FosB Is Essential for the Enhancement of Stress
Tolerance and Antagonizes Locomotor Sensitization
Yoshinori N. Ohnishi, Yoko H. Ohnishi, Masaaki Hokama, Hiroko Nomaru, Katsuhisa Yamazaki,
Yohei Tominaga, Kunihiko Sakumi, Eric J. Nestler, and Yusaku Nakabeppu
Background: Molecular mechanisms underlying stress tolerance and vulnerability are incompletely understood. The fosB gene is an
attractive candidate for regulating stress responses, because ?FosB, an alternative splice product of the fosB gene, accumulates after
transiently than ?FosB but exerts higher transcriptional activity. However, the functional differences of these two fosB products remain
Methods: We established various mouse lines carrying three different types of fosB allele, wild-type (fosB?), fosB-null (fosBG), and fosBd
allele, which encodes ?FosB but not FosB, and analyzed them in stress-related behavioral tests.
the absence of FosB, the function of FosB can be inferred from differences observed between these lines. The fosB?/dand fosBd/dmice
showed increased locomotor activity and elevated Akt phosphorylation, whereas only fosB?/dmice showed antidepressive-like behaviors
behavior and lower E-cadherin expression.
Conclusions: These findings indicate that FosB is essential for stress tolerance mediated by ?FosB. These data suggest that fosB gene
products have a potential to regulate mood disorder-related behaviors.
Key Words: Bipolar disorders, depression, fosB gene, knockout
mice, mania, mood disorder
latter encodes ?FosB protein, which lacks the C-terminal 101 aa
containing the transactivation domain in the full-length FosB pro-
tein (1). We have previously reported that FosB and ?FosB oppo-
sitely regulate Jun transactivity (1,2) and cell matrix adhesion (3) in
cultured cells; the proteins also regulate cell proliferation, differen-
with stress (9,12–17). ?FosB accumulates progressively after re-
peated or prolonged dopaminergic-related stimuli or convulsive
seizures reflecting its high level of stability, in contrast to FosB,
which, like c-Fos, is highly unstable and is expressed transiently
(12,18–23). However, functional roles of ?FosB and FosB in mood-
he fosB gene has an intron-like sequence in exon 4, which
transcripts, fosB and ?fosB messenger RNAs (mRNAs). The
in several brain areas appears to mediate decreased sensitivity to
the deleterious effects of chronic stress (13,24), while the influence
of FosB is completely unknown despite its much higher transactiv-
Here, we characterized new fosB mutant mice: 1) fosBd/dmice
with significantly enhanced expression of ?FosB but no FosB, 2)
?FosB. We found that fosB?/dmice serve as a model for endoge-
fosBd/dand fosB-null mice reflect the effects of enhanced ?FosB
between fosB?/dand fosBd/dmice revealed the influence of FosB
under ?FosB accumulating conditions.
Methods and Materials
Generation of Mutant Mice
See Supplement 1.
fosBd/dmice (F8–14) and fosBG/Gmice (F5–9) were obtained from
heterozygous intercrosses except for experiments involving halo-
peridol treatment. Control wild-type mice were littermates of each
mutant mouse line. No gross differences were apparent between
wild-type and mutant mice. Mice were housed in plastic mouse
cages with littermates with standard rodent chow and water ad
controlled, specific pathogen-free room with a 12-hour light/12-
hour dark cycle (light on at 8:00 AM and down at 8:00 PM). The
handling and killing of all animals were carried out in accordance
KS, YN), Department of Immunobiology and Neuroscience, Medical In-
stitute of Bioregulation, Kyushu University, Fukuoka, Japan; and Fish-
berg Department of Neuroscience (YNO, YHO, EJN), Mount Sinai School
of Medicine, New York, New York.
Address correspondence to Yusaku Nakabeppu, D.Sc., Kyushu University,
Medical Institute of Bioregulation, Department of Immunobiology and
Neuroscience, Division of Neurofunctional Genomics, 3-1-1 Maidashi
Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; E-mail: yusaku@bioreg.
Received Oct 26, 2010; revised Apr 23, 2011; accepted Apr 26, 2011.
BIOL PSYCHIATRY 2011;70:487–495
© 2011 Society of Biological Psychiatry
studies was granted by the Animal Experiment Committee of Ky-
See Supplement 1.
Western Blot Analysis
See Supplement 1.
See Supplement 1.
See Supplement 1.
Expression of FosB and ?FosB in Mutant Mice
fosB gene expression in mutant mice was analyzed with immu-
nohistochemistry and Western blotting. Brain immunohistochem-
istry of wild-type mice revealed that fosB gene expression is great-
bral cortex. The regional pattern of fosB gene expression in the
acids 245 to 315 of the C-terminus of FosB (1), detected FosB only
sparsely in NAc and dorsal striatum but substantially in cingulate
cortex (Figure S1C,D in Supplement 1). This suggests that the ma-
IR was undetectable in fosBd/dand fosBG/Gmice, and ?FosB IR was
undetectable in fosBG/Gmice (Figure S1E,F in Supplement 1).
We next performed quantitative Western blotting of striatal
nuclear extracts using anti-FosB (Figure 1D–F). Significantly en-
hanced expression of ?FosB and ?2?FosB in fosBd/dand to a
lesser extent in fosB?/dmice was confirmed in comparison with
wild-type mice. Expression levels of FosB and variant FosB
(vFosB) in fosB?/dmice were apparently decreased in compari-
son with those in wild-type mice, and there was no detectable
FosB and vFosB in fosBd/dmice (Figure 1D,F). Variant FosB is
thought to be ?1FosB or ?2FosB (Figure S1A in Supplement 1).
