrain:

lmmunocytochemical evidence for neurotoxicity. J Neurosci 8:2788-2803, 1988.

O’Hearn, E., and Molliver, M.E. Organization of raphe-cortical projections in rat:

A quantitative retrograde study. Brain Res Bull 13:709-726, 1984.

Olney, J.; Price, M.T.; Shahid Salles, K.; Labryere, J.; and Frierdich, G. MK-801

powerfully protects against the N-methyl aspartate neurotoxicity. Eur J

Pharmacol 141:357-361, 1987.

Ricaurte, G.A.; Forno, L.S.; Wilson, M.A.; DeLanney, L.E.; Irwin, I.; Molliver, M.E.;

and Langston, V.W. (+/-)3,4-Methylenedioxymethamphetamine selectively

damages central serotonergic neurons in nonhuman primates. JAMA 260:51-55,

1988.

Roussel, S.; Pinard, E.; and Seylaz, J. The acute effects of MK-801 on cerebral

blood flow and tissue partial pressures of oxygen and carbon dioxide in

conscious and alpha chloralose anaesthetized rats. Neuroscience 47:959-985,

1992.

Ryan, L.J.; Linder, J.C.; Martone, M.E.; and Groves, P.M. Histological and

ultrastructural evidence that D-amphetamine causes degeneration in

neostriatum and frontal cortex of rats. Brain Res 518:67-77, 1990.

Scallet, A.C.; Lipe, G.W.; Ali, S.F.; Holson, R.R.; Frith, C.H.; and Slikker, W., Jr.

Neuropathological evaluation by combined immunohistochemistry and

degeneration-specific methods: Application to

methylenedioxymethamphetamine. Neurotoxicology 9:529-538, 1988.

Schmidt, C.J. Neurotoxicity of the psychedelic amphetamine,

methylenedioxymethamphetamine. J Pharmacol Exp Ther 240:1-7, 1987.

Schmidt, C.J.; Black, C.K.; Abbate, G.M.; and Taylor, V.L.

Methylenedioxymethamphetamine-induced hyperthermia and neurotoxicity

are independently mediated by 5-HT2 receptors. Brain Res 529:85-90, 1990.

Schmidt, C.J.; Black, C.K.; and Taylor, V.L. L-dopa potentiation of the serotonergic

deficits due to a single administration of 3,4-methylenedioxymethamphetamine,

p-chloramphetamine or methamphetamine to rats. Eur J Pharmacol

203:41-49, 1991.

Schmidt, C.J.; Ritter, J.K.; Sonsalla, P.K.; Hanson, G.R.; and Gibb, J.W. Role

of dopamine in the neurotoxic effects of methamphetamine. J Pharmacol Exp

Ther 233:539-544, 1985.

148
Schmidt, C.J., and Taylor, V.L. Depression of rat brain tryptophan hydroxylase

activity following the acute adminstration of

methylenedioxymethamphetamine. Biochem Pharmacol 36:4012-4095, 1987.

Schmidt, C.J.; Wu, L.; and Lovenberg, W. Methylenedioxymethamphetamine: A

potentially neurotoxic amphetamine analogue. Eur J Pharmacol 124:175-178,

1988.

Seiden, L.S. Neurotoxicity of methamphetamine: Mechanisms of action and

issues related to aging. In: Miller, M.A., and Kozel, N.J., eds.

Methamphetamine Abuse: Epidemiologic Issues and Implications. National

Institute on Drug Abuse Research Monograph 115. DHHS Pub. No.

(ADM)91-1836. Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1991.

pp. 24-32.

Singh, V.B.; Corley, K.C.; Phan, T.H.; and Boadle-Biber, M.C. Increases in the

activity of tryptophan hydroxylase from rat cortex and midbrain in response to

acute or repeated sound stress are blocked by adrenalectomy and restored

by dexamethasone treatment. Brain Res 516:66-76, 1990a.

Singh, V.B.; Onaivi, E.S.; Phan, T.H.; and Boadle-Biber, M.C. The increases in

rat cortical and midbrain tryptophan hydroxylase activity in response to acute

or repeated sound stress are blocked by bilateral lesions to the central

nucleus of the amygdala. Brain Res 530:49-53, 1990b.

Sonsalla, P.K.; Nicklas, W.J.; and Heikkila, R.E. Role for excitatory amino acids

in methamphetamine-induced nigrostriatal dopaminergic toxicity. Science

243:398-400, 1989.

Steinbush, H.W.M. Distribution of serotonin-immunoreactivity in the central

nervous system of the rat-cell bodies and terminals. Neuroscience 6:557-

618, 1981.

Stone, D.M.; Hanson, G.R.; and Gibb, J.W. Differences in the central

serotonergic effects of methylenedioxymethamphetamine (MDMA) in mice

and rats. Neuropharmacology 26:1657-1661, 1991.

Stone, D.M.; Johnson, M.; Hanson, G.R.; and Gibb, J.W. Role of endogenous

dopamine in the central serotonergic deficits induced by 3,4-

methylenedioxymethamphetamine. J Pharmacol Exp Ther 247:79-87, 1988.

Stone, D.M.; Merchant, K.M.; Hanson, G.R.; and Gibb, J.W. Immediate and

long-term effects of 3,4-methylenedioxymethamphetamine on serotonin

pathways in brain of rat. Neuropharmacology 26:1677-1683, 1987.

Weihmuller, F.B.; O’Dell, S.J.; Cole, B.N.; and Marshall, J.F. MK-801 attenuates

the dopamine-releasing but not the behavioral effects of methamphetamine:

An in vivo microdialysis study. Brain Res 549:230-235, 1991.

Wilson, M.A.; Ricaurte, G.A.; and Molliver, M.E. Distinct morphologic classes

of serotonergic axons in primates exhibit differential vulnerability to the

psychotropic drug 3,4-methylenedioxymethamphetamine. Neuroscience

28:121-137. 1989.

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DISCUSSION

Q (Molliver): Are you suggesting that this degeneration after MDMA is due to

a neurotoxic effect of the drug?

A: Yes.

Q (Molliver): I feel uncomfortable with that. What is the mortality of your

animals, given the huge doses you are giving?

A: We get very little mortality with the MDMA.

Q (Molliver): With dosages that high, you can get hyperthermia, hypertension,

ischemia, and breakdown of the blood-brain barrier, in addition to pulmonary

hypertension with hypoxia. At doses that high, one wonders whether you are

seeing a specific drug effect. It is like the effects of cocaine, d-amphetamine,

and methamphetamine, where people found vasculitis and ischemia, etc., with

such high doses.

A: Certainly the fact that the doses are so high brings in the possibility about the

specificity of the drug, in the sense of how it is being mediated-what kinds of

things are kicking in at that kind of dose?

Q (Molliver): That is the dose range that is used to model experimental

hypertension.

A: Right. But the time course, I think, what we are seeing is unlikely to be

due to those kinds of secondary effects. The fact that we can see it after two

doses? So it is within 12 to 16 hours.

Q (Molliver): You can get cerebral infarcts after one dose.

A: Right, but these rats are clearly healthy, by all criteria.

Q (Tilson): Wouldn’t you expect the pattern to be more diffuse if that was the

case?

A (Molliver): Not necessarily.

Q (Jensen): Aren’t things like the hippocampus more susceptible to those kinds

of infarcts?

A (Molliver): That’s true; the hippocampus is more susceptible to ischemia.

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Q (Tilson): How consistent are the changes you see in a population of rats?

The slides that you have shown-are those representative of populations of

rats?

COMMENT (Landfield): All slides are typical examples. (Laughter)

A: There is a certain degree of variability within a given dose group or in a

given survival time. The degree of overlap between different doses is minimal,

at least within the ranges that we’ve used. So that we can distinguish different

dose levels, I would say, fairly consistently.

Q (Tilson): I was thinking about a pattern within the dose group, where there is

always that area of the cortex, always. . .

A: Yes. Absolutely. In fact, the degree of damage that changes from dose is

an increase in size of the area. The area itself never changes.

Q (Molliver): Did you look at the levels of neurotransmitters in the cortex, for

example, serotonin?

A: Yes, we did.

COMMENT (Miller): Also, the hyperthermia and heat retention aren’t as high

when rats are in wire bottom cages,

Q (Molliver): I know, but did you measure temperature?

A (Miller): We have. If they are in plastic cages with wood chip bedding, they

go up to 42° core temperature. If they are in wire bottom cages, they usually

don’t go any higher than 38°.

Q (Molliver): The main effect that I am concerned about is the sympathetic

response due to peripheral release of catecholamines. Rats are sensitive to

pulmonary hypertension, and the pulmonary vasculature can vasoconstrict.

Q (Jensen): Can they be protected?

A (Molliver): Possibly by an extensive sympathectomy and adrenalectomy. I

think that Jim Gibb did that study years ago, and he found that adrenalectomy

protected animals against MDA toxicity.

A (Gibb): If you give reserpine beforehand, you don’t get the effect because

you don’t have anything to cause the effect.

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COMMENT (Seiden): On the other hand, we’ve potentiated with reserpine.

COMMENT (Gibb): I am not sure when you gave yours. Did you give yours

before? How long before?

COMMENT (Seiden): About 24 hours.

COMMENT (Ricaurte): I think the experiment has been done with AMT

[alphamethyl tyrosine], where animals pretreated with AMT and then given

a high dose of methamphetamine will not show silver degeneration in the

striatum or the somatosensory cortex. In support of your idea, I would just like

to reinforce that, because there is no question that the silver methods have a

great potential for detecting degeneration. As you have emphasized, a major

question is how much of that degeneration is specific in nature so that you

could relate it to the catecholamine and monoamine systems and how much

is related to nonspecific effects (ischemia, seizures, hyperthermia)‘? My

suspicion is that some of what has been shown in the cortex is in fact

“nonspecific.” It may be an interesting phenomenon in and of itself in terms

of how it is mediated. But it is not the kind of serotonergic or dopaminergic

neurotoxicity that is produced at lower doses. In the approaches that we use,

one was with AMT and the other was combining the silver method with the

pharmacological approach whereby we would pretreat animals with a dopamine

reuptake blocker which we knew protected against the dopamine depletion

induced by methamphetamine. We could show that such treatment also

prevented the appearance of the silver degeneration in the striatum. My point

is that the onus is to really demonstrate the specificity.

A (Jensen): It is true. Every drug does, and the question is what is the

specificity and how is it. I should mention that fluoxetine will partially block the

degeneration. Significant portions of degeneration are blocked by fluoxetine.

COMMENT (Seiden): I would like to take issue about every drug. With

cocaine, in spite of approaching the doses that the animals can barely survive,

we don’t see neurotoxicity.

COMMENT (Gibb): Lew, that causes hyperthermia, right?

COMMENT (Molliver): It is very transient and is mild.

COMMENT (Gibb): Yes, but those doses that he is talking about cause

hyperthermia.

COMMENT (Molliver): I think Karl clearly sees degeneration. I think this is of

considerable interest. Perhaps it is even more of interest in terms of the clinical

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aspects of drug abuse-induced toxicity seen in the emergency rooms, where

the most common situation is vascular-mediated toxicity.

COMMENT (Landfield): As you yourself pointed out, these are new techniques,

and you have to determine whether these are temporary pathologies or

permanent. I would suggest possibly counting cells in some of these tissues.

We all need to begin to quantify just how much degeneration this staining

actually reflects.

COMMENT (Jensen): In fact, we are talking about doing an experiment

with MPTP [1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine], using standard

morphometric methods to count the number of cells in pars compacta and

seeing if that doesn’t account for a loss in corresponsive cells that we see

stained with cupric silver in the early time period. If there were major vascular

events at these high doses, I would expect, based on ischemia and stroke

models, to see pretty major cerebral gliosis. If you compromise the bloodbrain barrier in a fairly major way and let in a lot of lymphokines and astrocyte

mitogens, it would leave a fairly broad glial scar; we don’t see that, and I will

show you that with astrocytes. I think that one other thing that may be of equal

consideration is what Dr. Heimer alluded to with some of his examples. When

you have this extended damage, you almost have to be concerned about

transsynaptic effects as well. The proximate site of the toxicant may be one

set of cells, which upon degeneration will result in the damage of other cells.

So you may end up with a cascade, particularly with these high doses, that

may be dependent upon the presence of compound, but not directly related

to the toxicant.

Q (Molliver): Did you see any intercerebral hemorrhages?

A (Jensen): No, we perfuse.

ACKNOWLEDGMENTS

This document has been reviewed in accordance with U.S. Environmental

Protection Agency policy and approved for publication. Mention of trade names

or commercial products does not constitute endorsement or recommendation

for use. The work was supported by the National Institute on Drug Abuse

interagency agreement 89-4. Drs. Lynda Erinoff and Lewis Seiden provided

valuable discussion and comments. Julia Davis provided photographic

assistance.

153
AUTHORS

Karl F. Jensen, Ph.D.

Research Scientist

James O’Callaghan, Ph.D.

Research Scientist

Diane B. Miller, Ph.D.

Research Scientist

Health Effects Research Laboratory

Neurotoxicology Division

U.S. Environmental Protection Agency

Research Triangle Park, NC 27711

Jeanene Olin

Associate Scientist

Najwa Haykal-Coates

Associate Scientist

ManTech Environmental Technology Inc.

Research Triangle Park, NC 27709

José S. de Olmos, Ph.D.

Research Scientist

lnstituto de lnvestigación Médica

Casilla de Correo 389

500 Cordoba

ARGENTINA

154
Reactions of 5-HT Neurons to Drugs of

Abuse: Neurotoxicity and Plasticity

Mary Ann Wilson, Laura A. Mamounas, Kenneth H. Fasman,

Karen J. Axt, and Mark E. Molliver

INTRODUCTION

Many drugs of abuse that have mood-altering, mood-elevating, or hallucinogenic

effects have direct pharmacologic actions on serotonergic (5-HT) neurons,

particularly at 5-HT synaptic terminals (Aghajanian et al. 1970; McCall and

Aghajanian 1980; Trulson et al. 1981; Jacobs 1984; Glennon et al. 1984;

Rasmussen and Aghajanian 1986; Fuller 1986; Campbell et al. 1987). Several of

the psychotropic amphetamine derivatives, such as p-chloroamphetamine (PCA),

3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine

(MDMA), and fenfluramine, have been shown to release 5-HT from serotonergic

axon terminals (Gallager and Sanders-Bush 1973; Sanders-Bush and Martin

1982; Fattaccini et al. 1991; Johnson et al. 1991) and can also cause 5-HT

axon terminals to degenerate (Battaglia et al. 1987; O’Hearn et al. 1988;

Mamounas and Molliver 1988; Molliver and Molliver 1990). However, unlike 5,7-

dihydroxytryptamine (5,7-DHT), which is neurotoxic to all types of serotonergic

axons as well as serotonergic cell bodies, the amphetamine derivatives have

selective neurotoxic effects that are restricted to a particular subset of 5-HT

axons. Furthermore, the vulnerable and resistant axons can be distinguished

on the basis of anatomic characteristics (O’Hearn et al. 1988; Wilson et al. 1989;

Mamounas et al. 1991).

ANATOMIC FEATURES OF SEROTONERGIC PROJECTIONS

The serotonergic innervation of the forebrain arises from neurons in the raphe

nuclei, located in the mesencephalon and pons (Dahlstrom and Fuxe 1964;

Conrad et al. 1974; Bobillier et al. 1975; Moore and Halaris 1975; Moore et al.

1978; Azmitia and Segal 1978). Serotonergic axon terminals are found throughout

the forebrain; however, there are regional and laminar variations in the distribution

of 5-HT axons (Lidov et al. 1980; Morrison et al. 1982; Takeuchi and Sano 1983;

Takeuchi et al. 1983; Kosofsky et al. 1984). The morphology of serotonergic axons

has been examined in multiple species, and several morphologic axon classes

have been identified (Chan-Palay 1977; Köhler et al. 1980; Lidov et al. 1980;

Sano et al. 1982; Zhou and Azmitia 1986; Kosofsky and Molliver 1987; McLean

155
and Shipley 1987; Mulligan and Törk 1988). Thick, nonvaricose axons, which

are commonly found in white matter, are considered to be preterminal fibers.

The other two types of 5-HT axons are varicose, but they differ in caliber and

in the size of varicosities. fine axons with extremely small granular or fusiform

varicosities are the most abundant 5-HT axon type in most areas of forebrain.

Beaded axons, which have large, round varicosities and very fine intervaricose

segments, are found in more restricted locations. Kosofsky and Molliver (1987),

utilizing anterograde transport methods, have demonstrated that fine 5-HT axon

terminals arise from the dorsal raphe nucleus and beaded 5-HT axon terminals

arise from the median raphe nucleus. That study provided the first evidence that

the different classes of axon terminals belong to distinct projection systems, with

origins in different nuclei. The distribution of fine and beaded axon terminals has

recently been studied in detail in rats (Mamounas et al. 1991) and in primates

(Wilson et al. 1989; Hornung et al. 1990; Wilson and Molliver 1991). In most

areas of the forebrain, fine 5-HT axons form a dense terminal plexus with subtle

variations in density among different regions and cortical layers. However,

beaded axons are markedly restricted in distribution, occurring predominantly

in the outer neocortical laminae and in limited zones within particular structures

such as the hippocampus, amygdala, entorhinal cortex, and olfactory bulb

(figure 1). Beaded axons also form an extremely dense plexus lining the

ependymal surface of the cerebral ventricles in both species. In several species,

beaded axons have been observed to form pericellular arrays, or “baskets”

(Mulligan and Törk 1987, 1988; Hornung et al. 1990) intimately surrounding

GABA-ergic neurons in some locations within the cortex (Köhler 1982; Törk

et al. 1988; Törk and Hornung 1988; Freund et al. 1990; DeFelipe et al. 1991).

Although no other specific postsynaptic targets of fine or beaded axons have

yet been identified, the distinctive distributions of fine and beaded 5-HT axons

suggest that these two axon classes influence different postsynaptic targets

in the forebrain. For example, the markedly different laminar distributions of

these two types of 5-HT axons within the cerebral cortex suggest that fine

and beaded 5-HT axons are likely to terminate on different elements of

cortical circuitry. In addition, these 5-HT projections can be differentiated

pharmacologically: O’Hearn and colleagues (1988) have shown that fine and

beaded 5-HT axons are differentially vulnerable to the neurotoxic effects of

amphetamine derivatives such as MDA. These studies indicate that there are

two parallel systems of serotonergic projections, differing in axon morphology,

origin, distribution, and vulnerability to selective neurotoxins; they are likely to

have different postsynaptic targets and subserve different functions.

AMPHETAMINES AND AXON LOSS

The authors have attempted to determine what methods may be useful for

detecting and characterizing the neurotoxic effects of psychoactive compounds

such as amphetamine derivatives to develop and validate criteria of neurotoxicity

that are applicable to multiple neuronal systems, It is important to evaluate the

156
FIGURE 1. Fine and beaded 5-HT axons in the olfactory bulb are differentially

vulnerable to neurotoxic amphetamines. In control animals (left

panel), fine and beaded 5-HT axons are segregated within

different layers of the olfactory bulb: Fine axons ramify extensively

within the infraglomerular layers; beaded axons are localized

predominantly in the glomeruli where they form dense, convoluted

arrays. Neurotoxic amphetamines such as PCA or MDA cause

a marked loss of fine 5-HT axons, while sparing beaded axons

(right panel). After drug treatment, beaded 5-HT axons in the

glomerular layer remain intact, whereas fine axons degenerate,

leaving the infraglomerular layers nearly devoid of serotonergic

axons.

KEY: gl=glomerular layer; mi=mitral cell layer; ipl=internal plexiform layer;

5-HT immunocytochemistry, darkfield photomicrograph, bar=100 μm

SOURCE: Mamounas, L.A.; Mullen, C.A.; O’Hearn, E.; and Molliver, M.E. Dual

serotonergic projections to forebrain in the rat: Morphologically distinct

5-HT axon terminals exhibit differential vulnerability to neurotoxic

amphetamine derivatives. J Comp Neurol 314:558-586, 1991.

Copyright © 1991 by Wiley-Liss. Reprinted by permission of

Wiley-Liss, a division of John Wiley and Sons, Inc.

spectrum of neuronal and nonneuronal effects produced by these drugs in

terms of biochemical, pharmacologic, and morphologic criteria. These

parameters include neurotransmitter content and synthesis, the density and

activity of membrane proteins such as neurotransmitter uptake carriers, the

157
integrity of cytoskeletal elements, the morphologic characteristics of neuronal

cell bodies and processes, and the status of nonneuronal cells such as

microglia and astrocytes that may become activated or proliferate in response

to neuronal damage. It is also important to carefully examine the time course

of the effects of a particular drug and of the neuronal and nonneuronal

responses to damage. Drug-induced changes in pharmacologic parameters

such as neurotransmitter levels, synthesis, or uptake may be either transient

effects, from which neurons can readily recover, or persistent changes that

reflect damage to neuronal structure. In addition, certain phases of the

neuronal response to a neurotoxin, such as the period of active degeneration

of axons, may occur over a limited period; other effects, such as the induction

of certain microglial markers, may be delayed and may vary depending on the

nature of the damage that has occurred.

The response of fine serotonergic axons to PCA and related amphetamine

derivatives exhibits several temporal phases marked by particular functional

changes. Initially, a severe depletion of 5-HT content in forebrain occurs; after

a single, moderate drug dose, 5-HT content may recover to near-normal levels

(Sanders-Bush et al. 1975; Schmidt 1987). This acute recovery is transient,

and the ensuing loss of 5-HT content and other serotonergic markers persists

for weeks or months Although the initial reduction in 5-HT levels is thought to

result from release of 5-HT and inhibition of reuptake, degeneration of 5-HT

axons has been proposed as a likely explanation for the persistent decreases

in 5-HT levels observed after treatment with PCA or fenfluramine (Neckers et al.

1976a, 1976b; Harvey et al. 1975; Clineschmidt et al. 1978). However, others

have postulated that long-lasting deficits could be explained as depletion of

neurotransmitter caused by 5-HT release combined with inhibition of uptake

and synthesis (Sarkissian et al. 1990).

In a series of recent studies, both biochemical and anatomic results in animals

treated with several related amphetamine derivatives have provided evidence

in support of axonal degeneration. After animals are treated with PCA, MDA,

MDMA, or fenfluramine, there are reductions in a number of serotonergic

parameters that persist for weeks to months after treatment. These changes

include large, persistent reductions in:

Levels of 5-HT and its metabolite, 5-hydroxyindole-acetic acid (5-HIAA)

(Sanders-Bush et al. 1975; Neckers et al. 1976b; Clineschmidt et al. 1976;

Kleven and Seiden 1989)

The activity of the 5-HT synthetic enzyme, tryptophan hydroxylase

(Sanders-Bush et al. 1975; Neckers et al. 1976b Stone et al. 1986;

Schmidt and Taylor 1987)

158
The activity and density of 5-HT uptake sites, as determined in synaptosomal

or autoradiographic preparations (Sanders-Bush et al. 1975; Battaglia et al.

1987)

The density of 5-HT immunoreactive axons (O’Hearn et al. 1988; Mamounas

and Molliver 1988; Wilson et al. 1989; Appel et al. 1989; Molliver and Molliver

1990; Mamounas et al. 1991)

Retrograde axonal transport, demonstrated by labeling of raphe neurons with

a retrogradely transported fluorescent tracer (Fluoro-Gold [FG]) (figure 2)

(Mamounas and Molliver 1988; Fritschy et al. 1988; Molliver and Mamounas

1991; Axt and Molliver 1991)

These studies identify markers of axon viability that are lost after drug treatment:

neurotransmitter content and synthetic machinery, the mechanism for reuptake

of released neurotransmitter, and the cytoskeletal elements required for transport

of compounds from the axon terminal back to the parent cell body. In addition,

positive evidence of axonal degeneration is provided by morphologic studies

(figure 3), which reveal numerous extremely swollen, abnormally shaped, and

fragmented axon terminals at short survivals (1 to 3 days) after drug treatment

(O’Hearn et al. 1988; Wilson et al. 1989; Molliver and Molliver 1990; Axt et al.

1990, 1992). These abnormal morphologic features are characteristic of

degenerating axons. The time when degenerating axons are most numerous

corresponds with the onset of the long-lasting phase of serotonin loss noted

above; this transient phase of degeneration produces a persistent denervation.

Taken together, this constellation of abnormal findings observed after treatment

with amphetamine derivatives indicates that 5-HT axon terminals are damaged

by these compounds and subsequently degenerate.

The mechanism of the neurotoxic effects of amphetamine derivatives is as

yet unidentified; however, agents such as citalopram and fluoxetine, which

selectively inhibit 5-HT uptake, also block the degeneration of 5-HT axon

terminals after amphetamine treatment (Fuller et al. 1975; Schmidt and Gibb

1985). It has not been determined whether these amphetamine derivatives

have direct neurotoxic effects or whether a metabolite of the drug (or of released

monoamines) is responsible for the neurotoxicity observed in vivo. Mediation by

a toxic metabolite is suggested by in vitro studies (Molliver et al. 1988) and by

the lack of neurotoxicity observed after direct intracerebral injection of MDA,

MDMA (Molliver et al. 1986), or PCA (Berger et al. 1990). In addition, prior

depletion of both peripheral and central stores of 5-HT with reserpine and the

5-HT synthesis inhibitor parachlorophenylalanine blocks the neurotoxic effect

of PCA (Berger et al. 1989, 1992). These studies suggest that a toxic metabolite

of 5-HT or of a catecholamine (Schmidt et al. 1985; Commins et al. 1987; Stone

et al. 1988) may be formed after treatment with PCA and related compounds.

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FIGURE 2. Dorsal and median raphe projections to the olfactory bulb are

differentially vulnerable to neurotoxic amphetamines. Retrograde

axonal transport of the fluorescent dye FG was utilized to identfy

raphe neurons that project to the o/factory bulb. After large

injections of FG in both the glomerular and infraglomerular layers

of the olfactory bulb, retrogradely labeled raphe neurons were

mapped in control and PCA-treated rats (10 mg/kgx2;

administered 2 weeks prior to FG injections). In control rats,

many FG-labeled neurons (depicted by an X) are found in both

the dorsal and median raphe nuclei; most of these neurons are

serotonergic (an X surrounded by a box). Serotonergic axons

that are ablated by PCA do not transport the tracer to their cell

bodies; thus, changes in the distribution of FG-labeled cells in the

raphe nuclei after PCA treatment may be used to determine the

brain stem origins of damaged vs. spared axons. Administration

of PCA 2 weeks prior to FG injection reduces the number of

retrogradely labeled 5-HT neurons in the dorsal raphe (77-

percent reduction); the number of labeled neurons in the median

raphe remains unchanged. These results indicate that the fine

5-HT axons in the olfactory bulb, which are selectively damaged

by amphetamine neurotoxins such as PCA or MDA (see figure 1),

arise from the dorsal raphe nucleus, whereas the beaded 5-HT

axons, which are preferentially spared, arise from the median

raphe nucleus.

SOURCE: Molliver and Mamounas 1991

160
FIGURE 3. Abnormal serotonergic axons in the forebrain of squirrel monkeys

2 days after dexfenfluramine administration. Dexfenfluramine

(5 mg/kg, subcutaneous) was administered to squirrel

monkeys twice daily for 4 days. Two days later serotonin

immunocytochemistty revealed numerous 5-HT axons with

abnormal morphologic features that are characteristic of

degenerating axons. Panels A and B: engorged preterminal

axon stumps. Panels C through E: extremely swollen,

abnormally shaped, and fragmented axon terminals.

Brightfield photomicrographs, bar= 10 μm.

161
PCA has been shown to inhibit monoamine oxidase (Fuller 1966); this effect

may favor the formation of an abnormal, toxic metabolite by blocking the usual

mechanism for monoamine metabolism.

lmmunocytochemical studies, in addition to providing positive evidence of

axon degeneration, have revealed that fine and beaded 5-HT axons differ in

vulnerability to the effects of amphetamine derivatives. These two axon types

have different, partially overlapping distributions, and the local effect of

amphetamines on the 5-HT content in a particular region depends to a great

extent on the proportion of fine vs. beaded 5-HT axon terminals in that area.

Regions that are innervated primarily by fine 5-HT axons such as dorsal

neocortex, have proven especially vulnerable, whereas regions innervated

predominantly by beaded 5-HT axons, such as the amygdala, are relatively

resistant. Moreover, these studies suggest that compensatory changes within

surviving processes can mask the cytotoxic damage to fine axons when

assayed by means of biochemical markers such as neurotransmitter levels,

Because the nuclei of origin and the postsynaptic targets of fine and beaded

axons are different, it is likely that these two projections subserve different

functions; the functional effects of a chemical lesion restricted to one subset of

5-HT axons probably differ from the effects of a neurotoxin such as 5,7-DHT,

which causes both systems to degenerate. Thus, the morphologic information

provided by immunocytochemical methods may be useful in interpreting both

biochemical and behavioral data concerning the effects of these neurotoxic

compounds. Retrograde axonal transport methods also have proved useful and

highly sensitive in detecting neurotoxic drug effects on a set of axon terminals

that constitutes only a small proportion of the 5-HT afferents to a particular

region. For example, the dorsal raphe projection to the motor trigeminal nucleus

contributes a small proportion of the fibers to the dense plexus of 5-HT axon

terminals within the nucleus; their loss after PCA treatment is not readily

apparent in immunocytochemical preparations. However, the high anatomic

resolution and sensitivity provided by retrograde transport methods allowed

Fritschy and colleagues (1988) to detect the loss of the dorsal raphe projection

to the trigeminal motor nucleus. Thus, although anatomic data are more difficult

to quantify than biochemical measures, the spatial resolution and morphologic

information obtained with neuroanatomic methods can be helpful in detecting

and characterizing the neurotoxic effects of psychoactive compounds.

MORPHOMETRIC ANALYSIS OF AXON DENSITY

A method for quantitative analysis of neuroanatomic data is particularly useful

to provide high sensitivity for the detection of neurotoxic effects, obtain doseresponse curves, permit statistical analysis of results, and correlate these results

with other measures. To that end, the authors have undertaken the development

of a group of morphometric procedures for the semiautomated measurement of

axon density within terminal fields as seen in the light microscope. The goal of

162
this method is to quantify morphologic parameters of axon density, including

features such as the number of axon segments in a defined field, the cumulative

length of the observed axons, and ideally, a breakdown of these parameters

by axon type. This approach will then be applied to the problem of quantifying

anatomic changes in 5-HT axons following drug treatment or other experimental

perturbation. The results will be presented in a future report.

Although cell counting (and “blob” counting in general) is a well-examined

problem in quantitative microscopy with several workable solutions, there are

no accepted automated methods for measuring the morphologic features of

arborized axons. (Counting the number of axons in a cross-section through a

central nervous system [CNS] tract or a peripheral nerve has been explored,

but that problem is reduced to simple blob counting.) High magnification is

needed to resolve individual axons, but that introduces the optical limitation of

a narrow depth of field. Generalized line-counting techniques are inadequate

for dealing with tortuous axons that, at high magnification, move in and out of

the shallow focal plane.

The methods that we have been exploring for measuring axon density involve

the orderly application of a series of standard digital image-processing

techniques to digitized video images obtained directly from microscope slides

(figure 4, panel A). These algorithms have been modified for the specific

requirements of this task. Several heuristics based on the expected

characteristics of axons in the images are introduced at critical steps in the

image-processing procedures. For example, in spite of the tortuous path taken

by many cortical axons, they can be treated as locally linear objects. Thus, to

follow an axon through an image, small gaps are closed by keeping track of the

most recent tracing direction. Other processing steps take advantage of the

fact that the light intensity in these images varies sharply in the immediate

vicinity of an axon. Thus, the variance of intensity levels around an axon in

the image is a more useful segmentation measure than the binary intensity

thresholding that is commonly used in image analysis algorithms to separate

foreground objects from the image background. Techniques such as these

allow obvious background regions to be discarded automatically, permitting the

computationally intensive tracing algorithms to be restricted to areas that have

a high probability of containing axons (figure 4, panel B).

We are continuing to develop and refine these techniques for analysis of the

anatomic changes induced by the drugs under study. These methods will

provide quantitative information indicative of cumulative axon length as an

index of axon density. The accuracy of the methods is being evaluated based

on comparison with manually traced images obtained from control and treated

tissue sections.

163
FIGURE 4. Computerized image analysis of axon density: initial separation

of axons from background. Axon density will be determined in

video images obtained from light microscopic sections. This figure

shows an initial step in processing a digitized image to separate

stained axons from background. Panel A: Digitized image of

serotonergic axons in parietal cortex of a control rat. Because the

light intensity in these images varies sharply in the immediate

vicinity of an axon, the variance of intensity levels around an axon

in the image is a useful segmentation measure for separation of

foreground objects from the image background. Panel B: An

example of image segmentation based on local intensity variance:

processed image with background regions removed. Removing

large background regions from consideration greatly improves the

performance of axon tracing algorithms. Tracing methods are

considerably faster and more accurate when applied to the

remaining regions.

164
SPROUTING AND AGING OF SEROTONERGIC NEURONS AFTER

NEUROTOXIC DAMAGE

As discussed above, certain psychotropic amphetamine derivatives, such

as PCA and MDA, cause selective degeneration of one class of 5-HT axon

terminals. Although fine 5-HT axon terminals degenerate, beaded 5-HT axon

terminals are spared. In addition, preterminal 5-HT axons and 5-HT cell bodies

are spared. The degeneration caused by MDA or PCA is followed by axonal

sprouting, leading to slowly progressive reinnervation (Molliver et al. 1989;

Molliver and Axt 1990; Mamounas and Molliver 1991). This gradual, partial

reinnervation of forebrain proceeds over several months. The reinnervation

of cerebral cortex progresses from rostral to caudal in a pattern that reiterates

development: A bilaminar ingrowth of thick, straight axons in layers I and VI is

followed by arborization of axon terminals within all layers of cortex. This gradual

reappearance of fine 5-HT axon terminals and, in particular, the spatial pattern

of reinnervation, provide additional, compelling evidence that the preceding loss

of 5-HT axons was caused by a degenerative process and was not merely a

depletion of neurotransmitter. The newly formed axon terminals have the

morphologic features typical of fine 5-HT axons. If a second course of MDA is

administered at this point, many of the regenerated axons are vulnerable to the

neurotoxin (Wilson and Molliver 1990). Both the morphologic appearance of

the regenerated axon terminals and their vulnerability to a second course of

neurotoxin treatment provide support for the hypothesis that, after MDA-induced

degeneration, there is regenerative sprouting of fine dorsal raphe axons rather

than collateral sprouting of the spared, beaded median raphe axons.

Although amphetamine derivatives such as PCA and MDA damage 5-HT axon

terminals, a loss of neuronal cell bodies has not been detected in the raphe

nuclei. Furthermore, the monoaminergic neurons of the CNS exhibit an unusual

capacity to respond to injury with a regenerative response; if the 5-HT cell bodies

do not die, they can, through sprouting, give rise to new axonal projections.

