(3α,5α)3-Hydroxypregnan-20-one (3α,5α-THP) Regulation of the HPA Axis in the Context of Different Stressors and Sex

Abstract

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3α,5α-THP regulation of stress-induced hypothalamic CRF, CRF receptor type 1 (CRFR1), CRF binding protein (CRFBP), pro-opiomelanocortin (POMC), and glucocorticoid receptor (GR) by western blot and circulating corticosterone (CORT) by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague Dawley rats. Tissue was collected after rats were injected with 3α,5α-THP (15 mg/kg, IP) or vehicle 15 min prior to 30 min of restraint stress (RS), or 10 min of forced swim stress (FSS) and 20 min recovery. The initial exposure to a stress stimulus increased circulating CORT levels in both males and females, but 3α,5α-THP attenuated the CORT response only in females after RS. 3α,5α-THP reduced GR levels in male and females, but differently between stressors. 3α,5α-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3α,5α-THP reduced the CRFR1, CRFBP, and POMC increases after RS and FSS in males, but in females only after FSS. Our results showed different stress responses following different types of stressors: 3α,5α-THP regulated the HPA axis at different levels, depending on sex.

Keywords: 3α,5α-THP; CRF; CRFR1; HPA axis; allopregnanolone; corticosterone; forced swim stress; hypothalamus; neuro-steroids; restraint stress.

Figure 1

3α,5α-THP partially reduced the restraint stress-induced corticosterone increased in female rats. As expected, both acute restraint stress and forced swim stress induced an increase in CORT circulating levels, in male and female rats (Males VEH NRS: 130.9 ± 15.92 ng/mL vs. VEH RS: 779.1 ± 81.8 ng/mL, stress effect p < 0.0001; males VEH NFSS: 143.2 ± 32.8 ng/mL vs. VEH FSS: 380.5 ± 23.2 ng/mL, stress effect p < 0.0001. Females VEH NRS: 571.3 ± 80.5 ng/mL vs. VEH RS: 1684 ± 128.1 ng/mL, stress effect p < 0.0001; females VEH NFSS: 119.14 ± 22.2 ng/mL, vs. VEH FSS: 382 ± 23 ng/mL, stress effect p < 0.0001). Surprisingly, 45 min after the injection, 3α,5α-THP failed to attenuate the CORT stress response, in male and female rats, following restraint or forced swim stress (Males 3α,5α-THP NRS: 79.04 ± 13.1 ng/mL vs. 3α,5α-THP RS: 762.8.1 ± 70.32 ng/mL, stress effect p < 0.0001; males VEH RS: 779.1 ± 81.8 ng/mL vs. 3α,5α-THP RS: 762.8.1 ± 70.32 ng/mL, n.s.; males 3α,5α-THP NFSS: 185.7 ± 13.6 ng/mL vs. 3α,5α-THP FSS: 423 ± 25.8 ng/mL, stress effect p < 0.0001; male VEH FSS: 380.5 ± 23.2 ng/mL vs. 3α,5α-THP FSS: 423 ± 25.8 ng/mL, n.s. Females 3α,5α-THP NRS: 516.9 ± 54.3 ng/mL vs. 3α,5α-THP RS: 1164.6 ± 118 ng/mL, p < 0.001; females VEH RS: 1684 ± 128.1 ng/mL vs. 3α,5α-THP RS: 1164.6 ± 118 ng/mL, p < 001; females 3α,5α-THP NFSS: 218.5 ± 38.9 ng/mL vs. 3α,5α-THP FSS: 478.5 ± 27 ng/mL, stress effect p < 0.0001; females VEH FSS: 382 ± 23 ng/mL vs. 3α,5α-THP FSS: 478.5 ± 27 ng/mL, n.s.). Significant effects were found using Two-way ANOVA ^#### p < 0.0001, or following Tukey HSD test **** p < 0.0001, *** p < 0.001, ** p < 0.01. Data are represented as mean ± SEM. Abbreviations: VEH = rats treated with vehicle; 3α,5α-THP = rats treated with 3α,5α-THP; NRS = non-restraint stress; RS = restraint stress; NFSS = non-forced swim stress; FSS = forced swim stress.

