Results of this study demonstrate that in several behavioral paradigms female mice are more vulnerable to CUS than males. We found that loss of BDNF makes female mice more sensitive to some measures of anxiety and particular features of depression-like behaviors following CUS compared to littermate CTLs. In contrast, loss of BDNF in males fails to increase most measures of anxiety, anhedonia and depression-like behavior following CUS compared to wild type CTLs. This is not to say that there were no genotype effects observed following CUS in males, but rather that the effects of stress and genotype were more extensive in females than males. Collectively, these data suggest that loss of BDNF does not result in greater susceptibility to depression-related behavior per se
, but rather is linked to expression of these behaviors in a gender-specific manner in response to stress. CUS paradigms have produced alterations in locomotor activity, anxiety-like behavior, fur state, sucrose consumption, forced swim test, tail suspension tests, and corticosterone levels in rodents (25
). However, most robust CUS effects on these measures are in rats and effects in mice have been more difficult to ascertain suggesting that mice may be more resilient to chronic stress (25
). Most CUS studies have relied solely on males and our data would largely support the resiliency of male mice to stress in many of these behavioral paradigms. However, our data with female mice suggests they may have an increased vulnerability in some behavioral measures following CUS.
In females, we found that stress produced a significant decrease in total locomotor activity of inducible KOs compared to nonstressed CTLs, nonstressed KOs, and stressed CTLs suggesting that loss of BDNF in females exacerbated locomotor deficits. In males, we found that nonstressed inducible KOs were significantly hyperactive compared to nonstressed CTLs in agreement with previous findings (9
). Following CUS, male CTLs displayed significant hypoactivity compared to nonstressed CTLs while a similar trend, although not significant, was observed in BDNF KOs.
We assessed anxiety-related behavior using the OF test. In females, we found that CUS in BDNF KOs significantly reduced the time in the center of the arena and the number of entries to the center, indicative of an increase in anxiety-like behavior, compared to nonstressed KOs. In males, loss of BDNF did not alter anxiety related behavior following CUS. Our data is in contrast to previous findings of anxiolytic like effects of CUS in elevated plus maze (27
), however other reports utilizing chronic variable stress report anxiogenic like effects in this test (28
) and further studies suggest that these differences may be accounted for by the length of time between stress exposure and testing (29
We examined the effects of CUS in inducible KOs in paradigms that provide measures of depression-like behavior. We examined the fur state of animals to assess grooming behavior, SCT as a measure of anhedonia, and latency to feed in the NSF test in CTL and BDNF KO animals following CUS. In females, we found that in nonstressed conditions loss of BDNF did not alter fur score, sucrose intake or latency to feed in the NSF test compared to CTLs. Following CUS, we found that female BDNF KOs had a significantly poorer fur score and were more anhedonic than nonstressed KOs or stressed CTLs and displaying a strong trend towards an increase in latency to feed compared to nonstressed KOs. In male KOs, CUS did not produce significant differences in fur score or sucrose consumption compared to other groups, and in the NSF test BDNF KOs appeared less anxious following CUS than CTLs. Collectively, this data suggests that loss of BDNF in CUS females may increase anxiety related behavior and some measures of depression related behavior, however these effects are gender specific since similar effects were not observed in males.
We examined mice in the FST and TST, tests that are commonly used to assess antidepressant efficacy and, by extension, depression (30
). Surprisingly, we did not observe increased depression-like behavior as assessed by increased immobility in either FST or TST following CUS in either females or males independent of genotype. The FST and TST are often associated with depressive phenotypes in rodents following acute stress (24
). Our lack of a change in immobility in these paradigms may be due to the adaptive aspect of stress responses to CUS over time (31
). Recent interest has focused on uncovering the genetic mechanism behind the consistent observation that some animals display resilience to stress (32
). To this end, neither susceptible nor resilient mice display differences in the FST or TST after chronic social defeat stress (33
), although this finding is not unequivocal(34
). It is possible that the lack of a change in depression-like behavior as determined here could be due to the fact these tests are designed to predict antidepressant efficacy rather than as measures indicative of depression-like behavior (10
As a physiological measure of stress to support our behavioral findings, we assessed CORT concentration. Rather surprisingly, we found that under nonstressed conditions loss of BDNF is associated with decreased CORT levels in both males and females. Furthermore, we found that susceptibility for developing depression-related behaviors in female BDNF KO mice after CUS was correlated to a significant increase in CORT levels compared to nonstressed KOs and stressed CTLs. In contrast, in males we found that CUS did not significantly alter CORT levels compared to baseline levels of CORT in nonstressed CTLs and KOs. These findings suggest that under nonstressed conditions there is an interaction between BDNF and CORT in that loss of BDNF significantly reduced plasma CORT levels although this was not significantly correlated with decreased anxiety or depression like behavior. Following CUS, we found gender specific effects of the interaction between BDNF and CORT. Interestingly, a significant increase in CORT levels following CUS was observed in female BDNF KOs, the animals with the most pronounced anxiety and in some measures depression like behavior here.
We examined BDNF mRNA expression in all female and male groups. We found that under nonstressed conditions, both female and male KOs had a significant reduction in BDNF in hippocampus in agreement with previous data (9
). Following CUS, we found that both male and female CTLs showed a significant reduction in the amount of BDNF in hippocampus. Rather surprisingly, CUS did not further reduce BDNF levels in KOs compared to nonstressed conditions. This data suggests that there is a floor effect in the amount of BDNF reduction in hippocampus. Thus while there were significant behavioral differences in KOs following CUS, this was not directly correlated with the amount of BDNF mRNA.
Interestingly, studies examining the effect of gender on stress responses have shown that BDNF levels in dentate gyrus are reduced in females but not males after restraint stress in rats (36
), contrary to what we observed. Instead, our data suggests that dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis stress response between males and females may account for the gender differences in agreement with previous literature (37
). Recent studies have suggested that depression based gender differences may be the result of alterations in hormonal levels (39
) or in neuroanatomical differences between males and females (42
). It is for this reason that many laboratories utilizing female mice control for estrus cycle. However, in this study, we did not control for the estrous stage of females. While there may be concern that female mice cycling at different stages could impact the behavioral data and result in inconclusive data, we were able to observe significant behavioral effects even in spite of this concern. Our very large number of females in each group, which likely represents a sampling across all stages of cycling, may well have contributed to our ability to observe significant behavioral effects.
Intriguingly, a recent study has shown that in conditional BDNF KO mice there is evidence of increased depression-like behavior in females BDNF KOs but not males (9
), however it is difficult to make direct correlations with the data presented here and the previous study as the pattern of BDNF deletion in these various BDNF lines is quite different suggesting that regional pattern of BDNF deletion may influence depression based behavior. Future studies will be necessary to examine the mechanistic link between BDNF, gender differences, and the regional effect of BDNF in susceptibility to depression-related behavior.
Our findings suggest that loss of BDNF in forebrain contributes to some aspects of depression-like behavior in a complex manner with gender. The finding that BDNF deletion in males was not sufficient to produce alterations in many behaviors examined suggests that the neurotrophic hypothesis related to depression is more complicated than simply that loss of the gene triggers depression. The loss of forebrain BDNF increased vulnerability in aspects of depression-related behaviors in females after CUS suggesting a role for BDNF in mediating features of depression-related behavior in females.