Anxiety disorders affect more women than men. Because gonadal hormones like testosterone (T) play a key role in establishing many sex differences, T may underlie the sex differences in human anxiety. Indeed, androgens act as anxiolytics in humans and other species. In mice, this effect normally requires functional androgen receptors (AR) since T has no such anxiolytic effects in genetic males carrying a dysfunctional allele for AR. Cre-LoxP technology was used to recombine AR, producing a dysfunctional allele that resulted in androgen insensitivity in XY mice, a phenotype referred to as induced testicular feminization mutation (iTfm). These iTfm males show more anxiety related behavior than their wild type (WT) brothers in several tests, including the novel object test and elevated plus maze. When the mice were tested during the animals' resting phase (lights on period), iTfms did not differ from WT males in the light/dark box (L/D) test, replicating earlier results in our lab. However, when tested in the active phase, iTfm males appeared more anxious in the light-dark box than their WT brothers, consonant with the differences between these mice in other tests of anxiety. WT males castrated as adults and treated with T have a reduced HPA response to mild stress, as measured by corticosterone release, than untreated WT castrates. In contrast, iTfm males given T displayed a greater and more prolonged HPA response than WT males, indicating that AR activation normally attenuates the HPA axis. To determine whether ARs affect anxiety-related behaviors and HPA response by acting within the central nervous system (CNS), mice carrying a transgene utilizing the nestin promoter to drive expression of Cre recombinase were crossed with mice carrying a floxed allele of AR. Offspring carrying both constructs were designated NesARKO. Contradicting expectations, the NesARKO mice displayed only a partial knockout (KO) of AR expression in the brain: while there was a full KO in the hippocampus, medial prefrontal cortex (mPFC), bed nucleus of the stria terminalis (BNST), and periaqueductal grey (PAG), AR immunoreactivity was still seen in many cells in the amygdala and hypothalamus. No differences in anxiety-related behaviors or HPA function were seen between NesARKOs and WT males, demonstrating that full KO of AR in the mPFC and hippocampus had no effect on these behaviors. Consequently, those regions cannot be sites at which AR acts to modulate these behaviors in WT males. A subsequent study monitoring neuronal activity as reflected in cFos expression indicated that WT males with T have more cells respond in the basolateral amygdala (blAMY) and fewer cells respond in the suprachiasmatic nucleus of the hypothalamus (SCN) than either iTfms with T, or WTs without T. In WT males treated with T, approximately 65% of cells in the blAMY and SCN are AR positive, therefore AR may act directly on these cells to affect their response to mild stress. Together, these results demonstrate that T requires functional AR to modulate anxiety-related behavior and HPA function in mice, but does not act in the mPFC, hippocampus, BNST, or PAG to do so. T also acts through AR to affect the response of cells in the blAMY and SCN following mild stress, so these brain regions remain potential sites of action for T's anxiolytic effects in mice.
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