Figure 2

3α,5α-THP reduced restraint stress-induced hypothalamic GRs increase in male rats. Restraint stress, but not forced swim stress, induced an increase in hypothalamic GR expression, in both male and female rats (Males VEH RS: 129 ± 5.8% vs. VEH NRS, p < 0.05; Males VEH FSS: 93 ± 1.9% vs. VEH NFSS, n.s.. Females VEH RS: 41 ± 6.2% vs. VEH NRS, stress effect p < 0.0001; Females VEH FSS: 99.9 ± 5.2 vs. VEH NFSS, n.s.). 3α,5α-THP attenuated the restraint stress-induced enhancement in hypothalamic GR levels in both male and female rats (Males 3α,5α-THP NRS: 64 ± 5.5% vs. VEH NRS, p < 0.01; Males 3α,5α-THP RS: 63 ± 7% vs. VEH RS, p < 0.0001. Females 3α,5α-THP NRS: 87 ± 2.8% vs. VEH NRS, Females 3α,5α-THP RS: 114 ± 9.5% vs. VEH RS, treatment p < 0.05). 3α,5α-THP treatment resulted in a decrease in hypothalamic baseline GR levels in both males and females (Males 3α,5α-THP NFSS: 81 ± 4.8% vs. VEH NFSS, Males 3α,5α-THP FSS: 81 ± 6.1% vs. VEH FSS, treatment effect p < 0.01. Females 3α,5α-THP NFSS: 47 ± 5% vs. VEH NFSS, Females 3α,5α-THP FSS: 53 ± 7.7% vs. VEH FSS, treatment effect p < 0.0001). Significant effects were found using Two-way ANOVA ^#### p < 0.0001, ^## p < 0.01, ^# p < 0.05, or following Tukey HSD test **** p < 0.0001, ** p < 0.01, * p < 0.05. Data are represented as % ± SEM vs. VEH no stress. Abbreviations: VEH = rats treated with vehicle; 3α,5α-THP = rats treated with 3α,5α-THP; NRS = non-restraint stress; RS = restraint stress; NFSS = non-forced swim stress; FSS = forced swim stress. All % values are calculated vs. VEH no stress control group.

Figure 3

3α,5α-THP attenuated hypothalamic CRF stress-induced response in females following both stressors and in males following forced swim stress, but failed to reduce the restraint stress-induced increase in CRF in male rats. As expected, following restraint stress or forced swim stress, hypothalamic CRF levels increased in male and female rats (Males VEH RS: 176 ± 15% vs. VEH NRS, p < 0.001; Males VEH FSS: 155 ± 10.7% vs. VEH NFSS, p < 0.001. Females VEH RS: 244 ± 11.4% vs. VEH NRS, p < 0.0001; Females VEH FSS: 232 ± 18.5% vs. VEH NFSS, p < 0.0001). 3α,5α-THP reduced hypothalamic CRF stress-induced in males after forced swim stress (Males 3α,5α-THP NFSS: 76 ± 6.5% vs. VEH NFSS, n.s.; Males 3α,5α-THP FSS: 82 ± 3.7% vs. VEH FSS, p < 0.0001), but not after restraint stress (Males 3α,5α-THP NRS: 70 ± 9.9% vs. VEH NRS, n.s.; Males 3α,5α-THP RS: 251 ± 10.5% vs. 3α,5α-THP NRS, p < 0.000; Males 3α,5α-THP RS 56 ± 9.6% vs. VEH RS, p < 0.001). In female rats, 3α,5α-THP reduced hypothalamic CRF expression following both restraint stress and forced swim stress (Females 3α,5α-THP NRS: 122 ± 12% vs. VEH NRS, n.s.; females 3α,5α-THP RS: 154 ± 12.7 vs. VEH RS %, p < 0.0001; Females 3α,5α-THP NFSS: 76 ± 10.9%vs VEH NFSS, n.s.; Females 3α,5α-THP FSS: 56 ± 9.8% vs. VEH FSS, p < 0.0001). Significant effects were found using Two-way ANOVA, followed by Tukey HSD test, **** p < 0.0001, *** p < 0.001. Data are represented as % ± SEM vs. VEH no stress. Abbreviations: VEH = rats treated with vehicle; 3α,5α-THP = rats treated with 3α,5α-THP; NRS = non-restraint stress; RS = restraint stress; NFSS = non-forced swim stress; FSS = forced swim stress. All % values are calculated vs. VEH no stress control group.

Figure 4

3α,5α-THP reduced restraint stress-induced hypothalamic CRFR1 increase in male rats. Hypothalamic CRFR1 levels increased in male rats following restraint stress (Males VEH RS: 160 ± 12% vs. VEH NRS, p < 0.01), but not following forced swim stress (Males VEH FSS: 110 ± 4.8% vs. VEH NFSS, n.s.). 3α,5α-THP reduced hypothalamic CRFR1 stress-induced in males after restraint stress (Males 3α,5α-THP NRS: 93 ± 9.8% vs. VEH NRS, n.s.; Males 3α,5α-THP RS: 86 ± 11.6% vs. VEH RS, p < 0.001), and CRFR1 levels after forced swim stress (Males 3α,5α-THP NFSS: 101 ± 4.5% vs. VEH NFSS, n.s.; Males 3α,5α-THP FSS: 83 ± 6.5% vs. 3α,5α-THP NFSS, p < 0.05). Restraint or forced swim stress did not induce any changes in hypothalamic CRFR1 in female rats (Females VEH RS: 89 ± 7.2%, n.s. vs. VEH NRS; Females VEH FSS: 84 ± 5.4%, n.s. vs. VEH NFSS). However, 3α,5α-THP reduced hypothalamic CRFR1 with and without forced swim stress in female animals (Females 3α,5α-THP NFSS: 55 ± 4.6% vs. VEH NFSS, p < 0001; Females 3α,5α-THP FSS:61 ± 5.4% vs. VEH FSS, p < 0.05). Significant effects were found using Two-way ANOVA, followed by Tukey HSD test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. Data are represented as % ± SEM vs. VEH no stress. Abbreviations: VEH = rats treated with vehicle; 3α,5α-THP = rats treated with 3α,5α-THP; NRS = non-restraint stress; RS = restraint stress; NFSS = non-forced swim stress; FSS = forced swim stress. All % values are calculated vs. VEH no stress control group.

