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INTRODUCTION |
Immediate early genes
(IEGs)1 encode transcription
factors that are induced rapidly by extracellular signals and regulate
expression of target genes. IEGs have been implicated in many neuronal
functions including neuronal plasticity (1, 2). Interactions of these IEG products with specific DNA sequences in gene promoters either activate or suppress the initiation of gene transcription (3). AP-1
(TGACTCA) and CRE (TGACGTCA) motifs are two such DNA sequences that
are regulatory sites of the PKC- and PKA-mediated signal transduction
pathways, respectively (4-6).
The AP-1 DNA-protein complex is composed of heterodimers of members of
both the Fos and Jun IEG families (3, 4, 7-10). Homodimers of Jun
family members also bind with the AP-1 motif (11). The CRE motif is
constitutively bound with the CRE-binding protein (CREB) and the CRE
modulator protein (CREM) that are activated through phosphorylation
(12-15). The exact composition of the AP-1 and/or CRE DNA-protein
complex remains controversial. First, there appears to be cross-talk
between the AP-1 and CRE motifs (16). Jun proteins were found to bind
to the CRE motif (17, 18), and recent reports showed that CREB is also
a component of the AP-1 DNA-protein complex (19). Second, the
non-conserved 5'- and 3'- flanking regions of the consensus AP-1
sequences play an important role in determining binding affinity with
IEG proteins (20). Thus, gene specificity may occur in the composition
of Fos/Jun dimers binding to the AP-1 site. Third, DNA-protein binding activities show tissue specificity as well as developmental and excitation-dependent regulation that may be attributed to
IEG expression varying between brain regions and level of stimulation (5, 19, 21, 22).
Tyrosine hydroxylase (TH), the first and rate-limiting enzyme in the
biosynthesis of catecholamine neurotransmitters, is present in many
brain regions including the olfactory bulb. The rodent TH gene promoter
contains putative CRE and AP-1 motifs, the latter differing from the
consensus AP-1 only in the middle base (T versus C) (5,
23-26). The TH CRE was found to be required not only for basal
activity but also for cAMP-mediated up-regulation of TH promoter
activity in SK-N-BE(2)C and PC12 cells (27, 28). Depolarization-induced
up-regulation of TH promoter activity occurred through both the TH CRE
and AP-1 sites in PC12 cells (29). The latter study also demonstrated
that, whereas the CRE was solely responsible for cAMP-mediated
regulation of TH promoter activity, both CRE and AP-1 sites were
capable of mediating the effects of calcium influx on TH promoter
activity. However, the composition of either TH AP-1 or CRE DNA-protein
complex in any given brain region and cell line remains unclear.
TH expression has been used as a marker to study differentiation and
regulation of the large population of dopaminergic periglomerular neurons intrinsic to the olfactory bulb (30-33). These neurons receive
innervation from olfactory receptor cells and regulate the activities
of mitral and tufted cells (34). In the developing olfactory bulb, TH
phenotypic expression begins after precursor cells migrate from the
subventricular zone to the glomerular region where they receive sensory
afferent stimulation (32, 35). In adult rodents, either olfactory
deafferentation or odor deprivation, the latter produced by unilateral
naris closure, results in a significant loss of TH expression (30,
36-40). These observations indicate that olfactory input from receptor
cells is necessary for both the initiation and maintenance of TH
expression in the periglomerular neurons but do not define the
molecular mechanisms underlying TH gene regulation.
Lacking is evidence that supports a role for either TH AP-1 or CRE
activity in TH gene expression in the dopaminergic periglomerular neurons. Recent studies demonstrated that c-fos message and
c-Fos protein are partially colocalized with TH in the glomerular layer (41, 42). In parallel to the decreased TH expression, drastic down-regulation of c-fos expression occurred in
periglomerular neurons in the odor-deprived olfactory bulb (42),
suggesting a correlation between the c-fos and TH gene
regulation. However, a direct interaction between the c-fos
gene product and the TH AP-1 motif remains to be demonstrated.
Moreover, it is unknown whether the CRE motif on the TH promoter has a
role in TH gene regulation in periglomerular neurons of olfactory bulb
in response to alterations in either primary afferent innervation or stimulation.