In the fosB?/Gmice, the levels of vFosB and ?2?FosB, but not
FosB and ?FosB, were significantly decreased in comparison
fosBG/Gmice (Figure 1E,F).
Thesedatasuggestthat fosB?/dmiceprovideamodelof ?FosB
accumulation over long periods of time and that the difference
under conditions of ?FosB accumulation. Additionally, the differ-
sections of brain prepared from wild-type, fosBd/d, and fosBG/Gmice were subjected to immunohistochemistry with anti-FosB(N). The FosB(N) antibody was
expression is not different between wild-type and fosBd/dmice, and there is no expression of fosB gene products in fosBG/Gmice. (D, E) Western blotting
analysis of fosB gene products in striatal nuclear extracts. Blotting membranes were reacted with a rabbit monoclonal anti-FosB (5G4, Cell Signaling
Technology, Inc. Danvers, Massachusetts). The black arrow indicates p43 (FosB), the open arrowheads indicate p32/36 (?FosB), the red arrow indicates p31
gene product was detected in fosBG/Gmice [FosB: F(2,11) ? 10.4, p ? .0046; ?FosB: F(2,11) ? 16.1, p ?.0011; vFosB: F(2,11) ? 37.6, p ? .0001;
?2?FosB: F(2,11) ? 39.1, p ? .0001]. *p ? .05 in Dunnett’s multiple comparison test.#p ? .05 in t test. St, striatum; HP, hippocampus.
488 BIOL PSYCHIATRY 2011;70:487–495
Y.N. Ohnishi et al.
Higher Spontaneous Locomotor Activity infosBd/dMice and
Lower Activity in fosBG/GMice
To infer the function of endogenous ?FosB and FosB in the
central nervous system, we analyzed the two lines of mutant mice
and their wild-type littermates in several behavioral assays (Figure
2A–H). Spontaneous locomotor activity was followed through 1
week without any disturbance of individually housed mice in their
home cages. The activity of all mice tended to decrease gradually
because of the loss of interaction with littermates (Figure S2 in
higher locomotor activity compared with wild-type mice and
fosB?/dmice (Figure 2A). On the other hand, fosBG/Gmice showed
significantly lower locomotor activity compared with wild-type
tor activity at the start of the sleep cycle. These data suggest that
?FosB facilitates spontaneous motor behavior during periods of
wakefulness but does not introduce abnormal motor behavior in-
dependent of the circadian cycle. Despite accumulation of compa-
normalize basic locomotion.
We next examined the locomotor activity of mice housed inde-
pendently for 1 month. Spontaneous locomotion was dramatically
decreased in all the genotypes studied. Nevertheless, the locomo-
tor activity of fosBd/dmice was still much higher; in fact, the differ-
locomotor activity than wild-type mice, suggesting that accumu-
isolation may settle down to basal levels without effects of social
interaction. fosB?/Gmice showed significantly higher locomotor
activity than wild-type mice, suggesting that the relatively in-
creased expression level of ?FosB among all fosB gene products in
fosB?/Gmice may make an effect on the basal locomotor activity
Higher Exploratory Activity in BothfosBd/dand fosBG/GMice
but Opposite Behavioral Responses to Fear-Eliciting Stimuli
of anxiety-like behavior, both fosBd/dand fosBG/Gmice showed sig-
nificantly higher exploratory activity than wild-type mice (Figure
2C,G), suggesting that FosB may increase anxiety-like responses or
suppress intrinsic curiosity. Though all the mice showed habitua-
Figure 2. Spontaneous locomotor activity, open field, and elevated plus maze tests in fosB mutant mice. Littermates obtained from mating of fosB?/d?
accumulation type). Lower panels represent data of line G (fosB-null type). (A, B, E, F) Spontaneous locomotor activity during a 12-hour period in the night.
Data in panels (A) and (E) were obtained in the night of the third day after isolation, those in (B) and (F) after 1 month of isolation. Bars represent locomotor
[F(2,51) ? 8.82, p ? .001], while fosBG/Gmice showed significantly lower locomotor activity compared with wild-type mice [F(2,60) ? 3.87, p ? .026]. fosB?/d
intheopenarmsthanwild-typemice(H ?9.79,p?.007,Kruskal-Wallistest),and fosBG/Gmicespentsignificantlylesstimeintheopenarms[F(2,45) ?4.91,
p ? .01]. In (A) to (H),#p ? .05;##p ? .01; statistical difference between homozygous mutant and wild-type mice. *p ? .05; **p ? .01; statistical difference
between heterozygous mutant and wild-type mice.§p ? .05; statistical difference between heterozygous and homozygous mutant mice, using analysis of
variance (Dunnett’s post hoc test).
Y.N. Ohnishi et al.
BIOL PSYCHIATRY 2011;70:487–495 489
second day and fosB?/Gmice on the third and fourth days, respec-
tively, exhibited significantly higher locomotor activity than did
spontaneous locomotor activity (Figure 2B,F).
In the elevated plus maze, another test of anxiety-like behavior,
like behavior and that the presence of FosB may help to normalize
it. There was no significant difference in the number of entries into
the open and closed arms, but fosBd/dmice showed a trend for
the open-field and elevated plus maze tests, mutant mice showed
Repeated Forced Swimming in the Morris Water Maze
ability and found some differences among the mouse lines tested
(Figure 3A,B). The swimming speed of the mutant mice showed no
difference through the training days (Figure S4C,D in Supplement
1). Interestingly, though the actual swimming time (Figure 3C,D)
and distance traveled (Figure S4A,B in Supplement 1) for arrival to
the platform were grossly normal, the time to reach the platform
was shorter in fosB?/dmice and longer in fosBG/Gmice, especially
around the middle of the training days (Figure 3A,B; Table S1 in
Supplement 1), because of immobility in the water during training
(Figure 3E,F; Table S1 in Supplement 1). In the probe test, all the
genotypes spent the longest time in the quadrant where the plat-
form had previously existed (Figure 3G,H; Table S1 in Supplement
that in wild-type mice because of immobility in the first quadrant
(white boxes in Figure 3G,H).
