7 Immediate-Early Genes as Activity Markers in the CNS

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Immediate-Early Genes

as Activity Markers in the CNS

George S. Robertson

I. Discovery of the Proto-Oncogenes

Genetic analysis of viruses capable of producing tumors in mice

led to the discovery of cancer-causing genes termed oncogenes.

The v-fos oncogene is responsible for the ability of the FBJ-MSV

virus to produce bone tumors (Finkel et al., 1966; Curran and Teich,

1982). Shortly after identification of v-fos, it became clear that this

oncogene had a normal cellular counterpart (Curran et al., 1984).

The normal cellular sequences from which the viral oncogene

(V- X) was derived is referred to as thefos proto-oncogene or c-fos.

The protein product of c-fos is a 55-kDa protein (Fos) that plays an

important role in the signal transduction events mediating cell

growth and division (Morgan and Curran, 1991). Proto-oncogenes

such as c-fos contain negative regulatory elements that prevent

overexpression (Sassone-Corsi et al., 1988; Gius et al., 1990). How-

ever, these expression-limiting elements are not present within

v-fos, enabling the FBJ-MSV virus to produce osteosarcomas (bone

tumors). Overexpression of oncogene products in virally infected

cells leads to tumor formation because the signal transduction

pathways specifying growth and division become overstimulated

(Carbone and Levine, 1990).

The c-fus and c-jun proto-oncogenes were identified as genes

whose rapid but transient transcription was activated by expo-

sure of cells to serum or growth factors that initiate the cell cycle

(Greenberg and Ziff, 1984; Lamph et al., 1988). A number of related

proto-oncogenes were discovered shortly thereafter, using probes

From Neuromethods vol 33 Cell Neurobiology Techm ques

Eds A A Boulton G B Baker and A N Bateson 0 Humana Press lnc

231



232 Robertson

based upon sequences found within these genes to screen cDNA

libraries constructed from serum-stimulated cells. These included

f&B and thefts-related antigens (fva-1 andfia-2) as well as jun-B

(Cohen and Curran, 1988; Lau and Nathans, 1987; Ryder et al.,

1988; Zerial et al., 1989; Mshina et al., 1990). Although jUM-D

expression was not markedly elevated by growth factors or

serum, its high constitutive expression in 3T3 cells permitted iso-

lation of this third member of the jun family (Hirai et al., 1989;

Ryder et al., 1989). The zinc finger-containing gene NGFI-A, also

known as zip68, krox-24 and egr-1, was identified as a gene that is

rapidly activated by growth factors or serum in 3T3 cells

(Milbrandt, 1987).

2, Regulation of c-fos Expression

2.7. c-fos is an Immediate-Early Gene

In most cells, basal expression of c-fos mRNA and protein is

low (Morgan et al., 1987; Sagar et al., 1988; Smeyne et al., 1992). In

such cell types, extracellular signals are required to elevate

expression of this proto-oncogene. It is now well established that

c-fos expression in the central nervous system (CNS) can be trig-

gered by a broad host of physiological and pharmacological treat-

ments that increase neuronal activity (Morgan and Curran, 1991;

Hughes and Dragunow, 1995). This has led to the wide spread

use of c-fos as a metabolic marker for mapping functional path-

ways in the CNS. These studies have shown that the time course

for induction of c-fos expression is similar in most cases. At the

transcriptional level, activation usually takes place within several

minutes and lasts for approx 20 min with peak increases in mRNA

occuring 30-45 min after stimulation (Muller et al., 1984). After

this time, mRNA levels rapidly decrease with a half-life of approx

12 min. Synthesis of Fos follows mRNA expression with peak

increases detectable approx l-2 h after the onset of stimulation,

thereafter Fos levels rapidly decline to basal levels by 6-8 h (Muller

et al., 1984; Curran et al., 1984; Sonnenberg et al., 1989). The

induction of c-J?OSranscription is not dependent on the synthesis

of new proteins and readily occurs m the prescence of protein

synthesis inhibitors (Lau and Nathans, 1987; Curran and Morgan,

1986). This indicates that the protems required for c-fos expres-

sion are present in unstimulated cells and that their activation is

mediated by posttranslational processes such as phosphorylation.



Immediate-Early Genes as Activity Markers

233

Since transcriptional induction in the presence of protein synthe-

sis inhrbitors is characteristic of viral immediate-early genes, c-fos

and other rapidly induced genes, are commonly referred to as cel-

lular immediate-early genes (Lau and Nathans, 1987; Curran and

Morgan, 1987).