To elucidate some of the cis- and trans-acting elements important in
regulating TH expression in the olfactory bulb periglomerular neurons,
the current studies employed immunocytochemical, Western blot, and gel
shift analyses to investigate the composition of the AP-1 and CRE
binding complexes.
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EXPERIMENTAL PROCEDURES |
Animals--
Adult male CD-1 mice were purchased from Charles
River Breeding Laboratory (Kingston, NY) and housed under a 12/12-h
light/dark cycle. Unilateral naris closure was produced with a
spark-gap electrocautery under pentobarbital anesthesia (30 mg/kg
Nembutal). Closure was confirmed visually at the time of sacrifice.
Studies were carried out at least 2 months post-closure. All procedures were performed under protocols approved by the Cornell University Institutional Animal Care and Use Committee and conformed to National Institutes of Health guidelines.
Materials--
Polyclonal antibodies directed against c-Fos,
Fos-B, and Jun-D were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Additional polyclonal c-Fos antisera were obtained from
Genosys Biotechnologies (Woodlands, TX) and Oncogene Research Products (Cambridge, MA). Polyclonal antibodies for CREB, pCREB, and CREM were
purchased from Upstate Biotechnology (Lake Placid, NY). Polyclonal TH
antisera were prepared in the laboratory (43).
Immunocytochemistry--
Mice were perfused under deep
pentobarbital anesthesia with saline containing 0.5% sodium nitrite
and 10 units/ml heparin followed by 4% buffered (0.1 M
sodium phosphate, pH 7.2) formaldehyde. Following 1 h of
post-fixation, olfactory bulbs were infiltrated overnight with 30%
sucrose. Frozen sections (40 µm) were incubated with antisera
overnight at room temperature followed by incubation with the
appropriate biotinylated secondary antiserum, the Vector Elite kit
(Vector Laboratories; Burlingame, CA), and with
3,3'-diaminobenzidine-HCl (0.05%) and hydrogen peroxide (0.003%) as
chromogen (38).
Extraction of Nuclear Proteins--
Nuclear extracts were
isolated from olfactory bulbs according to the method of Roy et
al. (44). For each extraction, 0.3-0.5 g was employed,
representing about 20 olfactory bulbs. Samples were aliquoted and
stored at
70 °C until use. Protein concentrations were determined
with a protein assay kit (Bio-Rad).
Probe Selection and Labeling--
The oligonucleotides
containing the TH AP-1 and CRE motifs (synthesized by Gene Link;
Thornwood, NY) were identical to the regions of the TH 5' upstream
sequence ranging from
189 to
212 and from
32 to
54,
respectively, and they had the following sequences: AP-1,
5'-TGAGGGTGATTCAGAGGCAGGTGC-3' and
3'-ACTCCCACTAAGTCTCCGTCCACG-5'; CRE,
5'-GAGGGGCTTTGACGTCAGCCTGG-3' and
3'-CTCCCCGAAACTGCAGTCGGACC-5'. The sense and antisense
oligonucleotides were mixed in a 1:1 molar ratio and end-labeled with
32P using bacteriophage T4 polynucleotide kinase
(Boehringer Mannheim) according to Sambrook et al. (45) as
described previously. The specific activity of the probes was routinely
~150,000 cpm/ng. To measure changes in AP-1 binding activity induced
by naris closure, optical density of the film autoradiograms was
determined on an automated image analysis system (IBAS, Thornwood, NY)
as described previously (46).
Gel Retardation Assays--
DNA-protein reaction mixtures (20 µl) were established as follows: buffer (5 mM Hepes, pH
7.9, 10% glycerol, 25 mM KCl, 0.05 mM EDTA,
0.125 mM phenylmethylsulfonyl fluoride), 15 µg of nuclear extract, 1 µg of poly(dI-dC), 0.28 ng of radiolabeled probe in presence or absence of molar excess cold probe. Mixtures were incubated
at 22 °C for 20 min. Samples were loaded onto nondenaturing high
ionic strength polyacrylamide gels (8% bisacrylamide). After running
at 100 V for 3 h, the gels were dried and exposed to X-Omat films
(Kodak; Rochester, NY) with one intensifying screen. For supershift
assays, nuclear extracts were mixed with appropriate antibodies
(anti-c-Fos, -Fos-B, -Jun-D, -CREB, and -CREM, 0.1 µg of each). The
mixtures were incubated on ice for 20 min and then subjected to gel
retardation assay as described above.