The forced swim test is used widely as a measure of stress re-
sponsiveness, with immobility reflecting greater stress vulnerabil-
these data suggest that fosB gene products do not affect learning
and such activity requires the presence of FosB.
Repeated Forced Swim Test in Inescapable Condition
Next, to further assess the behavioral responses of fosB mutant
mice to inescapable stress, we used a conventional forced swim
days of the test in wild-type, fosBd/d, and fosBG/Gmice, but not in
fosB?/dmice (Figure 4). fosBd/dand fosBG/Gmice showed signifi-
cantly decreased swimming time on the second and third days,
respectively. Additionally, we injected the mice with paroxetine
test on the fourth day. Paroxetine increased the swimming time
well beyond levels seen on the first day in all types of mice except
not reach the level of the first day, and this recovery ratio was
significantly lower than that seen in wild-type mice.
Responsivity to Dopamine Signals in Mutant Mice
normal circadian pattern (Figure S2 and Figure S4A in Supplement
1) but appeared less anxious or fearful in the elevated plus maze
(Figure 2D). These behavioral phenotypes are somewhat reminis-
cent of the symptoms of attention-deficit/hyperactivity disorder
(27). Attention-deficit/hyperactivity disorder patients can be
treated successfully with methylphenidate (MPH). Methylpheni-
centration in synaptic clefts, which leads to hyperlocomotion in
we examined whether MPH exerted therapeutic-like effects in
fosBd/dmice compared with effects of other dopaminergic drugs
(Figure 5A–D; Figure S5 in Supplement 1). We found that MPH
induced transient hyperlocomotion (Figure 5A,B), which settled
types of fosB mutant mice and their wild-type littermates (Figure
S5C,D in Supplement 1). In particular, fosB?/dand fosBd/dmice
exhibited more hyperactivity in response to MPH than wild-type
mice. The transient hyperlocomotion exhibited by fosB?/dand
dark phase after MPH treatment was decreased in fosB?/dmice
(Figure 5A,C). These data suggest that accumulated ?FosB drives
locomotion during the dark phase, while the existence of FosB
prevents it (Figure S5C in Supplement 1). fosB?/Gand fosBG/Gmice
exhibited equivalent reactions to MPH as wild-type mice, suggest-
ing that normal low levels of ?FosB do not contribute to basal
dopamine sensitivity and that only accumulated ?FosB can facili-
tate it (Figure 5B; Figure S5B,D in Supplement 1).
Basal ganglia circuitry regulates motor function via two path-
ways, termed the direct and indirect pathways. More than 90% of
tion neurons. About half of them project directly to the midbrain
and express dynorphin and the D1 dopamine receptor; activation
of these neurons increases locomotion and induces fosB gene ex-
pression. The other half indirectly project to the midbrain via the
globus pallidus and subthalamic nucleus and express enkephalin
and the D2 dopamine receptor; activation of these neurons de-
creases locomotion (28). D2 receptor signaling in these neurons
inhibits such downregulation of locomotion and suppresses fosB
gene expression (29).
in direct pathway neurons increased daily running compared with
control littermates, whereas ?FosB overexpression predominantly
in indirect pathway neurons decreased it. At first, we hypothesized
that spontaneous hyperlocomotion and higher responsivity to
MPH in fosB?/dand fosBd/dmice are dependent on increased D1
receptor signaling sensitivity. However, locomotor responses to a
D1 receptor agonist (2 mg/kg SKF81297) showed no differences
among all the fosB mutant and wild-type mice (Figure 5A,B; Figure
onist (1 mg/kg haloperidol), which is reported to induce fosB gene
expression in D2-containing neurons, as well as hypolocomotion
(29,31). The total locomotor activity over 4 hours after haloperidol
treatment was significantly higher in fosBd/dmice and lower in
fosBG/Gmice compared with wild-type mice (Figure 5A,B; Figure
S5G,H in Supplement 1). This finding, along with the pattern of
results in the elevated plus maze (Figure 2D,H) and spontaneous
locomotion test on the second and third day after isolation from
ute to the increased locomotion and reduction in anxiety-like be-
havior induced by ?FosB, with FosB negatively regulating the ef-
fects of accumulated ?FosB.
We detected no behavioral differences in the effects of a D2
mice (Figure 5A,B,; Figure S5I,J in Supplement 1). These data sug-
gest that, in the mutant mice, negative feedback via D2 receptors
on dopamine neurons in the midbrain may function normally (32).
490 BIOL PSYCHIATRY 2011;70:487–495
Y.N. Ohnishi et al.
Figure 3. Repeated forced swimming in the Morris water maze in fosB mutant mice. Left panels represent data of line d (?FosB accumulation type). Right
mean ? SEM.#p ? .05;##p ? .01; statistical difference between homozygous mutant and wild-type mice. *p ? .05; **p ? .01; statistical difference between
heterozygous mutant and wild-type mice.§p ? .05;§§p ? .01; statistical difference between heterozygous and homozygous mutant mice, using analysis of
Y.N. Ohnishi et al.