2.2. The Calcium Response Element

The first demonstration that neurotransmitters can activate

immediate-early gene (IEG) expression came from studies show-

ing that depolarization of rat PC 12 pheochromocytoma cells by

exposure to nicotine produces a rapid elevation of c-f& expres-

sion (Greenberg et al., 1986). Elegant studies performed by

Greenberg and colleagues have yielded significant insights into

the signal transduction events that mediate c-fos activation by

depolarizing neurotransmitters. A key step in this process is the

influx of Ca2+ ons through specialized channels embedded in the

plasma membrane. In neurons, calcium entry may occur by way

of at least two types of Ca2+ channels: voltage sensitive calcium

channels (VSCCs) and N-methyl N-aspartate (NMDA) receptors.

In the case of VSCCs, channel opening is triggered by membrane

depolarization. In contrast, NMDA receptors are ligand-gated ion

channels that require both occupation by ligand and membrane

depolarization to open. The subsequent rise in intracellular Ca2+

induces c-fos transcription (Morgan and Curran, 1986; Curran and

Morgan, 1986). A Ca*+ response element (CaRE) locateld 60 nucle-

otides from the 5’ initiation site for c-fos mRNA synthesis plays

an important role in mediating the c-fos response to VSCC acti-

vation (Sheng et al., 1990). The CaRE (-TGACGTTT-) is similar

in sequence to a consensus CAMP response element (CRE)

(-TGACGTCA-) located within the regulatory regions of a variety

of genes that become activated when cells are exposed to agents

such as forskolin that activate adenylate cyclase and stimulate the

production of CAMP (Montminy et al., 1986). Placement of the

c-fos CaRE/CRE into the promotor of genes that fail to respond to

forskolin or VSCC activators endows the ability to respond to these

agents (Sheng et al.,

1990; Sheng et al., 1991). Constitutively

bound to the -60 CaRE is the calcium response element binding

protein (CREB) that is converted into a positive transcriptional

factor by phosphorylation at a critical regulatory site, serine 133

(Sheng et al., 1990). A crucial role for serine 133 phosphoryla-

tion in the activation of CREB’s transcriptional stimulating



234 Robertson

activity is indicated by the loss of this function when serine 133

is mutated to alanine (Sheng et al., 1991; Gonzales and

Montminy, 1989; Schwaninger et al., 1993). Serine 133 of CREB

may be phosphorylated by calcium/calmodulin-dependent

protein kineses (CaM kinases) as well as CAMP-dependent ki-

nase (PKA); both of these classes of kinases are thought to play

a role in mediating the induction of c-fos expression by VSCC

activators (Ghosh and Greenberg, 1995).

2.3. The Serum Response Element

A second key regulatory element in the c-fos promotor that

confers second messenger inducibility of this IEG is the serum-

response element (SIRE). The SRE was originally described as a

protein-binding site required for the induction of c-fos expression

by serum and growth factors (Treisman, 1992). The SRE, together

with flanking DNA sequences, binds an assembly of multiprotein

complexes that include the serum-response factor, Elk-l, and sev-

eral other transcriptional regulating factors (Shaw et al., 1989;

Hipskind et al., 1991; Hill et al., 1993). At present, the precise

mechanism responsible for activation of c-fos transcription via the

SRE is unclear, but likely involves a Ras-dependent mechanism

that culminates in phosphorylation of Elk-l by microtubule-

associated protein (MAP-11 (Marais et al., 1993). The development

of techniques enabling transfection of primary neurons has

assisted analysis of the relative roles of the CaRE and SRE in

NMDA receptor-mediated signaling. In hippocampal neurons,

NMDA receptor activation does not trigger significant amounts

of CaRE-dependent transcription (Bading et al., 1993). Neverthe-

less, Ca*+ influx through the NMDA receptor does result in the

induction of C--OS s well as several other IEGs in hippocampal

neurons indicating mediation by a non-CaRE element such as

the SRE. Consistent with this proposal, transfection studies per-

formed on cultured hippocampal neurons have shown that

NMDA receptor activation of C- X requires phosphorylation of

the SRE. In contrast to NMDA-mediated c-f0.s expression, acti-

vation of VSCCs by elevation of extracellular KC1 concentrations

promotes c-fos transcription by phosphorylation of CREB and

occurs in the absence of the SRE (Bading et al., 1993). These find-

ings suggest that Ca*+ influx through VSCCs and NMDA recep-

tors leads to the activation of c-fos expression via distinct

signalling pathways.



