National Institute on Drug Abuse

RESEARCH

MONOGRAPH SERIES

Activation of Immediate

Early Genes by Drugs

of Abuse

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

125
Activation of Immediate

Early Genes By Drugs of

Abuse

Editors:

Reinhard Grzanna, Ph.D.

Roger M. Brown, Ph.D.

NIDA Research Monograph 125

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 “Activation of Immediate Early Genes by Drugs of Abuse” held on

June 3-4, 1991, in Rockville, MD. The technical review was sponsored by the

National Institute on Drug Abuse (NIDA).

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Citation of the source is appreciated.

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

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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.

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

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Abstracts,” and “Psychopharmacology Abstracts.”

National Institute on Drug Abuse

NIH Publication No. 93-3504

Printed 1993

i i
Contents

Page

Introduction 1

Reinhard Grzanna and Roger M. Brown

Regulation of Immediate Early Gene Expression 3

Brent H. Cochran

Everything Activates C-fos- How Can It Matter? 25

Steven E. Hyman, Barry E. Kosofsky, Tuong V. Nguyen,

Bruce M. Cohen, and Michael J. Comb

Immediate Early Genes: Their Involvement in Physiological

and Pathological Responses in the Nervous System 39

Michael O. Hayward, Tom Curran, and James I. Morgan

Immediate Early Gene Activation and Long-Term Changes

in Neural Function: A Possible Role in Addiction? 54

Harold A. Robertson

Acute Effects of Psychomotor Stimulant Drugs on Gene

Expression in the Striatum 72

Ann M. Graybiel

Functional Organization of the Striatum: Relevance to Actions

of Psychostimulant Drugs of Abuse 82

Charles R. Gerfen

iii
Regulation of Neural Gene Expression in Opiate and

Cocaine Addiction 92

Eric J. Nestler, Clare M. Bergson. Xavier Guitart,

and Bruce T. Hope

C-Fos and Fos-Related Antigens as Markers for Neuronal

Activity: Perspectives From Neuroendocrine Systems

Gloria E. Hoffman, Wen-Sen Lee, M. Susan Smith, Rula Abbud,

Michelle M. Roberts, Alan G. Robinson, and Joseph G. Verbalis

117

Mechanisms of Opioid-Mediated Antinociception: Correlation

of Fos Expression and Behavior 134

Kathleen R. Gogas, Jon D. Levine, and Allan I. Basbaum

The Ontogeny of Immediate Early Gene Response to Cocaine:

A Molecular Analysis of the Effects of Cocaine on Developing

Rat Brain 181

Barry E. Kosofsky and Steven E. Hyman

NMDA Receptor Blockade Prevents Translation, but Not Transcription,

of the C-fos Gene Following Stimulation With Multiple Extracellular

Signals in Cultured Cortical Neurons: Implications for Plasticity and

Molecular Memory 172

Frank R. Sharp, Kinya Hisanaga, and Stephen M. Sagar

Induction and Suppression of Proto-Oncogenes in Rat

Striatum After Single or Multiple Treatments With Cocaine

or GBR-12909 181

Michael J. ladaroia, Eric J. Chuang, Choh-Lun Yeung, Yin Hoo,

Mayme Silverthorn, Jun Gu, and Gaetano Draisci

List of NIDA Research Monographs 212

iv
Introduction

Reinhard Grzanna and Roger M. Brown

A major goal in drug abuse research is to determine the neurobiological

mechanisms by which drugs of abuse produce tolerance, dependence,

and addiction. These behavioral manifestations of drug abuse have been

attributed to longlasting neuroadaptations in central nervous system (CNS)

neurons. The nature and extent of these neuroadaptations remain to be

characterized, but investigators agree that they are the result of drug-induced

alterations in neuronal gene expression.

The recent discovery that cocaine and amphetamine rapidly and transiently

induce immediate early genes (IEGs) provided the most direct indication

that drugs of abuse can profoundly influence gene expression. Following

their description a few years ago, IEGs and their protein products became

recognized as important links by which extracellular signals can produce

alterations in gene transcription. Turning on IEG expression by drugs of

abuse may be the initial step by which drugs alter the expression of late

genes to produce longlasting changes in neuronal functions. Thus, studies

of the effects of drugs of abuse on IEGs may hold the key to providing the

answer to how drugs of abuse produce long-term changes in neurons.

Studies of this class of genes also should provide a powerful approach to

explore correlations between drug-induced changes in behavior and the

neuronal systems in which drugs permanently alter gene expression.

Studies of the effects of drugs of abuse on IEGs are greatly facilitated by the

availability of methods to visualize the activity states of these genes in tissue

sections by immunohistochemistry and by in situ hybridization histochemistry.

This has opened a new and promising approach to define drug-induced,

permanent changes in the CNS and to identify the neuronal circuitries in

which they occur.

The chapters in this volume were presented at a conference on June 3-4, 1991,

in Rockville, MD, organized by the National Institute on Drug Abuse. The main

goal of this conference was to assess the potential impact of studies of IEGs in

the field of drug abuse research. The contributors to this volume reviewed

1
current developments in the field and discussed to what extent

studies of the effects of drugs of abuse on IEGs are likely to provide new

insights into the molecular underpinning of drug-seeking behavior.

Among the topics discussed were the specificity of the activation of IEGs

by drugs of abuse, the nature of the long-term alterations following the

induction, and the possible relationship between the observed changes in

IEG activity and behavior. Many of the questions discussed remain unresolved.

Yet, there was excitement about the remarkable specificity in the patterns of

IEG activation induced by different drugs in the CNS. The participants

expressed confidence about the prospects of linking drug-induced IEG

induction to specific alterations in peptide gene expression in identified

populations of CNS neurons.

This volume reflects the considerable optimism among drug abuse researchers

that studies of IEGs offer a new path to identify the long-term effects of drug

exposure in the CNS. The chapters reveal a sense of confidence that studies

of the action of drugs of abuse on IEGs will greatly facilitate efforts to identify

the neuronal systems critically involved in the development of drug addiction

and the nature of the changes produced within these systems.

AUTHORS

Reinhard Grzanna, Ph.D.

Senior Staff Fellow

Roger M. Brown, Ph.D.

Chief

Neuroscience Research Branch

National Institute on Drug Abuse

Parklawn Building, Room 11A-33

5600 Fishers Lane

Rockville, MD 20657

2
Regulation of Immediate Early Gene

Expression

Brent H. Cochran

INTRODUCTION

Recently, much excitement has been generated by the finding that various

immediate early genes (IEGs) are expressed in neurons in response to a

variety of neurotropic stimuli. Part of the reason these findings have generated

so much interest is that earlier work has suggested that long-term changes in

nervous system behavior require changes in gene expression (Goelet et al.

1986). Thus, since changes in IEG expression are coupled to neuronal activity

and neurotransmitter release, it may now be possible, using in situ techniques

that provide resolution at the single-cell level, to correlate the physiological state

of a neuron with behavioral and physiological outputs. This chapter reviews

what is currently known about IEGs and speculates on what roles these genes

might be playing in the operation of the nervous system.

IEGs (sometimes called early-response genes or primary-response genes)

were first identified not in the nervous system but through the study of cell

growth regulation. During the 1970s it became apparent that polypeptide

growth factors were key modulators of cell growth and differentiation of

multicellular organisms. Early studies had suggested that the mitogenic effects

of serum growth factors were mediated in part by the ability of these factors to

regulate gene expression (Smith and Stiles 1981). To identify such genes,

several investigators set out to clone them by differential or subtractive cDNA

cloning. The first of such genes cloned were those induced by the plateletderived growth factor (PDGF) in 3T3 fibroblasts (Cochran et al. 1983). The

genes cloned in this study displayed regulatory properties that are characteristic

of most early-response genes now known. These genes are induced rapidly

within 1 hour of growth factor treatment and are regulated at the level of

transcription (Cochran et al. 1988). They are induced by polypeptide growth

factors that bind to cell surface receptors in the absence of new protein

synthesis. Therefore, the induction of these genes is a primary response to

events at the cell surface and not secondary to other waves of gene expression

or changes in growth state of the cell. The ability of early-response genes to be

3
induced in the absence of new protein synthesis distinguishes this set of genes

from other genes, which may be called late-response genes (those that are

expressed only if protein synthesis is allowed). Late-response genes are likely

to be targets of transcription factors induced in the early part of the response.

Subsequently, it was found that some of the IEGs are the cellular homologs

of various retroviral oncogenes, including those for c-myc, c-fos, and c-jun

(Cochran et al. 1984; Greenberg and Ziff 1984; Kelly et al, 1983; Lamph et al.

1988; Muller et al. 1984; Ryder and Nathans 1988). Studies using antisense

RNAs or oligonucleotides or antibodies have shown that the expression of c-fos

and proto-oncogenes is necessary for the entry of quiescent fibroblasts into the

cell cycle (Heikkila et al. 1987; Holt et al. 1988, 1988; Nishikura and Murray

1987; Wickstrom et al. 1988). Microinjection or overexpression of c-myc by

itself can potentiate the response of fibroblasts to other growth factors (Armelin

et al. 1984; Kaczmarek et al. 1985). However, the finding that some IEGs can

also be induced in cells such as neurons that have terminally differentiated

indicates that the IEGs may have roles to play in processes other than cell

growth regulation (Bravo et al. 1985; Curran and Morgan 1985; Greenberg

et al. 1985; Kruijer et al. 1985).

A wide variety of early-response genes have been identified from studies of

serum growth factor-stimulated fibroblasts (Almendral et al. 1988; Cochran et al.

1983; Lau and Nathans 1985, 1987; Lim et al. 1987). The total number of such

genes is unclear but is probably in the range of 50 to 100. It can be seen from

table 1, which lists some of these genes, that there are a wide variety of gene

products encoded by early-response genes. The two best characterized

classes of such genes are transcription factor genes and secreted gene

products. However, these are not the only type of gene products represented.

Others such as ß-actin and rho-B encode cytoskeletal and signal transduction

molecules, respectively (Greenberg and Ziff 1984; Jahner and Hunter 1991 a).

The best characterized of these genes are transcription factors, which can be

subdivided into several distinct categories. C-Fos and c-Jun are DNA-binding

proteins that form heterodimers via their leucine zipper domains (Bohmann et

al. 1987; Chiu et al. 1988; Landschulz et al. 1988; O’Shea et al. 1989; Rauscher

et al. 1988a, 1988b; Vinson et al. 1989). Each of these genes has several

related homologs (i.e., jun-B, jun-D, fra-1, and fos-B), which all can form

heterodimers with opposite members of the family (Hai and Curran 1991;

Halazonetis et al. 1988; Nakabeppu et al. 1988). In addition, c-jun can form a

homodimer. Thus, the number of dimeric complexes formed in the cells by this

family of gene products is larger than the number of genes that encode them.

Almost all these homodimeric and heterodimeric complexes can bind to the

activator protein-1 (AP-1) consensus site (TGACTCA), although the affinity of

a given complex for a given site can vary (Hai and Curran 1991; Kovary and

Bravo 1991; Ryseck and Bravo 1991). Different fos/jun complexes have

4
TABLE 1. A list of representative early-response genes*

Gene Possible Function References

C-myc Transcriptional modulator?

Helix loop helix

Kelly et al. 1983; Murre et al. 1989

JE Cytokine

KC, MGSA, gro Cytokine

Cochran et al. 1983; Rollins et al.

1988

C-fos Transcription factor

Leucine zipper

Anisowicz et al. 1987; Cochran et al.

1983; Richmond et al. 1988

Cochran et al. 1984; Greenberg and

Ziff 1984; Kruijer et al. 1984; Muller

et al. 1984

Fra-1 Transcription factor

Leucine zipper

Cohen and Curren 1988

Fos-B Transcription factor

Leucine zipper

Zerial et al. 1989

Zif/268, egr-1 , Transcription factor Christy et al. 1988; Lemaire et al.

TIS8, KROX24, Zinc finger 1988; Milbrandt 1987; Sukhatme

NGFIA et al. 1988

KROX20, egr-2 Transcription factor

Zinc finger

Chavrier et al. 1989

NGFIB, TlS1,

nur77

C-jun

Steroid receptor

Superfamily

Transcription factor

Leucine zipper

Hazel et al. 1988; Milbrandt 1988

Jun-B Transcription factor

Leucine zipper

Lamph et al. 1988; Ryder and

Nathans 1988

Ryder et al. 1988

ß-actin

SRF

2’-5’ oligoadenylate

synthetase

Cytoskeletal component

Transcription factor

Enzyme

Greenberg and Ziff 1984

Norman et al. 1988

Garcia-Blanco et al. 1989

Rho-B Ras-like G protein Jahner and Hunter 1991 a

*For a more comprehensive list, see Herschman (1991).

5
different effects on transcription, with some, such as c-jun, being more

positively acting and others, such as jun-B, tending to be more negatively

acting (Bohmann et al. 1987; Chiu et al. 1988, 1989; Nakabeppu and Nathans

1991; Schonthal et al. 1989; Schutte et al. 1989). In addition, many of these

complexes can be modified posttranslationally, especially by phosphorylation,

and these phosphorylations can affect the function of the complex (Barber and

Verma 1987; Boyle et al. 1991; Lamph et al. 1990; Ofir et al. 1990; Binetruy

et al. 1991). Thus, through combinatorial action, the fos/jun gene family can

potentially exert a broad range of effects on the transcriptional program of the

cell.

However, the fos/jun family are not the only transcription factors that are earlyresponse genes, Other early-response genes that appear to be transcription

factors are zif/268, which encodes a zinc finger protein (Christy et al. 1988);

nur77, which is related to the steroid receptor family of transcription factors

(Hazel et al. 1988; Milbrandt 1988); and the c-myc gene, which has both a

helix-loop-helix domain and a leucine zipper (Murre et al. 1989). All these

genes are likely to be involved in the regulation of late-response genes, which

then presumably alter physiology and function of the cell. Secreted proteins

that are IEGs are KC/gro, which appears to be a melanocyte growth factor

(Anisowicz et al. 1987; Richmond et al. 1988), and JE, which is a member of a

large family of monocyte chemotractant factors (Oquendo et al. 1989; Rollins et

al. 1989). The functions of these secreted factors are not specific to fibroblasts

because expression of JE and KC has been detected in monocytes (Orlofsky

and Stanley 1987).

In addition to studies on fibroblasts, investigators have cloned genes from

other cell types that are induced early in response to various growth and

differentiation stimuli. Typically, it has been found that many of the genes

induced in T cells by mitogens, in liver cells by partial hepatectomy, or by

nerve growth factor (NGF) in PC12 cells are the same ones that are induced

by PDGF in 3T3 cells (Greenberg et al. 1985; Kujubu et al. 1987; Thompson

et al. 1986; Zipfel et al. 1989). Genes that are commonly induced in many

different cell types by many different agents typically include c-fos, c-jun, and

the c-myc proto-oncogenes (Bartel et al. 1989; Greenberg et al. 1985; Kelly et

al. 1983; Mechta et al. 1989; Thompson et al. 1986). In addition, in each cell

type there appears to be a class of early-response genes that have a pattern

of expression restricted to a particular cell type. For instance, in activated T

cells, there is a rapid induction of interleukin-2 (IL-2) and its receptor, both of

which are gene products that are specifically expressed in T cells (Greene et

al. 1989; Taniguchi 1988). Similarly, in PC12 cells there are unique cytoskeletal

proteins that are induced early in the response to NGF (Leonard et al. 1988).

6
REGULATION OF EARLY-RESPONSE GENES

Although the typical early-response gene is induced rapidly and transiently,

the magnitude and the time course of induction can vary. For instance, c-fos

transcription can be detected within 5 minutes of stimulation, but expression is

typically shut off at the transcriptional level by 1 hour. The messenger RNA

for c-fos is unstable, and the persistance of the message is less than 2 hours

(Greenberg and Ziff 1984; Muller et al. 1984). This could be compared with the

JE gene in fibroblast, which is transcriptionally induced rapidly by 30 minutes,

but which is still being transcribed up to 6 hours postinduction (Cochran et

al. 1988). Absolute levels of induction of these genes can vary greatly; for

example, the c-myc gene is induced only to a level of 30 to 50 copies per cell,

whereas the c-fos gene is present transiently at 20 times that level (Cochran

et al. 1988; Kelly et al. 1983). For individuals of the same gene family such as

the fos-related genes, the time courses can also vary. The time course of the

fra genes appears to be slower and longer than those of fos; this may have a

physiological significance for the type of AP-1 complexes present in the cell at

a given time, since it has been shown that the subunits of AP-1 complex can

interchange rapidly in solution (Kovary and Bravo 1991; Ryseck and Bravo

1991; Sonnenberg et al. 1989a). The mechanisms responsible for the transient

expression of IEGs also may vary. One mechanism by which transient

expression is maintained is autorepression of a promoter by its own gene

product. For instance, c-fos and in some cases c-myc can autoregulate their

own synthesis (Penn et al. 1990; Sassone-Corsi et al. 1988a). Another

mechanism by which expression may be shut off may be simply the downregulation of the second signal stimuli, either at the level of the receptor or at

the level of second messenger.

Most IEGs are capable of responding to a variety of extracellular stimuli.

In fibroblasts these agents include not only growth factors such as PDGF,

fibroblast growth factor, and epidermal growth factor (EGF), but also agents

such as phorbol esters and bombesin that can induce IEG expression

through non-tyrosine-kinase-type receptors (Lim et al. 1987; Rabin et al.

1986). Calcium ionophores and agents that affect cyclic AMP (cAMP) levels

within the cell can induce expression of some IEGs (Almendral et al. 1988;

Morgan and Curran 1986; Ran et al. 1986; Sheng et al. 1988). In PC12 cells, it

has been shown that IEGs can be induced by neurotransmitters, by membrane

depolarization, and by various manipulations of extracellular ions and ion

channels (Curran and Morgan 1985; Greenberg et al. 1985; Kruijer et al. 1985;

Morgan and Curran 1986). Moreover, a variety of recent studies have shown

that activated oncogenes such as ras and src can induce many of these same

early-response genes (Jahner and Hunter 1991b; Stacey et al. 1987). This

observation is not surprising considering that many of the activated oncogenes

are components of signal transduction pathways.

7
Not all IEGs are coordinately regulated. Different stimuli can elicit different

patterns of expression of the IEGs in the same cell type. C-fos is induced

readily by phorbol esters in fibroblasts, but the JE and KC genes are not (Hall

and Stiles 1987). Conversely, in 3T3 fibroblasts, interleukin-1 can induce JE

and KC but have little effect on c-fos (Hall et al. 1989). Moreover, a given

stimulus can have dramatically different effects on different cell types. In PC12

cells, c-fos is induced by calcium ionophores as well as by EGF (Greenberg et

al. 1985; Kruijer et al. 1985). However, in Balb/c-3T3 cells, calcium ionophores

and EGF are very weak inducers of fos (Hall and Stiles 1987). In addition, it is

clear that different stimuli can induce a similar set of genes in a given cell and

yet have different effects. This can be dramatically visualized in studies of

PC12 cells, for which EGF is a mitogen and NGF can induce differentiation.

Both of these factors can induce c-fos and c-myc in these cells, but only NGF

produces neurite outgrowth (Greenberg et al. 1985). The difference between

these responses must result from differing second signals generated by these

two factors, even though some of the second signals are likely to overlap.

Differences in signals could include the activation of different kinases and

phosphotylation of different substrates as well as activation of genes uniquely

by one factor or the other. To understand the complex regulation of IEGs, it will

be necessary to understand both the second signals generated by any given

stimulus and the regulatory elements that control the expression of each of

these genes.

Second messenger systems to which early-response genes can respond are

numerous and varied. The fact that phorbol esters such as phorbol myristoy

acetate can induce c-fos and c-jun indicates that activation of protein kinase

C is one of the pathways by which IEGs are induced (Lamph et al. 1988; Rabin

et al. 1986). The observation that a calcium ionophore such as A23187 can

induce c-fos in some cells indicates that calcium can be a second messenger

for the induction of some early-response genes, In PC12 cells there is

evidence to suggest that the opening of a voltage-gated calcium channel can

induce c-fos (Morgan and Curran 1986). IEGs such as c-fos and jun-B can

respond to agents such as forskolin and isobutyl methyl xanthine, which

increase intracellular cAMP concentrations (Berkowitz et al. 1989; Fisch et al.

1989a; Sassone-Corsi et al. 1988b). In addition, it is clear that second signal

pathways activated by other kinases can induce early-response genes. This

is indicated by the fact that cotransfection or microinjection of genes such as

c-raf and activation of a temperature-sensitive c-src gene can induce a broad

spectrum of early-response genes (Jahner and Hunter 1991b; Jamal and Ziff

1990; Kaibuchi et al. 1989). Moreover, activated ras, a G protein, can induce

at least c-fos (Fukumoto et al. 1990; Gauthier et al. 1990; Stacey et al. 1987).

Thus, the ras-activated signaling pathway appears to be another mechanism

by which some early-response genes can be activated.

8
Physiological activation of early-response genes is likely mediated by several of

these second signal pathways operating simultaneously in a ceil. For instance,

the PDGF receptor is known to activate a variety of these pathways and directly

phosphorylate on tyrosine several proteins involved in signaling pathways

(Williams 1989). PDGF stimulates phosphatidyl inositide turnover, thereby

activating protein kinase C. The PDGF receptor can also bind to GAP, a

modulator of the ras pathway, and in addition can activate c-raf (Molloy et al.

1989; Morrison et al. 1989). Thus, a network of second signals leads to the

activation of the appropriate set of early-response genes in any given cell type

in response to a given stimulus. Most of those pathways have not been worked

out in great detail and are likely to be quite complicated. This is the case even

in the unicellular yeast Saccharomyces cerevisiae. Mating pheromones are

extracellular peptides that regulate gene expression in yeast. A variety of

second signaling molecules similar to those found in mammalian cells have

been implicated in the yeast signal transduction process; these include G

proteins and a variety of protein kinases, including one related to the CDC28

kinase (for a review, see Herskowitz 1989). However, even with the power of

yeast genetics, these pathways are not yet fully understood.

TRANSCRIPTIONAL REGULATORY ELEMENTS OF IEGs

Ultimately, a complete understanding of the induction of IEGs will have to

come from the study of transcriptional regulatory elements of these genes

and the factors that interact with them. Currently, the c-fos proto-oncogene is

the best characterized of the early-response genes at the level of transcriptional

regulation. Analysis of the upstream regulatory elements of the c-fos gene

reveals a complex array of regulatory elements that can respond to one or

more second messenger signals. A diagram of the c-fos promoter is shown in

figure 1. Upstream of the transcriptional start site is a TATA consensus box to

which the transcription factor TFIID should bind (Buratowski et al. 1988; van

Straaten et al. 1983). This element is characteristic of most RNA polymerase II

promoters. Upstream of the TATA box at -65 is a Ca/CRE box, which serves at

least three functions in the promoter. This element contributes to basal levels

of expression (Fisch et al. 1987; Gilman et al. 1986; Runkel et al. 1991), can

respond to increases in cAMP and in PC12 cells can mediate responses to

intracellular calcium (Berkowitz et al. 1989; Fisch et al. 1989a; SassoneCorsi et al. 1988b; Sheng et al. 1988). A variety of transcription factors could

bind to this site, but the best evidence would suggest that the CREB factor,

which was initially isolated as a cAMP response element-binding protein, can

likely mediate both the response to cAMP and calcium (Sassone-Corsi et al.

1988b; Sheng et al. 1990). Recent work has shown that this factor gets

phosphotylated by the CaM II kinase in PC12 cells that have undergone

depolarization (Dash et al. 1991; Sheng et al. 1991).

9
FIGURE 1. The structure of the c-fos promoter. See text for explanation of

each element.

Upstream of this element at approximately -100 are two direct repeat elements

that also have multiple functions. There is evidence that these elements are

involved in maintenance of basal expression of c-fos (Fisch et al. 1987; Gilman

et al. 1986; Runkel et al. 1991). In addition, sequences that contain these

elements have been shown to be responsive to a cotransfected retinoblastoma

gene. Overexpression of the retinoblastoma gene can repress transcription

of fos through this site (Robbins et al. 1990). Recently, it has been suggested

that the transcription factor E2F, which was originally characterized as a factor

that bound upstream of adenovirus early genes that are transactivated by the

adenovirus transforming gene E1A, can bind to the retinoblastoma responsive

element (Chellappan et al. 1991). This is of interest since it has been shown

that E2F can form a complex with the retinoblastoma gene product.

Furthermore, E2F has a serum-stimulated DNA-binding activity and is involved

in the regulation of other serum-inducible genes in fibroblasts, including c-myc

and dihydrofolate reductase (Blake and Azizkhan 1989; Mudryj et al. 1990).

However, although these two intriguing factors interact with this site, it has

not yet been shown that this element plays a crucial role in the response of cfos to extracellular stimuli such as growth factors, phorbol esters, and cAMP.

Moreover, it is quite clear that c-fos is readily inducible in cells that contain

a functional retinoblastoma gene product and that only overexpression of

retinoblastoma causes a repression of fos in a transient assay. Further

upstream, at -200 and -290, are two potential AP-1 sites. The one at -290

has been shown to be functional and can bind proteins other than the Fos/

Jun family complex (Fisch et al. 1989b; Velcich and Ziff 1990).

Centered around -310 of the human c-fos promoter is a region of dyad

symmetry that has been termed the “serum response element” (SRE). It

has been shown that this element is sufficient by itself to mediate induction

of a heterologous gene by serum and phorbol esters (Gilman et al. 1986;

Greenberg et al. 1987; Treisman 1985). The serum response factor (SRF)

is a 67-kd phosphoprotein that binds to the core of the dyad symmetry element

(Gilman et al. 1986; Norman et al. 1988; Treisman 1987). This core region has

been called a CArG box and is also found upstream of several other earlyresponse genes as well as in muscle-specific enhancers (Miwa and Kedes

10
1987; Phan-Dinh-Tuy et al. 1988; Rivera et al. 1990; Walsh 1989). In A431

cells the SRF has a mildly inducible DNA-binding activity (Prywes and Roeder

1986), but in general this factor appears to be constitutively bound to the

CArG box (Herrera et al. 1989; Treisman 1986). Although the SRF is a

phosphoprotein, to date no phosphorylation changes have been shown that

correlate with the induction of the c-fos gene. It is likely that the SRF is the

anchoring member of a complex of factors that bind to the SRE. One such

factor that has been characterized is p62TCF (for ternary complex factor), which

appears to bind to the SRE only in conjunction with the SRF and makes

contacts with the 5’ side of the SRE (Schroter et al. 1990; Shaw et al. 1989a).

Mutations in the 5’ flank of the dyad symmetry region (outside the CArG box)

reduce the ability of the SRE to confer responses to phorbol esters, but these

same mutant elements still retain the ability to respond to serum (Graham

and Gilman 1991; Shaw et al. 1989a). In addition, the SRE can mediate

autorepression of c-fos transcription (Konig et al. 1989; Sassone-Corsi et

al. 1988a; Shaw et al. 1989b). Cotransfection of a constitutively produced

c-fos gene with a reporter gene that is driven by the SRE greatly reduces the

expression of a linked reporter gene. Thus, the rapid burst of transcription of

c-fos can be explained by the fact that the activation of the c-fos promoter

stimulates production of the c-Fos protein, which is then able to repress its own

transcription, It is not yet known whether this repression is a direct or indirect

effect. The C terminus of the c-Fos protein is required for this repression and is

often mutated in the viral form of the gene (Ofir et al. 1990). Understanding the

transcription factor complexes that bind near and around the SRE is essential

for understanding the regulation of c-fos and several other early-response

genes (de Belle et al. 1991).

Upstream of the SRE at -346 is a sequence called the sis/PDGF-inducible

factor (SIF) element. A transcription factor with an inducible DNA-binding

activity specifically binds to this element after treatment of 3T3 cells with PDGF

(Hayes et al. 1987). Genomic footprinting indicates that in A431 cells treated

with EGF there is also an inducible DNA-binding activity that interacts with this

element (Herrera et al. 1989). The SIF element confers induction onto the c-fos

promoter in the absence of the SRE but responds specifically to PDGF and not

to serum or phorbol esters (Wagner et al. 1990).

Most of these regulatory elements are capable of showing some activity outside

of the context of the fos promoter. However, the context within which they lie is

important as well. For instance, the SIF element requires sequences between

-222 and -100 of the c-fos gene to respond to PDGF. Furthermore, moving the

SRE closer to the promoter weakens its response to PDGF and phorbol esters

but not its response to serum (Wagner et al. 1990). Moreover, there are cell

type-specific differences in the ability of these elements to respond to a given

11
stimulus. It has been shown that there are differences between the response

of the SRE to TPA in 3T3 cells and Hela cells (Siegfried and Ziff 1989).

In addition to these upstream transcription factors, there is evidence from at

least two of the IEGs that there is regulation of transcriptional termination. A

transcription termination site has been found at the end of the first exon of both

c-fos and c-myc (Bentley and Groudine 1986; Lamb et al. 1990; Nepveu et al.

1967; Nepveu and Marcu 1986). The mechanisms by which transcriptional

termination is regulated in mammalian cells is not clear, but recent studies of

the human immunodeficiency virus Tat protein indicate that there are promoterspecific mechanisms for regulating transcriptional termination of mammalian

genes (Marciniak et al. 1990). However, it is not known how these termination

sites are regulated by external cellular signals,

SUMMARY

From this brief overview of the regulation of the c-fos promoter, it can be seen

that the regulation of early-response genes is a complex affair. Therefore, it is

not easy to predict from the upstream sequence of a given early-response gene

exactly which elements are responsible for responding to what signals in a

given cell type. However, from studies of other early-response genes, it is

clear that several of the elements found upstream of c-fos appear frequently

and are important in the regulation of other early-response genes. For instance

the zif/268 gene has four separate CArG boxes that are similar to fos. These

CArG boxes can function in the serum and TPA response, but interestingly,

they are not imbedded in a region of dyad symmetry as in c-fos (Christy and

Nathans 1989). Upstream of the c-jun oncogene is an AP-1 site that can

modulate the expression and induction of this gene and is responsive to TPA

(Angel et al. 1988). Moreover, other studies suggest that cAMP-mediated

signals can repress induction of c-jun through this element (de Groot et al.

1991; Mechta et al. 1989). This would explain why in certain circumstances,

such as depolarization of PC12 cells or in the striatum in response to cocaine,

there is an uncoupling of the induction of c-jun and jun-B. Depolarization

induces c-fos and jun-B but not c-jun; however, growth factors such as NGF

can induce all three genes in the same cell (Bartel et al. 1989). Upstream of

the jun-B gene there does not appear to be an SRE, but there is a new element

that can be responsive to both cAMP and phorbol esters (de Groot et al. 1991).

Genes such as c-myc, JE, and KC have no consensus SREs upstream, and the

regulatory elements responsible for the induction of these genes have not been

clearly identified (Rollins et al. 1988). However, there is some evidence from

the c-myc gene that the E2F binding sites are important for its regulation by

serum (Mudryj et al. 1990; Sacca and Cochran 1990). In addition, there are two

SIF sites upstream of the c-myc proto-oncogene (B.H. Cochran and T.E. Hayes,

12
unpublished results). Upstream of the nur-77 gene there are no SREs, but

there are four potential calcium/CRE-like elements (Watson and Milbrandt

1989). Thus, although most of these early-response genes can be isolated

from a differential screening of cDNA library in response to a single inducer

such as PDGF, the regulation of each is unique. In addition, there is much

more to understand about how some of the early-response genes such as that

of IL-2 or the IL-2 receptor are expressed as both tissue-specific and IEGs. It is

likely that the tissue-specific expression of early-response genes is due to a

combination of chromatin accessibility of the gene and interaction with universal

regulatory elements as well as tissue-specific regulatory factors (Greene et al.

1989; Phan-Dinh-Tuy et al. 1988).

The finding that many early-response genes are induced in the nervous system

in response to neurotransmitters and other physiological stimuli may indicate

that these genes have an important role to play in the function of the nervous

system. (For review, see Sheng and Greenberg [1990] and other chapters in

this monograph.) The functions of the early-response genes are undoubtedly

as varied as the gene products. However, many of these genes do appear to

be transcriptional regulatory factors and therefore are likely to modulate the

expression of other genes. Work on identifying the targets of these transcription

factors is still in the early stages. Interestingly, there is already evidence that

two important neurotransmitter genes, the proenkephalin and the prodynorphin

genes, may be regulated by an AP-1 complex (Naranjo et al. 1991; Sonnenberg

et al. 1989b). Thus, one of the roles of early-response genes in the nervous

system may be to regulate the levels of neurotransmitters in a particular neuron.

One role that has been suggested for these genes is that they mediate between

short-term signaling and long-term changes in the nervous system (Morgan

and Curran 1989). This is especially intriguing, since many studies have

implicated cAMP in the formation of long-term memory through transcriptional

changes in the nervous system (Goelet et al. 1986), tiowever, as the induction

of IEGs is transient, it is not yet clear how these genes would mediate such

long-term changes. They could be regulating the expression of structural

genes that could contribute to the formation of synapses and other structural

changes within neurons. Alternatively, early-response genes could serve as

a reset mechanism that functions to replenish and restore supplies of

neurotransmitters that have been utilized. Another possible role for earlyresponse genes would be to modulate in the intermediate term the temporal

patterns of spike trains of given neurons. Since these genes are expressed

on a time scale of hours, they could contribute to short-term adaptive changes

in the activity of a given neuron. Obtaining the answers to questions like these

should be the focus of future research.

13
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ACKNOWLEDGMENTS

This work was supported by the Human Frontiers in Science Program and by

National Institutes of Health award PO1 CA-40263.

AUTHOR

Brent H. Cochran, Ph.D.

Associate Professor of Biology

Center for Cancer Research and Department of Biology

Massachusetts Institute of Technology

Building El 7-517

77 Massachusetts Avenue

Cambridge, MA 02139

24
Everything Activates C-fos—How Can

It Matter?

Steven E. Hyman, Barry E. Kosofsky, Tuong V. Nguyen, Bruce

M. Cohen, and Michael J. Comb

INTRODUCTION

This chapter addresses often-raised questions concerning the specificity and

biological significance of drug-induced immediate early gene (IEG) expression,

especially c-fos expression, in the nervous system. The definition of cellular

IEGs and basic information about their induction are described elsewhere in

this volume and are well reviewed in Morgan and Curran (1991) and Sheng

and Greenberg (1990).

lmmunohistochemical detection of c-Fos protein has become a widely used

tool to map activation of cells in the nervous system in response to many

drugs and other stimuli. The widespread use of this approach has been

facilitated by the availability of antisera recognizing c-Fos and, more particularly,

by the properties of the c-fos gene, mRNA, and protein. IEGs, including c-fos,

are activated rapidly by multiple intracellular second messenger systems and,

therefore, by multiple primary neural stimuli. Under basal (unstimulated)

conditions, many IEGs, including c-fos, have nearly undetectable levels of

mRNA and protein; this lack of background expression contributes to the

sensitivity of the technique, making inductions readily detectable by visual

inspection. Given its simplicity, sensitivity, cellular resolution, and utility with

respect to diverse stimuli, c-Fos immunohistochemistry has been applied to

many paradigms, including several relevant to the field of substance abuse

such as administration of opiates (Chang et al. 1988; Chang and Harlan 1990),

cocaine (Graybiel et al. 1990; Young et al. 1991; Hope et al., in press), and

amphetamine (Graybiel et al. 1990; Nguyen et al., in press). In addition,

induction of c-fos mRNA has been studied in opiate withdrawal (Hayward

et al. 1990).

Given the many stimuli that activate c-fos expression, questions have

often been raised about the biological specificity and significance of

immunohistochemically detected c-Fos. Such concerns have been worsened

25
on occasion by the implication made by some investigators that induction

of c-fos mRNA or protein indicates significant phenotypic alterations in the

fos-expressing neurons. Much of this latter concern could be alleviated if

researchers were conceptually clear as to whether they were using c-fos as

a marker of activation of second messenger pathways, whether they were

investigating its biological functions within neurons, or both.

Like all intracellular signaling molecules, the c-fos gene product may have a

significant biological effect depending on the state of many other molecules in

the cell at the time of its induction. Moreover, under circumstances of normal

stimulation, the functions of IEGs such as c-fos are likely to be involved more

often in homeostasis than in change (e.g., coupling new synthesis of peptides

to secretion) (Morgan and Curran 1991). However, even mechanisms that are

primarily homeostatic under certain circumstances may contribute to significant

functional plasticity in the nervous system under others. For example, one

important hypothesis in the drug abuse field is that the state of dependence

that occurs with repetitive administration of opiates or psychostimulants reflects

adaptive changes within target neurons that result in altered cellular and

synaptic function. An important candidate mechanism for the adaptive changes

underlying the cellular basis of dependence is that repetitive drug administration

drives intracellular signaling systems, including expression of IEGs, in such a

fashion that the overall pattern of gene expression within the target neuron is

altered in ways that significantly alter cellular function.

C-FOS EXPRESSION MAY BE ACTIVATED BY MULTIPLE PATHWAYS

C-fos has been shown to be induced by a variety of stimuli relevant to signal

transduction within the nervous system, including growth factors (Fisch et al.

1987; Rivera and Greenberg 1990), and multiple intracellular messenger

pathways such as those activated by depolarization (Sheng et al. 1988, 1990)

and calcium entry (Fisch et al. 1987; Sheng et al. 1988), the cyclic AMP

(cAMP)/protein kinase A pathway (Sassone-Corsi et al. 1988; Fisch et al.

1989), the protein kinase C pathway (Fisch et al. 1987; Gilman 1988), and

others. C-fos expression appears to be blocked by agents that facilitate the

hyperpolarization of neurons (Morgan et al. 1987). Since the stimulation of

many neurotransmitter receptor types within the nervous system leads to

activation of calcium-dependent protein kinases, protein kinase A, or one or

more isoforms of protein kinase C, it is not surprising that many neural stimuli

or drugs that affect excitatory or G protein-linked synaptic transmission also

activate c-fos. Moreover, the activation of more than one of these pathways

may produce synergistic activation of gene expression (Sheng et al. 1990).