Because the spatial pattern of axonal ingrowth during regeneration reiterates

that seen in perinatal development and the relative axon density observed in

different forebrain areas is similar to that seen in untreated rats, it is plausible

that there is partial recovery of functional serotonergic innervation. However, it

has not yet been determined whether the new axon terminals form appropriate

connections with their targets.

The process of reinnervation has been examined further in long-term studies

of PCA-treated rats (Mamounas and Molliver 1991) where it exhibits several

unexpected features. These studies indicate that, although fine 5-HT axons

undergo regenerative sprouting during the first few months after treatment, the

innervation density does not reach control levels in caudal areas of neocortex

6 months after drug treatment (Mamounas and Molliver 1991). Moreover, at

longer survival times after PCA administration (6 to 12 months), the sprouting

165
appears to terminate, and there is a subsequent decrease in the density of fine

5-HT axons in cerebral cortex. Concomitant with this delayed decrease in axon

density is the appearance of structurally abnormal 5-HT axons that are relatively

thick and form highly tortuous endings. These aberrant tortuous 5-HT axons are

not seen during the early period of recovery after PCA; however, 1 year after

PCA administration, aberrant tortuous axons form a large proportion of the 5-HT

axon plexus in cortex. Aberrant axons of this type are not seen in young control

rats but appear with increasing frequency, thickness, and tortuousness in aged

untreated rats (from 1 to 2 years old). Quantitative analysis indicates that, 1 year

after PCA treatment, there is a twofold to threefold increase in the number of

these aberrant tortuous 5-HT axons in neocortex when compared with agematched control rats. Thus, despite the initial regenerative response of 5-HT

neurons after PCA-induced injury, the normal 5-HT innervation pattern is never

completely reestablished. Moreover, as noted above, this regenerative response

is followed by a second phase of axon loss. This phenomenon of delayed

axon loss, accompanied by the development of axon tangles, may represent

accelerated aging of 5-HT projections. There are other possible causes for

these late regressive changes, such as failure of regenerated axons to contact

appropriate targets.

There are several different meanings implied by the term “neurotoxicity,” as

used by various authors and investigators. In this discussion, the term refers

to structural damage to nerve cells that results in degeneration within one or

more compartments of a neuron, for example, the axon terminal. As described

above, psychoactive amphetamine derivatives have multiple effects on 5-HT

neurons, and it is important to distinguish between the acute pharmacological

effects of these drugs and the irreversible degeneration of 5-HT axon terminals

that may ensue. Acutely, these compounds evoke profound release of 5-HT

combined with inhibition of neurotransmitter synthesis and uptake, all of which

lead to depletion of the transmitter from axon terminals. These immediate

effects on neurotransmitter levels appear to be reversible. Moreover, if the

usual sequence of drug effects is interrupted by administration of an uptake

inhibitor such as citalopram within a few hours of amphetamine administration,

the affected neurons can be protected and rescued: Neurotransmitter levels

return to normal, and there is no morphologic or biochemical evidence for a

persisteni neurotoxic effect. However, in the absence of such pharmacologic

intervention, the acute drug-induced depletion is followed by a relentlessly

progressive degeneration of 5-HT axon terminals. During this second phase,

swollen, fragmented nerve fibers provide positive morphologic evidence

demonstrating that 5-HT axons degenerate after amphetamine treatment. The

resulting denervation causes persistent decreases in multiple markers for viable

5-HT projections. These long-lasting consequences of amphetamine treatment

reflect structural damage to the axonal compartment of a specific set of neurons

and constitute a neurotoxic effect. Thus, the authors apply the term neurotoxicity

to a drug effect that causes structural damage to neurons, whether it involves

166
degeneration of the cell body or is limited to degeneration of neuronal processes

(e.g., axon, dendrites). This concept of neurotoxicity applies to any compound

that produces neuronal degeneration, even though damaged processes may

subsequently be replaced through compensatory sprouting.

RESPONSES OF NONNEURONAL CELLS TO NEURONAL DAMAGE

Astrocytic Response

Many studies have demonstrated that astrocytic scarring occurs after mechanical

or severe degenerative damage in the CNS. In recent years, increases in levels

of the intermediate filament protein glial fibrillary acidic protein (GFAP), a major

cytoskeletal component in astrocytes, have been observed in association with

reactive astrocytosis (Graeber and Kreutzberg 1986; Brock and O’Callaghan

1987; Tetzlaff et al. 1988; Gilmore et al. 1990; Zini et al. 1990; Wang et al. 1991;

Zimmer et al. 1991). Biochemical assays of GFAP levels, measurement of GFAP

mRNA levels, and immunocytochemical detection of GFAP have been used to

measure astrocytosis (Brock and O’Callaghan 1987; Haj s et al. 1990; Tanaka

et al. 1991; Kost-Mikucki and Oblinger 1991; Poirier et al. 1991); these measures

have been proposed for use as an index of neurotoxicity (Brock and O’Callaghan

1987; Haglid et al. 1991; O’Callaghan and Miller, this volume). Marked increases

in the size of astrocytes, the intensity of GFAP immunocytochemical staining,

and the number of astrocytic processes have been observed after mechanical

lesions and after neurotoxin exposure that produces substantial brain lesions.

For example, the retrograde reaction seen in the facial nucleus after transection

of the facial nerve (VII) evokes a substantial increase in GFAP synthesis within

1 week of the injury (Graeber and Kreutzberg 1986, 1988). O’Callaghan and

colleagues (1990) have observed increases in GFAP levels after degeneration of

the dense striatal dopamine plexus brought about by 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine. Hence, there is good evidence to support an increase in

GFAP levels associated with several types of neuronal injury. However, the

astrocytic response may not be a sufficiently sensitive marker for detection of

all types of neuronal damage or of less severe injuries such as the selective

degeneration of sparsely distributed 5-HT axon terminals observed after PCA

treatment.

In a series of experiments carried out in the authors’ laboratory, the astrocytic

reaction was evaluated using GFAP immunostaining in rats that had been

treated with PCA (10 or 20 mg/kg, once per day for 2 days), at survival times

ranging from 3 days to 3 weeks, Although PCA treatment produced extensive

degenerative changes and a substantial loss of fine 5-HT axon terminals

throughout the forebrain, no consistent alterations in GFAP immunoreactivity

were observed with respect to the number of astrocytes, their staining intensity,

size, or number of processes. These studies suggest that astrocytic responses

visualized with GFAP staining may have limited sensitivity for the detection of

167
damage to sparsely distributed, fine axonal processes: moreover, a modest loss

of fine axon terminals may not be a sufficient stimulus to evoke an astrocytic

reaction, However, Frankfurt and colleagues (1991) have reported increases

in both the number of GFAP-labeled astrocytes and GFAP levels after injections

of the selective neurotoxin 5,7-DHT in the hypothalamus of adult rats. The latter

lesion causes extensive degeneration of preterminal axons as well as axon

terminals.

To further evaluate the usefulness of GFAP assays for detection of neurotoxic

effects on monoaminergic projections, Dr. Karen Axt has recently carried out a

series of studies in this laboratory, utilizing low-dose intraventricular injections

of 5,6-dihydroxytryptamine (5,6-DHT) and 5,7-DHT. These potent neurotoxins

selectively ablate 5-HT axons, especially around the ventricles and in parietal

and occipital cortex. In addition, both compounds produce a toxic effect on

some dopamine projections in the striatum. The damage to dopamine terminals

caused by 5,7-DHT can be prevented by pretreating the rats with nomifensine,

a catecholamine uptake blocker. Thus, three types of lesions can be produced

by injection of 5,6-DHT or 5,7-DHT: nonspecific mechanical damage along

the path of the injection cannula, selective neurotoxic damage to 5-HT axon

terminals, and damage to striatal dopamine axons. In animals treated with 5,6-

DHT or 5,7-DHT alone, an increase in GFAP immunostaining was observed in

the zone of mechanical damage along the cannula tract and in a region of the

caudal striatum that exhibited a marked loss of dopamine axons. In other

regions of forebrain that do not receive a prominent dopaminergic innervation

(notably in parietal and occipital cortex), striking losses of 5-HT axons occurred

without a notable increase in GFAP staining. Furthermore, when the neurotoxic

effects of 5,7-DHT on dopaminergic axons were selectively blocked by

pretreating with nomifensine, only minor changes in GFAP staining were

found in the striatum apart from the region of mechanical damage produced

by the injection cannula.

The finding that GFAP expression is not increased in regions with extensive

5-HT axon loss and is increased in the striatum primarily in response to a loss of

dopaminergic axons suggests that the astrocytic reaction may not be detectable

in all cases of neurotoxic effects on axons. Increases in GFAP staining are

observed in regions of neuronal death, retrograde reaction of cell bodies, and

degeneration of dense axonal projections. However, GFAP staining appears

to have limited use in detecting damage to sparsely distributed, fine axonal

projections such as the 5-HT projections to most areas of forebrain. Although

an astrocytic reaction is often associated with neuronal death, the failure to

detect astrocytosis or an increase in GFAP does not provide sufficient evidence

to exclude neuronal injury or neurotoxicity.

168
Microglial Responses

Microglial cells are a heterogeneous class of cells in the CNS that respond

to neuronal damage and have the capacity to proliferate and to become

phagocytic. They were first detected in the brain with heavy metal stains by

del Rio-Hortega (1932), but there has been controversy as to their origin and

even whether they are normally resident in brain tissue. In the past several

years there has been an increased interest in microglia, based on immunologic

studies demonstrating that a number of antigenic markers expressed by

peripheral macrophages and lymphocytes are found in brain cells with the

same morphology as classic microglia, or “dendritic cells” (Perry and Gordon

1991). There is evidence that some phagocytic cells in the brain are derived

from microglia that, in the past, may have been misidentified as astrocytes

(Graeber et al. 1988a). Current evidence indicates that microglia are extremely

numerous in the normal brain, are activated by neuronal injury, and may

participate in the elimination of damaged neuronal processes and somata

by phagocytosis.

Microglia exhibit several characteristic features that are altered in response

to CNS injury. Activated microglia undergo morphologic changes and

upregulation or novel expression of macrophage/lymphocyte antigens: under

certain circumstances, they may proliferate or become phagocytic. The

response to a particular injury varies, depending on the nature of the injury,

for example, neuronal death vs. axotomy (Streit et al. 1989).

To assess the response of microglia to neuronal damage, it is necessary to

develop reliable methods for identification of microglia in the resting state and

after activation. A number of anatomic methods have been utilized to detect

microglial activation. One of the most widely used is staining with either a

specific lectin such as that derived from Griffonia simplicifolia seeds (Streit

and Kreutzberg 1988) or with antibodies directed against monocytic markers.

A monoclonal antibody directed against the type 3 complement receptor on the

plasma membrane of macrophages, MRC OX-42 (Serotec), has been applied

in several studies of microglial activation (Graeber et al. 1988b, 1989; Konno et

al. 1989; Rinaman et al. 1991; Castellano et al. 1991; Sedgwick et al. 1991).

Our initial findings with this antibody confirm that OX-42 is a highly sensitive

marker for microglia in the resting and activated states. In rats treated with

PCA, there is a notable increase in the OX-42 staining intensity of microglia

and alterations in the morphology of microglial processes that are especially

evident in hippocampus, striatum, and neocortex (Wilson and Molliver 1992).

To further characterize the responses of microglia to neurotoxic damage and

identify indices of different states of glial activation following neuronal injury,

the authors are evaluating the utility of other microglial markers. The following

antisera have been selected for study: the Serotec mouse monoclonals OX-42,

169
described above; OX-1, directed against the rat leukocyte common antigen

CD45; OX-6, directed against major histocompatibility complex (MHC) Class II

antigen (la): OX-18, directed against an MHC Class I antigen; ED-l, directed

against a monocyte/macrophage cytoplasmic antigen that may be a marker for

lysosomal activation: and W3/25, directed against the rat homolog of the human

CD4 antigen.

A well-characterized experimental system for the study of microglial activation,

the facial nerve model (Graeber et al. 1988b), has been used to optimize

staining parameters and examine the response of microglia to motor neuron

axotomy. A retrograde cellular response (chromatolysis) is produced in the

motor neurons of the facial nucleus, without damage to the blood-brain barrier,

by transection of the facial nerve where it exits from the stylomastoid foramen.

The motor neurons of the facial nucleus undergo a characteristic retrograde

reaction indicative of degenerative changes and neuronal injury, but the cell

bodies do not die; rather, they survive, and the transected axons may later

regenerate.

The authors have examined the responses of microglial cells in the facial

nucleus 4 to 8 weeks after facial nerve transection. Staining of the six

microglial antigens noted above is increased in the facial nucleus on the side

of the lesion when compared with the unlesioned side. In this experimental

model, the antiserum revealing the largest differences vs. controls is OX-6,

which is directed against the MHC Class II (la) antigen. Despite the lack of

apparent cell death in this model, there is punctate staining on the lesioned

side with the ED-1 antiserum, which may indicate lysosomal activation.

The other antisera examined also appear to be useful markers for microglial

activation, and we are evaluating all six markers for the detection of neurotoxic

damage to 5-HT axon terminals. The nature and time course of microglial

responses have been reported to vary after different kinds of lesions; therefore,

it is important to utilize a wide variety of microglial markers and different survival

times when attempting to characterize the microglial response to a particular

class of injury. Preliminary results indicate that changes in the profile of

microglial markers may be useful for detecting neuronal injury and may be

more sensitive than measures of astrocytic activation for detecting selective

damage to a small subset of the axons in the neuropil.

REFERENCES

Aghajanian, G.K.; Foote, W.E.; and Sheard, M.H. Action of psychotogenic

drugs on single midbrain raphe neurons. J Pharmacol Exp Ther 171:178-187,

1970.

170
Appel, N.M.; Contrera, J.F.; and De Souza, E.B. Fenfluramine selectively and

differentially decreases the density of serotonergic nerve terminals in rat

brain: Evidence from immunocytochemical studies. J Pharmacol Exp Ther

249:928-943, 1989.

Axt, K.J., and Molliver, M.E. Dorsal and median raphe projections to rat

hippocampus are differentially affected by p-chloroamphetamine. Third IBRO

World Congress Neurosci Abstr, 1991. p. 287.

Axt, K.J.; Mullen, C.A.; and Molliver, M.E. Cytopathologic features indicative

of 5-hydroxytryptamine axon degeneration are observed in rat brain after

administration of both d- and I-methylenedioxyamphetamine. Ann N Y Acad

Sci 648:244-247, 1992.

Axt, K.J.; Teune, T.M.; and Molliver, M.E. Direct morphological evidence for

degeneration of serotonergic axons following administration of amphetamine

derivatives: dl-p-Chloroamphetamine, d-methamphetamine, and d- and Ifenfluramine. Soc Neurosci Abstr 16:1032, 1990.

Azmitia, E.C., and Segal, M. An autoradiographic analysis of the differential

ascending projections of the dorsal and median raphe nuclei in the rat.

J Comp Neurol 179:641-668, 1978.

Battaglia, G.; Yeh, S.Y.; O’Hearn, E.; Molliver, M.E.; Kuhar, M.J.; and De

Souza, E.B. 3,4-Methylenedioxymethamphetamine (MDMA) and 3,4-

methylenedioxyamphetamine (MDA) preferentially destroy serotonin

terminals in rat brain: Quantification of neurodegeneration by measurement

of 3H-paroxetine-labeled serotonin uptake sites. J Pharmacol Exp Ther

242:911-91 6, 1987.

Berger, U.V.; Grzanna, R.; and Molliver, M.E. Depletion of serotonin using pchlorophenylalanine (PCPA) and reserpine protects against the neurotoxic

effects of p-chloroamphetamine (PCA) in the brain, Exp Neurol 103:111-115,

1989.

Berger, U.V.; Grzanna, R.; and Molliver, M.E. The neurotoxic effects of pchloroamphetamine in rat brain are blocked by prior depletion of serotonin.

Brain Res 578:177-185, 1992.

Berger, U.V.; Molliver, M.E.; and Grzanna, R. Unlike systemic administration

of pchloroamphetamine, direct intracerebral injection does not produce

degeneration of 5-HT axons, Exp Neurol 109:257-268, 1990.

Bobillier, P.; Petitjean, F.; Salvert, D.; Ligier, M.; and Sequin, S. Differential

projections of the nucleus raphe dorsalis and nucleus raphe centralis as

revealed by autoradiography. Brain Res 85:205-210, 1975.

Brock, T.O., and O’Callaghan, J.P. Quantitative changes in the synaptic vesicle

proteins synapsin 1 and p38 and the astrocyte-specific protein glial fibrillary

acidic protein are associated with chemical-induced injury to the rat central

nervous system. J Neurosci 7:931-942, 1987.

Campbell, M.J.; Lewis, D.A.; Foote, S.L.; and Morrison, J.H. Distribution of

choline acetyltransferase-, serotonin-, dopamine-beta-hydroxylase-, tyrosine

hydroxylase-immunoreactive fibers in monkey primary auditory cortex.

J Comp Neurol 261:209-220, 1987.

171
Castellano, B.; Gonzalez, B.; Jensen, M.B.; Pedersen, E.B.; Finsen, B.R.;

and Zimmer, J. A double staining technique for simultaneous demonstration

of astrocytes and microglia in brain sections and astroglial cell cultures.

J Histochem Cytochem 39:561-568, 1991.

Chan-Palay, V. Cerebellar Dentate Nucleus-Organization, Cytology and

Transmitters. New York: Springer-Verlag, 1977. pp. 401-443.

Clineschmidt, B.V.; Totaro, J.A.; McGuffin, J.C.; and Pflueger, A.B.

Fenfluramine: Long-term reduction in brain serotonin (5-hyroxytryptamine).

Eur J Pharmacol 35:211-214, 1976.

Clineschmidt, B.V.; Zacchei, A.G.; Totaro, J.A.; Pflueger, A.B.; McGuffin, J.C.;

and Wishousky, T.I. Fenfluramine and brain serotonin. Ann N Y Acad Sci

305:222-241, 1978.

Commins, D.L.; Axt, K.J.; Vosmer, G.; and Seiden, L.S. Endogenously

produced 5,6-dihydroxytryptamine may mediate the neurotoxic effects of

parochloroamphetamine. Brain Res 419:253-261, 1987.

Conrad, L.C.A.; Leonard, C.M.; and Pfaff, D.W. Connections of the median

and dorsal raphe nuclei in the rat: Autoradiographic and degeneration study.

J Comp Neurol 156:179-206, 1974.

Dahlstrom, A., and Fuxe, K. Evidence for the existence of monoaminecontaining neurons in the central nervous system. I. Demonstration of

monoamines in cell bodies of brain neurons. Acta Physiol Stand 62(Suppl

232]:1-55, 1964.

DeFelipe, J.; Hendry, S.H.C.; Hashikawa, T.; and Jones, E.G. Synaptic

relationships of serotonin-immunoreactive terminal baskets on GABA

neurons in the cat auditory cortex. Cereb Cortex 1:117-133, 1991,

Del Rio-Hortega, P. Microglia. In: Penfield, W., ed. Cytology and Cellular

Pathology of the Nervous System. Vol. 2, Sec. X. New York: P.B. Hoeber,

1932. pp. 481-534.

Fattaccini, C.M.; Gozlan, H.; and Hamon, M. Differential effects of dfenfluramine and p-chloroamphetamine on H75/12-induced depletion of

5-hydroxytryptamine and dopamine in the rat brain, Neuropharmacology

30:15-23, 1991.

Frankfurt, M.; O’Callaghan, J.; and Beaudet, A. 5,7-Dihydroxytryptamine

injections increase glial fibrillary acidic protein in the hypothalamus of adult

rats. Brain Res 549:138-140, 1991.

Freund, T.F.; Gulyas, A.I.; Acsadi, L.; Gorcs, T.; and Toth, K. Serotonergic

control of the hippocampus via local inhibitory interneurons. Proc Natl

Acad Sci U S A 87:8501-8505, 1990.

Fritschy, J.-M.; Lyons, W.E.; Molliver, M.E.; and Grzanna, R. Neurotoxic effects

of p-chloroamphetamine on the serotoninergic innervation of the trigeminal

motor nucleus: A retrograde transport study. Brain Res 473:261-270, 1988.

Fuller, R.W. Serotonin oxidation by rat brain monoamine oxidase: Inhibition by

4-chloroamphetamine. Life Sci 5:2247-2252, 1966.

Fuller, R.W. Biochemical pharmacology of the serotonin system. Adv Neurol

43:469-480, 1986.

172
Fuller, R.W.; Perry, K.W.; and Molloy, B.B. Reversible and irreversible phases

of serotonin depletion by 4-chloroamphetamine, Eur J Pharmacol 33:119-124,

1975.

Gallager, D.W., and Sanders-Bush, E. In vivo measurement of the release of

5-hydroxytryptamine (5HT) from the hippocampus of the rat: Effect of RO4-

1284, pargyline and p-chloroamphetamine (PCA). Fed Proc 32:303, 1973.

Gilmore, S.A.; Sims, T.J.; and Leiting, J.E. Astrocytic reactions in spinal gray

matter following sciatic axotomy. Glia 3:342-349, 1990.

Glennon, R.A.; Titeler, M.; and McKenney, J.D. Evidence for 5-HT2 involvement

in the mechanism of action of hallucinogenic agents. Life Sci 35:2505-2511,

1984.

Graeber, M.B., and Kreutzberg, G.W. Astrocytes increase in glial fibrillary acidic

protein during retrograde changes of facial motor neurons. J Neurocytol

15:363-373, 1986.

Graeber, M.B., and Kreutzberg, G.W. Delayed astrocyte reaction following

facial nerve axotomy. J Neurocytol 17:209-220, 1988.

Graeber, M.B.; Streit, W.J.; and Kreutzberg, G.W. Axotomy of the rat facial

nerve leads to increased CR3 complement receptor expression by activated

microglial cells. J Neurosci Res 21:18-24, 1988b.

Graeber, M.B.; Streit, W.J.; and Kreutzberg, G.W. Identity of ED2-positive

perivasculat cells in rat brain. J Neurosci Res 22:103-106, 1989.

Graeber, M.B.; Tetzlaff, W.; Streit, W.J.; and Kreutzberg, G.W. Microglial cells

but not astrocytes undergo mitosis following rat facial nerve axotomy.

Neurosci Lett 85:317-321, 1988a.

Haglid, K.G.; Wang, S.; Hamberger, A.; Lehmann, A.; and Moller, C.J. Neuronal

and glial marker proteins in the evaluation of the protective action of MK 801.

J Neurochem 56:1957-1961, 1991.

Haj s, F.; Kálmán, M.; Zilles, K.; Schleicher, A.; and S tonyi, P. Remote

astrocytic response as demonstrated by glial fibrillary acidic protein

immunohistochemistry in the visual cortex of dorsal lateral geniculate nucleus

lesioned rats. Glia 3:301-310, 1990.

Harvey, J.A.; McMaster, S.E.: and Yunger, L.M. p-Chloroamphetamine:

Selective neurotoxic action in brain. Science 187:841-843, 1975.

Hornung, J.-P.; Fritschy, J.-M.; and Törk, I. Distribution of two morphologically

distinct subsets of serotoninergic axons in the cerebral cortex of the

marmoset. J Comp Neurol 297:165-181, 1990.

Jacobs, B.L. Postsynaptic serotonergic action of hallucinogens. In: Jacobs,

B.L., ed. Hallucinogens: Neurochemical, Behavioral, and Clinical

Perspectives. New York: Raven Press, 1984. pp. 183-202.

Johnson, M.P.; Conarty, P.F.; and Nichols, D.E. [

3H]Monoamine releasing and

uptake inhibition properties of 3,4-methylenedioxymethamphetamine and

p-chloroamphetamine analogues. Eur J Pharmacol 200:9-16, 1991.

Kleven, M.S., and Seiden, L.S.D-, L- and DL-Fenfluramine cause long-lasting

depletions of serotonin in rat brain. Brain Res 505:351-353, 1989.

173
Köhler, C. On the serotonergic innervation of the hippocampal region: An

analysis employing immunohistochemistry and retrograde fluorescent tracing

in the rat brain. In: Palay, S.L., and Chan-Palay, V., eds. Cytochemical

Methods in Neuroanatomy. New York: Alan R. Liss, 1982. pp. 387-405.

Köhler, C.; Chart-Palay, V.; Haglund, L.; and Steinbusch, H.W.M.

lmmunohistochemical localization of serotonin nerve terminals in the lateral

entorhinal cortex of the rat: Demonstration of two separate patterns of

innervation from the midbrain raphe. Anat Embryol 160:121-129, 1980.

Konno, H.; Yamamoto, T.; Iwasaki, Y.; Suzuki, H.; Saito, T.; and Terunuma,

H. Wallerian degeneration induces la-antigen expression in the rat brain.

J Neuroimmunol 25:151-159, 1989.

Kosofsky, B.E., and Molliver, M.E. The serotoninergic innervation of cerebral

cortex: Different classes of axon terminals arise from dorsal and median

raphe nuclei. Synapse 1:153-168, 1987.

Kosofsky, B.E.; Molliver, M.E.; Morrison, J.H.; and Foote, S.L. The serotonin

and norepinephrine innervation of primary visual cortex in the Cynomolgus

monkey (Macaca fascicularis). J Comp Neurol 230:168-178, 1984.

Kost-Mikucki, S.A., and Oblinger, M.M. Changes in glial fibrillary acidic protein

mRNA expression after corticospinal axotomy in the adult hamster.

J Neurosci Res 28:182-191, 1991.

Lidov, H.G.W.; Grzanna, R.; and Molliver, M.E. The serotonin innervation of the

cerebral cortex in the rat-an immunohistochemical analysis. Neuroscience

5:207-227, 1980.

Mamounas, L.A., and Molliver, M.E. Evidence for dual serotonergic projections

to neocortex: Axons from the dorsal and median raphe nuclei are differentially

vulnerable to the neurotoxin p-chloroamphetamine (PCA). Exp Neurol

102:23-36, 1988.

Mamounas, L.A., and Molliver, M.E. Aberrant reinnervation of rat cerebral

cortex by serotonergic axons after denervation by p-chloroamphetamine

(PCA). Soc Neurosci Abstr 17:1181, 1991.

Mamounas, L.A.; Mullen, CA.; O’Hearn, E.; and Molliver, M.E. Dual

serotonergic projections to forebrain in the rat: Morphologically distinct 5-HT

axon terminals exhibit differential vulnerability to neurotoxic amphetamine

derivatives, J Comp Neurol 314:558-586, 1991.

McCall, R.B., and Aghajanian, G.K. Hallucinogens potentiate responses to

serotonin and norepinephrine in the facial motor nucleus. Life Sci 26:1149-

1156, 1980.

McLean, J.H., and Shipley, M.T. Serotonergic afferents to the rat olfactory

bulb. I. Origins and laminar specificity of serotonergic inputs in the adult rat.

J Neurosci 7:3016-3028, 1987.

Molliver, D.C., and Molliver, M.E. Anatomic evidence for a neurotoxic effect of

(±)fenfluramine upon serotonergic projections in the rat. Brain Res 511:165-

168,1990.

174
Molliver, M.E., and Axt, K.J. Specific regional patterns of serotonergic

innervation in ventral areas of forebrain in the rat: Selective denervation

and reinnervation after administration of p-chloroamphetamine (PCA).

Soc Neurosci Abstr 16:1032, 1990.

Molliver, M.E., and Mamounas, L.A. Dual serotonergic projections to rat

olfactory bulb: p-Chloroamphetamine (PCA) selectively damages dorsal

raphe axons while sparing median raphe axons. Soc Neurosci Abstr 17:1180,

1991.

Molliver, M.E.; Mamounas, L.A.; and Carr, P. Reinnervation of cerebral cortex

by 5-HT axons after denervation by psychotropic amphetamine derivatives.

Soc Neurosci Abstr 15:417, 1989.

Molliver, M.E.; O’Hearn, E.; Battaglia, G.; and De Souza, E.B. Direct

intracerebral administration of MDA and MDMA does not produce serotonin

neurotoxicity. Soc Neurosci Abstr 12:1234, 1986.

Molliver, M.E.; Stratton, K.; Carr, P.; Grzanna, R.; and Baraban, J. Contrasting

in vitro and in vivo effects of p-chloroamphetamine (PCA) on 5-HT axons:

lmmunocytochemical studies in hippocampal slices. Soc Neurosci Abstr

14:210, 1988.

Moore, R.Y., and Halaris, A.E. Hippocampal innervation by serotonin neurons

of the midbrain raphe in the rat. J Comp Neurol 164:171-184, 1975.

Moore, R.Y.; Halaris, A.E.; and Jones, B.E. Serotonin neurons of the midbrain

raphe: Ascending projections. J Comp Neurol 180:417-438, 1978,

Morrison, J.H.; Foote, S.L.; Molliver, M.E.; Bloom, F.E.; and Lidov, H.G.W.

Noradrenergic and serotonergic fibers innervate complementary layers in

monkey primary visual cortex: An immunohistochemical study. Proc Natl

Acad Sci U S A 79:2401-2405, 1982.

Mulligan, K.A., and Törk, I. Serotonergic axons form basket-like terminals in

cerebral cortex. Neurosci Lett 81:7-12, 1987.

Mulligan, K.A., and Törk, I. Serotoninergic innervation of the cat cerebral cortex.

J Comp Neurol 270:86-110, 1988.

Neckers, L.M.; Bertilsson, L.; and Costa, E. The action of fenfluramine and pchloramphetamine on serotonergic mechanisms: A comparative study in rat

brain nuclei. Neurochem Res 1:29-35, 1976a.

Neckers, L.M.; Bertilsson, L.; Koslow, S.H.; and Meek, J.L. Reduction of

tryptophan hydroxylase activity and 5-hydroxytryptamine concentration in

certain rat brain nuclei after p-chloroamphetamine. J Pharmacol Exp Ther

196:333-338, 1976b.

O’Callaghan, J.P.; Miller, D.B.; and Reinhard, J.F., Jr. Characterization of the

origins of astrocyte response to injury using the dopaminergic neurotoxicant,

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Brain Res 521:73-80, 1990.

O’Hearn, E.; Battaglia, G.; De Souza, E.B.; Kuhar, M.J.; and Molliver, M.E.

Methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine

(MDMA) cause selective ablation of serotonergic axon terminals in forebrain:

lmmunocytochemical evidence for neurotoxicity. J Neurosci 8:2788-2803,

1988.

175
Perry, V.H., and Gordon, S. Macrophages and the nervous system. Int Rev

Cytoll 25:203-244, 1991.

Poirier, J.; Hess, M.; May, P.C.; and Finch, C.E. Astrocytic apolipoprotein E

mRNA and GFAP mRNA in hippocampus after entorhinal cortex lesioning.

Mol Brain Res 11:97-106, 1991.

Rasmussen, K., and Aghajanian, G.K. Effect of hallucinogens on spontaneous

and sensory-evoked locus coeruleus unit activity in the rat: Reversal by

selective 5-HT2 antagonists. Brain Res 385:395-400, 1986.

Rinaman, L.; Milligan, C.E.; and Levitt, P. Persistence of Fluoro-Gold following

degeneration of labeled motoneurons is due to phagocytosis by microglia and

macrophages. Neuroscience 44:765-776, 1991.

Sanders-Bush, E.; Bushing, J.A.; and Sulser, F. Long-term effects of pchloroamphetamine and related drugs on central serotonergic mechanisms.

J Pharmacol Exp Ther 192:33-41, 1975.

Sanders-Bush, E., and Martin, L.L. Storage and release of serotonin. In:

Osborne, N.N., ed. Biology of Serotonergic Transmission. New York: Wiley,

1982. pp. 95-118.

Sano, Y.; Takeuchi, Y.; Kimura, H.; Goto, M.; Kawata, M.; Kojima, M.;

Matsuura, T.; Yamada, H.; and Ueda, S. lmmunohistochemical studies on the

processes of serotonin neurons and their ramification in the central nervous

system-with regard to the possibility of the existence of Golgi’s rete nervosa

diffusa. Arch Histol Jpn 45:305-316, 1982.

Sarkissian, C.F.; Wurtman, R.J.; Morse, A.N.; and Gleason, R. Effects of

fluoxetine or D-fenfluramine on serotonin release from, and levels in, rat

frontal cortex. Brain Res 529:294-301, 1990.

Schmidt, C.J. Neurotoxicity of the psychedelic amphetamine,

methylenedioxymethamphetamine. J Pharmacol Exp Ther 240:1-7, 1987.

Schmidt, C.J., and Gibb, J.W. Role of the serotonin uptake carrier in the

neurochemical response to methamphetamine: Effects of citalopram and

chlorimipramine. Neurochem Res 10:637-648, 1985.

Schmidt, C.J.; Ritter, J.K.; Sonsalla, P.K.; Hanson, G.R.; and Gibb, J.W. Role of

dopamine in the neurotoxic effects of methamphetamine. J Pharmacol Exp

Ther 233:539-544, 1985.

Schmidt, C.J., and Taylor, V.L. Depression of rat brain tryptophan hydroxylase

activity following the acute administration of

methylenedioxymethamphetamine. Biochem Pharmacol 36:4095-4102, 1987.

Sedgwick, J.D.; Schwender, S.; Imrich, H.; Dörries, R.; Butcher, G.W.; and

Ter Meulen, V. Isolation and direct characterization of resident microglial cells

from the normal and inflamed central nervous system. Proc Natl Acad Sci

U S A 88:7438-7442, 1991.

Stone, D.M.; Johnson, M.; Hanson, G.R.; and Gibb, J.W. Role of endogenous

dopamine in the central serotonergic deficits induced by 3,4-

methylenedioxymethamphetamine. J Pharmacol Exp Ther 247:79-87, 1988.

176
Stone, D.M.; Stahl, D.C.; Hanson, G.R.; and Gibb, J.W. The effects

of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-

methylenedioxyamphetamine (MDA) on monoaminergic systems

in the rat brain. Eur J Pharmacol 128:41-48, 1986.

Streit, W.J.; Graeber, M.B.; and Kreutzberg, G.W. Expression of la antigen on

perivascular and microglial cells after sublethal and lethal motor neuron

injury. Exp Neurol 105:115-126, 1989.

Streit, W.J., and Kreutzberg, G.W. Response of endogenous glial cells to motor

neuron degeneration induced by toxic ricin. J Comp Neurol 268:248-263,

1988.

Takeuchi, Y.; Kimura, H.; Matsuura, T.; Yonezawa, T.; and Sano, Y. Distribution

of serotonergic neurons in the central nervous system: A peroxidaseantiperoxidase study with anti-serotonin antibodies. J Histochem Cytochem

31:181-185, 1983.

Takeuchi, Y., and Sano, Y. lmmunohistochemical demonstration of serotonin

nerve fibers in the neocortex of the monkey (Macaca fuscata). Anat Embryol

166:155-168, 1983.

Tanaka, H.; Araki, M.; and Masuzawa, T. Differential response of three

astrocyte-specific proteins to fimbrial transection of the rat brain:

lmmunohistochemical observations with antibodies against glial fibrillary

acidic protein, glutamine synthetase and S-100 protein. Acta Histochem

Cytochem 24:11-19, 1991.