Immedate-Early Genes as Actwity Markers

23.5

2.4. Regulatory Elements in the c-fos Promotor

Operate in an Interdependent Fashion

The previously described studies that were performed in cul-

tured cells suggest that the regulation of

c-fos

expression is medi-

ated by individual response elements (CaRE and SRE) that act

independently in response to extracellular stimuli. However, in

the intact organism, it is unlikely that the independent actions of

individual regulatory elements can account for the broad range

of stimuli which can induce c-fos expression. This issue was

recently addressed using a Fos-1acZ transgenic mouse in which

expression of the bacterial J3-galactosidase gene is driven by the

c-fos promoter. The Fos-1acZ transgene directs inducible expres-

sion of a fusion protein consisting of 315 N-terminus amino acids

from c-fos and 1015 C-terminus amino acids from /3-galactosidase

(Schilling et al., 1991; Smeyne et al., 1992). The Fos-1acZ fusion

protein retains the nuclear localization signal for Fos, and

P-galactosidase is exclusively revealed in nuclei. Employing the

histochemical detection of P-galactosidase activity encoded by this

gene, Smeyne et al. (1992) have shown that the Fos-1acZ construct

recapitulates both tissue- and stimulus-specific regulation of

c-fos expression in vivo. In order to determine the role of the CaRE

and SRE sites in controlling c-fos expression in the intact organ-

ism, transgenic animals have been created in which these

regulatory elements in the Fos-1acZ construct were rendered non-

functional by the introduction of clustered point mutations. Con-

sistent with transient transfection experiments in cultured

hippocampal neurons, mutation of the CaRE abolished Fos-1acZ

induction in primary neuronal cultures by KC1 (Robertson et al.,

199513).However, mutation of the SRE also eliminated KCl-induced

c-fos expression suggesting that multiple elements are necessary

and none sufficient for the complete activation of the gene by KCl.

A similar finding occurred in vivo. For instance, the induction of

c-fos in response to kainate-induced seizures is thought to involve

several transduction pathways, particularly Ca*+ influx through

VSCCs. However, mutation of either the CaRE or SRE completely

abolished karinate-induced c-fos expression in the majority of neu-

rons (Robertson et al., 1995b). Interestingly, the excitatory effects of

both KC1 and kainate on neuronal c-fos expression were also lost

after selective mutation of the &-inducible element (SIE). The SIE

is a regulatory element found within the c-fos promoter that is



236 Robertson

thought to confer inducibility of this gene to platelet derived

growth factor (PDGF) (Wagner et al., 1990). Consequently, the

CaRE, SRE, and SIE were required in combination for induction

of c-fos expression in many neurons. These findings suggest that,

at least within the context of the c-fos promoter, physiological sig-

nals in the CNS are not transduced in a linear fashion, resulting in

activation of a single response element but rather by interdepen-

dent networks of transcriptional regulating factors that require

multiple regulatory elements to operate properly (Robertson et

al., 1995b).

3. IEGs as Activity Markers in the CNS

3. I. Overview

Although basal levels of c-fos mRNA and protein are low in the

CNS, neuronal expression of this IEG is rapidly but transiently

elevated by a broad array of extracellular stimuli. Since a com-

plete description of the physiological and pharmacological treat-

ments that induced c-fos expression is beyond the scope of the

present review, I will focus upon those examples which are of

relevance to neuropsychopharmacology. As indicated previously,

the c-fos promoter contains regulatory elements that are activated

by the second messengers AMP and Ca2+. Since generation of

these second messengers is linked to stimulation of the extra-

cellular receptors for a broad range of neurotransmitters, detection

of c-fos mRNA and protein has proven to be a quick, inexpensive,

and reliable method for the identification of putative neuronal

targets for various classes of neuropharmacological agents.

Moreover, double labeling Fos-positive neurons with classic

neurochemical markers or retrograde tracers has permitted char-

acterization of the phenotypic and connection character of neu-

rons that express this IEG. Lastly, inhibition of c-fos expression

using antisense DNA technology has revealed some of the physi-

ological targets for this transcriptional regulating factor.

3.2. EC Induction in the Forebrain

by Treatments that Induce Seizures

Administration of pentylenetetrazole (PTZ) to rodents results

in the rapid onset of seizures and convulsions that last for approx

30 min. Within minutes of PTZ-induced seizures, a synchronous




237

wave of IEG expression occurs that is localized to those brain

regions which display seizure activity such as the hippocampus,

cortex and limbic system (Morgan et al., 1987; Saffen et al., 1988;

Sonnenberg et al., 1989a,b; Le Galle La Salle and Naquet, 1990).

PTZ-induced seizures result in the elevation of c-fos, c-jun, jun-B,

jun-D, and NGF-A in all of these brain regions. In addition to PTZ,

elevated neuronal IEG expression has been reported after the

induction of seizures by kindling (Dragunow and Robertson,

1987), electroconvulsive shocks (Hope et al., 1994a), audogenic

stimulation (Le Galle La Salle and Naquet, 1990), kainic acid

(Popovici et al., 1988), bicuculline (Gass et al., 1992a), or pilocarpine

(Barone et al., 1993). Seizure-induced IEG expression is paralleled by

an elevation of AP-l-like DNA binding that persists for up to 8 h

after the initiation of seizures (Sonnenberg et al., 1989a,b). Western

blotting performed with antisera that recognizes all known mem-

bers of the Fos family indicates that Fos production peaks 1-2 h

after the onset of seizures and returns to basal levels by 6-8 h.

