The results showed that stress exposure led to the reduced OAT and OAE percentage as the markers of anxiety-like behavior. SPS affected entrance latency in fear conditioning and extinction. Also, stress significantly impacted prefrontal BDNF. However, the reduction in serum BDNF following SPS was not statistically significant. Fluoxetine lessened SPS-induced alterations in prefrontal BDNF, fear conditioning, and extinction. However, this drug had no significant effect on OAT and OAE. Exercise alone or in combination with fluoxetine restored the malevolent effects of SPS on anxiety-like behavior, prefrontal and serum BDNF, fear conditioning, and extinction.

The present study confirmed that SPS is able to induce anxiety-like behavior and other PTSD-associated alterations such as BDNF changes and fear conditioning and extinction impairment [27] in female rats. Comparison between present results with our previous findings have shown no sex difference in SPS-induced impairments in behavior, BDNF, fear conditioning, and extinction. Also, therapeutic interventions resulted in somewhat similar effects on both sexes. Some sex differences included greater effect of combination therapy than monotherapies on prefrontal BDNF in males and a greater efficacy of combination therapy than fluoxetine alone on 4th extinction in female rats. Also, unlike male rats, fluoxetine and exercise did not improve OAT in female rats. The findings of our present and past studies [26] have been summarized in the Table 1 to more easily compare male and female rats.

Effects of exercise, fluoxetine, and a combination of both on prefrontal and serum BDNF in female PTSD rats

The results showed that SPS led to the BDNF reduction in the prefrontal cortex in female rats. The findings were similar to that in male PTSD animals in our previous study [26]. BDNF changes after stress have been frequently reported in several studies [28,29,30,31] and our study confirms these findings. Since BDNF has been shown to have an essential role in mediating stress-related pathophysiological changes in the brain [32] these findings are expectable. Impairment in fear extinction is a PTSD marker and BDNF has been reported to be involved in fear memories in the amygdala, hippocampus, and prefrontal cortex [33]. Owing to high sensitivity of the prefrontal cortex to stress and its modulation role in many behavioral and physiological responses to stress [34], BDNF alterations in the prefrontal cortex after SPS is an important finding.

Comparison between male and female rats showed no sex difference in BDNF reduction after SPS. Although some studies such as that of Aykaç revealed that BDNF alteration after SPS has sex differences [28, 30], our studies showed similar results in prefrontal BDNF between male and female rats. This finding was partly in agreement with the results of a systematic review and meta-analysis study that showed no significant effect of sex on peripheral BDFN in PTSD patients [35].

In Aykaç and colleagues' study, no significant difference was found between males and females in cortical BDNF levels after the social isolation test, however, female rats had lower amygdala BDNF than males after the test [28]. Based on the mentioned study, it is probable that the difference in findings between males and females originated from the region of the brain collected for BDNF measurement. In other words, stress-induced BDNF alteration in the distinct brain parts differs in a sex-dependent manner [28]. Since the prefrontal region has been shown to be one of the involved parts in PTSD, the present study assessed prefrontal BDNF and similar results were found in males and females.

Fluoxetine and exercise individually or in combination were able to restore prefrontal BDNF in female animals. These findings were similar to those in male animals [26]. However, there were some sex differences between males and females in the therapeutic potency of fluoxetine alone, exercise alone, and the combination of both. In female PTSD rats, there was no difference between the effect of monotherapies and combination therapy on prefrontal BDNF while in male PTSD rats, combination therapy was more potent than monotherapies.

It has been shown that both the antidepressant drug fluoxetine and exercise are able to increase BDNF expression in the brain exposed to stress [26, 28, 36], and our results are consistent with these findings. In this regard, exercise induces BDNF enhancement during extinction consolidation and, consequently, reduces threat expectancies following reinstatement in PTSD [37]. Also, it is asserted that the likely role of drugs such as antidepressants on fear extinction improvement is modulated through the BDNF-TrkB system [33]. Taken together, in studies designed for PTSD treatment with antidepressants and exercise, BDNF modulation is considered as one of the involved mechanisms.

Although sex differences in the therapeutic effects of fluoxetine or exercise on BDNF have been reported in some studies [28, 38, 39], in our studies, fluoxetine or exercise led to similar therapeutic outcomes in both sexes. The only sex difference in prefrontal BDNF was that the combination of fluoxetine and exercise was more potent than monotherapies in male but not in female rats. Interestingly, the combination of fluoxetine and exercise was able to enhance prefrontal BDNF to a higher level than the normal range in male SPS rats (data was not shown). In Aykaç and colleagues' study, the fluoxetine effects on BDNF were different between the two groups depending on the assessed region of the brain. For example, fluoxetine was able to restore BDNF in the female’s hippocampus but not in the male’s. Similar results have been reported by Mitic and colleagues [38]. Accordingly, it has been asserted that fluoxetine effects on stressed subjects are also in a brain region-specific manner [40]. Therefore, it is likely that inconsistency in the results of BDNF in male and female subjects is due to differences in the assessed regions of the brain.

