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Molecular mechanisms underlying stress tolerance and vulnerability are incompletely understood. The fosB gene is an attractive candidate for regulating stress responses, because ΔFosB, an alternative splice product of the fosB gene, accumulates after repeated stress or antidepressant treatments. On the other hand, FosB, the other alternative splice product of the fosB gene, expresses more transiently than ΔFosB but exerts higher transcriptional activity. However, the functional differences of these two fosB products remain unclear.
We established various mouse lines carrying three different types of fosB allele, wild-type (fosB+), fosB-null (fosBG) and fosBd allele which encodes ΔFosB but not FosB, and analyzed them in stress -related behavioral tests.
Since fosB+/d mice show enhanced ΔFosB levels in the presence of FosB, and fosBd/d mice show more enhanced ΔFosB levels in the absence of FosB, the function of FosB can be inferred from differences observed between these lines. fosB+/d and fosBd/d mice showed increased locomotor activity and elevated Akt phosphorylation, whereas only fosB+/d mice showed antidepressive-like behaviors, and increased E-cadherin expression in striatum, compared to wild-type mice. In contrast, fosB-null mice showed increased depression-like behavior and lower E-cadherin expression.
These findings indicate that FosB is essential for stress tolerance mediated by ΔFosB. These data suggest that fosB gene products have a potential to regulate mood disorder-related behaviors.
The fosB gene has an intron-like sequence in exon 4, which allows alternative splicing and the production of two distinct transcripts, fosB and ΔfosB mRNAs. The latter encodes ΔFosB protein which lack the C-terminal 101 aa containing the transactivation domain in the full -length FosB protein (1). We have previously reported that FosB and ΔFosB oppositely regulate Jun trans-activity (1, 2) and cell matrix adhesion (3) in cultured cells; the proteins also regulate cell proliferation, differentiation, and death (4–8). Interestingly, ΔFosB can act, in vivo and in vitro, as a transcriptional activator or repressor (9, 10). An example of the latter is feedback repression of the c-fos and fosB gene promoters (1, 11).
ΔFosB has been shown to be a key modulator of reward-related behaviors in response to drugs of abuse, natural reward, and coping with stress (9, 12–17). ΔFosB accumulates progressively after repeated 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 ΔFosB and FosB in mood-related behaviors are incompletely understood. Induction of ΔFosB 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 trans-activity (1).
Here, we characterized new fosB mutant mice: (i) fosBd/d mice with significantly-enhanced expression of ΔFosB but no FosB, (ii) fosB-null (fosBG/G) mice lacking both FosB and ΔFosB, and (iii) fosB+/d mice with reduced expression of FosB and enhanced expression of ΔFosB. We found that fosB+/d mice serve as a model for endogenous ΔFosB accumulating conditions, and that differences between fosBd/d and fosB-null mice reflect the effects of enhanced ΔFosB expression in the absence of FosB. In addition, differences observed between fosB+/d and fosBd/d mice revealed the influence of FosB under ΔFosB accumulating conditions.
See Supplement 1.
Two to five month-old male mice were used in most experiments. fosBd/d mice (F8-14) and fosBG/G mice (F5-9) were obtained from heterozygous intercrosses except for experiments involving haloperidol 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 libitum. Animals were maintained in an air-conditioned, light time-controlled, specific-pathogen-free room with a 12:12 hr light and dark cycle (light on at 8:00 A.M. and down at 8:00 P.M.). The handling and killing of all animals were carried out in accordance with the national prescribed guidelines, and ethical approval for the studies was granted by the Animal Experiment Committee of Kyushu University.
See Supplement 1.
See Supplement 1.
See Supplement 1.
See Supplement 1.
fosB gene expression in mutant mice was analyzed with immunohistochemistry (IHC) and Western blotting. Brain IHC of wild-type mice revealed that fosB gene expression is greatest in the olfactory bulb, nucleus accumbens (NAc), dorsal striatum, anterior cingulate cortex, dentate gyrus of hippocampus, and cerebral cortex. The regional pattern of fosB gene expression in the fosBd/d mice is the same as wild-type mice, but the immunoreactivity (IR) is much higher (Figure 1A–C, Figure S1G, H in Supplement 1). In wild-type mice, the anti-FosB(C), which was raised against amino acids 245–315 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 majority of the fosB gene expression signal is derived from ΔFosB. FosB IR was undetectable in fosBd/d and fosBG/G mice, and ΔFosB IR was undetectable in fosBG/G mice (Figure S1E, F in Supplement 1).