This indicates that Fos does not participate in Al?-1 complexes

detected at these later time points. In contrast, several lower

molecular weight proteins (35 kDa and 46 kDa) detected with this

antisera, termed Fos-related antigens (Fras), display prolonged

induction kinetics with respect to Fos. Subsequent studies have

shown that the 46-kDa Fra is actually FosB whereas the 35-kDa

Fra corresponds to a truncated version of FosB known AFosB

(Hope et al., 1994b). Using FosB and AFosB-selective antibodies,

it has been demonstrated that after seizure onset FosB expres-

sion peaks by 2-4, whereas AFosB levels are maximal at 4 h and

remain elevated for at least 8 h. Hence, seizures stimulate the form-

ation of dynamic AP-1 complexes whose composition changes over

time. Initially, seizure-induced Al?-1 complexes consist primarily

of Fos/ Jun dimers, but are replaced by FosB/ Jun and AFosB/Jun

dimers at later time points.

MK-801 is a noncompetitive antagonist of the N-methyl-

D-aspartate (NMDA) subclass of excitatory aminoacid receptors.

The distribution of NMDA receptors in the brain closely matches

the distribution of neurons that express Fos-like immunoreactiv-

ity after administration of PTZ suggesting that activation of these

receptors mediates c-fos induction by seizure activity (Morgan et

al., 1987). Consistent with this proposal, glutamate receptor ago-

nists increase c-fos expression in the brain whereas MK-801 reduces

IEG activation after kindling-induced seizures (Sonnenberg et al.,



238 Robertson

1989b; Page and Everitt, 1993; Sugimoto et al., 1993). The failure

of MK-801 to block IEG expression produced by electroshock (Cole

et al., 1990a) and lindane-induced seizures (Vendrell et al., 1992)

suggests that the activation of non-NMDA receptors and/or

VSCCs may also play an important role in this process. In addi-

tion to seizures, MK-801 has been reported to reduce IEG expres-

sion produced by more physiologically relevant stimuli. For

instance, NMDA receptor blockade prevents light-induced c-jiis

and NGFI-A mRNA expression m the retina (Gudehithlu et

a1.,1993). MK-801 also abolishes the high constitutive expression

of zif268 (NGFI-A) in the cortex indicating that expression of this

IEG is driven by natural synaptic activity (Worley et al., 1991).

3.3. IEG Induction in the Spinal Cord

by Nonnoxious and Painful Stimulation

Physiological stimulation of rat primary sensory neurons by hair

brushing or gentle joint manipulation promotes a modest eleva-

tion of Fos-like immunoreactivity in nuclei of postsynaptic neu-

rons of the dorsal horn (Hunt et al., 1987). These increases occur

mainly in layers II-IV which are innervated by low-threshold

AGcutaneous afferents. In contrast, painful chemical or heat stimu-

lation of the hind paws markedly increases Fos-like immunoreac-

tivity in layers I and II of the dorsal horn, which receive excitatory

input from nociceptive afferent terminals (Hunt et al., 1987).

Noxious peripheral stimulation also results in the appearance of

Fos expression in thalamic regions known to process painful

stiumulation (Bullitt, 1989). Consistent with the notion that these

increases are related to the activation of pain pathways, adminis-

tration of morphine substantially reduces the induction of Fos-like

immunoreactivity in superficial layers of the dorsal horn by

noxious stimulation of the hind paw (Tolle et al., 1990).

3.4. IEG Induction in the Striatum by Dopaminergic Drugs

Basal c-fos expression in the striatum in very low but is rapidly

elevated by systemic administration of cocaine and d-amphetamine,

stimulants that greatly enhance extracellular concentrations of

dopamine (Robertson et al., 1989b; Graybiel et al., 1990). In con-

trast, levodopa and directly acting Dl-like receptor agonists such

as SKF 38393 and CY 208-243 only weakly increase Fos-like

immunoreactivity in the intact striatum (Robertson et al., 1989a,b;




239

Robertson et al., 1992). Destruction of the nigrostriatal pathway

with 6-OHDA, however, endows levodopa and Dl-like agonists

with the ability to dramatically enhance c-fos expression in the

denervated striatum (Robertson et al., 1989a,b; Paul et al., 1992;

Morelli et al., 1993). These increases are completely blocked by

the selective Dl-like receptor antagonist SCH 23390 indicating that

activation of Dl-like receptors plays an important role in levodopa-

and Dl-like agonist-induced c-fos expression (Robertson et al.,

1989b, Morelli et al., 1993). The failure of levodopa and Dl-like

agonists to increase Fos-like immunoreactivity in the striatum of

intact animals suggests that the development of denervation-induced

supersensitivity is responsible for the large increases in c-fos

expression produced by these compounds in the 6-OHDA-denervated

striatum. This is consistent with the proposal that postsynaptic

changes in the striatum may contribute to the development of

levodopa-induced dyskinesias in Parkinson’s disease (Chase et

al., 1993; Obseo et al., 1994).

