Replacement with GABAergic steroid precursors restores the acute ethanol withdrawal profile in adrenalectomy/gonadectomy mice

Abstract

The neurosteroid allopregnanolone (ALLO) is a progesterone metabolite that is one of a family of neuroactive steroids (NAS) that are potent positive allosteric modulators of gamma-aminobutyric acid(A) (GABA(A)) receptors. These GABAergic NAS are produced peripherally (in the adrenals and gonads) and centrally in the brain. Peripherally produced NAS modulate some effects of ethanol intoxication (e.g., anxiolytic, antidepressant, and anticonvulsant effects) in rodents. We have found that NAS also may be involved in the rebound neural hyperexcitability following a high ethanol dose. Removal of the adrenals and gonads (ADX/GDX) increased withdrawal severity following 4 g/kg ethanol, as measured by handling-induced convulsions (HICs) in male and female DBA/2J mice. NAS are produced through the metabolism of progesterone (PROG), deoxycorticosterone (DOC), or testosterone, which can be blocked with the administration of finasteride (FIN), a 5alpha-reductase enzyme inhibitor. The current investigation was undertaken to clarify the step(s) in the biosynthetic NAS pathway that were sufficient to restore the acute ethanol withdrawal profile in ADX/GDX mice to that seen in intact animals. Male and female DBA/2J mice underwent ADX/GDX or SHAM surgery. After recovery, separate groups of animals were administered PROG, DOC, PROG+FIN, DOC+FIN, FIN, ALLO, ganaxalone (a synthetic ALLO derivative), corticosterone, or vehicle. Animals were then administered a 4 g/kg ethanol dose and allowed to undergo withdrawal. HICs were measured for 12 h and again at 24 h. The results indicate that replacement with PROG and DOC restored the withdrawal profile in ADX/GDX animals to SHAM levels, and that this effect was blocked with co-administration of FIN. Administration of FIN alone increased the withdrawal profile in both SHAM and ADX/GDX males. These findings indicate that the increase in acute withdrawal severity after ADX/GDX may be due to the loss of GABAergic NAS, providing insight into the contribution of endogenous GABAergic NAS to ethanol withdrawal severity.

Figure 1

Two doses of 50 mg/kg finasteride (FIN) reliably decreased allopregnanolone (ALLO) production over an 8 hour period. Separate groups of male and female DBA/2J animals were dosed with either 50 mg/kg FIN or 20% cyclodextrin at 24 hours before and immediately prior a 4 g/kg dose of EtOH. Different groups of animals were euthanized at 15 minutes, 2 hours and 8 hours following the EtOH injection. FIN administration significantly decreased ALLO production during the entire 8 hour period when compared to VEH treated animals. Bars represent the mean +/− the SEM for 8-10 animals/group. * p<0.05.

Figure 2

The effects of steroid replacement on acute ethanol (EtOH) withdrawal severity in male DBA/2J mice, measured by hourly handling-induced convulsions (HICs). Overall, in ADX/GDX males (panels A and B), replacement with progesterone (PROG) or deoxycorticosterone (DOC) decreased withdrawal severity. Co-administration of FIN blocked this effect. In SHAM males (panels C and D), administration of PROG or DOC increased withdrawal severity, and co-administration of FIN blocked this effect. Points represent the mean +/− the SEM for 9-16 for treatment groups and 36-40 for VEH treated groups that were collapsed across passes.

Figure 3

The effects of steroid replacement on acute EtOH withdrawal severity in female DBA/2J mice, measured by hourly HICs. Overall, in ADX/GDX females (panels A and B), replacement with PROG or DOC decreased withdrawal severity. Co-administration of FIN blocked this effect. In SHAM females (panels C and D), there was no effect of any drug treatment on acute withdrawal severity. Points represent the mean +/− the SEM for 12-16 for treatment groups and 28-40 for VEH treated groups that were collapsed across passes.

Figure 4

The effect of steroid replacement on acute EtOH withdrawal severity in DBA/2J mice, measured by AUC. In ADX/GDX males (panel A), administration of PROG and DOC decreased AUC, co-administration with FIN abolished this effect, and administration of FIN alone increased AUC. In SHAM males (panel B), PROG and DOC administration increased AUC, while co-administration with FIN abolished this effect. FIN administration alone significantly increased AUC, and corticosterone (CORT) administration tended to increase AUC. In ADX/GDX females (panel C), administration of PROG and DOC significantly decreased AUC, and administration of ALLO tended toward a decrease. Co-administration of FIN abolished these effects. In SHAM females (panel D), there were no effects of any drug administration on AUC. Bars represent the mean +/− the SEM for the animals depicted in Figures 2 and 3. + p< 0.10, * p<0.05 vs. respective VEH treated group.