The main finding of the current study is that subchronic treatment with fluoxetine prevented stress-induced changes in LDE test behavior in male C57BL/6J mice. A similar subchronic treatment regimen with the mood stabilizer, lithium, had similar, although somewhat less clear-cut, effects. By contrast, acute pre-test treatment with the antipsychotic haloperidol, the anti-ADHD drug methylphenidate, or the benzodiazepine anxiolytic chlordiazepoxide, failed to reverse the behavioral effects of stress.
The principal behavioral readout of the ten-day restraint stress procedure we employed was a significant increase in exploration (time spent in and/or entries) of the aversive light compartment of the LDE test. This profile was consistently seen across six separate experiments in the current study and replicates one of the main findings of Mozhui et al. (Mozhui et al., 2010
). Mozhui et al. also reported that this stress regimen increased time spent in the aversive open arms of the elevated plus-maze (EPM) in C57BL/6J mice, but did not affect immobility behavior in the FST (as also replicated here). Collectively, this pattern of data suggests that restraint stress produced behavioral alterations that generalize across anxiety-related but not ‘depression-related’ tests. A dissociation between anxiety- and depression-related measures is not unexpected considering examples in the literature where other repeated stressors, such as social defeat, produce assay- or domain-specific behavioral effects in C57BL/6J mice and other strains (e.g., Krishnan et al., 2007
; Strekalova et al., 2004
It can sometimes be difficult to parse a change in anxiety-related behavior from a change in locomotor activity in exploration-based assays for anxiety-like behavior, including the LDE test because more or less exploration of the (larger) light compartment typically drives up the overall amount of distance traveled in tandem (Holmes, 2001
). However, a number of observations argue that the stress-induced profile of C57BL/6J mice is unlikely to be a generalized hyperactivity effect. First, stress failed to alter measures of locomotor activity in the open field (this study) or the elevated plus-maze (Mozhui et al., 2010
). Second, a generalized hyperactivity effect of stress would have been expected to decrease forced swim test immobility, but this was not found either in the current study or in Mozhui et al. (Mozhui et al., 2010
). Third, of the four experiments where total distance traveled in the LDE could be measured via videotracking, there an effect of stress on this measure only in one experiment (lithium). Given the increase in distance traveled in the lithium experiment, and despite the arguments to the contrary above, the possibility that stress-induced increase in light compartment exploration in this experiment was confounded by hyperactivity cannot be discounted.
Another interpretation of the current results is that the stress-induced increase in the aversive light compartment of the LDE test reflect a decrease in anxiety-like behavior. This would be logical given a similar profile is produced by anxiolytic drugs in non-stressed subjects (Crawley, 1981
; Cryan and Holmes, 2005
), but paradoxical in the sense that stress is a risk factor for anxiety. However, the stress-induced profile of C57BL/6J mice cannot simply be explained by stress-resilience in this strain because stressed mice reliably showed a significant loss of body weight - a measure typically used to confirm the efficacy of restraint stress in rats and mice (Surget et al., 2009
; Willner, 1997
). Furthermore, stressed C57BL/6J mice showed significantly elevated serum corticosterone response to a novel (swim) stressor, demonstrating that the HPA-axis had been sensitized by restraint. Similar HPA-axis sensitization is seen with other repeated stressors in C57BL/6J mice (e.g., Krishnan et al., 2007
). Thus, increased LDE light compartment exploration occurred in the context of peripheral and neuroendocrine alterations classically associated with increased stress. We therefore suggest that C57BL/6J mice respond to restraint stress with an ‘active coping strategy’ that contrasts with inhibited behavioral responses exhibited by other strains such as DBA/2J and BALB/cJ (Mozhui et al., 2010
; Uchida et al., 2011
). Clearly, further studies will be needed to elucidate the precise nature of this ‘paradoxical’ response to stress in the C57BL/6J strain.
The major finding of the current study, which was that stress-induced LDE behavior and corticosterone sensitization (although not body weight loss) was ‘rescued’ by subchronic treatment with the clinically efficacious anxiolytic and antidepressant fluoxetine, may also be in line with an increase, rather than resilience, to stress. Subchronic treatment with the prototypical mood stabilizer lithium chloride also partially reversed stress-induced alterations in the LDE test, although this effect was less robust than that produced fluoxetine. Because both drugs were administered during stress exposure as well as LDE testing, it is unclear whether they prophylactically prevented a stress effect from developing, or blocked the expression of the behavioral effect. It is difficult to design an experiment to parse these possibilities because pre-test subchronic administration of fluoxetine is needed to mimic their efficacy in the clinic, and it is unclear whether the effect of stress in the LDE would be robustly manifest if a two-week treatment interval was interposed between stress and testing. Notwithstanding, the main conclusion from these data is stress-induced LDE behavioral is reversible by these clinically relevant drugs.