D2-like receptor antagonists such as haloperidol and raclopride

increase c-fos expression in the intact striatum (Dragunow et al.,

1990; Deutch et al., 1992; Robertson and Fibiger, 1992). The distri-

bution of increased Fos-like immunoreactivity produced by these

neuroleptics very closely matches the distribution of striatal D2

receptors (Boyson et al., 1986; Robertson and Fibiger, 1992). The

close topographical relationship between neuroleptic-induced

Fos-like immunoreactivity and D2 receptors suggests that halo-

peridol and raclopride increase Fos-like immunoreactivity in strr-

atal neurons that express D2 receptors, that is, in striatopallidal

neurons (Gerfen and Young, 1988; Gerfen et al., 1990). The fact

that enkephalin is utilized principally by striatopallidal neurons

as a neurotransmitter, enabled us to preferentially label striato-

palhdal with an oligonucleotide probe complementary to mRNA

encodmg enkephalin. Thus, by combining Fos-like immunohis-

tochemistry with the detection of proenkephalin mRNA by tn situ

hybridization histochemistry, we were able to demonstrate that

D2 antagonists elevate Fos-like immunoreactivity in striatopallidal

neurons (Robertson et al., 1992). Furthermore, neuroleptic-induced

Fos-like immunoreactivity was seldom found in striatonigral neu-

rons retrogradely labeled with fluoro-gold from the substantia

nigra pars reticulate (Robertson et al., 1992). These findings indi-

cate that D2-like receptor antagonists elevate c-fos expression pri-

marily in striatopallidal neurons. Moreover, they are consistent



240

Robertson

with neurochemical and neurophysiological studies showing that

dopamine inhibits striatopallidal activity (Pan et al., 1985; Gerfen

et al, 1990. Carlson et al., 1990).

Using retrograde tracing techniques, we have demonstrated that

the Dl-like receptor agonist SKF 38393 elevates Fos-like immu-

noreactivity in striatonigral neurons (Robertson et al., 1990). This

result is consistent with 2-deoxyglucose uptake studies showing

that Dl-like receptor agonists activate striatonigral neurons ipsi-

lateral to the 6-hydroxydopamine-lesioned substantia nigra

(Trugman and Wooten, 1987). In order to determine whether SKF

38393-induced Fos-like immunoreactivity was also present in

striatopallidal neurons, we combined Fos-like immunohistochem-

istry with the detection of proenkephalin mRNA by in situ

hybridization histochemistry. Dl-like receptor-activated Fos-like

immunoreactivity was infrequently found in striatopallidal neu-

rons identified with the proenkephalin oligonucleotide probe

indicating that it is preferentially localized in striatonigral neu-

rons (Robertson et al., 1992). This finding is in agreement with

autoradiographic and in sztu hybridization studies which report

that Dl receptors are expressed predominantly by striatonigral

neurons (Harrison et al., 1990; Gerfen et al., 1990; Le Moine et al.,

1991). Taken together, these results suggest Dl-like agonist-induced

c-fos expression may be utilized as a measure of striatonigral

activation.

In contrast to levodopa and Dl-like agonists, D2-like receptor

agonists such as quinpirole fail to elevate Fos-like immunoreac-

tivity in the 6-OHDA-denervated striatum (Robertson et al., 1989b;