26
If carefully interpreted, this convergence of multiple signaling pathways on

activation of c-fos may, in fact, prove to be advantageous in investigations of

neural circuitry in that the particular neurotransmitter receptor types mediating

the signal need not be known ahead of time. However, in analyzing the neural

circuitry and receptor types involved with IEG activation, both synergy and

cross-talk among second messenger pathways within cells must be taken into

account.

IF EVERYTHING ACTIVATES C-FOS, HOW CAN IT BE SIGNIFICANT?

IEGs and their protein products can be conceptualized as components of a

molecular cascade that transduces signals from cell surface receptors to the

nucleus. Thus, IEGs such as c-fos have been termed “third messengers” in

signal transduction cascades if neurotransmitters are designated as intercellular

“first messengers” and small molecules such as cAMP as intracellular “second

messengers” (Morgan and Curran 1991); alternatively, IEGs have been termed

“fourth messengers” if second messenger-dependent protein kinases are

designated as the intervening “third messengers” (Hyman and Nestler, in

press). In either case, characterizing the biological actions of IEGs within

particular neurons has many analogies with understanding the functions of

other molecules within signal transduction cascades, such as the second

messenger, cAMP, a ubiquitous molecule, the biological significance of

which is rarely questioned. Thus, for example, in considering the biological

specificity and function of cAMP within a cell, it is necessary to determine

the threshold for activating cAMP-dependent protein kinase, the available

substrates for this kinase within the cell, the basal phosphorylation status

of these substrates, the activity of specific phosphodiesterases and

phosphoprotein phosphatases, cross-talk with other second messenger

systems, and subcellular compartmentalization of cAMP and protein kinase

A. Thus, although many neurotransmitters activate cAMP, based on these

parameters its cellular effects are specific as well as biologically critical to

cellular function.

Similarly, investigations of the biological specificity and function of IEGs must

address such issues as what particular cells the IEG is induced in, which

potential target genes are transcriptionally active in those cells, what partners

exist within those cells for dimer formation with the protein product of the

IEG, what other proteins in the cell will compete for binding to DNA, and what

the effect is of the dimers formed on target gene expression. Experiments

that would address these issues include colocalization of IEGs coinduced

with putative target genes within identified neurons (Naranjo et al. 1991),

demonstration that the temporal appearance of the activator protein-1 (AP-1)

proteins of interest precede induction of the putative target gene in response to

27
the relevant stimulus (Sonnenberg et al. 1989a; Naranjo et al. 1991),

demonstration that the proteins of interest bind specifically to the target

gene with high affinity in vitro, and demonstration that the IEG activates

(or represses) the target gene following cotransfection (Sonnenberg et al.

1989a; Naranjo et al. 1991). Because of difficulties in culturing and transfecting

many primary cell types within the nervous system, evidence that a particular

IEG plays a role in the cell type of interest has been difficult to obtain to date.

Additional experiments that could strengthen the interpretation of a causal

interaction between IEG and target might include inhibition of expression of

specific IEGs using antisense oligonucleotides or inhibition of transcription

factor function by microinjection of specific antibodies into cells prior to

stimulation.

THE PROENKEPHALIN GENE IS A POTENTIAL TARGET FOR IEGMEDIATED ACTIVATION

The proenkephalin gene is widely investigated as a model of stimulus-induced

gene expression in brain and as a potential target gene for regulation by IEGs.

Hyman and colleagues (1988), Comb and colleagues (1988), and Sonnenberg

and colleagues (1989a) have demonstrated that the AP-1 transcription factor

(of which c-fos is a component; see Morgan and Curran 1991) binds the

proenkephalin promoter and that AP-1 proteins can positively and negatively

regulate proenkephalin gene expression (Sonnenberg et al. 1989a; Kobierski

et al. 1991).

The proenkephalin enhancer has been resolved into three closely spaced

DNA regulatory elements (figure 1) by detailed mutational analysis and DNAse I

footprinting with affinity purified protein (Hyman et al. 1988, 1989; Comb et al.

1988). Within the enhancer, the element most distal from the TATA box is

called ENKCRE-1. It contains the sequence TGGCGTA and binds NF-l-like

proteins (Chu et al. 1991). The element that is closest to the TATA box

contains the sequence CCGCCGGC and binds nuclear factor AP-2 (Comb

ENKCRE-1 ENKCRE-2 AP-2

FIGURE 1. The proenkephalin second messenger-inducible enhancer. The

sequence of the enhancer is shown; the sequences of the boxed

regulatory elements were determined by mutational analysis.

28
et al. 1988; Hyman et al. 1989). The middle element, ENKCRE-2, contains

the sequence TGCGTCA. Multimers of this sequence can confer cAMP and

Ca++ induction on a minimal proenkephalin promoter in a fashion similar to the

observed regulation of the endogenous proenkephalin gene in many cell types

(Nguyen et al. 1990; Kobierski et al. 1991). The ENKCRE-2 sequence is similar

to the consensus binding site for CREB (TGACGTCA) and for AP-1 proteins

(TGAC/GTCA). Although ENKCRE-2 contains the CGTCA motif that is required

for cAMP regulation in many genes (Hyman et al. 1988), it binds AP-1 proteins

with very high affinity (Hyman et al. 1988; Comb et al. 1988; Kobierski et al.

1991).

Stimulus paradigms in brain, in which both IEGs and the proenkephalin gene

are known to be regulated, include seizures (White and Gall 1987; White et

al. 1987; Gall 1988), antipsychotic drug administration (Sabol et al. 1983;

Tang et al. 1983; Sivam and Hong 1986; Romano et al. 1987), and precipitated

opiate withdrawal (Lightman and Young 1987). Seizures induced by electrical

or chemical means (e.g., pentylenetetrazol) produce dramatic effects on

expression of proenkephalin in the dentate gyrus of the hippocampus and in

entorhinal cortex. Seizures increase levels of enkephalin immunoreactivity

and decrease levels of dynorphin immunoreactivity in mouse hippocampus (Gall

1988). Electrical stimulation has also been shown to increase proenkephalin

mRNA levels and to decrease prodynorphin mRNA levels in rat hippocampus

(Morris et al. 1988). Because electrical stimulation and seizures also induce

c-fos and other IEGs (Morgan et al. 1987; White and Gall 1987; Dragunow

and Robertson 1987) with a time course that precedes proenkephalin mRNA

accumulation, the proenkephalin gene has been investigated as a model of

IEG-target gene interaction in seizure paradigms. Based on the temporal

relationship of induction, on the observation that AP-1 binding activity is induced

by seizures, and on activation of the proenkephalin promoter in undifferentiated

F9 cells by cotransfected c-fos and c-jun, Sonnenberg and colleagues (1989a)

concluded that proenkephalin is likely to be a target gene for c-Fos and c-Jun

induced in hippocampal neurons by seizures.

Although heterodimers of c-Fos and c-Jun may regulate proenkephalin in

F9 cells, the many proteins that can regulate gene expression via binding to

AP-1 sites (including Fos, Jun, and ATF family members) suggests that for

AP-1 -regulated genes such as proenkephalin, the relevant transcriptional

regulators may differ from cell type to cell type and, even within a given cell

type, may vary under different circumstances. It is also important to recall

that not all proteins that bind to AP-1 sites are IEGs. Some, such as jun-D,

are constitutively expressed, with little if any change in level of expression

following stimulation. This is relevant in the present context because it has

recently been shown that jun-D can positively regulate proenkephalin gene

29
expression in response to cAMP and Ca++ in a fashion that mimics physiologic

regulation in many cell types (Kobierski et al. 1991).

THE PROENKEPHALIN GENE CAN BE REGULATED AS AN IEG IN ITS

OWN RIGHT

The cAMP- and Ca++-inducible ENKCRE-2 site within the proenkephalin gene

is quite similar to the cAMP- and Ca++-inducible calcium-response element

that is involved with the rapid induction of c-fos in response to depolarization

(Sheng et al. 1988). Indeed, in transformed cell types, such as C6 glioma cells

and PC12 cells, the induction of proenkephalin gene expression by second

messengers is quite similar to the induction of c-fos: It is rapid, transient, and

independent of new protein synthesis. In fact, in such cell types the regulation

of proenkephalin gene expression in response to second messengers is

essentially indistinguishable from that of c-fos and other IEGs.

Activation of the proenkephalin promoter has been studied in C6 glioma cell

lines, which also express the endogenous rat proenkephalin gene (Yoshikawa

and Sabol 1986), by stably transfecting the plasmid pENKAT-12, which

contains human proenkephalin gene regulatory sequences (Comb et al. 1986).

In pENKAT-12, human proenkephalin gene sequences from nucleotides -193 to

+70 (with respect to the mRNA cap site), containing the proenkephalin second

messenger-inducible enhancer, are fused to the bacterial chloramphenicol

acetyltransferase (CAT) transcription unit. The best characterized of the C6

glioma stably transfected lines is the C6-D2 line (Nguyen et al. 1990). When

C6-D2 cells are treated with forskolin (10 μM) and the phosphodiesterase

inhibitor 3-isobutyl-1-methylxanthine (IMX) (0.5 mM), CAT mRNA correctly

initiated from the proenkephalin promoter is detectable by S1 analysis within

15 minutes (figure 2); mRNA levels peak by 90 to 120 minutes and then

rapidly decline. By 6 hours after addition of forskolin, levels of correctly initiated

mRNA have returned to basal levels, Moreover, the protein synthesis inhibitors

cycloheximide (100 μM) or anisomycin (10 μM) added to the cells just prior to

addition of forskolin and IMX have no effect on onset of mRNA appearance,

consistent with activation being caused by a rapid posttranslational modification

of preexisting proteins (figure 3). This pattern of activation is consistent with

second messenger-dependent posttranslational modification (phosphorylation)

of a preexisting transcription factor such as jun-D (Kobierski et al. 1991).

IEG-TARGET INTERACTIONS IN CHRONIC STIMULATION PARADIGMS

MUST TAKE DESENSITIZATION INTO ACCOUNT

Proenkephalin mRNA and peptides have also been shown to be induced by

antipsychotic drugs in rat striatum following 2 to 3 weeks of treatment (Sabol

30
FIGURE 2. S1 analysis showing the time course of accumulation of correctly

initated mRNA from pENkAT-12 in stably transfected C6 glioma

ceils (C6-D2). The protected band is 70 bp as expected for this

probe. The numbers represent time in hours after replacing the

growth media with low serum media (0.1 percent fetal bovine

serum) containing 10 μM forskolin and 0.5 mM IMX. Rapid

transcriptional changes are more easily seen with pENKAT- 12

than with the endogenous proenkephalin gene because the CAT

mRNA is relatively unstable; therefore, basal levels of mRNA are

low, leaving changes unmasked.

et al. 1983; Tang et al. 1983; Romano et al. 1987; Le Moine et al. 1950).

Because haloperidol has also been shown to induce expression of c-fos, zif/

268, and other IEGs in striatum (Dragunow et al. 1990; Miller 1990; Robertson

and Fibiger 1992; Nguyen et al., in press), a role for IEGs in the regulation of

proenkephalin gene expression is under investigation. However, in such

chronic stimulation paradigms an additional difficulty must be taken into

account: IEG expression is transient, and over a time course of minutes to

hours, the composition of AP-1 complexes is altered (Sonnenberg et al. 1989b).

Indeed, not only does c-fos expression rapidly return to basal levels, it also

becomes refractory to further activation (desensitization). The same is true of

cAMP-inducible proenkephalin gene expression in C6 glioma cells. As shown

in figure 2, forskolin and IMX-stimulated levels of pENKAT-12 mRNA decline to

undetectable levels by 6 hours after the onset of stimulation. To determine

whether expression had been desensitized, either the stable cAMP analog cpt31
FIGURE 3. Independence from new protein synthesis. S1 analysis showing

the time course of accumulation of correctly initiated mRNA from

pENKAT-in C6-D2 cells in response to forskolin (10 μM) and

IMX (0.5 mM). Anisomycin (10 μM) was added just before the

forskolin and IMX. Anisomycin causes superinduction of

proenkephalin mRNA levels, consistent with inhibition of synthesis

of a cAMP-inducible transcriptional repressor or inhibition of

synthesis of a labile mRNAse.

cAMP (200 μM) or the phorbol ester 12-0-tetradecanoyl-phorbol-14 acetate

(TPA) (50 nM) was added 90 minutes after the addition of forskolin and IMX,

by which time transcriptional activation appears to have peaked (figure 4).

Cpt-cAMP, which is a potent activator of proenkephalin gene expression (figure

4 inset), had no effect on proenkephalin expression when cells were pretreated

with forskolin and IMX for 90 minutes (i.e., the addition of cpt-cAMP did not

produce a new peak of mRNA synthesis or in any way prolong the time course

of mRNA accumulation). On the other hand, TPA, which activates protein

kinase C rather than the cAMP-dependent protein kinase, did produce a new

32
FIGURE 4. Desensitization of proenkephalin gene expression. S1 analysis in

which two parallel sets of plates of C6-D2 cells were treated and

assayed. In both sets of plates, forskolin (10 μM) and IMX (0.5

mM) were added at time 0. In the set of plates represented by

the first 8 lanes (times 0 to 6 hours from the left), cpt-cAMP (200

μM) was added 1.5 hours after forskolin and IMX. The addition

of cpt-cAMP has no effect on the normal time course of mRNA

accumulation (compare with figure 2); thus, by the 6-hour time

point (4.5 hours after addition of cpt-cAMP), no correctly initiated

mRNA is observed. In the set of plates represented by the last

3 lanes, TPA (50 nM) was added instead of cpt-cAMP 1.5 hours

after forskolin and IMX. The time points shown are for 3, 4, and

6 hours total (1.5, 2.5, and 4.5 hours after addition of TPA,

respectively). Compared with the corresponding cpt-cAMPtreated plates, there is a new burst of transcription with maximal

RNA accumulation 2.5 hours after the addition of TPA and

appreciable mRNA remaining after 6 hours total (4.5 hours after

the addition of TPA). The inset demonstrates the effectiveness of

cpt-cAMP (200 μM) on induction of pENKAT-12 in C6-D2 cells not

pretreated with forskolin or IMX after 1.5 and 2 hours.

33
burst of mRNA synthesis. These data argue that the signal transduction

pathway for cAMP-inducible proenkephalin gene expression has become

desensitized.

With these problems in mind, the authors have begun studying IEG-target

interactions in chronic drug treatment paradigms, which are critical to the field

of substance abuse research. Although studies of IEG-target interactions

in response to cocaine are in progress, we have begun with the study of

haloperidol because it is known to regulate the proenkephalin gene, whereas

other target genes are likely to be relevant to cocaine administration. In rats

given daily intraperitoneal injections of haloperidol, 2 mg/kg for 2 weeks, the

proenkephalin gene is induced twofold to threefold, as previously reported

(Sabol et al. 1983; Tang et al. 1983). However, at this time point c-fos mRNA is

undetectable, and jun-D mRNA has diminished 20 to 40 percent compared with

saline-treated animals (Nguyen et al., unpublished data). The phosphorylation

status of constitutively expressed AP-1 proteins in this paradigm are currently

being investigated; however, at the present time the mechanism by which

proenkephalin gene expression is activated in brain by a chronic drug stimulus

remains unknown.

SUMMARY

It is probably safe to say that the regulation of c-fos expression by drug

treatment and other stimulus paradigms has biological specificity and

mechanistic significance. However, as this brief essay makes clear, the

immunohistochemical detection of c-fos is only the tip of the biological iceberg.

IEGs deserve to be studied as critical components of the intracellular signaling

machinery that may transduce intercellular signals, including those produced

by drugs of abuse, into longer term changes in cellular function. However, the

study of IEG expression must be related to biologically significant target genes

and must be complemented by the study of phosphorylation of constitutively

expressed transcription factors. Moreover, as illustrated by the proenkephalin

gene, the regulation of genes that play important roles in differentiated cell

function may differ depending on which cell types and stimulus conditions

are investigated. Finally, attention must be paid to the complexities of gene

regulation under conditions of repetitive and chronic stimulation if phenomena

such as the neural substrates of drug dependence are to be understood.

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immediate early genes in the nervous system. Neuron 4:477-485, 1990.

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proenkephalin by Fos and Jun. Science 246:1622-1625, 1989a.

Tang, F.; Costa, E.; and Schwartz, J.P. Increase of proenkephalin mRNA and

enkephalin content of rat striatum after daily injection of haloperidol for 2 to 3

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37
AUTHORS

Steven E. Hyman, M.D.

Assistant Professor of Psychiatry

Director

Divisions on Addiction

Tuon Van Nguyen, M.D., Ph.D.

Research Fellow in Psychiatry

Molecular Neurobiology Laboratory

Massachusetts General Hospital

Department of Psychiatry

Harvard Medical School

Boston, MA 02115

Barry E. Kosofsky, M.D., Ph.D.

Instructor in Neurology

Michael J. Comb, Ph.D.

Assistant Professor of Neurology

Molecular Neurobiology Laboratory

Massachusetts General Hospital

Department of Neurology

Harvard Medical School

Boston, MA 02115

Bruce M. Cohen, M.D., Ph.D.

Associate Professor of Psychiatry

Department of Psychiatry

Harvard Medical School

Boston, MA 02115

Associate General Director

Mailman Laboratories

McLean Hospital

Belmont, MA 02138

38
Immediate Early Genes: Their

Involvement in Physiological and

Pathological Responses in the

Nervous System

Michael D. Hayward, Tom Corran, and James I. Morgan

INTRODUCTION

The Cellular Immediate Early Gene Response

The application of an extracellular stimulus to a cell has two types of general

consequences: a rapid, usually short-lived, response that is protein synthesis

independent and a more persistent action that frequently requires gene

transcription. The latter finding led to a search for the signaling molecules that

couple a stimulus to the transcriptional machinery of the cell. A quantum leap

in understanding this problem came with the discovery that several protooncogenes, including c-fos, c-jun, and c-myc, are rapidly (within minutes)

induced by a diverse array of extracellular stimuli. Since these genes encode

nuclear proteins, it was supposed that they might be involved in stimulusdependent regulation of transcription (Curran and Morgan 1987; Lau and

Nathans 1987). Subsequent studies have confirmed and expanded this basic

notion

Cellular lmmediate Early Genes and Learning

For the neurobiologist, alterations in gene expression have long been

supposed to underlie several key aspects of neurophysiology and, indeed,

neuropathology. For example, there are many studies dating back to the early

1960s indicating that protein synthesis is essential for memory acquisition in

both vertebrates and invertebrates (for reviews, see Barondes 1965, 1970,

1975; Agranoff 1972; Goelet et al. 1986). Indeed, based on the prevailing

literature of the time, Barondes (1975) suggested that memory acquisition was

composed of two processes. The first process began immediately upon training

(i.e., it had no measurable lag), lasted for hours, and involved posttranslational

39
modification of existing substrates. However, he went on to state that, “Process

II begins within minutes after training and augments for hours or longer;

Process II is mediated by the synthesis of proteins.” Although the properties of

Process II fit those of cellular immediate early (CIE) genes, it required another

decade until this notion could be extended by the specific suggestion that genes

such as c-fos might mediate this process (Goelet et al. 1986). Several studies

have been aimed at testing this hypothesis in mammalian models of learning,

notably long-term potentiation (LTP) (Douglas et al. 1988; Cole et al. 1989;

Wisden et al. 1990). Although it was thought initially that c-fos expression did

correlate with the induction of LTP, subsequently conditions could be defined

whereby LTP could be established in the absence of an induction of c-fos (e.g.,

Cole et al. 1989). However, another CIE gene, egr-1 (also known as NGFI-A,

zif/268, and krox-24), was consistently induced in the hippocampus by the

stimulus that elicits LTP (Cole et al. 1989; Wisden et al. 1990). Since many CIE

genes have been identified, it may be no simple matter to determine which, if

any, are critical in a process such as LTP. Indeed, until strategies become

available in vertebrates that permit specific interference with CIE gene

responses, the absolute involvement of particular genes of this class in

neurophysiological responses must remain an open issue.

CIE Genes and Kindling

Although it has drawn the most attention, learning is by no means the only

example in the nervous system of a process that requires a window of protein

synthesis concomitant with a stimulus for the establishment of a long-lasting

effect. For example, kindling is a widely used model of human complex partial

epilepsy that appears to require protein synthesis for its establishment. The

kindled state is induced by repeated administration of subconvulsant electrical

stimuli to various regions of the central nervous system (CNS). Treatment

with protein synthesis inhibitors during periods of stimulation either blocks or

attenuates the development of kindling (Cain et al. 1980; Jonec and Wasterlain

1979). Unlike the genesis of LTP, the induction of CIE genes has a close

temporal and spatial correlation with the kindling stimulus and the establishment

of the kindled state (Dragunow and Robertson 1987, 1988; Shin et al. 1990).

Indeed, both kindling and related seizure paradigms have suggested potential

target genes and biological consequences for CIE gene induction (reviewed in

Morgan and Curran 1991a, 1991b).

Kindling is known to bring about alterations in the levels of several

neuromodulatory peptides, neurotransmitter receptors, and neurotrophic

factors. These alterations and their relationship to CIE gene products have

provided several clues to CIE gene function. First, the overall transcriptional

40
changes in hippocampus elicited by seizures are consistent with the activation

of mechanisms that would tend to counteract the transduction of excitatory

signals (reviewed in McNamara et al. 1987) (i.e., inhibitory neurotransmission

is augmented and excitatory neurotransmission is depressed). For example,

there appears to be an up-regulation of aminobutyric acid (GABA) receptors

and a down-regulation of muscarinic receptors. There is not yet any direct

evidence to suggest that CIE genes mediate the transcription of these particular

genes, although appropriate increases in specific CIE genes have been

observed in the neuronal populations in which these changes occur. However,

in a second situation there are biochemical data to support the involvement of

CIE gene products in the regulation of neuropeptide gene expression in

hippocampus. It is known that proenkephalin transcription is increased in

the granule cells of the dentate gyrus following seizures (for reviews, see

McNamara et al. 1987; Morgan and Curran 1991a, 1991b). The timing of

this induction occurs just after Fos and Jun increase in these same cells

(Sonnenberg et al. 1989a, 1989b). Furthermore, the proenkephalin promoter

can be cooperatively transactivated in transient transfection assays by Fos

and Jun (Sonnenberg et al. 1989b). These data have been interpreted in

terms of a homeostatic response, whereby recruitment of the CIE genes

replenishes enkephalin stores that would be depleted by the high levels of

firing encountered during seizures (i.e., stimulus-transcription coupling is an

integral component of stimulus-secretion coupling).

CIE Genes and Neurotrophic Factors

A third rapid consequence of seizure is the induction, or increased transcription,

of neurotrophic factors such as nerve growth factor (NGF) and brain-derived

neurotrophic factor (BDNF). This again appears to occur in the same cells in

which there is an elevation of CIE gene expression. In addition to the relatively

rapid responses cited above, seizures and excitatory stimuli also elicit axonal

sprouting in the CNS (Sutula et al. 1988). This has led to the idea that there is

a general trophic response to stimulation in brain that has, as one of its early

components, the induction of CIE genes and growth factors (reviewed in

Morgan and Curran 1991b). In this model, neurotrophic factors such as BDNF

may be either CIE genes (as may be the case for NGF) or targets for CIE gene

products (Gall and lsackson 1989; Hengerer et al. 1990). It is supposed that

these genes underlie the physical growth response in the nervous system

following seizure (Sutula et al. 1988; Gall and lsackson 1989; Morgan and

Curran 1991b). In a wider sense, such an activity-driven growth response in the

nervous system might be viewed as a general process that provides for longterm neuronal plasticity in both normal and neuropathological situations.

41
FOS, JUN, AND AP-1 IN THE NERVOUS SYSTEM

The CIE genes c-fos and c-jun encode nuclear phosphoproteins, Fos and Jun,

respectively, that are components of the transcription factor activator protein-i

(AP-1) (reviewed in Curran and Franza 1988). Fos and Jun dimerize via a

leucine zipper-motif, an a-helical domain within both proteins that contains a

heptad repeat of leucine residues (Landschultz et al. 1988). The dimeric

complexes bind cooperatively to the AP-1 DNA consensus sequence

(TGACTCA) via domains rich in basic amino acids. Fos and Jun both contain

such a domain; therefore, each protein in the complex provides a half site for

DNA binding. The AP-1 sequence is known to be essential for both basal and

stimulated expression of some genes, and AP-1 binding at this element can

modulate transcriptional activity (reviewed in Curran and Franza 1988; Morgan

and Curran 1991a).

Several genes have been described that are related closely to either c-fos or

c-jun (e.g., fra-1, fra-2, fos-B, jun-B, jun-D). The protein products can also

participate in AP-1 dimeric complexes (reviewed in Kerppola and Curran 1991).

In general, all Jun family members can dimerize with all Fos family members,

In addition, all Jun (but not Fos) family members can form heterodimers and

homodimers with themselves, although the apparent affinity of these complexes

for the AP-1 site is lower than that of Fos family-Jun family heterodimers. The

situation has become more complicated with the discovery that the leucinezipper and basic DNA-binding motifs are present in several other families of

DNA-binding proteins. This so-called basic-zipper family includes such proteins

as the cyclic AMP response element binding proteins (CREBs) and the

activating transcription factors (ATFs). These proteins interact with DNA

sequences other than the AP-1 consensus, such as the cyclic AMP response

element (CRE) (TGACGTCA) (Hai and Curran 1991; Kerppola and Curran

1991). Furthermore, CREBs and ATFs form dimers with themselves as well as

with selective members of the Fos and Jun families (Hai and Curran 1991).

Thus, in some cases members of the Fos and Jun families may be components

of an AP-1 complex, whereas in other circumstances they may be part of a

complex interacting with a CRE. Obviously, this situation provides for a

considerable degree of mixing and matching and may be one way that diversity

is generated within the CIE response.

A second feature of the CIE gene response is that it has a temporal component.

In the rodent nervous system, pentylenetetrazole (PTZ) seizures elicit a rapid

and transient increase in the mRNAs encoding c-fos and c-jun (Morgan et al.

1987; Sonnenberg et al. 1989a, 1989b); fos and jun mRNAs reach maximum

values within 30 to 60 minutes following seizure and subsequently decline to

basal values by 2 to 3 hours. As anticipated, Fos accumulates in the brain for

42
1 to 2 hours and then disappears by about 4 hours postseizure. Although AP-1-

like DNA-binding activity rises during the first hour after seizure, as would be

expected, the levels remain elevated for at least 8 hours, despite the absence

of Fos. This additional AP-1 activity is contributed by a series of inducible Fosrelated proteins that appear with a delay following seizure and disappear with a

slower kinetic (Sonnenberg et al. 1989a). This means that AP-1 complexes

have a dynamic composition over time and suggests that the CIE gene

response is part of a temporally regulated program that may span many hours

following even a brief stimulus. The precise functional significance of the

different AP-1 complexes is not known at present; however, it may provide

a way of targeting the expression of different sets of genes over time or

sequentially inducing and repressing a set of genes (reviewed in Morgan

and Curran 1989).

FOS MAPPING STUDIES OF DRUGS OF ABUSE

These considerations have resulted in the widespread use of Fos

immunohistochemistry as a mapping technique to identify the pathways and

cells involved in particular responses in vivo (reviewed in Morgan and Curran

1991 a). It has even been proposed that Fos immunohistochemistry provides a

form of activity map analogous to that obtained with 2-deoxyglucose (Sagar et

al. 1988; Morgan and Curran 1991a). Of particular relevance has been the

application of Fos mapping to the study of drugs of abuse. Here, the general

concept has been that drug dependence, a long-term phenomenon, is elicited

by repetitive administration of a stimulus (the drug) and in some senses is

analogous to learning (i.e., do drugs of abuse induce a CIE response and could

CIE gene products be involved in the establishment of the dependent state?).

Morphine Regulation of C-fos

Acute administration of morphine induces Fos-like immunoreactivity (FLI) in

the caudate putamen of rat (Chang et al. 1988), a region rich in μ-type opiate

receptors. In addition, FLI can be detected in the ventromedial nucleus of

the hypothalamus (Chang and Harlan 1990); in contrast to the caudate, the

hypothalamus contains predominantly -type opiate receptors and few μ-type

receptors (Mansour et al. 1987). Thus, both classes of opiate receptors may

be capable of transducing signals that can induce c-fos. Another morphinesensitive brain structure, the locus coeruleus (LC), does not respond to acute

administration of morphine with an induction of c-fos. This is not surprising

since morphine depresses the firing rate of neurons in LC (Aghajanian 1978).

However, during chronic morphine treatment, firing rates in LC recover to

normal levels; whereafter, abrupt cessation of morphine or administration of

naloxone precipitates a withdrawal, and firing rates increase severalfold above

43
those seen in control and chronically treated animals (Aghajanian 1978; Christie

et al. 1987). The withdrawal results in an induction of fos and jun in LC and

several other regions of the CNS (Hayward et al. 1990). The increased firing

of neurons within LC during withdrawal is compatible with the notion that c-fos

is an activity-driven gene. However, 2-deoxyglucose uptake does not change

in other brain regions where fos is also induced by opiate withdrawal, showing

that there is not an absolute coincidence between activity, as measured by

2-deoxyglucose, and fos induction (Wooten et al. 1982; Kimes and London

1989; reviewed in Morgan and Curran 1991a). What role CIE genes play

during withdrawal is not known at present. However, several possibilities

are suggested. For example, the tyrosine hydroxylase (TH) gene can be

regulated in some systems by Fos and Jun (Gizang-Ginsberg and Ziff 1990).

Furthermore, the regulation of TH levels in a few highly specific regions of the

CNS has been implicated in the physical effects of morphine (and cocaine)

(Guitart et al. 1990; Beitner-Johnson and Nestler 1991). Indeed, morphine

induces changes in TH in several brain regions, including LC (Hayward et al.

1990). Thus, AP-1 complexes may provide a mechanistic link that couples

morphine to alterations in TH levels and thereby catecholamine biosynthesis

Cocaine and Amphetamine Regulatlon of C-fos

The psychomotor stimulants, methamphetamine and cocaine, can act indirectly

as dopamine agonists to elevate prodynorphin-derived peptides in the striatum

(Hanson et al. 1987; Sivam 1989). Since it had been suggested that Fos and

Jun act in the regulation of expression of several neuropeptide genes, including

preprodynorphin and preproenkephalin (White and Gall 1987; Draisci and

ladarola 1989; Sonnenberg et al. 1989b), several studies have undertaken the

analysis of immediate early (IE) gene expression following treatment with

amphetamines and cocaine (e.g., Graybiel et al. 1990; Young et al. 1991).

Acute cocaine induces FLI and c-fos mRNA in striatum, olfactory tubercle, the

islands of Calleja, and nucleus accumbens. Since this induction was blocked

by SCH 23390, it is inferred that cocaine is acting via D1-dopamine receptors

(Young et al. 1991). Likewise, amphetamine, as well as two D1-dopamine

receptor agonists, SKF 38393 and CY 208-243, induce fos in the striatum via

the D1-dopamine receptor (Robertson et al. 1989; Graybiel et al. 1990). D2-

dopamine receptor agonists such as LY 171555 do not induce fos in striatum

(Robertson et al. 1989), although this agent, like D1-dopamine agonists, can

elicit rotational behavior in rats bearing unilateral lesions in the substantia nigra.

That is, activation of D1-dopamine, but not D2-dopamine, receptors results in

fos induction in striatum. Furthermore, rotational behavior can be separated

from an induction of c-fos. Surprisingly, haloperidol, a neuroleptic with

dopamine antagonist properties, also induces c-fos in striatum (Miller 1990).

Since the action of haloperidol is blocked by the D2-dopamine receptor agonist

44
LY 171555, it is inferred that inhibition of D2-dopamine receptors either in

striatum or in neurons that project to it elicit fos induction, perhaps by a

disinhibition of striatal neurons,

Although both amphetamine and cocaine induced fos in striatum, they did so

with different anatomical distributions (Graybiel et al. 1990). Furthermore, there

were pharmacological distinctions in the mechanisms linking the two agents to

fos expression, suggesting that they acted via the same transmitter system but

in different cell populations. For example, in the caudatoputamen, acute and

chronic reserpine pretreatment suppressed subsequent c-fos induction by

cocaine but not amphetamine, suggesting that the two drugs utilize different

stores of monoamines (Graybiel et al. 1990).

Fos-IacZ Transgenic Mice

To advance the use of fos mapping, a line of transgenic mice were bred that

carry a fos-IacZ transgene (Smeyne and colleagues 1992). There were several

reasons for doing this. First, the technique would be less time consuming

and costly than immunohistochemistry and in situ hybridization. Second, it

would avoid the ambiguity of cross-reaction of antisera with Fos-related

proteins. Third, it would serve to define the regulatory elements required for cfos expression in vivo. Fourth, it would provide the first step in devising an

inducible gene system for use in mice. Fifth, by mutational analysis of the fos

promoter in the context of a fusion gene, it may be possible to derive insights

into the second messengers and transcription factors critical in specific neurons

following a particular challenge.

A fusion gene was constructed that comprised bacterial IacZ (ß-galactosidase)

fused in-frame into exon 4 of c-fos that was tested by stable transfection into

B104 neuroblastoma cells (Schilling et al. 1991). The gene contained all the

known 5’-upstream regulatory sequences of c-fos as well as all introns and

exons (which also contain intragenic regulatory sequences) and several

kilobases of 3’ flanking genomic DNA. Thus, the fusion gene also includes the

polyadenylation consensus sequence from c-fos and the Shaw-Kamen element

believed to confer the rapid degradation of c-fos mRNA (Schilling et al. 1991).

Furthermore, since the fusion is in the fourth exon of c-fos, the fusion protein

retains the leucine-zipper motif and other sequences that direct the protein to

the nucleus. It was expected that inducible nuclear ß-galactosidase activity

would be observed from the construct. This was deemed important since it

would greatly improve the sensitivity and resolution of the technique when

applied in animo. In a stably transfected B104 neuroblastoma cell line,

inducible nuclear (ß-galactosidase activity could be demonstrated following

treatment with serum, phorbol ester, and dibutyryl cyclic AMP, as well as

45
adrenergic agonists (Schilling et al. 1991). Thus, activation of both

the protein kinase A and protein kinase C pathways are efficiently coupled to

expression of the fusion gene. The induction of c-fos and the fusion gene was

both rapid and transient, but the fusion protein had a slightly greater half-life

than endogenous Fos (Schilling et al. 1991). Therefore, it was concluded that

this fusion gene, when tested in culture, behaved like endogenous fos and was

appropriate for use in transgenic mice.

Several lines of transgenic mice bearing the fos-IacZ transgene have been

derived (Smeyne et al. 1992). Three areas of research have been performed

to date and these are summarized here.

Constitutive Sites of Fos-LacZ Expression

In unstimulated mice, several tissues express the transgene constitutively.

Such tissues include the skin, root sheath cells of the hair follicle, ossifying

chondrocytes, and cells that give rise to the nail. This suggested that

constitutive expression of the transgene (and c-fos) is associated with cells

that are in terminal differentiation and are destined to die. Subsequently,

this notion has been extended by the observation of constitutive transgene

expression in other cells that exhibit programed cell death in vivo. What the

relationship is between expression of c-fos and cell death is unknown at the

present.

Expression of Fos-LacZ In the Developing Nervous System

During postnatal development, constitutive expression of fos-LacZ is readily

detected in the nuclei of neurons in several regions of the CNS. Notable

examples include, but are not limited to, mitral cells of the developing olfactory

bulb, CA1 pyramidal neurons of the hippocampus, neurons in the cingulate

cortex, and neurons within the parafascicular nucleus of the thalamus. Many

of these sites are undergoing active synaptogenesis at the times that this

expression is observed. However, it is unknown whether Fos is involved in,

or responsive to, the synaptogenesis or plasticity of these neurons during this

period.

Induced Expression of Fos-LacZ in the Nervous System

There are several sites of expression of the transgene in unstimulated mice

during the daytime, such as the raphe and reticular formation (Smeyne et al.

1992). It has been suggested that these sites of expression might be related to

the state of arousal/sleep (Smeyne et al. 1992). However, following several

forms of neural stimulation there ensues a rapid and transient induction of the

46
transgene that can be detected as nuclear ß-galactosidase activity. For

example, both PTZ and kainic acid seizures evoke a widespread, transient

induction of the transgene in overlapping, but not identical, populations of

neurons. These differences in distribution were not so apparent when studied

by Fos immunohistochemistry since the antibodies react with related proteins

that are induced to high levels over protracted periods following seizure

(Morgan et al. 1987; Sonnenberg et al. 1989a, 1989c). The transgene product,

like Fos, has a relatively short half-life, and so a precise map of Fos expression

is obtained. This has also pointed to the surprising fact that, in some neurons,

Fos-like proteins must be induced with little or no Fos expression. That

differences in patterns of Fos induction exist between related stimuli should

come as no surprise; in fact, it would be surprising if such differences did not

exist. It has only been the lack of specificity of the reagents that has heretofore

obscured these differences.

Recently, the analysis of gene induction in the fos-IacZ transgenic mouse has

been extended to examine other forms of stimulation, Light exposure during

presumptive night will induce the transgene in the suprachiasmatic nucleus

of the hypothalamus, as it does cognate c-fos. Thus, the transgene is capable

of responding to a physiological stimulus. The fos-lacZ transgene is also

responsive to drugs of abuse. In preliminary experiments both cocaine and

amphetamines have induced the gene in caudatoputamen; however, the

latter agent is the more efficient of the two. In addition, amphetamine, but not

cocaine, elicits a considerable induction of the gene in other regions of the CNS

that are presently being investigated. Therefore, future studies will be aimed at

establishing whether the transgenic mice can be useful in the assessment of

dependence and withdrawal in chronic models of drug abuse.