A stress-preventing effect of fluoxetine on LDE behavior in C57BL/6J extends similar findings examining other monoaminergic antidepressants, mouse strains and stress paradigms. For example, imipramine treatment five weeks into a nine-week chronic unpredictable mild stress (UCMS) regimen prevented anxiety-related and HPA-axis alterations in a number of strains including BALB/c and C57BL/6 (Ibarguen-Vargas et al., 2008
). More recently, Isingrini and colleagues reported that BALB/c mice exposed to UCMS for seven weeks showed reduced grooming, coat degradation and anhedonia that was blocked by fluoxetine treatment from the second week onward (Isingrini et al., 2010
) (see also Yalcin et al., 2008
). Similarly, fluoxetine concomitantly administered with low dose corticosterone over four weeks blocked corticosterone-induced coat degradation and anxiety-like behavior in C57BL/6NTac mice (David et al., 2009
). Taken together with the current findings, these data demonstrate that fluoxetine is effective in preventing effects of various chronic stressors.
In contrast to the effectiveness of fluoxetine and lithium, acute treatment with the antipsychotic haloperidol, the anti-ADHD medication methylphenidate, and the benzodiazepine chlordiazepoxide all failed to normalize stress-induced LDE behavior. Methylphenidate reduced light compartment time regardless of stress, while haloperidol and chlordiazepoxide were inactive in both stressed and non-stressed mice. The haloperidol dose used is known to be effective in other behavioral settings (Karlsson et al., 2008b
). The null chlordiazepoxide profile was also likely a true negative, as we showed in a series of additional experiments that, in non-stressed C57BL/6J mice, chlordiazepoxide was without effect in the EPM, novel open field and another variant of the LDE test. Moreover, a previous study also found no anxiolytic-like effects in these tests in C57BL/6JOIaHsd mice (Rodgers et al., 2002
). Thus, our negative data with these three drugs indicate that the stress-rescuing effects of fluoxetine (and to a lesser extent, lithium) are not reproduced by an antipsychotic, anti-ADHD or benzodiazepine anxiolytic medication. An important caveat in making this comparison across drugs is that fluoxetine and lithium were administered chronically during stress and LDE testing, whereas haloperidol, methylphenidate and chlordiazepoxide were given acutely prior to testing. Thus, whether chronic treatment with these drugs would rescue the effect of stress remains to be tested.
Another issue to be resolved stems from our observation that chronic fluoxetine treatment produced a general decrease in LDE behavior and increased FST immobility in non-stressed mice, and to a lesser degree, stressed mice. This was unexpected given similar regimens exert therapeutic efficacy in human patients, and prior studies demonstrate that from three injections to four weeks of treatment produces anxiolytic-like and antidepressant-related effects (Dulawa et al., 2004
; Holick et al., 2008
; Isingrini et al., 2010
; Karlsson et al., 2008a
; Mombereau et al., 2010
; Oh et al., 2009
; Richardson-Jones et al., 2010
). The apparent inconsistency between these studies and our current data may be explained by genetic background. Chronic treatments typically produce anxiolytic-like and antidepressant-related effects in BALB/cJ or ′129′ strains, or mixed genetic backgrounds, whereas, similar to our finding, C57BL/6J are often either unresponsive or found to exhibit non-specific sedation (Balu et al., 2009
; David et al., 2009
; Dulawa et al., 2004
) (but see Chen et al., 2006
; Oh et al., 2009
). These data suggest an atypical behavioral response to prolonged fluoxetine treatment in the C57BL/6J strain, and confound interpretation of stress-related effects of this treatment in the current study.
In summary, the current study found that repeated restraint stress produces behavioral and HPA-axis alterations that are prevented by subchronic fluoxetine treatment, and to some extent, subchronic lithium. Acute treatment with haloperidol methylphenidate or chlordiazepoxide prior to LDE testing failed to block the effect of stress. Together, these experiments demonstrate treatment-selective rescue of stress-induced behavior in C57BL/6J mice. The precise nature of this stress-induced phenotype and its potential relevance to specific human disease classes remains to be determined, but could prove useful for studying the genetic and neural mechanisms underlying individual differences in responses to stress. In this context, we and others have shown that various stressors have convergent effects on corticolimbic glutamatergic neurotransmission and expression of glutamate-related plasticity genes (Krishnan et al., 2007
; Mombereau et al., 2010
; Mozhui et al., 2010
; Surget et al., 2009
). Future work will seek to extend these findings by elucidating the role of the glutamate system in the current stress model.