Robertson et al., 1992; Paul et al., 1992). Instead, quinpirole elevates

Fos-like immunoreactivity in the ipsilateral globus pallidus. Elec-

trophysiological studies have reported that D2-like receptor acti-

vation increases the activity of pallidal neurons and that

6-hydroxydopamine lesions of the nigrostriatal pathway potenti-

ate this increase (Carlson et al., 1990). The increase in Fos-like

immunoreactivity in the globus pallidus ipsilateral to the

6-OHDA-denervated striatum is consistent with the enhanced

ability of quinpirole to activate pallidal neurons. The small amount

of D2 receptor mRNA in the globus pallidus (Meador-Woodruff

et al., 1989; Mengod et al., 1989) suggests that the excitatory effects

of quinpirole on pallidal neurons are indirect. Given that D2

receptors reside on striatopallidal neurons and function to inhibit

these neurons, it is possible that this pathway is involved in the



Immedmte-Early Genes as Activity Markers

247

stimulatory actions of quinpirole on pallidal activity. By inhibit-

ing striatopallidal neurons, quinpirole would presumably decrease

the release of such inhibitory neurotransmitters as GABA and

enkephalin from striatopallidal terminals resulting in a disinhibi-

tion of pallidal neurons. The ability of quinpirole to elevate c-fos

expression in the globus pallidus after 6-OHDA lesions may there-

fore be a reflection of D2 receptor supersensitivity in the striatum

rather than the globus pallidus. If this is the case, D2-like

receptor-mediated increases in c-fos expression in the globus

pallidus may serve as an excitatory index of both pallidal and

striatopallidal activity.

Zifl68

(also known as NGFI-A, egr-1 and krox-24) is a transcrip-

tional regulatory factor encoded by the IEG 268 (Changelin et

al., 1989). Like c-fos, ~ 268 is considered to be an activity marker

for certain neurons (Worley et al., 1991; Cole et al., 1992). How-

ever, unlike c-fos, there is high basal expression of zfl68 in the

striatum. In a recent report, constitutive expression of ~ 268

mRNA was detected in medium-sized striatal neurons (Mailleux

et al., 1992). Basal expression of ~ 268 appears to be driven by

natural synaptic activity (Worley et al., 1991). ~27268can therefore

be used to measure reductions in neuronal activity. For example,

administration of the selective Dl-like receptor antagonist SCH

23390 reduces basal ~ 268 expression in the intact striatum

(Mallieux et al., 1992). Given that Dl receptors in the striatum are

located predominantly on striatonigral neurons, this observation

suggests that SCH 23390 reduces the activity of striatonigral neu-

rons. Reductions in ~ 268 expression may therefore be used to

study decreases in striatonigral activity. Furthermore, striatal

~ 268 expression is elevated by systemic administration of D2-like

antagonists; whereas its expression is reduced by the D2-like ago-

nist quinpirole (Nguyen et al., 1992; Keefe and Gerfen, 1995). These

changes occur primarily in enkephalin neurons suggesting that

zif268 can also be used to measure both increases and decreases

in the activity of striatopallidal neurons.

4. Members of the Fos and

Jun Family Dimerize to Form the AP- 1 Complex

Fos and Jun are thought to function as transcriptional regulat-

ing factors that couple extracellular signals to alterations in cellu-

lar phenotype by regulating the expression of specific target genes



242 Robertson

(Morgan and Curran, 1991; Hughes and Dragunow, 1995). In

order to bind to DNA and regulate gene expression, eachfos

family protein must first dimerize with a protein product of

the iun family. Dimerization of Fos and Jun proteins is medi-

ated by hydrophobic interactions between an a-helical domain

containing a heptad repeat of five leucine residues common to

both partners-the so-called leucine zipper (Gentz et al, 1988;

Turner and Tjian, 1989). Protein products of thefos and @n fami-

lies can form heterodimers while members of the IWZ family

can also associate with themselves forming homodimers

(Nakabeppu et al., 1988; Cohen et al., 1989; Zerial et al., 1989;

Nishina et al., 1990). Heterodimers consisting of members of

the fun andfos families are commonly referred to as Al?-1 com-

plexes which bind to a specific sequence of DNA known as the

AP-1 site (Franz et al., 1988; Rauscher et al., 1988). The leucine

zipper motif permits the formation of a large number of differ-

ent Al?-1 complexes. For example, proteins encoded by each

member of thefis family (Fos, FosB, Fra-1 and Fra-2) can dimer-

ize with each member of the lun family (Jun, JunB, and JunD)

Accumulating evidence indicates that the wide variety of

potential dimer combinations serves as a mechanism for fine

transcriptional regulation.

5. Regulation of Neuropeptide

Gene Expression by Immediate-Early Genes

The wide spread inducibility of c-fos expression in the CNS has

led to the search for downstream genes that are regulated by Fos

(Hughes and Dragnow, 1995). AP-l-like binding sites have been

identified in the promoters of genes encoding the neuropeptides

enkephalin, dynorphin, cholecystokinin, and neurotensin, suggest-

ing that their expression may be regulated by Fos (Sonnenberg et

al., 1989; Monstein, 1993; Naranjo et al., 1991, Kislaukis et al., 1988).

Indeed, transient cotransfection studies indicate that Fos and Jun

can enhance proenkephalin and prodynorphin expression

(Sonnenberg et al., 1989). However, until recently it had not been

clear as to whether neuropeptide genes were in fact physiological

targets for Fos.