PERSPECTIVE ON THE APPLICATION OF IE GENES IN THE STUDY OF

DRUGS OF ABUSE

A combination of Fos immunohistochemistry, in situ hybridization, and now

histochemistry for ß-galactosidase in transgenic mice provides a novel and

precise method for assessing certain aspects of the physiology of drugs of

abuse. These include identifying sites within the CNS that show an induction

of the IE response as a result of acute drug administration as well as sites that

exhibit a response upon withdrawal. Since Fos mapping does not necessarily

equate with activity mapping as determined by 2-deoxyglucose, this may be

important in directing attention to regions of the nervous system that were

previously unsuspected of playing any role in drug responses and dependence.

The use of conventional neuropharmacologicai reagents in these systems can

resolve some aspects of the synaptology of the IE response to drugs. Thus, it

may provide a means to understand some of the signaling molecules and

47
pathways involved in drug responses. In addition, it points to potential target

genes that might underlie dependence, such as neuropeptide genes (e.g.,

proenkephalin and prodynorphin), neurotransmitter enzymes (e.g., TH),

neurotrophic factors and their receptors (e.g., NGF, BDNF, and trkB, as well

as genes encoding proteins involved in modifying the extracellular matrix

(e.g., collagenase and stromelysin). At the structural level these genes may

contribute to a general synaptic growth response that modifies the behavior

of a neural circuit over the long term (i.e., the CIE genes might elicit a chain of

transcriptional events that culminate in the sprouting of axons with concomitant

changes in synaptic density, size, and efficiency).

Several criticisms have been leveled at the significance of CIE genes. First,

many researchers find it difficult to see any specificity in the system since it is

apparently induced quite ubiquitously in most cell types by a diverse array of

agents (reviewed in Morgan and Curran 1991a). Thus, it might be argued that

this is merely a cellular stress mechanism, analogous to the induction of heat

shock proteins, designed to protect neurons when they are at risk for neurotoxic

damage. This argument is becoming less valid. First, these genes are induced

by normal physiological stimuli, not just the pharmacological and traumatic

stimuli used in early experiments. Second, many new CIE genes have been

discovered, some of which show restricted sites of expression and are

preferentially induced by certain agents in a particular cell type. Third, the

complex, differential, posttranslational modification of CIE gene products,

combined with their staggered induction patterns and multiple dimerization

properties, provides a startling array of diversity. That is, it is likely that diversity

is generated at one level by a combination of a relatively few restrictively

induced CIE gene products interacting with many more ubiquitously induced

members. In addition, specificity is determined by various protein-protein

interactions between induced and constitutive proteins as well as their

subsequent posttranslational modification. The presence or absence of

particular kinases, resident transcription factors, as well as specific signaling

pathways will dictate this latter aspect of the response.

Second, although the scenario outlined above nicely accounts for several

aspects of neurophysiology and neuropathology, it is unproven except for

correlations. Herein lies a fundamental difficulty in pursuing the function

of CIE genes in higher vertebrates, namely, the ability to specifically eliminate

them or their action in vivo. Since many of the CIE genes encode proteins

that are expressed naturally during development, it is likely that if they

were to be eliminated by homologous recombination the animals would die

in utero. Therefore, more subtle ways must be designed to impair their

function or expression. At one level this could be viewed as a novel branch

of pharmacology (i.e., designing agents that specifically impair the CIE

48
response while leaving short-term responses to a stimulus intact). Such

agents might target one of the later elements in the signal transduction

cascade between the stimulus and the CIE gene promoter. For instance, it

has been shown that certain types of benzodiazepines can greatly augment

the IE response, whereas calcium channel blockers and calmodulin antagonists

suppress it (Curran and Morgan 1985; Morgan and Curran 1986; reviewed in

Morgan and Curran 1991a, 1991b). Other agents might target the CIE gene

product by, for example, impeding their binding to DNA or a partner protein or

modifying posttranslational events (Curran and Morgan 1986). The use of

transdominant suppressor molecules in transgenic mice might provide such

an avenue. Until such issues are clarified, the precise role that the CIE genes

fulfill and the utility of regulating their expression and function in vivo will remain

an open question.

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52
AUTHORS

Michael D. Hayward, B.A.

Roche-Columbia University Graduate Program

Tom Curran, Ph.D.

Chairman

Department of Molecular Oncology and Virology

James I. Morgan, Ph.D.

Laboratory Head

Department of Neuroscience

Roche Institute of Molecular Biology

Roche Research Center

Nutley, NJ 07110

53
Immediate Early Gene Activation

and Long-Term-Changes in Neural

Function: A Possible Role in

Addiction?

Harold A. Robertson

INTRODUCTION

Psychostimulants such as cocaine and amphetamine have a range of

pharmacological effects on the central nervous system (CNS). The most

important of these effects, those related to addictive behavior, appear to

depend on dopaminergic mechanisms (Wise 1984). Thus, a receptor for

cocaine has been described (Ritz et al. 1987) and sequenced (Shimada et al.

1991; Kilty et al. 1991) that appears to be a site on the dopamine transporter

on dopamine nerve terminals. Furthermore, there is evidence from animal

studies to suggest that dopamine function in the nucleus accumbens plays an

important part in intracranial self-stimulation and self-administration of cocaine

(Roberts and Koob 1982). Finally, there is evidence that the dopaminergic

mechanisms in the nucleus accumbens may be a final common pathway for

many, if not all, substances that produce addictive behavior (Di Chiara and

lmperato 1988). Therefore, it seems clear that dopaminergic mechanisms play

a central role in the addictive properties of cocaine and other psychostimulant

drugs. These compounds have their effects on dopamine either by increasing

its release into the synapse (amphetamine) or by blocking the reuptake of

dopamine from the synapse (cocaine), the net effect in either case being an

increase in dopamine levels in the synapse. It should be noted that this

explanation for the actions of cocaine and amphetamine is almost certainly

incomplete. Other agents (e.g., tricyclic antidepressants, tyramine) share

these actions and do not produce addiction. The obvious question is, What

does the dopamine accumulation following cocaine and/or amphetamine do

that produces addiction? It seems reasonable to assume that dopamine

produces some sort of change in neural systems that leads to a long-term

change in function. It is known that the changes that occur are robust and

persistent. In animals, the sensitization and reverse tolerance produced by

54
psychostimulant drugs last many weeks and perhaps months. In humans, the

effects of psychostimulant use are still evident after years of abstinence (Koob

and Bloom 1988).

The mechanism underlying long-term changes in the brain is unknown. The

phenomenon of long-term potentiation is interesting but, in general, only lasts

for a few weeks at the most and decays over that period. By contrast, the

phenomenon of kindling in animals is generally regarded as representing a

permanent change in neuronal function. However, the biological basis of

kindling is largely unknown, although there are suggestions that it could

involve activation (or inhibition) of gene expression in parts of the limbic

system, including the hippocampus. Kindling is a process whereby repeated

subthreshold stimulation (electrical or chemical) will lead to a gradual increase

in response. Thus, repeated subconvulsant doses of a chemical convulsant

agent will also induce kindling (for reviews on kindling, see Goddard 1983

and Cain 1989). In the case of kindled seizures, this process culminates

in a major motor seizure. Most importantly, once an animal is kindled, it

apparently remains in the kindled state for the remainder of its life (about

33 months is the longest known duration). Moreover, the kindled state, unlike

long-term potentiation, appears to depreciate little with time (for a review, see

Cain 1989).

Kindling can also be induced chemically by agents such as cocaine. The

kindling-like effects of cocaine have been known longer than researchers

have been aware of kindling (Downs and Eddy 1932). Post and colleagues

(1975) drew attention to the idea that the local anesthetic actions of cocaine

might produce a process of “pharmacological kindling” during repeated highdose drug administration. In many ways, electrophysiological kindling was

reminiscent of the behavioral sensitization that is characteristic of cocaine.

Cocaine-induced behavioral sensitization is longlasting and shows crosssensitization to other stimuli and shows, at least in part, a dependence on

the interval between injections just as kindling does (Post and Contel 1981;

Post and Weiss 1988). The search for a mechanism to explain the persistent

changes that are characteristic of electrophysiological kindling led to a

consideration of the possible role of gene activation in this process. Since

electrophysiological kindling is a permanent change in neuronal function, it

was reasoned that the genetic material might be involved. Experiments

demonstrated that the immediate early gene (IEG) c-fos was rapidly and

transiently expressed in the dentate gyrus and other parts of the hippocampus

after a kindling stimulus (Dragunow and Robertson 1987a) or following

induction of seizure activity by chemical convulsants (Morgan et al. 1987;

Dragunow and Robertson 1987b). Subsequent work has shown that induction

of IEGs is a consequence of neuronal activation in many parts of the CNS

55
(see reviews by Robertson and Dragunow 1990; Morgan and Curran 1991;

Robertson et al. 1991 a).

IEG INDUCTION AND THE BRAIN

Activation of IEGs is not a unique property of neurons; indeed, IEGs were first

characterized in nonneuronal cells during attempts to identify genes that might

respond to growth factors (Cochran et al. 1983). This resulted in the description

of a class of genes whose transcription was rapidly (in minutes) but transiently

(usually for a few hours at most) activated following growth factor application.

Using differential screening of cDNA libraries from growth-factor stimulated

cells, many IEGs have been characterized, and the number of these, now at

about 100, continues to grow. In the absence of stimulation, the expression

of IEGs is generally low. Following stimulation, transcriptional activation is

rapid and transient, and the mRNAs transcribed from these genes often have

short half-lives. The subsequent shutoff of transcription requires new protein

synthesis; IEGs are therefore superinduced in the presence of protein synthesis

inhibitors (Sheng and Greenberg 1990). The crucial step linking these growthactivated IEGs to neuronal events was accomplished when several groups at

about the same time demonstrated that not only growth factors but also

neurotransmitters and neurotransmitter-related events such as depolarization

and calcium influx will induce transcription of IEGs both in cells in culture

(Greenberg et al. 1985, 1986; Morgan and Curran 1986) and in the brain in

vivo (Morgan et al. 1987; Hunt et al. 1987; Dragunow and Robertson 1987a).

Since c-fos was originally thought to be involved in cell division, studies

demonstrating c-fos induction in differentiated PC-12 cells and the discovery

of fos activation in brain was important in suggesting that IEGs could have a

function in postmitotic cells (Greenberg et al. 1986; Morgan et al. 1987; Hunt

et al. 1987; Dragunow and Robertson 1987a).

It is now clear from many studies that neurotransmitters and neuronal

growth factors in several systems will induce expression of IEGs (table 1).

Rapid c-fos induction occurs in PC-12 cells following application of elevated

K

+

, the calcium channel agonist BAY K8644, or external Ba++ (Curran and

Morgan 1985; Morgan and Curran 1986; Sheng et al. 1988), suggesting that

Ca++, entering through voltage-dependent Ca++ channels and/or L-type Ca++

channels, is the major second messenger regulating IEG expression. There

is also evidence that Ca++-calmodulin-dependent protein kinases (CaM kinases)

I and II play a role in the transduction of electrical signals to the nucleus, and

CREB (the adenosine 3’,:5’cyclic phosphate [cAMP] response element-binding

protein) may function to integrate Ca++ and cAMP signals (Morgan and Curran

1986; Greenberg et al. 1985, 1986; Sheng et al. 1991). In addition, recent

evidence suggests that AP-1 proteins may also have an important part to play

56
TABLE 1. Some neurotransmitters involved in activation of IEGs

Neurotransmitter Cell or Tissue Reference

Glutamate Cerebellar neurons Szekely et al. 1989

Cholinerglc (nicotinic) PC-12 Greenberg et al. 1985

Cholinergic (muscarinic) PC-12 Morgan and Curran 1986

Dopamine (D1, D57) Striatal neurons Robertson et al. 1989a, 1989b

Adenosine Neuron-glia hybrids Gubits et al. 1990

Nerve growth factor (NGF) PC-12 Greenberg et al. 1985

Calcitonin gene-related peptide Rat astrocytes Haas et al. 1991

in activation and inhibition of gene expression by cAMP. Thus, Jun-D and

Jun-B may be important in the regulation of the proenkephalin expression

by the cAMP response element (Kobierski et al. 1991), emphasizing the

importance of differential regulation of IEGs in control of gene expression.

IEGs: DIFFERENTIAL REGULATION

The situation is clearly more complicated in that many IEGs are simultaneously

activated by physiological stimulation, neurotransmitters, and growth factors. It

has been suggested that various combinations of IEGs could confer specificity

of cell response to different stimuli (Sonnenberg et al. 1989; Sheng and

Greenberg 1990; Wisden et al. 1990; Moratalla et al. 1992; Rusak et al., in

press), although there is little or no evidence for this idea.

The best known example of regulatory interaction between IEGs is the

interactions between the fos and jun families. Protein products of members

of these two IEG families can interact with each other to form heterodimeric

transcriptional factor complexes via a conserved dimerization domain called

the leucine zipper. Briefly, the protein c-Fos dimerizes with c-Jun to form a

c-Fos/c-Jun transcriptional factor that binds with high affinity and specificity

to DNA elements of the consensus sequence TGACTCA (the AP-1 site)

and, thus, leads to the transcription of nearby promoters by an unknown

mechanism (Curran and Franta 1988). However, it is clear that the situation

is even more complicated than many IEGs activating transcription because

some heterodimeric combinations are transcriptional inhibitors. The

heterodimer formed by c-Fos and Jun-B (the protein product of the jun family

member, Jun-B) represses transcription of genes with AP-1 sites in the

promoter region (Chiu et al. 1989; Schutte et al. 1989). Moreover, the situation

is made more complicated by the fact that c-Jun is an efficient activator of the

c-jun promoter, whereas Jun-B inhibits activation of the c-jun promoter. Thus,

57
Jun-B is a negative regulator of c-jun (Chiu et al. 1989). Since there are three

mammalian Jun proteins (c-Jun, Jun-B, Jun-D) and at least four Fos family

members (c-Fos, Fos-B, Fra-1 , Fra-2) to which the Jun proteins can bind,

there exist many possible combinations of Fos-Jun proteins. In addition,

there is also the possibility of Jun-Jun and Fos-Fos homodimers with

unknown potentials. Other members of the two families also probably

have unrealized potentials. For example, it has recently been shown that

a truncated form of Fos-B inhibits the transcriptional activation of Fos/Jun

heterodimers (Nakabeppu and Nathans 1991). Similarly, Fra-1 can combine

with Jun and bind to the AP-1 recognition element, raising the possibility that

this Fos family member might play a role in regulation at the AP-1 site (Cohen

et al. 1989). Finally, it is now becoming apparent that Fos/Jun can regulate

other receptors; for example, Fos and Fos/Jun can inhibit the glucocorticoid

receptor (Touray et al. 1991), and in turn, other factors, such as retinoic acid

(Schule et al. 1991), can regulate AP-1 -responsive genes.

The issue is further confused because in many studies, especially for neural

tissue, stimulation leads to activation of a variety of Fos and Jun family

members (in addition to other IEGs). For example, stimulation with D1

dopamine agonists can cause rapid expression of c-fos, c-jun, and jun-B in

striatum. Are these IEGs all in the same cell, or more attractively, are c-fos

and c-jun expressed in one type of cell leading to transcriptional activation

while in another cell c-fos and jun-B expression is producing transcriptional

inhibition? It is clear that the answers to such questions are of great

importance. However, in some tissues, such as the dentate granule cells after

seizure activity, it seems clear that c-fos, c-jun, and jun-B activation all occur in

the same cells (B.J. Chiasson and H.A. Robertson, unpublished observations).

In addition to this complexity, it is now suspected that the so-called flanking

sequences to the AP-1 site play an important role in the binding of Jun proteins

to the AP-1 site (Ryseck and Bravo 1991). It is important to note that AP-1 -like

sites are sometimes common, and it is not immediately obvious which sites

might be active (e.g., Hengerer et al. 1990). It is also clear that differential

regulation of IEGs does occur, and there are several instances in which c-fos

is expressed in the absence of c-jun or together with jun-B rather than c-jun

(Bartel et al. 1989; Naranjo et al. 1991). There are instances in which the c-jun

message is expressed following stimulation but no c-Jun protein appears to

be expressed, suggesting, among other possibilities, that IEG protein levels

might be regulated at the transcription level (Rusak et al., in press).

In summary, it is now clear that the picture of activation of IEGs that encode

nuclear proteins that regulate gene expression is misleading. The more

accurate picture has been described as regulation by committee, a series of

58
IEGs, members of various families, all interacting with one another and with

other second and third messengers to affect several transcriptional regulating

sites and, thus, altering gene expression. What has become clear is that

these genes probably play a role in postmitotic tissue, the best example

being neurons in the brain. However, it should be kept in mind that some

IEGs may perform not as messengers in tightly linked signaling systems but

as “housekeeping” genes, regulating the response of cellular metabolism to

particular alterations in conditions. For example, it is remarkable to note the

similarity between the activation of c-fos in cerebral cortex ipsilateral to a

lesion (Dragunow and Robertson 1988; Herrera and Robertson 1989, 1990a)

and the activation of the heat shock protein, HSP-71 (Gonzalez et al. 1989).

However, having raised this caution, it is clear from several examples in the

brain that there is good circumstantial evidence that c-fos and other IEGs

play an important role in the transduction of neuronal stimulation into specific

neuronal changes (plasticity); this neuronal stimulation can be elicited by

physiological (e.g., light, pain) or pharmacological means.

IEG ACTIVATION: A MAPPING TOOL FOR THE BRAIN?

It has been apparent since the work of Hunt and colleagues (1987) on the

effects of pain on the induction of c-fos in spinal cord that, apart from the

importance that the IEGs might play in cellular function in brain systems, the

robust response of the IEGs might be generally useful as an indication of

transsynaptic activation and in the mapping of polysynaptic pathways in the

brain. This usefulness has generally been confirmed in many subsequent

studies (e.g., Sagar et al. 1988; Rusak et al. 1990, in press; MacDonald et

al. 1990). This does not detract from the significance of IEGs as possibly

extremely important “third or fourth messengers” in signal transduction.

However, it does add an extremely important dimension to the uses of IEG

activation.

EXPRESSION OF IEGs IN THE MAMMALIAN BRAIN: KINDLING,

LONG-TERM POTENTIATION, AND SEIZURES

Induction of IEGs has now been demonstrated in several systems in the brain

and in association with several neurotransmitter systems. The initial studies

that suggested an important role for IEGs in the brain described activation of

c-fos and other IEGs in the brain following drug-induced seizure activity

(Morgan et al. 1987; Dragunow and Robertson 1987a; Saffen et al. 1988;

Le Gal La Salle 1988; Sonnenberg et al. 1989). A single electrical stimulus

to amygdala or hippocampus (at an intensity known to lead to the longlasting

effect known as kindling) will also produce an increase in c-Fos protein in the

hippocampal formation, with an especially high concentration in the dentate

59
granule cells (Dragunow and Robertson 1987a; White and Gall 1987; Dragunow

and Robertson 1987b). There is a continuing discussion over the possible

role of c-fos and other IEGs in the phenomenon of long-term potentiation

(LTP), which is the persistent (up to several weeks) increase in the excitatory

postsynaptic potential elicited at synapses following a high-frequency

stimulation of afferent neurons, Early studies suggested that LTP could

develop in the absence of c-fos induction (Douglas et al. 1988), and this

was confirmed by Cole and colleagues (1989) and Wisden and coworkers

(1990) who did, however, demonstrate a correlation between LTP and

induction of NGFI-A. Surprisingly, LTP induced using “burst” stimulation

involves c-fos induction, but LTP induced by the same number of stimulations

evenly spaced over the same train duration (i.e., LTP induced by the same

number of stimulations but with different spacing) is not associated with

increases in Fos protein (Dragunow et al. 1989). However, all earlier studies

were done on anesthetized animals, and more recent studies on the

development of LTP in unanesthetized animals revealed that, in this situation,

c-fos induction accompanies LTP induction (Jeffery et al. 1990). From all this

work, several points are clear. First, it is already known that LTP develops in

milliseconds, whereas c-fos mRNA only appears between 10 and 15 minutes

later, with c-Fos protein appearing shortly after. The same is true for other

IEGs. Thus, although it remains possible that IEGs play a role in maintenance

of LTP (but see Jeffery et al. 1990), it seems unlikely that they play any role in

induction of LTP. Second, some stimulation parameters produce LTP with c-fos

activation, but others produce LTP without c-fos activation, which suggests (as

was already suspected) that there are different types of LTP, differing perhaps

in persistence or other properties.

The kindling model is interesting in that, unlike LTP, induction of kindling

appears to be a permanent change in the brain (for a review of the differences

between kindling and LTP, see Cain 1989). A kindling stimulus will activate

c-fos in the dentate granule cells and in the hippocampal pyramidal neurons

(Dragunow and Robertson 1987a; White and Gall 1987). Other IEGs

(c-jun, jun-B, NGFI-A) are also activated by the kindling stimulus (H.A.

Robertson, unpublished data). It is known that kindling stimuli lead to

synaptic reorganization in the molecular layer of the dentate gyrus of the

hippocampus (Sutula et al. 1988). Kindling has also been shown to produce

increased synthesis of neurotrophic factors in the dentate gyrus (Ernfors

et al. 1991; B.J. Chiasson and H.A. Robertson, unpublished observations),

and Funabashi and colleagues (1988) demonstrated that injections of

antibody against NGF into the cerebral ventricles during stimulation would

prevent kindling. In at least one other system, IEGs appear to play a role

in the regulation of NGF synthesis (Hengerer et al. 1990, see below). Thus,

it is reasonable to suggest that IEG induction may be the first step in a

60
sequence of changes involving secretion of neurotrophic factors and

synaptic reorganization, culminating in a permanent alteration in the

susceptibility of this part of the brain to seizures.

DOPAMINE AND IEG ACTIVATION

The neurotransmitter dopamine appears to play a pivotal role in regulation

of movement by the striatum. Loss of dopamine-containing neurons of

the substantia nigra leads to a profound loss of motor control as seen in

Parkinson’s disease. Dopamine is also thought to play a similar role in

higher functions, and disorders of dopaminergic neurotransmission are

generally assumed to lie behind the schizophrenias and may also contribute

in large part to the disturbances of both thought and movement seen in

Huntington’s chorea. The dopaminergic system also appears to play a

central role in cocaine and amphetamine addiction (Roberts and Koob 1982;

Koob and Bloom 1988; Di Chiara and lmperato 1988). Activation of c-fos in

an animal model for Parkinson’s disease was first demonstrated using the

antiparkinsonian drug L-dopa (the immediate precursor of dopamine) and

drugs selective for the D1 -dopamine receptor (Robertson et al. 1989a, 1989b,

1991b; Paul et al., in press). After L-dopa or a D1-dopamine agonist, c-Fos

protein was found only in the regions of the brain (striatum) that were

experimentally depleted of dopamine and where dopamine receptors were

known to be supersensitive. Significantly, activation of IEGs appears

to be a D1-dopamine receptor-linked function (Robertson et al. 1989b).

Compounds that act indirectly to release dopamine (such as cocaine and

amphetamine) also increase expression of c-fos and other IEGs in striatum

via D1 dopamine receptors (Robertson et al. 1989b; Graybiel et al. 1990;

Young et al. 1991; Moratalla et al. 1992). It is most important to note that,

whereas amphetamine and cocaine activate IEGs in naive, untreated animals,

directly acting D1 dopamine receptor agonists only induce IEG transcription in

supersensitive tissue. Thus, even high doses of a D1 agonist or apomorphine

will not produce IEG expression in animals that have not been depleted of

their dopamine content.

Although D2 dopamine agonists apparently have little effect on IEG activation,

it is significant that the combination of a D1 and a D2 dopamine agonist has

synergistic effects on induction of c-fos, c-jun, jun-B, and NGFI-A (Paul et al.

1990, in press; Robertson et al. 1991 b) reminiscent of the synergism seen in

studies on locomotion in animal models for Parkinson’s disease (Robertson

and Robertson 1986, 1989). Moreover, although c-fos activation in the

striatum following D1 agonist stimulation is spread over the entire striatum,

the combination of D1 and D2 agonists produces a pattern that reveals the

compartmentalization of this brain region (Paul et al. 1990; Robertson et al.

61
1991b; Paul et al., in press). This once again illustrates the fact that IEG

activation is a potent tool for studying the anatomical organization of neuronal

structures.

Dopaminergic and glutamatergic mechanisms interface in the striatum as

two control mechanisms central to regulation of striatal function (Clow and

Jhamandas 1989). Glutamatergic neurotransmission appears to play some

role in dopamine-mediated activation of IEG expression. N-methyl-D-aspartate

(NMDA) receptor antagonists reverse amphetamine-induced activation of c-fos

(Johnson and Robertson 1989; Aronin et al. 1991; Snyder-Keller 1991; Paul et

al., in press; Berretta et al., in press), suggesting that glutamatergic and

dopaminergic mechanisms also interact at the level of regulation of gene

expression in striatum.

It was known that dopamine agonists induce expression of c-fos, and it

was at the same time surprising and potentially important to note that c-fos

induction also occurred following administration of D2 dopamine receptor

antagonists such as haloperidol (Dragunow et al. 1990; Miller 1990) and

clozapine (Robertson and Fibiger 1991; Hiroi et al. 1991). These drugs are

widely used in the treatment of mental disorders such as schizophrenia. The

finding of IEG activation by these drugs in clinically relevant doses suggests

the possibility that either the beneficial effects or some of the side effects of

these drugs might be mediated by changes in gene expression. Both the

symptoms of schizophrenia and the development of some side effects of

these drugs have time courses consistent with alterations in gene expression,

Thus, selective blockade of dopamine receptors may, like selective activation

of dopamine receptors, be associated with long-term changes in neuronal

function (table 2). The evidence at this time suggests that, although dopamine

D1 agonists exert a stimulatory effect on c-fos expression, D2 receptors appear

to mediate a tonic inhibitory effect on c-fos expression,

TABLE 2. Some possible associations between drug-induced activation of

lEGs and pathophysiological effects of dopaminergic drugs

Effect IEG Activator Drug Class Reference

Dystonias L-dopa D1 dopamine agonists Robertson et al.

1989a, 1989b

Tardive dyskinesias Haloperidol D2 dopamine antagonists Dragunow et al.

1990

Drug addiction Amphetamine, Psychostimulants Graybiel et al.

cocaine 1900; Young et al.

1991

62
IEGs AND CONTROL OF CIRCADIAN RHYTHMS

In mammals, the suprachiasmatic nucleus (SCN) is the site of the central

control mechanism for the regulation of circadian rhythms (Rusak and

Zucker 1979). The “clock” in the SCN is “set” (entrained to the environmental

lighting) by neurons in the retina that project to the SCN. Exposing an animal

to light during its “dark” phase will alter or shift the circadian rhythm. Exposing

rats or hamsters to light during the night will result in the induction of c-fos

and NGFI-A (Rusak et al. 1990) and other IEGs (Rusak et al., in press).

Most important, light will induce c-fos only during the “expected” dark phase

in animals kept in total darkness. Such animals continue to maintain the

circadian rhythm established before they were placed in total darkness.

During the “expected” light phase, exposure to light will not lead to the

induction of c-fos and other IEGs (Rusak et al. 1990, in press). There is

also a good correlation between the intensity of the light required to produce

the physiological effect (a shift in the circadian rhythm) and the induction

of mRNA production (Kornhauser et al. 1990). There is some evidence that

the photic induction of IEGs in the SCN is regulated by glutamate receptors

of the NMDA type, at least in part (Abe et al. 1991). The mechanism that

prevents light stimulation from inducing the IEGs during the subjective day is

pretranscriptional but otherwise unknown, Feasible mechanisms include the

possibilities that the retinal ganglion cells do not transmit the information or

that the receptors in the SCN exhibit rhythmic sensitivity. It appears that the

NMDA type of glutamate receptor is at least involved, but another possibility

is that the sensitivity of the NMDA receptor is being regulated via alterations

in the depolarization state of the SCN neurons by another endogenous or

exogenous factor.

DAMAGE-INDUCED ACTIVATION OF IEGs

Damage to tissues often produces homeostatic responses that include the

release of growth factor and induction of cell proliferation. Therefore, it is

not surprising that such brain damage will produce widespread activation of

c-fos (Dragunow and Robertson 1988). Indeed, early experiments attempting

to look at the activation of IEG synthesis in slices had been compromised by

the fact that the process of preparing the slice produced a maximum activation

of IEG expression (S.P. Hunt, personal communication, November 1987).

Similarly, when first reported, damage-induced activation of IEGs in brain in

vivo was (and is) a serious complication for in vivo experimentation. It meant

that introduction of a cannula or an electrode into brain, by itself, produced

significant IEG expression, Furthermore, induction of IEGs was seen not only

adjacent to the site of damage but also in parts of the cortex remote to the

damage (Dragunow and Robertson 1988; Herrera and Robertson 1989, 1990a,

63
1990b). For c-fos, this activation was confined to the damaged hemisphere and

was sensitive to NMDA receptor antagonists (Herrera and Robertson 1989,

1990a, 1990b; Dragunow et al. 1990; Traboulsee et al. 1991). There is at least

some circumstantial evidence that damage-induced IEG activation in cerebral

cortex is partially the result of spreading neuronal depression (Herrera and

Robertson 1990b).

IEGs AND CHANGES IN GENE EXPRESSION

The impetus for studying IEG activation came from the conviction that changes

in gene expression lay behind long-term biological change. The excitement

associated with the discovery of IEG activation in the instances cited above

(and many others as well) is that there is now a key for the problem of how

neuronal activity might alter gene expression. However, there is relatively little

direct evidence for such changes, and what evidence there is is circumstantial.

For example, a kindling stimulus activates c-fos expression, but there is no

direct evidence that c-fos expression is absolutely required for kindling to occur.

Similarly, light exposure during the dark phase leads to IEG expression in the

SCN, and in every instance studied so far, there is a relationship between IEG

activation and the phase shift in the circadian rhythms. But this cannot be

construed as other than circumstantial. On the positive side, there are several

situations in which it can be said that IEG expression can lead to changes in

gene expression. For example, the availability of mice with a transgenic c-fos

linked to a metallothionine reporter has been used by Thoenen and colleagues

(Hengerer et al. 1990) to demonstrate that activation of c-fos in fibroblasts in

sciatic nerve precedes NGF synthesis. Furthermore, they were able to show

that an AP-1 site in the first intron of the NGF promoter is necessary for NGF

synthesis. It IS interesting to note that there are eight AP-1 sites in the promoter

region of NGF in the mouse, but only one, apparently, is essential for NGF

synthesis. There is also a suggestion that the proenkephalin gene may be

up-regulated via an AP-1 site present in its promoter (Sonnenberg et al. 1989).

However, such regulation of proenkephalin is probably complex, involving

several other control elements (Comb et al. 1988; Kobierski et al. 1991).

Clearly, it is important to define the nature of the late-response genes. Are

there any long-term changes that result from activation of IEGs? What are

these changes, and how are they carried out? The other task is to show

conclusively that the IEGs are involved (or not involved) in long- or short-term

changes in function.

64
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ACKNOWLEDGMENTS

The work described in this chapter was supported wholly by the Medical

Research Council of Canada (grants MT 10402 and MT 10644) and the Human

Frontier Science Organization. Drs. Ben Rusak, Stephen Hunt, and Ann

Graybiel provided helpful discussion.

AUTHOR

Harold A. Robertson, Ph.D.

Professor

Department of Pharmacology

Dalhousie University

Sir Charles Tupper Medical Building

Room 601

Halifax, Nova Scotia B3H 4H7

CANADA

71
Acute Effects of Psychomotor

Stimulant Drugs on Gene Expression

in the Striatum

Ann M. Graybiel

INTRODUCTION

Long-term changes in behavior leading to drug addiction imply concomitant

drug-induced changes in central nervous system (CNS) processing.

A large body of behavioral work implicates the mesolimbic, mesostriatal,

and mesocortical systems as potential sites for such modifiability (Carr et

al. 1989; Koob and Goeders 1989; Koob and Bloom 1988; Kuhar et al.

1991). At the cellular level, changes ranging from alterations in receptor

expression and receptor interactions with intracellular signaling molecules

by pretranslational and posttranslational modification to alterations in

expression or activity of such molecules have been demonstrated following

repeated drug exposure, Both presynaptic and postsynaptic changes have

been suggested (Robinson and Becker 1986; Post and Contel 1983; Graybiel

1990; Terwilliger et al. 1991; Beitner-Johnson and Nestler 1991). Recently,

interest has focused also on the possibility that regulation of immediate early

gene (IEG) expression in neurons could lie at the basis of some or many of

these changes.

Interest in IEG regulation as part of the cellular basis of addiction is based

on at least two characteristics of these genes. First, IEGs are positive and

negative regulators of the transcription of other genes and can be regulators

of their own transcription as well (Sheng and Greenberg 1990; He and

Rosenfeld 1991). Thus, affecting IEG transcription could start a cascade of

further changes that could have enduring effects on CNS function. Second,

IEGs can act as bridges between the short-term effects of transient stimuli

impinging on cells and long-term changes resulting from these stimuli. Just

such considerations led to the proposal that transcriptional regulation is at the

basis of long-term memory in neural circuits (Goelet et al. 1986; Berridge 1986;

Morgan and Curran 1989).

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What evidence is there that alterations in transcriptional regulation occur

following exposure to drugs of abuse? For drugs that affect dopamine and

other biogenic amines, including cocaine and amphetamine, there is now direct

evidence that acute drug exposure induces expression of IEG mRNAs in the

striatum, certain sites in the limbic system, and certain cortical areas (Graybiel

et al. 1990; Young et al. 1991; Berretta et al. 1991a, in press; Moratalla et al.

1991, in press, submitted for publication; Cole et al. 1990; Dilts et al. 1990;

Grimes et al. 1990; Robertson et al. 1991). There is also evidence that

exposure to amphetamine and cocaine elicits expression of nuclear proteins

detected with antisera to Fos and related IEG protein products (Graybiel et al.

1990; Young et al. 1991). Thus, transcription and/or mRNA stabilization and

translation of IEG protein products are induced by cocaine and amphetamine.

PSYCHOMOTOR STIMULANTS ACTIVATE IEGs IN REGIONALLY

SPECIFIC PATTERNS

What characteristics of the IEG induction suggest specificity in the neural

circuits showing this response to psychomotor stimulants? First, amphetamine

and cocaine acutely activate IEGs in different macroscopic patterns in the

rostral caudatoputamen and also in the ventral striatum (Graybiel et al. 1990;

Fuxe et al. 1991). Expression of Fos-like protein is concentrated more strongly

in the striosomes than in the matrix of the rostral caudatoputamen in rats

acutely treated with amphetamine. By contrast, acute cocaine exposure

induces Fos-like proteins in both striatal compartments in the rostral

caudatoputamen. Combinations of D1-like and D2-like dopamine agonists

given in low dose (Paul et al. 1991, in press) and chronic apomorphine

(Grimes et al. 1990) also induce Fos-like protein expression in patterns that

respect striosome/matrix compartments in at least parts of the dopaminedepleted striatum.

These findings raise the possibility that there is differential neural activation

of the striosomal and matrix compartments following exposure to certain

amphetamine-like psychomotor stimulants. Such differential activation

could contribute to differences in behavioral responses to these drugs,

because the striosomes and matrix have different input and output connections

(Graybiel 1990). It is not clear how these effects of acute drug treatment relate

to the effects of chronic drug exposure. Conceivably, these patterns could

be related to the apparently differential toxicity induced in striosome- and

matrix-innervating nigrostriatal fibers by continuous high-dose amphetamine

(Ryan et al. 1988). It is also interesting that the Fos-like protein induction in

the striatum that is elicited by acute psychostimulant exposure is influenced by

concomitant morphine treatment and that this drug interaction is also reported

to be compartmental (Fuxe et al. 1991). All these lines of evidence suggest that

73
striosome/matrix compartmentation should be considered in addition to regional

subdivisions of the striatum in assessing the neural response to psychomotor

stimulants.

The selectivity of these drug effects on the induction of Fos-like proteins

extends to the cellular level (Berretta et al. 1991a, 1991b, in press). It has

been shown by double immunostaining that amphetamine and cocaine both

induce Fos-like proteins in striatal medium-sized neurons expressing the

phosphatase inhibitor DARPP-32 (dopamine and cyclic AMP [cAMP]-regulated

phosphoprotein) (Berretta et al. 1991a, 1991b, in press). In sharp contrast,

neither amphetamine nor cocaine induces detectable Fos-like protein in the

enkephalin-containing neurons in the caudatoputamen, even though many of

these neurons coexpress DARPP-32. Nor does either stimulant induce Fos

in striatal interneurons. These experimental findings establish that when Foslike protein induction does occur in the striatum in response to psychomotor

stimulants, the induction is only in a subset of the neurons present in the

regions of activation. As discussed below, the subset of responsive neurons

may have special functional characteristics that are related to the behavioral

effects of the stimulants.

It seems highly likely that these cell-specific patterns of IEG induction by

psychomotor stimulants are related to the differential expression of D1 -like and

D2-like dopamine receptors by striatal neurons. The D1-selective dopamine

receptor blocker SCH23390 blocks the induction of immunodetectable Fos-like

protein by either amphetamine or cocaine (Graybiel et al. 1990; Young et

al. 1991; Berretta et al., in press). However, this antagonist blocks 5-HT2

receptors as well as D1-like dopamine receptors so that a role for serotonin

in the induction is not yet to be discounted. Control experiments designed to

test for this have so far been negative (Graybiel et al. 1990). That the Fosresponsive neurons are immunoreactive for DARPP-32 agrees well with the

apparent D1-like dopamine receptor selectivity of the induction, for DARPP-32

is considered to be a marker for cells expressing D1-like dopamine receptors

(Walaas et al. 1983). The lack of induction in enkephalinergic striatal neurons

also is reasonable because these neurons are known to express D2-like

dopamine receptors (Le Moine et al. 1990) and are thought either not to

express D1 -like receptors in large numbers (Gerfen et al. 1991) or to include

a considerable subpopulation coexpressing D1-like and D2-like receptors

(Meador-Woodruff et al. 1991). In the latter case, the D2-like dopamine

receptors may block the adenylate cyclase signaling pathway activated by the

D1-like receptor activation (Stoof and Kebabian 1981; Berretta et al., in press).