BIOL PSYCHIATRY 2011;70:487–495 491
E-Cadherin Expression Levels Parallel Stress Responses and
fosB Gene Expression
To further elucidate how fosB gene products regulate complex
behavior, we selectively picked several postulated target proteins
from previous reports (3,14,33) and then performed Western blot-
ting with striatum samples (Figure S6A in Supplement 1). E-cad-
lated in fosB?/Gand fosBG/Gmice; in other words, the expression
level of E-cadherin paralleled the behavioral phenotype of stress
tolerance seen with forced swimming. Similar tendencies were de-
tected in hippocampus (data not shown). In contrast, there was no
difference in the expression levels of cyclin-dependent kinase 5,
the latter two are binding partners of E-cadherin) among fosB mu-
tant and wild-type mice (Figure S6A in Supplement 1). The level of
and wild-type mice (data not shown), suggesting that the upregu-
lation of E-cadherin is regulated by posttranslational mechanisms.
Akt Phosphorylation Is Increased by?FosB Accumulation
Finally, we checked the level of Akt and its phosphorylation in
the striatum of fosB mutant mice (Figure S6B in Supplement 1).
Total Akt protein levels showed no difference among mutant and
wild-type mice, but the phosphorylation state of Akt (T308) was
significantly elevated in fosB?/dand fosBd/dmice.
In this study, we provide evidence that accumulated ?FosB
increases locomotor activity and that FosB antagonizes this effect
similar to findings from in vitro cellular assays (1). On the other
hand, stress tolerance appears to be the sum of ?FosB and FosB.
These effects may be partly mediated via E-cadherin, an indirect
target of fosB gene products. Together, these data suggest distinct
patterns of behavioral abnormalities among the mutant mouse
lines examined (Figure 6). We propose that fosBG/Gmice exhibit
behaviors that resemble depression, including decreased locomo-
tion, increased immobility during forced swimming, and increased
anxiety-like responses. In contrast, fosB?/dmice exhibit behaviors
locomotion, increased stress tolerance, and reduced anxiety-like
responses. Interestingly, fosBd/dmice exhibit significantly higher
dopamine sensitivity and most of the altered behaviors seen in
present a picture of blended responses perhaps reminiscent of
work for validation, including studying these molecular findings in
the human disorders.
the repeated forced swim test revealed the stress vulnerability of
fosBd/dmice more clearly than the water maze, the difference be-
tween wild-type and fosB?/dmice was more apparent in the water
to detect initial stress vulnerability, while the water maze may be
paroxetine suggest that fosB gene products are partly required for
antidepressant responses to the drug, as reported recently (24).
ment 1), elevated plus maze (Figure 2D), and sensitivity to a D2
FosB has much higher transactivity than ?FosB (1,34). These data
suggest that lower expression levels of FosB may be sufficient to
the different cell types or target genes involved. In ?FosB bitrans-
genic mice, where ?FosB expression is inducible and relatively
restricted to D1 neurons in NAc and dorsal striatum, Kelz et al. (14)
reported higher locomotor activity in a novel test chamber on the
second day but not on the first day. This finding corresponds to
observations in fosB?/dmice, which exhibited higher locomotor
activity with a similar pattern (Figure 2C), thus suggesting that
?FosB accumulation in striatum plays an important role in mediat-
ing locomotor activation via dopamine signaling.
(23). Because Akt phosphorylation is also induced by dopamine
ylation and that the extent of spontaneous dopaminergic activa-
tion reflects levels of Akt phosphorylation. Perrotti et al. (23) re-
ported that morphine induces ?FosB accumulation in NAc, and
Russo et al. (36) reported that overexpression of a constitutively
indirect dopaminergic mechanisms such as increased dopamine
in NAc by inhibiting dopamine reuptake. Overexpression of ?FosB
enhanced cocaine locomotor sensitization (39), suggesting that
enhanced Akt phosphorylation by accumulated ?FosB might facil-
itate locomotor sensitization by cocaine and by morphine. This is
Figure 4. Repeated forced swim test of fosB mutant mice in inescapable
condition. Conventional repeated forced swim test was performed once a
day for 4 consecutive days. Each trial was performed for 6 minutes, and
mg/kg) was injected 30 minutes before the fourth day trial. In fosBG/Gmice,
the recovery ratio of the swimming time after administration of paroxetine
and a given day in each mouse line.#p ? .05; statistical difference between
fosBG/Gand wild-type mice using analysis of variance (Dunnett’s post hoc
492 BIOL PSYCHIATRY 2011;70:487–495
Y.N. Ohnishi et al.
and enhanced locomotor sensitization by MPH observed in fosBd/d
mice (Figure 5A; Figure S6C in Supplement 1). The mechanism by
is not known, because ?FosB could affect the expression of any of
several regulators of Akt phosphorylation (40). One possibility is
that accumulated ?FosB may repress the expression of a protein
to be suppressed by D2 signaling in striatum via the activation of
this phosphatase (41). These and other possibilities now require
fosB-knockout (KO) mice had been established by Brown et al.
