Androgen Regulation of Corticotropin Releasing Factor Receptor 1 in the Mouse Brain

Abstract

Stress-related mood disorders like anxiety and depression are more prevalent in women than men and are often associated with hypothalamic-pituitary-adrenal (HPA) axis dysregulation. Androgen actions through androgen receptors (ARs) decrease HPA axis responses and stress-associated behaviors. Corticotropin releasing factor (CRF) and its binding to CRF receptor 1 (CRFR1) is also critical for regulation of the HPA axis, anxiety, and depression. We first determined CRFR1/AR co-localization patterns in male and female CRFR1-GFP mice. High co-localization was found within the paraventricular nucleus (PVN), dorsolateral and anteroventral subdivisions of the bed nucleus of the stria terminalis (BSTdl and BSTav), medial preoptic area (MPOA), and posterodorsal medial amygdala (MePD). We next determined whether the non-aromatizable androgen dihydrotestosterone (DHT) regulates CRFR1 expression and stress-induced activation of CRFR1-expressing cells. In the PVN, CRFR1-GFP cell number decreased following gonadectomy (GDX), but DHT treatment reversed this effect. GDX-DHT treated mice also had a decreased CRFR1-GFP cell number within the BSTdl compared to gonad intact and GDX-untreated groups. Following restraint stress GDX-blank mice showed fewer c-Fos/CRFR1 co-localized neurons in the MePD compared to gonad intact and GDX-DHT groups indicating decreased stress-induced activation of CRFR1 neurons following GDX. Higher plasma corticosterone (CORT) was found in GDX males compared to GDX-DHT and sham males following restraint stress, with a negative correlation between PVN CRFR1+ neurons and corticosterone levels 30- and 90-min following restraint. Together these findings show androgens can directly alter CRFR1 levels in the brain which may have implications for sex differences in regulation of the HPA axis and stress-related behaviors.

Keywords: Stress; androgen; androgen receptor; corticotropin releasing factor; hypothalamic–pituitary–adrenal axis; sex difference.

Figure 1.. Anatomical locations within which CRFR1-GFP+ and co-labeled cells were quantified.

Plate numbers correspond to regions of interest with locations determined by the Allen Mouse Brain Reference Atlas (https://mouse.brain-map.org/static/atlas). Approximate ROI shapes for the BSTdl (A), BSTav (A), MPOA (B), PVN (C) and MePD (D). AC; anterior commissure, OT; optic tract, 3V; third ventricle.

Figure 2.. Co-localization of CRFR1-GFP and AR in regions of the male and female mouse brain.

Quantification of CRFR1-GFP/AR co-labeled neurons, the percentage of CRFR1-GFP neurons co-expressing AR, AR+, and CRFR1-GFP+ neurons are shown in the PVN (A-D), BSTdl (E-H), BSTav (I-L), MePD (M-P), and MPOA (Q-T). * Indicates statistical significance p≤0.05, ** p≤0.01 compared to other groups. (PVN data were published in part in a previous publication (Rosinger et al., 2019b), although the data presented reflect an increased N and re-analysis.)

Figure 3.. Images of CRFR1-GFP/AR co-labeling.

Representative images of CRFR1-GFP (brown), AR (black nuclear), and co-localized cells (brown with black nuclear label) are shown from the (A) PVN, (B) BSTdl, (C) BSTav, (D) MePD, and (E) MPOA of a male mouse. High magnification images of each region are shown directly below in F-J. Purple arrows indicate CRFR1-GFP only, red indicate AR only, and blue indicate examples of co-expressing neurons. 3V, 3^rd ventricle; AC, anterior commissure; OT, optic tract.

Figure 4.. Androgen regulation of corticosterone levels at baseline (0), and 30- and 90-minutes following restraint stress onset.

No differences were found at baseline (BSL), but GDX-Blank mice showed significantly elevated levels of corticosterone at 30 and 90 minutes after the onset of restraint stress. * Indicates p≤0.05, ** p≤0.001 for GDX-Blank compared to sham and GDX-DHT groups at the same timepoint. # Indicates p≤0.001 for sham compared to GDX-DHT mice at the 30-minute timepoint.

Figure 5.. Androgen regulation of CRFR1-GFP levels in the PVN, BSTdl, BSTav, MePD, and MPOA.

(A-D) In the PVN, GDX-Blank mice showed reduced CRFR1-GFP cell number compared to sham and GDX-DHT groups. (E-H) In the BSTdl, GDX-DHT mice showed a reduced number of CRFR1-GFP neurons relative to sham and GDX-Blank groups. No significant differences were found in the (I) BSTav, (J) MePD, or (K) MPOA. * Indicates p≤0.05, ** p≤0.01 compared to other groups. AC, anterior commissure.

Figure 6.. CRFR1-GFP/c-Fos co-localization in male mice following restraint stress.

(A-C) In the MePD, GDX-Blank mice showed fewer CRFR1-GFP/c-Fos co-localized cells compared to sham and GDX-DHT groups. No significant differences were found in the PVN (D-F), BSTav (G-I), BSTdl (J), or MPOA (K). CRFR1-GFP, green; c-Fos, magenta. * Indicates p≤0.05, ** p≤0.01 compared to other groups. OT, optic tract. Arrows indicate examples of co-labeled neurons.

Figure 7.. Correlations between corticosterone levels at 30- and 90-minutes after restraint onset and CRFR1-GFP and CRFR1/c-Fos levels in various brain regions.

Dark gray cells in the box (A) indicate significant positive or negative correlations (p≤ 0.05). Statistically significant correlations are also graphed in B-D.