Sex differences in activated corticotropin-releasing factor neurons within stress-related neurocircuitry and hypothalamic-pituitary-adrenocortical axis hormones following restraint in rats

Abstract

Women may be more vulnerable to certain stress-related psychiatric illnesses than men due to differences in hypothalamic-pituitary-adrenocortical (HPA) axis function. To investigate potential sex differences in forebrain regions associated with HPA axis activation in rats, these experiments utilized acute exposure to a psychological stressor. Male and female rats in various stages of the estrous cycle were exposed to 30min of restraint, producing a robust HPA axis hormonal response in all animals, the magnitude of which was significantly higher in female rats. Although both male and female animals displayed equivalent c-fos expression in many brain regions known to be involved in the detection of threatening stimuli, three regions had significantly higher expression in females: the paraventricular nucleus of the hypothalamus (PVN), the anteroventral division of the bed nucleus of the stria terminalis (BSTav), and the medial preoptic area (MPOA). Dual fluorescence in situ hybridization analysis of neurons containing c-fos and corticotropin-releasing factor (CRF) mRNA in these regions revealed significantly more c-fos and CRF single-labeled neurons, as well as significantly more double-labeled neurons in females. Surprisingly, there was no effect of the estrous cycle on any measure analyzed, and an additional experiment revealed no demonstrable effect of estradiol replacement following ovariectomy on HPA axis hormone induction following stress. Taken together, these data suggest sex differences in HPA axis activation in response to perceived threat may be influenced by specific populations of CRF neurons in key stress-related brain regions, the BSTav, MPOA, and PVN, which may be independent of circulating sex steroids.

Figure 1

HPA axis hormone concentrations immediately prior to (Before Stress) and following (After Stress) 30 min of restraint stress in Experiment 1. All values represent group means ± 1 SEM. (A,B) Plasma concentrations of ACTH (A) and CORT (B) in intact male and female rats. * P < 0.05 compared to males at the same timepoint. (C,D) Plasma concentrations of ACTH (C) and CORT (D) in females across three stages of the estrous cycle. *P < 0.05 compared to stress condition within the same estrous cycle stage.

Figure 2

All values represent group mean ± 1 SEM. A: Plasma estradiol concentrations on the day of stress in Experiment 1. *P < 0.05 compared to males. #P < 0.05 compared to DI. B,C,D: Relative c-fos mRNA expression immediately after stress in Experiment 1 within the BSTav (B), the MPOA (C), and the PVN (D). *p < 0.05 for all females compared to males. Abbreviations: BSTav – Bed Nucleau of the Stria Terminalis; MPOA – Medial Preoptic Area; PVN – Paraventricular Nucleus of the Hypothalamus; DI – diestrus; PRO – proestrus; EST – estrus.

Figure 3

Representative photomicrographs displaying the effect of sex on c-fos mRNA expression of female (A,B,C) and male (D,E,F) brains; within the anteroventral region of the bed nucleus of the stria terminalis (BSTav; A,D), the medial preoptic area (MPOA; B,E), and the paraventricular nucleus of the hypothalamus (PVN; C,F).

Figure 4

Representative photomicrographs of c-fos and CRF mRNA labeled neurons using dual FISH in the BSTav (A,D,G), MPOA (B,E,H) and PVN (C,F,I). A,B,C: Representative bregma images with red boxes showing the approximate location of photomicrographs in D–I. D,E,F: Magnified images from a female brain; Scale bar in D = 50μm. G,H,I: Images of a representative male brain at same magnification as in D,E,F. Blue=DAPI stain for nuclei; Red=CRF mRNA; Green=c-fos mRNA; Yellow= overlap of CRF and c-fos mRNA.

Figure 5

The effect of sex on CRF and c-fos mRNA containing cells as visualized by FISH in Experiment 1, and colocalization of both genes in the BSTav (A), the MPOA (B), and the PVN (C) after 30 min of restraint. * = p < 0.05 compared to males.

Figure 6

Experiment 2: Plasma hormone concentrations (means ± 1SEM) from intact males (n = 10) and females (n = 12) given sham surgery, and ovariectomized females with (OVX + E2; n = 10) or without (OVX; n = 10) estradiol replacement at baseline (Basal) and after 30 min of restraint (Stress). * = p < 0.05 compared to all other groups within the same stress condition. # = p < 0.05 compared to intact males and OVX females within that stress condition.