Investigating the mechanisms driving circadian and sex differences in the stress axis

Abstract

The stress response allows the body to adapt to an ever-changing environment. Stress results in the activation of corticotrophin releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of the hypothalamus. These neurons regulate the hypothalamic-pituitary-adrenal (HPA) axis which in turn controls the adrenal secretion of corticosteroids in response to stress. Previous research has shown a robust circadian rhythm in the secretion of corticosteroids from the adrenal gland with a peak occurring at the onset of the active period (Zeitgeber time (ZT) 12 in rodents). It has also been shown that females have a higher corticosteroid level across a 24-hour period compared with males. However, there has been very few studies investigating whether CRH peptide level and CRH neuronal excitability follows a circadian rhythm and if there are sex differences. This study aimed to investigate the mechanisms driving circadian and sex differences in the HPA axis by measuring CRH peptide level and CRH neuronal excitability across a 24-hour period in male and female mice. We used western blot to measure the CRH peptide level in the median eminence at four time points (ZT0, ZT6, ZT12 and ZT18) across a 24-hour period. Additionally, we measured CRH neuronal excitability at two time points (ZT0 and ZT12) using in vitro calcium imaging. We observed a statistically significant difference in the plasma corticosterone level across a 24-hour period with a peak occurring at ZT 12 (Two-way repeated measures ANOVA, F(3,29)=13.50, P<0.0001). The serum corticosterone level was significantly higher in females compared with males at ZT12 (Two-way repeated measures ANOVA with Sidak’s multiple comparisons test, males 47.53±5.83 ng/mL vs 93.59±18.61 ng/mL, F(1,29)=4.886, P=0.0351). However, we found no statistically significant differences in the CRH peptide level in the median eminence between the different time points (Mixed effects analysis, F(3,21)=0.3460, P=0.7924). At each time point, there were no statistically significant differences in the CRH peptide level between female and male mice (Mixed effects analysis, F(1,8)=0.5536, P=0.4781). Our calcium imaging experiments also revealed no statistically significant differences in PVN CRH neuronal excitability between different time points or between males and females (Mixed effects analysis, sex factor F(1,13)=0.01578, P=0.9020, time factor F(1,13)=2.081, P=0.1728). The results indicate that the CRH peptide levels in the median eminence and in vitro CRH neuronal excitability are constant throughout the day. These data suggest that the circadian and sex differences in corticosterone secretion are likely mediated by differences in other parts of the HPA axis.