(42) and actually have a potential to express ?3?FosB, an alterna-
tive-translation initiation product similar to ?2?FosB (Figure S1 in
Supplement 1). Hiroi et al. (12) and Zhu et al. (43) reported that
fosB-KO mice showed slightly higher locomotor activity when they
observation that fosBG/Gmice exhibit significantly higher locomo-
that FosB may suppress exploratory behavior in the novel environ-
ment independent of the presence of ?FosB. On the other hand,
ity to other types of stress paradigms (tail suspension test), and
Brown et al. (42) reported that the fosB-KO mice exhibit a defect in
nurturing and no difference in the Morris water maze test. These
findings are not consistent with our results, because fosBG/Gmice
exhibited higher stress vulnerability and no defect in nurturing
(data not shown). These differences could reflect the potential in-
the prior studies by Brown et al.  and C57BL6/J in the present
Interestingly, the expression level of E-cadherin and the stress
tolerance observed on repeated forced swimming paralleled the
combined expression of FosB and ?FosB. Only with regard to the
Figure 5. Locomotor activity induced by dopamine receptor agonists and antagonists in fosB mutant mice. Left panels represent data of line d (?FosB
accumulation type). Right panels represent data of line G (fosB-null type). Naive mice, without prior behavioral assays, were housed individually for 2 weeks
and then placed in a cage with an infrared automatic monitor system for 3 days. On the second day at 4:00 PM, the mice were injected with saline (SAL), and
on the third day at 4:00 PM, the mice were injected with 30 mg/kg methylphenidate (MPH), 2 mg/kg SKF81297 (SKF), 1 mg/kg haloperidol (HAL), or 1 mg/kg
locomotorcountsobservedduringthefirst4hoursfrom4:00 PMto8:00 PM(mean?SEM).fosB?/dandfosBd/dmiceexhibitedmorehyperactivityinresponse
to MPH than wild-type mice [F(2,23) ? 4.14, p ? .05]. The total locomotor activity immediately after haloperidol treatment was significantly higher in fosBd/d
Barsrepresentlocomotorcountsobservedduringthelast12hoursfrom8:00 PMto8:00 AMthenextday(mean?SEM).Thelocomotoractivityexhibitedinthe
dark phase after MPH treatment was decreased in fosB?/dmice [F(2,23) ? 3.78, p ? .05]. In (A) to (D),#p ? .05; statistical difference between homozygous
mutant and wild-type mice,§p ? .05; statistical difference between heterozygous and homozygous mutant mice, using analysis of variance (Dunnett’s post
hoc test). The number of mice is provided in Figure S5 in Supplement 1.
locomotor activity and stress tolerance. FosB and accumulated ?FosB am-
plify stress tolerance and E-cadherin expression cooperatively. Accumu-
lated ?FosB facilitates Akt phosphorylation and locomotor activity thor-
ough its dopaminergic sensitization, while FosB antagonizes the effects.
Y.N. Ohnishi et al.
BIOL PSYCHIATRY 2011;70:487–495 493
not antagonize but coordinates with ?FosB. ?FosB has been re-
ported to accumulate after chronic stress in several brain regions
gray promotes active coping responses against stress, such as
forced swimming (13). More recently, overexpression of ?FosB in
the NAc suppresses the deleterious effects of social defeat (24).
like effects of ?FosB require the presence of FosB.
E-cadherin is an important cell-cell adhesion molecule and influ-
ences synapse formation in neuronal cells (44). In rodent models of
ments reverse these abnormalities (45–47). Here, we found that the
nism underlying the stress tolerance induced by fosB gene products.
FosB might inhibit E-cadherin degradation via transactivation of an
inhibitory binding protein against Hakai, which is an E3-ligase of E-
growth factor-? signaling, which is increased in fosB?/dand fosBd/d
embryonic stem cells (3). Future studies are needed to explore these
Cyclin-dependent kinase 5 and GluR2, putative ?FosB targets
identified in bitransgenic mouse models (14,33,49), are expressed
al., unpublished data). McClung and Nestler (9) reported that the
expression levels of various ?FosB targets in these mice switched
in fosB gene expression in our mutant mice might not result in
altered expression of cyclin-dependent kinase 5 and GluR2.
fosB?/dand fosBd/dmutants differentially express aberrant levels of
lines exhibit behavioral abnormalities mimicking different types of
mood disorders. Further studies of these mice will shed light on the
mechanisms controlling mood disorder-related behaviors and thus
contributing to the development of improved treatments for these
This work was supported by grants from Core Research for Evolu-
tional Science and Technology, Japan Science and Technology
Agency, the Ministry of Education, Culture, Sports, Science, and Tech-
We thank Dr. M. Katsuki (National Institutes of Natural Sciences,
for Technical Support, Medical Institute of Bioregulation, for the DNA
The authors report no biomedical financial interests or potential
FosB that inhibits Fos/Jun transcriptional activity. Cell 64:751–759.
2. Nakabeppu Y, Nathans D (1989): The basic region of Fos mediates
specific DNA binding. EMBO J 8:3833–3841.
3. Ohnishi YN, Sakumi K, Yamazaki K, Ohnishi YH, Miura T, Tominaga Y,
FosB and ?FosB/?2?FosB encoded by alternatively spliced forms of
4. Kurushima H, Ohno M, Miura T, Nakamura TY, Horie H, Kadoya T, et al.
(2005): Selective induction of ?FosB in the brain after transient fore-
brain ischemia accompanied by an increased expression of galectin-1,
and the implication of ?FosB and galectin-1 in neuroprotection and
Regulation of the neuronal fate by ?FosB and its downstream target,
6. Nakabeppu Y, Oda S, Sekiguchi M (1993): Proliferative activation of
quiescent Rat-1A cells by ?FosB. Mol Cell Biol 13:4157–4166.
alteration with an increased expression of a novel processed form of
galectin-1 in the rat 3Y1 embryo cell line. J Biochem 131:653–661.
8. Tahara K, Tsuchimoto D, Tominaga Y, Asoh S, Ohta S, Kitagawa M, et al.
cell proliferation in the Rat1a embryo cell line.Cell Death Differ10:496–
9. McClung CA, Nestler EJ (2003): Regulation of gene expression and co-
caine reward by CREB and ?FosB. Nat Neurosci 6:1208–1215.