74
Neurons Expressing Fos-Like Protein Have a Special Functional Status in

the Basal Ganglia

The known biochemical anatomy of the striatum suggests that most of the

neurons expressing Fos in response to psychomotor stimulants are in the

class of striatal output neurons projecting into the movement-releasing

pathways of the basal ganglia-the neurons expressing substance P-like

tachykinin and dynorphin. Current models of the basal ganglia suggest that

when these neurons are activated, thalamocortical pathways are disinhibited,

and hence, regions of premotor cortex become active (Albin et al. 1989;

Alexander and Crutcher 1990; Graybiel 1991a). Substance P/dynorphincontaining striatal output pathways also lead to the substantia nigra pars

reticulata, which in turn results in disinhibition of the superior colliculus. By

contrast, the enkephalinergic neurons are striatal output neurons that project

into the pathway leading to the subthalamic nucleus, which is thought to

decrease movement release by the basal ganglia. Thus, these two sets of

neurons-those exhibiting an acute Fos response to psychomotor stimulant

exposure and those not doing so-are thought to have antagonistic roles in

movement control. The responsive neurons are thought to facilitate the

occurrence of movements, whereas the nonresponsive neurons are thought

to damp movements. It is not yet known whether the IEG response is related

to, or paralleled by, changes in the membrane potential of the responsive

neurons. if the transcription factor activation is correlated with neural activation,

however, results on induction of Fos-like protein (Graybiel 1991b; Berretta et al.,

in press) would suggest the following hypothesis: that some of the activating

properties of psychomotor stimulants could stem from an imbalance between

the activation of movement-releasing and movement-inhibiting pathways of the

basal ganglia.

THE IEG RESPONSE ELICITED BY PSYCHOMOTOR STIMULANTS IS

SELECTIVE TO SUBSETS OF IEGs

The induction of IEGs in striatal neurons following acute exposure to cocaine

and amphetamine is not limited to expression of c-fos, but findings to date

suggest that the response is nevertheless selective (Moratalla et al. 1990,

1991, submitted for publication; Cole et al. 1990). Judging from Northern

blotting and in situ hybridization analysis, mRNAs corresponding to two of the

IEGs of the fos/jun family so far tested, c-fos and jun-B, are strongly induced by

cocaine and amphetamine. Although c-jun mRNA is often coordinately induced

with c-fos, it is not induced by acute amphetamine or cocaine stimulation.

Some other mRNAs of fos/jun genes are also unresponsive and are also not

detectable by Northern blotting (Moratalla et al., submitted for publication). That

other Fos-related antigens may be induced has been suggested by Young and

75
colleagues (1991). In addition to the c-fos and c-jun induction, there is strong

induction of the zinc-finger gene NGFI-A (zif/268, egr-1) (Moratalla et al., in

press). These differential patterns of gene induction are interesting because

the DNA-binding proteins coded by these genes function as parts of dimers

in conjunction with other binding proteins of the same transcription factor

family, and these dimers in turn are parts of binding complexes that influence

transcription. As a result, a wide range of control is in principle possible,

depending on the particular genes that are coinduced and the levels and

kinetics of their induction (Struhl 1991; Sheng and Greenberg 1990; He and

Rosenfeld 1991; Morgan and Curran 1991).

ACTIVATION OF INTRACELLULAR SIGNALING PATHWAYS BY

PSYCHOMOTOR STIMULANTS: CLUES TO LONG-TERM CHANGES IN

NEURAL PROCESSING

The finding of selective patterns of IEG induction by psychomotor stimulants

should give clues to the signaling pathways leading to the induction and,

ultimately, to the consequences of the particular patterns found. For example,

in PC12 cells it has been shown that membrane depolarization activates c-fos

and jun-B, but not c-jun, and this pattern has been found in fibroblasts treated

with cAMP analogs (Bartel et al. 1989; Mechta et al. 1989). The D1-like

receptor selectivity of the psychomotor stimulant activation is probably

responsible for the selective patterns of fos/jun induction that have been

found (Moratalla et al., submitted for publication), because the c-fos promoter

has a calcium-and-cAMP responsive element (Berkowitz et al. 1989; Sheng

et al. 1988, 1990), and the jun-B promoter contains a novel repeat element

that can be activated by protein kinase A. By contrast, protein kinase A may

mediate an inhibition of c-jun directly or indirectly (de Groot et al. 1991; Chiu

et al. 1989; Schutte et al. 1989; Ryseck and Bravo 1991). Signaling pathways

leading from stimulation of adenylate cyclase and cAMP-dependent protein

kinase A would then need to be considered as prime targets of the acute effects

of psychomotor stimulant drugs. Nestler and colleagues find that molecules in

the cAMP/protein kinase A pathway are regulated during prolonged exposure to

cocaine and morphine (Guitart and Nestler 1989; Nestler et al. 1990; E. Nestler,

personal communication, January 1992). The convergence of evidence for

cAMP/protein kinase A mediation of responses to acute and long-term exposure

to psychomotor stimulants fits well with the hypothesis that short-term induction

of IEGs by these drugs of abuse could be implicated in the long-term responses

that follow their repeated use (e.g., Graybiel et al. 1990; Young et al. 1991).

What would be the characteristics of such neural modifications? For first

analysis, the problem can be broken down into three parts: the initial signaling

paths leading to changes in IEG induction, transcription, and translation; the

76
effects of the DNA-binding proteins on transcription of other genes; and the

downstream effects of such changes (for example, insertion of new receptor

proteins into the ceil membrane or regulation of neurotransmitter expression).

Some progress has been made in identifying the signaling pathways likely to be

involved in the induction of IEGs by acute exposure to psychomotor stimulants,

but there is little firm information about the effects of this induction. It is known

that neurotransmitters and neuropeptides as well as receptors and messenger

molecules undergo altered expression with prolonged exposure to dopamine

agonists and antagonists (see Graybiel 1990; Grimes et al. 1990; Gerfen et al.

1991). Several neuropeptide genes have recently been shown to contain AP-1-

like DNA binding motifs in the upstream regions of their promoters. Thus, the

signaling pathways may be in place for direct effects of Fos-like transcription

factors on neuropeptide expression. With transgenic mice it may be possible to

test this possibility directly and also to test whether regulatory control is exerted

through particular combinations of IEGs on expression of neurotransmitters,

receptors, and messenger molecules that could bring about long-term changes

in neural function.

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ACKNOWLEDGMENTS

The work from the author’s laboratory summarized in this chapter was

supported by the Human Frontier Science Program, the Javits Award from the

National Institutes of Health (NIH) NS-25529, the United Parkinson Foundation,

the Seaver Institute, and NIH Biomedical Research Support grant BRSG 2SO7

RR07047-26.

AUTHOR

Ann M. Graybiel, Ph.D.

Professor of Neuroanatomy

Department of Brain and Cognitive Sciences

Massachusetts Institute of Technology

Building E25, Room 618

Cambridge, MA 02139

81
Functional Organization of the

Striatum: Relevance to Actions of

Psychostimulant Drugs of Abuse

Charles R. Gerfen

Recent advances in understanding the functional organization of the basal

ganglia may provide some insight into the cellular and molecular action

of certain drugs of abuse. For example, the psychomotor stimulant effects

of cocaine (e.g., euphoria, locomotion, stereotypy) are thought to be mediated

primarily by dopaminergic mechanisms in brain regions such as the striatum,

including the nucleus accumbens (DeWit and Wise 1977; Pettit et al. 1984;

Roberts et al. 1977). Dopaminergic afferents to the striatum are directed

principally to the medium spiny neurons (Freund et al. 1984), which constitute

90 to 95 percent of the striatal neurons and give rise to the major striatal

efferent pathways (Somogyi et al. 1981). On the basis of their axonal

projections, striatal outputs are divided into two types: those that project to

the globus pallidus and those that project to the entopeduncular nucleus/

substantia nigra complex (Kawaguchi et al. 1990). Neurons contributing to

both pathways contain glutamic acid decarboxylase (Kita and Kitai 1988) and

use -aminobutyric acid (GABA) as a transmitter (Chevalier et al. 1985), but

each contains different neuropeptides. For example, the majority of striatonigral

neurons contain dynorphin and substance P (Brownstein et al. 1977; Get-fen

and Young 1988; Vincent et al. 1982), whereas the majority of striatopallidal

neurons contain enkephalin (Haber and Nauta 1983). Pharmacological and

lesion studies have demonstrated that dopamine differentially modulates the

expression of these peptides as determined by changes in immunoreactivity

(Hong et al. 1978, 1985; Li et al. 1987, 1988; Voorn et al. 1987) or mRNA levels

(Bannon et al. 1986; Gerfen et al. 1991; Morris et al. 1988; Young et al. 1986).

The contrasting effects of dopamine on peptide mRNA regulation appear to be

dependent on the differential expression of the D1 and D2 dopamine receptors

on striatonigral and striatopallidal neurons, respectively (Gerfen et al. 1990).

Thus, dopamine-induced alterations in striatal peptide mRNA levels provide a

paradigm to study receptor-mediated regulation of specific subtypes of striatal

output neurons.

82
Since cocaine increases dopaminergic transmission in the striatum (Hurd and

Ungerstedt 1989; Moore et al. 1977), the effects of this indirect agonist on the

mRNA levels of the striatal peptides dynorphin, substance P, and enkephalin

using in situ hybridization histochemistry (ISHH) (Hurd et al. 1992) were

examined. A treatment schedule was used in which rats were given unlimited

access to intravenous cocaine for a period of 7 days to examine effects that

may be related to the abuse of this drug. In this study, the self-administration

of cocaine at an average daily rate of 58.8 mg/day (approximately 200 mg/kg/

day) resulted in significant regionally specific alterations in the levels of mRNA

encoding the peptides dynorphin, substance P, and enkephalin in the striatum

(table 1). Both dynorphin and substance P mRNA levels show substantial

cocaine-induced increases in the dorsal striatum, whereas in the nucleus

accumbens, these increases are relatively modest, particularly in the case of

substance P (figure 1). Enkephalin mRNA is elevated only in the nucleus

accumbens. Since dynorphin and substance P mRNA are expressed by most

striatonigral neurons and enkephalin mRNA is expressed predominantly by

striatopallidal neurons (Gerfen and Young 1988), cocaine self-administration

differentially alters gene regulation in these two striatal output systems. The

most pronounced effect of cocaine is an elevation of both dynorphin and

substance P mRNA levels in the dorsal striatum, whereas enkephalin mRNA

levels are not significantly altered in this region. Thus, in the dorsal striatum

there is a difference in gene regulation in striatonigral and striatopallidal

TABLE 1. Average dynorphin, substance P, and enkephalin ISHH labeling

(measured in arbitrary optical density units) in the dorsal striatum

and nucleus accumbens in control, cocaine self-administering

animals and saline-infused animals that received restricted food

to match the weight of cocaine-treated animals

Area

Dorsal

striatum

Dynorphin ISHH Substance P ISHH Enkephalin ISHH

Control Cocaine Control Cocaine Control Cocaine

(Food (Cocaine vs. (Food (Cocaine vs. (Food (Cocaine vs.

Restricted) Control) Restricted) Control) Restricted) Control)

154.0±1.9 183.0±2.1 150.4±1.4 173.6±2.3 161.9±1.1 162.4±1.4

(154.9±2.3) +29.0* (150.2±2.1) +23.2* (164.6±1.4) +0.5

Nucleus 176.2±2.5 192.0±1.0 152.4±1.0 160.92±0.6 159.9±0.9 164.3±1.1

accumbens (177.6±2.3) +16.4* (151.0±1.1) +8.6* (161.3±0.9) +4.4*

*p>0.05

SOURCE: Hurd et al. 1992

83
FIGURE 1. Autoradiographic film images of (A, B) dynorphin (DYN), (C, D)

substance P (SP), and (E, F) enkephalin (ENK) ISHH labeling in

the striatum in a control (A, B, C) and cocaine self-administration

animal (B, D, F). CP=caudate putamen, ACG=nucleus

accumbens.

SOURCE: Hurd et al. 1992

84
neurons, In the ventral striatum, which includes the nucleus accumbens, the

cocaine-induced effects are more subtle and complex. Here dynorphin mRNA

levels show the largest increase compared with the other peptide mRNAs, but

this increase is considerably less pronounced than in the dorsal striatum. Both

substance P and enkephalin mRNA levels show a slight, significant, cocaineinduced elevation in the nucleus accumbens. Ventral striatal neurons that

express substance P project to the ventral pallidum (Haber and Nauta 1983);

however, it has not been established whether these neurons belong to a set

that coexpresses either enkephalin or dynorphin and that provides axon

collaterals to both the ventral pallidum and substantia nigra. Nonetheless, the

elevation in dynorphin, substance P, and enkephalin mRNA levels suggests

that gene regulation is increased, albeit only modestly, in most output neurons

in the nucleus accumbens during cocaine self-administration. This is in contrast

to the dorsal striatum, where cocaine self-administration results in a rather

selective and substantial increase in gene regulation in striatonigral neurons,

thus generating a relative imbalance between striatonigral and striatopallidal

neurons compared with the control condition.

As has been demonstrated with other pharmacologic manipulations of the

striatal dopaminergic system, cocaine appears to alter the relative balance of

function of striatopallidal and striatonigral neurons. A model for dopamine’s

functional effects in the dorsal striatum has been proposed that suggests that

this neurotransmitter modulates the balance of activity in the striatopallidal

and striatonigral output systems (Albin et al. 1989; Gerfen et al. 1990). The

consequences of altering the balance in these output pathways are related to

the ultimate effects on the activity of GABAergic neurons in the entopeduncular

nucleus and substantia nigra. Increased striatopallidal activity results in an

increase in the activity of nigral GABAergic neurons by way of the intervening

subthalamic nucleus (Kita and Kitai 1987). Conversely, increased striatonigral

activity directly inhibits nigral GABAergic neurons (Chevalier et al. 1985). The

behavioral consequences of opposed modulation of GABAergic nigral neurons

are generally modeled on the concept that the tonic inhibitory output of these

neurons is interrupted by inhibitory inputs to these neurons from the striatum

during movements (Chevalier et al. 1985; Deniau and Chevalier 1985). This

disinhibitory process has been best characterized for eye movements by

Hikosaka and Wurtz (1983a, 1983b, 1983c, 1983d) who, in a series of elegant

studies, showed that visual-, memory-, and reward-contingent eye movements

are correlated with pauses in the tonic activity of nigral GABAergic neurons and

increased activity in superior colliculus neurons. Akinesia that accompanies

Parkinson’s disease has been related to increased striatopallidal activity and

the resultant increase in excitatory subthalamic inputs to nigral GABAergic

neurons, which then predominates over the disinhibitory mechanisms required

85
for the generation of movements (Albin et al. 1989; Mitchell et al. 1989). This

model is supported by the report that lesions of the subthalamic nucleus reverse

akinesia in monkeys made Parkinsonian with 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP)-induced lesions (Bergman et al. 1990).

Following cocaine self-administration, there appears to be an increase in the

relative function of striatonigral vs. striatopallidal output systems in the dorsal

striatum, which, according to the above model, would result in a facilitation of

movement-related behavior. Whether such alterations are directly or indirectly

related to the effects of cocaine is unclear. A model that relates alterations in

the relative activity of ventral striatal output systems to specific behavioral

responses is yet to be developed. However, the present results suggest that

although cocaine self-administration may not produce as great an imbalance in

the output systems of the nucleus accumbens, the fact that both systems show

elevated gene regulation indicates that normal activity in this striatal subregion

is also altered by cocaine.

Although it has been proposed that the differential effects of dopamine on

striatonigral and striatopallidal neurons are mediated by the specific expression

of D1 and D2 dopamine receptors by these two types of output neurons,

respectively (Gerfen et al. 1990), the cocaine-induced effects reported here

do not necessarily indicate a predominant D1 receptor-mediated response.

In a previous study, peptide mRNA levels in striatonigral and striatopallidal

neurons were shown to be specifically altered by D1- or D2-selective agonists,

respectively (Gerfen et al. 1990). However, those drug treatments were

given to animals with lesions of the nigrostriatal dopamine pathway and thus

examined the consequence of activation mediated by one receptor type in the

absence of the other. In animals self-administering cocaine, both types of

dopamine receptors would presumably be activated simultaneously. In this

circumstance, there is considerable evidence that there are complex synergistic

interactions between D1 and D2 dopamine receptors (Carlson et al. 1985; Clark

and White 1987; Walters et al. 1987; Wieck and Walters 1987). One

such interaction is the synergistic effect of D1 and D2 dopamine receptors,

which has been well documented (Carlson et al. 1985; Clark and White 1987;

Walters et al. 1987). The cellular basis of interactions between the dopamine

receptor subtypes has not been established, but at least three likely

mechanisms may be postulated. First, although the majority of striatal

projection neurons express only one of the D1 and D2 receptor subtypes,

there may be a subset of neurons that express both. Second, there may be

interactions between neurons expressing different receptor subtypes through

local axon collaterals. Third, striatal interneurons may mediate interactions

between D1- and D2-expressing neurons. An example of this type of

interaction may involve acetylcholine modulation of D2-containing striatopallidal

86
neurons, acetylcholine release being induced by substance P (Arenas et al.

1991; Petitet et al. 1991) released from D1-containing striatonigral neurons

and acting on substance P receptors that are expressed by cholinergic neurons

(Gerfen 1991). These possible mechanisms mediating interactions among

striatal neurons are not exhaustive. Thus, the direct action of dopamine on

D1- and D2-containing striatonigral and striatopallidal neurons should be

considered an important, but not the only, mechanism by which dopamine

modulates the balance in the activity of these two striatal output pathways.

Changes in levels of mRNA-encoding peptides in striatopallidal and

striatonigral neurons provide an instructive assay of functional changes

induced by pharmacologic manipulation of the striatal dopamine system.

However, changes in peptide mRNA levels often take several days of repeated

treatments to develop to significant levels. The induction of transcription factors

such as c-fos in striatal neurons following a single pharmacologic treatment

provide an assay that supplies information concerning the acute actions of

dopamine receptor activation. Such studies have provided data that are

consistent with data obtained by using peptide mRNA assays in identifying

D1 receptor activation as specifically targeting striatonigral neurons (Robertson

et al. 1990). The use of c-fos induction has also proved instructive in studying

the acute actions of cocaine and amphetamine on striatal neurons (Graybiel

et al. 1990). This approach, when combined with identification of the neurons

in which the transcription factors are induced, promises to provide significant

new information about the cellular and molecular actions of cocaine and other

psychostimulant drugs.

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6-hydroxydopamine lesioned rats: D1/D2 coactivation induces potentiated

responses. Brain Res 405:234-246, 1987.

Young, W.S. III; Bonner, T.I.; and Brann, M.R. Mesencephalic dopaminergic

neurons regulate the expression of neuropeptide mRNAs in the rat forebrain,

Proc Natl Acad Sci U S A 83:9827-9831, 1986.

90
AUTHOR

Charles R. Gerfen, Ph.D.

Laboratory of Cell Biology

National Institute of Mental Health

National Institutes of Health

Building 36, Room 2D-10

Bethesda, MD 20892

91
Regulation of Neural Gene Expression

in Opiate and Cocaine Addiction

Eric J. Nestler, Clare M. Bergson, Xavier Gultart, and

Bruce T. Hope

INTRODUCTION

Although some aspects of drug addiction can occur relatively rapidly in

response to acute administration of a drug of abuse, most changes in brain

function associated with addiction occur gradually over time in response to

prolonged drug exposure. These gradually developing changes can persist for

days, sometimes much longer, after cessation of chronic drug administration.

The prominence of such delayed, progressively developing, and persistent

adaptations in brain function during drug addiction suggests that long-term

changes in neuronal gene expression are likely to be important in mediating

addictive phenomena.

Mechanisms Regulating Gene Expression

It is increasingly apparent that the processes controlling the expression of

neural genes, even in the fully differentiated adult brain, are extraordinarily

complex. However, in recent years, much has been learned about the

molecular mechanisms that control the expression of specific genes in the

central nervous system (CNS). Attention has focused primarily on a class

of DNA-binding proteins, termed “transcription factors,” that bind to specific

sequences of DNA (response elements) in the regulatory regions of certain

genes and thereby increase or decrease the rate at which those genes are

transcribed (for review, see Mitchell and Tjian 1989; Goodman 1990; Montminy

et al. 1990; Morgan and Curran 1991). The mechanism for this transcriptional

activation/repression is not fully understood but likely involves direct or indirect

interactions of transcription factors with the RNA polymerase II transcriptional

complex (Ptashne and Gann 1990).

A large and increasing number of transcription factors are known, and these

exhibit distinct structural, functional, and regulatory properties. A list of some

prominent classes of transcription factors in the CNS is shown in table 1.

Based on the few neural genes that have been characterized to date, it would

92
appear that many, and perhaps all, genes contain multiple response elements

such that the rate of expression of a given gene at any given point in time is

dependent on the integrated effects of many transcription factors.

Transcription factors contain a DNA-binding domain (e.g., zinc finger or

homeodomain) that is rich in positively charged amino acids. Many

transcription factors, such as the Fos/Jun family, also contain an additional

structural motif (e.g., LZ) that enables them to dimerize and thereby form an

active DNA-binding protein complex. In fact, different members of the Fos/Jun

family have been shown to dimerize with each other, and such heterodimers

TABLE 1. Examples of transcription factors and their DNA-binding sites

Transcription Factor Binding Site Response Element Structural Motif

CREB family CRE TGACGTCA LZ

Fos/Jun family AP-1 TGACTCA LZ

Zif/268 GC rich ? ZF

SP1 GC box GGGCGG ZF

Steroid receptor

family (e.g., GR)

GRE GGTACAN3TGTTCT ZF

POU family

(e.g., OCT, Plt)

POU ATTTGCAT HD

AP-2

(protein unidentified)

AP-2 CCCCAGGC ?

SRF SRE GATGTCCATA- ?

TTAGGACATC

HSF HSE ? ?

KEY: LZ=leucine zipper; ZF=zinc finger: HD=homeodomain; GR=glucocorticoid

receptor; SRF=serum response factor; HSF=heat shock factor; GC=guanosine

and cytosine; GRE=glucocorticoid response element; SRE=serum response

element; HSE=heat shock response element

SOURCE: Adapted from Mitchell and Tjlan 1989; Goodman 1990; Morgan and Curran

1991

93
appear to possess different DNA-binding properties, transcriptional activities,

stability, etc. (Morgan and Curran 1991; Ryseck and Bravo 1991). Such

formation of heterodimers has significant consequences as it dramatically

increases the number of functional transcriptional complexes that can be

formed from a finite number of factors. In addition, transcription factors

contain an activation domain, which is thought to be the region of the proteins

responsible for regulation of transcription (Ptashne and Gann 1990). Each

class of transcription factor has been shown to bind to a particular DNA

response element. However, these DNA-binding sites, such as those shown

in table 1, represent only “consensus sequences” in that the actual sites to

which the factors bind in different genes can vary. For example, functional

cyclic AMP response elements (CREs) in various neural genes are not all

“TGACGTCA”; in fact, only the last five nucleotides appear to be constant

among all known CREs (Goodman 1990; Montminy et al. 1990). In addition,

it can be seen from table 1 that certain response elements share considerable

homology; for example, the CRE and activator protein-1 (AP-1) sites. These

observations raise the possibility that the specificity of the cyclic AMP (cAMP)

response element binding protein (CREB) and Fos/Jun families for CREs and

AP-1 sites, respectively, is not absolute, with Fos/Jun potentially able to bind

CRE sites and vice versa. It has even been reported that members of the

Fos/Jun and CREB families, both of which contain the LZ motif, can crossdimerize and thereby form heterodimers with unique DNA-binding and

regulatory properties (Hai and Curran 1991). These examples of crossreactivity between different classes of transcription factors illustrate the

enormous complexity of possible mechanisms underlying the regulation of

neural gene expression.

Most forms of transcription factors are highly regulated, and it is through such

regulatory processes that important aspects of neural plasticity (including drug

addiction) would appear to be achieved. Four mechanisms appear to be most

prominent. First, the total amounts of certain transcription factors can be

dramatically altered via changes in their own transcription. This is the case

for the Fos/Jun family; the expression of these proteins is regulated in the

adult CNS in response to diverse types of stimuli (Morgan and Curran 1991).

Second, the ability of transcription factors to regulate gene expression can

be altered via their phosphorylation by diverse types of protein kinases. The

prototypical example for this is CREB, whose ability to regulate transcription

is controlled primarily through its phosphotylation by cAMP-dependent or

calcium-dependent protein kinases (Goodman 1990; Montminy et al. 1990;

Sheng et al. 1990, 1991; Van Nguyen et al. 1990). It appears that CREB

phosphorylation does not dramatically alter its DNA-binding activity but instead

increases the ability of CREB, bound to DNA, to activate the RNA polymerase II

transcriptional complex. Third, the ability of transcription factors to bind DNA

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can be regulated by their binding to other molecules. An example of this would

be inhibitory variants of transcription factors that do not bind DNA but rather

dimerize with the active, DNA-binding forms of the protein and thereby inhibit

their transcriptional activity. Such variant forms appear to exist for most

transcription factors, including Fos/Jun and CREB (Yen et al. 1991; Foulkes

et al. 1991). Fourth, the levels of transcription factors in the nucleus can be

regulated by altering the rate at which they are translocated from the cytoplasm

(where they are synthesized) to the nucleus. The prototypical example of this

are the steroid hormone receptors, which are normally cytoplasmic proteins

that, on binding their specific hormone, translocate into the nucleus where they

act as transcription factors (Evans and Arriza 1989). These four mechanisms

are not mutually exclusive; most transcription factors are probably regulated by

more than one of them. This is illustrated by the steroid hormone receptors,

which are regulated not only by hormone binding and nuclear translocation

but also by their phosphorylation by several types of protein kinases and by

regulation of their total amount (e.g., Evans and Arriza 1989; Hoeck et al. 1989;

Denner et al. 1990).

A Strategy To ldentify Changes In Gene Expression That Underlie Drug

Addiction

The complexity of genetic regulatory mechanisms suggests that the complete

identification of the precise steps by which drugs of abuse alter gene

expression in the brain to produce addiction is an extremely difficult task.

In an attempt to overcome these obstacles, the authors’ laboratory has

followed a particular strategy to begin to understand this process, This

strategy, summarized in the list below, involves the study of anatomically

well-defined, discrete brain regions where behavioral pharmacological

studies have indicated an important role in addictive behaviors and where

electrophysiological studies have identified some functional changes in

specific neurons that underlie the addictive behaviors. In this context,

researchers at the authors’ laboratory have set out first to identify the

specific proteins whose altered expression may underlie the functional

changes observed electrophysiologically. These target proteins are then

used to study the genetic mechanisms underlying their altered expression.

The advantage of this strategy is that it focuses biochemical and molecular

studies on phenomena that are directly relevant to drug addiction. The strategy

for establishing the role of transcription factors in drug addiction is as follows:

1. Identify proteins whose altered levels of expression contribute to functional

aspects of drug addiction in identified neuronal cell types.

2. Study drug regulation of candidate transcription factors that could be

involved.

95
3. Provide direct evidence for a causal role of a specific transcription factor by

transfection or viral infection studies.

Together, these studies establish the precise molecular steps through which

a drug of abuse alters the expression of some target protein and thereby

produces addictive changes in the CNS.

IDENTIFICATION OF BIOCHEMICAL ADAPTATIONS INVOLVED IN DRUG

ADDICTION

Studies in the Locus Coeruleus

The locus coeruleus (LC) is the major noradrenergic nucleus in brain, located

on the floor of the fourth ventricle in the rostral pons. The electrophysiological

effects of opiates in the LC are well established. Acutely, opiates inhibit LC

neurons via the activation of a K+

 channel and the inhibition of a slowly

depolarizing Na+

 channel (Aghajanian and Wang 1987; North et al. 1987).

Both actions are mediated via pertussis toxin-sensitive G proteins (G1

 and/or

Go (Aghajanian and Wang 1986; North et al. 1987), and regulation of the Na+

channel is mediated by reduced levels of neuronal cAMP and of activated

cAMP-dependent protein kinase (Wang and Aghajanian 1987, 1990; Alreja

and Aghajanian 1991). Chronically, LC neurons develop tolerance to these

acute inhibitory actions as neuronal firing rates recover toward control levels

(Aghajanian 1978; Christie et al. 1987). The neurons also become dependent

on opiates after chronic exposure in that abrupt cessation of opiate treatment,

such as through administration of an opiate receptor antagonist, leads to a

severalfold elevation in LC firing rates above control levels in vivo (Aghajanian

1978; Rasmussen et al. 1990). A variety of pharmacological and behavioral

studies have indicated that such regulation of LC neuronal excitability

contributes to physical aspects of opiate addiction, namely, physical

dependence and withdrawal (Rasmussen et al. 1990).

The tolerance and dependence that occur in LC neurons during chronic opiate

exposure cannot be explained by changes in opiate receptors (Christie et al.

1987) and this led our laboratory several years ago to consider the possibility

that intracellular messenger pathways may be involved. Since that time, it has

been demonstrated that chronic administration of opiates leads to a dramatic

up-regulation of the cAMP system at every major step between receptor and

physiological response. Chronic opiates increase levels of (Nestler

et al. 1989), adenylate cyclase (Duman et al. 1986), cAMP-dependent protein

kinase (Nestler and Tallman 1988), and several phosphoprotein substrates

for the protein kinase (Guitar-t and Nestler 1989, 1990), including tyrosine

hydroxylase (Guitart et al. 1990), the rate-limiting enzyme in the biosynthesis

96
of catecholamine neurotransmitters. These adaptations require chronic

exposure to the opiates and are blocked by concomitant adminstration of

an opiate receptor antagonist, indicating that persistent activation of opiate

receptors is involved.

The up-regulation of adenylate cyclase and cAMP-dependent protein kinase

can be viewed as a compensatory, homeostatic response of LC neurons to

persistent opiate inhibition of the cells (figure 1) (Nestler and Tallman 1988).

According to this view, up-regulation of the cAMP system increases the intrinsic

excitability of LC neurons and thereby accounts, at least in part, for opiate

tolerance, dependence, and withdrawal. That is, in the opiate-dependent state,

the combined presence of the opiate and the up-regulated cAMP system would

return LC firing rates to control levels, whereas removal of the opiates would

leave the up-regulated AMP system unopposed, which would lead to withdrawal

activation of the neurons, Several lines of evidence support this scheme.

First, cAMP and agents that elevate cAMP levels excite LC neurons via the

activation of cAMP-dependent protein kinase and the subsequent activation

of the slowly depolarizing Na+ channel (Wang and Aghajanian 1990). In fact,

the basal firing rate of LC neurons appears to be entirely dependent on the

activity of the cAMP system and of this channel (Alreja and Aghajanian 1991).

Second, the time course by which certain components of the up-regulated

cAMP system revert to normal during opiate withdrawal parallels the time

course by which withdrawal activation of LC neurons and various behavioral

signs of withdrawal recover (Rasmussen et al. 1990). Third, recordings from

LC neurons in brain slices in vitro, where most synaptic connections of the

neurons have been severed, have indicated that LC neurons from dependent

animals exhibit basal firing rates more than twofold greater than those from

control animals (Kogan et al. 1992). This finding establishes that intrinsic

mechanisms contribute to opiate dependence in these cells. Moreover, LC

neurons from dependent animals show a dramatically greater maximal

responsiveness to cAMP analogs, providing functional evidence for an upregulated cAMP system during dependence (Kogan et al. 1992). Taken

together, these findings demonstrate that up-regulation of the cAMP system

represents one mechanism by which opiates induce addiction in these neurons.

Opiate-induced up-regulation of the cAMP system in the LC occurs at both the

level of protein and messenger RNA (Nestler et al. 1989; Guitart et al. 1990;

Nestler 1992), consistent with the possibility that the changes arise through the

regulation of gene expression. As described below, opiate regulation of these

proteins offers a model system in which to study the mechanisms by which

opiates alter gene expression and produce addictive changes in target neurons.

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ACUTE OPIATE ACTION IN THE LC

CHRONIC OPIATE ACTION IN THE LC

FIGURE 1. Schematic illustration of the mechanisms of acute and chronic

opiate action in the LC. See text for discussion.

VIP-R=vasoactive intestinal polypeptide receptor

SOURCE: Modified from Nestler 1992. Copyright 1992 by Society for

Neuroscience (New York).

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A General Role for G Proteins and the cAMP System in Opiate Addiction

Alterations in levels of G proteins and the cAMP system may represent a

general mechanism by which certain types of neurons within the CNS respond

to opiates and develop addiction (Terwilliger et al. 1991a). Thus, chronic

morphine has been shown to increase levels of (1) adenylate cyclase and

cAMP-dependent protein kinase in dorsal root ganglion/spinal cord cocultures,

amygdala, and nucleus accumbens and of (2) the protein kinase alone in the

thalamus (Makman et al. 1988; Terwilliger et al. 1991a). As in the LC, upregulation of the cAMP system in dorsal root ganglion/spinal cord cocultures

could account for some of the electrophysiological changes associated with

opiate tolerance, dependence, and withdrawal in these neurons (Crain and

Shen 1990).

Opiate regulation of G proteins is more regionally variable, with increased

levels of observed in the amygdala (as seen in the LC) and

decreased levels observed in dorsal root ganglion/spinal cord cocultures and

nucleus accumbens (Attali and Vogel 1989; Terwilliger et al. 1991a). Whether

levels of these G proteins increase or decrease in response to chronic morphine

may be related to the development of homologous vs. heterologous forms of

desensitization, respectively, in the specific neuronal cell types (Terwilliger

et al. 1991a).

Studies of Morphine and Cocaine Action in the Mesolimbic Dopamine

System

The nucleus accumbens (NAc), together with dopaminergic neurons in the

ventral tegmental area (VTA) that innervate the NAc, is implicated in mediating

psychological aspects of addiction, namely, drug reinforcement and craving,

for opiates and many other drugs of abuse (Wise and Bozarth 1987; Koob

and Bloom 1988; Clouet et al. 1988). Thus, it was of particular interest to

test whether chronic cocaine might produce changes similar to morphine in

G proteins and the cAMP system in the VTA-NAc pathway. Indeed, chronic

cocaine was found to decrease levels of (Nestler et al. 1990) and to

increase levels of adenylate cyclase and cAMP-dependent protein kinase in

the NAc (Terwilliger et al. 1991 a). Morphine and cocaine regulation of these

intracellular messenger proteins, summarized in figure 2, was not observed in

the other major dopaminergic system in the brain, the nigrostriatal system; nor

was such regulation seen in response to other classes of psychotropic drugs

that lack reinforcing properties. These biochemical actions of cocaine could

account for the supersensitivity of D1-dopamine receptor function observed in

this brain region in response to chronic cocaine in electrophysiological studies

(Henry et al. 1989; Henry and White 1991). Although the electrophysiological

99
FIGURE 2. Common adaptations in G proteins and the cAMP system in the

NAc in response to chronic morphine and chronic cocaine. G1

immunoreactivity of subunit; A cyclase, adenylate cyclase

activity; CA kinase, cAMP-dependent protein kinase activity

in particulate (P) and soluble (S) fractions. Data represent

means±SEM (n=8 to 74). All changes shown in the figure are

statistical/y significant by test.

SOURCE: Data from Terwilliger et al. 1991a

effects of chronic morphine action on NAc neurons have not yet been studied,

similar effects compared with chronic cocaine would be predicted based on

the biochemical findings.

Morphine and cocaine were also shown to exert common chronic actions on

several phosphoproteins in the VTA-NAc pathway, including increased levels

of tyrosine hydroxylase in the VTA, lower levels of active tyrosine hydroxylase

in the NAc, and lower levels of several forms of neurofilament proteins in the

VTA (Beitner-Johnson and Nestler 1991; Beitner-Johnson et al., in press).

Regulation of these morphine- and cocaine-regulated phosphoproteins

(MCRPP), like the changes in the G proteins and the cAMP system, showed

regional and pharmacological specificity.

In addition to their regional and pharmacological specificity, further evidence to

support the idea that morphine and cocaine regulation of G proteins, adenylate

cyclase, cAMP-dependent protein kinase, and the various MCRPPs is related

to drug reward mechanisms includes the findings that differences in these

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intracellular messenger proteins exist in the VTA-NAc pathway of Lewis and

Fischer 344 rats. Lewis rats self-administer opiates, cocaine, and alcohol at

much higher rates than Fischer rats (George and Goldberg 1989; Suzuki et al.

1988) and also develop greater degrees of conditioned place preference to

morphine and to cocaine (Terwilliger et al. 1991b), Interestingly, the NAc of

Lewis vs. Fischer rats contains lower levels of G,, higher levels of adenylate

cyclase and cAMP-dependent protein kinase, and lower levels of tyrosine

hydroxylase, whereas the VTA of Lewis vs. Fischer rats contains higher levels

of tyrosine hydroxylase and lower levels of neurofilaments (Beitner-Johnson

et al. 1991; Guitart et al. 1991; Terwilliger et al. 1991b), In each case, levels

of these specific intracellular signaling proteins in the VTA-NAc pathway of

Lewis rats, compared with Fischer rats, resemble morphine- and cocaineinduced changes in these proteins in outbred Sprague-Dawley rats.