Tetzlaff, W.; Graeber, M.B.; Bisby, M.A.; and Kreutzberg, G.W. Increased glial

fibrillary acidic protein synthesis in astrocytes during retrograde reaction of

the rat facial nucleus. Glia 1:90-95, 1988.

Törk, I., and Hornung, J.-P. Interaction of serotonin and GABA in cerebral

cortex and hippocampus. Soc Neurosci Abstr 14:678, 1988.

Törk, I.; Hornung, J.-P.; and Somogyi, P. Serotonergic innervation of

GABAergic neurons in the cerebral cortex. (Abstract.) Neurosci Lett

30[Suppl]:S131, 1988.

Trulson, M,E.; Heym, J.; and Jacobs, B.L. Dissociations between the effects of

hallucinogenic drugs on behavior and raphe unit activity in freely moving cats.

Brain Res 215:275-293, 1981.

Wang, S.; Lees, G.J.; Rosengren, L.E.; Karlsson, J.-E.; Stigbrand, T.;

Hamberger, A.; and Haglid, K.G. The effect of an N-methyl-D-aspartate lesion

in the hippocampus on glial and neuronal marker proteins. Brain Res

541:334-341, 1991.

Wilson, M.A., and Molliver, M.E. Regeneration in the CNS following MDAinduced neurotoxicity: Pharmacologic evidence for selective reinnervation by

fine serotonergic axons. Soc Neurosci Abstr 16:1032, 1990.

Wilson, M.A., and Molliver, M.E. The organization of serotonergic projections to

cerebral cortex in primates: Regional distribution of axon terminals.

Neuroscience 44:537-553, 1991.

Wilson, M.A., and Molliver, M.E. Microglial responses to neurotoxic ablation of

serotonergic axon terminals. Soc Neurosci Abstr 18:960, 1992.

177
Wilson, M.A.; Ricaurte, G.A.; and Molliver, M.E. Distinct morphologic classes

of serotonergic axons in primates exhibit differential vulnerability to the

psychotropic drug 3,4-methylenedioxymethamphetamine. Neuroscience

28:121-137, 1989.

Zhou, F.C., and Azmitia, E.C. Induced homotypic sprouting of serotonergic

fibers in hippocampus. II. An immunocytochemistry study. Brain Res 373:337-

348, 1986.

Zimmer, C.; Sampaolo, S.; Sharma, H.S.; and Cerv s-Navarro, J. Altered glial

fibrillary acidic protein immunoreactivity in rat brain following chronic hypoxia.

Neuroscience 40:353-361, 1991.

Zini, I.; Zoli, M.; Grimaldi, R.; Pith, E.M.; Biagini, G.; Fuxe, K.; and Agnati, L.F.

Evidence for a role of neosynthetized putrescine in the increase of glial

fibrillary acidic protein immunoreactivity induced by a mechanical lesion in the

rat brain, Neurosci Lett 120:13-16, 1990.

DISCUSSION

Q: Have you seen a microglial reaction along the needle track?

A (Molliver): Yes, very striking.

Q (Landfield): Only the astrocytes have processes like those you showed.

A: Those are identical to the cells that del Rio Hortega identified as microglia,

and they do not costain with GFAP, so they are not astrocytes.

Q (Landfield): The localization that you have shown in the hippocampus is very

similar to the astrocyte localization.

A: In the hippocampus, astrocytes have a similar pattern

Q (Landfield): The microglia are truncated cells, and they are just not that

frequent. There are many more astrocytes in the hippocampus, and the

distribution you show looks much more like a stain for astrocytes. So I think

there may be some cross-reactivity in your stain.

A: We were puzzled about that at first, because I did not believe that there

were that many resident microglia in the brain. Now, there is much re-interest

in microglia, and there are many recent papers. A number of laboratories,

particularly G. Kreutzberg’s group in Germany and several laboratories in

England and the United States, have demonstrated abundant microglia with

monoclonal antibodies directed against MHC markers.

COMMENT (Landfield): That is a problem with antibodies, I think, because at

the EM [electron microscopy] level it is clear that is precisely what they are.

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They have been well described: The microglia are dark cells, they are more

rounded, and they don’t have the long extensive processes you show. Also, If

you don’t see an astrocyte reaction, then you probably don’t have degenerating

axons. I would like to bring this up again in terms of what we were talking about

before, about the need to have a secondary method to validate a decrease in

staining; as Lew Seiden pointed out, a decrease in neurochemistry doesn’t

necessarily mean you have degeneration. In fact, if you did have such massive

degeneration, I would be astounded if you didn’t have loss of cells in the raphe

itself. Why don’t you have loss of cells? Why don’t you have an astrocyte

reaction? I would say that you may have a definite decrease in staining, and

a decrease in neurochemistry, but it is still an open question whether you have

degeneration. And I tend to doubt it from these data.

COMMENT (Molliver): That is an interesting point, and I am certainly open to

discussion about this. Recently it has been found that there are two distinct

populations of glial cells in the brain, and you can distinguish in the same

section or in adjacent sections-those which stain with GFAP and those which

stain with OX-42 or a battery of other monocyte markers for microglia. Recent

studies from Kreutzberg and colleagues have shown that the microglia, rather

than astrocytes, are the main glial cells that respond to degeneration.

COMMENT (Landfield): With semithin sections or EM, you can see all the cells

that are there.

COMMENT (Olney): But you can’t trace the whole process because they are

so thin.

COMMENT (Landfield): No, you can’t trace the whole process. But in many

sections, the astrocytes may be very elongated, whereas you never or rarely

see that in the dark cells: dark cell processes are just not that frequent. I can

show you in any section of semithins, and you can clearly see where long

processes are distributed. And the distribution shown here is like that for

astrocytes.

COMMENT (Molliver): That is the point that I was trying to make. Based on

the localization of distinct immune markers on microglia, we should consider

a revision of the concept of these two cell types. For example, there is now a

great deal of evidence that the AIDS virus selectively affects microglia and that

it replicates in these cells. We see that microglia, not astrocytes, stain for these

immune markers, common to macrophages.

As far as the degeneration process goes, several parameters need to be

considered. I agree that the loss of staining by itself is not evidence for

degeneration. However, consider, first, what the biochemical factors are.

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There is a loss of transmitter, a loss of tryptophan hydroxylase activity, a loss

of serotonin uptake. Those are independent factors. Uptake is a function of

the plasma membrane; transmitter storage is a function of the synaptic vesicles;

and synthesis is a function of the cytoplasmic protein, tryptophan hydroxylase.

There is loss of axon transport, both anterograde and retrograde, indicating that

cytoskeletal function is impaired. There appears to be no transport, storage, or

uptake, and there is hardly any evidence for viability of 5-HT axons. Even that

itself is not sufficient. These enormous, swollen, globular, fragmented axons

that are 10 to 20 times larger than normal provide positive evidence for

degeneration. One of the most striking results is seen in sagittal sections:

5-HT axons in the hypothalamus form large, bulbous, swollen endings, which

have been described by Cajal after axotomy and by Bjorkland and Hokfelt after

neurotoxic lesions of catecholamine axons. The combination of the loss of all

evidence of viability, the presence of enormously swollen, fragmented axons,

of growth cones with filopodia coming off, and the gradual pattern of sprouting

over 8 to 12 months makes most comfortable with the interpretation that the

5-HT axon terminals have degenerated after treatment with PCA or MDA.

COMMENT (Landfield): There are more direct ways to study this. I am just

saying this type of evidence reminds me a little bit of the early aging studies

with Golgi silver stains. It was originally thought that the brains were massively

degenerating in normal aging, but it has all turned out not to be true. In fact,

you find growth of dendrites rather than the loss of dendrites in normal aging in

some cases. Only in the very extreme cases of Alzheimer’s disease have they

seen clear attrition. So, many things that you see with staining are affected by

tissue quality and stainability.

I am probably preaching to the converted, for most people here. Staining of

fibers is interesting, but again, an important question is, do the raphe cell bodies

stain? Do these lose staining? Because the answer may tell us whether the

cells are degenerating.

COMMENT (Molliver): The raphe cell bodies seem to be normal, although we

haven’t yet obtained optimal morphology. George Ricaurte has reported

evidence of inclusion bodies in the raphe cell bodies in the primate. We are

continuing to look for other changes in these cells.

COMMENT (Landfield): If you could locate the cell bodies, you should be able,

at the EM level or with other methods, to follow the axons.

COMMENT (Molliver): It is not technically feasible to follow axons over

several regions in the EM in sections that are 700 angstroms. I am basing

my interpretation of degeneration on the composite set of data such as loss of

5-HT uptake, loss of anterograde transport, loss of retrograde transport, and

abnormal morphology.

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COMMENT (Landfield): All those data also can be consistent with massive

excitation-induced depletion of these cells and with fatigue or an exhaustive

state.

COMMENT (Molliver): For several months?

COMMENT (Landfield): But on the other hand, it is very unusual to see this

kind of massive axon degeneration and no change in the cell bodies and no

astrocyte reaction.

COMMENT (Molliver): Damage to the trochlear nucleus is being studied by

Hazel Murphy at the Medical College of Pennsylvania, as a model of the

neuronal response to injury. They have found that the degree of injury is a

function of the distance from the cell body that the axon is cut. If the axon is

cut near the terminals, there is little reaction in the cell body. As you cut the

axon closer to the cell body, you begin to see some changes. If you cut at the

decussation of the trochlear nerve in the isthmus, then the cell bodies show

degeneration. More distally they don’t degenerate, but they reinnervate the

superior oblique muscle.

COMMENT (Landfield): Did you study the loss of axon all the way back?

COMMENT (Molliver): The only axon loss that we detect is in the axon

terminals. The preterminal axons in the MFB [medial forebrain bundle] are not

only intact, but the axons are slightly thickened, as if there were damming of

transported material.

Q (Jensen): Mark, given the amount of depletion that has taken place, how

much information do you think there actually would be in histochemistry? In

other words, if you’re labelling the serotonin, that is gone. Right?

A: Only in terminals.

Q: Okay, only in terminals, but . . .

A: There are increased amounts in the preterminal axons.

Q: Right. But in the cortex, you get the depletion probably before you get

degeneration. Right?

Q: Would you say they are concomitant?

A: No, there is a specific sequence of transmitter change that has been

eloquently described by Chris Schmidt: but we and several other people have

seen it. Within 15 minutes after you give the drug, there is massive release of

181
serotonin from the terminals: then over the next 24 hours, there is recovery of

5-HT levels. The cells recover, the axons contain serotonin, and then from 1

to 3 days later, the axons become enormously swollen and fragmented. The

sequence is that there is release, depletion, and 1 to 2 days later, degeneration

of the terminals, followed by gradual regeneration months later.

Q: So you are saying that the depletion, the long-term depletion, is dependent

upon degeneration?

A: The long-term depletion is likely due to degeneration; the short-term

depletion is not. We have been able to take hippocampal slices and

incubate them-remove them from an animal and incubate them in artificial

cerebrospinal fluid. If you treat an animal with PCA or MDMA and remove

the brain rapidly, the brain slices are depleted of serotonin; if you then put

the slices in an incubation unit, serotonin synthesis occurs and the serotonin

becomes restored if you sacrifice the animals at 2 hours, 4 hours, 6 hours,

or up to 12 hours after the drug. At about 18 hours you no longer get

restoration, and that coincides with the time that you see swollen, abnormallooking axons. Thus, during the first 12 hours, the axons are intact and

recovery of 5-HT is seen. At 18 hours, there is degeneration.

Q: Are you suggesting then that you blocked the initial release and it has no

effect upon degeneration?

A (Molliver): No. The two mechanisms are dissociated. Ray Fuller and Chris

Schmidt have demonstrated that, and we have also confirmed it. You get initial

depletion of transmitter, and then you wait, depending on the drug, up to 6 to

12 hours when you can give an uptake blocker (citalopram or fluoxetine) which

prevents degeneration. You get depletion dissociated from degeneration.

Presumably the amphetamine derivatives are binding to the 5-HT uptake site;

if you displace them, within 6 to 12 hours, depending on whether it is MDA or

PCA, you can get substantial protection against toxicity.

COMMENT (Ricaurte): Can I just make a comment referring back to what Dr.

Landfield was discussing? While I agree with Mark in his interpretation of the

data, I think the questions you raise are superb from the standpoint of the level

of evidence that we should demand to arrive at a conclusion that indeed we are

dealing with a neurotoxic action. The question that I would have for you is,

what would it take to convince you that you are dealing with a neurotoxic drug?

A (Landfield): Having done stains for many years and knowing their variability,

I always feel a little uncomfortable with them. What I prefer is EM and semithin

sections. You can see every element. The observer then can see every type of

cell that is there.

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COMMENT (Ricaurte): With regard to amphetamine neurotoxicity, Phil Groves

has published EM immunocytochemical evidence demonstrating that

amphetamine is toxic to striatal dopamine titers.

COMMENT (Landfield): I think you have to see them actually degenerate.

There is another thing that bothers me about this interpretation: The sprouting

usually takes place in about 15 days in many systems, If only the terminals are

damaged, it is unprecedented to see decreases in neurochemistry for this long,

because it is also unprecedented for the terminals to take so long to sprout.

So there is something very unusual happening here, very interesting.

COMMENT (Molliver): Actually this sprouting begins at 2 to 4 weeks, in regions

close to the cell bodies. But it extends to the cortex only over a period of

months.

Q: I also had a question regarding your implication of degeneration and

regeneration since you only use a positive stain here-immunochemistry. It

would seem to me that you are implying degeneration, but you should use

degeneration staining to really show that in the first place. And in the second

place, you should really count cells, cell bodies. Because I have done an entire

series of MPTP-treated animals, I see the entire phenomenon that you see,

including the swellings. But the swellings turn out to be swollen axons that stain

with the Fink-Heimer method. However, after a period of time, they are gone;

and later still, the cell bodies are gone, totally gone. Other studies have shown

that it can take up to a year or 18 months for long-axon systems to degenerate

all the way. I think that many of your data should be supplemented by some

of the silver methods and counting some of the cell bodies.

A (Molliver): I agree with you. We have resisted counting cell bodies,

COMMENT: I understand why.

COMMENT (Seiden): It may be possible that in a highly collateralized system

like the 5-HT system, unless you damage some of the collaterals, the cell

bodies will not degenerate.

COMMENT (Appel): There are data that I can offer from experiments we did

at the Addiction Research Center of NIDA in Baltimore to support what Mark

says and might answer some of your criticisms because you asked about other

approaches. The approach that we used was in vitro receptor autoradiography.

We looked at the 5-HT uptake site. Using paroxetine binding, I did a study after

fenfluramine treatment at high dose, and George Battaglia did a study after

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MDMA or MDA treatment that is in press now in Synapse. Basically, what the

three different protocols showed was a decrease in the density of 5-HT uptake

sites throughout the frontal cortex, striatum, hippocampus, our hypothesis

being that the uptake site is on the axon. If the axon degenerates, there is no

substrate for the uptake site. The uptake site disappeared and was not seen

immediately or 2 weeks following treatment. However, there wasn’t a change

in the density of these uptake sites on the dorsal and medial raphe, the site of

the cell bodies from which regeneration could occur later on.

That is one set of studies. It was neurochemically specific, because

what we also did (and what George did) was to use mazindol to look at

catecholaminergic uptake sites vs. serotonin to show that those sites did

not change at all. Rob Zaczek did a study with fenfluramine and long-term

recovery (and these are now homogenate assays) looking for serotonin and

also serotonin uptake sites with paroxetine binding. The serotonin uptake

sites showed a very slow recovery. The study was carried out only to 32 weeks

or 8 months. There was a slow recovery; it never reached 100 percent. I think

at the 30-week time it was still at about 70 percent for the 5-HT uptake sites.

This is just a homogenate of frontal cortex, so it is general. What was

interesting was with the 5-HIAA and the serotonin content (these were

measured in hippocampus and in frontal cortex). There was a transitory

increase in serotonin and 5-HIAA. At 8 weeks, there was a transitory increase

in hippocampus; then it dropped back down. At 16 weeks, it was a transitory

increase in cerebral cortex, and then it dropped back down. What I was

wondering when you were showing these little diagrams is if there could be

a corresponding time course, where you have these large bulbs (for lack of

a better word)-the transitory appearance of them. Then they disappear,

because you start to get sprouting of your smaller fibers, your filopodia. And

then you were having your transient increase, or maybe the production that is

blocking up at the end of these damaged axons. When regeneration starts

occurring, 5-HT starts moving on, and maybe the levels drop back down.

Rob’s study was done only in fenfluramine-treated animals and was carried

out only as far as the 32 weeks. So this problem has been looked at with

more than just immunochemistry. I suffered the same “slings and arrows”

when I looked at fenfluramine. You are trying to stain for a neurotransmitter

that is not there. So there are other approaches.

Q (Seiden): Wasn’t there a 64-week study?

A (Appel): I don’t think so; there is a MDMA study approaching 1 year.

1Synapse 8(4):249-260, 1991.

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COMMENT (Ricaurte): George Battaglia and Errol De Souza did a 12-month

study with MDMA. After the 12 months, the uptake sites were completely

recovered. However, serotonin was only recovered to 50 percent of control.

COMMENT (Appel): There are still some rats sitting back at Baltimore;

everybody has left, but there are still a couple of rats. (Laughter)

COMMENT (Landfield): I think that it is clear that there are major

neurochemical effects of these treatments. But I still would like to see

some more done to directly visualize degenerating serotonergic synapses

and cells.

COMMENT (O’Callaghan): Perhaps with a good TPH [tryptophan hydroxylase]

antibody, one could stain the terminal fields that are degenerating. In the early

stages of a loss of immunoreactivity of serotonin in that terminal field, could you

double label with neurofilament antisera and see the progression of loss?

COMMENT (Molliver): Yes, you could. We have tried that. The problem is that

fine, unmyelinated axons are very few and contain little or no neurofilament

protein.

Q (Cho): Has anybody studied those with EM?

A (Molliver): We have tried to. The problem is that once you get to sections

that are a fraction of a micron thick, the incidence of affected axons is quite

small. There actually is one report recently by Constantine Sotello in Paris

who has shown EM evidence of degenerating axons after fenfluramine.

Q (Seiden): What is the best alternative hypothesis?

A (Landfield): The best alternative hypothesis I would think is that there is

something about the uptake of these drugs that blocks axon transport of the

transmitter serotonin, so that you have a loss of transmitter in terminals for 1 or

2 months; at least the synapses are nonfunctional. Something like that would

account for why there is no loss of serotonin all the way back in the cell body.

What are the uptake sites? They are proteins, which may be altered by

treatment. They may be phosphorylated, or changed in conformation so that

they may not bind. There could be all sorts of changes in uptake or transport

mechanisms.

COMMENT (Seiden): It is not only the binding aspect of serotonin uptake sites

that is compromised. If you do the synaptosomal preparation and you look at

the uptake of labelled serotonin, you will find that there is a change in Bmax.

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COMMENT (Landfield): I am not arguing that that is not true. I just saw a

paper where calbindin immunostaining totally disappeared after 15 minutes of

exposure to calcium. The treatment simply changed the conformation of the

protein so it was no longer visible by immunostaining. So one could imagine

many ways to inactivate binding to channels or uptake sites.

COMMENT (Molliver): Calbindin was found in the cell bodies of calbindinpositive and GABA [gamma aminobutyric acid] cells. When their axons are

transected close to the cell body, the cells stop producing transmittersynthesizing enzymes. In this study, the raphe cells are still producing

transmitter-synthesizing enzymes, which are accumulating along the axon.

That is not a comparable situation.

COMMENT (Landfield): I am just using that as an example of the sensitivity

of immunostaining and how it can change, just like that.

COMMENT (Appel): I can offer you still another approach which is going on

right now. We have treated some animals with fenfluramine or saline, let them

recover for 2 weeks, then injected tritiated proline into their dorsal raphe, and

are looking for anterograde transport or the lack of transport. I guess what we

are looking for is not so much a smoking gun as a gun with a stuffed muzzle.

I can tell you that I developed the injection sites-all the animals received

their tritiated proline and a first set of films has been developed after 6 weeks.

It is going to take a lot longer than 6 weeks to know the results, but here

is still another approach, and this type of approach is not based on the

neurotransmitter-it is based on the locus from which regeneration occurs.

Q (Landfield): I think that’s a good approach.

Q (Heimer): I was thinking why not try PHAL method.

A (Appel): I didn’t have an iontophoretic apparatus.

Q (Heimer): Using the PHAL method, you will be able to see the morphology

of these fibers very nicely, which you can’t do in autoradiography.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service research grants

DA-04431 and NS-15199 and National Institute on Drug Abuse contract

271-90-7408.

186
AUTHORS

Mary Ann Wilson, Ph.D.

Fellow

Department of Neuroscience

Laura A. Mamounas, Ph.D.

Research Associate

Department of Neuroscience

Kenneth H. Fasman, Ph.D.

Assistant Professor

Departments of Neuroscience and Biomedical Engineering

Karen J. Axt, Ph.D.

Research Associate

Department of Neuroscience

Mark E. Molliver, M.D.

Professor

Departments of Neuroscience and Neurology

Johns Hopkins University School of Medicine

725 North Wolfe Street

Baltimore, MD 21205

187
Quantification of Reactive Gliosis

as an Approach to Neurotoxicity

James P. O’Callaghan and Diane B. Miller

INTRODUCTION

If handed a bottle containing a pure chemical, told its molecular structure and

melting or boiling point, and then asked whether this substance was neurotoxic,

most scientists probably would not even hazard a guess. However, the U.S.

Environmental Protection Agency (EPA) must face this “real world” scenario on

a daily basis. For example, under the Toxic Substances Control Act (15 USC

2601 et seq. 1976), the EPA’s Pre-Manufacturing Notification Process requires

companies to give notification of their intent to manufacture a new chemical or

mixture. Within 90 days of receipt of this information, the EPA must decide

whether there are sufficient grounds to deny permission to manufacture the

compound. This decision is to be based on “the potential risk of injury to health

or the environment” posed by exposure to the compound (15 USC 2601 et seq.

1976). Ideally, risk of injury to health posed by exposure to a given compound

should include not only the effects linked to the most widely known “icities,”

carcinogenicity and teratogenicity, but neurotoxicity as well. In practice,

however, it is unlikely that the compound’s potential to cause nervous system

damage would be considered, because there is no widely accepted means for

assessing neurotoxicity. Thus, in the absence of prior knowledge of the potential

of a given chemical or mixture to produce neurotoxic effects, there would be no

reason to bar its production. This is a problem not just for the introduction of

new chemicals. Of the 65,000 chemicals in EPA’s existing chemical inventory,

few have been screened for neurotoxicity (National Research Council 1983;

U.S. Congress 1990; Tilson 1990). Clearly, from the perspective of the EPA,

the sheer volume of potentially neurotoxic substances dictates the need for

development and application of broadly applicable approaches for assessing

neurotoxicity. The purpose of this chapter is to show how the application of one

such approach, quantification of reactive gliosis, can be used to assess the

neurotoxic effects of broad classes of chemicals found in the environment. In

this context, drugs of abuse will be considered as “environmental pollutants”

to illustrate how this approach can be extended to compounds of interest to

another Federal agency, the National Institute on Drug Abuse (NIDA).

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Why is assessing neurotoxicity so difficult? A major reason is that targets of

neurotoxic insult are diverse and unpredictable. In comparison to other organ

systems, the mammalian central nervous system (CNS) is extremely complex.

It is composed of a vast array of different types of neurons and glia. At the

molecular level, this cell-type heterogeneity is reflected in the diversity of distinct

brain mRNAs (messenger RNAs) and their respective proteins. As many as

30,000 mRNAs are thought to exist, and 20,000 of these may be brain specific

(Sutcliffe 1988). These diverse cell types provide potentially equally diverse

targets of chemical-induced injury. Indeed, the classic textbook on experimental

neurotoxicology by Spencer and Schaumburg (1980) documents the multiplicity

of “selectively vulnerable” targets of neurotoxicity. As exemplified by the

dilemma faced by the EPA, often there is no database that points to the targets

affected by a suspect neurotoxicant. It seems obvious that this situation has led

neurotoxicologists to rely heavily on morphological evaluations for assessing

neurotoxicity (for a discussion, see Switzer 1991; Beltramino et al., this volume).

Morphological measures undeniably remain the “gold standard” for neurotoxicity

assessments. Without prior knowledge of the actions of a given compound,

morphologically based approaches serve as the only means for locating affected

regions and cell types. Moreover, because morphological changes generally are

taken as evidence of structural alterations, they often are thought to be indicative

of irreversible damage or, at the least, of a pathological process. Therefore, it is

not surprising that chemical-induced changes in nervous system morphology

are widely regarded as being indicative of an undesirable and, hence, adverse

or “neurotoxic” effect. It can be argued that biochemical, physiological, and

behavioral endpoints also can be used as indicators of neurotoxicity. Should

permanent changes in any of these measures occur as a result of chemical

exposures, they could be taken as indications of adverse (i.e., neurotoxic)

effects on the nervous system. Reversible changes in these measures are

more difficult to interpret. Although they could be indicative of damage to

specific brain regions or cell types, such changes instead may reflect reversible

pharmacological actions of the compound being studied. As noted by Switzer

(1991), it is not sensible to describe the change in a particular endpoint without

determining whether that change is related to destruction or damage of the

anatomical entity that produces or controls that endpoint. This does not imply

that reversible pharmacological effects should not be considered adverse or

perhaps even neurotoxic (Tilson 1990). Nevertheless, it seems likely that a

particular endpoint will gain broader acceptance as an indicator of neurotoxicity

if it can be linked to neural damage, given that nervous system damage is almost

universally accepted as an adverse effect. If neural damage is to be taken as

the most common basis for defining the neurotoxic state, then it follows that

there should be a set of criteria for defining this condition. Such criteria do

indeed exist.

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CRITERIA FOR DEMONSTRATION OF NEURAL DAMAGE

There are at least three widely accepted morphological criteria for establishing

the presence of neural damage. They are (1) cell loss, (2) argyrophilia (silver

staining), and (3) gliosis. Demonstration of any of the three is presumptive

evidence for neural damage. In the context of this chapter, the term “damage”

does not necessarily connote cell destruction. Thus, argyrophilia and gliosis

(criteria 2 and 3) can be used as indicators of damage in the the absence of

evidence for cell loss (criterion 1).

In theory, application of any of the above three criteria to neurotoxicity

assessment will aid in identifying damaged neural targets. In practice, although

application of the first criterion may be suitable for determining chemical-induced

cell loss in regions with large cell types organized in easily recognized layers

(e.g., hippocampus and cerebellum), damage to other vulnerable regions may

go undetected (O’Callaghan and Jensen 1992; Beltramino et al., this volume).

Only recently have unbiased cell-counting methods been developed in an

attempt to overcome this problem (Moller et al. 1989). The second criterion,

an argyrophilic reaction to neural damage, has proven to be an earlier and more

sensitive indicator of the neurotoxic condition (Balaban et al. 1988; Switzer 1991;

O’Callaghan and Jensen 1992; Balaban 1992; Beltramino et al., this volume).

Moreover, unlike the classic basic dyes used to detect cell loss (e.g., cresyl

violet and H and E), silver stains can reveal damage to nerve terminals as well

as damage or loss of neuronal perikarya (Balaban et al. 1988; O’Callaghan and

Jensen 1992; Beltramino et al., this volume). The use of silver degeneration

stains in assessments of neurotoxicity is reviewed elsewhere in this monograph

by Beltramino and colleagues and by Jensen and colleagues. The focus of the

remainder of this chapter is on the application of the third criterion, gliosis, for

assessing toxicant-induced injury of the CNS.

APPLICATION OF GLIAL FIBRILLARY ACIDIC PROTEIN ASSAYS TO

DETECT AND QUANTIFY GLIOSIS

Convinced of the need for a broadly applicable approach to neurotoxicity

assessment linked to neural damage, the authors and colleagues wanted to

utilize an endpoint that would reveal damage anywhere in the CNS following

exposure to any type of neurotoxicant. This endpoint needed to be a sensitive

indicator of neural damage as well as one that could be readily quantified.

Gliosis, a condition otherwise known as astrogliosis or reactive gliosis, satisfies

all these requirements.

Reactive gliosis occurs following nervous system damage induced by a

variety of insults, including physical damage, disease, or chemicals (Eng 1988;

O’Callaghan 1991 a, 1991b, 1992; Hatten et al. 1991; Norton et al. 1992). It is

characterized by hypertrophy and, rarely, hyperplasia of astrocytes, a subtype

190
of CNS glia (O’Callaghan 1991a, 1991b; Norton et al. 1992). Damage to any

neuronal and glial cell type (including astrocytes themselves) appears to elicit

reactive gliosis anywhere in the CNS. Classically, reactive astrocytes were

distinguished by their increased size, longer and thicker processes, and

increased content of glial filaments (Norton et al. 1992).

The development of antibodies to glial fibrillary acidic protein (GFAP), the

major protein of astrocyte intermediate filaments (Eng 1985) has led to the

widespread application of GFAP immunocytochemistry for studying reactive

gliosis (Lindsay 1986; Eng 1988). Although the results of these studies have

firmly established gliosis as a dominant response to traumatic injury of the

CNS, only a few investigations have been devoted to an examination of gliosis

following chemical exposures (O’Callaghan 1991a, 1991b, 1992; O’Callaghan

and Jensen 1992; Norton et al. 1992). Moreover, most attempts to quantify

gliosis have been limited to counts of GFAP-positive cells in immunostained

sections, Not only is this approach tedious, but it is hampered by a lack of

sensitivity as well as fixation artifacts (Shehab et al. 1990; Norton et al. 1992).

Because antibodies to GFAP are widely available through commercial sources,

it was reasoned that development of a GFAP radioimmunoassay offered the

most viable option to achieve sensitive and rapid quantification of GFAP as

an indicator of the degree of reactive gliosis.

The authors have developed two assays for GFAP: a solid phase immunoassay

(O’Callaghan and Miller 1985; Brock and O’Callaghan 1987) and a sandwich

enzyme-linked immunosorbent assay (ELISA) (O’Callaghan 1991c). The former

procedure is a modification of the nitrocellulose-based, dot-immunobinding

procedure of Jahn and colleagues (1984). The latter is a novel microtiter plate

assay employing both monoclonal and polyclonal antibodies to GFAP in a

sandwich format (O’Callaghan 1991 c). Both procedures share the advantage

that they can be used to assay detergent homogenates of nervous tissue directly

without further fractionation or purification. Thus, the data obtained are indicative

of effects that occurred in intact tissue, not in an extract of an operationally

defined membrane or cytosol fraction. It is likely that the sandwich ELISA will

prove to be more widely applicable than the dot-immunobinding procedure

because it is cheaper and faster and does not require the use of radioactive

reagents (O’Callaghan 1991c). When both GFAP assays are used to analyze

the same samples, essentially identical results are obtained (O’Callaghan

1991c).

VALIDATION OF GFAP AS A BIOCHEMICAL INDICATOR OF

NEUROTOXICITY

If reactive gliosis is a common response to neural damage, homogenates of

brain regions damaged by known neurotoxicants should contain an increased

concentration of GFAP. To test this hypothesis, the authors examined the effects

191
of prototype neurotoxicants known to damage diverse cell types in different

regions of the CNS. Both adult and developing rodents were used in these

validation experiments. The results of these studies have been reviewed

(O’Callaghan 1988, 1991a, 1991b, 1992; O’Callaghan and Miller 1989;

O’Callaghan and Jensen 1992). Quantitative increases in GFAP that result

from brain injury induced by agents or conditions other than those employed in

our investigations also have been reported. These studies have been reviewed

(Hatten et al. 1991; Norton et al. 1992).

Some of the prototype neurotoxicants employed, all of which resulted in an

increase in GFAP, were trimethyltin (TMT), kainate, triethyltin, ibotenate,

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine,

bilirubin (Gunn rat), 5,7-dihydroxytryptamine (5,7-DHT), cadmium, colchicine,

3-acetylpyridine, methylmercury, methamphetamine, iminodipropionitrile,

methylazoxymethanol, and ethanol. Compounds chosen were known to damage

neurons in several different brain regions to determine whether astrocyte

responses could be observed in response to injury of any area of the CNS.

Compounds also were chosen that ranged from those that caused overt cell loss

(e.g., TMT and bilirubin) (Brock and O’Callaghan 1987; O’Callaghan and Miller

1985) to those that caused damage to subcellular elements that would not be

detected in routine neuropathological examinations. An example of a compound

in the latter category is MPTP, a substituted pyridine known to damage

dopaminergic nerve terminals (Hallman et al. 1985; Heikkila et al. 1984). In our

validation scheme, we first emphasized studies of compounds that caused

outright cell loss. Overt neuron loss manifested at the light microscopic level is

not a subtle effect. Therefore, if large increases in GFAP were not seen with

compounds in this category, the utility of GFAP as an indicator of neural damage

would be limited. We were not disappointed. Compounds that caused readily

recognizable destruction of neuronal perikarya, such as TMT (Brock and

O’Callaghan 1987), resulted in large (as much as 6,000 percent) increases in

GFAP in the affected brain region (Brock and O’Callaghan 1987). The results of

these initial studies provided the impetus to examine the effects of compounds

where damaged neural components (as revealed by a permanent loss of a

specific marker protein) would not be evident from Nissl staining (e.g., MPTP).

Here, too, large increases in GFAP (350 percent of control) were found in brain

regions known to be damaged by the compound being used (O’Callaghan et al.

1990a, 1990b).

A number of features associated with chemical-induced gliosis emerged from

our studies of prototype neurotoxicants. A review of these features illustrates

the advantages and limitations of the use of GFAP as a biochemical indicator

of neural damage. A list and brief synopsis of our observations follows:

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1. Large, dose-dependent increases in GFAP result from acute exposure to

chemicals that destroy neuronal perikarya. Thus, increases in GFAP occur

under conditions associated with outright cell loss as evidenced by loss of

Nissl staining.

2. Large, dose-dependent increases in GFAP result from acute exposure to

chemicals that damage cellular components not revealed by Nissl staining.

Thus, assays of GFAP are sensitive enough to reflect damage to cellular

elements under conditions where there is no Nissl-staining evidence of cell

death or damage, a level of sensitivity shared by reduced silver methods for

detecting neuronal degeneration (O’Callaghan and Jensen 1992; Beltramino

et al., this volume; Jensen et al., this volume).

3. Targets of chemical-induced neural damage are revealed by increases in

GFAP. Trauma-induced gliosis can spread to an entire brain hemisphere

following a stab lesion (Mathewson and Berry 1985). In contrast, chemicalinduced lesions appear to be confined to the site of damage (Balaban et al.

1988; O’Callaghan 1991a, 1991b, 1992). Thus, assays of GFAP should be

useful for localizing sites of chemical-induced brain damage. However, this

will be the case only if the increase in GFAP is large enough to be detected

within the sample of tissue subjected to assay. Ensuring that toxicantinduced increases in GFAP are not masked by the baseline concentration

of GFAP in unaffected tissue requires dissection of the brain into several

regions. Dividing the brain into six to eight regions (O’Callaghan 1991b)

has proven sufficient to locate areas of gliosis. To reiterate, assays of GFAP

will not reveal sites of reactive gliosis if such sites represent only a small

percentage of the total tissue sampled.