Western Blot--
Nuclear proteins (10~30 µg) were separated
by SDS-polyacrylamide gel (10% bisacrylamide) electrophoresis
according to the method of Laemmli (47) and electrophoretically
transferred onto a nitrocellulose membrane (48). Blots were incubated
in blocking solution (BS; 0.1 M phosphate-buffered saline,
5% non-fat milk, 0.5% Tween 20) at room temperature for 15 min. Blots
were then incubated with polyclonal antibodies (1:2000 diluted in BS)
for 2 h. After three 5-min washes in BS, blots were incubated in
the peroxidase-conjugated secondary antibody (1:1000 diluted in BS) for
1 h. Blots were washed four times, 5 min each, in 0.1 M phosphate-buffered saline containing 0.5% Tween 20 and
detected with ECL reagents (Amersham; Arlington Heights, IL) according
to manufacturer instructions.
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RESULTS |
Tissue Localization and Regulation of TH and IEGs--
As
previously shown (38), in the control olfactory bulb TH was
concentrated in cell bodies and processes of periglomerular neurons
located in the glomerular layer of olfactory bulb (Fig. 1A). TH expression was
drastically reduced in the olfactory bulb ipsilateral to the naris
closure (Fig. 1B). The IEG proteins, c-Fos (Fig. 1,
C and D) and Fos-B (Fig. 1, E and
F), were localized to nuclei distributed in the glomerular,
mitral, and granule cell layers. The number of both c-Fos and Fos-B
immunopositive neurons showed dramatic reductions in the periglomerular
cells of olfactory bulb ipsilateral to the closure. c-Fos expression
was absent in the glomerular layer (Fig. 1D). Fos-B
expression, which was more widely distributed, showed a 50% reduction
in response to odor deprivation (mean number of cells per unit
area ± S.E.: contralateral to naris closure 287.3 ± 11.97 versus ipsilateral to naris closure 155.2 ± 6.06;
p < 0.001, n = 4). Expression of these
early genes also was reduced in the granule, but not the mitral, cell
layer. Double label immunofluorescence studies showed that a
subpopulation of Fos-B immunoreactive cells in the glomerular layer
also expressed TH (data not shown).

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Fig. 1.
Light microscopic immunocytochemical analysis
of tyrosine hydroxylase, c-Fos and Fos-B expression in olfactory bulbs
contralateral (A, A', C, C', E, E') and ipsilateral
(B, B', D, D', F, F') to naris closure. A',
B', C', D', E', and
F' are higher magnification images of A,
B, C, D, E, and
F, respectively. Bar = 130 µm in
A-F and 50 µm in
A'-F'.
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In contrast to c-Fos and Fos-B, naris closure did not alter the
expression of CREB (Fig. 2, A
and B) and Jun-D (Fig. 2, E and F) in
the olfactory bulb. Whereas c-Fos, Fos-B, and Jun-D were present
primarily in neurons, CREB (Fig. 2, A and B)
displayed a broader distribution, that is, this IEG product appeared to be expressed in both neurons and glia. The phosphorylated form of CREB,
pCREB, showed a primarily neuronal localization in the olfactory bulb.
Levels of pCREB were variably increased by odor deprivation (Fig. 2,
C and D).

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Fig. 2.
Light microscopic immunocytochemical
demonstration of CREB (A, B), phosphorylated
CREB (C, D) and Jun-D (E,
F) expression in olfactory bulbs contralateral (A,
A', C, C', E, E') and ipsilateral (B, B', D, D', F,
F') to naris closure. A', B',
C', D', E', and F' are
higher magnification images of A, B, C, D, E, and
F, respectively. Bar = 130 µm in
A-F and 50 µm in A'-F'.