In the present study, unlike the prefrontal BDNF, serum BDNF did not show a statistically significant difference between SPS and non-SPS rats. Due to difficulty in crossing the blood–brain barrier [41], peripheral BDNF may not be an accurate biomarker to reflect slight alterations in brain BDNF [42]. However, according to the results of a meta-analysis study [35], it was probable that measuring plasma BDNF instead of serum BDNF would produce more conclusive results.

Effects of exercise, fluoxetine, and a combination of both on fear extinction in female PTSD rats

Deficient fear extinction is considered as one of the hallmarks of PTSD symptoms and the present study showed that SPS impaired the extinction of conditioned fear in female rats. Entrance latency decreased in all intervention groups, including fluoxetine alone, exercise alone, and fluoxetine plus exercise over time. These results suggest that both monotherapy and combination therapy were able to facilitate fear memory extinction in female SPS rats. Comparison between male and female SPS rats indicated somewhat similar results in terms of the effectiveness of the interventions. However, it is mentionable that in female SPS rats, unlike in males, combination therapy showed greater efficacy in reducing entrance latency on the 4th day than fluoxetine alone.

Although some studies showed that stress-exposed female rats have more disrupted performance in the fear extinction learning [5, 29], our study did not reveal a distinct difference between males and females in the impact of interventions, except greater efficacy of combination therapy than fluoxetine alone in female rats.

The present study showed that the interventions were able to enhance BDNF in the prefrontal regions in both male and female SPS rats. Besides, they improved fear extinction over time in both sexes. These findings support the documents reporting that the positive effect of interventions such as exercise on fear extinction learning is mediated by increasing BDNF [37, 43, 44]. Since BDNF plays a role in the fear extinction enhancement, targeting the impaired extinction in anxiety disorders such as PTSD via BDNF signaling may be an important and novel way to enhance treatment efficacy [33].

There was a slight sex difference in terms of the therapeutic effect of fluoxetine on the 4th extinction, but not the extinction index. The results in female SPS rats showed that the effect of fluoxetine was satisfying but it was less than that of combination therapy. Previous studies also reported that fluoxetine resulted in a different effect between male and female groups [9, 10].

Effects of exercise, fluoxetine, and a combination of both on anxiety-like behaviors in female PTSD rats

PTSD is an anxiety disorder and SPS is known as an valid rodent model of it [45]. The results of EPM tests showed that SPS was able to induce anxiety-like behavior in female rats. SPS-exposed rats showed fewer entries into the open arms and lower percentage of open arm time in comparison to non-SPS rats.

Fluoxetine alone could not show a significant therapeutic effect on anxiety-like behaviors assessed by EPM in female rats. However, exercise was able to improve the percentage of open arm entries but had no significant effect on the percentage of open arm time in female SPS rats. Combination therapy was more potent than monotherapies and improved both parameters including OAT and OAE in the female rats as it did in male rats. Comparison of the present results with our findings of the previous study showed that fluoxetine alone and especially exercise alone led to better effects in male rats than that in females. These results can be supported to some extent by Whitworth and colleagues’ study that showed active men have significantly lower PTSD symptoms than active women [6]. Morgan and colleagues’ study showed exercise-induced anxiety behaviors in young female mice [15]. Regarding the role of gonadal hormones on the stress-related pathways such as the prefrontal cortex–amygdala pathway [46], it was not surprising to see some different findings between males and females. Although the present study has shown many similarities between male and female rats regarding the effects of treatments, some inconsistent findings support the opinion of the role of sex and hormones on stress management and the need for individualized treatments considering sex effects [7, 15, 47, 48].

Fluoxetine alone was not able to induce statistically significant improvement in anxiety behavior in female rats, however, it resulted in a subtle increase in OAE and OAT. Therefore, it may show a synergic effect if co-administrated with other treatments, as this synergic effect was revealed in combining fluoxetine with exercise. We can claim the same for exercise alone that slightly increased OAE. Potentiation of the therapeutic effect of fluoxetine in combination with exercise was also reported in Gobinath and colleagues' study on postpartum depression in female rats. Their study showed that fluoxetine augmented neurogenesis only when combined with exercise [13]. Taken together, in some parameters, the greater therapeutic effects were shown in male SPS rats than in female rats. These findings can partly assert sex-specific psychopharmacology in psychological disorders. While there were some sex differences in response to fluoxetine alone or exercise alone, combination therapy was able to restore all parameters affected by PTSD in both sexes.

It is worth mentioning that although some sex differences in the effect of interventions on stress-exposed subjects [6, 28] have been reported, the accurate conclusion needs to compare intervention-induced changes with the normal scores in males and females separately. It is probable that the baseline score is different in a sex-specific manner and consequently leads to different final results between males and females. For example, in our study OAT and OAE percentage in sham groups, as the baseline scores, were different between males and females (data was not shown). Regarding these different findings in the present study, the changes after SPS induction and following intervention have been compared with the base scores in each sex separately.