Immediate-Early

Genes
as Activity Markers in the CNS
George

I. Discovery

S. Robertson

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). However, 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 exposure 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

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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
isolation 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
2.7.

of c-fos Expression

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 triggered by a broad host of physiological
and pharmacological
treatments 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
pathways 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?OStranscription
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 expression are present in unstimulated
cells and that their activation
is
mediated
by posttranslational
processes such as phosphorylation.

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Since transcriptional induction in the presence of protein synthesis inhrbitors is characteristic of viral immediate-early genes, c-fos
and other rapidly induced genes, are commonly referred to as cellular 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 showing that depolarization of rat PC 12 pheochromocytoma cells by
exposure to nicotine produces a rapid elevation of c-f& expression (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+ions 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 nucleotides from the 5 initiation site for c-fos mRNA synthesis plays
an important role in mediating the c-fos response to VSCC activation (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 phosphorylation in the activation of CREBs transcriptional
stimulating

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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
kinase (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 serumresponse 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 several 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 microtubuleassociated 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). Nevertheless, Ca*+ influx through the NMDA receptor does result in the
induction of C--OSas 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 performed 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, activation 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 findings suggest that Ca*+ influx through VSCCs and NMDA receptors leads to the activation of c-fos expression via distinct
signalling pathways.

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2.4. Regulatory Elements in the c-fos Promotor

Operate in an Interdependent
Fashion
The previously described studies that were performed in cultured cells suggest that the regulation of c-fos expression is mediated 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 expression 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 organism, transgenic animals have been created in which these
regulatory elements in the Fos-1acZ construct were rendered nonfunctional by the introduction of clustered point mutations. Consistent 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 neurons (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

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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 signals in the CNS are not transduced in a linear fashion, resulting in
activation of a single response element but rather by interdependent 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 complete description of the physiological and pharmacological treatments 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 extracellular 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 characterization of the phenotypic and connection character of neurons that express this IEG. Lastly, inhibition of c-fos expression
using antisense DNA technology has revealed some of the physiological 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

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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 members 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 expression peaks by 2-4, whereas AFosB levels are maximal at 4 h and
remain elevated for at least 8 h. Hence, seizures stimulate the formation 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-methylD-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 immunoreactivity 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 agonists increase c-fos expression in the brain whereas MK-801 reduces
IEG activation after kindling-induced
seizures (Sonnenberg et al.,

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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 addition to seizures, MK-801 has been reported to reduce IEG expression 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 elevation of Fos-like immunoreactivity
in nuclei of postsynaptic neurons 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 stimulation of the hind paws markedly increases Fos-like immunoreactivity 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, administration 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 contrast, 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;

Immediate-Early

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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 levodopaand 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 Parkinsons 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 distribution 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 haloperidol and raclopride increase Fos-like immunoreactivity
in strratal 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 striatopalhdal with an oligonucleotide probe complementary to mRNA
encodmg enkephalin. Thus, by combining Fos-like immunohistochemistry 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 neurons retrogradely labeled with fluoro-gold from the substantia
nigra pars reticulate (Robertson et al., 1992). These findings indicate that D2-like receptor antagonists elevate c-fos expression primarily in striatopallidal neurons. Moreover, they are consistent

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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 immunoreactivity 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 ipsilateral 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 immunohistochemistry with the detection of proenkephalin
mRNA by in situ
hybridization histochemistry. Dl-like receptor-activated Fos-like
immunoreactivity
was infrequently found in striatopallidal neurons identified with the proenkephalin
oligonucleotide
probe
indicating that it is preferentially localized in striatonigral neurons (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 immunoreactivity 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. Electrophysiological studies have reported that D2-like receptor activation increases the activity of pallidal neurons and that
6-hydroxydopamine
lesions of the nigrostriatal pathway potentiate 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

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stimulatory actions of quinpirole on pallidal activity. By inhibiting striatopallidal neurons, quinpirole would presumably decrease
the release of such inhibitory neurotransmitters
as GABA and
enkephalin from striatopallidal terminals resulting in a disinhibition of pallidal neurons. The ability of quinpirole to elevate c-fos
expression in the globus pallidus after 6-OHDA lesions may therefore 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 transcriptional 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). However, 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 neurons. 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 agonist 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 regulating factors that couple extracellular signals to alterations in cellular phenotype by regulating the expression of specific target genes

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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 mediated 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 families 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 complexes 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 different Al?-1 complexes. For example, proteins encoded by each
member of thefis family (Fos, FosB, Fra-1 and Fra-2) can dimerize 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, suggesting 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

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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 dorsolateral striatum suggesting that an increase in synthesis is responsible 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 intracellular 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 expression 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 AP1 binding site has been identified in the NT/N promoter that contributes 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. Selectivity of the antisense effect was confirmed by establishing that
expression of a nontargeted immediate-early gene cc-iun) and neuropeptide (enkephalin), located in striatal neurons that would otherwise have displayed haloperidol-induced
FL1 and c-f& mRNA,
were not altered by the antisense ODN. In this way, we demonstrated 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 neuronal activation in the CNS, FL1 is limited by the fact that it cannot 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 alternative 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 transcript (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 factor 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 initiated by Fos (Hope et al., 1992). Consistent with this proposal,
repeated administration of the mixed Dl /D2 receptor agonist apomorphine 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 experiments 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 neurotransmission
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, recognizes just FosB (FosB[C]) (Nakabeppu
and Nathans, 1991;
Nakabeppu et al., 1993). In a first series of studies, we demonstrated that decreasing striatal D2 receptor stimulation by either
chronic administration of haloperidol or dopaminergic denervation produced a prolonged elevation of FosB-like immunoreactivity detected with the FosB(N) antibody. In contrast, the FosB(C)
antibody failed to demonstrate an increase in FosB-like immunoreactivity 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 recognizes 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 outputs 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 persistently upregulate excitatory transmembrane
signalling in
striatopallidal neurons. In a second series of studies, we investigated 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 neurons. 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 studies 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 populations 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 correlated with increases in gene signalling activity, it may be
appropriate to view AFosB as a marker of chronic neuronal activation. 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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