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TH AP-1 and CRE Binding Activities--
Because naris closure
produced down-regulation in expression of some IEG products in parallel
to that observed for TH in periglomerular neurons, gel retardation
assays were performed to determine whether TH AP-1 and CRE binding
activities in olfactory bulb were altered by odor deprivation. TH AP-1
and CRE probes formed distinctive DNA-protein complexes with nuclear
extracts isolated from control olfactory bulbs (Fig.
3). The TH AP-1 complex was identified as a single band. Competition assays demonstrated that this binding activity was completely blocked by excess cold AP-1 probe. Also, the
AP-1 binding activity was blocked by excess cold TH CRE probe. Compared
with the normal olfactory bulb, nuclear extracts from the odor-deprived
olfactory bulb showed a 32% decrease (mean optical density in
arbitrary units ± S.E.: ipsilateral, 0.094 ± 0.003 versus contralateral, 0.140 ± 0.006; p < 0.003, n = 5) in AP-1 binding activity in parallel
with the decline in Fos protein found immunocytochemically.

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Fig. 3.
Gel shift analyses demonstrating the distinct
DNA-protein complexes formed between TH AP-1 and CRE probes and nuclear
extracts isolated from olfactory bulbs ipsilateral (cl) and
contralateral (op) to naris closure. Competition
assays employed excess cold AP-1 and CRE probes.
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Interactions of nuclear extracts with the TH CRE probe produced two
DNA-protein complexes with distinctive migration rates during gel
electrophoresis (Fig. 3). The intensity of the lower band was higher
than the upper band. As predicted, excess cold CRE probe abolished the
CRE binding activity. Excess cold AP-1 probe only partially blocked the
CRE/protein binding. In contrast to the AP-1 binding activity which was
down-regulated by odor deprivation and in agreement with the
immunocytochemical findings, CRE binding activity remained unchanged
with differing olfactory experience.
Components of TH AP-1 and CRE Complexes--
Supershift assays
were performed to identify the nuclear proteins that formed complexes
with TH AP-1 and CRE motifs. Polyclonal antibodies for c-Fos, Fos-B,
Jun-D, CREB, and CREM were used to examine the AP-1 complex. Antibodies
for Fos-B and Jun-D produced a shift of the AP-1 complex to species
with lower migration rates (Fig. 4).
Fos-B antibody shifted the whole AP-1 complex, whereas the Jun-D
antibody shifted a major portion of the complex. Relative to the
control, the Fos-B- as well as the Jun-D-associated binding activities
decreased in the olfactory bulb ipsilateral to the naris closure. In
contrast, antibodies for c-Fos, CREB, or CREM did not produce a
supershift from the AP-1 complex (Fig. 4).

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Fig. 4.
Supershift analyses to demonstrate the
composition of the AP-1 complex in nuclear extracts of olfactory bulbs
both ipsilateral (cl) and contralateral (op) to
naris closure employing antibodies to Fos-B, Jun-D, c-Fos, CREB, and
CREM. Arrow indicates the supershifted band produced by
the Jun-D antibody.
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Antibodies for CREB, CREM, Fos-B, and Jun-D, but not c-Fos,
supershifted the CRE-protein complexes (Fig.
5). Although the CREB antibody was
somewhat more efficient than the CREM antibody, they produced similar
supershift patterns retarding all of the upper band and part of the
lower band. The antibody for Fos-B supershifted part of the lower band,
resulting in a band overlapping the original upper band. The Jun-D
antibody supershifted only a small portion of the CRE complexes (Fig.
5). Finally, c-Fos antibodies did not retard the CRE-binding
complexes.

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Fig. 5.
Supershift analyses to demonstrate the
composition of the CRE complex in nuclear extracts of olfactory bulbs
both ipsilateral (cl) and contralateral (op) to
naris closure employing antibodies to Fos-B, Jun-D, c-Fos, CREB,
and CREM. Arrows indicate the supershifted bands
produced by Fos-B and Jun-D antibodies.
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Specificity of Antibodies--
Western blot analyses were
performed to demonstrate the specificity of polyclonal antibodies that
produced retardation in the supershift studies. Fos-B antibody
recognized a protein with a molecular mass of 40-42 kDa (Fig.