4. Chemical-induced increases in GFAP are not permanent. The astrocyte

response to traumatic injury or disease often is manifested by a persistent

glial scar that is GFAP positive (Reier 1986). In contrast, we found that all

chemical-induced injuries of the CNS examined to date result in an increase

in GFAP that abates with time. The rate of onset and the subsequent

decline in GFAP levels vary markedly from toxicant to toxicant (O’Callaghan

1991a, 1991b; Norton et al. 1992). Some chemicals cause an increase in

GFAP that is slow to develop and slow to decline. For example, with TMT

the peak increase does not occur until 5 weeks postdosing, and the return to

baseline level is not reached until 12 weeks (Brock and O’Callaghan 1987).

Other chemical injuries result in a rapid onset and a rapid decline in GFAP.

For example, with MPTP the peak increase occurs within 48 hours and the

return to control occurs by 21 days (O’Callaghan et al. 1990a, 1990b). The

reason for these disparate time-effect relationships remains unknown. Our

current hypothesis is that sustained cellular damage is required to maintain a

continued elevation of GFAP (see O’Callaghan et al. 1990a for a discussion

of this issue). Sustained damage may reflect the continued presence of the

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5.

toxic insult (O’Callaghan et al. 1990a) or, as emphasized by Heimer and

coworkers (Beltramino et al., this volume), a “cascading effect” involving

pathways “recruited into the pathophysiologic process.” Regardless of

the mechanisms responsible for induction and maintenance of the GFAP

response to chemical-induced injury, our findings indicate that assessments

of neurotoxicity using assays of GFAP should employ multiple time points

to prevent false-negative results.

Hypertrophy, not hyperplasia, is the dominant response to chemical-induced

injuries of the CNS. Early studies by Cavanagh (1970) suggested that

astrocytes divide in response to penetrating injuries of the CNS. This

hyperplastic response is purported to be the basis of the glial scar (Reier

1986). A study using [

3H]thymidine autoradiography combined with GFAP

immunocytochemistry confirmed that stab injuries cause astrocytes to divide

in the region surrounding the wound site (Latov et al. 1979); this study often

serves as the basis of the contention that massive astrocytic hyperplasia

accompanies all brain injuries. However, careful examination of the data

indicates that stab injuries result in astrocyte proliferation only in the vicinity

of the wound site (Latov et al. 1979; Hatten et al. 1991; Norton et al. 1992).

Moreover, even at the site of injury, only a small percentage of astrocytes

proliferate (Latov et al. 1979). Both at the wound site and especially in the

surrounding tissue, hypertrophy is the dominant astrocytic reaction (Norton et

al. 1992). In studies of prototype neurotoxicants, the authors confirmed that

astrocytic proliferation can occur (Brock and O’Callaghan 1987) but, again,

only on a limited basis. Less than 1 percent of total astrocytes were found to

undergo division (Brock and O’Callaghan 1987). Given that blood contains

astrocyte mitogens (Giulian et al. 1988) it is not surprising that astrocytes

divide in the vicinity of stab wounds. However, when the blood-brain barrier

remains intact, as is the case following exposure to MPTP (O’Callaghan et

al. 1990a), large increases in GFAP occur apparently without the proliferation

of astrocytes (Brock and O’Callaghan 1987; O’Callaghan 1991a). If our data

for MPTP and other prototype neurotoxicants can be applied to the general

case, we do not expect astrocytic hyperplasia to play a major role in the

observed increase in GFAP. Given this probability, it is doubtful that GFAP

immunocytochemistry-based cell counts will be of much value in the

assessment of general neurotoxic responses because more GFAP per

astrocyte, not more astrocytes, will be the predominant neurotoxic response.

6. Pharmacological agents at therapeutic dosages do not cause an increase

in GFAP. One of the assumptions underlying the authors’ use of GFAP

assays to detect and quantify neural damage is that drugs administered

at therapeutic dosages would not cause an increase in GFAP. Although

comprehensive dose- and time-effect studies have not been attempted,

compounds from several pharmacological classes have been examined.

None of these agents affected GFAP levels in any brain region examined

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(O’Callaghan 1991a, 1991b, 1992). These findings are consistent with

our view that increases in GFAP occur as a consequence of chemicalinduced neural damage but do not result from drug exposures at therapeutic

dosages. This does not mean that drug dosages above the pharmacological

range would not result in neural damage (see below).

In aggregate, these findings indicate that assays of GFAP can be used to

quantify dose-, time-, and region-dependent patterns of toxicant-induced

astrogliosis (O’Callaghan 1991a, 1991b). Using this response as an indirect

indicator of chemical-induced neural damage, we have been able to detect

effects at toxicant dosages below those that would result in light microscopic

evidence of neurotoxicity. As expected, pharmacological agents at therapeutic

dosages do not appear to elicit this generalized response to nervous system

damage.

APPLICATION OF GFAP ASSAYS TO DETECT AND QUANTIFY THE

NEUROTOXIC EFFECTS OF SUBSTITUTED AMPHETAMINES

Various substituted amphetamines (e.g., amphetamine, methamphetamine,

methylenedioxyamphetamine, methylenedioxymethamphetamine [MDMA],

fenfluramine, parachloroamphetamine) are considered to be neurotoxic

because they produce long-term (several days to several months) decrements

in indicators of brain dopaminergic and serotonergic function (Fuller 1989, 1978,

1985; Seiden and Kleven 1989; Gibb et al. 1990; Molliver et al. 1990; Schmidt

and Kehne 1990; De Souza et al. 1990). Included among these effects are

decreases in brain amines (Ricaurte et al. 1980, 1984a; Wagner et al. 1980;

Commins et al. 1987; Schmidt 1987), decreases in the rate-limiting enzymes

responsible for amine biosynthesis (Hotchkiss and Gibb 1980; Stone et al. 1991;

Schmidt and Taylor 1987; Lovenberg and Bruckwick 1978), and decreased

ligand binding to high-affinity uptake sites for dopamine and serotonin (Commins

et al. 1987; Appel et al. 1989; Kovachich et al. 1989; Schmidt 1987; Battaglia et

al. 1987). At higher dosages of these compounds, evidence of neural damage

has been observed using silver degeneration stains (Commins and Seiden 1986;

Commins et al. 1987; Ricaurte et al. 1982, 1984a, 1984b; Scallet et al. 1988;

Jensen et al., this volume). In the examination of these compounds, the intent

was to establish whether the previously observed effects on dopaminergic and

serotonergic systems were accompanied by the astrocytic (GFAP) response

characteristic of neural damage. Where an increase in GFAP was observed,

a related goal was to establish whether damaged dopaminergic or serotonergic

neurons served as a stimulus for this effect. Silver degeneration stains also

were employed as an alternate means of detecting sites of damage (see

Jensen et al., this volume). Two amphetamine derivatives, methamphetamine

and MDMA, were evaluated. These compounds were chosen because they

represent prototype amphetamines for producing long-term deficits in

dopaminergic and serotonergic systems, respectively (Gibb et al. 1990).

195
EFFECTS OF MPTP AND METHAMPHETAMINE IN THE C57BL/6J MOUSE

Recently, Sonsalla and colleagues (1989) compared the effects of MPTP and

methamphetamine on the nigrostriatal dopaminergic system of the C57BI/6J

mouse. Both compounds produced deficits in dopamine levels and in the activity

of tyrosine hydroxylase (TH), a marker enzyme of the nigrostriatal dopaminergic

pathway. These effects were equated with the “neurotoxic” actions of these

compounds. Pretreatment with the noncompetitive glutamate receptor

antagonist, MK-801, blocked the effects of methamphetamine but not those

of MPTP.

From previous work on MPTP in the mouse, it is known that long-term

decrements in striatal dopamine are accompanied by apparently permanent

decrements in the concentration of TH holoenzyme protein (O’Callaghan et al.

1990a, 1990b). Moreover, a large (350 percent of control) increase in GFAP

is associated with the loss of TH protein (O’Callaghan et al. 1990a) as is an

argyrophilic reaction in the nigrostriatal pathway (O’Callaghan and Jensen 1992).

Taken together, these data are viewed as strong evidence for a neurotoxic action

of MPTP on nigrostriatal dopaminergic neurons. Thus, MPTP-induced depletion

of striatal dopamine in the mouse can be considered a neurotoxic effect, because

this action of the compound is associated with changes in other endpoints that

are indicative of neural damage. The effects of methamphetamine on striatal

dopamine in the mouse may also be indicative of damage to this pathway,

but indicators of neural damage, such as loss of TH protein or an increase in

GFAP, have not been examined. To address this question, we adopted the

methamphetamine-dosing regimen for the mouse that was developed by

Sonsalla and coworkers (1989). MPTP served as a positive control and, as

in the study by Sonsalla and colleagues (1989) the potential neuroprotective

effects of MK-801 were evaluated.

As demonstrated previously (O’Callaghan et al. 1990a), a single subcutaneous

(SC) injection of MPTP (12.5 mg/kg) results in a large (300 percent of control)

increase in striatal GFAP at 2 days postdosing (figure 1). A greater-than50-percent reduction in the concentration of TH (protein) and dopamine

accompanies the increase in GFAP. None of these effects of MPTP were

altered by pretreatment and posttreatment with MK-801 (2x1 mg/kg; see

Sonsalla et al. 1989). Administration of methamphetamine (4x5 mg/kg in

1 day; see Sonsalla et al. 1989), like administration of MPTP, caused a large

increase in striatal GFAP at 2 days postdosing (figure 2). A greater-than50-percent reduction in the concentration of TH (protein) and dopamine

accompanied the increase in GFAP. In contrast to the effects of MPTP, the

methamphetamine-induced increase in GFAP and the decrease in TH and

dopamine were completely antagonized by MK-801. All the effects of MPTP

and methamphetamine observed at 2 days postdosing also were observed at

a 7-day time point (data not shown). These data are in complete agreement

196
FIGURE 1. Effects of MPTP alone or in combination with MK-801 on the

concentration of GFAP, TH (protein), and dopamine in striatum

of C57BI/6J mice. Mice received 12.5 mg/kg MPTP SC and

were killed at 48 hours postdosing. MK-801 (2x1 mg/kg) was

administered according to the regimen described by Sonsalla

and colleagues (1989). GFAP, TH, and dopamine were

determined as described in O'Callaghan (1990a).

* Significantly different from corresponding control, p<0.05

with those of Sonsalla and colleagues (1989). We conclude that both MPTP

and methamphetamine damage nigrostriatal dopaminergic neurons in the

mouse and that the effects of the latter compound may be mediated through

a glutaminergic mechanism.

EFFECTS OF METHAMPHETAMINE IN THE LONG-EVANS RAT

Methamphetamine is viewed as a dopaminergic neurotoxicant in the rat as

well as in the mouse. In comparison to the mouse, however, rats require

considerably higher dosages to achieve equivalent depletions of striatal

dopamine or reductions in TH activity (Sonsalla et al. 1986). In an attempt to

achieve decrements in striatal dopamine in a time equivalent to that of the

mouse model, we administered four dosages of 50 mg/kg SC to the Long-Evans

197
FIGURE 2. Effects of methamphetamine alone or in combination with MK801 on the concentration of GFAP, TH (protein), and dopamine

in striatum of C57BI/6J mice. Mice received 5 mg/kg

methamphetamine, 4 times in 1 day SC and were killed at

48 hours postdosing. The methamphetamine dosing regimen

was as described by Sonsalla and colleagues (1989). MK-801

(2x1 mg/kg) was administered according to the regimen

described by Sonsalla and colleagues (1989). Measurements

were determined as described in the legend to figure 1.

* Significantly different from corresponding control, p<0.05

rat on a schedule identical to that used for the mouse studies, To prevent

mortality with this high-dose regimen, rats were housed individually in wirebottom cages with a 1 -week acclimation period (D.B. Miller and J.P. O’Callaghan,

unpublished observation). As in the mouse studies, we also evaluated the

neuroprotective effect of MK-801. At 2 days postdosing, the concentration of

striatal GFAP was slightly elevated; however, the concentration of TH was

unaffected (figure 3). In contrast, the concentration of striatal dopamine was

reduced by more than 50 percent, that is, to levels equivalent to those seen in

the mouse at 1/10 the dosage. MK-801 pretreatment and posttreatment (2x1

mg/kg) SC did not alter the effects of methamphetamine on GFAP and dopamine

(figure 3). The effect of methamphetamine on striatal dopamine was transient;

at 7 days postdosing, dopamine levels had returned to control values (data not

198
FIGURE 3. Effects of methamphetamine alone or in combination with MK801 on the concentration of GFAP, TH (protein), and dopamine

in striatum of Long-Evans rats. Rats received 50 mg/kg

methamphetamine, 4 times in 1 day SC and were killed at 48

hours postdosing. The dosing schedules for methamphetamine

and MK-807 (2x1 mg/kg) were the same as used for the mouse

experiments (figure 2). Measurements were determined as

described in the legend to figure 1.

* Significantly different from corresponding control, p<0.05

shown). At the 7-daytime point, the concentration of GFAP and TH also did not

differ between the control and methamphetamine groups (data not shown).

Our findings suggest that the mouse and rat respond in different manners to

methamphetamine. By comparing the GFAP and TH data obtained from the

two species, it would not be speculative to conclude that methamphetamine is,

at best, weakly neurotoxic to rat nigrostriatal neurons. At the least, our findings

suggest that the transient methamphetamine-induced decrease of striatal

dopamine in the rat is not an adequate predictor of underlying damage to

nigrostriatal neurons. Indeed, the transient effects of methamphetmine on

striatal dopamine in the rat may be more of a reflection of the influence of

environmental temperature (Bowyer et al. 1992) rather than an indication of

199
neuronal damage. The fact that an effect of methamphetamine on GFAP and

TH in the rat was not observed cannot be viewed as a failure of these endpoints

to reveal underlying damage to the nigrostriatal pathway. lntracisternal

administration of the dopaminergic neurotoxicant, 6-hydroxy (6-OH) dopamine,

to the rat results in a large increase in striatal GFAP (250 percent of control) and

a large and permanent decrease in striatal TH protein (J.P. O’Callaghan and

G.R. Breese, unpublished observation). Thus, loss of TH protein in association

with an increase in striatal GFAP appears to provide an adequate reflection of

nigrostriatal damage in the rat. None of our data rule out the possibility that

methamphetamine-induced brain damage in the rat may be obtained with

dosing regimens that induce long-term decrements in the transmitter content

of specific neuronal pathways (Ricaurte et al. 1980). However, even when the

methamphetamine dosage was increased to as much as 150 mg/kg, twice daily

for 2 days, we failed to see marked increases in GFAP at time points ranging

from 2 to 9 days postdosing (data not shown).

EFFECTS OF MDMA IN THE LONG-EVANS RAT

MDMA is widely viewed as being neurotoxic to serotonergic neurons (for

reviews, see McKenna and Peroutka 1990, 1991). As little as a single

administration of 20 mg/kg to the rat results in long-lasting (several weeks)

decreases in 5-hydroxytryptamine (5-HT) levels and tryptophan hydroxylase

activity in the rat cerebral cortex (Schmidt 1987; Schmidt and Taylor 1987).

Long-term decrements in the density of 3H-paroxetine-labeled 5-HT uptake

sites, findings suggestive of serotonergic nerve terminal damage, also have

been observed with MDMA after repeated administration of 20 mg/kg, twice

daily for 4 days (Battaglia et al. 1987). Multiple high-dose regimens of MDMA

result in evidence of terminal degeneration based on reduced silver degeneration

stains (Commins et al. 1987). Most of these data have been obtained from

studies in the rat because serotonergic neurons apparently are not affected by

MDMA in the mouse (Stone et al. 1991; McKenna and Peroutka 1991).

We began our examination of the effects of MDMA by administering a single

20 mg/kg dose. Cortex, striatum, and hippocampus, regions known to show

5-HT depletion with this dose of MDMA, then were assayed for GFAP. Levels

of GFAP were not changed at time points ranging from 4 hours to 4 weeks

postdosing (figure 4). Because the degree of neurotoxicity associated with

the administration of MDMA is purported to be related to the number as well

as the size of the doses received (McKenna and Paroutka 1991), we reasoned

that a multiple-dose regimen might be required to see an MDMA-induced

increase in GFAP. Therefore, twice daily administrations of 5 to 30 mg/kg of

MDMA were given for 7 days, and GFAP and 5-HT levels in cortex, hippocampus,

and striatum were assayed. The data obtained at 2 days after the last dose of

MDMA are shown in figure 5. Again, no increase was seen in GFAP in any of

these regions, although large decrements in 5-HT were found. GFAP also was

200
FIGURE 4.

Days After MDMA (20 mg/kg)

Effects of a single administration of MDMA (20 mg/kg SC) on the

concentration of GFAP in cortex, hippocampus, and striatum of

the Long-Evans rat. GFAP was determined as described in the

legend to figure 1.

201
FIGURE 5. Effects of MDMA (10 to 30 mg/kg, twice daily for 7 days) on

the concentration of GFAP and 5-HT in cortex, striatum, and

hippocampus of the long-Evans rat. Rats were killed 2 days

after the last dose of MDMA. GFAP and 5-HT data were

generated from samples prepared from the same rats. GFAP

was determined as described in the legend to figure 1. 5-HT

was determined by high-performance liquid chromatography

(HPLC) coupled with in-line fluorescence detection.

* Significantly different from control, p<0.05

202
not affected at 7 or 60 days postdosing (data not shown). 5-HT levels, however,

remained reduced in striatum and hippocampus but not in cortex at 60 days

postdosing (data not shown). These data indicated that an MDMA-dosage

regimen sufficient to produce a large and long-lasting decrease in 5-HT was

not sufficient to induce an astrocyte reaction characteristic of neural injury.

Finally, at an even higher total dosage of MDMA, we were able to see an

increase in GFAP (figure 6). Twice-daily administrations of 75 to 150 mg/kg of

MDMA for 2 days resulted in a dose-dependent increase in the levels of GFAP

in cortex and striatum at 2 days postdosing. Surprisingly, this extremely highdose regimen resulted in a U-shaped dose-response curve with respect to levels

of 5-HT; the highest total dosage of MDMA caused the least decrease in 5-HT

(figure 6). Thus, evidence for MDMA-induced neural damage, as revealed by

an increase in GFAP, was not necessarily linked to the propensity of the

compound to cause decreases in levels of 5-HT.

In comparison with the data obtained for the dopaminergic neurotoxicants,

MPTP, methamphetamine, and 6-OH dopamine, the data obtained from our

studies of the putative serotonergic neurotoxicant MDMA were more difficult to

interpret. With the dopaminergic neurotoxicants, increments in the concentration

of GFAP in conjunction with decrements in TH (but not dopamine) could be

used to estimate damage to nigrostriatal neurons, the putative targets of the

neurotoxic effects of these compounds. Similarly, it would have been useful to

determine whether MDMA-induced increases in GFAP were associated with

decrements in the amount of tryptophan hydroxylase as well as the observed

decrements in 5-HT to obtain an estimate of damage to serotonergic neurons.

Our attempts to quantify tryptophan hydroxylase have not been successful;

therefore, we do not know whether the increase in GFAP that accompanies the

high-dose regimen of MDMA is associated with loss of serotonergic terminals or

axons. However, because the 5-HT uptake inhibitor, fluoxetine, causes a partial

blockade of 5-HT depletions as well as the increase in GFAP, it is likely that at

least a portion of the GFAP increase emanates from an astrocyte response to

damaged serotonergic neurons (O’Callaghan et al. 1991).

MPTP and 6-OH dopamine were used as positive controls in our examination

of the potential for methamphetamine to induce a GFAP increase in the

neostriatum of the mouse and rat, respectively. These controls were

incorporated to ensure that assays of GFAP would reveal damage to

dopaminergic fibers in the striatum. To achieve a similar level of confidence

in the results of our MDMA studies, the serotonergic neurotoxicant 5,7-DHT

was used as a positive control for damage to serotonergic neurons of the rat.

Following pretreatment with desmethylimiprimine to protect catecholaminergic

neurons, we gave bilateral intraventricular injections of 5,7-DHT (125 μg on

each side). At 7 days after dosing, this regimen resulted in an increase (25 to

60 percent) in the concentration of GFAP in all brain areas surveyed (figure 7).

5-HT levels in these same brain regions (prepared from the same animals)

203
FIGURE 6. Effects of MDMA (75 to 150 mg/kg, twice daily for 2 days) on

the concentration of GFAP and 5-HT in cortex, striatum, and

hippocampus of the Long-Evans rat. Rats were killed 2 days

after the last dose of MDMA. GFAP and 5-HT data were

generated from samples prepared from the same rats. GFAP

and 5-HT were determined as described in the legends to

figure 1 and figure 5, respectively.

* Significantly different from control, p<0.05

204
FIGURE 7. Effects of 5,7-DHT (125 μg/side, bilateral intraventricular

injections) on the concentration of GFAP and 5-HT in cortex,

striatum, and hippocampus of the Long-Evans rat. Rats were

killed 7 days after dosing. GFAP and 5-HT data were generated

from samples prepared from the same rats. GFAP was

determined by the method of O’Callaghan (1991b). 5-HT was

determined by HPLC with electrochemical detection.

* Significantly different from uninjected control, p<0.05

205
were reduced by 60 to 80 percent. The concentration of GFAP also was

increased in most brain areas 3 days after administration of 5,7-DHT (data

not shown). In general, the magnitude of the GFAP increase observed at

3 days postdosing was smaller than that observed at 1 week. Moreover, the

decrements in 5-HT also were smaller at the 3-day time point in comparison to

those observed at 1 week (data not shown). These data indicate that the

neurodegenerative effects of the prototype serotonergic neurotoxicant 5,7-DHT

result in an astrogliotic reaction as revealed by an increase in GFAP. These

findings are consistent with previous observations of an increase in GFAP

concentration and immunoreactivity following intrahypothalamic administration

of 5,7-DHT (Frankfurt et al. 1991). The effects of 5,7-DHT on GFAP stand in

contrast to the absence of any effect on GFAP with an MDMA dosage regimen

that resulted in 5-HT decrements equivalent to or greater than those seen with

5,7-DHT (see figure 5). Thus, the fact that MDMA-dosing regimens that cause

long-lasting decreases in 5-HT do not result in astrogliosis (figure 5) suggests

that such regimens may lead to down-regulation of serotonin biosynthesis

without causing nerve terminal or axonal degeneration.

UNRESOLVED ISSUES AND DATA GAPS

Based on the results of studies of a large variety of prototype neurotoxicants,

the authors feel confident that assays of GFAP represent a sensitive means

for detecting and quantifying neural damage. Silver staining will be revived as

another approach for assessing toxicant-induced damage to the nervous system

(Switzer 1991; Beltramino et al., this volume; Jensen et al., this volume). Each

of these methods has its advantages, The GFAP-based approach is simple

and quantitative, but it provides only an indirect indication of neural damage.

Reduced silver methods, although more time consuming and qualitative, provide

a means for the direct identification of the damaged pathways. Where GFAP

assays have been combined with reduced silver methods to assess neural

damage, results obtained with both techniques have proven to be similar in a

number of respects (O’Callaghan and Jensen 1992). The similar sensitivity of

the two procedures may be the most important shared feature for interpreting

the potential neurotoxic effects of substituted amphetamines. High dosages of

substituted amphetamines were required to induce an argyrophilic reaction in

the affected regions of the rat brain (Commins and Seiden 1986; Commins et al.

1987; Ricaurte et al. 1984b; Jensen et al., this volume). Likewise, high dosages

of substituted amphetamines were required to cause an increase in GFAP in the

damaged brain areas of this species (figures 3 and 6). Clearly, this “high-dose”

requirement does not indicate that the two approaches are not sensitive. A low

dosage of MPTP in the mouse results in a large increase in striatal GFAP and

an accompanying argyrophilic reaction in the nigrostriatal pathway (O’Callaghan

and Jensen 1992). Similarly, a relatively modest methamphetamine-dosage

regimen in the mouse also causes a marked increase in GFAP (figure 2), an

effect that we predict will be accompanied by an argyrophilic reaction.

206
What then are we to make of our data for the effects of amphetamines in the

rat? The simple conclusion is that high dosages of these compounds are

required to cause neural damage in this species. Long-lasting effects of

substituted amphetamines on a number of constituents of serotonergic and

dopaminergic neurons have been described over the years (Gibb et al. 1990).

However, one implication of these data is that these effects, when observed

in the rat, may not reflect amphetamine-induced damage to serotonergic or

dopaminergic pathways. These arguments are not meant to imply that most

amphetamine derivatives are not detrimental to dopaminergic and serotonergic

function. The long-lasting amphetamine-induced deficits in the biochemical

machinery associated with these transmitter systems are suggestive, minimally,

of a long-term deficit in function. Such effects can be considered adverse or

“neurotoxic” to the affected pathways. Our findings emphasize the need to

establish the dosage requirements for such effects in relation to the dosage

requirements needed to cause neural damage. Given the differences between

mouse and rat responses to methamphetamine, the cross-species generality of

these dosage requirements also needs to be established for other substituted

amphetamines.

Finally, we note that both astrogliosis and argyrophilia appear to represent

common and, apparently, equally sensitive responses to all types of neurotoxic

exposures, This observation is suggestive of the existence of a common set of

“damage” signals responsible for initiating these general neurotoxic responses.

No such signals have as yet been identified (O’Callaghan 1991a, 1991b; Norton

et al. 1992). Elucidation of the mediators responsible for induction of astrogliosis

and argyrophilia may provide the earliest and most sensitive markers for the

detection and characterization of neural damage.

REFERENCES

Appel, N.M.; Contrera, J.F.; and De Souza, E.B. Fenfluramine selectively and

differentially decreases the density of serotonergic nerve terminals in rat brain:

Evidence from immunocytochemical studies. J Pharmacol Exp Ther 249:928-

943, 1989.

Balaban, CD. The use of selective silver degeneration stains in neurotoxicology:

Lessons from studies of selective neurotoxicants. In: Issacson, R.L., and

Jensen, K.F., eds. The Vulnerable Brain and Environmental Risks, Vol. 1,

Malnutrition and Hazard Assessment. New York: Plenum, 1992. pp. 223-238.

Balaban, C.D.; O’Callaghan, J.P.; and Billingsley, M.L. Trimethyltin-induced

neuronal damage in the rat: Comparative studies using silver degeneration

stains, immunocytochemistry and immunoassay for neuronotypic and gliotypic

proteins. Neuroscience 26:337-361, 1988.

207
Battaglia, G.; Yeh, S.Y.; O’Hearn, E.; Molliver, M.E.; Kuhar, M.J.; and

De Souza, E.B. 3,4-Methylenedioxymethamphetamine and 3,4-

methylenedioxyamphetamine destroy serotonin terminals in rat brain:

Quantification of neurodegeneration by measurement of [

3H]paroxetinelabeled serotonin uptake sites. J Pharmacol Exp Ther 242:911-916, 1987.

Bowyer, J.F.; Tank, W.A.; Newport, G.D.; Slikker, W.F., Jr.; Ali, SF.; and Holson,

R.R. The influence of environmental temperature on the transient effects of

methamphetamine on dopamine levels and dopamine release in rat striatum.

J Pharmacol Exp Ther 260:817-824, 1992.

Brock, T.O., and O’Callaghan, J.P. Quantitative changes in the synaptic vesicle

proteins synapsin I and p38 and the astrocyte-specific protein glial fibrillary

acidic protein are associated with chemical-induced injury to the rat central

nervous system. J Neurosci 7:931-942, 1987.

Cavanagh, J.B. The proliferation of astrocytes around a needle wound in the

rat brain. J Anat 106:471-487, 1970.

Commins, D.L., and Seiden, L.S. Alpha-methyltyrosine blocks

methylamphetamine-induced degeneration in the rat somatosensory cortex.

Brain Res 365:15-20, 1986.

Commins, D.L.; Vosmer, G.; Virus, R.M.; Woolverton, W.L.; Schuster, CR.;

and Seiden, L.S. Biochemical and histological evidence that

methylenedioxymethylamphetamine (MDMA) is toxic to neurons in the

rat brain. J Pharmacol Exp Ther 241:338-345, 1987.

De Souza, E.B.; Battaglia, G.; and Insel, T.R. Neurotoxic effects of MDMA on

brain serotonin neurons: Evidence from neurochemical and radioligand

binding studies. In: Whitaker-Azmitia, P.M., and Peroutka, S.J., eds. The

Neuropharmacology of Serotonin. Ann N Y Acad Sci 600:682-698, 1990.

Eng, L.F. Glial fibrillary acidic protein (GFAP): The major protein of glial

intermediate filaments in differentiated astrocytes. J Neuroimmunol 8:203-214,

1985.

Eng, L.F. Regulation of glial intermediate filaments in astrogliosis. In: Norenberg,

M.D.; Hertz, L.; and Schousboe, A., eds. Biochemical Pathology of Astrocytes.

New York: Alan R. Liss, 1988. pp. 79-90.

Frankfurt, M.; O’Callaghan, J.; and Beaudet, A. 5,7-Dihydroxytryptamine

injections increase glial fibrillary acidic protein in the hypothalamus of adult

rats. Brain Res 549:138-140, 1991.

Fuller, R.W. Neurochemical effects of serotonin neurotoxins: An introduction, In:

Jacoby, J.H., and Lytle, L.D., eds. Serotonin Neurotoxins. Ann N Y Acad Sci

305:178-181, 1978.

Fuller, R.W. Persistent effects of amphetamine, p-chloroamphetamine, and

related compounds on central dopamine and serotonin neurons in rodents.

Psychopharmacol Bull 21:528-532, 1985.

208
Fuller, R.W. Recommendations for future research on amphetamines

and related designer drugs. In: Asghar, K., and De Souza, E., eds.

Pharmacology and Toxicology of Amphetamine and Related Designer Drugs.

National Institute on Drug Abuse Research Monograph 94. DHHS Pub.

No. (ADM)89-1640. Washington, DC: Supt. of Docs., U.S. Govt. Print. Off.,

1989. pp. 341-357.

Gibb, J.W.; Johnson, M.; Stone, D.; and Hanson, G.R. MDMA: Historical

perspectives. In: Whitaker-Azmitia, P.M., and Peroutka, S.J., eds. The

Neuropharmacology of Serotonin. Ann N Y Acad Sci 600:601-612, 1990.

Giulian, O.; Woodward, J.; Young, D.G.; Krebs, J.F.; and Lachman, L.B.

Interleukin-1 injected into mammalian brain stimulates astrogliosis and

neovascularization. J Neurosci 8:2485-2490, 1988.

Hallman, H.; Lange, J.; Olson, L.; Stromberg, I.; and Jonsson, G.

Neurochemical and histochemical characterization of neurotoxic effects of 1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine on brain catecholamine neurons in

the mouse. J Neurochem 44:117-127, 1985.

Hatten, M.E.; Liem, R.K.H.; Shelanski, M.L.; and Mason, C.A. Astroglia in CNS

injury. Glia 4:233-243, 1991.

Heikkila, R.E.; Hess, A.; and Duvosin, R.C. Dopaminergic neurotoxicity of 1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. Science 224:1451-1453,

1984.

Hotchkiss, A.J., and Gibb, J.W. Long-term effects of multiple doses of

methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase

activity in rat brain. J Pharmacol Exp Ther 214:257-262, 1980.

Jahn, R.; Schiebler, W.; and Greengard, P. A quantitative dot immunobinding

assay for protein using nitrocellulose membrane filters. Proc Natl Acad Sci

U S A 81:1684-1687, 1984.

Kovachich, G.B.; Aronoson, C.E.; and Brunswick, D.J. Effects of high-dose

methamphetamine administration on serotonin uptake sites in rat brain

measured using [

3H]cyanoimipramine autoradiography. Brain Res 505:123-

129, 1989.

Latov, N.; Nilaver, G.; Zimmerman, E.A.; Johnson, W.G.; Silverman, A.;

Defendini, R.; and Cote, L. Fibrillary astrocytes proliferate in response to

brain injury. Dev Biol 72:381-384, 1979.

Lindsay, R.M. Reactive gliosis. In: Fedoroff, S., and Vernadakis, A., eds.

Astrocytes: Cell Biology and Pathology of Astrocytes. Vol. 3. Orlando, FL:

Academic Press, 1986. pp. 231-262.

Lovenberg, W., and Bruckwick, E. Enzyme changes as an index of neurotoxin

specificity. In: Jacoby, J.H., and Lytle, L.D., eds. Serotonin Neurotoxins.

Ann N Y Acad Sci 305:182-189, 1978.

Mathewson, A.J., and Berry, M. Observations on the astrocyte response to a

cerebral stab wound in adult rats. Brain Res 327:61-69, 1985.

McKenna, D.J., and Peroutka, S.J. Neurochemistry and neurotoxicity of 3,4-

methylenedioxymethamphetamine (MDMA, “ecstasy”). J Neurochem

54:14-22, 1990.

209
McKenna, D.J., and Peroutka, S.J. Serotonin neurotoxins: Focus on MDMA (3,4-

methylenedioxymethamphetamine, “ecstasy”). In: Peroutka, S.J., ed. Serotonin

Receptor Subtypes. Basic and Clinical Aspects. New York: Wiley-Liss, 1991.

pp. 125-146.

Moller, A.; Strange, P.; and Gundersen, H.J.G. Efficient estimation of cell volume

and number using the nucleator and the dissector. J Microsc 159:61-71, 1989.

Molliver, M.E.; Berger, U.V.; Mamounas, L.A.; Molliver, D.C.; O’Hearn, E.; and

Wilson, M.A. Neurotoxicity of MDMA and related compounds: Anatomic studies.

In: Whitaker-Azmitia, P.M., and Peroutka, S.J., eds. The Neuropharmacology

of Serotonin. Ann N Y Acad Sci 600:640-664, 1990.

National Research Council. Risk Assessment in the Federal Government:

Managing the Process. Washington, DC: National Academy of Sciences, 1983.

Norton, W.T.; Aquino, D.A.; Hozumi, I.; Chiu, F.-C.; and Brosnan, C.R.

Quantitative aspects of reactive gliosis: A review. Neurochem Res 17:877-885,

1992.

O’Callaghan, J.P. Neurotypic and gliotypic proteins as biochemical markers

of neurotoxicity. Neurotoxicol Teratol: 10:445-452, 1988.

O’Callaghan, J.P. Assessment of neurotoxicity: Use of glial fibrillary acidic protein

as a biomarker. Biomed Environ Sci 4:197-206, 1991a.

O’Callaghan, J.P. The use of glial fibrillary acidic protein in first-tier assessments

of neurotoxicity. J Am Coil Toxicol 6:719-726, 1991b.

O’Callaghan, J.P. Quantification of glial fibrillary acidic protein: Comparison

of slot-immunobinding assays with a novel sandwich ELISA. Neurotoxicol

Teratol 13:275-281, 1991c.

