Quantification of ten neuroactive steroids in plasma in Withdrawal Seizure-Prone and -Resistant mice during chronic ethanol withdrawal

Rationale: The rapid membrane actions of neuroactive steroids, particularly via an enhancement of γ-aminobutyric acid_A receptors (GABA_ARs), participate in the regulation of central nervous system excitability. Prior evidence suggests an inverse relationship between endogenous GABAergic neuroactive steroid levels and behavioral changes in excitability during ethanol withdrawal. Objectives: Previously, we found that ethanol withdrawal significantly decreased plasma allopregnanolone (ALLO) levels, a potent GABAergic neuroactive steroid, and decreased GABA_AR sensitivity to ALLO in Withdrawal Seizure-Prone (WSP) but not in Withdrawal Seizure-Resistant (WSR) mice. However, the effect of ethanol withdrawal on levels of other endogenous GABA_AR-active steroids is not known. Methods: After validation of a gas chromatography-mass spectrometry method for the simultaneous quantification of ten neuroactive steroids, we analyzed plasma from control male WSP-1 and WSR-1 mice and during ethanol withdrawal. Results: We quantified levels of nine neuroactive steroids in WSP-1 and WSR-1 plasma; levels of pregnanolone were not detectable. Basal levels of five neuroactive steroids were higher in WSR-1 versus WSP-1 mice. Ethanol withdrawal significantly suppressed five neuroactive steroids in WSP-1 and WSR-1 mice, including ALLO. Conclusions: Due to lower basal levels of some GABA_AR-active steroids in WSP-1 mice, a withdrawal-induced decrease in WSP-1 mice may have a greater physiological consequence than a similar decrease in WSR-1 mice. Because WSP-1 mice also exhibit a reduction in GABA_AR sensitivity to neuroactive steroids during withdrawal, it is possible that the combined decrease in neuroactive steroids and GABA_AR sensitivity during ethanol withdrawal in WSP-1 mice represents a neurochemical substrate for severe ethanol withdrawal.