Reproductive steroid receptors and actions in the locus coeruleus of male macaques: Part of an aggression circuit?

• NE neurons contain ERα and ERβ, but not AR.
• Androgens increase tyrosine hydroxylase (TH) in dendrites of locus coeruleus (LC).
• Androgens increase dopamine-β-hydroxylase (DBH) axon density in the dorsal raphe.
• Metabolism of T to estrogen (E) increases CRF axon density in the LC.
• Androgens increase androgen receptor (AR) expression in the LC, but not ERα or ERβ.

This study was initiated to determine whether the noradrenergic (NE) neurons of the locus coeruleus (LC) could mediate the stimulatory action of androgens on serotonin-related gene expression in male macaques. These experiments follow our observations that serotonin neurons lack androgen receptors (ARs), and yet respond to androgens. Male Japanese macaques (Macaca fuscata) were castrated for 5–7 months and then treated for 3 months with [1] placebo, [2] T (testosterone), [3] DHT (dihydrotestosterone; non-aromatizable androgen) plus ATD (steroidal aromatase inhibitor), or [4] FLUT (Flutamide; androgen antagonist) plus ATD (n = 5/group). The noradrenergic (NE) innervation of the raphe was determined with immunolabeling of axons with an antibody to dopamine-β-hydroxylase (DBH). Immunolabeling of tyrosine hydroxylase (TH) dendrites and corticotropin releasing hormone (CRH) axons innervating the LC was also determined. Due to the longer treatment period employed, the expression of the cognate nuclear receptors was sought. Androgen receptor (AR), estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) immunostaining was accomplished. Quantitative image analysis was applied and immunopositive neurons or axons with boutons were measured. Double-label of NE neurons for each receptor plus TH determined whether the receptors were localized in NE neurons. Androgens with or without aromatase activity significantly stimulated DBH axon density in the raphe (ANOVA, p = 0.006), and LC dendritic TH (ANOVA, p < 0.0001), similar to serotonin-related mRNA expression in the raphe. There were significantly more AR-positive neurons in T- and DHT + ATD-treated groups compared to placebo or FLUT + ATD-treated groups (ANOVA, p = 0.0014). There was no difference in the number of positive-neurons stained for ERα or ERβ. The CRH axon density in the LC was significantly reduced with aromatase inhibition, suggesting that CRH depends on estrogen, not androgens (ANOVA, p = 0.0023). Double-immunohistochemistry revealed that NE neurons did not contain AR. Rather, AR-positive nuclei were found in neighboring cells that are likely neurons. However, > 80% of LC NE neurons contained ERα or ERβ. In conclusion, the LC NE neurons may transduce the stimulatory effect of androgens on serotonin-related gene expression. Since LC NE neurons lack AR, the androgenic stimulation of dendritic TH and axonal DBH may be indirectly mediated by other neurons. Estrogen, either from metabolism of T or from de novo synthesis, appears necessary for robust CRH innervation of the LC, which differs from female macaques.