The trldecapeptlde neurotensm (NT) IS widely distributed

throughout the CNS, where it is thought to function as a classical

neurotransmitter or neuromodulator (Iversen et al., 1978; Kitabgi



/mmediate-Early Genes as Actrvity Markers

243

et al., 1977; Uhl, 1982). A single injection of the prototypical

antipsychotic haloperidol produces a dramatic elevation of

neurotensin/neuromedin N (NT/N) mRNA levels in the dorso-

lateral striatum suggesting that an increase in synthesis is respon-

sible for the subsequent enhancement of NT concentrations

(Govoni et al., 1980; Merchant et al., 1991; 1992a,b). Several lines

of evidence suggest that c-fos may participate in those intracellu-

lar events, responsible for haloperidol-induced proneurotensin

mRNA expression in the dorsolateral striatum. Several lines of

evidence suggest that c-f& may participate in those intracellular

events responsible for haloperidol-induced NT/N mRNA expres-

sion in the dorsolateral striatum. First, haloperidol dramatically

elevates c-f& mRNA and Fos-like immunoreactivity (FLI) in the

dorsolateral striatum, with peak increases occurring before those

in NT/N mRNA (Merchant et al., 1992b; Robertson and Fibiger,

1992). Thus, there is a temporal relationship between the c-fos and

NT/N induction. Second, a remarkable correspondence between

the distribution of haloperidol-induced c-fos mRNA, FL1 and

NT/N mRNA in the striatum has been noted suggesting that these

increases occur in the same population of neurons (Deutch et al.,

1992; Merchant et al., 1992a, Robertson et al., 1992). Third, an AP-

1 binding site has been identified in the NT/N promoter that con-

tributes to the inducibility of this gene by nerve growth factor in

PC-12 cells (Kislaulcis and Dobner, 1990). Consistent with this

proposal, we have recently utilized antisense DNA technology to

demonstrate that c-fos induction is necessary for the subsequent

elevation of proneurotensin mRNA in the dorsolateral striatum

by haloperidol (Robertson et al., 1995a). Haloperidol-induced c-fas

expression was selectively blocked by microinjection of an

antisense phosphorothioate oligodeoxyribonucleic (ODN) to this

immediate-early gene into the dorsal striatum. Inhibition of c-fos

expression by the antisense ODN attenuated haloperidol-induced

neurotensin gene expression in the dorsolateral striatum. Selec-

tivity of the antisense effect was confirmed by establishing that

expression of a nontargeted immediate-early gene cc-iun) and neu-

ropeptide (enkephalin), located in striatal neurons that would oth-

erwise have displayed haloperidol-induced FL1 and c-f& mRNA,

were not altered by the antisense ODN. In this way, we demon-

strated that the antisense ODN diminished haloperidol-induced

neurotensin gene expression by selectively preventing c-fos

expression.



244 Robertson

6. AfosS as a Chronic Marker of Neuronal Activation

Although immunohistochernical detection of FL1 has proven

to be a very useful technique for the identification of acute neu-

ronal activation in the CNS, FL1 is limited by the fact that it can-

not be used to study neuronal populations that are activated by

chronic stimulation. For instance, chronic administration of

antipsychotics such as haloperidol or clozapine results in a rapid

desensitization of the acute increases in both c-f& mRNA and FL1

produced by these drugs (Coppers et al., 1995; Merchant et al.,

1995; Sebens et al., 1995). Downregulation of the c--&s response is

a general phenomenon that has been reported to occur with

repeated exposure to a variety of treatments (Winston et al., 1990,

Hope et al., 1994a; Rosen et al., 1994). In contrast, levels of the IEG

product AFosB are enhanced by chronic exposure to treatments

that acutely elevate Fos (Hope et al., 1994b, Doucet et al., 1996;

Vahid-Ansari et al., 1996). These studies suggest that it may be

possible to use AFosB as a marker for chronic neuronal activation

6.1. AfosB is Produced by Alternative Splicing of fosB

Two different forms of f&B mRNA are generated by alterna-

tive splicing of the transcript from a singlefosB gene (Dobrzanski

et al., 1991; Mumberg et al., 1991; Nakabeppu and Nathans, 1991;

Yen et al., 1991). The longer transcript f”&B) encodes a protein

338 amino acids in length called FosB, whereas the shorter tran-

script (AfosB) encodes a truncated form of FosB known as AFosB.

AfosB mRNA is produced by deletion of 140 bases from the fosB

transcript. This deletion shifts the reading frame by a single base,

creating the stop codon TGA. As a result, AFosB is only 237 amino

acids long and lacks the last 101 amino acids found in FosB. Present

within this truncated region is the hepatoproline sequence (amino

acids 257-263) that functions as an activation domain in FosB.

AFosB is therefore a much weaker transcriptional activating fac-

tor than FosB but displays prolonged induction kinetics compared

to Fos and FosB.