Figure 5

3α,5α-THP attenuated the forced swim stress-induced hypothalamic CRFBP response in male rats. In male rats, restraint stress induced a decrease of hypothalamic CRFBP levels (Males VEH RS: 71 ± 4.6% vs. VEH NRS, stress effect p < 0.0001), while forced swim stress induced an increase of hypothalamic CRFBP expression (Males VEH FSS: 129 ± 6.8% vs. VEH FSS, p < 0.01). 3α,5α-THP treatment did not change the decrease of CRFBP levels in males due to restraint stress (Males 3α,5α-THP NRS: 77 ± 4.9% vs. VEH NRS, treatment effect p < 0.01; Males 3α,5α-THP FSS: 99 ± 6.5% vs. VEH FSS, p < 0.01). In female rats, restraint stress or 3α,5α-THP administration did not change hypothalamic CRFBP levels (Females VEH RS 81 ± 5.5% vs. VEH NRS, n.s.; Females 3α,5α-THP NRS 102 ± 4.9% vs. VEH NRS, n.s.; Females 3α,5α-THP RS 100 ± 7.3% vs. VEH NRS). 3α,5α-THP treatment resulted in a decrease in CRFBP levels in female animals following forced swim stress (Females 3α,5α-THP NFSS: 61 ± 5.5% vs. VEH NFSS, treatment effect p < 0.0001; Females 3α,5α-THP FSS: 64 ± 4.4% vs. VEH FSS, treatment effect p < 0.0001). Significant effects were found using Two-way ANOVA ^#### p < 0.0001, ^## p < 0.01 or following Tukey HSD test ** p < 0.01. Data are represented as % ± SEM vs. VEH no stress. Abbreviations: VEH = rats treated with vehicle; 3α,5α-THP = rats treated with 3α,5α-THP; NRS = non-restraint stress; RS = restraint stress; NFSS = non-forced swim stress; FSS = forced swim stress. All % values are calculated vs. VEH no stress control group.

Figure 6

3α,5α-THP attenuated the restraint stress and forced swim stress-induced hypothalamic POMC increase in male rats, but did not induce any change in female rats. Male rats exposed to restraint stress or forced swim stress showed an increase in hypothalamic POMC levels (Males VEH RS: 135 ± 5.4% vs. VEH NRS, p < 0.01; Males VEH FSS: 125 ± 3.4% vs. VEH NFSS, p < 0.01). 3α,5α-THP attenuated the stress-induced increase in hypothalamic POMC levels, following both restraint stress or forced swim stress in males (Males 3α,5α-THP RS: 87 ± 7.3% vs. VEH RS, p < 0.0001; Males 3α,5α-THP FSS: 88 ± 7.3%, p < 0.0001). Hypothalamic POMC levels increased following restraint stress in female rats (Females VEH RS: 198 ± 20% vs. VEH NRS, stress effect p < 0.0001), but 3α,5α-THP did not attenuate this increase (Females 3α,5α-THP RS: 226 ± 21% vs. 3α,5α-THP NRS, stress effect p < 0.0001; Females 3α,5α-THP RS: 226 ± 21% vs. VEH RS, n.s.). Forced swim stress failed to increase hypothalamic POMC levels in female rats, but 3α,5α-THP administration decreased POMC levels in the absence of stress (Females 3α,5α-THP NFSS: 75 ± 4.5% vs. VEH NFSS, p < 0.001). Significant effects were found using Two-way ANOVA ^#### p < 0.0001 or following Tukey HSD test **** p < 0.0001, *** p < 0.001, ** p < 0.01. Data are represented as % VEH no stress ± SEM. Abbreviations: VEH = rats treated with vehicle; 3α,5α-THP = rats treated with 3α,5α-THP; NRS = non-restraint stress; RS = restraint stress; NFSS = non-forced swim stress; FSS = forced swim stress.