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

Abstract

Rationale The rapid membrane actions of neuroactive steroids, particularly via an enhancement of γ-aminobutyric acidA receptors (GABAARs), 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 GABAAR 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 GABAAR-active steroids is not known. Methods After validation of a gas chromatography-mass spectrometry method for the simultaneous quantification of 10 neuroactive steroids, we analyzed plasma from control male WSP-1 and WSR-1 mice and during ethanol withdrawal. Results We quantified levels of 9 neuroactive steroids in WSP-1 and WSR-1 plasma; levels of pregnanolone were not detectable. Basal levels of 5 neuroactive steroids were higher in WSR-1 versus WSP-1 mice. Ethanol withdrawal significantly suppressed 5 neuroactive steroids in WSP-1 and WSR-1 mice, including ALLO. Conclusions Due to lower basal levels of some GABAAR-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 GABAAR sensitivity to neuroactive steroids during withdrawal, it is possible that the combined decrease in neuroactive steroids and GABAAR sensitivity during ethanol withdrawal in WSP-1 mice represents a neurochemical substrate for severe ethanol withdrawal.

Antiseizure Activity of Midazolam in Mice Lackingδ-Subunit Extrasynaptic GABAAReceptors

“…To provide direct support for the lack of involvement of neurosteroids in the antiseizure action of midazolam, plasma and brain levels of the neurosteroid allopregnanolone were determined by liquid chromatography-mass spectrometry in animals pretreated with midazolam. Allopregnanolone levels are commonly measured by mass spectrometry assay (Reddy et al, 2004;Porcu et al, 2009;Snelling et al, 2014). Allopregnanolone levels were determined 30 minutes after injection with an antiseizure dose of midazolam (1 mg/kg i.p.)…”

Ethanol withdrawal-induced dysregulation of neurosteroid levels in plasma, cortex, and hippocampus in genetic animal models of high and low withdrawal

“…Endogenous neurosteroids that rapidly enhance γ-aminobutyric acid A receptor (GABA A R)-mediated inhibition (e.g., Belelli and Lambert, 2005; Carver and Reddy, 2013; Paul and Purdy, 1992) are formed by the 5α-/5β- and then 3α-reduction of the parent steroids progesterone, deoxycorticosterone (DOC), testosterone, and dehydroepiandrosterone (DHEA; e.g., Finn et al, 2004; Porcu et al, 2009, 2016; Snelling et al, 2014). The progesterone metabolites allopregnanolone (ALLO; 3α,5α-THP or tetrahydroprogesterone) and pregnanolone (3α,5β-THP) and the DOC metabolite tetrahydrodeoxycorticosterone (3α,5α-THDOC) are the three most potent neurosteroids characterized to date, as they enhance GABA A R-mediated inhibition with nanomolar (nM) potencies, directly activate GABA A Rs with micromolar potencies, and exert effects on other ligand gated ion channels with micromolar potencies (Belelli and Lambert, 2005; Belelli et al, 1990; Carver and Reddy, 2013; Paul and Purdy, 1992; Purdy et al, 1990; Rupprecht and Holsboer, 1999; Veleiro and Burton, 2009).…”

“…We and others found that WD is associated with a decrease in GABA A R inhibition mediated by a variety of factors that includes functional changes in GABA A R properties, a reduction in the steroidogenic effect of acute ethanol administration, and a decrease in endogenous ALLO levels (see Finn et al, 2004; Kumar et al, 2009). In rodents, monkeys, and humans, WD decreases ALLO levels in plasma and several brain regions (Beattie et al, 2017; Cagetti et al, 2004; Hill et al, 2005; Maldonado-Devincci et al, 2014; Romeo et al, 1996; Snelling et al, 2014; Tanchuck et al, 2009). In small cohorts of male and female alcoholics, the decrease in ALLO and 3α,5α-THDOC levels corresponded to an increase in the subjective ratings of anxiety and depression during days 4 – 5 of WD, versus controls (Hill et al, 2005; Romeo et al, 1996).…”

“…We recently modified a gas chromatography-mass spectrometry (GC-MS) method (Porcu et al, 2009) to simultaneously quantify the GABA A R-active 3α,5α-/3α,5β-reduced metabolites of progesterone, DOC, testosterone and DHEA as well as the precursors pregnenolone and DHEA in plasma from control WSP and WSR mice from the first genetic replicate (WSP-1 and WSR-1) and at 8 h WD from 72 h continuous ethanol vapor exposure (Snelling et al, 2014); there was no line difference in the WD-induced suppression in plasma DHEA, ALLO, 3α,5α/3α,5β-androstanediol, and 3α,5α-androsterone levels or increase in plasma corticosterone (CORT) levels. While the pattern of changes in steroid levels imply that ethanol WD is exerting independent effects at several steps of the steroidogenic pathway in plasma (discussed in Snelling et al, 2014), it is not known if similar results would be observed in discrete brain regions.…”

“…While the pattern of changes in steroid levels imply that ethanol WD is exerting independent effects at several steps of the steroidogenic pathway in plasma (discussed in Snelling et al, 2014), it is not known if similar results would be observed in discrete brain regions. So, cortex and hippocampus were examined in the present studies, based on evidence that basal neurosteroid levels in plasma do not fully reflect levels in cortex and hippocampus (Caruso et al, 2013) and that divergent changes in ALLO immunoreactivity were observed during WD in mPFC and CA3 (Maldonado-Devincci et al, 2014).…”