Sex matters: females in proestrus show greater diazepam anxiolysis and brain‐derived neurotrophin factor‐ and parvalbumin‐positive neurons than males

In humans and animal models, sex differences are reported for anxiety‐like behavior and response to anxiogenic stimuli. In the current work, we studied anxiety‐like behavior and response to the prototypical anti‐anxiety drug, diazepam. We used 6th generation outbred lines of adult Long Evans rats with high and low anxiety‐like behavior phenotypes to investigate the impact of proestrus on the baseline and diazepam‐induced behavior. At three doses of diazepam (0, 0.1, and 1.0 mg/kg, i.p.), we measured anxiogenic responses on the elevated plus maze of adult male and female rats. We assessed parvalbumin and brain‐derived neurotrophin protein levels in forebrain and limbic structures implicated in anxiety/stress using immunohistochemistry. At baseline, we saw significant differences between anxiety lines, with high anxiety lines displaying less time on the open arms of the elevated plus maze, and less open arm entries, regardless of sex. During proestrus, high anxiety females showed less anxiety‐like behavior at 0.1 mg/kg, while low anxiety females displayed less anxiety‐like behavior at 0.1 and 1.0 doses, relative to males. Brain‐derived neurotrophin protein was elevated in females in the medial prefrontal cortex and central amygdala, while parvalbumin‐immunoreactive cells were greater in males in the medial prefrontal cortex. Parvalbumin‐positive cells in high anxiety females were higher in CA2 and dentate gyrus relative to males from the same line. In sum, when tested in proestrus, females showed greater anxiolytic effects of diazepam relative to males, and this correlated with increases in neurotrophin and parvalbumin neuron density in corticolimbic structures. Using outbred lines of animals phenotyped as showing high (HAn) and low anxiety (LAn)‐like behavior, we confirmed HAn F6 offspring were less active on the open arms of the elevated plus maze (EPM) at baseline. HAn and LAn females were more active on the open arms of the EPM after diazepam treatment. Using a graphic user interphase, we counted a greater number of positively stained parvalbumin neurons (i.e., that stain a subset of GABA neurons) in the mPFC.