10. Renthal W, Kumar A, Xiao G, Wilkinson M, Covington HE, 3rd, Maze I, et
al. (2009): Genome-wide analysis of chromatin regulation by cocaine
reveals a role for sirtuins. Neuron 62:335–348.
after chronic amphetamine exposure. J Neurosci 28:7344–7349.
mutant mice: Loss of chronic cocaine induction of Fos-related proteins
and heightened sensitivity to cocaine’s psychomotor and rewarding
13. Berton O, Covington HE 3rd, Ebner K, Tsankova NM, Carle TL, Ulery P, et
al. (2007): Induction of ?FosB in the periaqueductal gray by stress pro-
motes active coping responses. Neuron 55:289–300.
sensitivity to cocaine. Nature 401:272–276.
15. Nestler EJ (2008): Transcriptional mechanisms of addiction: Role of
al. (2008): The influence of ?FosB in the nucleus accumbens on natural
reward-related behavior. J Neurosci 28:10272–10277.
al. (2006): An essential role for ?FosB in the nucleus accumbens in
morphine action. Nat Neurosci 9:205–211.
18. Hiroi N, Marek GJ, Brown JR, Ye H, Saudou F, Vaidya VA, et al. (1998):
Essential role of the fosB gene in molecular, cellular, and behavioral
actions of chronic electroconvulsive seizures. J Neurosci 18:6952–6962.
19. Herdegen T, Leah JD (1998): Inducible and constitutive transcription
factors in the mammalian nervous system: Control of gene expression
by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res Brain Res Rev
20. McClung CA, Ulery PG, Perrotti LI, Zachariou V, Berton O, Nestler EJ
21. Nestler EJ, Kelz MB, Chen J (1999): ?FosB: A molecular mediator of
long-term neural and behavioral plasticity. Brain Res 835:10–17.
22. Perrotti LI, Hadeishi Y, Ulery PG, Barrot M, Monteggia L, Duman RS,
Nestler EJ (2004): Induction of?FosB in reward-related brain structures
after chronic stress. J Neurosci 24:10594–10602.
23. Perrotti LI, Weaver RR, Robison B, Renthal W, Maze I, Yazdani S, et al.
(2008): Distinct patterns of ?FosB induction in brain by drugs of abuse.
24. Vialou V, Robison AJ, Laplant QC, Covington HE 3rd, Dietz DM, Ohnishi
YN, et al. (2010): ?FosB in brain reward circuits mediates resilience to
stress and antidepressant responses. Nat Neurosci 13:745–752.
extinction trials in the water maze: Relationship with measures of anxi-
ety in aged and adult rats. Neurobiol Learn Mem 87:309–323.
26. Schulz D, Topic B, De Souza Silva MA, Huston JP (2004): Extinction-
induced immobility in the water maze and its neurochemical concom-
494 BIOL PSYCHIATRY 2011;70:487–495
Y.N. Ohnishi et al.
27. van der Kooij MA, Glennon JC (2007): Animal models concerning the
role of dopamine in attention-deficit hyperactivity disorder. Neurosci
28. Gerfen CR, Engber TM, Mahan LC, Susel Z, Chase TN, Monsma FJ Jr,
(1996): Chronic alterations in dopaminergic neurotransmission pro-
duce a persistent elevation of ?FosB-like protein(s) in both the rodent
and primate striatum. Eur J Neurosci 8:365–381.
30. Werme M, Messer C, Olson L, Gilden L, Thorén P, Nestler EJ, Brené S
(2002): ?FosB regulates wheel running. J Neurosci 22:8133–8138.
induce distinct programs of transcription factor expression in the stria-
32. Tang L, Todd RD, O’Malley KL (1994): Dopamine D2 and D3 receptors
33. Chen J, Zhang Y, Kelz MB, Steffen C, Ang ES, Zeng L, Nestler EJ (2000):
Induction of cyclin-dependent kinase 5 in the hippocampus by chronic
electroconvulsive seizures: Role of ?FosB. J Neurosci 20:8965–8971.
34. Ulery PG, Nestler EJ (2007): Regulation of ?FosB transcriptional activity
by Ser27 phosphorylation. Eur J Neurosci 25:224–230.
35. Brami-Cherrier K, Valjent E, Garcia M, Pagès C, Hipskind RA, Caboche J
striatal neurons: A new route to cAMP response element-binding pro-
tein phosphorylation. J Neurosci 22:8911–8921.
al. (2007): IRS2-Akt pathway in midbrain dopamine neurons regulates
behavioral and cellular responses to opiates. Nat Neurosci 10:93–99.
37. Leone P, Pocock D, Wise RA (1991): Morphine-dopamine interaction:
Ventral tegmental morphine increases nucleus accumbens dopamine
involvement. Gen Pharmacol 23:1221–1225.
39. Ulery-Reynolds PG, Castillo MA, Vialou V, Russo SJ, Nestler EJ (2009):
Phosphorylation of ?FosB mediates its stability in vivo. Neuroscience
40. FrankeTF(2008):PI3K/Akt:Gettingitrightmatters. Oncogene27:6473–
41. Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR,
Caron MG (2005): An Akt/beta-arrestin 2/PP2A signaling complex
mediates dopaminergic neurotransmission and behavior. Cell 122:
42. Brown JR, Ye H, Bronson RT, Dikkes P, Greenberg ME (1996): A defect in
nurturing in mice lacking the immediate early gene fosB. Cell 86:297–
43. Zhu H, Lee M, Agatsuma S, Hiroi N (2007): Pleiotropic impact of consti-
tutive fosB inactivation on nicotine-induced behavioral alterations and
stress-related traits in mice. Hum Mol Genet 16:820–836.
Cadherin regulates dendritic spine morphogenesis. Neuron 35:77–89.
45. McEwen BS (2005): Glucocorticoids, depression, and mood disorders:
Structural remodeling in the brain. Metabolism 54:20–23.
46. Norrholm SD, Ouimet CC (2001): Altered dendritic spine density in ani-
Neurobiology of depression. Neuron 34:13–25.