We next performed quantitative Western blotting of striatal nuclear extracts using anti-FosB (Figure 1D–F). Significantly enhanced expression of ΔFosB and Δ2ΔFosB in fosBd/d and to a lesser extent in fosB+/d mice was confirmed in comparison to wild-type mice. Expression levels of FosB and "variant FosB" ("vFosB") in fosB+/d mice were apparently decreased in comparison to those in wild-type mice, and there was no detectable FosB and vFosB in fosBd/d mice (Figure 1D, F). vFosB is thought to be Δ1FosB or Δ2FosB (Figure S1A in Supplement 1). In the fosB+/G mice, the levels of vFosB and Δ2ΔFosB but not FosB and ΔFosB were significantly decreased in comparison to those in wild-type mice, and none of those were detected in fosBG/G mice (Figure 1E, F).
These data suggest that fosB+/d mice provide a model of ΔFosB accumulation over long periods of time, and that the difference between fosBd/d and fosB+/d mice can disclose the function of FosB under conditions of ΔFosB accumulation. Additionally, the differences observed between fosBd/d and fosBG/G mice, and shared traits between fosBd/d and fosB+/d mice, reflect the results of ΔFosB accumulation.
To infer the function of endogeneous ΔFosB 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 one week without any disturbance of individually housed mice in their homecages. The activity of all mice tended to decrease gradually due to the loss of interaction with littermates (Figure S2 in Supplement 1). By the third night, fosBd/d mice showed significantly higher locomotor activity compared to wild-type mice and fosB+/d mice (Figure 2A). On the other hand, fosBG/G mice showed significantly lower locomotor activity compared to wild-type mice (Figure 2E). All mutant mice showed normal levels of locomotor activity at the start of the sleep cycle. These data suggest that ΔFosB facilitates spontaneous motor behavior during periods of wakefulness, but does not introduce abnormal motor behavior independent of the circadian cycle. Despite accumulation of comparable levels of ΔFosB in fosB+/d mice, the mice showed lower levels of locomotion compared to that seen in fosBd/d mice, suggesting that FosB suppresses the effect of accumulated ΔFosB and helps to normalize basic locomotion.
We next examined the locomotor activity of mice housed independently for one month. Spontaneous locomotion was dramatically decreased in all of the genotypes studied. Nevertheless, the locomotor activity of fosBd/d mice was still much higher; in fact, the difference between the activities of fosBd/d and wild-type mice was larger than that seen earlier. fosB+/d mice also showed significantly higher locomotor activity than wild-type mice, suggesting that accumulated ΔFosB enhances spontaneous locomotion (Figure 2B). On the other hand, the difference between fosBG/G and wild-type mice was abolished, suggesting that the level of locomotion after one month of isolation may settle down to basal levels without effects of social interaction. fosB+/G mice showed significantly higher locomotor activity than wild-type mice, suggesting that the higher expression ratio of ΔFosB versus FosB in fosB+/G mice may make an effect on the basal locomotor activity (Figure 2F).
On the first day in the open field test, which provides a measure of anxiety-like behavior, both fosBd/d and fosBG/G mice showed significantly higher exploratory activity than wild-type mice (Figure 2C, G), suggesting that FosB may increase anxiety-like responses or suppress intrinsic curiosity. Though all of the mice showed habituation after the second day of the test, fosB+/d and fosBd/d mice on the second day, and fosB+/G mice on the third and fourth days, respectively, exhibited significantly higher locomotor activity than did wild-type mice, suggesting that the difference reflects spontaneous locomotor activity (Figure 2C, G).
In the elevated plus maze, another test of anxiety-like behavior, fosBd/d mice stayed significantly longer in the open arms than wild-type mice, and fosBG/G mice spent significantly less time in the open arms (Figure 2D, H), suggesting that ΔFosB might suppress anxiety-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/d mice showed a trend for increased transitions (Figure S3A, C in Supplement 1). In contrast to the open-field and elevated plus maze tests, mutant mice showed no differences in the light-dark test (Figure S3B, D in Supplement 1).