The common chronic actions of morphine and cocaine in the mesolimbic

dopamine system are particularly striking since the two drugs exert opposite

electrophysiological effects on VTA neurons acutely (Henry et al. 1989), yet

both are clearly addicting. Therefore, the common chronic actions could

represent a general mechanism by which these drugs of abuse exert some of

their long-term reinforcing actions in this neural pathway. The findings in Lewis

and Fischer rats raise the additional, exciting possibility that similar mechanisms

may contribute to individual genetic vulnerability to drug addiction (Nestler, in

press).

GENETIC MECHANISMS UNDERLYING BIOCHEMICAL ADAPTATIONS IN

THE cAMP SYSTEM

General Scheme

Based on the knowledge of opiate and cocaine action in specific brain regions

and of the mechanisms underlying the regulation of gene expression, one can

formulate a working scheme to explain how these drugs of abuse might alter

the expression of genes for specific target proteins in particular neuronal cell

types. Two possible regulatory mechanisms involving the CREB and Fos/Jun

families of transcription factors are shown in figure 3. These two mechanisms

are not mutually exclusive, nor are they the only types of mechanisms that

could be involved, because any of the transcription factors, or a combination

of factors, shown in table 1 could conceivably play a role. However, evidence

for neurotransmitter regulation of transcription factors is best established for

the CREB and Fos/Jun families. Moreover, several of the known target genes

for opiates and cocaine (e.g., cAMP-dependent protein kinase, tyrosine

hydroxylase, neurofilaments) are known to contain cAMP and/or AP-1

response elements (Cambi et al. 1989; Reeben et al. 1991; S. McKnight,

personal communication, September 1990).

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FIGURE 3. Schematic illustration of the intracellular messenger pathways

through which diverse extracellular signals, including drugs of

abuse such as opiates, could regulate gene expression in target

neurons. The figure illustrates two general types of mechanisms

that could be involved. In one mechanism, opiates, through the

regulation of second messengers and protein kinases, regulate

the phosphorylation of a CREB-like transcription factor. This

alters the transcriptional activity of CREB and leads to altered

expression of specific target proteins. In the second mechanism,

regulation of CREB phosphorylation leads to altered expression of

a fos-like transcription factor. Altered levels of fos, in turn, lead to

altered expression of specific target proteins.

SOURCE: Modified from Nestler 1992. Copyright 1992 by the Society for

Neuroscience (New York).

102
As discussed in the introduction, CREB proteins are generally thought to be

constitutively expressed in neurons, with their transcriptional activity regulated

primarily via their phosphorylation by cAMP- or calcium-dependent protein

kinases. According to the scheme shown In figure 3, opiates in the LC, via

their initial effects on the cAMP pathway (via inhibition of adenylate cyclase)

(Duman et al. 1988) or calcium pathway (via inhibition of neuronal firing)

(Aghajanian 1978; Christie et al. 1987) could lead to decreases in the

phosphorylation state of CREB and to subsequent changes in gene expression.

Because this mechanism involves the regulation of a transcription factor that is

already present in the neurons, it could begin to occur very rapidly (i.e., within

minutes). The specificity for opiate action in this scheme would reside in (1)

the presence of opiate receptors, (2) the ability of the activated receptors to

regulate the cAMP and calcium pathways, and (3) the particular genes

accessible to CREB, in a given neuronal cell type.

In contrast to CREB, the Fos/Jun family of transcription factors tends to be

expressed at low levels in neurons under basal conditions. In response to

diverse types of extracellular signals, high levels of expression of these

transcription factors are induced via increases in rates of transcription and

mRNA translation (for review, see Morgan and Curran 1991). It appears that

phosphorylation of CREB, or a CREB-like protein, by cAMP- or calciumdependent protein kinases mediates the effects of such extracellular signals

on Fos expression (Sheng et al. 1990); similar mechanisms may underlie the

regulation of the other transcription factors in the Fos/Jun family. Thus,

regulation of Fos and Fos-like transcription factors represents an additional

mechanism by which opiates could regulate the expression of other genes via

alterations in the cAMP or calcium systems (figure 3). Because this mechanism

requires protein synthesis, it is slower than the first mechanism described

above, with earliest changes in Fos/Jun target gene expression expected

perhaps 30 to 60 minutes following the initial stimulus.

The two regulatory schemes presented in figure 3 represent a first attempt to

explain the actions of opiates on gene expression in the LC and other target

neurons. As more is learned about the regulation of the CREB, Fos/Jun, and

other families of transcription factors, more complete hypotheses can be

formulated to account for the actions of opiates, cocaine, and other drugs of

abuse on neural gene expression.

Regulation of Specific Transcription Factors by Drugs of Abuse

Regulation by Opiates. As an initial attempt to study the transcription

factors that mediate opiate action in the LC, Hayward and colleagues (1990)

studied regulation of Fos expression in response to acute and chronic opiate

103
administration and during opiate withdrawal. It was found that acute opiates

decrease levels of Fos mRNA and protein in this brain region. This reduced

level of Fos expression persists with chronic opiate treatment, whereas

precipitation of opiate withdrawal by an opiate receptor antagonist produces

a dramatic induction of Fos at the mRNA and protein levels (figure 4). The

observed changes in Fos levels are consistent with the possibility that

increased levels of expression of cAMP-dependent protein kinase,

and tyrosine hydroxylase in the LC during chronic opiate treatment could be

mediated to some extent via decreased levels of Fos and that the return of

levels of expression of these intracellular messenger proteins toward control

levels during withdrawal could be achieved as a result of elevated Fos

expression. Induction of Jun mRNA was also observed during opiate

withdrawal (Hayward et al. 1990), indicating that several members of the

Fos/Jun family could be involved in these processes.

The induction of Fos and Jun during opiate withdrawal was observed in several

brain regions in addition to the LC, including the NAc, VTA, neostriatum, and

frontal cortex, but not in other regions, notably hippocampus and whole pons

(figure 5) (Hayward et al. 1990). Regulation of Fos expression in the NAc and

VTA indicates that this class of transcription factor could contribute to opiate

actions on the G proteins and the cAMP system in the mesolimbic dopamine

system via a mechanism similar to the one suggested above for the LC.

What does induction of Fos during withdrawal reflect? It has been suggested

that induction of Fos can be used as a molecular measure of neural activity

(Morgan and Curran 1991), and indeed, depolarizing stimuli would be

expected to induce Fos via activation of the calcium pathway. However,

such a relationship between Fos expression and neural activity is not absolute,

since Fos can be induced by a cAMP signal (Sheng et al. 1990; Morgan and

Curran 1991) and cAMP is known to exert negative effects on the firing rate

of particular neurons (see below for cocaine). Thus, a rigorous interpretation

of these data is that induction of Fos can be used to map those neurons in the

brain in which the intracellular cAMP and/or calcium systems become activated

during opiate withdrawal or other drug treatments.

The recent development of a back phosphorylation and immunoprecipitation

procedure has made it possible to study morphine regulation of CREB

phosphorylation in the rat LC (Guitart et al. 1992). This procedure results in the

specific immunoprecipitation of a 43-kD phosphoprotein that represents CREB:

The protein comigrates with purified, phosphorylated CREB on one- and twodimensional gel electrophoresis and yields identical phosphopeptide maps as

does the purified protein. By use of this procedure, it has been demonstrated

that acute morphine administration decreases the phosphorylation state of

104
FIGURE 4. Autoradiograms showing regulation of Fos mRNA in the LC in

response to acute and chronic morphine treatment and opiate

withdrawal. A: Control, subcutaneous (SC) administration of

normal saline to control rats; tolerant, SC administration of saline

to morphine-tolerant rats; and withdrawal, SC administration of

naltrexone to morphine-tolerant rats. Animals were sacrificed 1

hour after the injections. B: Control, SC administration of normal

saline to control rats and acute, SC administration of morphine

to control rats. Animals were sacrificed 90 minutes after the

injections, and levels of Fos mRNA were analyzed (using 5 mg

of total RNA) by Northern blotting.

SOURCE: Hayward et al. 1990. Copyright 1990 by Elsevier Science

Publishers (Amsterdam).

105
FIGURE 5. Autoradiograms showing regulation of Fos mRNA in various

regions of rat brain in response to opiate withdrawal. Opiate

withdrawal was precipitated in morphine-tolerant rats by SC

administration of naltrexone. Animals were sacrificed 1 hour

after the initiation of withdrawal, and levels of Fos mRNA were

analyzed (using 5 to 10 mg of total RNA) by Northern blotting.

AM, amygdala; VT, ventral tegmental area; NA, nucleus

accumbens; NS, neostriatum (caudate/putamen); CT whole

cerebral cortex; PNS, whole cross-sections of anterior pons

from which LC nuclei had been excised; PGI, nucleus

paragigantocellularis; HP, hippocampus; DR, dorsal raphe;

PAG, periaqueductal gray.

SOURCE: Hayward et al. 1990. Copyright 1990 by Elsevier Science

Publishers (Amsterdam).

CREB in the LC, an effect that diminishes after chronic exposure to the

opiate. In contrast, precipitation of opiate withdrawal increases CREB

phosphorylation in this brain region (Guitart et al. 1992). This regulation

of CREB phosphorylation is consistent with the known effects of acute and

chronic opiates and opiate withdrawal on the activity of the cAMP system in

the LC.

These studies of opiate regulation of transcription factors, although preliminary,

highlight the utility of the LC as a model system in which to study transcription

factor regulation. The LC is a system in which specific candidate target genes

have been identified and in which changes in those genes have been shown to

106
be physiologically important. Through increasingly mechanistic studies of this

system, it will be possible to delineate the precise molecular steps by which

opiates regulate the expression of specific intracellular messenger proteins and,

as a result, induce aspects of tolerance, dependence, and withdrawal in these

neurons.

Regulation by Cocaine. In a series of related experiments, the authors’

laboratory has begun to study the effects of acute and chronic cocaine on

Fos expression in the mesolimbic dopamine system. Hope and Nestler

(1991) and Hope and colleagues (in press) have replicated the findings of

other groups (Graybiel et al. 1990; Young et al. 1991) showing that acute

administration of cocaine (at a dose of 15 mg/kg intraperitoneal [IP]) induces

Fos mRNA levels in the NAc (figure 6) and the caudate/putamen 45 minutes

after drug administration (Hope and Nestler 1991; Hope et al., in press).

Moreover, it was shown that acute cocaine elicits a similar induction in mRNA

levels for Jun and zif. (Zif is a transcription factor that, although binding to a

distinct response element, is regulated in several circumstances in a fashion

similar to Fos and Jun.) As systemic administration of cocaine is known to

inhibit a majority of neurons in the NAc (White et al. 1987), cocaine induction

of Fos and related transcription factors represents one example, alluded to

above, where these proteins can be induced in the absence of an increase in

neuronal activity. One possible mechanism of cocaine action, which can be

tested directly, is that the drug induces Fos via the indirect activation of D1-

dopamine receptors and the subsequent activation of the cAMP pathway,

which also exerts inhibitory effects on these cells electrophysiologically. An

alternative possibility that the authors do not favor but cannot exclude is that

cocaine induces Fos in the minority of neurons in the NAc that are activated

by systemic administration of the drug.

Chronic administration of cocaine (15 mg/kg IP b.i.d. for 14 days) almost

completely abolishes the ability of a subsequent acute dose of cocaine

(administered 18 hours after the previous dose) to induce Fos mRNA levels

in these two brain regions (figure 6) (Hope and Nestler 1991; Hope et al., in

press). A similar “desensitization” was observed for the ability of cocaine to

induce jun and zif.

To further investigate this apparent desensitization, Hope and Nestler (1991)

and Hope and colleagues (in press) studied acute and chronic regulation of

AP-1 binding activity in the NAc using the gel shift assay (figure 6). It was

found that acute cocaine dramatically induces AP-1 binding activity in this

brain region 2 hours following drug administration, indicating that induction of

Fos and Jun mRNA is associated with an increase in the functional activity of

these or related transcription factors. However, chronic cocaine administration

107
FIGURE 6. Regulation of Fos mRNA and of AP-1 binding activity by acute

and chronic cocaine in the rat NAc. Control rats received twice

daily IP injections of saline for 14 days and an additional saline

injection on day 15; as with the controls, acute rats received

saline injections for 14 days but received a single injection of

cocaine hydrochloride (15 mg/kg) on day 15; chr+ac rats received

twice daily injections of cocaine for 14 days and an additional

cocaine injection on day 15. Animals were sacrificed 45 minutes

108
(for analysis of Fos mRNA levels) or 2.5 hours (for analysis of AP1 binding activity) after their last injection on day 15. A: Levels

of Fos mRNA in the NAc of control and treated rats analyzed by

Northern blotting. B: Levels of AP-1 binding activity in the NAc

of control and treated rats analyzed by gel shift assays. Data

represent means±SEM (n=4 to 8).

SOURCE: Data from Hope and Nestler 1991; Hope et al., in press

appears to produce a persistent increase in AP-1 binding activity in the NAc:

Levels of AP-1 binding are elevated 18 hours after the last chronic dose of

cocaine whether or not an additional acute dose is administered. These

observations indicate that chronic cocaine leads to a persistent increase in

Fos- and/or Jun-like proteins.

If confirmed, these findings suggest that chronic cocaine produces a gradual

change in the protein composition of the AP-1 complex in NAc neurons.

For example, chronic cocaine not only could produce a desensitization to

Fos responsiveness but also a concomitant accumulation of various Fos- or

Jun-related transcription factors. Such a change in the composition of the

AP-1 complex could alter its transcriptional activity and/or specificity (e.g.,

see Ryseck and Bravo 1991) and thereby lead to some of the changes in gene

expression seen in response to chronic cocaine in this brain region. In other

words, such a change in the composition of the AP-1 complex could represent

a “molecular switch” underlying some of the chronic actions of cocaine (Hope

et al., in press). To test this possibility directly, it will be necessary to develop

antibodies that distinguish the numerous individual members of the Fos and

Jun family in immunocytochemical and immunoblotting studies. The

immunocytochemical studies offer the additional possibility of identifying

subsets of NAc and striatal neurons that exhibit differential immediate early

gene responses to acute and chronic cocaine administration.

As a first attempt to identify the specific proteins that account for the increased

AP-1 binding activity observed under acute and chronic cocaine treatments,

levels of Fos and Jun protein in the NAc by immunocytochemical techniques

are now being studied in collaboration with Drs. Barry Kosofsky and Steven

Hyman at Harvard. Acute cocaine treatment induces a dramatic increase in

levels of Fos-like immunoreactivity, as seen for Fos mRNA, in the NAc and

caudate/putamen. However, similar to the observations with northern blotting,

preliminary evidence suggests that chronic cocaine decreases the inducibility

of Fos-like immunoreactivity (Hope et al., in press). In the striatum, this

desensitization in Fos induction shows an interesting medial to lateral gradiant

109
indicating that subpopulations of neurons respond differentially to chronic

cocaine.

Future Studies

The types of studies described above have been and will continue to be

useful in identifying specific types of transcription factors that could be involved

in opiate and cocaine regulation of gene expression in specific target brain

regions, However, at best, these studies can offer only circumstantial evidence

for the possible role of a given transcription factor in mediating drug action on

the expression of specific target proteins. Ultimately, more direct evidence is

required to establish a causal relationship between a given transcription factor

and target gene. Such direct evidence could theoretically be obtained by

monitoring the transcriptional activity of a target gene under conditions where

the activity of a specific transcription factor (or of a second messenger/protein

phosphorylation pathway involved in the functioning of that factor) is specifically

altered.

Our laboratory is testing the use of a nonpathogenic strain of herpes simplex

virus-1 (HSV-1) (Dobson et al. 1990) as a vehicle for introducing DNA into

specific neuronal cell types in vivo. In this procedure, the recombinant virus is

injected in the vicinity of a brain region of interest, where it appears to be able

to infect a certain percentage of the nearby neurons (C.M. Bergson and E.J.

Nestler, unpublished observations, June 1991). An example of how this

procedure could be used to more directly study possible transcriptional

mechanisms involved in drug addiction is described below for the case of

opiate regulation of tyrosine hydroxylase gene expression in the LC. A fusion

gene subcloned into the recombinant HSV-1, consisting of part of the regulatory

region of the tyrosine hydroxylase gene coupled to a reporter gene-any gene

whose expression can be easily monitored (e.g., genes for ß-galactosidase,

chloramphenicol acetyltransferase, or luciferase)—can be introduced into LC

neurons via HSV-1 infection. If chronic morphine regulation of tyrosine

hydroxylase in the LC occurs at the level of gene expression as inferred from

the mRNA data (see above), treatment of rats with chronic morphine would

also be expected to increase expression of the reporter gene. Next,

introduction of fusion genes with mutated response elements in the tyrosine

hydroxylase promoter could then be used to determine which response

element (or combination of response elements) is required for such

transcriptional regulation. The direct involvement of a specific transcription

factor in this process could also be tested by introducing DNA that, when

transcribed, encodes an RNA strand that is antisense for a given transcription

factor mRNA. The antisense RNA would hybridize with the sense mRNA and

thereby prevent it from being translated into the transcription factor under

110
investigation. Ultimately, similar types of experiments could be carried out

using transgenic animals, where the constructed genes would be introduced

into all somatic cells, including the relevant neurons through germ line

transmission. At very early stages of investigation, these examples illustrate

how the precise mechanisms by which drugs of abuse regulate gene

expression can be delineated using molecular biological techniques.

CONCLUSIONS

This chapter described the experimental strategy that the authors’ laboratory is

using to investigate the role of changes In gene expression in the development

of opiate and cocaine addiction. The strategy involves the study of discrete

brain regions that are known to contribute to behavioral aspects of drug

addiction and in which electrophysiological studies provide insight into the

functional changes at the neuronal level that underlie the addictive behaviors,

This makes it possible to understand biochemical and molecular actions of the

drugs within a functional context. The focus is on identifying intracellular

signaling proteins that are regulated by chronic drug treatments at the level

of gene expression. These target proteins are then used to investigate the

mechanisms by which the drugs of abuse alter the expression of the genes

encoding them. To date, it has been possible to identify alterations in G

proteins and the cAMP system in specific regions of the CNS in response to

chronic opiates and/or chronic cocaine. Of particular interest is the upregulation of the cAMP system observed in the LC in response to chronic

opiates (biochemical adaptations shown to contribute to opiate tolerance,

dependence, and withdrawal exhibited by these neurons) and a similar upregulation of the cAMP system observed in the mesolimbic dopamine system

in response to chronic opiates and chronic cocaine, which could be related to

drug reinforcement or craving mediated via this neural pathway. Current

studies are aimed at investigating opiate and cocaine regulation of transcription

factors in these discrete brain regions. Ultimately, it will be possible to expand

on the correlative data resulting from these studies with more direct analyses

that make use of in vivo methods for detecting changes in target gene

expression. Together, these studies of opiate and cocaine action will help

define the precise mechanisms, at the molecular level, by which these drugs

of abuse alter the expression of specific genes in particular neuronal cell types

and thereby produce physical and psychological aspects of addiction.

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ACKNOWLEDGMENTS

This work was supported by US. Public Health Service grants DA-05490,

DA-07359, and DA-04060; by the McKnight Endowment Fund for the

Neurosciences; and by the Abraham Ribicoff Research Facilities of the

Connecticut Mental Health Center, State of Connecticut Department of Mental

Health.

AUTHORS

Eric J. Nestler, M.D., Ph.D.

Elizabeth Mears and House Jameson Associate Professor of Psychiatry and

Pharmacology

Clare M. Bergson, Ph.D.

Postdoctoral Fellow

Xavier Guitart, Ph.D.

Postdoctoral Fellow

Bruce T. Hope, Ph.D.

Postdoctoral Fellow

Laboratory of Molecular Psychiatry

Departments of Psychiatry and Pharmacology

Yale University School of Medicine and Connecticut Mental Health Center

34 Park Street

New Haven, CT 06506

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C-Fos and Fos-Related Antigens

as Markers for Neuronal Activity:

Perspectives From Neuroendocrine

Systems

Gloria E. Hoffman, Wen-Sen Lee, M. Susan Smith, Rula Abbud,

Michelle M. Roberts, Alan G. Robinson, and Joseph G. Verbalis

INTRODUCTION

The alterations in neuronal function underlying drug abuse are complex, yet

important. Several specific neuronal systems have been implicated in drugseeking behavior, addiction, and withdrawal, but none has emerged as key.

What may be needed is an approach to mark neurons throughout the brain that

are influenced by drugs of abuse. Once targeted neurons are identified, studies

can be extended to define their phenotype and projections and to determine

their functions in drug-related processes. The use of immediate early gene

(IEG) products may provide an important tool for labeling neurons whose

activity has changed as a result of drug treatment. As is detailed below, c-Fos

and Fog-related antigens (FRAs) serve as markers for identifying neuronal

systems whose activity is increased or decreased in response to a stimulus.

In situ hybridization of IEG messenger RNAs (mRNAs) as well as

immunocytochemical localization of protein products are useful for addressing

this issue. The use of the protein products in examining specific systems can

have advantages over the study of mRNA. First, molecular probes are not

available for all IEGs, whereas antisera have been generated that recognize

the protein products of some genes not yet cloned. Second, IEG products

such as c-Fos, c-Jun, and Zif/268, when present, concentrate within the

cell nucleus (Sheng and Greenberg 1990). This feature makes it possible to

label these proteins along with cytoplasmic markers for the neurotransmitter

(Ceccatelli et al. 1969; Hoffman et al. 1990; Jacobson et al. 1990) and/or

retrogradely transported tracers (Menetrey et al. 1969) using standard double

immunocytochemical techniques. For heterogenous neuronal populations, or

diffusely organized neuronal systems, this capability is imperative. In contrast,

mRNA is located in the cytoplasm. Techniques for double labeling of either two

117
mRNAs or one mRNA and an immunocytochemically identified marker are

not yet sufficiently reliable to be easily applied; generally, there is great

compromise in the quality of either the mRNA label, the immunocytochemical

marker, or both. Another advantage to localizing the proteins is that their

appearance is delayed by at least 30 to 45 minutes after the stimulus is

delivered, unlike the mRNA, which increases within 5 to 10 minutes after

stimulation. This delay allows the investigator to move or manipulate the

animal prior to sacrifice without concern that such handling will result in IEG

expression. The case is well illustrated for studies of light activation. In this

instance, for study of mRNA changes, animals must be sacrificed under

safelight conditions, whereas immunocytochemical analysis would allow

an animal to be anesthetized and brought into the light for perfusion

without allowing sufficient time for the light stimulus to be translated into

new IEG proteins. A disadvantage in using IEG products localized by

immunocytochemistry rather than by in situ hybridization of their mRNA is

the lack of precise quantitation inherent in the immunocytochemical methods.

In this chapter, the authors present evidence that, in spite of this problem,

localization of IEG products with standard immunohistochemical techniques

permits assessment of relative changes in gene expression. The studies

presented in this chapter use natural stimuli and document that the expression

of the IEGs is not only induced by drug treatment or other experimental

manipulation but also is involved in normal homeostatic function.

C-FOS AS A MARKER FOR NEURON ACTIVATION: LUTEINIZING

HORMONE-RELEASING HORMONE NEURONS AS THE MODEL

Most of the data presented here focus on the authors’ studies of neuroendocrine

regulation. In particular, study of the regulation of reproductive function has

been most revealing. The study of neuroendocrine-adenohypophyseal function

has presented challenges to physiologists in that conventional means of

assessing neuronal activity are impractical owing to the small size and scattered

distribution of the neuroendocrine neurons regulating the anterior pituitary. The

case in point is well illustrated for one neuroendocrine system: the luteinizing

hormone-releasing hormone (LHRH) (also known as gonadotropin-releasing

hormone or GnRH) neurons, which stimulate the release of both luteinizing

hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary.

LHRH neurons are small, few in number, and widely scattered. The rat brain

contains only 1,200 LHRH neurons (Wray and Hoffman 1986a, 1986b), 70

percent of which send their axons to the median eminence to effect LH and FSH

release (Merchanthaler et al. 1989). The LHRH cells are scattered over the full

rostral-to-caudal extent of the forebrain and generally do not reside within classic

cytoarchitectonic boundaries (Hoffman and Gibbs 1982; Silverman et al. 1982),

To make matters worse, the LHRH somata typically measure only 8 to 10 μm in

diameter: even in sites where the LHRH neurons are most numerous, they are

116
interspersed among non-LHRH neurons, making the possibility of successfully

locating the cells and then recording from them remote.

Means have been devised for monitoring LHRH secretory activity. The most

direct method involves cannulation of the tiny portal blood vessels that carry

LHRH released at the median eminence of the hypothalamus to the anterior

pituitary. With this approach, during an LH surge the output of LHRH increases

(Sarkar et al. 1976). However, the necessity for anesthesia in performing these

studies precludes accurate quantitative assessment of changes in LHRH that

occur in the awake state. In sheep, portal blood collection is possible in the

awake animal, and measurements of LHRH reveal increases in LHRH release

that accompany LH pulses in the basal state, with dramatic increases during an

LH surge (Moenter et al. 1990). Yet the methods employed in sheep cannot be

easily adapted to smaller mammals. An alternative has been the use of pushpull cannulae placed either in the median eminence or in the anterior pituitary.

LHRH measured by push-pull techniques shows pulsatility during baseline

states and moderate, but clear, increases in LHRH output during an LH surge

(Levine and Ramirez 1982; Park and Ramirez 1989). Unfortunately, damage

to the axons that are under investigation invariably accompanies the collection

of samples. Moreover, although data from push-pull experiments convincingly

link increases in LHRH secretion with increases in LH secretion, they tell little

about the stimulation of LHRH neurons at a cellular level. Measurement of

changes in IEG expression provided the first direct evidence that cellular LHRH

activity accompanied LH surges (Hoffman et al. 1990; Lee et al. 1990a, 1990b).

LHRH neurons normally do not express c-Fos. However, c-Fos expression is

induced within 45 minutes following electrochemical stimulation and during

induced or spontaneous LH surges (Hoffman et al. 1990; Lee et al. 1990a,

1990b). An analysis of the location of stimulated LHRH neurons expressing

c-Fos during the peak of an LH surge identifies a subpopulation of LHRH

neurons in the preoptic area in the vicinity of the organum vasculosum of the

lamina terminalis (OVLT), below the anterior commissure, which extends into

the anterior hypothalamus (figure 1). Interestingly, the LHRH neurons located

above the anterior commissure and rostral to the OVLT remain “quiet” during

an LH surge. Yet these same cells will express c-Fos after electrochemical

stimulation. During an LH surge, the activated LHRH population may have

been targeted by selective innervation of LHRH neurons, which, when

contacted by catecholamine or neurotensin axons (Hoffman 1985), are

distributed in a pattern similar to that observed for c-Fos expression in LHRH

neurons during a surge (Hoffman et al. 1990; Lee et al. 1990a, 1990b).

In the authors’ studies, female rats were cannulated on the morning of the

proestrus LH surge, and blood samples were monitored throughout the day at

30- to 60-minute intervals until the time of sacrifice. These were assayed by

radioimmunoassay for LH. Each animal then was anesthetized and perfused

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FIGURE 1. Distribution of c-Fos-activated LHRH cells. The panels illustrate the location of LHRH neutrons that

express c-Fos and those that are devoid of c-Fos immunoreactivity throughout the rostra/

forebrain. (A) The components of the LHRH cell field scattered within the vertical limb of the diagonal

band of Broca, rostral to the OVLT, do not express c-Fos during an LH surge. (B) At a Ievel close to the

OVLT, the LHRH neurons are most numerous, and approximately half express c-Fos. (C) More

caudalty, within the preoptic area, LHRH neurons express c-Fos; again, approximate half the cells are

stimulated. (D) At the junction of the preoptic area and anterior hypothalamus, c-Fos activation within

LHRH neurons continues.

SOURCE: Modified from Lee et al. 1990b
transaortically first with saline containing 2 percent sodium nitrite and followed

by 4 percent buffered paraformaldehyde (pH 6.8) containing 2 to 2.5 percent

acrolein (EM grade). The brains were removed, sunk in 25 percent aqueous

sucrose solution, and cut into 12 series of 25 μm sections, which were stored

in cryoprotectant solution (Watson et al. 1966) until immunocytochemical

procedures for c-Fos and LHRH were initiated. Immunocytochemical staining

was accomplished with specific antisera for LHRH (LR-1) and antisera

generated against the amino acids within the N-terminus of c-Fos (Cambridge

Research Biochemicals sheep anti c-Fos, OA11-821; Dr. Tom Curran’s anti-alu

Fos). Sequential staining of first c-Fos followed by LHRH was performed with

the ABC “elite” procedure allowing primary antisera concentrations to be

1:44,000-50,000 for the c-Fos antisera and 1:100,000 for anti-LHRH (when

double immunoperoxidase methods were used; if immunofluorescence was

used instead, the concentration of anti-LHRH was increased to 1:30,000).

The c-Fos staining following visualization of peroxidase activity with a nickel

diaminobenzidine chromogen appeared blue black; LHRH reactivity was

revealed with either diaminobenzidine (immunoperoxidase reactions), which

stained golden brown, or Texas Red (immunofluorescence procedure), which

fluoroesced red after excitation in the green range. An example of LHRH

neurons activated during an LH surge stained with the immunofluorescent

procedure is shown in figure 2.

On analysis of plasma LH levels, the authors noted that rats whose plasma

LH levels were high 30 to 60 minutes before the time of sacrifice had greater

levels of c-Fos expression within their LHRH neurons than did rats whose

LH levels were low prior to sacrifice (figure 3). Analysis of plasma LH and

c-Fos immunoreactivity in LHRH neurons revealed a highly significant linear

relationship (figure 4), indicating that the amplitude of LH secretion reflects the

number of LHRH neurons activated. Further investigation of the relationship

between LHRH stimulation and LH secretion was aimed at the use of a model in

which the amplitude of an LH surge was attenuated by prevention of the actions

of progesterone. Intact female rats were treated at 12:30 p.m. on the afternoon

of proestrus with the progesterone antagonist RU 486 (5 mg, subcutaneously

[SC]) and compared with untreated proestrus rats. In a separate series of

experiments, castrated female rats treated with estradiol or with progesterone

provided a second similar model. Rats were ovariectomized and 2 weeks

later were administered 1 μg estradiol benzoate subcutaneously (SC) at 9:00

a.m. Twenty-four hours later a second injection of estradiol benzoate (50 μg

SC) was administered (also at 9:00 a.m.) followed by an SC injection of

progesterone (5 mg) or vehicle at 12:30 p.m. Plasma LH was monitored as

described above. In animals treated with RU 486 and in castrates receiving

only estrogen, the blunting of the LH peak was accompanied by a decrease in

the degree of LHRH c-Fos expression (figure 5) (Lee et al. 1990b). Close

examination of the LHRH neurons revealed that, in addition to showing fewer

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FIGURE 2. C-Fos expression in LHRH neurons. C-Fos (arrows) is expressed

in LHRH neurons during an LH surge. (A) Bright-field image

shows that the nuclei of two LHRH neurons in the field contain

c-Fos. (B) The same section viewed under fluorescence

microscopy reveals immunofluorescent LHRH neurons (arrows)

close to the OVLT that express c-Fos in their nuclei.

LHRH neurons activated when the actions of progesterone were prevented or

blocked, progesterone-deprived rats showed a reduced intensity of c-Fos

protein immunoreactivity within their nuclei (figure 6). Thus, progesterone

appeared not only to recruit more LHRH neurons into the active state but also

increased the extent of stimulation of individual cells. More generally, these

data indicate that c-Fos expression is graded to the intensity of the stimulus

and, as was revealed by progesterone deprivation, is not all or none. Further

confirmation of this feature has been obtained for hypovolemic stimulation of

magnocellular neurons of the hypothalamus (Roberts et al., submitted for

publication), in which, following delivery of increasing hemorrhagic stimuli, c-Fos

immunoreactivity within vasopressin and oxytocin neurons increased both in

terms of the number of neurons expressing c-Fos and the intensity of the

staining within each stimulated neuron. An example showing the general

changes in c-Fos expression in the paraventricular nucleus is shown in figure 7.

The changes in c-Fos expression paralleled the changes in hormone secretion.

FRAs AS MARKERS FOR BASELINE ACTIVITY: THE

TUBEROINFUNDIBULAR DOPAMINE NEURONS AS THE MODEL SYSTEM

The analysis of LHRH, vasopressin, and oxytocin neurons, although

demonstrating that c-Fos can effectively serve as a marker for stimulated

122
FIGURE 3. C-Fos activation in LHRH neurons during the rising phase of

an LH surge. A plot from an animal whose plasma LH had

only begun to rise within the hour prior to sacrifice (A) is

compared with a plot from a rat whose plasma LH had nearly

reached peak values (B). LHRH neurons expressing c-Fos are

depicted as closed squares LHRH neurons devoid of c-Fos

immunoreactivity are shown as open squares Note that the

higher degree of c-Fos activation in LHRH is associated with a

greater elevation of plasma LH.

SOURCE: Lee et al., in press, copyright 1992, by permission of Oxford

University Press (Oxford, England).

activity, did not allow the authors to analyze changes in baseline activity.

Screening antisera that might allow a broader picture of IEG expression and

examining other neuroendocrine systems provided some insight into this

problem. Recently, Dragunow and Faull (1990) as well as Jacobsen and

colleagues (1990) observed that staining with antisera generated against the

M-peptide region of c-Fos revealed many nuclei in the cerebral cortex of control

animals, whereas staining with an N-terminally directed c-Fos antiserum (or in

situ hybridization of c-Fos mRNA) showed little or no baseline c-Fos expression.

Since Western blots of the M-peptide antiserum indicated recognition of multiple

proteins, it was reasoned that, in baseline conditions, some FRAs, but not

c-Fos, are expressed. Consequently, the authors sought to determine if the

basal FRA expression could be manipulated in a fashion to allow the

examination of decreases in activity following stimulus delivery.

The test system was the tuberoinfundibular dopamine system. This

neuroendocrine system, located within the arcuate nucleus of the

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FIGURE 4. Relationship between the percent of LHRH neurons that

expressed c-Fos and plasma LH values 30 to 60 minutes prior

to sacrifice on the afternoon of proestrus. Each point represents

one animal. R=0.957; p<0.001.

SOURCE: Modified from Lee et al., in press, copyright 1992, by permission of

Oxford University Press (Oxford, England).

hypothalamus, tonically inhibits prolactin release. Following the onset

of suckling, prolactin release markedly increases, due at least in part to

an inhibition of the dopamine’s inhibitory tone (Moore 1987). Groups of

lactating rats (postpartum day 12) continually suckling their pups and

diestrus rats were compared for the presence or absence of FRA proteins

within dopamine neurons of the arcuate nucleus. An antiserum generated

against the M-peptide region of c-Fos (generated by Dr. Michael ladarola)

served as the FRA marker and was used at a concentration of 1:15,000.

Staining for the enzyme tyrosine hydroxylase (TH) with an anti-TH serum

served as a marker for dopamine in the hypothalamic neurons. Staining

124
FIGURE 5. Effects of progesterone on the magnitude of the LH surge and

activation of LHRH neurons. (A) Effect of administration of the

progesterone antagonist RU 486 on the amplitude of the LH

surge (shaded bar) and degree of c-Fos expression within LHRH

neurons (striped bar) at the time of the peak of the LH surge. For

comparison, the values of the RU 486-treated rats are depicted

along with untreated proestrus rats. (B) Effect of administration

of estrogen only or estrogen plus progesterone to ovariectomized

rats on the magnitude of the LH surge (shaded bar) and degree

of c-Fos expression (striped bar) in LHRH neurons at the time of

the peak of the LH surge.

SOURCE: Lee et al. 1990a

125
FIGURE 6. Effect of removal of progesterone’s influence on intensity of

c-Fos protein in activated LHRH neurons. In addition to

reducing the number of neurons activated during an LH surge,

ovariectomized rats replaced with estrogen only showed a

marked reduction in the intensity of c-Fos staining in LHRH

neurons. (A) Ovariectomized rat treated with estrogen and

progesterone; (B) ovariectomized rat treated only with estrogen.

Similar results were obtained following treatment of intact cycling

rats with RU 486.

SOURCE: Lee et al. 1990a

126
FIGURE 7. Responses of neurons of the paraventricular nuclei to graded

hemorrhage, Increasing stimuli to the magnocellular neurons of

the paraventricular nuclei resulted in increasing levels of c-Fos

expression. (A) Control, (B) 2 cc blood removed, (C) 4 cc blood

removed, (D) 6 cc blood removed.

127
strategies were identical to those described above for LHRH and c-Fos.

Approximately half the dopamine neurons within the arcuate nucleus of diestrus

rats expressed FRA immunoreactivity (figure 8, panel A). In contrast, FRA

immunoreactivity was absent in arcuate dopamine neurons from lactating rats

suckling their pups (figure 8, panel B). These data demonstrate that FRA

immunoreactivity can reveal reductions in activity as a result of the suckling

stimulus within a specific inhibitory neuron population. The suckling stimulus,

or consequences of hormonal changes resulting from suckling, appears to

affect a great many brain systems in addition to the dopamine neurons.

Analysis of FRA immunoreactivity throughout the brain, including the neocortex

and hippocampus, revealed a marked reduction in baseline levels in lactating

rats compared with cycling rats. An example of the pattern of FRA staining in

FIGURE 8. FRA expression in the dopamine neurons of the arcuate nucleus

in (A) a diestrus and (B) a lactating female rat. FRA proteins,

normally expressed within the dopamine neurons, are no longer

expressed in suckling rats.

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FIGURE 9. Baseline FRA expression in the somatomotor cortex. (A) In the

diestrus rat many neurons thoughout all cell layers express FRAs.