6). Consistent with the
immunocytochemical findings and gel retardation assays, Fos-B
immunoreactivity decreased in the olfactory bulb ipsilateral to the
closed naris. The Fos-B antibody also recognized two polypeptides with
molecular masses of 27 and 14 kDa. These two polypeptides might
represent alternative splicing products of the Fos-B gene and/or
proteolytic products of the Fos-B protein. The Fos-B antibody did not
recognize c-Fos, which in mouse had a molecular mass of 56 kDa when
detected with several c-Fos antibodies (data not shown). The Jun-D
antibody recognized a doublet with molecular masses of 39 and 43 kDa.
The CREB antibody recognized a 45-kDa polypeptide, and the CREM
antibody recognized 44- and 32-kDa polypeptides.

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Fig. 6.
Western blot analyses to illustrate the
specificity of the antisera that produce supershifts in the gel
mobility assays. The specific bands corresponding to the reported
molecular masses (kDa) for the proteins are indicated by the
arrows.
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DISCUSSION |
Regulation of TH gene expression in the rodent olfactory bulb
displayed unique activity dependence (30, 36-40, 49, 50). TH
expression was rapidly down-regulated following unilateral naris
closure, a procedure that prevents odorant access to the olfactory
receptor epithelium resulting in reduced stimulation of the olfactory
bulb. Previous studies revealed both cell line- (27, 51-53) and CNS
region-dependent TH gene regulation (54, 55), suggesting
differences in either expression or binding of transcription factors to
specific cis-acting elements. The current study investigated the
composition and specificity of binding complexes with putative AP-1 and
CRE sites in the regulation of TH gene expression in the olfactory bulb.
The present study demonstrated that Fos-B, CREB, pCREB, and Jun-D are
present in the periglomerular cells of the glomerular layer and likely
colocalize with TH as previously shown for c-Fos (41, 42). Consistent
with the idea that Fos expression reflects neuronal activity, both
c-Fos and Fos-B immunoreactivities were down-regulated in the olfactory
bulb ipsilateral to the naris closure. The parallel reductions in Fos
proteins and TH suggested that these two IEGs may be involved in TH
gene regulation in an activity-dependent manner. In
contrast, naris closure had no effect on immunoreactivities for either
Jun-D or CREB and may actually have induced pCREB. The presence of
these IEGs may be necessary for the activation of cis-acting elements
in the TH gene promoter, but may not directly induce TH gene
expression. Thus, the unique expression of the IEGs in the glomerular
layer suggested that they may play different roles in regulating gene
expression in these dopaminergic neurons.
Gel mobility shift assays in the present study revealed that Fos-B is
the only member of the Fos family present in the TH AP-1-protein
complex in the olfactory bulb. Fos-B may form heterodimers with both
Jun-D and other members of the Jun family because Jun-D antibody only
partially supershifts the AP-1-protein complex. The specificity of the
TH AP-1 motif for Fos-B may result from the specific 5'- and
3'-flanking regions of the AP-1 sequence, previously shown to be
critical for binding activity (20). Transcription factor affinity with
cis-acting elements mediates in part the differential promoter
sensitivity of genes to the same extracellular signals. The reductions
in the TH AP-1 binding activity and Fos-B immunoreactivity, shown in
tissue staining and Western blots in the naris-closed olfactory bulb,
suggest a significant role of Fos-B in the TH gene regulation in this
brain region. Although c-Fos is present in the periglomerular neurons
and down-regulated by naris closure, this IEG product does not appear
to contribute to the TH AP-1-protein complex. The Western blot analyses
showing that the Fos-B antibody does not cross-react with c-Fos, which has a molecular mass of 56 kDa, further supports the concept that Fos-B, but not c-Fos, may be involved in down-regulation of the TH gene
in periglomerular dopaminergic neurons in the naris-closed olfactory bulb.