Next, we used the Morris water maze to evaluate spatial learning 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+/d mice, and longer in fosBG/G mice especially around the middle of the training days (Figure 3A, B, and Table S1 in Supplement 1), due to immobility in the water during training (Figure 3E, F, and Table S1 in Supplement 1). In the probe test, all of the genotypes spent the longest time in the quadrant where the platform had previously existed (Figure 3G, H and Table S1 in Supplement 1). However, in fosB+/G and fosBG/G mice, the time was shorter than that in wild-type mice due to immobility in the first quadrant (white boxes in Figure 3G, H).
The forced swim test is used widely as a measure of stress responsiveness, with immobility reflecting greater stress vulnerability. Not surprisingly, the Morris water maze was reported recently to provide useful measures of swim stress responses (25, 26). Together, these data suggest that fosB gene products do not affect learning ability, however, accumulated ΔFosB decreases stress vulnerability, and such activity requires the presence of FosB.
Next, to further assess the behavioral responses of fosB mutant mice to inescapable stress, we used a conventional forced swim test. We found a trend for decreased swimming time over repeated days of the test in wild-type, fosBd/d, and fosBG/G mice, but not in fosB+/d mice (Figure 4). fosBd/d and fosBG/G mice showed significantly decreased swimming time on the second and third day, respectively. Additionally, we injected the mice with paroxetine (10 mg/kg), a standard antidepressant, 30 min before the swim test on the fourth day. Paroxetine increased the swimming time well beyond levels seen on the first day in all type of mice except fosBG/G mice. In fosBG/G mice, the swimming time recovered but did not reach the level of the first day, and this “recovery ratio” was significantly lower than that seen in wild-type mice.
fosBd/d mice showed hyperlocomotion in the active phase with a 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 reminiscent of the symptoms of attention deficit hyperactivity disorder (ADHD) (27). ADHD patients can be treated successfully with methylphenidate (MPH). MPH inhibits dopamine reuptake and increases the dopamine concentration in synaptic clefts, which leads to hyperlocomotion in healthy individuals. However, ADHD patients calm down in paradoxical reactions to MPH. Therefore, we examined whether MPH exerted therapeutic-like effects in fosBd/d mice 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 down to baseline within 4 hr after intraperitoneal injection in all types of fosB mutant mice and their wild-type littermates (Figure S5C, D in Supplement 1). In particular, fosB+/d and fosBd/d mice exhibited more hyperactivity in response to MPH than wild-type mice. The transient hyper-locomotion exhibited by fosB+/d and fosBd/d mice were similar, but the locomotor activity exhibited in the dark phase after MPH treatment was decreased in fosB+/d mice (Figure 5A, C). These data suggest that accumulated ΔFosB drives locomotion during the dark phase, while the existence of FosB prevents it (Figure S5C in Supplement 1). fosB+/G and fosBG/G mice exhibited equivalent reactions to MPH as wild-type mice, suggesting that normal low levels of ΔFosB do not contribute to basal dopamine sensitivity, and that only accumulated ΔFosB can facilitate it (Figure 5B, Figure S5B, D in Supplement 1).
Basal ganglia circuitry regulates motor function via two pathways, termed the direct and indirect pathways. More than 90% of striatal neurons are medium spiny GABAergic projection 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 expression. 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 decreases locomotion (28). D2 receptor signaling in these neurons inhibits such down regulation of locomotion, and suppresses fosB gene expression (29).
Werme et al. reported that ΔFosB overexpression selectively in direct pathway neurons increased daily running compared with control littermates, whereas ΔFosB overexpression predominantly in indirect pathway neurons decreased it (30). At first, we hypothesized that spontaneous hyperlocomotion and higher responsivity to MPH in fosB+/d and fosBd/d mice 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 of the fosB mutant and wild-type mice (Figure 5A, B, and Figure S5E, F in Supplement 1). Next, we administered a D2 receptor antagonist (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 hr after haloperidol treatment was significantly higher in fosBd/d mice, and lower in fosBG/G mice, compared to wild-type mice (Figure 5A, B and 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 littermates (Figure S2 in Supplement 1), suggests that accumulated ΔFosB suppresses the D2 receptor signal. This in turn may contribute to the increased locomotion and reduction in anxiety-like behavior induced by ΔFosB, with FosB negatively regulating the effects of accumulated ΔFosB.
We detected no behavioral differences in the effects of a D2 agonist (1 mg/kg LY 171555) among the fosB muant and wild-type mice (Figure 5A, B, and Figure S5I, J in Supplement 1). These data suggest that, in the mutant mice, negative feedback via D2 receptors on dopamine neurons in the midbrain may function normally (32).