(B) In the lactating rat the baseline FRA expression in the cerebral

cortex is markedly reduced.

the neocortex of a diestrus rat and a lactating rat is shown in figure 9. These

changes are consistent with the attenuation of many responses to stress

observed in lactating rats and offer another example of the use of FRA staining

as a means of identifying neuronal populations whose activity diminishes in

response to neuronal stimuli and/or hormonal changes,

REPRODUCTIVE STATE AS A VARIABLE IN STUDYING BRAIN

ACTIVATION: LACTATION AND STIMULATION BY NMA

Lactating rats, in addition to showing blunted behavioral changes to stress, fail

to possess spontaneous pulses of LH release. In investigating the possible

mechanisms of this reproductive quiescence, the authors treated lactating

animals with the excitatory amino acid N-methyl-aspartate (NMA) at doses that

stimulate LH secretion in cycling rats (Smith and Lee 1990). Attention in these

studies was returned to c-Fos as a marker for stimulated activity. Lactating rats

and a group of diestrus female rats were fitted with jugular cannulae, and four

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FIGURE 10. Induction of c-Fos in the cerebral cortex of an NMA-treated rat

(A) Diestrus female rats show strong induction of c-Fos in the

somatomotor cortex following subconvulsive doses of NMA. (B)

Similar treatment of lactating rats fails to induce c-Fos in the

cerebral cortex. The patterns of c-Fos expression appeared to

mirror the behavioral effects of NMA.

pulses of 40 mg/kg NMA were administered at 10-minute intervals. Ninety

minutes following the last dose of NMA, the rats were anesthetized and

perfused for localization of c-Fos. In the diestrus rats, administration of NMA

elicited signs of hyperactivity, but no seizures. In contrast, administration of

NMA to lactating rats did not induce behavioral changes. Examination of c-Fos

revealed marked induction of c-Fos in neurons throughout the neural axis of the

diestrus rats, whereas the same treatment of lactating rats did not induce c-Fos

immunoreactivity. Examples of c-Fos staining within the neocortex of a diestrus

and lactating rat treated with NMA are shown in figure 10. Initial studies

exploring the recovery of excitability in lactating rats reveal that both the

suckling stimulus as well as progesterone participate in the dampening of NMA

responsiveness (Abbud et al. 1991). These studies not only provide another

example of IEG products as effective tools for studying neuronal activation but

also illustrate the importance of the animal’s endocrine state in determining the

effects of drugs on the nervous system.

130
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neocortical refractoriness to NMA stimulation in the lactating rat as revealed

by c-Fos expression: Recovery after pup removal and blockade of

progesterone receptors. Soc Neurosci Abstr 17:907, 1991.

Ceccatelli, S.; Villar, M.J.; Goldstein, M.; and Hokfelt, T. Expression of c-fos

immunoreactivity in transmitter characterized neurons after stress. Proc Nat/

Acad Sci U S A 86:9569-9573, 1989.

Dragunow, M., and Faull, R.L.M. MK-801 induces c-fos protein in thalamic

and neocortical neurons of rat brain. Neurosci Lett 11:39-45, 1990.

Hoffman, G.E. Organization of LHRH cells: Differential apposition of

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1985.

Hoffman, G.E., and Gibbs, F.P. LHRH pathways in the rat brain:

Deafferentiation spares a subchiasmatic LHRH projection to the median

eminence. Neuroscience 7:1979-1993, 1982.

Hoffman, G.E.; Lee, W.; Attardi, B.; Yann, V.; and Fitzsimmons, M.D. LHRH

neurons express c-fos after steroid activation. Endocrinology 126: 1736.

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Jacobson, L.; Sharp, F.R.; and Dallman, M.F. Induction of fos-like

immunoreactivity in hypothalamic corticotropin-releasing factor neurons

after adrenalectomy in the rat. Endocrinology 126:1709-1719, 1990.

Lee, W.-S.; Smith, M.S.; and Hoffman, G.E. Progesterone enhances the surge

of luteinizing hormone by increasing the activation of luteinizing hormonereleasing hormone neurons, Endocrinology 127:2604-2606, 1990a.

Lee, W.-S.; Smith, M.S.; and Hoffman, G.E. Luteinizing hormone-releasing

hormone (LHRH) neurons express c-Fos during the proestrous LH surge.

Proc Natl Acad Sci U S A 87:5163.5167, 1990b.

Lee, W.-S.; Smith, MS.; and Hoffman, G.E. c-Fos activity identifies recruitment

of LHRH neurons during ascending phase of proestrous luteinizing hormone

surge. J Neuroendocrinol, in press.

Levine, J.E., and Ramirez, V.D. Luteinizing hormone-releasing hormone

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G.E. c-Fos expression in vasopressin and oxytocin neurons reveals

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ACKNOWLEDGMENTS

The studies reported in this manuscript were supported by National Institutes of

Health grants NS-27014, NS-28730, HD-14643, and HD-21350 and National

Science Foundation grant BNS-8919953. Thomas C. Waters provided

photographic assistance.

AUTHORS

Gloria E. Hoffman, Ph.D.

Associate Professor

132
Wen-Sen Lee, Ph.D.

Research Associate

M. Susan Smith, Ph.D.

Professor

Rula Abbud

Department of Physiology

University of Pittsburgh School of Medicine

El 440 Biomedical Science Tower

3500 Terrace Street

Pittsburgh, PA 15261

Michelle M. Roberts, M.D.

Associate Professor

Alan G. Robinson, M.D.

Professor

Joseph G. Verbalis, M.A.

Professor

Department of Medicine

University of Pittsburgh School of Medicine

El 140 Biomedical Science Tower

3500 Terrace Street

Pittsburgh, PA 15261

133
Mechanisms of Opioid-Mediated

Antinociception: Correlation of Fos

Expression and Behavior

Kathleen R. Gogas, Jon D. Levine, and Allan I. Basbaum

INTRODUCTION

Recent studies have emphasized the utility of monitoring the expression of

immediate early genes (IEGs) to map functionally relevant pathways in the

central nervous system (CNS) (Hunt et al. 1987; Morgan et al. 1987; Sager

et al. 1988). The authors’ laboratory has been studying the expression of the

c-fos gene to characterize the circuitry underlying the transmission of noxious

(i.e., painful) information into the spinal cord (Menétrey et al. 1989; Presley

et al. 1990; Gogas et al. 1991). Despite the rapid growth of this new field,

many questions are still unsettled as to the appropriateness of c-fos message

or Fos protein as a marker of neuronal activity and as to the stimulus specificity

of Fos expression. Thus, the goals of this chapter are twofold. First, it will be

discussed how the expression of c-fos can be used in the author’s system to

identify which populations of spinal cord neurons are activated by noxious

stimulation and to evaluate the effects of known analgesics on noxious

stimulus-evoked Fos expression. Second, the question of stimulus specificity

will be addressed by comparing the pattern of c-fos expression produced by

nonnoxious as well as noxious stimuli.

SPINAL CORD NOCICEPTIVE CIRCUITRY

The authors’ analyses of the expression of the c-fos gene has been done

primarily in the spinal cord dorsal horn. This region is the first CNS site for the

transfer of nociceptive (i.e., pain) messages from the periphery and, thus, plays

a key role in nociception. The dorsal horn has been divided on cytoarchitectural

grounds into several discrete laminae, numbered I through VI from dorsal to

ventral. High threshold (i.e., nociceptive) myelinated A-S and unmyelinated C

primary afferent fibers carry nociceptive information from the periphery to the

dorsal horn; A-o fibers synapse on neurons in lamina I, the outer portion of

lamina II, and lamina V; C-fibers predominantly synapse on neurons in laminae

134
I and outer II (Light and Perl 1979; Sugiura et al. 1987). Consistent with the

anatomical projection of the primary afferent fibers, electrophysiological studies

have established that neurons in laminae I and Ilo respond predominantly to

noxious input; lamina V neurons respond to both noxious and nonnoxious

stimuli and are thus referred to as wide-dynamic range neurons (for a review,

see Besson and Chaouch 1987). Cells in the nucleus proprius (laminae III, IV)

and in lamina VI are predominantly driven by nonnoxious stimulation. Finally,

many neurons in laminae VII and VIII of the ventral horn and in lamina X,

around the central canal, also respond to noxious stimulation.

Although they provide important information about the functional properties of

spinal neurons, electrophysiological techniques have limitations. Specifically,

since only one cell is analyzed at a time, it is not possible to monitor the

responses of large populations of neurons to noxious stimulation. Thus,

sample size is typically small. Furthermore, since the animals are almost

always anesthetized and/or spinalized, it is not possible to correlate physiology

with behavioral responses to stimulation. Even when recording is done in

awake animals (e.g., Collins 1987), one is still limited by the number of cells

that can be recorded.

THE USE OF FOS EXPRESSION TO ASSESS THE ACTIVITY OF NEURONS

IN THE CNS

Hunt and colleagues (1987) were the first to show that immunocytochemical

localization of the Fos protein product of the c-fos protooncogene can be

used to monitor the “activity” of large populations of neurons in the spinal cord

dorsal horn following either noxious or nonnoxious stimulation. As discussed in

detail elsewhere in this monograph, c-fos is the cellular homolog of a retroviral

transforming factor that induces osteosarcoma in susceptible strains of mice.

Transcription of the c-fos gene is among the earliest nuclear events that occur

when a cell is stimulated (Curran and Morgan 1985). Activation of transcription

can be produced by a variety of external stimuli, including neurotransmitters

and membrane depolarization (Curran and Morgan 1985; Greenberg et al.

1986; Morgan and Curran 1986). The latter stimuli in turn increase intracellular

calcium, cyclic AMP (cAMP), diacylglycerol, and other second messengers,

which, either directly or indirectly, stimulate transcription of the c-fos gene.

Although increased Fos expression is associated with cellular proliferation

and differentiation, its function in postmitotic neurons has not been established.

However, it is clear that the expression of c-fos as well as other IEGs (e.g.,

c-jun) acts as a link between the excitation of neurons and long-term adaptive

responses in the cell. Since the c-fos message appears within 5 to 10 minutes

of stimulation and the Fos protein within another 10 to 20 minutes, it has been

135
proposed that the expression of IEGs, either by immunocytochemistry or in situ

hybridization, provides a sensitive assay for assessing the activity of neurons in

the CNS.

Since the basal levels of Fos protein in the spinal cord are low, stimulusinduced increases in Fos expression are readily detected. In studies of c-fos

expression after noxious peripheral stimulation, it was first demonstrated that

the pattern of Fos immunoreactivity is consistent with the anatomical location

of nociresponsive neurons in the dorsal and ventral horn. Thus, following

noxious stimulation in anesthetized rats, Hunt and colleagues (1987) reported

that Fos immunoreactivity was concentrated in regions that contain neurons

that respond to noxious stimulation, including the superficial laminae (I and Ilo)

and in lamina V of the dorsal horn, Two important questions arose from that

study. First, would the pattern of Fos expression be similar in awake animals?

Second, would the magnitude of the behavioral responses to noxious stimuli

correlate with the numbers of Fos-immunoreactive neurons? To address these

questions, the authors developed a model whereby they could administer a

noxious stimulus to a rat, record its behavioral response to the stimulus, and,

most important, correlate the behavior with the levels of Fos expression in the

cord.

FOS EXPRESSION AFTER SOMATIC NOXIOUS STIMULATION

In the authors’ initial studies, Gogas and colleagues (1991) used the formalin

pain model in which a dilute concentration of formalin is injected into the

hindpaw of the rat. The formalin model has been well characterized both

electrophysiologically and pharmacologically (Dickenson and Sullivan 1987a,

1987b; Hunskaar et al. 1986; Drower et al. 1987). Formalin injection produces

a characteristic behavioral response that lasts for as much as 4 hours and can

be readily quantified in terms of intensity (Dubuisson and Dennis 1977). The

basic protocol was to inject the formalin into the plantar surface of the hindpaw.

The rats were then placed into an observation chamber so that their behavior

could be observed and recorded over a 1-hour period (Gogas et al. 1991).

The animals were then anesthetized and perfused, with the spinal cord tissue

removed and postfixed as previously described (Presley et al. 1990; Gogas et

al. 1991). The tissue was processed immunocytochemically with the avidinbiotin procedure of Hsu and colleagues (1981) using commercially available

kits (Vectastain ABC; Vectorlabs, Burlingame, CA). We have used a rabbit

polyclonal antiserum directed against an in vitro translated product of c-fos

(provided by Dr. Dennis Slamon of the University of California, Los Angeles).

An important advantage of Fos immunocytochemistry is that the results can be

studied quantitatively. To this end, the outlines of three sections through the

136
lumbar enlargement were made with a camera lucida attachment under

dark-field illumination; then under bright-field conditions the distribution of

Fos-positive cells was plotted with a 4X objective and a camera lucida.

To quantitate the anatomical results, the spinal cord drawings were divided

into four segments: (1) the superficial laminae (laminae I, Ilo, and Iii), (2) the

nucleus proprius (laminae Ill and IV), (3) the base of the dorsal horn (laminae

V and VI), and (4) the intermediate zone and the ventral horn (laminae VII,

VIII, IX, and X). The number of Fos-positive cells in each of these regions was

counted and averaged so that each animal had a mean value for regional Fos

expression. In addition, the sum of the mean regional values (i.e., superficial

Fostnucleus proprius Fos+neck Fos+ventral Fos) was used as a measure

of the total number of Fos-immunoreactive neurons in each animal. The

investigator responsible for plotting and counting the Fos neurons had no

knowledge of the drug treatment of each animal.

In light of the controversy concerning the factors that induce Fos expression,

a significant finding using the formalin stimulus is that the evoked Fos

immunoreactivity is largely found ipsilateral to the stimulated hindpaw

(figure 1). This finding reinforced the idea that the increased number of

Fos-positive cells was not related to handling or stressing of the animal during

the protocol. In addition, the rostral-caudal distribution of the formalin-evoked

Fos immunoreactivity was restricted to the lumbar cord (figure 2) with maximal

staining in the lumbar enlargement (L4-L5), the area that receives the densest

primary afferent innervation from the portion of the sciatic nerve territory at the

site of hindpaw injection. The greatest number of Fos-immunoreactive neurons

was seen in the superficial laminae of the cord (laminae I and Ilo), in the neck

of the cord (lamina V and VI), and in lamina VII and around the central canal.

Each of these areas has previously been shown to contribute to the processing

of nociceptive information in the cord (Besson and Chaouch 1987).

The time course of the Fos expression also closely mirrored the time

course of the behavioral response (figure 3). Thus, the maximal levels of

Fos immunoreactivity were seen at 1 to 2 hours post-formalin injection, with

decreasing activity over the next 6 hours; by 24 hours, almost no Fos staining

remained in the cord. Taken together with the finding that the level of basal

Fos expression in the cord is low, these results led to the conclusion that the

Fos immunoreactivity in the cord is related to the noxious nature of the formalin

stimulus and not to nonspecific factors secondary to the animal’s perception of

the stimulus (e.g., stress).

FOS EXPRESSION AFTER VISCERAL NOXIOUS STIMULATION

Further evidence in support of this hypothesis is derived from studies of the

pattern of Fos immunoreactivity evoked by a noxious visceral stimulus

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FIGURE 1. This photomicrograph illustrates the pattern of hindpaw formalinevoked Fos immunoreactivity in neurons of the L4-L5 spinal cord

of a rat that received an intraperitoneal (P) injection of saline.

There is dense labeling in the superficial layers (laminae I and II)

and in the neck of the dorsal horn (V). There are no Fosimmunoreactive neurons in the motoneuron (MN) pool. The

densest labeling is in the medial part of the superficial dorsal

horn; the lateral part (asterisk) does nor receive primary afferent

input from the plantar hindpaw. SG=substantia gelatinosa.

SOURCE: Presley et al. 1990. Copyright 1990 by the Society for

Neuroscience (New York).

(Menétrey et al. 1989). Since the pattern of visceral primary afferent

termination in the cord is different from that of somatic afferents (Cervero

1985; Sugiura et al. 1989), it was expected that the pattern of Fos expression

following visceral stimulation with formalin would differ. For this set of

experiments, rats were anesthetized and injected (IP) with acetic acid in the

right lower quadrant of the abdomen, which provides a model of visceral

stimulation (Taber et al. 1969). Several important differences were found

138
FIGURE 2. Camera lucida drawings showing the rostrocaudal distribution

of Fos-immunoreactive neurons 2 hours after formalin

injection into the ipsilateral plantar hindpaw. The most intense

immunoreactivity is noted at the L3-L5 segments, the major site

of termination of afferents from the plantar hindpaw. Labeling in

the superficial dorsal horn is concentrated in the L3-L5 segments,

whereas labeling in the neck of the dorsal horn and ventromedial

gray is present throughout the lumbar enlargement.

SOURCE: Presley et al. 1990. Copyright 1990 by the Society for

Neuroscience (New York).

139
FIGURE 3. Camera lucida drawings taken from the L4-L5 level of rats killed

at various times after plantar hindpaw formalin injection. Maximal

Fos immunoreactivity is present at 2 to 4 hours postinjection and

then it disappears slowly. Staining is barely above control by 16

hours postinjection.

SOURCE: Presley et al. 1990. Copyright 1990 by the Society for

Neuroscience (New York).

140
between the pattern of Fos immunoreactivity evoked by the visceral stimulus

and that observed in response to hindpaw formalin stimulation (figure 4). First,

the densest staining was in the caudal thoracic rather than the lumbar cord, as

was seen following formalin. Second, the staining following visceral stimulation

was largely restricted to laminae I and V of the dorsal horn and to the area

around the central canal. This different topographic distribution further argues

that the induction of Fos immunoreactivity in spinal neurons is not secondary

to stress but is related directly to the particular stimulus. Third, there was a

far more extensive rostral caudal distribution of the Fos immunoreactivity,

particularly in lamina I, where staining from the upper cervical to the sacral cord

was found. This is consistent with the known anatomy of the primary afferent

fibers that carry noxious visceral information; these fibers terminate over many

segments rostral and caudal to their major termination point in the cord (Sugiura

et al. 1989). The absence of Fos immunoreactivity in lamina II is also

remarkably consistent with the central termination pattern of visceral afferents

(Cervero 1985; Sugiura et al. 1989).

FOS EXPRESSION AFTER NONNOXIOUS STIMULATION

To further validate the use of Fos immunocytochemistry to monitor activity in

pain pathways in the CNS, it was important to show that the pattern of Fos

immunoreactivity in the cord following a nonnoxious stimulus was different from

that seen following a noxious stimulus. Initial work by Hunt and colleagues

(1987) suggested that this would be difficult to document. In their study,

nonnoxious stimuli produced only minimal increases in Fos immunoreactivity,

albeit in appropriate regions of the dorsal horn. However, Gogas and

colleagues (1990) found that allowing an animal to walk on a slow-turning

rotarod device for 1 hour evokes widespread Fos immunoreactivity in the

cord. Fos expression was notably lacking in laminae I and Ilo of the cervical

and lumbar segments. However, the inner portion of lamina II as well as

laminae III and IV, areas that contain neurons that respond exclusively to

nonnoxious stimulation (Bennett et al. 1980; Light et al. 1979), contained many

labeled cells. Additional staining was found in the medial portion of laminae V

and VI, an area that receives inputs from joint afferents (Brown and Fyffe 1978,

1979), and in lamina IX, where many Fos-immunoreactive MNs were found.

These results are important since they provide considerable support for the

hypothesis that Fos protein expression is stimulus specific and can, therefore,

be used to study those populations of neurons in the cord that are activated by

noxious stimulation. Furthermore, the modulation of noxious stimulus-evoked

Fos expression by pain-relieving drugs such as opioids can also be studied,

which can provide information about the mechanisms by which these

compounds produce analgesia.

141
FIGURE 4. These schematics illustrate the cervical (C5) through

lumbrosacral (L6-S1) distribution of Fos-immunoreactive cells in

response to noxious visceral stimulation. Note that the densest

staining is at the thoracolumbar junction (T13-L1), and the most

extensive rostrocaudal distribution of labeled cells was found in

the superficial dorsal horn.

SOURCE: Menérey, D.; Gannon, A.; Levine, J.D.; and Basbaum, A.I. 1989.

Journal of Comparative Neurology. Copyright © 1989 by WileyLiss. Reprinted by permission of Wiley-Liss, a division of John

Wiley and Sons, Inc.

142
THE EFFECTS OF ANALGESICS ON NOXIOUS STIMULUS-EVOKED FOS

EXPRESSION

Opioids produce their analgesic effects by acting at opioid receptors

(for a review, see Martin 1983) that are located at both supraspinal and spinal

levels (Basbaum and Fields 1978, 1984). At supraspinal sites, opioids are

believed to activate neurons in the midbrain periaqueductal gray, which in turn

excite neurons of the medullary nucleus raphe magnus. The latter neurons

project to and inhibit spinal cord nociresponslve neurons. Opioids also act at

opioid receptors in the spinal cord where they can directly inhibit incoming

nociceptive transmission in the dorsal horn (Yaksh and Noueihed 1985). If

formalin-evoked Fos immunoreactivity in the spinal cord is, as proposed,

related to the activity of nociresponsive neurons, then doses of an opioid,

such as morphine, that produces behavioral analgesia should also inhibit

noxious stimulus-evoked Fos expression.

THE EFFECTS OF SYSTEMIC MORPHINE ON FORMALIN-EVOKED FOS

EXPRESSION

Presley and colleagues’ (1990) initial studies using systemic morphine injection

supported the authors’ hypothesis. Systemic administration of morphine, 10

minutes prior to formalin injection, produced a dose-related inhibition of both

the pain behaviors and Fos immunoreactivity evoked by the formalin (figure

5). At the highest dose tested (10 mg/kg, IP), morphine produced a complete

suppression of the formalin-evoked behaviors and greatly attenuated the levels

of formalin-evoked Fos immunoreactivity; however, labeled cells could still be

detected in the superficial dorsal horn. The effects of this dose of morphine

were reversed by the opiate receptor antagonist, naloxone, consistent with

activity of morphine at an opiate receptor. The finding that morphine could

produce complete behavioral analgesia without completely blocking noxious

stimulus-evoked Fos expression at the level of the cord is important.

Apparently, analgesia can be achieved without completely “shutting off

the activity of dorsal horn neurons in the spinal cord.

As described above, it is important to show not only that the pattern of Fos

immunoreactivity is stimulus specific but also that the levels of Fos protein in

the cord are related to the expression of an overt, recordable behavior. In fact,

the number of Fos-immunoreactive neurons and the magnitude of the animal’s

behavioral response to formalin are closely related; animals that exhibit the

highest levels of pain-related behaviors have many Fos-immunoreactive

neurons in the spinal cord (figure 6). Conversely, animals that are completely

analgesic exhibit much lower levels of Fos immunoreactivity. This finding

provides further evidence that Fos expression is a valid measure of neuronal

activity.

143
FIGURE 5. Camera lucida drawings of representative L4-L5 sections showing

a dose-related, naloxone-reversible inhibition of formalin-evoked

Fos-like immunoreactivity (FLI) by morphine. The labeling in the

ventral gray matter is profoundly suppressed by 2.5 mg/kg; at any

given dose, the region with the most residual staining is the

superficial dorsal horn.

SOURCE: Presley et al. 1990. Copyright 1990 by the Society for

Neuroscience (New York).

144
FIGURE 6. Correlation between noxious stimulus-evoked “pain” behavior

and spinal cord FLI. The mean area-under-the-curve (AUC)

scores for behavior (abscissa) are plotted against the overall

mean FLI values (i.e., the mean sum of the superficial+nucleus

proprius +neck+ventral FLI; ordinate) for each of the six

intracerebroventricular (ICV) drug treatments used (0.006,

0.02, 0.06, 0.20, 0.60 μg DAMGO (D-Ala2

 NMe—Phe4, Gly-OL5-

enkephalin) or saline; n=3 per group). The expression of pain

behavior was significantly correlated with the number of neurons

expressing FLI (Pearson’s r=0.84, p<0.001).

SOURCE: Reprinted with permission from Neuroscience 42, Gogas,

K.R.; Presley, R.W.; Levine, J.D.; and Basbaum, A.I. The

antinociceptive action of supraspinal opioids results from an

increase in descending, inhibitory control: Correlation of

nociceptive behavior and c-fos expression, 1991, Pergamon

Press, plc.

That the number of Fos-immunoreactive neurons in individual regions of the

cord can be quantified further allows the differential regulation by morphine of

the activity of neurons in functionally distinct areas of the cord to be determined

(figure 7). Importantly, the dose-response curve for the effects of morphine

145
FIGURE 7. Dose of morphine vs. the number of Fos-immunoreactive neurons

in different laminar regions of the spinal gray matter (meant

SEM), including the supeficial dorsal horn (laminae I and II),

the nucleus proprius (laminae lIl and IV), the neck of the dorsal

horn (laminae V and VI), and the ventral gray (laminae VII, VIII,

and X). There was a statistically significant suppression of Fosimmunoreactive neurons after doses of morphine 2.5-10 mg/kg

(*p<0.05, **p<0.01) in the superficial dorsal horn, in the neck of

the dorsal horn, and in the ventral gray. The superficial dorsal

horn consistently contained the greatest number of residual

labeled cells; this was particularly evident at higher doses of

morphine (5.0 and 10.0 mg/kg). In contrast, labeling in the ventral

gray was eliminated by these doses. The dose-response curve

for the nucleus proprius was flat, with no statistically significant

suppression of labeling.

SOURCE: Presley et al. 1990. Copyright 1990 by the Society for

Neuroscience (New York).

146
on Fos expression in the nucleus proprius, the region that contains primarily

nonnociresponsive neurons, was flat; that is, no significant inhibition of Fos

expression at any of the morphine doses tested was found. This is consistent

with the fact that morphine predominantly inhibits nociresponsive neurons (Le

Bars et al. 1976a, 1976b). By contrast, the inhibition of Fos immunoreactivity

in the superficial dorsal horn, laminae V and VI, and the ventral horn of the cord

was dose related. The dose-response curves in laminae V and VI and

the ventral horn were steeper than in the superficial laminae. However, even

at the highest dose tested, which produced 100 percent inhibition of behavioral

analgesia, there was still significant residual "activity” in all areas, particularly

the superficial cord. Since a major component of the residual staining was

found in the marginal layer (lamina I) of the superficial dorsal horn, a region

that contains neurons that respond almost exclusively to noxious stimulation,

this finding is of particular interest. However, lamina I contains a mixture of

projection and interneurons. In a double labeling study, Menétrey and

colleagues (1969) found that approximately 45 percent of marginal layer

neurons that express Fos following noxious stimulation project to regions

of the brain that have been implicated in the rostral transmission of nociceptive

information. Conceivably, opioids preferentially inhibit the population of lamina I

projection neurons; that is, the residual labeling may be concentrated in a

population of interneurons that do not contribute to the “pain”-related response.

That retrograde labeling and Fos immunocytochemistry can be combined

(figure 8) makes it possible to determine whether opioids produce a selective

reduction in the number of retrogradely labeled marginal cells that express

Fos immunoreactivity. This possibility is being tested. Again, it is important

to emphasize that this son of experiment could not be done without using Fos

immunoreactivity as a marker for populations of neurons activated by noxious

stimulation.

THE EFFECTS OF SUPRASPINALLY ADMINISTERED OPIOIDS ON

NOXIOUS STIMULUS-EVOKED FOS EXPRESSION

Since systemically administered morphine produces analgesia via an action

at both spinal and supraspinal sites (Yaksh and Noueihed 1985; Basbaum

and Fields 1984), we could not distinguish the relative contribution of either

site to the inhibitory effects found. One approach to addressing this issue was

to separately test the effects of supraspinal and intrathecal (IT) administration

of morphine on formalin-evoked Fos expression in the cord. It was found

that administration of morphine into the third ventricle (ICV) or directly

onto the spinal cord (IT) inhibits both formalin-evoked behaviors and Fos

immunoreactivity (K.R. Gogas, G.I. Batchkine, H.J. Cho, and A.I. Basbaum,

manuscript in preparation). Since morphine is relatively nonselective for μ-,

receptors (Martin 1983), it is still not clear which opioid receptor subtype

147
FIGURE 8. This bright-field photomicrograph illustrates Fos-immunoreactive

and retrogradely labeled neurons in a 40-μm thick horizontal

section through laminaI of the lumbar dorsal horn. The

retrograde tracer WGAapoHRP-AU was injected into the

contralateral brain stem and thalamus. Fos-positive cells

have a uniformly stained nucleus Projection neurons

contain punctate cytoplasmic label that results from silver

intensification of the colloidal gold in the tracer; the nuclei

of single-labeled projection neurons are unstained

Double-labeled cells have a densely stained nucleus

and cytoplasmic silver precipitate.

SOURCE: Menétrey, D.; Gannon, A.; Levine, J.D.; and Basbaum, A.I. 1989.

Journal of Comparative Neurology. Copyright © 1989 by WileyLiss. Reprinted by permission of Wiley-Liss, a division of John

Wiley and Sons, Inc.

148
morphine is acting on to produce its effects. There is considerable controversy

concerning the specific circuitry by which oploids such as morphine produce

antinociception (e.g., Fang et al. 1986; Roerig and Fujimoto 1989; Heyman et

al. 1987). In addition, it is not clear whether activation of receptors

produces analgesia.

This pharmacological question is ideally suited to analysis with Fos

immunocytochemistry. Specifically, by studying the effects of selective

agonists for either opioid receptors on both formalin-evoked pain

behavior and on spinal cord Fos immunoreactivity, not only can it be determined

whether these compounds are analgesic, but also the circuitry through which

they produce their analgesic effects can be examined. We started this series

of experiments by studying the effects of supraspinal administration of the μselective agonist DAMGO on pain behaviors and on spinal cord Fos expression

evoked by noxious stimulation in rats. For ICV drug infusions, guide cannulae

were stereotaxically implanted in the third ventricle of rats. After surgery, the

animals were allowed to recover for 7 to 10 days prior to behavioral testing.

For dose-response studies, ICV injections were made 10 minutes prior to

injection of formalin into the hindpaw. Immediately thereafter, the animal

was placed into a Plexiglas chamber so that the animal’s movements could

be viewed and its behavior recorded over a 1-hour period as discussed above.

The laminar distribution of formalin-evoked Fos expression in the spinal

cord was similar to that seen previously (figure 9, panel A). DAMGO (ICV)

produced a dose-related, naloxone-reversible suppression of both the

nociceptive behaviors and spinal cord Fos immunoreactivity evoked by

formalin (figure 9). Since the dose-response curve for ICV DAMGO spanned

two orders of magnitude (0.006 to 0.60 μg), the authors were able to calculate

estimates of the dose required to produce a 50-percent reduction (i.e., ED50)

in both pain behaviors and Fos immunoreactivity. It was found that ICV

DAMGO exhibits similar potencies for inhibition of pain behavior (ED50

=0.06 μg) and overall Fos immunoreactivity (ED50=0.05 μg); this suggests

that there is a similar mechanism of action for the two measures. There was

a significant correlation between the expression of behavioral nociception and

overall Fos immunoreactivity produced by formalin (figure 6), again supporting

the contention that noxious stimulus-evoked Fos immunoreactivity can be used

as a marker for nociceptive neurons.

Although the potencies for DAMGO-mediated inhibition of formalin-evoked

pain behaviors and Fos immunoreactivity were similar, the maximal levels of

inhibition (i.e., Emax) for each measure were different. Specifically, a dose

of DAMGO that produced 100-percent inhibition of behavioral nociception

produced only 81 percent overall inhibition of Fos immunoreactivity. When the

149
FIGURE 9. Photomicrographs showing the pattern of formalin-evoked FLI

in neurons from sections of the lumbar enlargement of rats that

received ICV injections of saline (A), 0.06 μg DAMGO (B), 0.60

μg DAMGO (C), or 0.60 μg DAMGO plus 1.0 μg naloxone (D).

150
In saline-treated rats (A) there is dense labeling in laminae I and

II as well as in the neck of the dorsal horn and the ventral horn.

Pretreatment with 0.06 μg ICV DAMGO (B) produced 50-percent

inhibition of overall FLI (i.e., superficial FLI+nucleus proprius

FLl+neck FLl+ventral FLI) A quantitative analysis of the number

of neurons expressing FLI in the superficial, nucleus proprius,

neck, or ventral regions of the cord showed that this dose

produced close to 50-percent inhibition in the neck and ventral

regions but had minimal effect on the level of FLI in the superficial

layers. Pretreatment with 0.80 μg DAMGO (C) produced

approximately 84-percent inhibition of the FLI in the neck and

ventral regions but only 64-percent inhibition of the FLI in the

superficial dorsal horn. The effects of 0.60 μg DAMGO were

completely reversed by naloxone (D). Calibration bars=250 μm;

dc=dorsal column; Ili=inner laminae II.

SOURCE: Reprinted with permission from Neuroscience 42, Gogas, K.R.;

Presley, R.W.; Levine, J.D.; and Basbaum, A.I. The antinociceptive

action of supraspinal opioids results from an increase in

descending, inhibitory control: Correlation of nociceptive

behavior and c-fos expression, 1991, Pergamon Press, plc.

potencies of ICV DAMGO were considered in different laminae (figure 10), it

was found that there was approximately a fourfold difference in the estimated

ED,, values for the laminae V/VI or ventral horn vs. the superficial laminae;

thus, at doses of 0.04 to 0.06 μg ICV there was an approximate 50-percent

decrease in both the behavioral response and the Fos expression in laminae

V-X. By contrast, the dose-response curve for DAMGO-mediated inhibition of

Fos immunoreactivity in the superficial cord was shallow. In fact, at the ED,,

doses for inhibition of Fos expression in the neck and ventral regions of the

cord, there was less than 1 O-percent inhibition of Fos in the superficial dorsal

horn.

These data are consistent with previous findings with systemic morphine

(Presley et al. 1990), which also showed that complete behavioral analgesia

could be produced without eliminating the Fos immunoreactivity in the

superficial cord. On the other hand, our studies with both systemic and ICV

morphine showed that a dose of morphine that produced approximately

50 percent inhibition of Fos immunoreactivity in the neck and ventral cord

produced a comparable suppression in the superficial dorsal horn Fos

immunoreactivity. This contrasts with our findings using DAMGO in which

doses that produced a 50-percent reduction in Fos immunoreactivity in the

151
FIGURE 10. Effects of ICV DAMGO on noxious stimulus-evoked FLI in the

spinal cord, Animals (n=3 per dose) were treated and the tissue

processed as described in the text Results are expressed as

mean (±SEM) number of neurons per 50-μm section expressing

FLI in either the superficial (•), nucleus proprius neck

or ventral portions of the ipsilateral spinal cord. Drug

treatments are indicated on the abscissa. The potency for

CV DAMGO-mediated inhibition of FLI in the superficial region

(ED50=0.18 μg) was approximately four times less than for

either the neck or ventral regions (ED50=0.04 and 0.05 μg,

respectively). The curve for inhibition of the nucleus proprius

was flatter than for the other regions of the cord, with significant

inhibition only at 0.60 μg ICV DAMGO. Note that the highest

dose of ICV DAMGO tested (0.60 μg) produced 100-percent

inhibition of pain-related behaviors without producing

comparable levels of inhibition in FLI at the level of the cord.

SOURCE: Reprinted with permission from Neuroscience 42, Gogas, K.R.;

Presley, R.W.; Levine, J.D.; and Basbaum, A.I. The antinociceptive

action of supraspinal opioids results from an increase in

descending, inhibitory control: Correlation of nociceptive behavior

and c-fos expression, 1991, Pergamon Press, plc.

152
neck and ventral cord had almost no effect in the superficial dorsal horn. We

hypothesize that the more profound suppression of Fos immunoreactivity in the

superficial dorsal horn seen following systemic doses of morphine results from

the fact that after systemic administration there Is both a supraspinal and spinal

contribution to analgesia.

Since DAMGO is a potent and selective μ-opioid receptor agonist (Handa

et al. 1981), and since morphine, when given supraspinally, is also thought to

act preferentially at the F-receptor (Fang et al. 1986; Roerig and Fujimoto

1989), the contrast between the levels of superficial suppression following

ICV morphine and DAMGO is particularly interesting. Thus, morphine (ICV)

produced a maximal inhibition (i.e., 65 percent) of superficial dorsal horn

Fos immunoreactivity at the lowest dose tested (2.5 μg); there was no greater

inhibition when higher doses were tested. By contrast, the dose-response

curve for ICV DAMGO-mediated Fos suppression in the superficial cord

was shallow. In fact, significant inhibition occurred only at the highest dose

tested (0.60 μg), If one considers this difference in terms of the relationship

between inhibition of superficial dorsal horn neuronal activity and behavioral

antinociception, it can be seen that doses of ICV morphine and DAMGO that

produce 50-percent inhibition of behavioral nociception produce 65-percent and

cl O-percent inhibition of superficial Fos immunoreactivity, respectively. Since

DAMGO is thought to be a much more efficacious agonist than morphine,

it is highly unlikely that the differences between the two compounds relate to

differences in activity at the v-receptor. Rather, these differences suggest

that ICV morphine acts at another site in addition to the p-receptor, possibly

the -receptor. Alternatively, since some researchers have provided evidence

for the existence of subpopulations of μ-opioid receptors in brain (Wolozin

and Pasternak 1981; Rothman et al. 1983), it is possible that the μ-receptor

populations with which morphine and DAMGO interact are different.

Regardless of the receptor(s) through which morphine acts to produce its

effects, it is clear that by monitoring the regulation of Fos protein expression

by these compounds, in addition to monitoring behavioral effects, much new

information on the mechanisms through which these drugs work can be

obtained.

THE EFFECT OF DORSOLATERAL FUNICULUS LESIONS ON OPIOIDMEDIATED INHIBITION OF NOXIOUS STIMULUS-EVOKED FOS

IMMUNOREACTIVITY

It is commonly believed that analgesic compounds, such as morphine,

activate descending systems within the brain stem, which in turn inhibit spinal

cord nociresponsive neurons (Basbaum and Fields 1978, 1984). For example,

supraspinal microinjection of morphine attenuates behavioral responses in

153
nociceptive tests, such as the tail-flick or hot-plate test (Jensen 1988; Tung and

Yaksh 1982), and intracerebral injection of morphine inhibits the firing of dorsal

horn neurons that respond selectively to noxious stimuli (Bennett and Mayer

1979; Du et al. 1984; Gebhart et al. 1989). Furthermore, the antinociceptive

effects of systemic or intracerebral injection of morphine are attenuated by

lesions of the brain stem or spinal cord (Barton et al. 1980; Basbaum et al.