The absence of c-Fos in the TH AP-1-protein complex in the olfactory
bulb may reflect tissue specificity of TH gene regulation and/or
differential affinities of the Fos proteins with the TH AP-1 motif. It
has been recently reported that the AP-1 motif is involved in the
induction of TH gene transcription by reserpine treatment in the rat
adrenal medulla but not in the sympathetic ganglia, suggesting
tissue-specific regulatory mechanisms for this gene (56). Cell line
specificity of TH gene transcription also was shown to involve the AP-1
motif (51, 52). Alternatively, the inability to detect c-Fos in the
DNA-protein complex may be because of low affinity of this IEG product
with the TH AP-1 motif. It is possible that c-Fos can bind to the TH
AP-1 motif only when it is overexpressed. Both c-Fos and Fos-B
expression as well as AP-1 binding activity can be increased by
activation of glutamate receptors in the brain (57) and by nerve growth
factor treatment in PC12 cells (58). In PC12 cells, c-Fos was
identified in the TH AP-1-protein complex shortly after growth factor
treatment, but it was replaced gradually by Fos-B which was induced by
prolonged treatment (58), indicating a transient effect of c-Fos and a long-lasting effect of Fos-B on TH gene transcription. Indeed, Fos-B
and Fos-B-like proteins have been implicated in the long-term biochemical adaptations observed following chronic treatment (59, 60).
In sum, the demonstration that the AP-1-protein complex is composed
primarily of Fos-B and Jun proteins in the adult mouse olfactory bulb
suggests that Fos-B may play an essential role in the TH gene
regulation in this brain region.
The present study demonstrated that excess TH CRE oligonucleotide
completely blocked TH AP-1 binding activity. One explanation for this
finding is that the TH CRE is also the binding site for the protein
complexes in the olfactory bulb extracts that bind to TH AP-1,
e.g. Fos/Jun. This competition for transcription factors may
occur because the consensus AP-1 and CRE motifs in many genes including
TH are very similar in structure, displaying only one nucleotide
difference. However, note that the putative TH AP-1 motif differs from
that in other genes by one nucleotide located in the middle of the
sequence (24). A second possibility is that CRE competes with AP-1 for
only certain components that are essential to formation of AP-1-protein
complexes. For instance, binding of Jun/CREB to the excess CRE added to
the binding reaction might deplete free Jun, thus resulting in a
decrease in availability of Fos/Jun complexes. Jun proteins were
localized to complexes binding to CRE sequences presumably as Jun/Jun
homodimers and/or Jun/CREB heterodimers (16, 18). More recently, it was
reported that CREB was present in the AP-1-protein complex (19). The finding that AP-1 oligonucleotide partially blocks CRE binding activity
suggested that some proteins such as CREB and CREM only bind to the TH
CRE. In contrast, the supershift assays show that Fos-B/Jun
heterodimers bound both TH AP-1 and CRE sequences. Although the effect
of binding of Fos-B/Jun to CRE motif on the TH promoter activity is
unknown, the ability of this protein complex to bind to both TH AP-1
and CRE motifs suggests cross-talk between these two cis-acting elements.
The present studies also suggested that the CRE motif may play only a
minor role in TH down-regulation in the olfactory bulb ipsilateral to
naris closure. The gel shift assays revealed no detectable change in
CRE binding activity between the normal and naris-closed olfactory
bulbs. Whereas gel supershift assays showed that CREB/CREM proteins are
major components of the TH/CRE protein complexes, immunocytochemistry
confirmed the binding data that CREB was unchanged in response to naris
closure. Although CREB, thought to constitutively bind to the CRE
motif, can be activated upon phosphorylation (12, 14, 15), pCREB was,
if anything, variably increased in response to naris closure. Although
Fos-B/Jun-D are present in the CRE-protein complexes, they represent
only minor components. Therefore, it is unlikely that naris closure reduces TH expression in the periglomerular neurons primarily through
the CRE motif.
In conclusion, the present study demonstrated unique
activity-dependent expression of immediate early genes in
the glomerular layer of mouse olfactory bulb. Fos proteins exhibited
reduced immunoreactivities in the periglomerular neurons in the
olfactory bulb ipsilateral to the naris closure. Gel mobility shift
assays demonstrated a decrease in the TH AP-1 binding activity. Fos-B was found to be a major component of the TH AP-1-protein complexes and
a minor component of the CRE-protein complexes, suggesting that Fos-B
may be involved in the activity-dependent gene regulation of TH in the olfactory dopaminergic neurons.