O’Callaghan, J.P. Assessment of neurotoxicity using assays of neuron- and

glia-localized proteins: Chronology and critique. In: Tilson, H., and Mitchell, C.,

eds. Neurotoxicology. New York: Raven Press, 1992. pp. 83-100.

O’Callaghan, J.P., and Jensen, K.F. Enhanced expression of glial fibrillary acidic

protein and the cupric silver degeneration reaction can be used as sensitive

and early indicators of neurotoxicity. Neurotoxicology 13:113-122, 1992.

O’Callaghan, J.P.; Jensen, K.F.; and Miller, D.B. Characterization of

methylenedioxymethamphetamine (MDMA) neurotoxicity using assays of

serotonin and glial fibrillary acidic protein (GFAP): Changes in specific

neocortical regions implicate serotonergic and non-serotonergic targets,

Soc Neurosci Abstr 17:1429, 1991.

O’Callaghan, J.P., and Miller, D.B. Cerebellar hypoplasia in the Gunn rat is

associated with quantitative changes in neurotypic and gliotypic proteins.

J Pharmacol Exp Ther 234:522-533,1985.

O’Callaghan, J.P., and Miller, D.B. Assessment of chemically-induced alterations

in brain development using assays of neuron- and glia-localized proteins.

Neurotoxicology 10:393-406, 1989.

O’Callaghan, J.P.; Miller, D.B.; and Reinhard, J.F., Jr. Characterization of the

origins of astrocyte response to injury using the dopaminergic neurotoxicant,

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Brain Res 521:73-80, 1990a.

210
O’Callaghan, J.P.; Miller, D.B.; and Reinhard, J.F., Jr. 1-Methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP)-induced damage of striatal dopaminergic

fibers attenuates subsequent astrocyte response to MPTP. Neurosci Lett

117:228-233, 1990b.

Reier, P.J. Gliosis following CNS injury: The anatomy of astrocytic scars and

their influences on axonal elongation. In: Fedoroff, S., and Vernadakis, A.,

eds. Astrocytes: Cell Biology and Pathology of Astrocytes. Orlando, FL:

Academic Press, 1986. pp. 263-324.

Ricaurte, G.A.; Guillery, R.W.; Seiden, L.S.; and Schuster, C.R. Nerve terminal

degeneration after a single injection of d-amphetamine in iprindole-treated

rats: Relation to selective long-lasting dopamine depletion. Brain Res

291:378-382, 1984a.

Ricaurte, G.A.; Guillery, R.W.; Seiden, L.S.; Schuster, C.R.; and Moore, R.Y.

Dopamine nerve terminal degeneration produced by high doses of

methylamphetamine in the rat brain. Brain Res 235:93-103, 1982.

Ricaurte, G.A.; Schuster, C.R.; and Seiden, L.S. Long-term effects of repeated

methylamphetamine administration on dopamine and serotonin neurons in

the rat brain: A regional study. Brain Res 193:153-163, 1980.

Ricaurte, G.A.; Seiden, L.S.; and Schuster, C.R. Further evidence that

amphetamines produce long-lasting dopamine neurochemical deficits by

destroying dopamine nerve fibers. Brain Res 303:359-364, 1984b.

Scallet, A.C.; Lipe, G.W.; Ali, SF.; Holson, R.R.; Frith, C.H.; and Slikker, W.

Neuropathological evaluation by combined immunohistochemistry

and degeneration-specific methods: Application to

methylenedioxymethamphetamine. Neurotoxicology 9:529-538, 1988.

Schmidt, C.J. Neurotoxicity of the psychedelic amphetamine,

methylenedioxymethamphetamine. J Pharmacol Exp Ther 240:1-7, 1987.

Schmidt, C.J., and Kehne, J.H. Neurotoxicity of MDMA: Neurochemical effects.

In: Whitaker-Azmitia, P.M., and Peroutka, S.J., eds. The Neuropharmacology

of Serotonin. Ann N Y Acad Sci 600:665-681, 1990.

Schmidt, D.J., and Taylor, V.L. Depression of rat brain tryptophan

hydroxylase activity following the acute administration of

methylenedioxymethamphetamine. Biochem Pharmacol 36:4012-4095, 1987.

Seiden, L.S., and Kleven, MS. Methamphetamine and related drugs: Toxicity

and resulting behavioral changes in response to pharmacological probes. In:

Asghar, K., and De Souza, E., eds. Pharmacology and Toxicology of

Amphetamine and Related Designer Drugs. National Institute on Drug Abuse

Research Monograph 94. DHHS Pub. No. (ADM)89-1640. Washington, DC:

Supt. of Docs., U.S. Govt. Print. Off., 1989. pp. 146-160.

Shehab, S.A.A.; Cronly-Dillon, J.R.; Nona, S.N.; and Stafford, CA. Preferential

histochemical staining of protoplasmic and fibrous astrocytes in rat CNS with

GFAP antibodies using different fixatives. Brain Res 518:347-352, 1990.

Sonsalla, P.K.; Gibb, J.W.; and Hanson, G.R. Roles of D1 and D2 dopamine

receptor subtypes in mediating the methamphetamine-induced changes in

monoamine systems. J Pharmacol Exp Ther 238:932-937, 1986.

211
Sonsalla, P.K.; Nicklas, W.J.; and Heikkila, R.E. Role for excitatory amino acids

in methamphetamine-induced nigrostriatal dopaminergic toxicity. Science

243:398-400, 1989.

Spencer, P.S., and Schaumburg, H.H. Experimental and Clinical

Neurotoxicology. Baltimore, MD: Williams & Wilkins, 1980.

Stone, D.M.; Hanson, G.R.; and Gibb, J.W. Differences in the central

serotonergic effects of methylenedioxymethamphetamine (MDMA) in mice

and rats. Neuropharmacology 26:1657-1661, 1991.

Sutcliffe, J.G. mRNA in the mammalian central nervous system. Annu Rev

Neurosci 11:157-198, 1988.

Switzer, R.C. Strategies for assessing neurotoxicity. Neurosci Biobehav Rev

15:89-93, 1991.

Tilson, H.A. Neurotoxicology in the 1990s. Neurotoxicol Teratol 12:292-300,

1990.

US. Congress, Office of Technology Assessment. Neurotoxicity: Identifying and

Controlling Poisons of the Nervous System. Washington, DC: Supt. of Docs.,

U.S. Govt. Print. Off., 1990.

Wagner, J.; Ricaurte, G.A.; Seiden, L.S.; Schuster, C.R.; Miller, R.J.; and

Westley, J. Long-lasting depletions of striatal dopamine and loss of dopamine

uptake sites following repeated administration of methamphetamine. Brain

Res 181:151-160, 1980.

DISCUSSION

Due to technical difficulties, there is no transcription available of the discussion

that followed Dr. O’Callaghan’s presentation.

ACKNOWLEDGMENTS

This chapter was prepared with support from NIDA interagency agreement ND89-4. Dr. Christopher J. Schmidt prepared the samples used to generate data

for figure 4.

AUTHORS

James P. O’Callaghan, Ph.D.

Senior Research Scientist

Diane B. Miller, Ph.D.

Senior Research Scientist

Neurotoxicology Division (MD-74B)

Health Effects Research Laboratory

U.S. Environmental Protection Agency

Research Triangle Park, NC 27711

212
Pharmacokinetic Approaches to the

Study of Drug Action and Toxicity

Arthur K. Cho, Masayuki Hiramatsu, Yoshito Kumagai,

and Nita Patel

INTRODUCTION

Pharmacokinetics refers to the events that occur following drug administration

to alter the availability of a drug by elimination or metabolism. These processes

contribute to the effects of a drug not only by removal from the body but,

in some instances, by forming pharmacologically or toxicologically active

compounds. It is therefore critical to understand the pharmacokinetics of

drugs when studying their effects in vivo and in cellular systems. Because

pharmacokinetics is based on measurements of the concentration of drugs and

metabolites in plasma and tissue, it requires bioanalytical chemical techniques.

The concentrations involved in the studies are usually in the micromolar range,

and when pharmacokinetic experiments are performed, the volumes involved

are small, commonly 0.1 to 0.2 mL. In the authors’ studies, we have found it

advantageous to perform experiments at the in vivo and in vitro levels to direct

subsequent experiments or interpret results from others. The in vitro incubation

mixtures usually contain cellular components, whereas the in vivo samples

are plasma or tissue homogenates so that the analytes are in complex matrices.

As a result, the analytical procedures require high sensitivity and specificity.

This chapter describes applications of these procedures to studies of the

pharmacology and toxicology of methylenedioxymethamphetamine (MDMA)-

and methylenedioxyamphetamine (MDA)-related compounds.

PHARMACOKINETIC AND PHARMACODYNAMIC STUDIES OF MDMA

MDMA is a substituted amphetamine with actions on the 5-hydroxytryptamine

(5-HT) and the dopamine (DA) systems (Schmidt 1987; Stone et al. 1988;

Johnson et al. 1988). The compound causes an initial release of DA as well as

5-HT but under appropriate regimens also causes a long-term depletion of 5-HT

and reduction in tryptophan hydroxylase activity (Stone et al. 1989). In addition

to these chemical changes, there are morphological changes consistent with a

loss of presynaptic terminals in different areas of the brain (Ricuarte et al. 1985;

213
Wilson et al. 1989). Early studies focused on MDMA’s temporal and metabolic

disposition in the whole animal as it related to the pharmacodynamics of

MDMA. Because MDMA is a lipid-soluble compound of moderate volatility,

it was analyzable by gas chromatography (GC) techniques. The more polar

catechol metabolites required liquid chromatography (LC). Therefore, the

analyses utilized GC/mass spectrometry (MS) and high-performance LC/

electrochemical detection (HPLC-ECD). MS is highly sensitive, and when

it was coupled to a capillary gas chromatograph, high specificity also was

achieved. Similarly, ECD, when coupled to a liquid chromatograph, also was

able to achieve high sensitivity and specificity for electroactive compounds.

The electrochemical detection was especially sensitive for easily oxidized

substrates such as the catechol function.

GC/MS With Selectedion Monitoring

A mass spectrometer is more commonly used to collect data resulting from

the fragmentation of the analyte in an electron beam. A series of charged

fragments is generated, and the mass spectrometer identifies the charge-tomass ratio of each fragment and determines its relative abundance. The

fragmentation pattern is characteristic of the analyte and may be used to

identify or partially identify its chemical structure. In the selected ion monitoring

mode, the mass spectrometer is focused on two fragments, one characteristic

of the analyte and the other for the internal standard, which is frequently an

isotopic variant of the analyte (Jenden and Cho 1979). Because the instrument

is focused on only two masses instead of scanning a range, the limits of

detectability are much lower. In the assay for MDMA, a fragment with mass/

charge (m/z) 154 was used for the analyte and a fragment of m/z 164 for the

internal standard (figure 1). The peak at m/z 154 is caused by ß-cleavage

of the trifluoroacetyl derivative and is the base peak or the most abundant

fragment in the spectrum. The internal standard was a deuterium-substituted

variant that contains two deuterium atoms in the ethyl side chain, On

fragmentation, this variant has one peak at m/z 155 and a second, less intense

peak (50 percent) at m/z 164. Although the m/z 155 peak would be more

sensitive, the nondeuterium-enriched (d0) analyte MDMA also has a small

(5 percent) quantity of fragment at this mass as well. To avoid overlap, the

signal at m/z 164 with less than 1 percent overlap was chosen.

Synthesis of Internal Standards

The internal standard was prepared by the route indicated in figure 1, utilizing

conventional synthetic procedures with lithium aluminum deuteride instead of

the hydride (Lindeke and Cho 1972).

214
FIGURE 1. The synthesis of deuterium-substituted MDMA and its conversion

to the trifluoroacetyl derivative. The fragmentation resulting in the

ions used for selected ion monitoring is shown.

Analytical Procedure

In the analysis, the internal standard was added to the sample; and the analyte,

MDMA, and the internal standard were extracted into an organic solvent. The

extract was concentrated by evaporation of solvent; the residue was treated with

trifluoroacetic anhydride and injected into the GC/MS. The mass spectrometer

was focused on the preset masses, and the appropriate gas chromatographic

peak was analyzed for ratios of signal from the analyte and internal standard.

Quantitation was achieved with a standard curve generated from mixtures of

fixed concentrations of internal standard and varying concentrations of analyte.

Results of the initial studies determining the pharmacokinetics of MDMA after

equimolar intravenous doses of (+) and (-) MDMA are shown in figure 2.

215
FIGURE 2. Time course of plasma MDMA and MDA after intravenous

administration of (+) and (-) MDMA (10 mg/kg)

In Vivo Pharmacokinetics

The MDMA was administered to groups of at least five rats that had been

implanted with Vascular Access Ports (Model SLA, Norfolk Medical Products,

Skokie, IL) (Cho et al. 1990). These ports permit drawing of blood from

unrestrained and conscious animals. Volumes of 0.5 mL of blood were collected,

and the plasma (0.2 mL) was separated and analyzed by the procedures

described above. The times chosen for sample collection were based on

preliminary experiments and the time course of plasma decay. The object was

to have data points that follow drug concentration changes with time so that

parameters for the function describing the plasma time curve could be estimated.

The function could then be used to (1) estimate the concentrations at time points

not analyzed, (2) obtain rate constants for intercompartment transfer, and

(3) estimate the area under the plasma time curve. (Intercompartment transfer

refers to processes such as absorption from the site of administration, distribution

throughout the body compartments, and elimination, either by excretion or by

metabolism.) The data were fitted to the pharmacokinetic model shown in

figure 3, an open two-compartment model, reflecting the movement of drug

from the central to the peripheral compartment (k12, k21), through metabolism

to MDA (k1, k4) and other pathways of elimination (k3) The fitting was

216
FIGURE 3. Compartment model used to analyze the pharmacokinetic data of

figure 2

accomplished by nonlinear regression procedures (Motulsky and Ransnas 1987)

using Dixon’s BMDP software program on a microcomputer (Dixon 1985). In this

regression procedure, the data are interactively fitted to an exponential function

that is the solution to the differential equation describing the plasma decay curve.

The function can be set up on a spreadsheet and the concentrations at any time

calculated by substituting the desired time. This method of data analysis

provides estimates of rate constants (table 1) together with estimates of the

errors in their values, The significance of the constants can be evaluated using

the F value (Motulsky and Ransnas 1987). The values of the individual rate

constants or microparameters are useful in evaluating the relative importance

of different metabolic processes, For example, the ratio of k1, the rate constant

for MDA formation, to k3, the rate constant representing all other pathways of

elimination, is about 10. This means that MDA formation represents only a

small proportion of the total drug elimination process (-10 percent), The relative

values of k3

 for the (+) and (-) isomers indicate a substantial enantiospecificity in

MDA formation. The effect of pharmacological manipulation of the rate constants

is shown in table 1 also. Phenobarbital pretreatment, which commonly induces

cytochrome P450 isozymes, did not appear to alter the rates of demethylation

but had a significant effect on k3

, the alternate pathways of elimination. At the

217
TABLE 1. Microscopic rate constants-MDMA

Treatment k1

 (min-1) k3

 (min-1) k3 (min-1)

(+)MDMA, control .0025 .0240* .0058

(-)MDMA, control .0006 .0190 .0062

(+)MDMA, phenobarbital .0026 .0354†.00103

(-)MDMA, phenobarbital .0011 .0378 .0093

(+)MDMA, SKF 525A .0022 .0202* .0058

(-)MDMA, SKF 525A .0004 -.0207†-.0059

* p<.01 vs. (-) control

†

p<.01 vs. (+) control

doses used, the cytochrome P450 inhibitor SKF 525A appeared to decrease the

values of k3

 for the (+) but not the (-) isomer.

The results show that the outcome of pretreatment is not always predictable.

Thus, phenobarbital induction did not increase levels of MDA as might be

expected. Instead of increasing the rate of MDA formation, induction increased

the rate of MDA elimination.

In Vitro Metabolism

The pharmacokinetic data showed that MDA formation is a relatively minor

pathway in the overall elimination pathways of MDMA (Hiramatsu et al. 1990).

To evaluate other pathways, in vitro experiments were conducted to obtain

quantitative data of regiospecificity (Kumagai et al. 1991a). These studies

required the analysis of highly polar catecholamines and their derivatives

(figure 4). To study the overall metabolism, the aforementioned GC/MS assay

was used along with an additional HPLC/ECD procedure (Hiramatsu et al. 1990).

The results, shown in table 2, reveal that the catecholamine dihydroxy

methamphetamine (DHMA) is the dominant metabolite, occurring at levels

10 times greater than the others (Kumagai et al. 1991a). In spite of the extensive

demethylenation in vitro, there was very little if any DHMA present in any of the

plasma samples assayed, including the early time points. An explanation for this

discrepancy was found in direct pharmacokinetic evaluation of -methyldopamine

( -MeDA) because the N-methyl analog was not available in sufficient quantity to

study in this manner. The pharmacokinetic parameters obtained after equimolar,

intravenous doses of a-MeDA and MDA are shown in table 3. The value of k2

for -MeDA, the rate constant for elimination, was found to be 10 times greater

218
FIGURE 4. Metabolic pathways of MDMA

TABLE 2. Metabolism of MDMA by liver microsomes

Species DHMA MDA MPA OHMDMA

Rat (+)* 5.37 0.21 0.12 .025†

Rat (-)* 3.35 0.31 0.05 .010†

* Nmols of product formed in 5-minute incubation of 30 nmols MDMA

†

approximate

KEYS: MPA=methylenedioxy phenylacetone;

OHMDMA=hydroxymethylenedioxy methamphetamine

than that for MDA. The rate constant for -MeDA is equivalent to a half-life of

about 2 minutes, compared with 22 minutes for MDA. Thus, even with a large

rate of formation, the high rate of elimination precludes accumulation of

significant levels of the catechol in plasma.

In Vitro Toxicity

Methods also are being developed to study the chemical mechanisms of

neurotoxicity utilizing a neuroblastoma glioma clonal cell line, NG 108-15

219
TABLE 3. Microscopic rate constants-MDA and -MeDA

Vd k21 k 2 k12 t1/2

Compound (1/kg)

-MeDA 0.16

MDA 0.76

(min-1) (min)

.059 .360 .048 1.92

.079 .032 .219 21.65

NOTE: The open, two-compartment parameters are: Vd is the volume of

distribution of the central compartment; k2

 is the rate constant for

elimination: k12, k21 are rate constants for fluxes to and from the central

compartment. The half-life, t1/2, was calculated from k2.

(Patel et al. 1991). This cell line has been described by Furuya and associates

(1985) as having features associated with the 5-HT nerve terminal such as the

ability to store, release, and accumulate 5-HT. The intention is to use such a

model to test metabolites of MDMA and other neurotoxins. These cells contain

high levels of glutathione (GSH), a peptide that protects cells against insult by

electrophiles, free radicals, and metals by undergoing addition, oxidation, and

complexing reactions, respectively (Reed 1985). The high levels of GSH in this

cell line and the availability of depleting agents permits the role of this agent in

protection against toxins to be examined experimentally.

The proposed metabolism of MDMA to potentially toxic metabolites is shown in

figure 4. In this sequence, MDMA can undergo demethylenation to the catechol

DHMA (Kumagai et al. 1991 a) or hydroxylation to the Ar-hydroxy compound. In

studies reported elsewhere (Kumagai et al. 1992), a ring-hydroxylated metabolite

of MDMA has been characterized, but the location of the hydroxyl group has not

been established. DHMA can be generated directly by a cytochrome P450-

dependent reaction or by a hydroxyl radical-dependent oxidation (Kumagai et

al. 1991b). Catechols are readily autoxidized to the o-quinone, and this reaction,

together with a rapid addition of GSH, has been reported (Hiramatsu et al. 1990).

However, in low-GSH conditions, the o-quinone can react with nucleophilic

centers in biological molecules and cause a biochemical lesion (Graham et al.

1978; Slaughter and Hanzlik 1991). Alternatively, the proposed Ar-hydroxy

metabolite can be generated, which also can undergo demethylenation to form

the highly reactive trihydroxy derivative. The position of ring hydroxylation of

MDMA has since been identified as the 6-position based on comparison of

HPLC retention times and GUMS data with authentic material. These functions

can cause cellular toxicity by reduction of oxygen to superoxide catalytically,

acting as an electron transfer agent between reduced nicotinamide adenine

dinucleofide phosphate and oxygen (Graham et al. 1978). Superoxide rapidly

220
disproportionates to oxygen and hydrogen peroxide, and its continuous formation

by the trihydroxy compound can result in hydroxyl radical formation through the

Haber Weiss reaction. Hydroxyl radical is highly reactive and thought to be the

basis for toxicity of a variety of oxidizing agents (Cohen 1984). GSH would

protect the cells against the electrophile- and radical-based insults shown above

so that its depletion should alter the toxicity of agents whose action proceeded

by this mechanism.

In initial studies, the interaction of the metabolite of MDA, MeDA, was

compared with DA for its toxic actions on the cell line (Patel et al. 1991).

Because both compounds contain the catechol moiety, toxic actions based on

o-quinone formation should be very similar, reflective of the availability of the

quinone. Toxicity was measured as cellular destruction and required fairly high

concentrations of the compounds. The results are shown in table 4 as EC50

values, the concentration necessary to kill 50 percent of the cells. The value

for DA was almost four times higher than that for -MeDA. The total quantities

of catechol accumulated, on the other hand, were much greater after DA (Patel

1990). When the GSH in the cells was depleted by the synthesis inhibitor,

buthionine sulfoximine (BSO), the toxicity of the two compounds increased,

but the increase was much greater for -MeDA. Thus, the results of the study

showed that, although DA is accumulated by the cells much more extensively,

it is much less toxic than its -methyl analog. Furthermore, its toxicity does

not increase in the same proportion on GSH depletion.

One explanation for this discrepancy is in the intracellular disposition of the two

compounds (Patel et al. 1991). DA is a neurotransmitter, and its removal from

cytoplasm by storage and monoamine oxidase-initiated metabolism resulted

in low availability. In support of this notion, DA accumulation was found to

TABLE 4. Interaction of catecholamines and NG 108-15 cells

EC50*

(mM)

Compound Control BSO Treated†

DA 24.5 10.5

-MeDA 6.8 0.6

* Concentration required to kill 50 percent of the cells after a 2-hour exposure

†

 Cells treated with buthionine sulfoximine (BSO, 2 mM) for 24 hours prior to

drug exposure (Patel et al. 1991)

221
decrease substantially in reserpine pretreated cells, whereas the accumulation

of -MeDA was not affected (Patel 1990). The metabolism of DA by monoamine

oxidase to dihydroxyphenylacetic acid and homovanillic acid is not available

to -MeDA, although the latter compound may be converted to its 3-methyl

derivative by catechol o-methyltransferase. The major transformation of -MeDA

appears to be the GSH adduct, originally found in incubates of liver preparations

(Hiramatsu et al. 1990). Although the GSH adducts are formed from both DA

and -MeDA, the early levels of the latter are much higher. The intracellular

concentration of GSH is in the millimolar range and far exceeds the concentration

of the catecholamines so that the rate of formation of GSH adduct is dependent

on catecholamine availability. Therefore, the concentration of -MeDA and its

quinone oxidation product must be higher and available for binding to cellular

nucleophiles. The GSH adducts appear to be excreted by the cell, and analysis

of the culture medium after incubation has shown that there are micromolar

levels of the GSH adduct of -MeDA present, whereas, similar to DA, GSH

concentration was below detectable limits. Thus, -MeDA is extensively

converted to the GSH adduct of the o-quinone, whereas DA appears to have

limited conversion. If the concentration of this adduct reflects that of the toxic

o-quinone, the greater toxicity of -MeDA can be accounted for by its greater

availability.

ACKNOWLEDGMENT

The research described in this chapter was supported by U.S. Public Health

Service grants DA-02411 and DA-04206 and a Merck Postdoctoral Fellowship

(YK).

REFERENCES

Cho, A.K.; Hiramatsu, M.; DiStefano, E.W.; Chang, A.S.; and Jenden,

D.J. Stereochemical differences in the metabolism of 3,4-

methylenedioxymethamphetamine in vivo and in vitro: A pharmacokinetic

analysis. Drug Metab Dispos 18:686-691, 1990.

Cohen, G. Oxy-radical toxicity in catecholamine neurons. Neurotoxicology 5:77-

82, 1984.

Dixon, W.J., ed. BMDP Statistical Software. Los Angeles: University of California

Press, 1985. pp. 388-412.

Furuya, S.; Sawada, M.; Nagatsu, T.; Suzuki, O.; and Higashida, H. Localization

of [3H]-serotonin in neuroblastoma X glioma hybrid cells. Brain Res 361:77-90,

1985.

222
Graham, D.G.; Tiffany, S.M.; Bell, W.R., Jr.; and Gutknecht, W.F. Autoxidation

versus covalent binding of quinones as the mechanism of toxicity of

dopamine, 6-hydroxydopamine, and related compounds toward Cl 300

neuroblastoma cells in vitro. Mol Pharmacol 14:644-653, 1978.

Hiramatsu, M.; Kumagai, Y.; Unger, S.E.; and Cho, A.K. Metabolism

of methylenedioxymethamphetamine (MDMA): Formation of

dihydroxymethamphetamine and quinone identified as its glutathione

adduct. J Pharmacol Exp Ther 254:521-527, 1990.

Jenden, D.J., and Cho, A.K. Selected ion monitoring in pharmacology.

(Commentary.) Biochem Pharmacol 28:705-713, 1979.

Johnson, M.; Hanson, G.R.; and Gibb, J.W. Effects of dopaminergic and

serotonergic receptor blockade on neurochemical changes induced

by acute administration of methamphetamine and 3,4-

methylenedioxymethamphetamine. Neuropharmacology 27:1089-1096, 1988.

Kumagai, Y.; Lin, L.Y.; Philpot, R.M.; Yamada, H.; Oguri, K.; Yoshimura, H.;

and Cho, A.K. Regiochemical differences in cytochrome P450 isozymes

responsible for the oxidation of methylenedioxyphenyl groups by rabbit liver.

MOl Pharmacol 42:695-702, 1992.

Kumagai, Y.; Lin, L.Y.; Schmitz, D.A.; and Cho, A.K. Hydroxyl radical mediated

demethylenation of methylenedioxyphenyl compounds. Chem Res Toxicol

4:330-334, 1991b.

Kumagai, Y.; Wickham, K.A.; Schmitz, D.A.; and Cho, A.K. Metabolism of

methylenedioxyphenyl compounds by rabbit liver microsomes: Participation

of different cytochrome P450 isozymes in the demethylenation reaction,

Biochem Pharmacol 42:1061-1067, 1991a.

Lindeke, B., and Cho, A.K. Specifically deuterated 1-phenylisopropylamines.

Synthesis of deuterium labelled (+)-amphetamine, (±)-pmethoxyamphetamine and (±) -methyltyramine. Acta Pharm Suec

9:363-372, 1972.

Motulsky, H.J., and Ransnas, L.A. Fitting curves to data using nonlinear

regression: A practical and nonmathematical review. FASEB J 1:365-374,

1987.

Patel, N.J. “Catecholamines as Mediators of 3,4-

Methylenedioxymethamphetamine (MDMA) and 3,4-

Methylenedioxyamphetamine (MDA) Toxicity: A Cell Culture Study.” Ph.D.

thesis, University of California, Los Angeles, 1990.

Patel, N.; Kumagai, Y.; Unger, S.E.; Fukuto, J.M.; and Cho, A.K.

Transformation of dopamine and a-methyldopamine by NG108-5 cells:

Formation of thiol adducts. Chem Res Toxicol 4:421-426, 1991.

Reed, D.J. Cellular defense against reactive metabolites. In: Anders, M.W., ed.

Bioactivation of Foreign Compounds. New York: Academic Press, 1985. pp.

71-108.

223
Ricuarte, G.; Bryan, G.; Strauss, L.; Seiden, L.; and Schuster, C. Hallucinogenic

amphetamine selectively destroys brain serotonin nerve terminals. Science

229:986-988, 1985.

Schmidt, C.J. Neurotoxicity of the psychedelic amphetamine,

methylenedioxymethamphetamine. J Pharmacol Exp Ther 240(1):1-7, 1987.

Slaughter, D.E., and Hanzlik, R.P. Identification of epoxide- and quinonederived bromobenzene adducts to protein sulfur nucleophiles. Chem Res

Toxicol 4:349-359, 1991.

Stone, D.M.; Hanson, G.R.; and Gibb, J.W. In vitro reactivation of rat cortical

tryptophan hydroxylase following in vivo inactivation by

methylenedioxymethamphetamine. J Neurochem 53:572-581, 1989.

Stone, D.M.; Johnson, M.; Hanson, G.R.; and Gibb, J.W. Role of endogenous

dopamine in the central serotonergic deficits induced by 3,4-

methylenedioxymethamphetamine. J Pharmacol Exp Ther 247:79-87, 1988.

Wilson, M.A.; Ricaurte, G.A.; and Molliver, M.E. Distinct morphologic classes of

serotonergic axons in primates exhibit differential vulnerability to the

psychotropic drug 3,4-methylenedioxymethamphetamine. Neuroscience

28:121-137, 1989.

DISCUSSION

Due to technical difficulties, there is no transcription available of the discussion

that followed Dr. Cho’s presentation.

AUTHORS

Arthur K. Cho, Ph.D.

Professor

Department of Pharmacology

University of California, Los Angeles, School of Medicine

Center for Health Sciences

10833 LeConte Avenue

Los Angeles, CA 90024-1735

Masayuki Hiramatsu, Ph.D.

Instructor

Department of Chemical Pharmacology

Meijo University

Faculty of Pharmaceutical Sciences

Tenpaku-ku

Nagoya 468

JAPAN

224
Yoshito Kumagai, Ph.D.

Staff Scientist

Research Team for Health Effects of Air Pollutants

The National Institute for Environmental Studies

P.O. Tsukuba

I baraki 305

JAPAN

Nita Patel, Ph.D.

Research Fellow

Department of Pharmacology

University of Rochester School of Medicine

601 Elmwood Avenue

Rochester, NY 14642

225
Mechanisms Mediating Biogenic

Amine Deficits Induced by

Amphetamine and Its Congeners

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

and Glen R. Hanson

INTRODUCTION

In other chapters of this monograph, evidence is presented that the neurotoxicity

of drugs of abuse can be assessed by monitoring behavioral, neurophysiologic,

and neuroanatomic parameters. In this chapter, evidence is cited that neurotoxicity

also can be assessed by determining the effects of drugs of abuse on

neurochemical elements. Because Gibb and colleagues at the University of

Utah have investigated primarily methamphetamine (METH) and its congeners

over the past two decades, this group of abused agents is used as an example.

In monitoring neurochemical parameters, one can measure neurotransmitter

concentrations ex vivo or by in vivo dialysis. Enzyme activity, either Vmax and/or

Km, can be determined. Loss of uptake sites (Battaglia et al. 1987) or alteration

of binding sites also can be assessed.

In 1971, Koda and Gibb (1971, 1973) attempted to simulate in rats the conditions

that exist in METH abusers who characteristically inject the drug intravenously

at frequent intervals over a period of 2 to 3 days. METH (10 to 15 mg/kg,

subcutaneously [SC]) was administered every 6 hours for 30 hours, and the

effect on tyrosine hydroxylase (TH) activity and on the concentrations of dopamine

(DA) and its metabolites, dihydroxyphenylacetic acid and homovanillic acid, in

the neostriatum was determined. Following drug treatment, these parameters

were decreased to approximately 60 percent of normal. Because it was known

that amphetamines release catecholamines, it was not surprising that the

concentrations of DA and its metabolites were depressed, but an explanation

for the decrease in TH activity was not immediately apparent. Induction of

TH in the adrenal gland, superior cervical ganglion, and selected areas of the

brain had been reported in stressed animals (Zigmond et al. 1980; Masserano

et al. 1981) but a decline in the activity of this enzyme was rather perplexing;

it was acknowledged that 6-hydroxydopamine (6-OHDA), a potent neurotoxin,

also decreased TH activity.

226
After a comparable dosing schedule, METH had similar effects on the serotonergic

system (Hotchkiss et al. 1979; Hotchkiss and Gibb 1980). Tryptophan hydroxylase

(TPH) activity was markedly decreased after a single dose of METH; a decrease

in concentrations of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid

(5-HIAA) was also observed. A major difference in the response of the 5-HT and

DA systems was the more rapid onset and more profound response observed in the

5-HT system compared with the DA system. The question then arose as to whether

these large, repeated doses of METH were neurotoxic to all neurotransmitter

systems or to selected neurons. The effect of METH on the cholinergic system

was assessed by measuring choline acetyltransferase activity in the neostriatum,

nucleus accumbens, and olfactory tubercle after METH; because this enzyme,

which is responsible for acetylcholine synthesis, was not altered, it was concluded

that cholinergic systems remained intact after METH (Hotchkiss et al. 1979).

Using a similar strategy, glutamic acid dehydrogenase was measured to determine

whether the -aminobutyric acid (GABA) system was affected; the activity of this

enzyme was unaffected. It appears, then, that the neurotoxic effect of large doses

of METH is limited to the dopaminergic and serotonergic systems.

Ricaurte and colleagues (1985) reported that an analog of amphetamine, 3,4-

methylenedioxyamphetamine (MDA), decreased concentrations of neostriatal 5-HT

and 5-HIAA but had no effect on DA or norepinephrine content. Morphological

changes, as demonstrated by the Fink-Heimer technique, also were observed:

moreover, 5-HT uptake sites were compromised. Stone and coworkers (1986)

examined the effects of MDA and 3,4-methylenedioxymethamphetamine (MDMA)

on the activity of both TPH and TH. Whereas METH characteristically decreases

both TH and TPH activity, MDA or MDMA depressed only TPH activity. The more

selective effect of MDMA or MDA on the serotonergic system is reminiscent of the

response to p-chloroamphetamine.

A compound cannot be assumed to be neurotoxic if the neurochemical changes

are transient: however, when neurochemical alterations persist, neurotoxicity is

suspected. Bakhit and colleagues (1981) and Stone and colleagues (1987)

administered multiple doses of METH or MDMA to rats, stopped the drug, and

waited for varying periods of time after the dosing was discontinued to measure

the neurochemical response. When five administrations of MDMA or METH

(10 mg/kg) were given, TPH activity and concentrations of 5-HT and 5-HIAA were

still depressed at 110 days. Because MDMA affects the serotonergic system

selectively, comparable results were obtained for the dopaminergic system only

when METH was administered. These data, coupled with the morphological

evidence from other laboratories (Ricaurte et al. 1984; Commins et al. 1987;

O’Hearn et al. 1988), are highly suggestive that large doses of METH are

neurotoxic to dopaminergic neurons, whereas MDMA is selectively neurotoxic

to the serotonergic system.

