Sex differences in neurotransmission parameters in response to repeated mild restraint stress exposures in intact male, female and ovariectomised female rats

The present study determined whether a repeated mild restraint stress exposure would differentially alter neuronal activity in male and female rats to gain insights into neurobiological substrates involved in sex differences in stress-induced behavioural responses. In our first set of experiments, we used Western blot analysis to determine whether alterations in several synaptic proteins were elicited by the repeated stress treatment. We found bidirectional changes in synaptophysin levels in female cerebral cortex and hippocampus that diverged between intact and ovariectomised females. There were persistent elevations in spinophilin levels in the male, but not female, hippocampus following the repeated mild restraint stress exposure. By contrast, levels of the NMDA receptor scaffolding protein, PSD-95, were altered only in intact female cerebral cortex and ovariectomised female hippocampus. We next used immunohistochemical evaluation of Fos expression as a marker for neuronal activation. We found significant increases in Fos immunoreactivity in all sex conditions across multiple brain regions in response to the repeated mild stress. Fos protein induction was greatest in the frontal cortex, piriform cortex and amygdala, with the degree of induction varying by sex condition. Fos induction was dramatically higher in amygdala and piriform cortex only in intact females following repeated stress compared to a single restraint stress exposure, suggestive of sensitisation rather than habituation. By contrast, the frontal cortex of intact and ovariectomised females showed habituation to the repeated stressor. Males displayed modest sensitisation in both the frontal cortex and dentate gyrus with no changes in other brain areas. Taken together, these findings show that exposure to a mild repeated stress results in sex differences in synaptic adaptations and patterns of brain activation that likely contribute to observed sex differences in stress-induced behaviours. This approach provides valuable insights into interactions between the hormonal milieu and responses to a repeated mild stress, and further supports the importance of considering hormonal status in treatment of stress-related disorders.