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 blotting with striatum samples (Figure S6A in Supplement 1). E-cadherin was significantly up-regulated in fosB+/d mice, and down-regulated in fosB+/G and fosBG/G mice, in other words, the expression level of E-cadherin paralleled the behavioral phenotype of stress tolerance seen with forced swimming. Similar tendencies were detected in hippocampus (data not shown). In contrast, there was no difference in the expression levels of Cdk5, GluR2, α-catenin, or β-catenin (the first two are putative ΔFosB targets identified in bitransgenic mice, and the latter two are binding partners of E-cadherin) among fosB mutant and wild-type mice (Figure S6A in Supplement 1). The level of E-cadherin mRNA in NAc also showed no difference among mutant and wild-type mice (data not shown), suggesting that the up-regulation of E-cadherin is regulated by post -translational mechanisms.
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+/d and fosBd/d mice.
In this study, we provide evidence that accumulated ΔFosB 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 ΔFosB 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/G mice exhibit behaviors that resemble depression, including decreased locomotion, increased immobility during forced swimming, and increased anxiety-like responses. In contrast, fosB+/d mice exhibit behaviors that in some ways resemble manic-like symptoms, including hyperlocomotion, increased stress tolerance, and reduced anxiety-like responses. Interestingly, fosBd/d mice exhibit significantly higher dopamine sensitivity, and most of the altered behaviors seen in fosB+/d mice except for increased stress tolerance. These mice thus present a picture of blended responses perhaps reminiscent of certain aspects of bipolar disorder. Clearly, these interpretations are based on initial analyses in rodent models and require much further work for validation, including studying these molecular findings in the human disorders.
The similar results obtained from fosBG/G mice in the water maze (Figure 3) and repeated forced swim test (Figure 4) indicate that the water maze has the potential to reveal responses to stress. Whereas the repeated forced swim test revealed the stress vulnerability of fosBd/d mice more clearly than the water maze, the difference between wild-type and fosB+/d mice was more apparent in the water maze, suggesting that the repeated forced swim test may be better to detect initial stress vulnerability, while the water maze may be better to detect stress tolerance that develops over time. Data with paroxetine suggest that fosB gene products are partly required for antidepressant responses to the drug, as reported recently (24).
In fosB+/d mice, the antagonistic relationship between FosB and accumulated ΔFosB, first seen in vitro (1), was captured in measures of spontaneous locomotor activity (Figure 2A, Figure S2A in Supplement 1), elevated plus maze (Figure 2D), and sensitivity to a D2 antagonist (Figure 5A, Figure S5G in Supplement 1). The expression level of ΔFosB in fosB+/d mice is much higher than that of FosB, but FosB has much higher trans-activity than ΔFosB (1, 34). These data suggest that lower expression levels of FosB may be sufficient to suppress the activity of accumulated ΔFosB, and thereby normalize some of the consequences of its accumulation (Figure S7 in Supplement 1). Such antagonism is not seen in all cases, perhaps reflecting the different cell types or target genes involved. In ΔFosB bitransgenic mice, where ΔFosB expression is inducible and relatively restricted to D1 neurons in NAc and dorsal striatum, Kelz et al. reported higher locomotor activity in a novel test chamber not on the first day but on the second day (14). This finding corresponds to observations in fosB+/d mice, which exhibited higher locomotor activity with a similar pattern (Figure 2C), thus suggesting that ΔFosB accumulation in striatum plays an important role in mediating locomotor activation via dopamine signaling.
We know that the fosB gene is induced by dopaminergic signals (23). Since Akt phosphorylation is also induced by dopamine (35), it is possible that accumulated ΔFosB enhances Akt phosphorylation, and that the extent of spontaneous dopaminergic activation reflects levels of Akt phosphorylation. Perrotti et al. reported that morphine induces ΔFosB accumulation in NAc (23), and Russo et al. reported that overexpression of a constitutively active form of Akt in NAc enhances locomotor sensitization induced by morphine (36). Locomotor sensitization by morphine is regulated by indirect dopaminergic mechanisms such as increased dopamine release in NAc (37, 38). Cocaine directly increases dopamine release in NAc by inhibiting dopamine reuptake. Overexpression of ΔFosB enhanced cocaine locomotor sensitization (39), suggesting that enhanced Akt phosphorylation by accumulated ΔFosB might facilitate locomotor sensitization by cocaine and by morphine. This is consistent with the significant up-regulation of Akt phosphorylation and enhanced locomotor sensitization by MPH observed in fosBd/d mice (Figures 5A, S6C in Supplement 1). The mechanism by which ΔFosB accumulation leads to increased Akt phosphorylation is not known, since ΔFosB could affect the expression of any of several regulators of Akt phosphorylation (40). One possibility is that accumulated ΔFosB may repress the expression of a protein phosphatase, such as PP2A, since Akt phosphorylation is known to be suppressed by D2 signaling in striatum via the activation this phosphatase (41). These and other possibilities now require direct investigation.