1976, 1977) and can be antagonized by IT injection of monoamine antagonists

(Proudfit and Hammond 1981; Jensen 1986). However, there is contradictory

neurophysiological evidence that indicates that intracerebral and ICV morphine

decreases, rather than increases, descending controls to produce analgesia

(Bouhassira et al. 1988; Dickenson and Le Bars 1987; Duggan et al. 1980).

The authors’ results provide strong evidence in support of the hypothesis that

both ICV morphine and DAMGO produce analgesia and suppression of spinal

cord Fos immunoreactivity by increasing descending controls.

To specifically test this hypothesis, Gogas and colleagues (1991) studied

the effects of the administration of supraspinal opioids in animals that

underwent bilateral lesions of the spinal cord pathway through which

opioids exert their effect (see the dorsolateral funiculus [DLF]). They predicted

that DLF lesions would totally block the opioid-mediated behavioral analgesia

and the suppression of spinal cord Fos immunoreactivity. In fact, the dose

of ICV DAMGO that produced 100-percent behavioral analgesia in normal

animals (0.60 μg) had no effect on either behavior or Fos immunoreactivity in

animals with DLF lesions (figure 11). This finding supports the conclusion that

supraspinal opioids produce their analgesic effects by increasing, rather than

decreasing, the activity of descending control systems. This experiment again

illustrates the advantage of monitoring large populations of neurons with Fos

immunocytochemistry to answer questions that previously could be addressed

only at the single-cell level.

CONCLUSION

The examples discussed in this report demonstrate the usefulness and validity

of monitoring Fos expression to assess the activity of populations of CNS

neurons. However, several important caveats need to be underlined when

interpreting the results of these experiments. First, there is no unequivocal

way of knowing whether a Fos-expressing neuron is, in fact, nociresponsive.

This assumption is based on electrophysiological characterization of neurons

in the different spinal cord laminae. Similarly, if a neuron does not express

the Fos protein, one cannot conclude that the unlabeled neuron is not

nociceptive. Also, since the function of the Fos protein in spinal cord neurons

is not presently known, no conclusions can be drawn regarding the biological

significance of a neuron expressing Fos immunoreactivity. In spite of these

154
FIGURE 11. Effects of DLF lesions on noxious stimulus-evoked "pain"

SOURCE:

behaviors and on overall FLI. Results are expresseb as mean

(±SEM, ordinate) behavioral AUC scores (top) or number of

neurons per 50-μm section expressing FLI (bottom) in rats

receiving either ICV saline (open bars) or DAMGO (0.60 μg;

filled bars). DAMGO significantly inhibited both the painresponse and FLI expression in both intact and sham rats.

These inhibitory effects were reversed in rats with DLF lesions.

The mean response for DAMGO-treated intact animals was

zero; this is indicated by + (top). **Indicates groups significantly

different from saline controls by either Dunnett‘s (top) or

Neuman-Keuls (bottom) post-hoc tests (p<0.01).

Reprinted with permission from Neuroscience 42, Gogas, K.R.;

Presley, R.W.; Levine, J.D.; and Basbaum, A.I. The antinociceptive

action of supraspinal opioids results from an increase in descending,

inhibitory control: Correlation of nociceptive behavior and c-fos

expression, 1991 , Pergamon Press, plc.

155
limitations, Fos expression can be used as a marker to tell whether and when

something happens to a particular neuron. Importantly, the findings (1) that

the distribution of noxious stimulus-evoked Fos immunoreactivity closely

parallels the known distribution of spinal nociceptors, (2) that there is a

significant correlation between the expression of noxious stimulus-evoked

pain behaviors and spinal cord Fos expression, and (3) that noxious stimulusevoked Fos immunoreactivity is suppressed by systemic as well as supraspinal

administration of opioid analgesics, such as morphine and DAMGO, strongly

suggest that this approach can be used to study the mechanisms by which

drugs produce analgesia.

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ACKNOWLEDGMENTS

This chapter was prepared with support from National Institutes of Health

training grant NS-07265, NRSA NS-08892-01, a postdoctoral fellowship from

the Pharmaceutical Manufacturers Association (KRG), NS-14627, and DE/NIDA

08973 (AIB).

AUTHORS

Kathleen R. Gogas, Ph.D.

Postdoctoral Fellow

Department of Anatomy and Keck Center for Integrative Neurosciences

159
Jon D. Levine, M.D., Ph.D.

Associate Professor

Departments of Medicine, Oral Surgery, and Anatomy

Division of Neurosciences

Allan I. Basbaum, Ph.D.

Professor

Departments of Anatomy and Physiology and Keck Center for Integrative

Neurosciences

University of California, San Francisco School of Medicine

Box 0452

San Francisco, CA 94143

160
The Ontogeny of Immediate Early Gene

Response to Cocaine: A Molecular

Analysis of the Effects of Cocaine on

Developing Rat Brain

Barry E. Kosofsky and Sfeven E. Hyman

EPIDEMIOLOGY

Current estimates suggest that between 13 and 17 percent of infants born

in urban hospitals in the United States have been exposed to cocaine in

utero (Frank et al. 1988). Multivariate analysis has shown that mothers

who abuse cocaine during gestation have infants with a 93-g lower birth

weight, a .17-centimeter decrement in length, and a .43-centimeter smaller

head circumference (Zuckerman et al. 1989). The dramatic decrease in

head size is a further indication of in utero effects of maternal cocaine on the

developing brain. Drug-induced changes in brain development are likely to

account for the hyperreflexia, increased irritability, alterations in tone and

coordination, and developmental delay, including fine motor and visual motor

skills, seen in some infants exposed to cocaine in utero (Kosofsky 1991).

CENTRAL NERVOUS SYSTEM MATURATION

Clinical data suggest that infants exposed to cocaine only during the first

trimester of gestation are normal size at birth but show a statistically significant

compromise of the Neonatal Behavioral Assessment Scale (Brazelton), with

poor orienting, poor motor ability, and abnormal reflexes (Chasnoff et al. 1989).

The implications are that the window of vulnerability to cocaine for developing

brain may begin early in gestation and that the toxicity ultimately manifested

may be dependent on the time of fetal exposure. The protracted timetable

of central nervous system (CNS) maturation confers a continuum of biologic

vulnerability to developing brain. Moreover, the developmental consequences

of a toxic CNS insult relate critically to the period during which the fetus is

exposed to drugs (Evrard et al. 1989).

161
CNS development requires a complex coordination of genetic factors

and environmental forces that direct brain maturation and shape infant

development in a reproduceable, yet individualized manner. The cellular

events underlying these processes require differential gene transcription.

The authors hypothesize that the toxic developmental effects of substances

of abuse may be mediated via perturbations of normal patterns of gene

expression. To address this question, we have conducted experiments in

an animal model, exposing developing rat brain to cocaine, permitting

identification and characterization of some of the earliest cellular events

(and targets) mediating drug effects. This analysis focuses on a class of

immediate early genes (IEGs) that act as transcription factors (IEGs c-fos,

c-jun, and zif/268) that may subserve one mechanism by which substances

of abuse alter genetic programs during development.

IMMEDIATE EARLY GENES

The molecular mechanisms underlying the neuronal response to cocaine

exposure remain elusive. Recently, IEG response has been used as a

marker of neuronal activation in various CNS stimulation paradigms, including

models of seizure (Morgan et al. 1987; Sonnenberg et al. 1989), nociceptive

stimulation (Draisci and ladarola 1989; Naranjo et al. 1991), and circadian

neurobiology (Aronin et al. 1990). IEGs are called proto-oncogenes because

they represent the normal cellular homoiogues of transforming viral oncogenes

(Morgan and Curran 1991). Nuclear proto-oncogenes function in cellular signal

transduction as transcription factors binding to the promoters of target genes,

thereby influencing transcription of specific gene products. Drug-induced

alterations in IEG expression can modify gene expression during development

and thereby alter cellular identity and neuronal repertoire. The IEGs c-fos,

c-jun, and zif/268 are of particular interest because they demonstrate rapid

and dramatic induction in specific brain areas of adult rats exposed to cocaine

or amphetamine (see other chapters in this volume). In most cell types, basal

levels of c-fos and c-jun mRNA and of c-Fos and c-Jun protein are relatively

low (Morgan and Curran 1991). in contrast, certain cells maintain relatively

high basal levels of zif/268 mRNA and Zif/268 protein (Worley et al. 1991).

The induction of c-fos, c-jun, and zif/268 in response to novel stimuli in

neurons follows a similar pattern; mRNA transcription occurs within 5 minutes

and continues for approximately 20 minutes, with peak steady-state mRNA

levels at 30 to 45 minutes and peak protein synthesis at about 2 hours

poststimulation (not shown).

162
Activation of IEGs by Cocaine

The constraints on drug-induced IEG repertoire imposed by cell phenotype

in adult rats were addressed by Young and colleagues (1991) and Graybiel

and colleagues (1990), who reported that cocaine-stimulated fos induction in

striatum could be completely blocked by pretreatment with a D1 antagonist

(SCH 23390). Interpretation of experiments employing the D2 antagonist

(sulpiride) was complex because D2 antagonists independently activate c-fos

expression. Coupled with the knowledge that In striatum different (though

not mutually exclusive) populations of neurons have D1 vs. D2 receptors

(Gerfen et al. 1990), one can conclude that receptor phenotype of striatal

neurons is one of the cellular determinants conferring cocaine inducibility

of IEG expression. This idea is supported by anatomic studies (Berretta et

al. 1991) that have identified the subset of neurons in adult rat striatum that

express c-Fos after cocaine administration as containing DARP-32, a marker

for neurons expressing D1 receptors. By analogy, the susceptibility of the

developing brain to be affected by cocaine may relate to the maturation of

presynaptic (e.g., aminergic projections), postsynaptic (e.g., D1 receptors),

and/or intrinsic (e.g., cellular signal transduction pathways) features specific

for a given cell type in a given brain region at a particular point in development.

EXPERIMENTAL RESULTS

The authors have performed northern gel analyses to characterize the acute

induction of the IEGs c-fos, c-jun, and zif/268 in three areas of rat brain:

dorsolateral neocortex, striatum, and cerebellum. Rats were examined at

four different ages: postnatal (P) day 8 (P8, roughly equivalent to term

for a human fetus), 2 weeks of age (P15), 4 weeks of age (P28), and at

maturity. Animals were injected with cocaine (40 mg/kg in .9 percent saline)

and sacrificed 45 minutes later, when regional brain dissections were

performed on ice. Total cellular RNA was prepared by a modification of

the method from Berger and Chirgwin (1989). Samples were homogenized

in 4M guanidinium isothiocyanate followed by centrifugation through a 5.7M

cesium chloride pad. RNA was quantitated, and 30-μpg samples were run on

a formaldehyde gel. RNA was transferred to a nylon membrane, cross-linked,

and hybridized with nick-translated probes for c-fos, c-jun, or zif/268 and for

cyclophilin (unregulated internal reference gene). Autoradiograms were

quantitated with densitometry and represented graphically. Figure 1 (P8),

figure 2 (P15), figure 3 (P28), and figure 4 (adult) depict severalfold induction

(vs. saline-injected controls) of IEGs by region, representing an average from

two experiments for each plot (three to four animals pooled for each condition,

for each experiment). There is considerable specificity evident with regard to

163
which IEG is activated in which brain area at what point in development. At the

earliest age examined (figure 1, P8), IEG activation is limited to the striatum,

where c-fos and zif/268 are Induced. By P15 (figure 2) and thereafter (P28,

figure 3; adult, figure 4) all three IEGs are induced in striatum and cortex. The

elevation of c-jun and zif/268 in cortex and striatum at these ages was modest

(1.2 to 2.2x) but consistent. C-fos induction was more significant in cortex (1.8

to 2.1x) and was dramatically increased in striatum (4.6 to 6.6x). At all ages

examined, striatal c-fos demonstrated the most significant cocaine-induced

IEG elevation. In cerebellum, c-jun and zif/268 were not induced at any age

examined. At this dose, but not at 30 mg/kg, c-fos was induced in cerebellum,

but only on or after P15 (data not shown).

FIGURE 1. Induction of c-fos mRNA (4.2-fold) and zif/268 mRNA (1.8/fold) in

striatum of P8 rats treated with cocaine, 40 mg/kg/intraperitoneal

(IP), vs. saline. Rats were sacrificed 45 minutes after injection,

At this age, changes in striatal c-jun were not evident, nor was

induction of c-fos, c-jun, or zif/268 evident in neocortex or

cerebellum.

164
FIGURE 2. Graph depicting induction (vs. saline) of c-fos, c-jun, and zif/268

mRNA in cortex and striatum of 15-day-old rat pups sacrificed 45

minutes after IP injection of cocaine, 40 mg/kg. All three IEGs are

induced in cortex and striatum.

To characterize the consequences of cocaine-induced striatal IEG activation,

the authors first performed gel-shift assays. We examined changes in the

level of AP-1 DNA binding activity evident in striata of animals at different

ages (P28 and adults) 2 hours after injection with cocaine, 30 mg/kg/lP.

Striatal tissue suspended in buffer was homogenized, and protein was isolated

as previously described (Nguyen et al., in press). Protein was quantitated and

samples preincubated on ice for 15 minutes with poly (dl-dC), with or without

cold competitor (100 ng). Samples were then incubated at room temperature

for 15 minutes with 1 to 1.5 ng of P

32-end-labeled double-stranded

oligonucleotide probe and electrophoresed through a 4-percent nondenaturing

polyacrylamide gel. On day P28 (figure 5) and in the adult (figure 6), extracts

prepared from saline-treated animals demonstrated basal levels of binding

activity to the AP-1 DNA sequence. However, cocaine administration

augmented AP-1 DNA binding activity 2- to 2.5-fold on day 28 and 3- to

3.5-fold in the adult (representing the average of two samples per condition).

165
FIGURE 3. Graph depicting the striatal and cortical induction (vs. saline) of cfos, c-jun, and zif/268 mRNA in 28-day-old rat pups. Cocaine, 40

mg/kg/lP, was injected 45 minutes prior to sacrifice. C-fos

elevation in cortex (2-fold) and striatum (4.6-fold) was greater

than that of c-jun or zif/268.

The specificity of cocaine-induced increases in AP-1 DNA binding activity

is demonstrated by competition with unlabeled double-stranded DNA

oligonucleotide containing the AP-1 sequence (figure 5, lanes 5 and 6;

figure 6, lanes 3 and 7), but absence of competition is demonstrated by

an oligonucleotide containing the AP-2 sequence (figure 6, lane 4).

CONCLUSIONS

The authors have demonstrated cocaine-induced changes in gene expression

during rat brain development at two levels of analysis: (1) regional and

temporal specificity of mRNA induction for the IEGs c-fos, c-jun, and zif/268

from northern gel experiments and (2) functional confirmation that in striatum

AP-1 DNA binding activity is increased in parallel with the elevation in IEG

mRNA described above. These experiments provide evidence that mRNA

changes occur within 45 minutes, and AP-1 DNA binding changes occur

166
FIGURE 4. Graph depicting induction (vs. saline) by cocaine (40 mg/kg/IP

injected 45 minutes prior to sacrifice) of c-fos , c-jun, and zif/268

in adult cortex and striafum. The most significant response is the

induction of c-fos mRNA (6.6-fold) in sfriatum.

within 2 hours after exposing developing brain to cocaine. One central

question concerns the identification of target genes that, by virtue of having

AP-1 (or AP-1 -like) DNA binding sequences in their regulatory regions,

may be altered in their expression. Candidates include synthetic enzymes

for neurotransmitters (e.g., tyrosine hydroxylase), peptide neurotransmitter

precursors (e.g., substance P), and growth factors (e.g., nerve growth factor)

among many other genes. The regional and temporal specificity of IEG

induction by cocaine identifies a mechanism by which selective alteration

of target gene expression can be altered during development consequent

to drug exposure.

Significance

Exposure of the developing brain to cocaine altered the expression of three

IEG transcription factors, which as third messengers may influence the

expression of cascades of genes. The implications of the regional and

167
FIGURE 5. Gel retardation assay performed on striatal extracts (20 μg) from

P28 rats prepared from two sets (A and B) of three animals

injected IP with saline (lanes 1 and 2) or cocaine 30 mg/kg (lanes

3, 4, 5, and 6) 2 hours before sacrifice. The arrow designates the

position of the specific band induced by cocaine administration.

Cocaine induces a 2- to 2.5.fold increase in specific AP-1 binding

acfivify (compare lane 1 vs. lane 2, and lane 3 vs. lane 4). The

specific band is competed by cold AP-1 oligonucleotide at 100-

fold excess over labeled probe (lanes 5 and 6).

temporal specificity of altered IEG expression by cocaine for nervous system

development are profound. Normal brain development requires complex and

finely tuned genetic coordination, where individual neurons undergo phenotypic

differentiation to assume unique chemical, physiologic, and connectional

features. The maturation of synaptic circuitry, chemical neurotransmission,

and neurophysiologic repertoires that subserve the normal acquisition of

developmental skills requires a complex orchestration of genetic regulation

and environmental influences. Drug-induced alterations during such critical

periods may irrevocably alter CNS form and function. The experiments

outlined characterize some of the phenomenology of IEG activation by drugs

of abuse in developing rat brain. The significance of this work relates to the

hypothesis that (1) as transcription factors, IEGs may play a critical role in

the genetic events that shape human behavior and (2) drug-induced IEG

dysregulation may have long-lasting structural and functional consequences

when perturbed during brain development.

168
FIGURE 6. P

32-end-labeled AP-1 oligonucleotide binding of striatal extracts

(12 μg) prepared 2 hours after IP injection of saline (lanes 1 and

5) or cocaine, 30 mg/kg (lanes 2, 3, 4, 6, and 7) in two sets of

adult animals (A and B). The arrow designates the position of the

specific band induced by cocaine administration. Basal levels of

AP-1 DNA binding activity (lanes 1 and 5) were induced 3- to 3.5-

fold by cocaine (lanes 2 and 6). The specificity of binding is

demonstrated by competition with cold AP-1 oligonucleotide

(lanes 3 and 7), but not by cold AP-2 oligonucleotide (lane 4),

both used at 65-fold excess over labeled probe.

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AUTHORS

Barry E. Kosofsky, M.D., Ph.D.

Research and Clinical Fellow

Molecular Neurobiology Laboratory

Department of Neurology

Massachusetts General Hospital

Harvard Medical School

Fruit Street

Boston, MA 02114

Steven E. Hyman, M.D.

Director of Research

Department of Psychiatry

Massachusetts General Hospital

Director

Divison on Addictions

Harvard Medical School

Boston, MA 02115

171
NMDA Receptor Blockade Prevents

Translation, but Not Transcription, of

the C-fos Gene Following Stimulation

With Multiple Extracellular Signals in

Cultured Cortical Neurons:

Implications for Plasticity and

Molecular Memory

Frank R. Sharp, Kinya Hisanaga, and Stephen M. Sagar

INTRODUCTION

Morgan and Curran (1985) first showed that ionic depolarization of neural crestderived PC12 cells induced the c-fos proto-oncogene and that massive brain

depolarization produced by metrazole-induced seizures induces c-fos in mouse

brain neurons (Morgan et al. 1987). Although metrazole induced Fos-like

immunoreactivity (FLI) in cortex, hippocampus, amygdala, and many other

structures, it did not induce Fos in all brain structures, nor did it induce Fos

in all neurons in each structure.

Hunt and colleagues (1987) showed that noxious sensory stimulation induced

FLI in spinal cord neurons. Dragunow and Robertson (1987) showed that

kindling induced c-fos in granule cells of the rat dentate gyrus. Sagar and

colleagues (1988) found that osmotic stimuli induced FLI in hypothalamic

paraventricular neurons (Sharp et al. 1991), cortical stimulation induced FLI in

cerebellar Purkinje cell neurons (Sharp et al. 1989a), and seizures induced FLI

in hippocampus. Sagar and coworkers (1988) suggested that c-fos expression

could be used to map activated neurons much like the 2-deoxyglucose method

has been used to map the central nervous system response to physiological

and pharmacological stimuli. Since then, multiple studies have confirmed

some of the initial promise of the c-fos “mapping” method (Cole et al. 1989).

However, several practical and theoretical problems with Fos have become

apparent.

172
First, it is not clear that c-fos can be induced in all neural cells. For example,

tactile whisker sensory stimulation does not induce c-fos in any whisker-related

pathway. However, section of the infraorbital nerve to the whiskers induces cfos in the spinal trigeminal nucleus pars caudalis and decreases c-fos

expression in whisker barrel cortex contralateral to the lesion (Sharp et al.

1989b). Therefore, depolarization of neurons in vivo may be necessary, but

it is not a sufficient stimulus to induce c-fos. This is supported by the finding

that light stimulation, which should activate most retinal neurons, induces Fos

in a small subpopulation of retinal neurons (Sagar and Sharp 1990). Since

depolarization alone cannot account for induction of c-fos, either specific

transmitters or the concurrent actions of several extracellular signals might be

required for the induction of c-fos in neurons. The concurrent activation of two

extracellular receptors has been shown to be important for induction of the c-fos

gene (Curran and Morgan 1985).

Mechanisms of c-fos induction in glia appear to be different. In most in vivo

studies, c-fos appears to be induced in neurons and not in glia. However,

Dragunow and Robertson (1988) showed that FLI was induced in glial-like cells

at the sites of cortical injury. This was confirmed by Sharp and colleagues

(1990). To better define the stimuli responsible for c-fos induction in glia

following injury, several groups have studied c-fos in cultured glia. Arenander

and coworkers (1989) found that mitogens and stellation agents induced

immediate early genes in cultured rat astrocytes. Hisanaga and coworkers

(1990a) showed that c-fos is induced in astrocytes with agents that promote

differentiation or proliferation, but c-fos is not induced in astrocytes with

depolarizing agents. Sagar and colleagues (1991) have shown that epidermal

growth factor (EGF) and transforming growth factor alpha induce c-fos mRNA

and Fos protein in identified Muller glial cells of the rabbit retina in vivo.

Therefore, it is possible that induction of c-fos in glial cells at sites of injury

is related to local release of trophic factors that act directly on glia.

Induction of c-fos in cortical neurons has been linked to activation of glutamate

receptors, Sonnenberg and colleagues (1989) showed that various glutamate

agonists, including N-methyl-D-aspartate (NMDA) and kainate, induce Fos, Fosrelated antigen, and activator protein-l DNA-binding activity in the mouse brain.

Dragunow and Robertson (1988) and Sharp and coworkers (1989b) showed

that cortical injury induced c-fos in neurons throughout ipsilateral cortex. Sharp

and coworkers (1989b) showed that injection of quinolinic acid, an NMDA

agonist, into cortex markedly induced Fos throughout the ipsilateral hemisphere

compared with saline-injected subjects. Prior injection of the noncompetitive,

NMDA receptor blocker, MK801, into the ventricle before local injury or

quinolinic acid injections prevented Fos induction in the ipsilateral hemisphere.

These data suggested that the NMDA receptor mediated cortical induction of cfos in neurons following cortical injury.

173
It is possible that the NMDA receptor plays an important role in c-fos induction

in cortical neurons via a variety of stimuli. For example, section of the

infraorbital nerve induced Fos in brain stem but decreased Fos in cortex (Sharp

et al. 1989c). The explanation for the decreases of Fos expression in cortex

was unclear. It is hypothesized that cortical induction of Fos in most rats

handled in the laboratory is related to stress of handling, injections, and surgery

(Sharp et al. 1991). If true, then the decreases in Fos expression in cortex

following infraorbital section could be explained by the fact that concurrent

activation of the following two receptors is required: (1) activation of stress

receptors via unknown pathways and transmitters and (2) concurrent activation

of NMDA receptors by thalamocortical fibers. To test the idea that induction of

c-fos in cortical neurons might require the concurrent action of two receptors,

studies of cultured cortical neurons were undertaken (Hisanaga et al., in press).

METHODS

Dissociated neocortical cells were prepared from 16- or 17-day-old SpragueDawley rats using methods previously described by Hisanaga and Sharp

(1990). Brains were removed and neocortex was dissected in L15 medium.

Following incubation for 40 minutes at 37 °C in 10 mL phosphate-buffered

saline (PBS) containing 100 U papain, 0.02 percent DNAase, 25 mM glucose,

and 1 mM cysteine, the tissue was rinsed in 30 percent heat-inactivated horse

serum and triturated with a plastic pipette in 8 mL of medium. Cells were

centrifuged at 600 g for 5 minutes. Following aspiration of medium, cells were

suspended in minimal essential medium (MEM) containing 0.5 mM glutamine,

20 mM glucose, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 percent

fetal calf serum. The cells were passed through an 80 μm mesh, counted, and

plated on poly-D-lysine (20 μg/mL)-coated plates at a density of 1x106

 cells per

cm2. Cells were maintained at 37°C in 5 percent CO2/95 percent air, The

medium was changed to serum-free MEM or serum- and magnesium-free MEM

4 to 5 hours following seeding. Cultured neurons were studied 3 days after

seeding.

Cultured cortical neurons were treated with (1) glutamate (10 μM), (2) zinc

chloride (100 μM), (3) carbachol (1, 10, 50 mM), (4) dibutyryl-cyclic AMP (dbcAMP) (1 mM), (5) 12-O-tetra-decanoylphorbol-13-acetate (TPA) (50 nM), (6)

potassium chloride (46 mM), (7) vasoactive intestinal polypeptide (VIP) (1 μM),

(8) somatostatin (1 μM), (9) neuropeptide Y (NPY) (1 μM), (10) basic fibroblast

growth factor (bFGF) (1 μg/mL), (11) nerve growth factor (NGF) (5 ng/mL), (12)

EGF (20 ng/mL), and (13) platelet-derived growth factor (PDGF) (1 U/mL).

NMDA receptor blockers (MK801, 0.1 μM; CPP, 10 μM; and APH, 30 to 100

μM) were added 15 minutes prior to each stimulant.

174
C-fos mRNA Northern blotting was performed according to Hisanaga and

coworkers’ (1990b) method, which is a modification of the method of Auffray

and Rougeon (1980). Following isolation of the RNA and electrophoresis,

the RNA was transferred to nylon membranes by capillary blotting. C-fos

riboprobe was prepared from full-length c-fos cDNA using SP6 RNA

polymerase and 32P-CTP. Radiolabeled RNA was purified over a Nensorb

column. The nylon membranes were hybridized with riboprobe in 1.5X SSC,

50 percent formamide, 1 percent SDS gel, 0.5 percent nonfat dry milk, 0.5 mg/

mL denatured salmon sperm DNA, and 0.2 mg/mL yeast tRNA at 65 °C for 24

to 36 hours. Membranes were washed twice at 65 °C for 30 minutes in 2X

SSC 0.1 percent SDS, 0.5X SSC 0.1 percent SDS, 0.2X SSC 0.1 percent

SSC. They were exposed to film at -70 °C with an intensifying screen.

For detection of Fos protein and Fos-related antigen proteins,

immunocytochemistry was performed. Neurons were fixed with

pacaformaldehyde/lysine/polylysine fixative 2 hours after each stimulation.

After two PBS washes, cultures were incubated for 20 minutes in PBS

containing 2 percent goat serum, 0.2 percent Triton X-100, and 0.1 percent

bovine serum albumin. Cultures were incubated with polyclonal antibody

to Fos and Fos-like antigens diluted 1:50 for 2 to 3 days at 4 °C. The first

antibody was detected using a biotin-conjugated secondary antiserum

(diluted 1:200), followed by the avidin-peroxidase complex reacted with

diaminobenzidine and hydrogen peroxide as previously described (Sharp

et al. 1990; Sagar et al. 1988).

Cultures stained for glial fibrillary acidic protein using monoclonal antibodies

were negative, whereas 1 to 2 percent of the cells in each well were

immunoreactive for vimentin (presumed glioblasts). The remaining 98 to 99

percent of the cells stained for alpha-tubulin and neuron-specific enolase,

demonstrating that the cultures were nearly purely neuronal.

RESULTS

Control cultures treated with saline, PBS, or vehicle showed that only a few

neurons (0.5 to 1.5 percent) stained for FLI, which was induced in a much

greater percentage of neurons (6 to 10 percent) following treatment with

glutamate, bFGF, zinc, VIP, K+, db-cAMP, and TPA. This occurred in

magnesium-containing and magnesium-free medium. NGF, EGF, PDGF,

somatostatin, NPY, and carbachol did not induce FLI in the cultured neurons,

Glutamate, bFGF, zinc, VIP, K+, db-cAMP, and TPA induced c-fos mRNA in

the cultured neurons. Pretreatment with NMDA receptor blockers prevented

glutamate and db-cAMP induction of c-fos mRNA. Pretreatment with NMDA

175
receptor blockers did not affect fibroblast growth factor (FGF) induction of c-fos

mRNA and only partially blocked K+, TPA, zinc, and VIP induction of c-fos

mRNA.

However, the most novel aspect of this study was the observation that

pretreatment of cultured cortical neurons with noncompetitive (MK801) and

competitive (CPP and APH) NMDA antagonists completely blocked the

induction of FLI by all agents tested. Treatment of cultured cortical neurons

with NMDA antagonists alone had no effect on c-fos mRNA or FLI. In addition,

treatment of cultures with NMDA antagonists had no effect on total protein

synthesis as measured by 3H leucine into trichloroacetic acid-precipitated

proteins. Since the polyclonal antibody used in this study detects both Fos

protein and two lower molecular weight Fos-related antigens (Aronin et al. 1990,

1991), the data demonstrate that the NMDA antagonists prevent induction of

Fos protein and Fos-related antigens.

DISCUSSION

Glutamate induces c-fos mRNA and Fos protein in a small subpopulation (8 to

10 percent) of cultured cortical neurons. This induction appears to occur via the

NMDA receptor since both competitive and noncompetitive NMDA receptor

blockers prevent glutamate induction of c-fos mRNA and Fos protein, Similar

results have been observed in cultured cerebellar granule cell neurons (Szekely

et al. 1989).

The data also show that K+, TPA, bFGF, zinc, VIP, and db-cAMP induce c-fos

mRNA and Fos protein in a similar small subpopulation (8 to 10 percent) of

cultured cortical neurons. Moreover, although NMDA receptor blockers prevent

c-fos mRNA induction by glutamate and db-cAMP, other agents like K+, TPA,

bFGF, zinc, and VIP all induce c-fos mRNA in the presence of NMDA receptor

blockers. These observations prove that induction of c-fos mRNA via these

signaling pathways is not linked to release of glutamate and/or activation of

the NMDA receptor. The data also show that several agents (including NGF,

PDGF, EGF, NPY, somatostatin, NMDA antagonists, and carbachol) do not

induce the c-fos gene in cultured cortical neurons.

The unexpected finding of this study was that NMDA receptor blockers do

prevent the induction of FLI by all agents tested, including K+, TPA, bFGF,

zinc, VIP, and db-cAMP as well as glutamate. The authors believe that the

results are not artifactual since (1) the agents did induce c-fos mRNA; (2)

three chemically different NMDA antagonists blocked induction of Fos protein,

but not c-fos mRNA; (3) total protein synthesis was not affected by the NMDA

antagonists; and (4) graded doses of NMDA antagonists produced graded

176
decreases in FLI when glutamate was used as the stimulus. However, it is

possible that the observed effect might be unique to cultured, fetal cortical

neurons and not be a general phenomenon of developing or mature cortical

neurons in vivo. However, preliminary data in the authors’ laboratory suggest

that NMDA antagonists can block translation, but not transcription, of the c-fos

gene in the intact mammalian brain.

Nevertheless, the mechanism by which NMDA receptors might inhibit induction

of Fos protein, but not c-fos mRNA, by VIP, FGF, zinc, and other stimuli is

potentially very interesting. There are at least three possible mechanisms.

First, it is conceivable that activation of the NMDA receptor is necessary for

posttranslational processing of Fos or for phosphorylation of Fos. If the authors’

polyclonal antibody does not recognize unprocessed or unphosphorylated

Fos protein, then the data would show that the NMDA receptor is essential

for the posttranslational processing or phosphorylation that would make it

possible to immunostain the protein in culture. This seems unlikely since other

polyclonal antibodies to Fos detect unphosphorylated and phosphorylated

Fos (Sonnenberg et al. 1989). Second, it is possible that the NMDA receptor

might downregulate protease activity in some undescribed fashion, For

example, NMDA receptor blockade could lead to overactivity of a protease

that leads to rapid degradation of the Fos protein so that it was not detectable

immunocytochemically. There is little precedent for this. Third, and most likely,

is the possibility that the NMDA receptor is somehow involved in translation of

c-fos mRNA to Fos protein. This could occur via calcium entry through the

NMDA channel. Regulation of translation does occur in other systems,

Expression of yeast transcription activator GCN4 (whose DNA binding site

is related to the Fos-Jun binding site) is regulated at the translational level.

External stimuli appear to activate a protein kinase that mediates translational

regulation of GCN4 mRNA (Hinnebusch 1990). In addition, one calciumcalmodulin kinase affects translation by phosphorylating the elongation factor

2 (Ryazanov et al. 1988).

If NMDA antagonists are shown to block induction of Fos protein, but not c-fos

mRNA, from a variety of stimuli, these results could have important implications.

NMDA antagonists cause learning impairment (Morris et al. 1986) and prevent

plasticity in cortex and other brain regions (McDonald and Johnston 1990). Fos

could play a role in NMDA-mediated plasticity by producing long-term changes

in gene expression related to a variety of extracellular signals. The authors’

results suggest that if neurotrophic factors and neurotransmitters mediate

plasticity by induction of c-fos mRNA, the expression of Fos protein by these

agents requires concurrent activation of the NMDA receptor. Therefore,

blockade of NMDA receptors not only prevents direct NMDA-mediated events

but also prevents induction of Fos protein from a variety of other extracellular

signals.

177
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ACKNOWLEDGMENTS

This work was supported by the Merit Review Program of the Department of

Veterans Affairs Research Service and by National Institutes of Health grants

NS-28167 and NS-24666 (FRS) and NS-27488 (SMS).

AUTHORS

Frank R. Sharp, M.D.

Professor of Neurology

Kinya Hisanaga, M.D., Ph.D.

Assistant Professor

Stephen M. Sagar, M.D.

Professor of Neurology

Department of Neurology (V127)

University of California at San Francisco

Department of Neurology

Veterans Affairs Medical Center (127)

4150 Clement Street

San Francisco, CA 94121

180
Induction and Suppression of ProtoOncogenes in Rat Striatum After

Single or Multiple Treatments With

Cocaine or GBR-12909

Michael J. ladarola, Eric J. Chuang, Choh-Lun Yeung, Yin Hoo,

Mayme Silverthorn, Jun Gu, and Gaetano Draisci

INTRODUCTION

Dopamine is well known as a neurotransmitter, but its concurrent role as

a regulator of gene expression in postsynaptic neurons is just beginning to

be fully appreciated. Alterations in gene expression are one way a neuron

can modify its complement of expressed proteins in response to an alteration

in synaptic input. Increases or decreases in gene expression can lead

to corresponding changes in cellular concentrations of neuropeptides,

neurotransmitter synthesizing enzymes, transmitter receptors, or intracellular

targets of second messengers. Plastic modifications in the transmitter signaling

apparatus in a key set of neurons subsequently may alter the functional status

of entire circuits of interconnected neuronal populations. Theoretically,

permanent or quasi-permanent alterations in behavior can result, be they

adaptive or maladaptive. The molecular mechanisms that effect a change in

the expression of a gene are not fully understood. However, understanding

events at the molecular level will be an essential component for determining

the basis of postnatal neuronal plasticity. How does the brain react to novel

or prolonged changes in stimuli? Certainly the changes induced by chronic

ingestion of central nervous system (CNS)-active drugs, be they abused or

not, can be conceptualized in terms of plastic or adaptive changes. Thus,

investigating changes in neuronal gene expression and the mechanisms

governing gene expression following acute and chronic administration of

abused drugs is a powerful new approach to understanding the neuromolecular

basis for the initiation and maintenance of drug addiction.

A longstanding precedent for studying gene expression in the context of the

dopamine system is found in studies of chronic dopamine D2 receptor blockade

with the antipsychotic drug haloperidol. In animals, chronic treatment with

181
haloperidol increases striatal proenkephalin gene expression as determined

by an elevation in striatal content of proenkephalin-derived peptides (Hong

et al. 1978) and preproenkephalin mRNA (Tang et al. 1983). Furthermore,

the increase in preproenkephalin gene expression seen in animals appears

to apply also to humans. Examination of proenkephalin-derived peptides in

cerebrospinal fluid (CSF) of human subjects treated chronically with haloperidol

shows that they are significantly elevated (ladarola et al. 1988a), suggesting

that the increase seen in striatal tissue in animals is reflected in an increase

in CSF content in humans. The relevance of the striatum to CSF peptides is

also supported by the observation of decreased CSF levels following striatal

lesions (ladarola and Mouradian 1989). In humans, acute and, in some

cases, permanent changes in behavior (e.g., parkinsonian-like side effects

and tardive dyskinesia, respectively) can occur following antipsychotic drug

therapy. Although a causal relationship between the increase in enkephalin

biosynthesis and the development of parkinsonian-like side effects has not

been established, the example illustrates that (1) chronic drug treatment can

modify neuronal gene expression and (2) results obtained in dopaminergic

neural circuits in animals may be applicable to humans. Thus, molecular

studies of the dopamine system and the effects of cocaine in animal models

may be particularly relevant to the human situation.