227
DOPAMINE IS ESSENTIAL FOR THE NEUROTOXIC RESPONSE

The mechanism by which the neurotoxic effect of the amphetamine analogs

occurs has been under consideration from the very beginning of Gibb and

colleagues’ studies. Buening and Gibb (1974) reported that the DA antagonists,

chlorpromazine and haloperidol, prevent the usual response to METH. In

subsequent studies, Sonsalla and coworkers (1986a) observed that either D1

or D2 antagonists block the effects on the DA system; the effects of METH on

the 5-HT system were blocked only by D1 antagonists.

Further evidence that DA is essential for the METH-induced neurotoxicity was

then obtained (Gibb and Kogan 1979; Hotchkiss and Gibb 1960; Schmidt et al.

1965). DA synthesis was blocked at the rate-limiting step by inhibiting TH by

administering -methyl-p-tyrosine (MT). The METH-induced alterations in both

the dopaminergic and the serotonergic systems were prevented by concurrent

administration of MT. When synthesis of DA was reinstated by administering

L-dopa concomitantly with the METH and MT, the neurochemical deficits returned.

Recently, it was found that when the nigrostriatal dopaminergic projections

are destroyed by prior injection of 6-OHDA into the substantia nigra, METH

(Johnson et al. 1967; Sonsalla et al. 1966b) or MDMA (Stone et al. 1968) does

not cause the characteristic decrease in striatal TPH activity. However, METH

or MDMA still decreases TPH activity in the cerebral cortex or hippocampus of

6-OHDA-pretreated animals. Thus, dopaminergic innervation is critical for the

METH or MDMA to cause the neurotoxic response.

Catecholamines were then depleted by prior administration of reserpine. In

animals so depleted, MDMA was ineffective in causing its usual deficit in

TPH activity (Stone et al. 1988). This observation provides further evidence that

DA is essential for the neurotoxicity observed after MDMA or METH. Additional

evidence for the necessity of DA in the neurotoxic process was recently provided

by Chris Schmidt (personal communication, May 1991), who demonstrated that

L-dopa enhanced the deficits in rat brain 5-HT content 1 week after administering

an otherwise ineffective single dose of MDMA or METH.

IS GLUTAMATE INVOLVED IN THE NEUROTOXICITY?

Sonsalla and colleagues (1989) recently reported that MK-801, an N-methylD-aspartate (NMDA) antagonist, protects against the METH-induced deficits in

the dopaminergic and serotonergic systems, which suggests that glutamate is

associated with the METH-induced neurochemical events that lead to toxicity.

Johnson and coworkers (1989) confirmed these observations but found that

MK-801 does not protect against the MDMA-induced neurotoxicity. Glutamate

appears to play a role in the METH-induced neurotoxicity but not in the MDMAinduced deficits.

228
ROLE OF AN OXIDATIVE PROCESS IN NEUROTOXICITY

The above data provide significant evidence that DA and/or its reactive

metabolites are essential for neurotoxicity. The question remains as to the

mechanisms by which DA causes the response. Stone and colleagues (1989)

have evidence that an oxidative process is responsible for the serotonergic

alterations associated with the neurotoxicity. It was reported earlier that TPH

activity is rapidly decreased within 15 minutes after a single dose of METH

(Bakhit and Gibb 1981). Schmidt and Taylor (1987) reported no change in the

Km for substrate or pterin cofactor, but a decrease in Vmax, was observed after

MDMA. TPH is activated by a variety of conditions, including exposure of the

enzyme to certain phospholipids, mild proteolysis, and phosphorylating

conditions (Hamon et al. 1981; Vitto and Mandell 1981; Garber and Makman

1987); under these conditions, the affinity of the enzyme is increased, and the

apparent Km for tryptophan and/or the pterin cofactor is decreased.

In contrast, a strong reducing environment (5 mM dithiothreitol [DTT] and

50 mM Fe2+ under a nitrogen atmosphere) increases the apparent Vmax of TPH

but does not alter the apparent Km (Hamon et al. 1978). It has been previously

reported that in vitro exposure of TPH to oxygen inactivates the enzyme (Kuhn

et al. 1980). The oxygen-inactivated TPH can be reactivated by incubating

the enzyme in an anaerobic milieu under reducing conditions. The similarity

between the inactivation by molecular oxygen and by MDMA prompted Stone

and colleagues (1989) to investigate the role of enzyme oxidation in the acute

decline of TPH by MDMA.

Rats were administered a single dose (10 mg/kg) of MDMA and killed 3 hours

later. Crude enzyme from the cerebral cortex (27,000 x g supernatant) from

saline- or MDMA-treated rats was placed in test tubes, and DTT and Fe2+ were

added. The preparation was incubated under nitrogen at 25°C for varying

periods of time, and TPH activity was assayed by a modified 14CO2-trapping

procedure (Ichiyama et al. 1970). The time course of in vitro reactivation is

depicted in figure 1. Addition of DTT and Fe++ to the enzyme from salinetreated rats prevented deterioration of the enzyme activity observed with

incubation in water. When the reducing mixture was incubated with TPH from

MDMA-treated rats, enzyme activity was restored from 40 percent of control to

normal over a 24-hour incubation period. Several other sulfhydryl-reducing

agents were examined for their ability to reactivate MDMA-inactivated TPH.

Although all compounds slightly elevated the inactivated enzyme, only DTT

and 2-mercaptothanol caused significant reactivation of MDMA-depressed

enzyme during the 24-hour preincubation period.

229
FIGURE 1. Time course of in vitro reactivation of MDMA-inactivated cortical

TPH. Enzyme solutions were prepared from rats killed 3 hours

after a single SC injection of MDMA (10 mg/kg) or saline (sal).

Reactivation mixture (DTT/Fe2+) or water was added to enzyme

preparations, which were then assayed immediately (zero time

point); the remainder was subsequently preincubated under N2

for up to 48 hours. Each point represents the mean of four

determinations.

SOURCE: Stone et al. 1989. Copyright 1989 by Journal of Neurochemistry.

Because MDMA decreases TPH activity by decreasing Vmax, rather than the

affinity for either the substrate or cofactor (Schmidt and Taylor 1987) the effect

of reducing conditions on the kinetics of the enzyme was determined (figure 2).

When cortical TPH from MDMA-treated rats was incubated under reducing

conditions, enzyme reactivation essentially doubled the Vmax of TPH, whereas

the Km for either tryptophan or the cofactor, 8-methyl-5,6,7,8-tetrahydropterin

(6-MPH4

), was not significantly altered.

Insight into the time course of the toxicity induced by MDMA in vivo could likely

be obtained by assessing the ability of reducing conditions to reactivate the

230
FIGURE 2. Double reciprocal plots of inactivated and reactivated TPH activity

from MDMA-treated rats, as a function of 6-MPH4 (A) or tryptophan

(trp) (B) concentrations. TPH solutions were prepared from rats

killed 3 hours after a single dose of MDMA (10 mg/kg, SC).

Following addition of reactivation mixture, inactivated and

reactivated enzyme solutions were preincuba ted for 24 hours in

room air at 0°C and under N2 at 25°C, respectively. The Km and

Vmax values are the means&EM derived from double reciprocal

plots of each of three enzyme samples (each sample was prepared

from the pooled cortices of five rats). The lines shown are based

on the mean Km and Vmax values; each point represents the mean

value for l/velocity at that substrate concentration.

SOURCE: Stone et al, 1989, Copyright 1989 by Journal of Neurochemistry.

231
enzyme at increasing times after administering the drug. If less and less

enzyme is reactivated as the time of in vivo exposure to MDMA increases,

this would suggest the development of irreversible drug-induced damage.

Rats were killed at varying times after MDMA administration, and the enzyme

was incubated under reactivating conditions (table 1). Neurotoxicity occurred

rapidly following MDMA (10 mg/kg).

Within 6 hours after MDMA was administered, <50 percent of inactivated TPH

could be reactivated; by 3 days, essentially all the MDMA-inactivated enzyme

(>80 percent) had been irreversibly altered.

TABLE 1. Cortical TPH activity from rats killed at various times after saline or

MDMA treatment, before and after preincubation under reactivating

conditions

Time

TPH Before Preincubation TPH After Preincubation

Saline MDMA Saline MDMA

Percent

Recovery

3 hours 98.0±5.9 54.4±10.3 91.5±6.8 93.1±9.0 103.8

(55.6±10.5) (101.7±9.8)†

6 hours 103.±8.2 38.0±5.1 100.4±6.8 69.8±6.6 51.7

(36.9±5.0) (69.5±6.5)†

3 days 99.6±3.1 48.6±4.0 91.1±4.9 51.8±4.9 15.8

(48.8+4.0) (56.9+5)

1 week 80.6±2.9 50.2±2.9 82.9±6.0 52.6+5.0 2.9

(62.4±3.6) (63.5±6.0)

30 days* 53.7±4.5 29.9±2.7 65.4±3.9 30.3±3.2 -21.2

(55.7±5.0) (46.3±4.9)

NOTE: Rats were killed at indicated times following a single saline or MDMA (10 mg/kg)

injection. Cortical TPH activity (nmol/g of tissue/hour) from each rat was

assessed following the addition of reactivation mixture, both before and after a

20- to 24-hour preincubation under N2. Results are presented as the

means&EM (n=5 to 7). Numbers in parentheses indicate percentage of saline

control. Percent recovery was calculated as: ([percent after preincubation -

percent before preincubation]/[100 - percent before preincubation]) X 100.

*30 days after multiple MDMA doses (10 mg/kg per dose: five doses, one every 6 hours)

†

p<0.05 vs. corresponding percentage before preincubation

SOURCE: Stone et al. 1989. Copyright 1989 by Journal of Neurochemistry.

232
Because congeners of MDMA also decrease TPH activity, it was determined

whether enzyme from animals injected with a single dose of METH (10 mg/kg),

p-chloroamphetamine (5 mg/kg), or MDMA (10 mg/kg) could be restored by

incubating under reducing conditions (figure 3). Incubation with DTT/Fe2+ for 24

hours reactivated the TPH from animals treated with each of the congeners. It

appears that oxidative inactivation is common to the neurotoxicity of all three

amphetamine congeners.

FIGURE 3. Comparison of cortical TPH activity, before and after reactivation,

from rats treated with MDMA, METH, or p-chloroamphetamine

(PCA). Rats were administered a single SC injection of MDMA

(10 mg/kg), METH (10 mg/kg), PCA (5 mg/kg), or saline and killed

3 hours later. Cortical TPH activity (nmol/g of tissue/hour) was

assessed following the addition of reactivation mixture, both before

and after a 24-hour preincubation under N2. Results are the

means&EM (n=6).

*p<0.05

**p<0.001 vs. time-matched saline

†Denotes a significant difference (p<0.01) between percentage after

preincubation and percentage before preincubation (percent=TPH activity from

drug-treated rats/TPH activity from time-matched saline-treated rats] X 100)

SOURCE: Stone et al. 1989. Copyright 1989 by Journal of Neurochemistry.

233
CONCLUSIONS

Amphetamine and its analogs cause neurochemical deficits in brain

dopaminergic and serotonergic systems. Because these deficits persist for long

periods after discontinuing the drug, neurotoxicity is inferred. Endogenous DA

is essential for neurotoxicity to occur. TPH, inactivated by administering

amphetamine congeners to rats, can be reactivated by incubating the enzyme

under reducing conditions; this suggests that the serotonergic deficits are

associated with oxidative reactions.

REFERENCES

Bakhit, C., and Gibb, J.W. Methamphetamine-induced depression of tryptophan

hydroxylase: Recovery following acute treatment. Eur J Pharmacol 76:229-

233,1981.

Bakhit, C.; Morgan, M.E.; Peat, M.A.; and Gibb, J.W. Long-term effects of

methamphetamine on the synthesis and metabolism of 5-hydroxytryptamine

in various regions of the rat brain. Neuropharmacology 20:1135-1140, 1981.

Battaglia, G.; Yeh, S.Y.; O'Hearn, E.; Molliver, M.; Kuhar, M.; and DeSousa,

E.B. 3,4-Methylenedioxymethamphetamine and 3,4-

methylenedioxyamphetamine destroy serotonin terminals in rat brain:

Quantitation of neurodegeneration by measurement of [

3H]paroxetine-labeled

serotonin uptake sites. J Pharmacol Exp Ther 242:911-916, 1987.

Buening, M.E., and Gibb, J.W. Influence of methamphetamine and neuroleptic

drugs on tyrosine hydroxylase activity. Eur J Pharmacol 28:30-34, 1974.

Commins, D.L.; Vosmer, G.; Virus, R.M.; Woolverton, W.L.; Schuster, C.R.; and

Seiden, L.S. Biochemical and histological evidence that

methylenedioxymethamphetamine (MDMA) is toxic to neurons in the rat

brain. J Pharmacol Exp Ther 241:338-345, 1987.

Garber, S.L., and Makman, M.H. Regulation of tryptophan hydroxylase activity

by a cyclic AMP-dependent mechanism in rat striatum. Mol Brain Res 3:1-10,

1987.

Gibb, J.W., and Kogan, F.J. Influence of dopamine synthesis on

methamphetamine-induced changes in striatal and adrenal tyrosine

hydroxylase activity. Naunyn Schmiedebergs Arch Pharmacol 310: 185-187,

1979.

Hamon, M.; Bourgoin, S.; Artaud, F.; and Nelson, D. Regulatory properties of

neuronal tryptophan hydroxylase. Adv Exp Med Biol 133:231-251, 1981.

Hamon, M.; Bourgoin, S.; Heryu, F.; and Simmonet, G. Characteristics of the

activation by dithiothreitol and Fe2+ of tryptophan hydroxylase from rat brain.

Neurochem Res 3:585-598, 1978.

Hotchkiss, A., and Gibb, J.W. Long-term effects of multiple doses of

methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase

activity in rat brain. J Pharmacol Exp Ther 214:257-262, 1980.

234
Hotchkiss, A.; Morgan, M.; and Gibb, J.W. The long-term effect of multiple doses

of methamphetamine on neostriatal tryptophan hydroxylase, tyrosine

hydroxylase, choline acetyl transferase and glutamate decarboxylase activities.

Life Sci 25:1373-1378, 1979.

Ichiyama, A.; Nakamura, S.; Nishizuka, Y.; and Hayaishi, O. Enzymic studies on

the biosynthesis of serotonin in mammalian brain. J Biol Chem 245:1699-1709,

1970.

Johnson, M.; Hanson, G.R.; and Gibb, J.W. Effect of MK-801 on the decrease in

tryptophan hydroxylase induced by methamphetamine and its methoxy analog.

Eur J Pharmacol 185:315-318, 1989.

Johnson, M.; Stone, D.M.; Hanson, G.R.; and Gibb, J.W. Role of the

dopaminergic nigrostriatal pathway in methamphetamine-induced depression

of the neostriatal serotonergic system. Eur J Pharmacol 135:231-234, 1987.

Koda, L.Y., and Gibb, J.W. The effect of repeated large doses of

methamphetamine on adrenal and brain tyrosine hydroxylase. Pharmacologist

13:253, 1971.

Koda, L.Y., and Gibb, J.W. Adrenal and striatal tyrosine hydroxylase activity after

methamphetamine. J Pharmacol Exp Ther 185:42-48, 1973.

Kuhn, D.M.; Ruskin, B.; and Lovenberg, W. Tryptophan hydroxylase: The role of

oxygen, iron, and sulfhydryl groups as determinants of stability and catalytic

activity. J Biol Chem 255:4137-4143, 1980.

Masserano, J.M.; Takimoto, G.S.; and Weiner, N. Electroconvulsive shock

increases tyrosine hydroxylase activity in the brain and TH adrenal gland of

the rat. Science 214:862-665, 1981.

O’Hearn, E.; Battaglia, G.; DeSousa, E.B.; Kuhar, M.J.; and Molliver, M.E.

Methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine

(MDMA) cause selective ablation of serotonergic axon terminals in forebrain:

lmmunocytochemical evidence for neurotoxicity. J Neurosci 8:2788-2803,

1988.

Ricaurte, G.; Bryan, G.; Strauss, L.; Seiden, L.; and Schuster, C. Hallucinogenic

amphetamine selectively destroys brain serotonin nerve terminals. Science

229:986-988, 1985.

Ricaurte, G.A.; Seiden, L.S.; and Schuster, C.R. Further evidence that

amphetamines produce long-lasting dopamine neurochemical deficits by

destroying dopamine nerve fibers. Brain Res 303:359-384, 1984.

Schmidt, C.J.; Ritter, J.K.; Sonsalla, P.K.; Hanson, G.R.; and Gibb, J.W. Role of

dopamine on the neurotoxic effects of methamphetamine. J Pharmacol Exp

Ther 233:539-544, 1985.

Schmidt, C.J., and Taylor, V.L. Depression of rat brain tryptophan hydroxylase

activity following the acute administration of methylenedioxymethamphetamine.

Biochem Pharmacol 36:4095-4102, 1987.

Sonsalla, P.K.; Gibb, J.W.; and Hanson, G.R. Roles of D1 and D2 dopamine

receptor subtypes in mediating the methamphetamine-induced changes in

monoamine systems. J Pharmacol Exp Ther 238:932-937, 1988a.

235
Sonsalla, P.K.; Gibb, J.W.; and Hanson, G.R. Nigrostriatal dopamine actions on

the D2

 receptors mediate methamphetamine effects on the striatonigral

substance P system. Neuropharmacology 25:1221-1229, 1986b

Sonsalla, P.K.; Nicklas, W.J.; and Heikkila, R.E. Role for excitatory amino acids

in methamphetamine-induced nigrostriatal dopaminergic toxicity. Science

243:398-400, 1989.

Stone, D.M.; Hanson, G.R.; and Gibb, J.W. In vitro reactivation of rat cortical

tryptophan hydroxylase following in vivo inactivation by methylenedioxy

methamphetamine. J Neurochem 53:572-581, 1989.

Stone, D.M.; Johnson, M.; Hanson, G.R.; and Gibb, J.W. Role of endogenous

dopamine in the central serotonergic deficits induced by 3,4-

methylenedioxymethamphetamine. J Pharmacol Exp Ther 247:79-87, 1988.

Stone, D.M.; Merchant, K.M.; Hanson, G.R.; and Gibb, J.W. Immediate and

long-term effects of 3,4-methylenedioxymethamphetamine on serotonin

pathways in brain of rat. Neuropharmacology 26:1677-1683, 1987.

Stone, D.M.; Stahl, D.C.; Hanson, G.R.; and Gibb, J.W. The effects of 3,4-

methylenedioxymethamphetamine (MDMA) and 3,4-

methylenedioxyamphetamine (MDA) on monoaminergic systems in the rat

brain. Eur J Pharmacol 128:41-48, 1986.

Vitto, A., and Mandell, A.J. Stability properties of activated tryptophan

hydroxylase from rat midbrain. J Neurochem 37:601-607, 1981.

Zigmond, R.E.; Chalazontis, A.; and Joh, T.H. Preganglionic nerve stimulation

increases the amount of tyrosine hydroxylase in the rat superior cervical

ganglion. Neurosci Lett 20:61-65, 1980.

DISCUSSION

Q: Methamphetamine, do you have a single dose?

A: Do I have a single dose? I don’t have one here, but we do have it.

You get more effect with amphetamine on the dopamine system than on the

serotonergic system. And so, obviously with one dose, you are not going to

see much effect on the dopamine system.

Q: You give high doses?

A: Yes.

Q (Seiden): I wonder if you could state whether you think that the dopamine is

exerting its effects by activating other neurons or is it exerting some effects on

the chemistry of the system independent of neuroactivity?

A: I don’t know the answer to your question, but my speculation would be that

the dopamine is auto-oxidized and that auto-oxidation results in the formation

of hydroquinone or in free radicals. I think those reactive metabolites might be

236
responsible for causing the neurotoxicity. At this point that’s my guess, and

only time will tell, after the appropriate experiments have been performed.

Q (Seiden): Just before I left for this meeting, I read a paper in which they

showed that 5,7-DHT could catalyze the reduction of glutathione.

A (Gibb): That should be explored.

Q (Ricaurte): Could you comment on the difference between an amphetamine

and methamphetamine? The basis for the question is that if dopamine release

is the key, and both amphetamine and methamphetamine are very good

dopamine releasers, both would be expected to have similar neurotoxic

properties. Yet there is no question that methamphetamine is much more

toxic than amphetamine to serotonin systems.

A: Yes

Q (Ricaurte): Why do you think that there is such a difference?

A: I don’t know. There are obviously some factors that still need some sorting

out. In our hands at least, we see toxicity with both. But there is more: I agree

with you. In our hands, compared to amphetamine, methamphetamine is more

toxic to the serotonergic than to the dopaminergic system.

COMMENT (Cho): Let me comment on that. We have done some studies

comparing methamphetamine- and amphetamine-induced release of dopamine

after an intravenous dose, and it is very clear that the rate at which dopamine

comes out is much greater with methamphetamine. I think that this is just a

question of the rapidity with which methamphetamine gets into the brain. The

volume of distribution of methamphetamine is much larger than amphetamine.

I think that is because the stuff gets in the brain more quickly and it is causing

release faster. So it’s like comparing morphine and heroin.

Q (Cho): I want to go back to your thiol protein recovery experiments. When

you did the thiol experiments, was this a whole cell preparation or was it cellfree?

A (Gibb): We homogenized and spun it down at 27,000 g. So this was

27,000 g supernatant.

Q (Cho): I guess what I am really asking is where is the enzyme that you are

activating? Is it the bottom of the cell particle?

A (Gibb): Yes, I understand your question. I don’t know the answer, but the

27,000 g supernatant still has cell particles.

237
COMMENT (Cho): Sometimes glutathione has trouble getting access to sites,

and that was why I was asking that question.

COMMENT (Gibb): In other words, you are saying that glutathione can’t get in.

Q (Cho): If you could get a soluble enzyme, you could go after it. The other

question is why you used iron 2 with the DTT?

A (Gibb): Thank you for bringing that up. I should have mentioned it. The

iron is not essential in the cortical preparation, but in the striatal preparation, it

enhances the response. I don’t know why. Perhaps there is enough iron bound

to the enzyme that you don’t need the iron. But if you get a more purified

enzyme, then iron is essential.

Q (Cho): What do you think iron is doing?

A: I don’t know.

COMMENT (Cho): If you have iron 2 and you continually generate it

(presumably you would with the thiol reagent present), you would have ideal

conditions for generating hydroxyl radical, because it is the iron 2 and oxygen

and peroxide that generate this stuff. So, in a way, within that system, it sounds

to me as if you were fighting the cell. That was the thing that should have

bothered me.

COMMENT (Gibb): I suppose there are enough protective capabilities within

the preparation that that is not occurring.

COMMENT (Cho): But then I looked at your cortical preparation, and you didn’t

get any better results in the absence of iron.

COMMENT (Gibb): And the only reason we included it, Art, was that in the

neostriatum it does seem to improve the situation to some extent. That has

been observed not only with MDMA but with oxygen as well.

Q: What do you think we should make of what looks like an increase in TPH

activity as you use agents that modify the dopamine release?

A (Gibb): Yes. That is an inconsistent finding, but I think that it is fascinating;

and I think that it demonstrates that there is possibly some interaction that is

going on between the dopamine and the serotonergic system.

Q: Maybe it is a basal interaction?

238
A (Gibb): Yes. I think that has been demonstrated neurophysiologically as well

as neurochemically. But what controls that interaction, I am not sure. But it is

very pronounced. We can get up to 130 percent of control when we give

reserpine or somehow interfere with the dopamine input to the serotonergic

system.

Q: It could be determined by your basal dopamine?

A (Gibb): Yes, but I think there are those projections, those interactions that are

very real.

Q (Seiden): Did you have any EDTA [ethylenediaminetetraacetic acid] present?

A: No, we didn’t.

Q (Olney): Why does MK801 protect?

A (Gibb): That is a good question. Based on some other experiments that

we have done, we think that there is a link between the dopamine and the

glutamate systems. Glen Hanson has done a lot of experiments with the

neuropeptides, which is another story and is very fascinating, and I think that

it offers another way that we can get at some of these problems. But it seems

that when we give NMDA, we have been aggravating the situation. It appears

that way, that maybe it is a sequence.

COMMENT (Olney): So there is an NMDA receptor involved?

COMMENT (Gibb): Yes. I am sure that you are happy about that.

COMMENT (Olney): Well, I am puzzled.

COMMENT (Gibb): So are we at this point,

COMMENT (Olney): I am also happy. Makes things nice and challenging

and complicated. Ordinarily, you would think of an NMDA receptor being on

dendrites or cell bodies; and if it is mediating any toxicity, it would be toxic to

the cell bodies and dendrites. That is the conventional arrangement. But you

are dealing here with an apparent toxic effect impinging on axons and axon

terminals.

COMMENT (Gibb): We are.

COMMENT (Olney): We don’t know of any way that NMDA receptors would

be involved in that kind of toxicity, but it is a fascinating possibility that you’re

putting your finger on a new type of excitotoxicity that hasn’t been invented

before.

239
COMMENT (Gibb): Right. We have found some very encouraging results,

John; but as I said about the neuropeptide story, we can simulate or at least

cause similar effects with NMDA as with methamphetamine on the increase in

neuropeptides that Hanson has observed. We thought that with MK801 we can

sort out those interrelationships better with the neuropeptides than we can

neurotoxicity. It may be a little more physiologic than the neurotoxicity.

COMMENT (Ricaurte): John, just in relation to this whole question, we have

envisioned that NMDA receptors are on DA terminals and hypothesized that

if NMDA receptor activation is involved in the toxicity of amphetamines, we

should be able to reproduce the toxicity by giving NMDA directly into the

striatum. When you inject NMDA into the striatum, you see damage, but it

appears to be nonspecific since DA levels per se are unaffected by high

doses of NMDA. There is no question that the competitive and noncompetitive

NMDA receptor blockers protect against methamphetamine toxicity. Just a

few weeks ago at a New York Academy of Sciences meeting, John Marshall

showed some very nice data obtained with in vivo microdialysis demonstrating

that a compound like MK801 was very effective in interfering with

methamphetamine-induced dopamine release; so indeed the basis of

protection may involve NMDA receptors, perhaps by modulating DA release.

COMMENT (Gibb): Somehow I don’t know which one comes first in the

sequence, but obviously, it appears to be a component.

COMMENT (Seiden): There is no question that this result is reproducible.

Q (Landfield): What calcium channel blockers did you try?

A (Gibb): Nimodipine, which makes it worse. I have forgotten the others, but

we tried quite a few.

COMMENT (Cho): I would just like to comment, because flunarizine is quite

different chemically from some of the other calcium channel blockers. You are

right; it is a dirty drug. But this goes back to what clearly represents my bias,

but it seems that we ought to know the effects of these multiple drugs on the

disposition of some of these compounds because part of the selectivity or part

of the discrepancies that one might see could be due to the disposition or

changes in the brain.

A (Gibb): We’ve certainly entertained that thought. It doesn’t seem to fit with

anything else. It could very well be that it is a disposition problem or it also has

other effects on other systems. I think the thing that we have found fascinating

about it is that it works selectively on one system and not the other. And that

came as a bit of a surprise. It just so happens that it is the opposite of the

MK801, and so it makes it kind of an interesting story.

240
AUTHORS

James W. Gibb, Ph.D.

Professor and Chairman

Michel Johnson, Ph.D.

Research Assistant Professor

Glen R. Hanson, D.D.S., Ph.D.

Professor

Department of Pharmacology and Toxicology

University of Utah

112 Skaggs Hall

Salt Lake City, UT 84112

Donna M. Stone, Ph.D.

Department of Neurosciences, MS #60

Genentech, Inc.

460 Point San Bruno

South San Francisco, CA 94080

241
Neurotoxicity and Drugs of Abuse:

Cannabinoid Interaction With Brain

Glucocorticoid Receptors

Philip W. Landfield and J. Charles Eldridge

INTRODUCTION

Abuse of psychoactive pharmacologic substances continues to be one of the

most difficult problems confronting the quality of human life in our society.

Although there is wide recognition of the socioeconomic and personal problems

created by alterations of behavior resulting from acute effects of drugs of abuse,

evidence is presented here to indicate that further consideration should be

given also to the deleterious physical effects of long-term exposure to these

substances. It is important to recognize that many older users have been

exposed chronically to drugs of abuse for many years and may believe that

there is little risk in moderate but continued exposure. For example, results of

a recent household survey conducted by the National Institute on Drug Abuse

suggest that 4.8 percent of the entire US. population smoke marijuana at least

once a month and that more than 3 million Americans use the drug “daily or

almost daily” (ADAMHA News 1992). Because marijuana achieved a popular

status during the 1960s it is likely that hundreds of thousands of middle-aged

Americans have been exposing themselves continuously to this drug for more

than two decades and perhaps their entire adult lives. Therefore, these users

could be at significant health risk if drugs such as cannabinoids possess toxic

effects with long-term exposure or if they are capable of interaction with the

aging process.

The authors have recently described neurotoxic responses in the hippocampal

regions of rats chronically administered tetrahydrocannabinol (THC), a major

psychoactive cannabinoid, and have proposed that the mechanism for this

toxicity may lie in similarity of cannabinoid structure and function to those of

glucocorticoid (GC) steroids (Eldridge and Landfield 1990; Landfield et al. 1988;

Eldridge et al. 1991) (figure 1). GC-mediated toxicity, particularly during brain

aging, has been seen in a number of studies (Landfield and Eldridge 1991;

Landfield et al. 1980) and evidence is reviewed in this chapter that brain-aginglike neurotoxicity can be exacerbated by continuous exposure to chemical

substances that interact with GC-sensitive systems in hippocampus.

242
Corticosterone

FIGURE 1. Chemical structures of corticosterone and THC

GC-MEDIATED NEUROTOXICITY IN STRESS AND AGING

It has been suggested that adrenal steroids could mediate aspects of peripheral

aging. The classic studies of Selye and associates led them to propose that

stress, by stimulating adrenal GC secretion, could augment the mammalian

aging process (Selye and Tuchweber 1976). In addition, early studies of

Robertson and Wexler demonstrated that Pacific salmon died normally from

the catabolic effects of elevated GC secretion associated with spawning

(Wexler 1976). Wexler (1976) further showed that elevated GC secretion

enhanced cardiovascular and other disease conditions often associated with

peripheral aging.

243
However, although genetic programing of cell death had been suggested as

a possible underlying cause of neural aging (Finch 1976), there were few

hypotheses in the mid-1970s that specifically addressed the mechanistic

bases for irreversible degenerative change in aged neurons. However, it

was recognized that the hippocampus and other brain regions are rich in GC

receptors (McEwen et al. 1968; Stumpf and Sar 1975), and it was also known

that the hippocampus is among the structures most vulnerable to normal brainaging changes and to senile dementia of the Alzheimer’s type (Tomlinson and

Henderson 1976; Wisniewski and Terry 1973). Therefore, a series of studies

was undertaken to test the hypothesis that GCs might modulate hippocampal

aging. These studies found correlations between GC secretion and quantitative

parameters of hippocampal aging (Landfield 1978; Landfield et al. 1978). It was

further found that adrenalectomy could retard development of morphologic

correlates of hippocampal aging (Landfield et al. 1981 a; Landfield 1987).

Although one study has reported a loss of hippocampal neuron number in young

animals administered high GC doses for a relatively short time (Sapolsky et al.

1985), it has remained difficult to perform long-term studies of elevated GC

levels because of rapid decline of health of the animals under these conditions

(Landfield 1987). Nevertheless, the findings have combined to suggest strongly

that GCs can play a role in the vulnerability of the aging hippocampus (Landfield

and Eldridge 1991; Landfield 1987; Sapolsky et al. 1985).

Additional studies of possible deleterious effects of endogenous GCs used

indirect indicators of differential exposure to chronic stress (Meany et al. 1988;

Uno et al. 1989). However, only recently were studies reported in which it was

possible to maintain a mild, nondamaging stress for prolonged periods (Kerr et

al. 1991). In these studies, it was found that several electrophysiologic and

morphologic markers of brain aging were accelerated by chronic stress in rats

of different ages. Furthermore, it appeared that the toxic impact of GCs on the

hippocampus increased with aging, suggesting that an age-related “susceptibility

factor” might increase the vulnerability of neurons to steroid-mediated toxicity.

Therefore, it is increasingly apparent that the development of pathology

associated with brain aging, particularly as observed in the hippocampus,

may be linked to the degree of exposure of the structures to GCs. The

deleterious effects of this exposure arise apparently from persistent exposure to

endogenous adrenal steroids and can be exacerbated by stressful environments,

Conceivably, then, chronic exposure to xenobiotic substances such as drugs of

abuse could also exert biologic or toxicologic effects on mammalian brain

through the GC receptor system.

CHRONIC CANNABINOID EXPOSURE AND DEVELOPMENT OF

NEUROPATHOLOGY

There are numerous reports in the literature that THC administration stimulates

pituitary-adrenal secretion, including corticosterone (CORT) release, in animals

(Dewey 1986; Martin 1986). Although accommodation of response has been

244
reported (Eldridge et al. 1991; Miczek and Dixit 1980), stimulation still can be

elicited after extended chronic exposure (Landfield et al. 1988). Other studies

have found that CORT stimulation following mild footshock was greater if

animals were treated with THC (Jacobs et al. 1979) and that this enhanced

secretion was likely caused by increased pituitary adrenocorticotropin (ACTH)

release (Dewey et al. 1970). Further support for involvement of central nervous

system mechanisms emerged from observations that barbiturate pretreatment

reduced THC-stimulated CORT and ACTH release (Dewey et al. 1970; Barry et

al. 1973).

Additional studies found that cannabinoid administration altered uptake of

radiolabeled CORT in rat brain (Drew and Slagel 1973; Johnson et al. 1978),

suggesting a mechanism involving GC receptors. It occurred to several

researchers that similarities of cannabinoid chemical structure to steroids

(figure 1) might cause the substances to substitute for GC binding in the brain

(Martin 1986; Dewey 1986). This interaction could, for instance, inhibit GCmediated negative feedback control of ACTH release, thereby activating

pituitary-adrenal secretion. Moreover, because the hippocampus contains a

dense population of GC receptors (McEwen et al. 1968; Stumpf and Sar 1975),

and because this brain region is believed to participate in the homeostatic

control of pituitary-adrenal secretion (De Kloet and Reul 1985; McEwen et

al. 1986; Jacobson and Sapolsky 1991), it seemed conceivable that the

hippocampus could be an important site of cannabinoid action. Therefore, it

was hypothesized that chronic THC administration might induce hippocampal

neuropathology, either by a direct agonist action through hippocampal GC

receptors or by stimulating release of endogenous CORT to neurotoxic levels

(Eldridge and Landfield 1990; Landfield et al. 1988; Eldridge et al. 1991).

Long-term administration studies have yielded data highly consistent with this

hypothesis (Landfield et al. 1988). In these studies, it was observed that THCtreated rats (8 to 10 mg/kg x 5 days/week x 8 months) exhibited decreased

neuronal density and increased glial cell reactivity in the hippocampus. The

changes appeared similar in degree to those observed in normally aged rats

of 24 months or older (Landfield et al. 1981b). However, the THC-treated

animals were only 11 to 12 months old at sacrifice, suggesting that THC might

have accelerated brain-aging-like activities. These changes are described

further below.