fosB-knockout (KO) mice had been established by Brown et al. (42), and actually have a potential to express Δ3ΔFosB, an alternative-translation initiation product similar to Δ2ΔFosB (Figure S1 in Supplement 1). Hiroi et al. reported that fosB-KO mice showed slightly higher locomotor activity when they were introduced into a novel test chamber (12, 43). This corresponds to our observation that fosBG/G mice exhibit significantly higher locomotor activity in the open field on the first day (Figure 2G), suggesting that FosB may suppress exploratory behavior in the novel environment independent of the presence of ΔFosB. On the other hand, Zhu et al. (43) reported that the fosB-KO mice exhibit lower sensitivity to other types of stress paradigms (tail suspension test), and Brown et al. (42) had reported that the fosB-KO mice are defect in nurturing, and no difference in the Morris water maze test. These findings are not consistent wit h our results, since fosBG/G mice exhibited higher stress vulnerability and no defect in nurturing (data not shown). These differences could reflect the potential influence of Δ3ΔFosB or the different genetic backgrounds (BALB-C in the prior studies by Brown et al. and C57BL6/J in the present investigations).
Interestingly, the expression level of E-cadherin and the stress tolerance observed upon repeated forced swimming paralleled the combined expression of FosB and ΔFosB. Only with regard to the particular expression pattern and behavioral phenotype does FosB not antagonize but coordinates with ΔFosB. ΔFosB has been reported to accumulate after chronic stress in several brain regions (22, 24), and accumulated ΔFosB in the ventrolateral periaqueductal gray promotes active coping responses against stress, such as forced swimming (13). More recently, overexpression of ΔFosB in the NAc suppresses the deleterious effects of social defeat (24). Findings from the present study suggest that such antidepressant-like effects of ΔFosB require the presence of FosB.
E-cadherin is an important cell-cell adhesion molecule, and influences synapse formation in neuronal cells (44). In rodent models of depression, the number of mature synapse buttons or dendritic spines is decreased for several neuronal cell types, and antidepressant treatments reverse these abnormalities (45–47). Here, we found that the expression level of E-cadherin in fosB mutant and wild-type mice parallels stress tolerance, suggesting that E-cadherin may be one mechanism 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-cadherin, or directly inhibit the transcription of Hakai mRNA (48). Accumulated ΔFosB might increase E-cadherin translation via TGF-β signaling, which is increased in fosB+/d and fosBd/d ES cells (3). Future studies are needed to explore these possibilities directly.
Cdk5 and GluR2, putative ΔFosB targets identified in bitransgenic mouse models (14, 33, 49), are expressed at similar levels among fosB mutants and wild type. McClung and Nestler reported that the expression levels of various ΔFosB targets in these mice switched from up- to down-regulated, or vice versa, between two and eight weeks after turning on ΔFosB expression (9). The constitutive changes in fosB gene expression in our mutant mice might not result in altered expression of Cdk5 and GluR2.
In summary, the fosBG/G mutant is a definitive fosB-null mouse, and fosB+/d and fosBd/d mutants differentially express aberrant levels of ΔFosB and FosB from the endogenous fosB gene. These three mouse l ines 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 disorders.
We thank Dr. M. Katsuki (National Institutes of Natural Sciences, Tokyo, Japan) for CCE ES cells, M. Otsu in the Laboratory for Technical Support, Medical Institute of Bioregulation for the DNA sequence analyses, A. Matsuyama and K. Nakabeppu for animal care, and S. Kitamura for tissue processing. We also thank all members of our laboratory for their helpful discussions. This work was supported by grants from CREST, Japan Science and Technology Agency, the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant number: 16012248), and the Japan Society for the Promotion of Science (Grants 16390119 and 18300124, 22221004).
The authors report no biomedical financial interests or potential conflicts of interest.
Supplementary material cited in this article is available online.