Androgen regulation of corticotropin-releasing hormone receptor 2 (CRHR2) mRNA expression and receptor binding in the rat brain

Abstract

Stress-induced affective disorders, such as depression and anxiety, are more prevalent in females than in males. The reduced vulnerability to these disorders in males may be due to the presence of androgens, which are known to dampen the stress response and reduce anxiety-like behaviors. However, a neurobiological mechanism for this sex difference has yet to be elucidated. Corticotropin-releasing hormone receptor 2 (CRHR2) has been implicated in regulating anxiety-type behaviors and is expressed in stress-responsive brain regions that also contain androgen receptors (AR). We hypothesized that androgen may exert its effects through actions on CRHR2 and we therefore examined the regulation of CRHR2 mRNA and receptor binding in the male rat forebrain following androgen administration. Young adult male Sprague/Dawley rats were gonadectomized (GDX) and treated with the non-aromatizable androgen, dihydrotestosterone propionate (DHTP) using hormone filled Silastic capsules. Control animals received empty capsules. Using quantitative real-time RT-PCR, CRHR2 mRNA levels were determined in block-dissected brain regions. DHTP treatment significantly increased CRHR2 mRNA expression in the hippocampus, hypothalamus, and lateral septum (p<0.01) when compared to vehicle-treated controls. A similar trend was observed in amygdala (p= 0.05). Furthermore, in vitro autoradiography revealed significantly higher CRHR2 binding in the lateral septum in androgen-treated males, with the highest difference observed in the ventral lateral region. Regulation of CRHR2 mRNA by AR was also examined using an in vitro approach. Hippocampal neurons, which contain high levels of AR, were harvested from E17-18 rat fetuses, and maintained in primary culture for 14 days. Neurons were then treated with dihydrotestosterone (DHT; 1 nM), DHT plus flutamide (an androgen receptor antagonist), or vehicle for 48 h. CRHR2 mRNA levels were measured using quantitative real-time RT-PCR. Consistent with in vivo studies, DHT significantly increased CRHR2 mRNA expression in hippocampal neurons (p<.02) compared to vehicle-treated controls. Flutamide treatment prevented the effect of DHT on CRHR2 mRNA indicating that DHT's effect on CRHR2 expression is AR-mediated. Thus, the CRHR2 gene appears to be a target for regulation by AR and these data suggest a potential mechanism by which androgen may alter mood and anxiety-related behaviors.

Fig. 1

Androgen treatment of gonadectomized male rats increases levels of CRHR2 mRNA in block dissected brain regions. CRHR2 mRNA levels are shown in lateral septum (A), hypothalamus (B), and hippocampus (C) as measured by real-time RTPCR. * indicates significant differences (p < 0.01) versus control group (n=6–8). Each bar represents the mean +/− SEM. DHTP, dihydrotestosterone propionate.

Fig. 2

(A) Photomicrographs of representative autoradiograms showing in vitro I¹²⁵-suavagine binding. Outlined areas were examined for quantitation by densitometry. (B) Androgen treatment of gonadectomized males significantly increased ¹²⁵I-sauvagine binding in the lateral septum. (C) Androgen treatment of gonadectomized males significantly increased ¹²⁵I-sauvagine binding within the caudal dorsal and ventral caudal subregions of the lateral septum. * indicates significant differences (p < 0.05) versus control group. Each bar represents the mean +/− SEM (lateral septum, n=8; hippocampus, n=3; VMH, n=5 per treatment group). LS, lateral septum; Hippo, hippocampus; VMH, ventral medial hypothalamus; DHTP, dihydrotestosterone propionate; cc, corpus callosum; cd, caudal dorsal; cv, caudal ventral; vc, ventral caudal; MS, medial septum; ac, anterior commissure; 3V, third ventricle; Arc, arcuate nucleus.

Fig. 3

Androgen treatment of primary hippocampal neurons in culture increases CRHR2 mRNA. Concomitant treatment of DHT with flutamide blocked the effects of DHT. CRHR2 mRNA levels were measured by real-time RTPCR. * indicates those groups that were significantly different from control (p < 0.001, n=10). Each bar represents the mean +/− SEM. DHT, dihydrotestosterone; flu, flutamide.