Assessment of Neurotoxicity

The paradigm of coupling long-term drug or hormone treatment with

quantitative neuroanatomy was developed initially to test hypotheses on

physiologic factors that may modify the development of morphological

correlates of brain aging as well as to study mechanisms that govern gradual

structural remodeling in the adult mammalian brain. It was clear that

experimental treatments such as hormonal manipulations or pharmacological

agents would need to be maintained for extended periods (e.g., 20 to 30

245
percent of the lifespan or 5 to 9 months in rats) and that extremely sensitive

techniques for measuring morphological and behavioral correlates of brain

aging would be required (see Landfield 1982 for methodological issues).

As a result, sensitive and reliable indices for quantifying subtle differences in

brain organization have been developed. The accuracy and reliability of the

methods are greatly enhanced by measurement of multiple morphometric

variables because these variables cross-validate one another and tend to

cancel out random variances (Landfield 1962).

Long-Term Exposure to THC

Using similar paradigms and procedures, long-term studies on the effects of

chronic treatment with THC were undertaken, based on extensive quantitative

light and electron microscopic (EM) studies. As mentioned, in the first study

(Landfield et al. 1988), chronic THC decreased hippocampal neuronal density

but not synaptic density (although another group of investigators has found

altered synaptic density in THC-treated rats [Scallet et al. 19871). THC also

increased the percentage of astrocyte cytoplasm occupied by inclusions at

the EM level (Landfield et al. 1988). In a second long-term (8 months) study,

effects were replicated, and it also was found, at the EM level, that THC

administration increased the cytoplasmic volume occupied by Golgi apparatus

(Cadwallader et al. 1989). These results were consistent with the possible

stimulation of genomic activity by GC agonist-like activation in these neurons.

The similarity of the THC-dependent pattern of neurotoxicity to the aging/

CORT-associated neuropathology pattern suggested an interaction of THC with

steroid systems in the induction of the pathological effects. In addition, during

acute restraint stress, plasma levels of both ACTH and CORT were significantly

elevated in rats treated chronically (4 months) with THC, compared with those

given pluronic vehicle alone or saline (Landfield et al. 1988) which confirmed

numerous reports, discussed above (Dewey 1986; Martin 1986), that THC

stimulates CORT release. The results also demonstrated that the endocrine

alterations did not completely adapt with prolonged exposure and therefore

could be sufficient to advance aging-like effects.

CANNABINOID INTERACTION WITH BRAIN GC RECEPTORS

As a result of suggestions that cannabinoids might be capable of direct

interaction with GC receptors in the brain, two possible mechanisms were

hypothesized: On the one hand, pituitary-adrenal activation could result from

cannabinoid antagonism of normal GC negative feedback suppression of ACTH

release, leading to increased CORT secretion; on the other hand, cannabinoids

could express direct agonist activity through GC receptors in the hippocampus,

to imitate or promote the same neuropathologic changes that appear to be

induced by CORT (Landfield 1987; Sapolsky et al. 1985; Kerr et al. 1991).

Either of these hypothesized mechanisms likely would require direct interaction

of cannabinoids with GC-receptor-binding events in brain.

246
These hypotheses were tested by preparing cytosol from hippocampal tissue

of adrenalectomized (ADX) rats (to remove all endogenous corticosteroids) and

incubating the cytosols with radiolabeled dexamethasone (DEX) plus or minus

various potential competing agents (for additional methodologic details, see

Eldridge et al. 1991; Eldridge and Landfield 1990). With inhibition of tracer

binding to receptors set at 100 percent by determination of displacement with

100-fold molar excess unlabeled DEX, it was observed that 100-fold molar

excess THC displaced 32 percent of tracer binding, whereas cannabidiol (CBD),

a nonpsychoactive constituent of marijuana, displaced only 16 percent of specific

radiolabeled DEX binding sites.

The rat hippocampus contains two specific types of GC receptor, and DEX

binds to both types in vitro (Reul and De Kloet 1985; Landfield and Eldridge

1989). In our studies, incubation of hippocampal cytosol with [

3H]-DEX plus

100-fold excess of RU-28362 (RU), a specific type II or GC receptor ligand

(Hermann et al. 1987; Raynaud et al. 1984), caused displacement of

81 percent of total binding capacity. Additional incubations of RU plus either

THC or CBD failed to displace more receptor binding than did RU alone,

indicating that cannabinoid displacement more likely affected the type II

population. Interaction with type I (mineralocorticoid receptor) binding would

have appeared as a further displacement when THC or CBD was added to RU.

Similar results to these were obtained when cytosols incubated with [

3H]-DEX

plus or minus RU, THC, or CBD were layered onto 5 to 20 percent sucrose

gradients and centrifuged to separate various binding components by molecular

weight. Cannabinoid displacement of GC binding occurred in the 8S region,

where the cytoplasmic GC receptor migrates.

Studies of the kinetics of cannabinoid competition also were performed

by incubation of cytosol with a range of doses of radiolabeled DEX in a

Scatchard-type assay (Eldridge and Landfield 1990; Eldridge et al. 1989a).

Plots of the data of high-affinity receptors should be linear, with the x-intercept

indicating binding capacity, or Bmax. Inhibitor agents, whether competitive or

noncompetitive inhibitors, shift the plot in relation to their ability to compete

against tracer binding. As in the previously described studies, RU-28362

competed against binding to type II sites (184 fmol/mg cytosol protein vs. a

total capacity of 210 fmol/mg; type I=the difference of 26 fmol/mg). Again,

THC at 100-fold molar excess was able to displace a significant, but incomplete,

portion of tracer binding (lower Bmax), and the Scatchard plot with THC added

was parallel to that of either total receptors or the type II fraction (figure 2).

If THC binds to the full population of type II GC receptors, a parallel slope

and lower Bmax, would be viewed as evidence that the THC interaction was

noncompetitive in nature. If the competition between competitor (THC) and

tracer (DEX) had occurred at the same binding site, the Scatchard plot would

have demonstrated the same Bmax as seen with incubation with unlabeled

DEX, but the slope (affinity) would have been different. However, no change

in affinity was seen in the THC plot; the x-intercept was lower, indicating that

247
FIGURE 2. Scatchard plot of cannabinoid competition with [

3H]-DEX binding

to hippocampal GC receptors. Aliquots of pooled cytosol

prepared from ADX rats (methodologies as described in Eldridge

and Landfield 1990 and Eldridge et al. 1989a) were incubated in

vitro with 1.25 to 20 nM radiolabeled DEX alone or plus 100-fold

molar excess of unlabeled DEX, RU-28362, THC, or CBD. DEX

plot indicates total specific displacement (210 fmol/mg protein),

and RU plot indicates type II receptor displacement (184 fmol/

mg). The slope of the THC plot (Kd=2. 18 nM) was approximately

parallel to those for DEX (3.11 nM) and RU (2.85 nM), but THC

displaced fewer sites (106 fmol/mg). A combination of RU and

THC, each at 100-fold molar excess over tracer concentration,

produced a plot not different from that produced by RU alone,

Cannabidiol displacement of [

3H]-DEX was very slight, not linear,

and could not be plotted.

SOURCE: Eldridge, J.C.; Murphy, L.L.; and Landfield, P.W. Cannabinoids and

the hippocampal glucocorticoid receptor: Recent findings and

possible significance. Steroids 56:226-231, 1991. Reprinted with

permission of Butterworth-Heinemann, Inc.

the capacity for radiolabeled DEX binding was reduced in the presence of THC.

Such a plot suggests a “noncompetitive” type of THC inhibition, wherein the

competitor associates with an allosteric site, distinct from the hormone binding

domain, that nevertheless alters the ability of the steroid to bind at its domain.

248
There is increasing evidence of allosteric binding sites on steroid receptors.

For example, Svec (1988) has described noncompetitive effects on

corticosteroid receptor binding by progesterone as well as by nonsteroidal

agents. However, substantially more work is necessary before the nature

of THC interaction with GC receptors is fully clarified.

THC-Mediated Down-Regulation of GC Receptors

One additional and critical test of the ability of THC to alter GC receptors was

conducted, which, in several ways, provided some of the clearer evidence of

a functionally significant interaction of THC and brain GC receptors. It is well

established that elevated secretion or administration of large doses of CORT

down-regulates GC receptor levels in the hippocampus (De Kloet and Reul

1985; McEwen et al. 1986; Tornello et al. 1982; Eldridge et al. 1989b). When

GC receptor content was analyzed in ADX rats maintained on a low level of

replacement steroid and given 10 mg/kg THC for 14 days (a dose that rats

tolerated in our long-term studies), down-regulation of GC receptor binding was

likewise observed in the THC-treated animals. Type I receptor content was not

affected. Previous studies have indicated that CORT administration can downregulate both receptor types (De Kloet and Reul 1985), so these results appear

to provide strong support for a functionally relevant and specific in vivo effect

of THC on the type II GC receptor. The occurrence of the response in ADX

animals suggests that the THC effect was mediated by direct interaction with

GC receptors rather than through secondary release of adrenal steroids.

The authors and colleagues have reported that down-regulation of brain GC

receptors was significantly impaired in aged animals exposed to daily stress

for 6 months (Eldridge et al. 19896) and that up-regulation of GC receptors

that normally occurs following adrenalectomy also was impaired in aged rats

(Eldridge et al. 1989a). The fact that hippocampal GC receptors down-regulate

less in intact aged rats has raised the possibility that this could increase the

vulnerability of hippocampal neurons to CORT-related degeneration during

aging (Landfield and Eldridge 1991). It is also conceivable that repeated

lifelong activation of brain GC receptors leads over time to reduced plasticity

and, thus, to increased vulnerability and neuronal loss. It therefore seems

possible that chronic exposure to THC could accelerate this process, perhaps

by increasing the vulnerability of hippocampal neurons.

Agonist or Antagonist Effects

Further evidence demonstrating direct agonist-like effects of cannabinoids on

GC-mediated response systems has recently been observed in the authors’

laboratories. Levels of mRNA for glial fibrillary acidic protein, which has been

shown to be specifically inhibited by GCs (Nichols et al. 1990), were also found

to be reduced in the hippocampus of ADX rats treated with THC (Eldridge et al.

1992). In addition, levels of calbindin-D28K, which have been shown to increase

specifically in hippocampus following GC exposure (lacopino and Christakos

249
1990), have been found in our laboratories to be increased in the hippocampus

of ADX rats treated with THC (Geddes et al. 1992).

Based on these findings, there appears to be considerable support for the

possibility that THC, which affects the neuroendocrine stress response, also

interacts directly with brain GC receptors and modulates the impact of

corticosteroids on the brain. It is clear that a substance need not be a steroid

in molecular structure to regulate GC receptor binding. Therefore, one result

of chronic exposure to cannabinoids could be allosteric modulation of brain

GC receptors such that one or more of the following occurs: (1) the effects of

CORT activation are mimicked; (2) endogenous CORT binding to GC receptors

is enhanced; (3) regulatory plasticity of GC receptors is impaired, making

neurons more susceptible to the toxic effects of CORT; (4) negative feedback

control of ACTH is partially blocked, resulting in higher CORT levels and

leading to greater neuronal degeneration; or (5) neuroendocrine control

mechanisms unrelated to GC receptors are altered, resulting in a persistent

hyperactivity of stress responses and higher CORT secretion. These

possibilities are, of course, not mutually exclusive, nor would they represent

all possible means through which drugs of abuse may alter or mimic CORT

impact on the brain.

INTERACTIONS OF OTHER DRUGS OF ABUSE WITH STRESS SYSTEMS

AND INDUCTION OF NEUROPATHOLOGY

To date, clear-cut quantitative neuroanatomical evidence of brain-aging-like

neuron loss in the hippocampus, or of brain GC receptor modulation, has been

found for chronic exposure to only one drug of abuse, that is, THC. However,

other types of neuropathology have been seen with several drugs of abuse,

particularly stimulants such as methamphetamine.

With only a few exceptions (e.g., in Seiden et al. 1976), neuropathological or

synaptic remodeling effects of major addicting drugs have not been examined

in long-term paradigms similar to those in which THC effects have been

found (e.g., 7- to 8-month exposure [Landfield et al. 1988]); the longest

treatment period in a neuropathology-cocaine study appears to have been

21 days (Seiden and Kleven 1989). Thus, if other drugs of abuse act in part

analogously to THC and/or interact with gradual brain-aging-like neurotoxic

mechanisms or if they induce wholly different, but nevertheless subtle, synaptic

remodeling changes, their brain-structural effects might not be detectable in

animals until after several months of exposure.

There also is growing evidence that drugs of abuse other than THC are

capable of interaction with brain neuroendocrine stress systems. For

example, several studies have shown that cocaine, amphetamines, and

methylenedioxymethamphetamine (MDMA) are strong stimulants of ACTH

and CORT release, under a variety of conditions; moreover, the site of action

appears to lie in the brain (Moldow and Fischman 1987; Rivier and Vale 1987).

250
There are also strong effects of morphine and other opioids on the adrenalpituitary axis, but the pattern of these effects is controversial; some studies

have found a decrease, whereas others find an increase, depending on

experimental conditions. This discrepancy may arise possibly because there

are multiple mechanisms and sites of opioid modulation of CORT release, one

inhibitory and one stimulatory (Bird et al. 1987; Cetera et al. 1988; Nikolarakis

et al. 1987). It is also clear that brain serotonin systems are intimately involved

in regulation of the pituitary-adrenal stress response (Azmetia and McEwen

1974; Jhanwar-Uniyal et al. 1987; Feldman et al. 1984; Lorens and Van de Kar

1987) and that amphetamines, cocaine, MDMA, and morphine strongly modify

serotonin systems (Seiden and Kleven 1989; Cunningham and Lakosky 1988;

Dworkin and Smith 1987). Thus, a wide range of drugs of abuse interacts with

the brain control mechanisms of the stress response.

In addition, there is recent intriguing evidence that drugs with some abuse

potential can directly modulate brain corticosteroid receptors. For example,

the tricyclic antidepressant imipramine, which, like cocaine, is a reuptake

blocker at catecholaminergic terminals, was found to alter GC binding in cells

of locus coeruleus and the raphe nuclei (Kitiyama et al. 1988). In addition,

MDMA reduced both serotonin and type II GC receptors in the striatum (Lowy et

al. 1989). These effects of both imipramine and MDMA were independent of

plasma CORT release and may reflect secondary modulation of GC receptors

by activation of catecholamine or serotonin receptors. These results are

consistent with the possibility that monoamine receptor activation can directly

regulate corticosteroid receptor concentration in the same neurons (Kitiyama et

al. 1988; Lowy et al. 1989). Although we have suggested that THC interacts

with GC receptors directly (Eldridge and Landfield 1990; Landfield et al. 1988;

Eldridge et al. 1991) and its molecular structure bears some similarity to that

of steroids (Martin 1986), it also should be noted that similarity of molecular

structure is clearly not a requisite for the potential modulation of brain GC

receptors by drugs of abuse that activate the stress response. That is,

corticosteroid receptors apparently contain at least one allosteric site at which

binding of ligands can be modified, both by other steroids (e.g., progesterone

and corticosterone) and by other agents (e.g., ketoconazole) that bear no

apparent similarity to steroids (Svec 1988).

Thus, it seems clear that a number of drugs of abuse can modulate GC

receptors, either by direct interaction or by secondary effects following

monoamine receptor activation. Furthermore, a range of drugs of abuse

modulate the adrenal-pituitary system that can, in turn, feed back onto brain

GC receptors. For these reasons, there appear to be multiple pathways

through which drugs of abuse can modulate the brain’s response to adrenal

stress hormones. In addition, there are reports that some drugs of abuse in

addition to THC can induce neurotoxicity in selected systems (Seiden et al.

1976; Nwanzee and Johnson 1981; Ricuarte et al. 1983, 1984; Lorenz 1981).

Therefore, it is conceivable that some aspects of this neurotoxicity may also

be mediated by effects on GC receptors.

251
REFERENCES

ADAMHA News 18:14-15, 1992.

Azmetia, E.C., and McEwen, B.S. Adrenocortical influence on rat brain

tryptophan hydroxylase activity. Brain Res 78:291-302, 1974.

Barry, H.; Kubena, R.K.; and Pernatch, J.L. Pituitary-adrenal activation and

related responses to -tetrahydrocannabinol. Prog Brain Res 39:323-329,

1973.

Bird, D.J.; Buckingham, J.C.; and Baker, B.I. Hypothalamo-pituitary-interrenal

responses to opioid substances in the trout. Gen Comp Endocrinol 68:33-39,

1987.

Cadwallader, L.B.; Vinsant, S.L.; and Landfield, P.W. Effects of chronic -THC

on hippocampal ultrastructure and adrenal hypertrophy. Sot Neurosci Abstr

15:288, 1989.

Cetera, B.; Bugajski, J.; and Gadek-Michalska, A. Involvement of central

histamine receptors in corticosterone secretion induced by intraventricular

administration of morphine. Agents Actions 23:250-253, 1988.

Cunningham, K.A., and Lakosky, J.M. Electrophysiological effects of cocaine

and procaine on dorsal raphe serotonin neurons. Eur J Pharmacol 148:457-

462, 1988.

De Kloet, E.R., and Reul, J.M.H.M. Feedback action and tonic influence of

corticosteroids on brain function: A concept arising from the heterogeneity of

brain receptor systems. Psychoneuroendocrinology 117:2505-2511, 1985.

Dewey, W.L. Cannabinoid pharmacology. Pharmacol Rev 38:151-201,1986.

Dewey, W.L.; Peng, T.C.; and Harris, L.S. The effect of 1-trans

tetrahydrocannabinol on the hypothalamo-hypophyseal-adrenal axis of rats,

Eur J Pharmacol 12:382-388, 1970.

Drew, W.G., and Slagel, D.E. Tetrahydrocannabinol: Selective impairment of

corticosterone uptake by limbic structures of the rat. Neuropharmacology

12:909-916, 1973.

Dworkin, S.I., and Smith, J.E. Neurobiological aspects of drug-seeking

behaviors. In: Thompson, T.; Dews, P.B.; and Barrett, J.E., eds. Advances in

Behavioral Pharmacology, Vol. 6: Neurobehavioral Pharmacology. Hillsdale,

NJ: Erlbaum, 1987. pp. 1-44.

Eldridge, J.C.; Fleenor, D.G.; Kerr, W.S.; and Landfield, P.W. Impaired upregulation of type II corticosteroid receptors in hippocampus of aged rats,

Brain Res 478:248-256, 1989a.

Eldridge, J.C.; Hu, H.-Y.: Extrom, P.J.; and Landfield, P.W. Interactions

between cannabinoids and steroids in the rat hippocampus. Soc Neurosci

Abstr 18:789, 1992.

Eldridge, J.C.; Kute, T.E.; Brodish, A.: and Landfield, P.W. Apparent agerelated resistance of type II hippocampal corticosteroid receptors to downregulation during chronic escape training. J Neurosci 9:3237-3242, 1989b.

Eldridge, J.C., and Landfield, P.W. Cannabinoid interactions with glucocorticoid

receptors in rat hippocampus. Brain Res 534:135-141, 1990.

252
Eldridge, J.C.; Murphy, L.L.; and Landfield, P.W. Cannabinoids and the

hippocampal glucocorticoid receptor: Recent findings and possible

significance. Steroids 56:226-231, 1991.

Feldman, S.; Melamed, E.; Conforti, N.; and Weidenfeld, J. Effect of central

serotonin depletion on adrenocortical responses to neural stimuli. Exp Neurol

85:661-666, 1984.

Finch, C.E. The regulation of physiological changes during mammalian aging.

Q Rev Biol 51:49-83, 1976.

Geddes, J.L.; Eldridge, J.C.; and Landfield, P.W. Glucocorticoid agonist-like

effect of delta-9-THC on immunoreactive calbindin upregulation in CA1

hippocampal neurons. Soc Neurosci Abstr 18:345, 1992.

Hermann, T.; Schramm, K.; and Ghraf, R. Photoaffinity labeling with [

3H]-RU28362: A powerful tool for the study of rat brain glucocorticoid receptors,

J Steroid Biochem 26:417-423, 1987.

lacopino, A.M., and Christakos, S. Corticosterone regulates calbindin-D28k

mRNA and protein levels in rat hippocampus. J Biol Chem 265:10177-10180,

1990.

Jacobs, J.A.; Dellarco, A.J.; Manfredi, R.A.; and Harclerode, J. The effects

of -tetrahydrocannabinol, cannabidiol and shock on plasma corticosterone

concentrations in rats. J Pharm Pharmacol 31:341-346, 1979.

Jacobson, L., and Sapolsky, R.M. The role of the hippocampus in feedback

regulation of the hypothalamic-pituitary-adrenal axis. Endocr Rev 12:118-134,

1991.

Jhanwar-Uniyal, M.; Renner, K.; Ballo, M.; Luine, V.N.; and Leibowitz, S.F.

Serotonin and 5-hydroxyindoleacetic acid levels in discrete hypothalamic

areas of the rat brain: Relation to circulating corticosterone. Neurosci Lett

79:145-150, 1987.

Johnson, K.M.; Dewey, W.L.; Ritter, KS.; and Beckner, J.S. Cannabinoid

effects on plasma corticosterone and uptake of 3H-corticosterone by mouse

brain. Eur J Pharmacol 47:303-308, 1978.

Kerr, D.S.; Campbell, L.W.; Applegate, M.D.; Brodish, A.; and Landfield, P.W.

Chronic stress-induced acceleration of electrophysiologic and morphologic

biomarkers on hippocampal aging. J Neurosci 11:1316-1324, 1991.

Kitiyama, I.; Janson, A.M.; Cintra, A.: Fuxe, K.; Agnati, L.F.; Ogret, S.O.;

Harfstrand, A.; Eneroth, P.; and Gustafsson, J.A. Effects of chronic

imipramine treatment on glucocorticoid receptor immunoreactivity in various

regions of the rat brain. J Neural Transm 73:191-203, 1988.

Landfield, P.W. An endocrine hypothesis of brain aging and studies on brainendocrine correlations and monosynaptic neurophysiology during aging. In:

Finch, C.E.; Potter, D.E.; and Kenny, A.D., eds. Aging and Neuroendocrine

Relationships. Parkinson’s Disease. Vol. 2. New York: Plenum, 1978. pp.

179-199.

Landfield, P.W. Measurement of brain aging. Conceptual issues and

neurobiological indices. In: Adelman, R., and Roth, G., eds. Endocrine and

Neuroendocrine Mechanisms of Aging. Boca Raton, FL: CRC Press, 1982.

pp. 183-207.

253
Landfield, P.W. Modulation of brain aging correlates by long-term alterations of

adrenal steroids and neurally-active peptides. In: De Kloet, E.R.; Wiegant,

V.M.; and De Wied, D., eds. Progress in Brain Research. Vol. 72.

Amsterdam: Elsevier, 1987. pp. 279-300.

Landfield, P.W.; Baskin, R.K.; and Pitler, T.A. Brain aging correlates:

Retardation by hormonal-pharmacological treatments. Science 214:581-584,

1981 a.

Landfield, P.W.; Braun, L.D.; Pitler, T.A.; Lindsey, J.D.; and Lynch G.

Hippocampal aging in rats: A morphometric study of multiple variables in

semithin sections. Neurobiol Aging 2:265-275, 1981b.

Landfield, P.W.; Cadwallader, L.B.; and Vinsant, S. Quantitative changes in

hippocampal structure following long-term exposure to

tetrahydrocannabinol: Possible mediation by glucocorticoid systems. Brain

Res 443:47-62, 1988.

Landfield, P.W., and Eldridge, J.C. Increased affinity of type II corticosteroid

receptor binding in aged rat hippocampus. Exp Neurol 106:110-113, 1989.

Landfield, P.W., and Eldridge, J.C. The glucocorticoid hypothesis of brain aging

and neurodegeneration: Recent modifications. Acta Endocrinol 125:54-64,

1991

Landfield, P.W.; Sundberg, D.K.; Smith, M.S.; Eldridge, J.C.; and Morris, M.

Mammalian aging: Theoretical implications of changes in brain and endocrine

systems during mid and late life. Peptides 1 (Suppl 1):185-196, 1980.

Landfield, P.W.; Waymire, J.L.; and Lynch, G.S. Hippocampal aging and

adrenoconicoids: Quantitative correlations. Science 202:1098-1102, 1978.

Lorens, S.A., and Van de Kar, L.D. Differential effects of serotonin (5-HT1A and

5-HT2) agonists and antagonists on renin and corticosterone secretion.

Neuroendocrinology 45:305-310, 1987.

Lorenz, H. Fluorescence histochemistry indicates damage of striatal dopamine

nerve terminals in rats after multiple doses of methamphetamine. Life Sci

28:911-916, 1981.

Lowy, M.T.; Nash, J.F., Jr.; and Meltzer, H.Y. Selective reduction of striatal type

II glucocorticoid receptors in rats by 3,4-methylenedioxymethamphetamine

(MDMA). Eur J Pharmacol 163:157-161, 1989.

Martin, B.R. Cellular effects of cannabinoids. Pharmacol Rev 38:45-106, 1986.

McEwen, B.S.; Weiss, J.M.; and Schwartz, L. Selective retention of

corticosterone by limbic structures in brain. Nature 220:911-912, 1968.

McEwen, B.S.; De Kloet, E.R.; and Rostene, W. Adrenal steroid receptors and

actions in the nervous system. Physiol Rev 66:1121-1188, 1986.

Meany, M.J.; Aitken, D.H.; Bhatnagar, S.; Van Berkel, C.; and Sapolsky, R.M.

Postnatal handling attenuates neuroendocrine, anatomical and cognitive

impairments related to the aged hippocampus. Science 238:766-768, 1988.

Miczek, K.A., and Dixit, B.N. Behavioral and biochemical effects of chronic

tetrahydrocannabinol in rats. Psychopharmacology 67:195-201, 1980.

Moldow, R.L., and Fischman, A.L. Cocaine-induced secretion of ACTH, betaendorphin and corticosterone. Peptides 8:819-822, 1987.

254
Nichols, N.R.; Ostberg, H.H.; Masters, J.N.; Millar, S.L.; and Finch, C.E.

Messenger RNA for glial fibrillary acidic protein is decreased in rat brain

following acute and chronic corticosterone treatment. Mol Brain Res 7:1-7,

1990.

Nikolarakis, K.; Pfeiffer, A.; Stalla, G.K.; and Herz, A. The role of CRF in the

release of ACTH by opiate agonists and antagonists in rats. Brain Res

421:373-376, 1987.

Nwanzee, E., and Johnson, G. Amphetamine neurotoxicity on dopamine nerve

terminals in the caudate nucleus of mice. Neurosci Lett 26:163-168, 1981,

Raynaud, J.P.; Ojasoo, T.; Jouquey, A.; Moguilewsky, M.; and Teutsch, G.

Probes for steroid receptors. In: Labrie, F., and Proulx, L., eds.

Endocrinology. New York: Elsevier, 1984. pp. 533-538.

Reul, J.M.H.M., and De Kloet, E.R. Two receptor systems for corticosterone in

rat brain: Microdistribution and differential occupation, Endocrinology

117:2505-2509, 1985.

Ricuarte, G.A.; Seiden, L.S.; and Schuster, C.R. Further evidence that

amphetamines produce long-lasting dopamine neurochemical deficits by

destroying dopamine nerve fibers. Brain Res 303:359-364, 1984.

Ricuarte, G.A.; Seiden, L.S.; and Schuster, C.R. Increased dopamine

metabolism in the rat neostriatum after toxic doses of d-methamphetamine.

Neuropharmacology 22:1383-1388, 1983.

Rivier, C., and Vale, W. Cocaine stimulates adrenocorticotropin (ACTH)

secretion through a corticotrophin-releasing factor (CRF)-mediated

mechanism. Brain Res 422:403-406, 1987.

Sapolsky, R.M.; Krey, L.C.; and McEwen, B.S. Prolonged glucocorticoid

exposure reduces hippocampal neuron number: Implications for aging.

J Neurosci 5:1222-1227, 1985.

Scallet, A.C.; Uemura, E.; Andrews, A.; Ali, S.F.; McMillan, D.E.; Paule, M.G.;

Brown, R.M.; and Slikker, W., Jr. Morphometric studies of the rat

hippocampus following chronic delta-9-tetrahydrocannabinol (THC). Brain

Res 436:193-198, 1987.

Seiden, L.S.; Fischman, M.W.; and Schuster, CR. Long-term

methamphetamine-induced changes in brain catecholamines in tolerant

rhesus monkeys. Drug Alcohol Depend 1:215-219, 1976.

Seiden, L.S., and Kleven, MS. Lack of toxic effects of cocaine on dopamine or

serotonin neurons in the rat brain. In: Clouet, D.; Asghar, K.; and Brown, R.

eds. Mechanisms of Cocaine Abuse and Toxicity. National Institute on Drug

Abuse Research Monograph 88. DHHS Pub. No. (ADM)89-1585.

Washington, DC: Supt. of Docs., US. Govt. Print. Off., 1989. pp. 276-289.

Selye, H., and Tuchweber, B. Stress in relation to aging and disease. In: Everitt,

A.V., and Burgess, J.A., eds. Hypothalamus, Pituitary and Aging. Springfield,

IL: Charles C Thomas, 1976. pp. 553-569.

Stumpf, W.E., and Sar, M. Anatomical distribution of corticosterone-containing

neurons in rat brain. In: Stumpf, W.E., and Grant, L.E., eds. Anatomical

Neuroendocrinology. Basel: Karger, 1975. pp. 254-261.

255
Svec, F. Differences in the interaction of RU-486 and ketoconazole with the

second binding site of the glucocorticoid receptor. Endocrinology 123:1902-

1906, 1988.

Tomlinson, B.E., and Henderson, G. Some quantitative cerebral findings in

normal and demented old people. In: Terry, R.D., and Gershon, S., eds.

Neurobiology of Aging. New York: Raven Press, 1976. pp. 183-204.

Tornello, S.; Orti, F.; De Nicola, A.F.; Rainbow, T.C.; and McEwen, B.S.

Regulation of glucocorticoid receptors in rat brain by corticosterone treatment

of adrenalectomized rats. Neuroendocrinology 35:411-417, 1982.

Uno, H.; Tarara, R.; Else, J.G.; Suleman, M.A.; and Sapolsky, R.M.

Hippocampal damage associated with prolonged and fatal stress in primates.

J Neurosci 9:1705-1711, 1989.

Wexler, B.C. Comparative aspects of hyperadrenocorticism and aging. In:

Everitt, A.V., and Burgess, J.A., eds. Hypothalamus, Pituitary and Aging.

Springfield, IL: Charles C Thomas, 1976. pp. 333-361.

Wisniewski, H.M., and Terry, R.D. Morphology of the aging brain, human and

animal. In: Ford, D.M., ed. Progress in Brain Research. Vol. 40. Amsterdam:

Elsevier, 1973. pp. 167-186.

DISCUSSION

Q: Did you see anything in dentate granule cells with THC?

A (Landfield): Well, we didn’t measure them because they are generally very

resistant to the loss of cells during brain aging.

Q: What about extrapolating your data to the humans?

A: Chronic stress is bad for you; chronic THC is bad for you; aging is bad for

you.

Q: So, should we retire early?

A: That might be more stressful for some.

Q: Does your THC regimen remove the thymus?

A: I don’t know. I think it does. But, no, I will take it back. We considered

measuring it, and I think we looked at a few animals, but we don’t have

systematic data on thymus involution.

Q: Have you had a chance to look at other receptors? I guess my question

is that THC is a rather dirty compound. Does it interact with a number of

receptors, not just glucocorticoid receptors?

256
A: There is a receptor termed the “cannabinoid receptor” that is also bound

by THC. My colleague, Chuck Eldridge, is now looking at the interaction of

glucocorticoid receptors with cannabinoid-like, or CP compounds. There is

a little bit of interaction, but it is very dependent upon whether it is studied in

membrane or cytosol. The cannabinoid receptor is membrane bound, and the

glucocorticoid receptor is cytosolic. However, it is certainly conceivable that

there is more than one THC site In the brain, given the multiplicity of effects that

THC has. I think it still needs to be determined more clearly whether the CP

site or the GC site is the site of greater psychoactive relevance, and in which

proportion. I suspect that the GC site is important for stress reduction effects

of THC, and certainly it may have neurotoxic relevance; but whether it has other

psychoactive relevance is something that we will have to study in the future.

ACKNOWLEDGMENTS

Studies conducted in the authors’ laboratories that are described here were

supported in part by National Institute on Drug Abuse grants DA-03637 and

DA-06218.

AUTHORS

Philip W. Landfield, Ph.D.

Professor and Chair

Department of Pharmacology

College of Medicine

University of Kentucky Chandler Medical Center, MS-305

Lexington, KY 40536-0084

J. Charles Eldridge, Ph.D.

Associate Professor

Department of Physiology and Pharmacology

Bowman Gray School of Medicine

Wake Forest University

Winston-Salem, NC 27157-1083

257
Nitric Oxide as a Mediator of

Neurotoxicity

Ted M. Dawson, Valina L. Dawson, and Solomon H. Snyder

INTRODUCTION

Nitric oxide (NO) is a newly identified neuronal-messenger molecule, first

recognized as endothelium-derived relaxing factor (EDRF) and as a modulator

of the tumoricidal and bactericidal actions of macrophages (Marletta 1989). The

brain NO synthase (NOS) has been purified to homogeneity as a 150-kilodalton

protein with an absolute requirement for calcium, calmodulin, and nicotinamideadenine dinucleotide phosphate (NADPH) (Bredt and Snyder 1990). It has been

cloned and has sequence homology to cytochrome P-450 reductase (Bredt et al.

1991a). The macrophage NOS also has been purified to homogeneity and is a

dimer of molecular weight 250 kilodalton (Stuehr et al. 1991). The macrophage

enzyme differs from the brain/endothelial enzyme in that it does not require

calcium and calmodulin but has a requirement for tetrahydrobiopterin (Tayeh

and Marletta 1989; Kwon et al. 1990; Yui et al. 1991; Stuehr et al. 1991). A

physiologic role for NO in the brain and nervous system has been established

by demonstrations that arginine derivatives, which are potent and selective

inhibitors of NOS, block neuronally mediated relaxation of the intestine (Bult

et al. 1990; Boeckxstaens et al. 1991) and stimulation of cyclic guanosine

monophosphate (cGMP) formation by glutamate in the cerebellum (Garthwaite et

al. 1988, 1989; Bredt and Snyder 1989). NOS has been immunohistochemically

localized to discrete regions of the brain and neurons in the periphery. It is

localized to populations of medium-to-large aspiny neurons in the cerebral

cortex and corpus striatum, basket and granule cells of the cerebellum,

pedunculopontine nucleus, and other areas (Bredt et al. 1990; Dawson et al.

1991a), In the periphery, it is localized to ganglion cells and their fibers in the

adrenal medulla, myenteric plexus neurons of the intestine, and nerve fibers in

the posterior pituitary that receive projections from NOS-containing neurons in

the supraoptic and paraventricular hypothalamic nuclei (Bredt et al. 1990;

Dawson et al. 1991a).