6.2. Dopaminergic Regulation of A fosB Expression

It is well known that chronic administration of dopaminergic

stimulants that acutely increase c-fos expression leads to a rapid

loss in the ability of these compounds to elevate c-fos expression

in the striatum (Hope et al., 1992; Iadarola et al., 1993; Rosen et



immediate-Early Genes as Activity Markers

245

al., 1994). Despite this desensitization, AP-1 binding remains

elevated suggesting that another member of the fos family is

responsible for the maintenance of transcriptional changes initi-

ated by Fos (Hope et al., 1992). Consistent with this proposal,

repeated administration of the mixed Dl /D2 receptor agonist apo-

morphine to 6-OHDA-lesioned rats or of cocaine (an indirect

dopamine agonist) to normal animals, produces a persistent

elevation of FLI, detected with an antibody that recognizes all

known members of thefus family, in the striatum (Zhang et al.,

1992; Hope et al., 1994a). Western blotting and gel-shift experi-

ments indicated that a 35 kDa Fos-related antigen is at least partly

responsible for the prolonged increase in Al?-1 binding produced

by chronic apomorphine or cocaine administration (Pennypacker

et al., 1992; Hope et al., 1994a).

Given that the truncated form of FosB (AFosB) is approx 35 kDa

in size and displays prolonged induction kinetics (Nakabeppu and

Nathans, 1991; Mumberg et al., 1991; Nakabeppu et al., 19931, we

examined the effects of chronic alterations in dopaminergic neu-

rotransmission on expression of this protein in the striatum

(Doucet et al., 1996). AFosB- and FosB-like immunoreactivity were

detected using two different affinity-purified rabbit polyclonal

antibodies (Nakabeppu and Nathans, 1991; Nakabeppu et al.,

1993). One antibody, raised against amino acids 79-131 of the

N-terminus of FosB, recognizes both FosB and AFosB (FosB[N]).

The second antibody, raised against a portion of the C-terminus

of FosB that is missing from AFosB (amino acids 245-3151, recog-

nizes just FosB (FosB[C]) (Nakabeppu and Nathans, 1991;

Nakabeppu et al., 1993). In a first series of studies, we demon-

strated that decreasing striatal D2 receptor stimulation by either

chronic administration of haloperidol or dopaminergic denerva-

tion produced a prolonged elevation of FosB-like immunoreac-

tivity detected with the FosB(N) antibody. In contrast, the FosB(C)

antibody failed to demonstrate an increase in FosB-like immunore-

activity after these treatments. These findings were confirmed by

Western blotting that demonstrated a preferential elevation of

AFosB-like proteins. Since the FosB(C) antibody selectively rec-

ognizes FosB, these results suggest that chronic elevations in D2

receptor-mediated signaling selectively elevate AFosB expression.

Using retrograde tract tracing techniques to label the major out-

puts from the striatum, we demonstrated that chronic haloperidol

administration and destruction of the nigrostriatal pathway



246

Robertson

selectively increase AFosB levels in striatopallidal neurons. This

observation is consistent with the ability of these treatments to per-

sistently upregulate excitatory transmembrane signalling in

striatopallidal neurons. In a second series of studies, we investi-

gated the effects of repeated administration of the Dl-like receptor

agonist CY 208-243 (1 mg/kg, injected subcutaneously twice daily

for 5 d) on striatal AFosB levels in rats that had sustained unilateral

lesions of the nigrostriatal pathway. Chronic administration of CY

208-243 produced a dramatic and selective elevation of AFosB

expression in the denervated striatum. Retrograde labelling revealed

that these increases occurred predominantly in striatonigral neu-

rons. This is in line with numerous studies showing that chronic Dl

receptor activation profoundly increases striatonigral gene expression

m the dopaminergically deafferenated striatum. Moreover, our stud-

ies suggest that the 35-kDa Fos-related antigen observed by others

after prolonged apomorphine or cocaine administration is actually

AFosB (Pennypacker et al., 1992; Hope et al., 199413).

In summary, our findings indicate that chronic alterations in

dopaminergic neurotransmission produce a prolonged elevation

of AFosB expression in the striatum. Cellular localization studies

indicate that enhanced AFosB expression occurs in neuronal popu-

lations that display increases in the expression of genes encoding

a variety of protein classes such as receptors, neuropeptides, and

synthetic enzymes. Inasmuch as these changes appear to be cor-

related with increases in

gene

signalling activity, it may be

appropriate to view AFosB as a marker of chronic neuronal acti-

vation. If this is the case, it should be possible to use AFosB as a

chronic activity marker in other paradigms in much the same way

that Fos has been used as an acute marker.

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7 Immediate-Early Genes as Activity Markers in the CNS

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