49. Peakman MC, Colby C, Perrotti LI, Tekumalla P, Carle T, Ulery P, et al.
(2003): Inducible, brain region-specific expression of a dominant nega-
Y.N. Ohnishi et al.
BIOL PSYCHIATRY 2011;70:487–495 495
Ohnishi et al.
Supplemental Methods and Materials
Generation of mutant mice. fosB+/d and fosB+/G embryonic stem (ES) cell clones were
injected into blastocysts prepared from C57BL/6J mice (Clea Japan Inc., Tokyo, Japan).
The fosBd allele contained three base substitutions just after the last codon of ΔFosB in
exon 4, which block alternative splicing and restrict the fosB gene product to ΔFosB
exclusively. The fosBG allele replaced the 3’ portion of exon 2 and 5’ portion of exon 3
with d2EGFP cDNA sequence in frame, following a stop codon. The fosBG allele
therefore encodes a non-functional N-terminal segment of FosB fused to d2EGFP,
however, expression of the fusion protein was undetectable presumably due to
nonsense-mediated mRNA decay (1). In both mutant alleles, neomycin resistant gene
cassettes for positive selection were removed by Cre/loxP-mediated recombination in
ES cells before blastocyst injection. Male chimeras derived from two independently
targeted ES cell clones were mated with C57BL/6J females to obtain germ line
transmission. Heterozygous (fosB+/d and fosB+/G) mice were backcrossed onto
C57BL/6J males several times and at least one time onto C57BL/6J females for
maintaining these strains. To yield experimental animals, fosB+/d (N7-13 generation) or
fosB+/G (N4-8 generation) mice were intercrossed. Genotyping was performed with
genomic polymerase chain reaction (PCR) with LGFFB-1
(5’-CTCGTTTAGGACACAGG CACAGT-3’) and FBEX2U (5’-ACGGTCACCGC
AATCACAAC-3’) in case of G type mouse line (Figure S8A in Supplement 1). In case of
d type mouse line, genotyping was performed with genomic PCR with UIP
(5’-CAATGCCCCCTTCTGCCCTTTA-3’) and LIP (5’-TGCTACTTGTGCCTC
GGTTTCC-3’) (Figure S8B in Supplement 1), and introduced mutation was confirmed
by DNA sequencing of genomic PCR products. Detailed information has been reported
Ohnishi et al.
Immunohistochemistry. fosB+/+, fosBd/d, and fosBG/G mice (5 months old) were deeply
anesthetized with pentobarbital (25 mg/kg, i.p.), and their brains were quickly removed
and fixed with 4% paraformaldehyde in PBS [pH 7.4] overnight at 4°C. Fixed tissues
were immersed sequentially in 20 and 30% sucrose in PBS at 4°C overnight for each
sucrose concentration, and frozen in optimal cutting compound. Immunostaining was
performed on free-floating cryostat sections (10 µm). Briefly, sections were pretreated
with 0.3% H2O2 in methanol for 30 min to block endogenous peroxidase activity,
following by incubating with Block Ace (Dainippon Seiyaku, Tokyo, Japan) and reacted
with rabbit polyclonal anti-FosB(N) antibody, which detects FosB and ΔFosB (3), at a
dilution of 1:400, or rabbit polyclonal anti-FosB(C), which detects only FosB (3), at a
dilution of 1:500, overnight at 4°C. After washing with PBS three times, sections were
incubated with mouse monoclonal anti-rabbit immunoglobulin antibody labeled with
biotin at a dilution of 1:2000 at room temperature for 40 min. Each antibody was diluted
in 10% Block Ace. After washing with PBS three times, labeled antibody was detected
with the avidin biotin peroxidase method.
Western blot analysis. Isolated striata from deeply anesthetized mice were
homogenized with dounce homogenizers in 200 µl lysis buffer (10 mM Tris-HCl [pH
7.5], 320 mM sucrose, 5 mM DTT, 1% protease inhibitor cocktail (Nacalai Tesque,
Japan), 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM orthovanadate,
and 20 mM β-glycerophosphate). The homogenates were transferred to Eppendorff
tubes and centrifuged at 1700 rpm at 4°C for 10 min. The supernatants were
considered cytosol extractions; the pellets (nuclei) were resuspended in 200 µl lysis
buffer with 0.5% NP-40, 1 mM EDTA, and 1 mM EGTA. After 10 min lytic reactions at
4°C, the samples were centrifuged at 1700 rpm at 4°C for 10 min. The supernatants
were discarded and the pellets (pure nuclei) were resuspended in 100 µl extraction
buffer (50 mM Tris-HCl [pH 7.5], 10% glycerol, 400 mM NaCl, 5 mM DTT, 0.5% NP-40,
Ohnishi et al.
1% protease inhibitor cocktail, 10 mM sodium pyrophosphate, 50 mM sodium fluoride,
1 mM orthovanadate, and 20 mM β-glycerophosphate) and kept at 4°C for 30 min.
Then, the samples were centrifuged at 15000 rpm at 4°C for 7 min. The supernatants
were frozen in liquid nitrogen and stored at -80°C as nuclear extracts. Protein
concentrations were quantified by the Bradford Assay. For gel electrophoresis,
samples were suspended in 2X SDS sample buffer with 2-mercaptoethanol and boiled.