NOS AND NEURONAL NADPH-DIAPHORASE

Dawson and colleagues (1991a) have shown that the distribution of NOS neurons

matches precisely with the distribution of NADPH-diaphorase-containing neurons,

258
NADPH-diaphorase-containing neurons were first identified histochemically by

Thomas and Pearse (1984, 1961) by the reduction of nitroblue tetrazolium by

NADPH to a formazan precipitate. Much interest in the function of NADPHdiaphorase stems from observations that these neurons are selectively resistant

to the degeneration in Huntington’s disease and survive ischemia as well as

destruction of neuronal tissue by excitatory amino acids (Ferrante et al. 1985;

Uemura et al. 1990; Beal et al. 1986; Koh et al. 1986;

Koh and Choi 1988).

During our initial studies of the distribution of NOS, the authors and coworkers

did not observe close colocalization with any known neurotransmitters or

neuronal markers. However, it became clear that the distribution of NOS

seemed to parallel the distribution of NADPH-diaphorase-containing neurons.

In the brain and periphery, NOS-containing neurons matched precisely with

the distribution of NADPH-diaphorase-containing neurons, In the brain, NOS

is colocalized with neuropeptide Y (NPY) and somatostatin-containing neurons

in the striatum and cortex (Dawson et al. 1991a), In the striatum, these are the

neurons that survive in Huntington’s disease (Ferrante et al. 1985). In contrast,

in the pedunculopontine nucleus, NOS-containing neurons colocalize with choline

acetyltransferase (ChAT)-containing neurons. In the pedunculopontine nucleus,

NOS-containing neurons do not colocalize with the somatostatin- and NPYcontaining neurons, and in the striatum, NOS does not colocalize with ChATcontaining neurons (Dawson et al. 1991a).

In the periphery, NOS colocalizes with NADPH-diaphorase-containing nerve

fibers in the posterior pituitary as well as with the NOS-containing neurons of the

supraoptic and paraventricular hypothalamic nuclei. In the adrenal medulla, NOS

colocalizes with NADPH-diaphorase-containing ganglion cells. The adrenal cortex

displays NADPH-diaphorase staining but no NOS immunoreactivity. Several

NADPH-requiring enzymes of the steroid biosynthesis pathway may account for

NADPH-diaphorase staining observed here. In the intestine, NOS-containing

neurons colocalize with NADPH-diaphorase-containing myenteric plexus neurons.

These neurons probably account for the relaxation of the intestine and stomach

by NO (Bult et al. 1990; Boeckxstaens et al. 1991).

By utilizing oligonucleotide probes to the cDNA for NOS, Bredt and colleagues

(1991b) have also colocalized the message for NOS to NADPH-diaphorase

neurons in the brain. The striking colocalization of NOS with NADPH-diaphorase

suggests that NOS catalytic activity accounts for NADPH-diaphorase staining.

By transfecting the whole open-reading frame of the cDNA for NOS into human

kidney 293 cells, encoded protein and catalytic activity were demonstrated in these

cells (Dawson et al. 1991a). These transfected cells exhibit NADPH-diaphorase

staining that is proportional to the amount of expressed NOS immunoreactivity.

In addition, the relative amount of NOS immunoreactivity and NADPH-diaphorase

staining is identical in individual neurons of several regions of the brain (table 1).

259
TABLE 1. Comparison of NOS immunoreactivity and NADPH-diaphorase

staining

Tissue NOS NADPH-Diaphorase

Recombinant NOS protein

Mock transfected cells

NOS transfected cells (1 μg cDNA)

NOS transfected cells (10 μg cDNA)

Cerebellum

Basket ceils

Granule cells

Purkinje cells

Cortex

Medium-large aspiny neurons

Hypothalamus

Paraventricular nucleus

Supraoptic nucleus

Pedunculopontine tegmental nucleus

Retina

Amacrine cells

Ganglion cells

Striatum

Medium-large aspiny neurons

0 0

4+ 4+

8+ 8+

2+

2+

0

4+

3+

3+

5+

2+

1+

4+

2+

2+

0

4+

3+

3+

5+

2+

1+

4+

NOTE: In 4 to 6 separate experiments in which 20 to 40 neurons were

examined per region, the density of NOS immunoreactivity is identical

to NADPH-diaphorase staining (1+=low; 8+=high). In kidney cells

transfected with NOS cDNA, the ratio of immunoreactive NOS and

NADPH-diaphorase staining is identical to that seen in positive

neurons.

SOURCE: Dawson et al. 1991a

These findings establish that NOS is responsible for NADPH-diaphorase staining

of neurons.

Another approach to establish that NOS accounts for NADPH-diaphorase staining

would be to purify the NADPH-diaphorase protein and determine whether it is

260
identical to the NOS protein. However, NADPH-diaphorase activity measured in

homogenates may not reflect the enzyme monitored by histochemical staining. In

efforts to purify NADPH-diaphorase by monitoring nitroblue tetrazolium reduction,

we have observed multiple protein bands that are distinct from NOS. Hope and

colleagues (1991) also have observed substantial NADPH-diaphorase catalytic

activity that could be separated from NOS activity. However, Hope and colleagues

(1991) did show that purified NOS does possess NADPH-diaphorase activity that

is similar to our own observations. As such, histochemistry reveals enzyme

activity that is highly concentrated in discrete neurons, whereas the catalytic

activity of homogenates may reflect the contribution of multiple enzymes with

“diaphorase” activity.

NO AND NEUROTOXICITY

The identity of NADPH-diaphorase-containing neurons with NOS suggested that

NO may be involved in modulating neurotoxicity, because these neurons survive

a variety of toxic insults. Dawson and colleagues (1991b) used rat primary

cortical cultures as described by Koh and Choi (1988) to investigate the effects

of inhibitors of NOS on glutamate neurotoxicity. Neurotoxicity was assessed by

the exclusion of trypan blue or by the measurement of lactate dehydrogenase. In

the primary culture system, N-methyl-D-aspartate (NMDA) displays a toxic LD50

of 280 μM. A 300 μM concentration of NMDA consistently destroys approximately

60 percent of the neurons (figure 1). Simultaneous application of 300 μM NMDA

with 100 μM nitroarginine (N-Arg), a NOS inhibitor, attenuates NMDA-induced

cell death by approximately 70 percent (figure 1). The addition of 1 mM L-arginine

(L-Arg), the endogenous substrate for NOS, reverses the protective effects of NArg (figure 1). To further ascertain whether NO is involved in NMDA neurotoxicity,

500 μM of reduced hemoglobin, which binds NO, was added simultaneously with

300 μM NMDA (figure 1). Hemoglobin completely prevents NMDA toxicity. The

500 μM concentration of reduced hemoglobin employed is similar to that required

for reduced myoglobin to prevent macrophage-mediated cell death (Stuehr and

Nathan 1989).

Cortical cultures possess NOS catalytic activity and protein that are approximately

10 percent that of cerebellar levels (figure 2). In addition, NMDA can stimulate

the formation of NO in cortical cultures. The formation of cGMP can be used as

an indirect measure of NO formation, because NO binds to the heme moiety of

guanylate cyclase and subsequently activates the enzyme (Bredt and Snyder

1989). NMDA stimulation of cGMP levels in cultures is antagonized by N-Arg,

and this antagonism is competitively reversed by L-Arg. Superoxide dismutase,

which stabilizes NO, enhances NMDA stimulation of cGMP. Hemoglobin, which

complexes NO, prevents NMDA-induced cGMP formation, Thus, as in cerebellar

slices, NO stimulates cGMP formation in cortical cell cultures (Dawson et al.

1991b).

261
FIGURE 1. Hoffman modulation photomicrographs of primary cortical

cultures illustrating the effect of manipulation of NOS on NMDA

neurotoxicity. Panel A: Control well of primary cortical neurons.

Panel B: 300 μM NMDA+ 10 μM glycine consistently yields 60

to 70 percent neuronal death as determined by trypan blue

exclusion. Dead neurons appear as black dots, and viable

neurons remain unstained. Panel C: 100 μM N-Arg protects

against NMDA toxicity. Panel D: 1 mM L-Arg reverses N-Arg

neuroprotection, thus demonstrating that NMDA toxicity is

mediated via NO. Panel E: Depletion of the culture media of

L-Arg with arginase or growing the cells in an L-Arg-free media

completely attenuates neurotoxicity, indicating an L-Arg

dependence for NMDA-mediated cell death. Panel F: 500 μM

reduced hemoglobin (Hb) that inactivates NO attenuates NMDA

neurotoxicity; thus, NO is released to mediate its toxicity.

Bar=100 μM.

SOURCE: Dawson et al. 1991b

262
FIGURE 2. NOS catalytic activity was assessed by measuring the conversion

of [

3H]arginine to [3H]citrulline in homogenized primary cortical

(CTX) neuron cultures and compared with cerebellar (CB)

homogenates. NOS catalytic activity in CTX was approximately

1/10 that of CB. Similar results were obtained by Western blot

analysis.

SOURCE: Dawson et al. 1991b

N-Arg inhibits NMDA-induced cell death with an EC50 of 20 μM. Methylarginine

(M-Arg), another NOS inhibitor that is less potent than N-Arg, inhibits NMDAinduced cell death with an EC50 of 170 μM (Dawson et al. 1991b), N-Arg does

not act directly on NMDA receptors because N-Arg has no effect on [

3H]MK-801

binding or on NMDA-induced currents measured in cortical cultures by wholecell patch-clamp analysis (figure 3).

Because L-Arg is able to reverse the neuroprotective effects of N-Arg against

NMDA neurotoxicity, we wondered whether L-Arg plays a role in NMDA

neurotoxicity. To determine that fact, arginase was added in amounts that

completely degraded L-Arg or treated sister cultures for 24 hours in a medium

without L-Arg, a procedure that fully depletes cells of L-Arg (Sessa et al. 1990).

In both preparations, 300 μM NMDA no longer causes cell death, and the LD50

of NMDA is increased more than twentyfold to 7.5 mM. Furthermore, there is

an absolute requirement of 100 mM L-Arg to obtain maximal NMDA

neurotoxicity.

Although NMDA-receptor activation presumably accounts for the majority of

neurotoxicity associated with synaptically released glutamate, activation of

263
N-Arg Does Not Affect

NMDA-Induced Currents

in Cultured Cortical Neurons

FIGURE 3. N-Arg has no effect on [

3H]MK-801 binding or NMDA

currents as assessed by whole-cell patch-clamping.

SOURCE: Dawson et al. 1991b

264
quisqualate and kainate receptors may also play a role (Koh et al. 1990). The

effects of N-Arg on cytotoxicity induced by quisqualate, kainate, and NMDA

were compared (table 2). Quisqualate neurotoxicity is diminished somewhat

by treatment with high concentrations of N-Arg, perhaps because at these

concentrations quisqualate influences NMDA receptors (Koh et al. 1990). N-Arg

provides no protection against kainate-induced cell death. Effects of glutamate

and its analogs are similar in cultures from the caudate, hippocampus, and

cerebral cortex.

The authors evaluated the specificity of L-Arg in reversing N-Arg-neuroprotective

effects against NMDA toxicity by examining various analogs of L-Arg. Homoarginine,

which can serve as a precursor of NO but has less affinity for NOS than L-Arg,

partially reverses the effects of N-Arg. The stereoisomer D-arginine is much

weaker than L-Arg. These results establish the specificity for L-Arg reversal of

N-Arg neuroprotection against NMDA neurotoxicity.

TABLE 2. Inhibition of glutamate neurotoxicity by N-Arg and reversal by

L-Arg in primary neuronal cultures

Compound

% Cell Death

Cortex

300 μM NMDA 62.0±2.5

+100 μM N-Arg 20.3±3.0*

+N-Arg+1 mM L-Arg 64.6±6.5

500 μM quisqualate

+500 μM N-Arg

+N-Arg+5 mM L-Arg

60.7±4.1

41.5±3.6*

63.0±4.5

100 μM kainate

+500 μM N-Arg

83.9±4.3

83.0±4.1

NOTE: Values represent the means±SEM for n>6, n<30. Cell death was

determined by 0.4 percent trypan blue exclusion by viable cells.

Significant overall F values were obtained by using a one-way,

between-groups analysis of variance. Specific comparisons on all

possible pairwise combinations were made with the Spjotvoll-Stoline

post hoc test.

*p<0.05

SOURCE: Dawson et al. 1991b

265
Sodium nitroprusside, which spontaneously releases NO, was used to determine

the direct effect of NO on cortical cells. A 5-minute application of sodium

nitroprusside elicits cell death in a concentration-dependent fashion with a 50-

percent maximal response at 50 μM and enhancement of cGMP levels in cortical

cells at a similar potency. The influence of sodium nitroprusside on cGMP levels

as well as cytotoxicity appears to involve NO, because these effects are blocked

by hemoglobin. In addition, the dose-response relationship and time-course of

NO-mediated neuronal death parallels NMDA-mediated neurotoxicity (Dawson

et al., in press).

The experiments described above indicate that NO mediates glutamate

neurotoxicity in neuronal cultures. Glutamate-receptor-mediated cell death is

prevented by inhibitors of NOS whose protective effects are proportional to

their potencies as inhibitors of NOS. The inhibition of cell death is competitively

reversible by L-Arg and is exerted also by various arginine derivatives in

proportion to their activities as substrates for NOS. Removal of arginine from

the culture media by arginine-free media or by arginase also prevents NMDAinduced cell death, demonstrating an absolute requirement of L-Arg for NMDAinduced neurotoxicity. Furthermore, sodium nitroprusside, which generates NO,

elicits cell death in a concentration-dependent fashion that parallels the formation

of cGMP. Reduced hemoglobin, which complexes NO, prevents both NMDAand sodium nitroprusside-induced neurotoxicity. N-Arg also diminishes infarct

volume following focal ischemia in mice, thus establishing the physiologic

relevance of NO in neurotoxicity (Nowicki et al. 1991).

NO previously has been shown to mediate cytotoxicity resulting from activated

macrophages that kill tumor cells and bacteria via NO. The exact source of

neurotoxic NO is not yet definitively established. Neurons that contain NOS are

a likely source, whereas microglia, which may contain a macrophage-like NOS,

are alternative candidates. If neuronal NOS mediates glutamate neurotoxicity,

then destruction of NOS-containing neurons should attenuate glutamate

neurotoxicity. Choi and coworkers (Koh and Choi 1988; Koh et al. 1986) have

shown that NADPH-diaphorase neurons, which are now known to be NOS

neurons, in primary cortical cultures resist NMDA neurotoxicity but are extremely

susceptible to kainate or quisqualate toxicity. Low-dose quisqualate treatment

of the cultures, which selectively kills NOS/NADPH-diaphorase neurons,

subsequently reduces the toxicity of NMDA, implying that these neurons mediate

NMDA killing of other neurons (Dawson et al., in press). Furthermore, neuronal

cell death tends to occur around the processes of NOS neurons that ramify

extensively, contacting the majority of neurons in the culture even though NOS

neurons comprise only about 2 percent of neurons in the culture. Ontogeny

studies show that the appearance of NOS immunoreactivity coincides with the

development of NMDA toxicity several days after the appearance of NMDAmediated currents (Dawson et al., in press). Whether there is an inducible

266
NOS in microglia is presently unknown, so that a role for microglia or resident

macrophages in NO-mediated toxicity is unclear.

CONCLUSIONS

Realization that NADPH-diaphorase neurons are NOS-containing neurons

and the discovery that NO may mediate glutamate neurotoxicity help clarify the

physiological role of these neurons in neurodegenerative disorders (figure 4).

Their resistance to NMDA neurotoxicity and exquisite sensitivity to kainate and

quisqualate neurotoxicity have long been a puzzle. The “diaphorase” portion of

NOS catalytic activity might account for their survival, because induction of

NAD(P)H: (quinone-acceptor) oxidoreductase (DT diaphorase) in a neuronal cell

line prevents glutamate toxicity (Murphy et al. 1991). Furthermore, these cells

may possess other unique protective mechanisms against NO. Manganesesuperoxide dismutase colocalizes with NADPH-diaphorase-containing neurons

in the striatum (Inagaki et al. 1991). Superoxide dismutase may be partly

responsible for their survival, because there is evidence that superoxide

dismutase treatment of cortical cultures can attenuate NMDA neurotoxicity

(Dawson et al., in press). How NO kills cells is unknown. However, NO is a

highly reactive free radical, and free-radical formation has been implicated in

various forms of toxicity (Choi 1990; Meldrum and Garthwaite 1990), In addition,

NO can react with a superoxide anion to form peroxynitrate, which is highly

reactive (Beckman et al. 1990). Furthermore, NO can complex with iron sulphur

center of enzymes to inactivate them (Stuehr and Nathan 1989; Hibbs et al.

1988). Many of these enzymes are in the mitochondrial-electron-transport chain

so that their inactivation would diminish the ability of cells to deal with oxidative

stress.

267
NMDA-R ACTIVATED NOS ACTIVITY

FIGURE 4. Schematic diagram of NMDA-mediated neurotoxicity. The "donor

cell" (NOS/NADPH-diaphorase-containing neurons) that activates

NOS via Ca2+ binding to calmodulin on NMDA receptor stimulation

releases NO to adjacent neurons. Under normal conditions within

the target neuron, NO activates guanylate cyclase to form cGMP

that activates cGMP-dependent cation channels and cGMPdependent protein kinases that phosphorylates various proteins.

Under conditions of sustained NMDA receptor stimulation,

excessive amounts of NO are released that may kill adjacent

“target” neurons. NO toxicity is probably mediated via the formation

of free radicals. In addition, NO may combine with superoxide

anion (O2-) to form peroxynitrate (ONOO-) that spontaneously

decomposes to the hydroxyl free radical (OH) and NO2 (Sessa et

al. 1990). Why NOS neurons survive is unknown, but they probably

possess “factors” that detoxify NO. Potential candidates include

the “diaphorase” portion of NOS as well as the enrichment of

manganese-superoxide dismutase in these neurons.

268
REFERENCES

Beal, M.F.; Kowall, N.W.; Ellison, D.W.; Mazurek, M.F.; Swartz, K.J.; and

Martin, J.B. Replication of the neurochemical characteristics of Huntington’s

disease by quinolinic acid. Nature 321:168-171, 1986.

Beckman, J.S.; Beckman, T.W.; Chen, J.; Marshall, P.A.; and Freeman, B.A.

Apparent hydroxyl radical production by peroxynitrite: Implications for

endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A

87:1620-1624, 1990.

Boeckxstaens, G.E.; Pelckmans, P.A.; Bult, H.; DeMan, J.G.; and Herman, A.G.

Evidence for nitric oxide as mediator of non-adrenergic non-cholinergic

relaxations induced by ATP and GABA in the canine gut. Br J Pharmacol

102:434-438, 1991.

Bredt, D.S.; Glatt, C.E.; Hwang, P.M.; Fotuhi, M.; Dawson, T.M.; and Snyder,

S.H. Nitric oxide synthase protein and mRNA are discretely localized in

neuronal populations of the mammalian CNS together with NADPH

diaphorase. Neuron 7:615-624, 1991b.

Bredt, D.S.; Hwang, P.M.; Glatt, C.; Lowenstein, C.; Reed, R.R.; and Snyder,

S.H. Cloned and expressed nitric oxide synthase structurally resembles

cytochrome P-450 reductase. Nature 351:714-718, 1991a.

Bredt, D.S.; Hwang, P.M.; and Snyder, S.H. Localization of nitric oxide synthase

indicating a neural role for nitric oxide. Nature 347:768-770, 1990.

Bredt, D.S., and Snyder, S.H. Nitric oxide mediates glutamate-linked

enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci U S A

86:9030-9033, 1989.

Bredt, D.S., and Snyder, S.H. Isolation of nitric oxide synthetase, a calmodulinrequiring enzyme. Proc Natl Acad Sci U S A 87:682-685, 1990.

Bult, H.; Boeckxstaens, G.E.; Pelckmans, P.A.; Jordaens, F.H.; Van Maercke,

Y.M.; and Herman, A.G. Nitric oxide as an inhibitory non-adrenergic noncholinergic neurotransmitter. Nature 345:346-347, 1990.

Choi, D.W. Cerebral hypoxia: Some new approaches and unanswered

questions. J Neurosci 10:2493-2501, 1990.

Dawson, T.M.; Bredt, D.S.; Fotuhi, M.; Hwang, P.M.; and Snyder, S.H. Nitric

oxide synthase and neuronal NADPH diaphorase are identical in brain and

peripheral tissues. Proc Natl Acad Sci U S A 88:7797-7801, 1991a.

Dawson, V.L.; Dawson, T.M.; Bartley, D.A.; Spirak, C.E.; Uhl, G.R.; and Snyder,

S.H. Mechanisms of nitric oxide mediated neurotoxicity in primary brain

cultures. J Neurosci, in press.

Dawson, V.L.; Dawson, T.M.; London, E.D.; Bredt, D.S.; and Snyder, S.H. Nitric

oxide mediates glutamate neurotoxicity in primary cortical culture. Proc Natl

Acad Sci U S A 88:6368-6371, 1991b.

Ferrante, R.J.; Kowall, N.W.; Beal, M.F.; Richardson, E.P., Jr.; Bird, E.D.; and

Martin, J.B. Selective sparing of a class of striatal neurons in Huntington’s

disease. Science 230:561-563, 1985.

269
Garthwaite, J.; Charles, S.L.; and Chess-Williams, R. Endothelium-derived

relaxing factor release on activation of NMDA receptors suggests role as

intercellular messenger in the brain. Nature 336:385-388, 1988.

Garthwaite, J.; Garthwaite, G.; Palmer, R.M.J.; and Moncada, S. NMDA

receptor activation induces nitric oxide synthesis from arginine in rat brain

slices. Eur J Pharmacol 172:413-416, 1989.

Hibbs, J.B., Jr.; Taintor, R.R.; Vavrin, Z.; and Rechlin, E.M. Nitric oxide: A

cytotoxic activated macrophage effector molecule. Biochem Biophys Res

Commun 157:87-94, 1988.

Hope, B.T.; Michael, G.J.; Knigge, K.M.; and Vincent, S.R. Neuronal NADPH

diaphorase is a nitric oxide synthase. Proc Natl Acad Sci U S A 88:2811-

2814, 1991.

Inagaki, S.; Suzuki, K.; Taniguchi, N.; and Takagi, H. Localization of Mnsuperoxide dismutase (Mn-SOD) in cholinergic and somatostatin-containing

neurons in the rat neostriatum. Brain Res 549:174-177, 1991.

Koh, J.-Y., and Choi, D.W. Vulnerability of cultured cortical neurons to damage

by excitotoxins: Differential susceptibility of neurons containing NADPHdiaphorase. J Neurosci 8:2153-2163, 1988.

Koh, J.-Y.; Goldberg, M.P.; Hartley, D.M.; and Choi, D.W. Non-NMDA receptormediated neurotoxicity in cortical culture. J Neurosci 10:693-705, 1990.

Koh, J.-Y.; Peters, S.; and Choi, D.W. Neurons containing NADPH-diaphorase

are selectively resistant to quinolinate toxicity. Science 234:73-76, 1986.

Kwon, N.S.; Nathan, C.F.; Gilker, C.; Griffith, O.W.; Matthews, D.E.; and Stuehr,

D.J. L-citrulline production from L-arginine by macrophage nitric oxide

synthase. J Biol Chem 265:13442-13445, 1990.

Marietta, M.A. Nitric oxide: Biosynthesis and biological significance. Trends Biol

Sci 14:488-492, 1989.

Meldrum, B., and Garthwaite, J. Excitatory amino acid neurotoxicity and

neurodegenerative disease. Trends Pharmacol Sci 11:379-387, 1990.

Murphy, T.H.; DeLong, M.J.; and Coyle, J.T. Enhanced NAD(P)H: Quinone

reductase activity prevents glutamate toxicity produced by oxidative stress.

J Neurochem 56:990-995, 1991.

Nowicki, J.P.; Duval, D.; Poignet, H.; and Scatton, B. Nitric oxide mediates

neuronal death after focal cerebral ischemia in the mouse. Eur J Pharmacol

204:339-340, 1991.

Sessa, W.C.; Hecker, M.; Mitchell, J.A.; and Vane, J.R. The metabolism of Larginine and its significance for the biosynthesis of endothelium-derived

relaxing factor: L-glutamine inhibits the generation of L-arginine by cultured

endothelial cells. Proc Natl Acad Sci U S A 87:8607-8611, 1990.

Stuehr, D.J.; Cho, H.J.; Kwon, N.S.; Weise, M.F.; and Nathan, C.F. Purification

and characterization of the cytokine-induced macrophage nitric oxide

synthase: An FAD- and FMN-containing flavoprotein. Proc Natl Acad Sci

U S A 88:7773-7777, 1991.

270
Stuehr, D.J., and Nathan, C.F. Nitric oxide. A macrophage product responsible

for cytostasis and respiratory inhibition in tumor target cells. J Exp Med

169:1543-1555, 1989.

Tayeh, M.A., and Marietta, M.A. Macrophage oxidation of L-arginine to nitric

oxide, nitrite, and nitrate. J Biol Chem 264:19654-19658, 1989.

Thomas, E., and Pearse, A.G.E. The fine localization of dehydrogenases in the

nervous system. Histochemistry 2:266-282, 1961.

Thomas, E., and Pearse, A.G.E. The solitary active cells. Histochemical

demonstration of damage-resistant nerve cells with a TPN-diaphorase

reaction. Acta Neuropathol 3:238-249, 1964.

Uemura, Y.; Kowall, N.W.; and Beal, M.F. Selective sparing of NADPHdiaphorase-somatostatin-neuropeptide Y neurons in ischemic gerbil striatum.

Ann Neurol 27:620-625, 1990.

Yui, Y.; Hattori, R.; Kosuga, K.; Eizawa, H.; Hiki, K.; and Kawai, C. Calmodulinindependent nitric oxide synthase from rat polymorphonuclear neutrophils.

J Biol Chem 266:12544-12547, 1991.

DISCUSSION

Q: The NMDA-mediated toxicity might be mitigated by hypothermia. Did you

do an analysis of the NADPH diaphorase temperature dependency?

A: You mean the temperature dependence of the enzyme?

Q: Right, the neuroprotective effects of MK801 are due to hypothermia, the

protection against ischemic damage.

A: That is very conceivable, but we haven’t looked at it.

COMMENT (Molliver): Your culture is temperature controlled.

A: At least in culture, the temperature is probably not important. In the in vivo

setting it is possible, but I think Dr. Olney’s work with the retina and the work of

other investigators have shown that MK801 can protect against neurotoxicity

despite all of these potential factors.

Q: You mentioned that sodium nitroprusside causes hypertension.

A: No, nitroarginine, the inhibitor of NO [nitric oxide] synthase. Actually, not

the work that we have done, but other investigators’ work has shown that

nitric oxide is probably one of the major regulators of blood pressure. Animals

treated with inhibitors of NO synthase develop severe hypertension. Thus, in

vivo studies examining the effect of NO synthase inhibitors on neurotoxicity will

be difficult to interpret, unless the animals are concurrently made normotensive

or in the event that a specific neuronal NO synthase inhibitor is available.

271
COMMENT (Ramsay): I would like to make a comment on something that

Dr. Dawson mentioned in passing, and that is the general toxic mechanism.

You mentioned that nitric oxide inhibits mitochondrial systems in general.

I think that the exquisitely specific effects of a lot of drugs of abuse and their

metabolites in the brain are often overlooked because they also have general

toxic effects and that mitochondria in particular are very sensitive to these

toxic effects. Mitochondria are essential for energy, not only for the regular

maintenance of the cells, but also for the defense mechanisms against radicals.

That means energy. When you compromise the energy, you also compromise

these mechanisms. Of the mitochondrial respiratory chain, the most sensitive

step is in Complex I, which is a very complex enzyme with 23 peptides, with

5 iron sulfur clusters, and so is probably a prime target for the nitric oxide. We

also know quite a lot about the effects of inhibition of Complex I from the work

on MPTP [1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine] and the ultimate toxin,

MPP+. You can titrate Complex I with MPP+ and see immediate effects in the

respiratory chain of the mitochondria. The second mitochondrial target that is

important in metabolites of the neuroactive compounds is Complex III, which,

though not rate limiting, is very sensitive to quinones; and quinones are very

often produced as metabolites by the P450 system. A good example of this in

general mechanisms of toxicity is that of acetaminophen metabolites, which

cause hepatoxicity. There the quinones have been shown to interact directly

with Complex III and inhibit the passage of electrons, thereby impairing the

energy metabolism of the cell to such an extent that even the lactate-producing

enzymes in the liver cannot support the cell. So I think that this is something

to keep in mind when dealing with complex metabolites in the brain. These

effects are easy to measure. You can measure them in isolated mitochondria.

You can add the brain homogenates in which you are looking for metabolites.

You can add them back to a mitochondria preparation. They can also be

measured in cell cultures, which is rather nice. Sanchez-Ramos did that

with MPP+ and showed that the heme groups of the later respiratory chain

complexes stayed oxidized and couldn’t be reduced, thereby showing that

the block of MPP+ was in the early stages of the respiratory chain. I think

that it is something to keep in mind when looking at toxicity in general.

ACKNOWLEDGMENTS

Ted M. Dawson is a Pfizer Postdoctoral Fellow and is supported by grants

from the French Foundation, Dana Foundation, the American Academy of

Neurology, and U.S. Public Health Service CIDA NS 01578-01. Valina L.

Dawson is a recipient of a Pharmacology Research Associate Training Program

Award (National Institute of General Medical Sciences). Solomon H. Snyder’s

research is supported by U.S. Public Health Service grant DA-00266 and

Research Scientist Award DA-00074.

272
AUTHORS

Ted M. Dawson, M.D., Ph.D.

Instructor

Departments of Neuroscience and Neurology

Solomon H. Snyder, M.D.

Director

Departments of Neuroscience,

Pharmacology and Molecular Sciences, and

Psychiatry and Behavioral Sciences

Johns Hopkins University School of Medicine

725 North Wolfe Street

Baltimore, MD 21205

Valina L. Dawson, Ph.D.

Postdoctoral Fellow

Addiction Research Center

Laboratory of Molecular Neurobiology

National Institute on Drug Abuse

P.O. Box 5180

Baltimore, MD 21224

273
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116 DRUG DISCRIMINATION: APPLICATIONS TO DRUG ABUSE

RESEARCH. Richard A. Glennon, Ph.D.; Torbjörn U.C. Jörbe, Ph.D.; and

Jerry Frankenheim, Ph.D., eds.

GPO Stock #017-024-01470-2 $13 NCADI #M116

117 METHODOLOGICAL ISSUES IN EPIDEMIOLOGICAL, PREVENTION,

AND TREATMENT RESEARCH ON DRUG-EXPOSED WOMEN AND THEIR

CHILDREN. M. Marlyne Kilbey, Ph.D., and Khursheed Asghar, Ph.D., eds.

GPO Stock #017-024-01472-9 $12 NCADI #M117

118 DRUG ABUSE TREATMENT IN PRISONS AND JAILS. Carl G. Leukefeld,

D.S.W., and Frank M. Tims, Ph.D., eds.

GPO Stock #017-024-01473-7 $16 NCADI #M118

119 PROBLEMS OF DRUG DEPENDENCE 1991: PROCEEDING OF THE

53RD ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS

OF DRUG DEPENDENCE, INC. Louis Harris, Ph.D., ed.

GPO Stock #017-024-01474-5 $22 NCADI #M119

120 BIOAVAILABILITY OF DRUGS TO THE BRAIN AND THE BLOOD-BRAIN

BARRIER. Jerry Frankenheim, Ph.D., and Roger M. Brown, Ph.D., eds.

GPO Stock #017-024-01481-8 $10 NCADI #M120

121 BUPRENORPHINE: AN ALTERNATIVE TREATMENT FOR OPIOID

DEPENDENCE. Jack D. Blaine, Ph.D., ed.

GPO Stock #017-024-01482-6 $5 NCADI #M121

279
123 ACUTE COCAINE INTOXICATION: CURRENT METHODS OF

TREATMENT. Heinz Sorer, Ph.D., ed.

GPO #017-024-01501-6 $6.50 NCADI #M123

124 NEUROBIOLOGICAL APPROACHES TO BRAIN-BEHAVIOR

INTERACTION. Joseph Frascella, Ph.D., and Roger M. Brown, Ph.D., eds.

GPO Stock #017-024-01492-3 $9 NCADI #M124

125 ACTIVATION OF IMMEDIATE EARLY GENES BY DRUGS OF ABUSE.

Reinhard Grzanna, Ph.D., and Roger M. Brown, Ph.D., eds.

GPO #017-024-01503-2 $7.50 NCADI #M125

126 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH.

VOLUME II: STRUCTURE, FUNCTION, AND EXPRESSION. Theresa N.H.

Lee, Ph.D., ed.

NCADI #M126

127 PROGRESS AND ISSUES IN CASE MANAGEMENT. Rebecca Sager

Ashery, D.S.W., ed.

GPO Stock #017-024-01490-7 $14 NCADI #M127

126 STATISTICAL ISSUES IN CLINICAL TRIALS FOR TREATMENT OF

OPIATE DEPENDENCE. Ram B. Jain, Ph.D., ed.

GPO Stock #017-024-01491-5 $6.50 NCADI #M128

129 INHALANT ABUSE: A VOLATILE RESEARCH AGENDA. Charles W.

Sharp, Ph.D.; Fred Beauvais, Ph.D.; and Richard Spence, Ph.D., eds.

GPO Stock #017-024-01496-6 $12 NCADI #M129

130 DRUG ABUSE AMONG MINORITY YOUTH: ADVANCES IN RESEARCH

AND METHODOLOGY. Mario R. De La Rosa, Ph.D., and Juan-Luis Recio

Adrados, Ph.D., eds.

NCADI #M130

131 IMPACT OF PRESCRIPTION DRUG DIVERSION CONTROL SYSTEMS

ON MEDICAL PRACTICE AND PATIENT CARE. James R. Cooper, Ph.D.;

Dorynne J. Czechowicz, M.D.; Stephen P. Molinari, J.D., R.Ph.; and Robert C.

Peterson, Ph.D., eds.

NCADI #M131

132 PROBLEMS OF DRUG DEPENDENCE, 1992: PROCEEDING OF THE

54TH ANNUAL SCIENTIFIC MEETING, THE COLLEGE ON PROBLEMS OF

DRUG DEPENDENCE, INC. Louis Harris, Ph.D., ed.

GPO Stock #017-024-01502-4 $23 NCADI #M132

280
133 SIGMA, PCP, AND NMDA RECEPTORS. Errol B. De Souza, Ph.D.; Doris

Clouet, Ph.D.; and Edythe D. Condon, Ph.D., eds.

NCADI #M133

134 MEDICATIONS DEVELOPMENT: DRUG DISCOVERY, DATABASES,

AND COMPUTER-AIDED DRUG DESIGN. Rao S. Rapaka, Ph.D., and

Richard L. Hawks, Ph.D., eds.

NCADI #M134

281
NIH Publication No 93-3644

Printed 1993