This study identified diurnal rhythms of immobility induced by FST in WT mice. Male WT mice reached their peak near late daytime (ZT10) with a nadir near midnight (ZT18), while female wild types reached their peak near early night (ZT14) with a nadir near early daytime (ZT2). Female WT mice showed characteristics very similar to those of negative mood in human studies, where peak depression also occurred in the early active period [2
]. However, due to the sample size, the character of participants, and the data collection methods, studies investigating a link between circadian rhythms and human mood have produced mixed results. In a study of working females, peaks for positive emotions were detected at noon and again in the evenings, while peaks in negative emotions were found at mid-morning and mid-afternoon [15
]. In a study of college students, the only peak for positive emotions occurred in the evening, while the only peak for negative emotions occurred in the early morning [2
]. In the present study, we profiled circadian rhythms of immobility in FST in both male and female mice. It is hypothesized that circadian clock dysfunction, which is primarily regulated by clock genes, leads to mood disorders [1
]. Here, we further investigated the role of core circadian genes in the regulation of circadian rhythms of immobility response in the FST.
We found that both male and female Clock
mutant mice lost their diurnal rhythms of immobility in FST, indicating its potential role in maintaining the circadian rhythms of depression-like behavior. There are a few studies revealing the possible roles of the Clock
gene in mood disorders. For example, chronic treatment with the antidepressant fluoxetine increases the expression of Clock
in the hippocampus [16
], while disruption of Clock
expression induces mania-like behavior [9
]. Human studies found that the Clock
gene with a polymorphism in the 3'-flanking region has been associated with greater severity of insomnia during antidepressant treatment [17
] and a higher recurrence rate of bipolar episodes [18
]. However, Benedetti et al.
failed to find a possible effect of the Clock
genotype on the regulation of perceived diurnal mood fluctuations during a major depressive episode [18
]. This observation is not implicitly conflicting with our findings for two reasons: firstly, the Clock
mutant mice used in our study possesses a deletion in exon 19 of the Clock
gene which is different from the human genotype; secondly, “behavioral despair” induced by FST in mice can’t entirely mimic the mood of depressive episode in humans.
We also found that mice that are homozygous for the targeted allele of either mPer1
showed distinct circadian changes of immobility induced by FST in comparison to WT mice. Per1Brdm1
female mice showed significant diurnal rhythms of immobility behavior with delays in both peak and nadir times when compared to female WT mice. The pattern of diurnal rhythms of immobility for male Per1Brdm1
mice was very similar to that of WT male mice. In contrast, neither male nor female Per2Brdm1
mice showed significant rhythmicity. These results indicate that mPer1
play important but distinct roles in the regulation of circadian rhythms in depression-like behavior. These genes also have distinct roles in circadian clock function [19
]. In addition, the circadian rhythm of circulating glucocorticoids differs between Per1Brdm1
mice show markedly elevated levels of circulating glucocorticoids lacking any circadian rhythm, whereas Per2Brdm1
mice demonstrate elevated but diurnally fluctuating serum glucocorticoids levels [20
]. Dysregulation in either the CLOCK system or the hypothalamic-pituitary-adrenal (HPA) axis may cause similar, pathologic manifestations by uncoupling circulating cortisol concentrations from tissue sensitivity to glucocorticoids [21
]. Therefore, further studies should investigate whether the complex crosstalk between the circadian CLOCK system and the HPA axis are involved in the effects of the Clock
genes on depression.
Anxiety and depression occur more frequently in females than in males in the human population [22
], indicating the existence of sex-specific differences in the mechanisms of depression. Most importantly, sex hormones affect many aspects of circadian responses, and there are significant sex-specific differences in rhythmicity [23
]. Here, we show that WT mice display diurnal rhythms of immobility with a sex-specific pattern. This implies that male–female differences also exist in the interaction of environmental factors and internal circadian rhythmicity. Furthermore, Per1Brdm1
mice showed circadian rhythms of immobility, but no sex-specific differences were observed. Our results implied that mPer1
regulates the rhythmicity of immobility in a sex-specific pattern.
Few studies have investigated sex-specific differences in response to stress and depression-like behavior. One study did investigate the influence of sex on depression-like behavior during different phases of the circadian cycle [24
]. Consistent with our results, they found that female rats had more immobility in the dark phase in comparison to male rats. However, they did not find a circadian phase effect in males [24
]. There may be several reasons for this result, which differs from ours. The experiments examined different model systems (mice vs.
rat). Additionally, the circadian rhythm of the rats was reset by reversing the light cycle, which may stress the animals, leading to potential effects on late behavioral testing. Finally, all behavioral testing of the rats was performed during a single time period, preventing analysis of the full range of circadian changes.
Our study reveals that there are distinct, sex-specific differences in the levels of immobility among the mouse strains deficient in core circadian genes. Per1Brdm1
female mice show higher levels of immobility in comparison to males, while female Per2
mutant mice show consistently lower levels of immobility in both day and night compared to males of the appropriate genotype. This finding may result partly from the complex crosstalk between the circadian CLOCK system and sex hormones. For example, the circadian genes, mPer1
, and mClock
are also involved in the regulation of the female reproductive and estrous cycles, which may further influence behaviors [25
]. Similarly, fluctuations in ovarian hormones have area-specific effects on the expression of Per2
in the brain [27
However, there are limitations in our study. The estrus cycle of the female mice was not considered. It had been reported that female rats in proestrous and estrous phases exhibit more immobility than animals in the diestrous phase [28
]. Another study, however, indicate that the estrous cycle did not significantly modulate behavioral outcomes tested by FST [29
]. In our study, female mice were grouped together in different estrous cycles, and mice in different estrous cycles were included in each group, which may balance the potential influences of the estrous cycle partly. Nevertheless, estrus cycles should be more carefully considered in our future studies. Another limitation in our study is we only tested immobility behavior induced by FST, the data need be further determined by another measure, such as the tail suspension test or learned helplessness.
In summary, our results highlight the differences in the circadian characteristics of immobility induced by FST in wild type, ClockΔ19, Per1 (Per1Brdm1), and Per2 (Per2Brdm1) mutant mice. All four genotypes showed sex-specific differences in the level of immobility, while sex-specific differences in circadian patterns only occurred in WT mice. This result indicates that there is no common behavioral profile associated with the disruption of individual core circadian genes, perhaps because of the complex crosstalk between the circadian CLOCK system and sex hormones. Our study supports the hypothesis that the disturbance of biological clocks contributes to depression and improves our understanding of the involvement of circadian genes in the regulation of mood disorders.