Samples were loaded onto 12.5% SDS-polyacrylamide gels, and transferred to PVDF
membranes. The membranes were immediately placed into TBST buffer (10 mM Tris
[pH 7.5], 100 mM NaCl and 0.1% Tween 20) with 5% milk powder, and immunoblotted
with primary antibodies (1:1000 anti-FosB (5G4), 1:1000 anti-Cdk5, 1:1000 anti-Akt,
1:1000 anti-Phospho-Akt (T308), 1:1000 anti-Phospho-Akt (Ser473) from Cell
Signaling Technology, Danvers, MA, 1:100 anti-E-cadherin, 1:200 anti-α-catenin, 1:100
anti-β-catenin, 1;500 anti-GluR2 from Santa Cruz Biotechnology, Santa Cruz, CA), and
then horseradish-peroxidase conjugated Protein A (Jackson ImmunoResearch, West
Grove, PA) or anti-rabbit IgG (DC03L, Merck Ltd., Japan) at 1:10000 dilution. The
reactions were washed with TBST buffer. Bound antibodies were visualized using an
enhanced chemiluminescence (ECL+plus) detection system (Amersham Biosciences,
Piscataway, NJ). The blots were stained with Ponceau S.
Behavior analyses. All behavior analysis was carried out with male mice (2-4 months
old) from 2:00 to 8:00 P.M. except measures of spontaneous locomotor activity, which
were determined throughout the day. Behavior experiments were initiated at least one
week after littermates were separated and housed independently, again except for
spontaneous locomotor activity measurements, which were determined one week after
separating littermates. All separation for single housing started after two months old.
Spontaneous locomotor activity was measured with an infrared beam sensor
(NS-AS01; Neuroscience Inc., Japan) placed about 200 mm above the center of a
Ohnishi et al.
homecage (27 x 14 x 15 cm), and analyzed by DAS-008 software. Measurements were
obtained for two time points, one of which was just after separation of littermates and
continued for one week, the other was more than one month after separation of
littermates and continued for 3 or 4 days.
Exploratory locomotor activity in a novel environment was measured in a
circular open field (60 cm in diameter, 30 cm deep). Locomotor activity was quantified
with an automated video tracking system, the video-image motion analyzer AXIS-90
(Neuroscience, Tokyo, Japan). The experimental mouse was placed in the center, and
distance and moving time were measured for 10 min. This experiment was carried out
four days in a row.
Elevated plus maze test was performed using an apparatus consisting of two
open arms (25 x 8 cm) and two closed arms of the same dimensions with black
Plexiglas walls 15 cm high, arranged such that both open and closed arms faced each
other with a quadrangular center (8 x 8 cm). The maze was placed 65 cm above the
floor and is made of white plastic. The maze floor was covered with ash gray expanded
polystyrene. The mouse was placed at the center of the maze with head facing an open
arm, and given 5 min to explore the plus maze. The number of times to enter into each
arm, dipping times on the open arms, and time spent in each arm were scored utilizing
Light and dark box was performed using an apparatus consisting of a lighted
white compartment (25 x 30 cm; 20 cm height) and a dark black compartment (25 x 15
cm; 20 cm height), which were connected by a lighted white compartment (7.5 x 7.5
cm; 20 cm height). For the assessment of anxiety related-behavior in the light and dark
box, mice were placed in the larger white compartment with their head facing a corner.
The time spent in the white compartment, and the number of entries into the white
compartment, were recorded for five min after first time entry into the black
Ohnishi et al.
Morris water maze test was performed using a pool (1.2 m in diameter, white
polypropylene plastic) with water maintained at 19°C. In the hidden-platform test, the
Plexiglas platform (10 cm in diameter) was submerged 1.5 cm below water level and
kept in the same location throughout training. The landmarks (four white polypropylene
plastic boards marked with triangle, rectangle, circle, or cross) were attached on the
edge of the pool. Swimming paths were tracked with a camera on the ceiling of the
room and stored in a computer (Target/2 system, Neuroscience Inc). Three starting
positions were used pseudo-randomly and the mice were trained with four trials per
day for ten days. After reaching the platform, the mouse was allowed to remain on it for
30 s before being returned to its home cage. If the mouse did not find the platform
within 60 s, the training trial was terminated without leading to the platform, and a
maximum score of 60 s was assigned. In a probe test, mice swam for 60 s in the pool
without the platform.
Repeated forced swim test in inescapable condition was examined in
transparent circular cylinder (diameter: 18 cm, height: 35 cm) filled with water
maintained at around 19°C (height: 12 cm). Each trial was performed for 6 min once a
day for 4 consecutive days, and the swimming time was counted for the last 4 min.
Paroxetine (Sigma-Aldrich, Japan K.K. Tokyo, 10 mg/kg) was injected intraperitoneally
30 min before the fourth day trial. Swimming time was monitored with the same
equipment as used for the water maze test.
Locomotor activity after pharmacological treatments was measured as follows.
Naive mice, without prior behavioral assays, were housed individually for two weeks,
and then placed in a cage with the infrared beam sensor for 3 days. On day 2, animals
received intraperitoneal injections of saline (SAL) as a negative control at 4:00 P.M. On
day 3, the mice were given an intraperitoneal injection of 30 mg/kg methylphenidate
(MPH, Sigma), 2 mg/kg SKF81297 (SKF, Sigma), 1 mg/kg haloperidol (HAL, Sigma) or
1 mg/kg LY 171555 (QNP, Sigma) at 4:00 P.M. and immediately returned to their home
Ohnishi et al. Download full-text
cage. Each mouse was used for only this experiment and only for one drug. We
followed the locomotor activity until 10:00 A.M. on the 4th day.
Statistical analysis. For the statistical analysis among different genotype mice, the
data were analyzed by one-way analysis of variance (ANOVA), or Kruskal-Wallis
one-way ANOVA on ranks. Dunnett’s test was used for multiple comparisons
procedures. In the case of repeated forced swim tests, Wilcoxon signed-rank test was
performed for comparing the swimming time between the first and other day in each
type of mice.