Generally, an increase in mRNA content reflects a change in gene transcription

and/or a modification of mRNA stability. Gene transcription is regulated, in part,

by interactions of sequence-specific DNA-binding proteins (Pans-acting factors

or transcription factors) with binding sites on DNA (cis-acting elements or

enhancer). It has recently been hypothesized that one class of transcription

factors, called cellular immediate early genes (IEGs), serves to couple synaptic

transmission at the plasma membrane to alterations in transcription in the cell

nucleus (for review, see Morgan and Curran 1991). It is hypothesized that

signals transduced by receptors, via second messengers, initially activate

transcription and translation of IEGs, which then translocate to the nucleus,

acting there as “third messengers” to modify the expression of a set or network

of secondary target genes. Thus, these genes may represent a requisite first

step in long-term modification of neuronal function. In vivo and in vitro

experiments have demonstrated that expression of IEGs is very malleable.

Expression of c-fos, the prototypical IEG, can be altered by several types of

highly specific stimuli in several different neuronal circuits (see other chapters,

this volume).

Initial experiments in spinal cord (Draisci and ladarola 1989) with c-fos and the

prodynorphin opioid peptide gene first suggested that dopamine might be an

activator of c-fos gene expression in striatum. In the spinal cord, a marked,

prolonged increase in prodynorphin gene expression was observed in dorsal

182
horn neurons following transsynaptic activation during peripheral inflammation

(ladarola et al. 1986, 1988b). The dynorphin gene codes for the protein

precursor of the dynorphin family of opioid peptides. In an initial attempt

to understand the transcriptional processes regulating dynorphin gene

expression, our attention was drawn to c-fos. Fos-immunoreactivity was

shown to be rapidly induced by nociceptive stimuli in the same regions of the

spinal cord as was dynorphin (Hunt et al. 1987). The authors’ laboratory

showed that an increase in dynorphin gene expression was preceded by an

increase in c-fos mRNA (Draisci and ladarola 1989) and its protein product

Fos, as well as several other Fos-immunoreactive proteins seen in Western

blots of spinal cord nuclear extracts (ladarola et al. 1989). Furthermore, we

also showed that both Fos-immunoreactive proteins and dynorphin peptide

immunoreactivity or preprodynorphin mRNA were colocalized within the

same spinal cord dorsal horn neurons (Noguchi et al. 1991). Given this

suggestive, albeit circumstantial, evidence, the authors and colleagues

hypothesized that Fos, a known transcription factor, might be involved in the

regulation of dynorphin gene expression. These data provided the basis for

our prediction (Young et al. 1991) that, in striatum, the increase in prodynorphin

gene expression seen following dopaminergic activation (Li et al. 1988; Sivam

1989; Gerfen et al. 1991) would be preceded by an increase in c-fos expression

(despite the disparate location and innervation of the two CNS regions). We

observed that a single injection of cocaine induced a rapid (within 30 minutes),

fivefold to eightfold elevation in striatal c-fos mRNA. GBR-12909, a longer

acting dopaminergic uptake blocker, also increased c-fos mRNA and striatal

Fos protein content. Similarly, but to a proportionately lesser extent, expression

of the NGFI-A gene (Milbrandt 1987), which encodes a zinc finger-containing

transcription factor, is elevated following cocaine injection.

Single-dose studies are undeniably informative but do not provide an accurate

model of human cocaine use in addicts (Gawin and Ellinwood 1988; Gawin

1991; Spotts and Schontz 1980). These individuals generally administer

cocaine by intravenous injection or by smoking “crack,” a preparation of the

free base form of cocaine. Both routes of administration provide nearly

equivalent blood levels (Foltin and Fischman 1991) and yield a rapid-onset

euphoria-of relatively short duration (less than 10 minutes) but very intense—

called a “rush” or “flash” (see case studies in Spotts and Shontz 1980). The

cocaine user administers the drug multiple times over the course of a drug use

session (during an evening or longer, up to several days in some cases) to

experience the rush numerous times. Although this is a very characteristic

human usage pattern, neurochemical or molecular studies of the effects of

cocaine in animals generally do not try to model this pattern. The authors

have employed a multiple administration schedule as a first approximation

for the multiple administrations seen in human cocaine abuse. We find that

183
cocaine, administered multiple times in a single day, has a dual effect on the

expression of IEGs: Cocaine administration can either stimulate or suppress

the expression of c-fos. A single injection stimulates c-fos expression but at

the same time triggers a process that inhibits c-fos expression. We refer to

the latter effect as suppression or refractoriness to induction. A single injection

produces a short-lived suppression of approximately 1 to 2 hours. However,

multiple injections produce a suppression that can last for days. Thus, the

duration of suppression is greatly magnified with repeated administration.

The net effect of the prolonged suppression of IEG expression is to functionally

uncouple dopaminergic neurotransmission from dopaminergic regulation of

gene expression.

MATERIALS AND METHODS

Treatments

Male Sprague-Dawley rats (300 g) were housed in groups of three, given

access to food and water ad libitum, and kept on a 12-hour light/dark

cycle. Cocaine hydrochloride (HCI) was dissolved in saline and administered

intraperitoneally (IP) unless specified otherwise, in a volume of 0.2 or 0.3 mL/g

of body weight. Rats were sacrificed at various times after injection by

exposure to CO,. The brain was rapidly removed, and the frontal cortex,

caudate-putamen, hippocampus, and cerebellum were dissected out on an

ice-cold plate. GBR-12909 HCI was dissolved as follows: 20 mg was added

to 40 μL of glacial acetic acid, followed by 60 μL of H2O, and allowed to

dissolve completely; next, 1.7 mL of 200 mM sodium carbonate was added

and mixed, followed by 200 μL of Tween 20. Rats were injected either IP or

intravenously (0.2 mL/100 g body weight). In some cases animals were

pretreated IP 30 minutes prior to cocaine with various doses of SCH-23390,

a dopamine D1 receptor antagonist, or sulpiride, a dopamine D2 receptor

antagonist (Young et al. 1991).

Analyses

RNA was extracted from caudate-putamen, frontal pole or cerebral cortex,

hippocampus, and cerebellum by the guanidine thiocyanate/cesium chloride

centrifugation method as described previously (Draisci and ladarola 1989).

RNA blots were prepared and probed for transcripts with [

32P]-labeled, cloned

rat cDNA c-fos insert (Curran et al. 1987) or oligonucleotide probes to c-fos,

NGFI-A, prodynorphin, cyclophilin, or ß-actin, the latter two being used for

standardization.

184
Western blots were prepared with proteins extracted from isolated striatal cell

nuclei by a modification of the method of Tata (1974). After electrophoresis

in a lo-percent polyacrylamide gel and transfer to polyvinyldifluoride (PVDF)

membranes, the membranes were blocked with 1 percent gelatin, 0.25 percent

Tween 20, 10 mM HEPES buffer pH 7.4, and 0.02 percent sodium azide for 60

minutes: incubated for 1 hour with rabbit polyclonal antisera; washed 2X times;

incubated with [125I]-protein A for 1 hour; and washed several times at room

temperature. Autoradiographs were then obtained.

Gel shift assays were performed on proteins extracted from the isolated

nuclei. A synthetic double-stranded 21 nucleotide oligomer from the gibbon

ape leukemia virus (GALV) enhancer containing an AP-1 (TGAGTCA)

consensus sequence at its core was used as a probe.

lmmunocytochemical analysis of Fos-positive neuronal cell nuclei was

performed with standard peroxidase-antiperoxidase method on 30-μ frozen

sections after perfusion of rats (under deep pentobarbital anesthesia) with 4

percent paraformaldehyde. Visualization of immune complexes involved

using diaminobenzidine as the chromogenic substrate. The anti-Fos antibody

was employed at a dilution of 1:10,000 or 1:20,000.

RESULTS

A single injection of cocaine has a powerful effect on c-fos gene expression.

A dose of 30 mg/kg IP produced a marked, fivefold to sevenfold increase in

striatal c-fos mRNA within 30 minutes (figure 1). The increase in mRNA content

was relatively short lived and did not substantially outlast the 30-minute time

point. The elevation in mRNA content was both dose dependent and regionally

specific, occurring in regions receiving a strong dopaminergic innervation (e.g.,

striatum) but not in hippocampus (figure 2). lmmunocytochemical staining for

Fos-immunoreactive proteins disclosed nuclear staining in neurons in striatum,

nucleus accumbens, olfactory tubercle, and the islands of Calleja that was weak

in saline-injected controls (figure 3) and very intense following treatment with

cocaine (figure 4) or GBR-12909 (figures 5 and 6). The effect of cocaine was

dose related in terms of the number of cells that could be counted and in terms

of the intensity of staining. We quantitated the number of stained cells for the

dose response at 2 hours postcocaine, a time at which the protein staining was

at a peak (figure 7, upper panel), using doses of 0.3 to 30 mg/kg (Young et al.

1991). There was some light staining in control sections, but this varied

between rats and generally was not of an intensity to be easily quantitated.

The number of cells was increased significantly at the 3.0 mg/kg dose and

tended to reach a plateau between 10 and 30 mg/kg (figure 7, lower panel).

What is not apparent in the cell counts is that at higher doses, although the

185
FIGURE 1. Time course of increase in striatal c-fos mRNA. Rats were

administered 40 mg/kg of cocaine IP. The main increase

(eightfold to tenfold) occurred at 30 minutes. The autoradiogram

is overexposed to show that only minor elevations of c-fos

transcript were detected between 2 and 48 hours compared

with saline (last lane). Densitometric scan of the corresponding

ethidium bromide-stained RNA gel is placed at the bottom and

indicates that the amount of RNA loaded in each lane is similar.

186
FIGURE 2. Upper panel: Dose-response of c-fos mRNA increase assessed

30 minutes after IP injection of cocaine at doses between 5 and

40 mg/kg. The increase is regionally selective, occurring in

striatum but not in hippocampus. Increases over control were

detected at all doses of cocaine. Lower panel: The blot was

reprobed for preprodynorphin mRNA. No change was seen in

the amount of this transcript at 30 minutes after cocaine in either

caudate or hippocampus. The blot was probed a third time for

ß-actin (not shown), which demonstrated that the amount loaded

was very similar across the 10 lanes.

187
FIGURE 3. Immunocytochemical staining for Fos-immunoreactive proteins. A

section through the head of the right caudate-putamen from a

saline-injected rat shows the weak staining of striatal neuronal

nuclei for Fos immunoreactivity. A small amount of lateral

ventricle is seen in the upper left. At this dilution of antiserum

(1:10,000) control sections were generally blank except for

scattered darkly stained neurons in pyriform cortex and olfactory

tubercle. (64X magnification)

188
FIGURE 4. Increase in striatal Fos immunoreactivity after cocaine treatment.

Dense nuclear staining can be seen for Fos-immunoreactive

proteins at 2 hours after IP injection of 30 mg/kg cocaine. The

lateral ventricle is on the right. (64X magnification; antibody

dilution, 1:10,000)

number of cells recruited was not that much greater, the intensity of staining

increased dramatically. The degree of staining intensity throughout the

affected regions went from light brown to darker brown to nearly black in

many cells as the dose of cocaine or GBR-12909 increased. The intensity

aspect was not quantitated and does not reproduce well in the black and white

photomicrographs but can be readily appreciated in the tissue sections. GBR12909 was also capable of increasing Fos protein content in caudate nucleus

189
FIGURE 5. Increase in Fos immunoreactivity 2 hours after IP injection of 20

mg/kg of GBR-12909. As formulated in this study, GBR-12909

produced essentially the same increases in c-fos mRNA and Fosimmunoreactive proteins that cocaine did. However, behaviorally,

the time course of GBR’s action was much longer than that of

cocaine. This aspect was not systematically examined for its

effect on immunocytochemical or molecular endpoints. GBR12909 treatment caused marked staining throughout the rostralcaudal and dorso-ventral extent of the striatum as illustrated in

the section through the posterior caudate. (64X magnification;

antibody dilution, 1:10,000)

and in ventral striatal areas (figures 5 and 6). We also observed a marked

increase in c-fos mRNA with GBR-12909 in striatum at the 20 mg/kg IP dose

when rats were sacrificed within 30 to 40 minutes of injection (not shown).

GBR-12909 was also effective when given intravenously: Increases in c-fos

190
FIGURE 6. GBR-12909 increases Fos immunoreactivity in the olfactory

tubercle and islands of Calleja. Generally, control sections (see

figure 4) are nearly blank except for the occasional darkly stained

neuronal nucleus in the o/factory tubercle and adjacent pyriform

cortex. After cocaine, large numbers of nuclei are densely

stained in both the tubercle and the islands. (64X magnification;

antibody dilution, 1:10,000)

mRNA were seen as rapidly as 10 minutes and continued to accumulate at 20

and 60 minutes to reach a level (fivefold to eightfold increase) comparable to

that obtained with the 30 mg/kg IP dose of cocaine (not shown). In general, our

molecular observations of GBR-12909 were similar to those of cocaine.

The rapidity and selective regional distribution of the mRNA increase and

subsequent increase in protein content suggest that treatment with a

dopamine reuptake blocker rapidly induces c-fos gene expression. To test

the involvement of dopamine receptors in mediating the effect of cocaine,

we pretreated the rats with several doses of either the D1 selective antagonist

SCH-23390 or the D2 antagonist sulpiride. Using immunocytochemistry and

counts of Fos-immunoreactive cell nuclei, we observed that SCH-23390

pretreatment at a dose of 10 μg/kg partially attenuated the effect of 30 mg/kg

of cocaine, and a dose of 50 μg/kg completely blocked the effect of cocaine

(figures 8 and 9). Pretreatment with sulpiride partially attenuated the effect of

191
FIGURE 7. Time course and dose response of the increase in Fosimmunoreactive (IR) cell nuclei following cocaine 30 mg/kg IP.

Upper panel: The peak elevation of Fos-immunoreactive proteins

is seen between 2 and 4 hours and returns to control over

approximately the next 24 hours. This contrasts with the increase

in c-fos mRNA, which peaked sharply at 30 minutes. Lower

panel: The increase in Fos-immunoreactive protein staining

occurred over the same dose increase in mRNA. Data were

collected 2 hours after injection. Stained nuclei were counted

regardless of intensity. However, the intensity increased

progressively, especially between 10 and 30 mg/kg. At the latter

dose many neurons were very dark brown, almost black, in color.

*p<0.05, **p<0.01.

SOURCE: Young et al. 1991

192
FIGURE 8. Blockade of Fos induction by SCH-23390. (A) Diagram of

forebrain section illustrating region of caudate shown in B through

F. (B) Saline-treated control. (C) 2 hours after cocaine 30 mg/kg

IP. (D through F) High-power micrographs of caudate from rats

2 hours after treatment with cocaine 30 mg/kg IP alone (D), or

pretreated 30 minutes prior to cocaine with the D1 antagonist

SCH-23390 at 10 μg/kg (E) and 50 μg/kg (F). The bar in all cases

equals 100 μm; v=lateral ventricle. Preadsorption of the antibody

with the antigenic peptide abolished all staining (not shown).

SOURCE: Young et al. 1991

cocaine, but there was not a clear dose-related inhibition of the cocaine-induced

elevation of Fos-immunoreactive nuclei (figure 9, lower panel). Administration

of the antagonists alone produced no significant effect on the number of Fosimmunoreactive cell nuclei. These data suggest that the main receptor

mediating the induction of c-fos in striatal neurons is the D1 receptor. Since

the D1 receptor is positively linked to adenylate cyclase, it suggests the cyclic

193
FIGURE 9. Comparison of blockade of cocaine-induced Fos immunoreactivity

produced by pretreatment with SCH-23390 or sulpiride. Neither

antagonist alone had a marked effect on striatal c-fos staining. A

dose-related decrease in the number of stained nuclei was seen

with the D1 antagonist SCH-23390, whereas a clear dose-related

decrease was not seen with the D2 antagonist sulpiride.

(sal=saline, coc=cocaine) *p<0.05

SOURCE: Young et al. 1991

194
AMP (cAMP) second-messenger system is an important element in the

induction of c-fos gene expression in these cells.

The antibody used in the immunocytochemistry studies was directed

against a c-fos sequence KVEQLSPEEEEKRRIRRERNKMAAA,

which is highly conserved between species and among the four distinct

members of the Fos/Fos-related antigen (Fra) family, which includes

Fos-B (REETLTPEEEEKRRVRRERNKLAAA) (Zerial et al. 1989), Fra-1

(PCEQISPEEEERRRVRRERNKLAAA) (Cohen and Curran 1988), and

Fra-2 (RDEQLSPEEEEKRRIRRERNKLAAA) (Nishina et al. 1990; Matsui et

al. 1990). This suggested that more than one protein species may be elevated

in the Fos-immunoreactive striatal nuclei as seen with immunocytochemistry.

If multiple proteins were detected via Western blot methodology, they might

correspond to one or more of the multiple Fos/Fra proteins or fragments thereof.

To investigate this possibility, we performed Western blot analysis of striatal

nuclear extracts (figure 10). After isolation of the nuclei by centrifugation

through a 2.1-M sucrose solution, the nuclear pellet was homogenized directly

in SDS sample buffer, assayed for protein content, and electrophoresed in a 4-

percent stacking/10-percent separating polyacrylamide gel along with molecular

weight markers. We observed a temporally distinct cascade of immunoreactive

protein bands with the immunoblot technique (figure 10). The basal state

contained a small but detectable amount of 55-kDa Fos protein (barely visible

on the autoradiogram) as well as several, more distinct, lower molecular weight

Fra proteins, especially one at 41 kDa. Within 2 hours an abundant amount of

the 55-kDa Fos protein was seen and was followed thereafter by increases

in the lower molecular weight Fra proteins (figures 10 and 11). We have

confirmed the identity of the 55-kDa protein by probing with an antibody raised

against an n-terminal peptide (DYEASSSRCSSASPAGDSLS) from the rat Fos

protein. In these blots (not shown), an increase in the 55-kDa nuclear protein

was detected as soon as 1 hour after a 30-mg/kg IP dose of cocaine. By 2

hours after cocaine treatment, and even more conspicuously after 4 hours,

increases were seen in the lower molecular weight Fra proteins (figure 11).

An analogous temporal pattern of Fos and Fra expression has been seen in

hippocampus with convulsant stimulation (Sonnenberg et al. 1989).

One of the hallmarks of IEGs is that they do not require protein synthesis to

undergo an increase in transcription, All the mechanisms and proteins needed

to activate IEG transcription preexist in the cell’s cytosol or nucleus. The

activation of c-fos expression by cocaine fits this criterion, We observed that

the potent protein synthesis inhibitor anisomycin, which crosses the blood-brain

barrier, did not prevent the increase in c-fos mRNA (figure 12). In fact, we

saw the phenomenon of superinduction, in which the stimulus produces an

exaggerated elevation in mRNA levels in the presence of a protein synthesis

195
FIGURE 10. Western blot analysis of Fos-immunoreactive proteins at

different times following cocaine 30 mg/kg IP. The numbers

at the left show the molecular weight markers (M. W. Stds in

kDa); those on the right, the molecular weights for the Fosimmunoreactive proteins as recognized by the broadly specific

anti-Fos antisera. The 55-kDa protein corresponds to the c-fos

gene product; those below, to the various Fra’s. An increase in

Fos protein is seen as early as 1 hour in further repetitions of

this experiment; the Fra proteins lag behind the increase in Fos

protein.

SOURCE: Young et al. 1991

inhibitor. Although this is interesting in itself, the main point of the anisomycin

treatment was to determine whether it blocked or attenuated the increase in cfos transcripts. We did not observe an attenuation, and this reinforces the idea

that the increase in c-fos seen with cocaine is part of a broader spectrum of IEG

activation in caudate-putamen and ventral striatal areas that involves not only

fos but other IEGs as well (Hope et al. 1992).

We have shown that an acute injection of cocaine induces c-fos mRNA, Fos

protein, and several other Fos-immunoreactive proteins (Fra’s) and that these

196
FIGURE 11. Densitometric analysis of the increase in Fos and Fra proteins. The early increase in the 55-kDa Fos

protein is clearly seen in the scans (first peak on the left in each scan record) and in the graph derived

from the scan data. The Fra proteins, composed of the 44-, 41-, and 35-kDa components, reach their

peak approximately 2 hours later than Fos.

SOURCE: Young et al. 1991
FIGURE 12. The increase in c-fos mRNA is independent of protein synthesis,

The bars represent results expressed as counts per minute

(cpm) of protected c-fos mRNA from an RNase protection

assay. Rats were treated with saline (sal); cocaine 30 mg/kg

IP (coc); or anisomycin 100 mg/kg given 20 minutes prior to

cocaine 30 mg/kg IP (coc+aniso). Anisomycin pretreatment

did not prevent the increase in c-fos mRNA induced by cocaine.

Anisomycin alone also stimulated an accumulation of c-fos

mRNA (not shown). In fact, the increase produced by

anisomycin alone was additive with that produced by cocaine.

At the 30 and 40 mg/kg doses of cocaine, an increase in c-fos

mRNA occurred in cerebellum. The cerebellar increase also

was not attenuated by anisomycin.

198
proteins are exclusively located in the cell nucleus. A further question is

whether the induced proteins lead to an increase in the amount of AP-1

complex formation in the cell nucleus. Fos is a member of the leucine

zipper family of transcription factors and is known to bind as a heterodimer

with members of the Jun transcription factor family to the AP-1 enhancer

element (but also with other proteins, see Hai and Curran 1991). The AP-1

consensus sequence is TGAGTCA. We used a small segment of the GALV

enhancer that contains the AP-1 consensus in a gel shift reaction with extracts

of nuclei isolated from normal and cocaine-induced striatum to determine

whether the elevation in Fos protein content led to an increase in AP-1

complex formation. The gel mobility shift assay yielded an AP-1 shift in

control striatum and an elevated amount of AP-1 complex within 1 hour

following cocaine injection (figure 13). In both cases, the primary AP-1

shift contained Fos-immunoreactive proteins since the entire complex could

be shifted further still by addition of the Fos antibody. The entire amount of

complex was further retarded, which suggests that a Fos-immunoreactive

protein was an element in all the complexes formed in both the control and

cocaine-induced conditions. Thus, the elevated capacity for striatal extracts

to form AP-1 complexes in the cocaine-treated condition is consistent with the

Northern blot, Western blot, and immunocytochemical data demonstrating an

increase in c-fos mRNA and Fos/Fra protein(s).

The most interesting effect of cocaine on IEG expression occurred when the

effects of a multiple dosing regimen were examined (figure 14). With several

doses given over the course of a single day (4 injections of 30 mg/kg IP, at

2-hour intervals), we observed a biphasic effect: The initial injection caused

the familiar marked (fivefold to sevenfold) increase in c-fos mRNA, but the

response to subsequent injections was nearly completely attenuated. Figure

15 shows that the c-fos induction was completely suppressed in response to

the fourth injection of a series of 30 mg/kg injections given 2 hours apart over

the course of a single day. We have mapped the time course of suppression

following a single acute cocaine injection and have found that a short-lived

period (approximately 1 to 2 hours) of suppression (or refractoriness to

induction) occurs following a single injection of cocaine (not shown). However,

the duration of suppression becomes much more prolonged with multiple

injections. In the case illustrated in figure 15, where four successive injections

were given, the duration of suppression lasted for 3 to 4 days. The long

duration required for recovery of full sensitivity to cocaine was mapped out by

giving challenge doses of cocaine at progressively greater intervals following

the subchronic series of injections (figures 16 and 17). Our data indicate that

the duration of suppression cumulates with multiple injections. Thus, multiple

injections of cocaine produce a long-term uncoupling of dopaminergic

neurotransmission from dopamine-regulated gene expression.

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FIGURE 13. Gel mobility shift analysis of AP-1 complex formation. In

addition to stimulation of c-fos mRNA and Fos and Fra proteins,

treatment with cocaine also increases the capacity for formation

of a Fos-containing protein DNA complex. A double-stranded

oligonucleotide from the GALV enhancer containing the AP-1

consensus was incubated with striatal nuclear extracts from

control and cocaine-treated rats. The location of the basal

AP- 1 complex is indicated. The entire complex can be shifted

further still by concurrent incubation with the Fos antibody (Ab),

which makes the complex larger and thus less mobile. The

super-shift indicates that Fos-immunoreactive proteins are in

the basal state AP-1 complex and are elevated after cocaine

induction. Higher concentrations of antibody disrupt the

complex completely via sequestration of Fos proteins; complex

formation could also be competed by an excess of unlabeled

AP-1 oligonucleotide but was unaffected by an excess of

unrelated oligonucleotide (not shown).

200
FIGURE 14. Multiple dosage regimen used to obtain suppression. Rats were

injected IP with a single dose of cocaine 30 mg/kg or with four

successive doses at 2-hour intervals. In both cases animals

were sacrificed 30 minutes after the last dose of cocaine. We

refer to the four successive injections as subchronic treatment.

The interplay between the induction and suppression mechanisms is

shown diagramatically in figure 18. In the acute situation activation of the

induction mechanism (shown by the upward deflection) is followed very

shortly by actuation of the suppression mechanism. The temporal kinetics

of the two are different: The induction is potent and short lasting, whereas the

suppression process has a longer time course, somewhat similar conceptually

to an action potential followed by a recurrent inhibitory postsynaptic potential.

The cumulation of the inhibition with repeated injections is shown in figure 18.

Here the induction process is eventually overwhelmed by the suppression,

which becomes progressively greater with each injection. The effects of the

suppression slowly reverse over the next several days, but complete recovery

is not achieved until 4 days postinjection.

DISCUSSION

The data presented show that cocaine induces a dual abnormality in

dopaminergic regulation of IEG expression. First, an acute injection induces

a large increase in c-fos gene expression. Second, a suppression mechanism

is induced, which renders the system refractory to induction by a subsequent

system. This period of refractoriness or suppression is variable and depends

on the frequency and number of multiple injections. Also, categorizing these

effects as “abnormalities” may be conceptually misleading. Since cocaine

blocks dopamine reuptake, it may be more accurate to think of cocaine as

201
FIGURE 15. Two representative RNA blots probed for c-fos (left) and a summary of the percent increase over

control from three separate experiments (right). An acute injection (Ac) produces a marked

(approximately sixfold) increase in c-fos mRNA in comparison to saline-injected controls (C). in

contrast, 30 minutes after the last injection in the subchronic series (Ch) no substantial increase is

seen (30 to 60 percent over control). The blots were reprobed for ß-actin, the c-fos mRNA to ß-actin

mRNA ratio was calculated, and the data from three experiments expressed as the mean&EM

percent of saline-injected controls are shown in the bar graph. The mutiple injections completely

suppress the ability of cocaine to induce an increase in c-fos mRNA.
FIGURE 16. Treatment schedule to determine time course of recovery

from suppression following the multiple injection subchronic

treatment. In all cases rats were sacrificed at 30 minutes after

the last injection in a series. The dose of cocaine for all

treatments was 30 mg/kg. Six groups of rats were treated:

(1) a single injection of saline or (2) cocaine; (3) the subchronic

series (i.e., four injections of cocaine at 2-hour intervals); (4)

the subchronic series and 24 hours later a Challenge dose of

cocaine; (5) the subchronic series with Challenge at 48 hours;

and (6) the subchronic series with challenge at 96 hours.

causing an exaggeration of ongoing dopaminergic activity, only potentiating

processes that are already in place. Thus, to some extent, induction and

suppression of IEG expression are likely to be normal components of the

biochemical repertoire accompanying dopaminergic neurotransmission. This

idea is supported by our Northern and Western blot data, which usually show a

low, but detectable level of c-fos mRNA and Fos-immunoreactive proteins in

basal state striatum. Modulation of expression either up (the usual case) or

down is possible. A similar situation is seen for NGFI-A (not shown). However,

the basal expression of NGFI-A mRNA is much higher than for c-fos, and large

amounts of NGFI-A protein can be detected in control brain nuclear extracts.

These data suggest that induction of NGFI-A, unlike c-fos, occurs on a fairly

high level of constitutive expression.

203
FIGURE 17. Northern blot analysis of c-fos mRNA induction, suppression, and recovery from suppression. The bar

graph shows the results from the six groups described in figure 16. RNA blots were prepared from

caudate-putamen and hippocampus and probed for c-fos and cyclophilin mRNAs. The bars represent

the ratio of c-fos mRNA to cyclophilin mRNA. Intensity of hybridization was determined with a

phosphor storage screen system and lasar readout. This method increases the linear range of

radiation detection several log units over x-ray film. The inset shows film autoradiograms for the

two transcripts. Note the large (~sixfold) increase due to acute cocaine treatment and the nearly

complete suppression at 6 hours following the subchronic series. The suppression is still evident at 1

and 2 days after the multiple injections. Full recovery is not seen until 4 days have elapsed after the

subchronic series of injections.
FIGURE 18. Diagrammatic representation of the induction and suppression

of c-fos expression by repeated cocaine injections. The

induction process is triggered by excess dopamine at the

synapse secondary to blockade of reuptake by cocaine.

The effect is mediated by D1 receptors positively /inked

to adenylate cyclase. This effect is transient but is

accompanied by a concurrent activation of the suppression

process. The mechanism(s) of suppression are not presently

known but appear to taper off slowly. The suppression

cumulates markedly with repeated cocaine administration,

thereby resulting in a prolonged uncoupling of dopaminergic

gene regulation from dopaminergic neurotransmission.

Pretreatment with antagonists of the D1 and D2 subtypes of dopamine

receptors demonstrated that the c-fos induction in response to cocaine was

blocked by the D1 antagonist. The D1 receptor is linked in a positive fashion to

adenylate cyclase, which suggests that c-fos gene expression is activated by a

cAMP-dependent process. In support of this, many in vitro studies have shown

that c-fos expression can be activated by stimulation of the cAMP secondmessenger system (for review, see Montminy et al. 1990). Furthermore, one

or more cAMP-responsive cis-acting elements are present in the promoter of

the c-fos gene. In these studies, sequences at -60 in the c-fos promoter confer

cAMP-dependent inducibility on a c-fos promoter-reporter construct in transient

expression assays (Sassone-Corsi et al. 1988). Deletions or mutations in the

cAMP response element (CRE) at -60 eliminate cAMP responsiveness in the

transient expression assay. The CRE is a binding site for a transcription factor

called CREB (Hoeffler et al. 1988), which is a member of the activating

transcription factor (ATF) family of leucine zipper trans-acting factors (Hai et al.

205
1989). Thus, a likely scenario for c-fos induction, and possibly induction of

other IEGs, by cocaine might be as follows: Cocaine enhances dopaminergic

activity via blockade of reuptake, thereby activating dopamine receptors.

Activation of the D1 receptor increases intracellular cAMP and the activity

of protein kinase A, which then phosphorylates susceptible substrates, one

of which may be CREB or another member of the ATF family. These in

turn rapidly act as positive regulators of c-fos gene expression via the c-fos

CRE sites. CREB is known to be phosphorylated by protein kinase A, and

phosphorylation affects the binding of CREB to the CRE in in vitro binding

assays (for review, see Montminy et al. 1990). Furthermore, CREB is

constitutively expressed, suggesting that no new protein synthesis is required

for the increase in c-fos gene expression. The latter conclusion is supported

by the authors’ data showing that the increase in c-fos mRNA still occurs in

the presence of a high degree of protein synthesis inhibition produced by

anisomycin.

GBR-12909, a cocaine analog without the susceptible ester bond found in

cocaine, was also able to rapidly induce c-fos mRNA and Fos-immunoreactive

proteins. Both compounds increased c-fos gene expression. Thus, by

biochemical criteria they appear similar; however, the duration of action is

longer for GBR-12909. This is exemplified by the duration of behavioral

activation. lntraperitoneal injection of GBR-12909 at 30 mg/kg produced an

intense stereotypy that lasted for at least 60 minutes. This is nearly four times

longer than the increase in locomotor activity produced by an equal dose

of cocaine. Although we did not formally analyze it, GBR produced more

stereotypy, such as intense sniffing directed at the hole for the water spout in

the cage, rather than locomotion. In comparison, cocaine produced stereotypic

head movements, but at most doses they were superimposed on an increase

in locomotion. Regardless of the behavioral differences, we observed an

abundant elevation in c-fos immunoreactivity throughout the striatum, nucleus

accumbens, olfactory tubercle, and islands of Calleja. The accumbens, in

particular, has been implicated in the reinforcing properties of cocaine (see

Kuhar et al. 1991 for review and Hope et al. 1992).

Several lines of evidence suggest cAMP-dependent mechanisms mediate

the increase in c-fos expression seen with cocaine. Fewer data are available

concerning the mechanisms underlying the suppression or refractoriness to

induction seen with multiple cocaine injections. We speculate that suppression

can be achieved at two levels of cellular organization. The first level is at the

plasma membrane and involves receptor desensitization. In vitro studies

with cells exposed to D1 agonists have shown acute and more prolonged

desensitization depending on the duration of exposure to agonists of the D1

receptor in cells that express the D1 receptor naturally (Barton and Sibley

206
1990). The second level is at the cell nucleus and involves transcriptional

mechanisms. Recently, a group of transacting factors that mediate downregulation of c-fos transcription had been elucidated. One of these, called

CREM (cAMP response element modulator), can act as an inhibitory

modulator of cAMP-induced activation of c-fos gene expression (Foulkes

et al. 1991a, 1991b). Thus, we propose that sequential activation of CREB

followed by CREM forms the basis of the induction and suppression of c-fos

expression following cocaine injection. Obviously, receptor desensitization

and transcriptional modulation can act in combination to cause IEG

suppression. Our data suggest that the suppression of IEG expression is a

powerful regulatory mechanism governing the production of protein from this

class of genes expression. The fact that, with repeated drug administration,

the suppression mechanism(s) override the induction process reinforces the

premium that the cell places on regulation of IEG expression. IEG expression

is allowed to reach only a certain degree and duration before it is actively

inhibited. Thus, the effective and prolonged suppression of IEG expression

seen with repeated cocaine administration may be viewed as providing a

protective or homeostatic function.

It is interesting to speculate on the meaning of some of these phenomena the

authors have observed with cocaine administration. The data illuminate both

the normal functioning of the dopaminergic system and its abnormal functioning

subsequent to cocaine abuse. Normal dopaminergic functioning probably

involves intermittent activation of c-fos and modulation of NGFI-A expression

either up or down from the basal state of expression. In humans this may

serve to couple positively rewarding or pleasurable stimuli to modifications in

gene expression. Since cocaine is one of the most rewarding drugs in animal

self-administration studies, it is tempting to suggest a relationship between

psychological processes such as behavior modification with positive reinforcers

and dopaminergic control of gene expression. C-fos, acting in concert with

other transcription factors, constitutes a molecular interface between behavioral

and environmental cues and gene regulation, whereby the IEGs integrate the

various inputs received at the cell membrane into a coherent alteration in

gene expression. The triggering stimulus may only briefly influence

neurotransmission (e.g., cell depolarization, transmitter release) but may

yield more permanent changes in neuronal biochemistry and function. A single

intravenous injection of cocaine causing a brief “rush” fits this model very well.

Subsequent regulation of target genes such as the dopamine D1 receptor or the

preprotachykinin gene, both of which have AP-1 consensus elements in their

promoter region, could lead to more permanent changes in neuronal function.

Obviously, optimal changes in target gene expression depend on the rate of

presentation and the intensity of the reinforcing cues to provide the optimal

amount and duration of IEG expression, Too great an intensity at too frequent

207
a rate of presentation leads to a rapid uncoupling of the system as occurs with

multiple injections of cocaine. Recent studies with once-daily injections suggest

that even with this more mild treatment schedule full uncoupling can occur if

cocaine is administered for a long enough duration (e.g., 14 days) (Hope et al.

1992).

The second area for speculation is the potential impact the induction and

suppression processes may have on the human abuse situation. Because

the uncoupling switches the cell from a transmitter-regulated mode of IEG

expression to a purely constitutive mode, the normal response to hedonic cues

at the molecular level is short circuited. Thus, certain gene products may

accumulate to abnormal levels or fail to accumulate as necessary and thereby

establish a new but abnormal complement of neuronal gene products. If the

drug is abused continually, then maintenance of the new state (constitutive

mode only) is relatively ensured, especially given the long duration of the

suppression effect. In fact, the suppression could carry over from one session

to another, even with a hiatus of 1 or more days as often occurs in human

abuse (Spotts and Shontz 1980; Gawin and Ellinwood 1988; Gawin 1991).

Such a scenario could yield a very prolonged state of dysregulation. Thus, it is

possible that one or more of the problems related to addiction and craving may

result, not from the prominent induction of IEGs seen with a single injection,

but from the prolonged blockade of regulated expression engendered by the

suppression process.

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AUTHORS

Michael J. ladarola, Ph.D.

Research Pharmacologist

Eric J. Chuang, B.S.

Choh-Lun Yeung, B.S.

Yin Hoo, M.D.

Mayme Silverthorn, B.A.

Jun Gu, Ph.D.

Gaetano Draisci, M.D.

R.

Neurobiology and Anesthesiology Branch

National Institute of Dental Research

National Institutes of Health

Building 30, Room B-l 0

Bethesda, MD 20892

211
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107 DRUG ABUSE PREVENTION INTERVENTION RESEARCH:

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114 METHODOLOGICAL ISSUES IN CONTROLLED STUDIES ON EFFECTS

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117 METHODOLOGICAL ISSUES IN EPIDEMIOLOGY, PREVENTION, AND

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120 BIOAVAILABILITY OF DRUGS TO THE BRAIN AND THE BLOODBRAIN BARRIER. Jerry Frankenheim, Ph.D., and Roger M. Brown, Ph.D., eds.

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121 BUPRENORPHINE: AN ALTERNATIVE TREATMENT FOR OPIOID

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122 RESEARCH METHODS IN WORKPLACE SETTINGS. Steven W. Gust,

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123 ACUTE COCAINE INTOXICATION: CURRENT METHODS OF

TREATMENT. Heinz Sorer, Ph.D., ed.

124 NEUROBIOLOGICAL APPROACHES TO BRAIN-BEHAVIOR

INTERACTION. Roger M. Brown, Ph.D., and Joseph Fracella, Ph.D., eds.

219
NIH Publication No. 93-3504

Printed 1993