Sex-specific hypnotic effects of the neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile are mediated by peripheral metabolism into an active hypnotic steroid.

BACKGROUND: The novel synthetic neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) blocks T-type calcium channels but does not directly modulate neuronal γ-aminobutyric acid type A (GABAA) currents like other anaesthetic neurosteroids. As 3ß-OH has sex-specific hypnotic effects in adult rats, we studied the mechanism contributing to sex differences in its effects. METHODS: We used a combination of behavioural loss of righting reflex, neuroendocrine, pharmacokinetic, in vitro patch-clamp electrophysiology, and in vivo electrophysiological approaches in wild-type mice and in genetic knockouts of the CaV3.1 T-type calcium channel isoform to study the mechanisms by which 3ß-OH and its metabolite produces sex-specific hypnotic effects. RESULTS: Adult male mice were less sensitive to the hypnotic effects of 3ß-OH compared with female mice, and these differences appeared during development. Adult males had higher 3ß-OH brain concentrations despite being less sensitive to its hypnotic effects. Females metabolised 3ß-OH into the active GABAA receptor positive allosteric modulator (3α,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3α-OH) to a greater extent than males. The 3α-OH metabolite has T-channel blocking properties with sex-specific hypnotic and pharmacokinetic effects. Sex-dependent suppression of the cortical electroencephalogram is more pronounced with 3α-OH compared with 3ß-OH. CONCLUSIONS: The sex-specific differences in the hypnotic effect of 3ß-OH in mice are attributable to differences in its peripheral metabolism into the more potent hypnotic metabolite 3α-OH.

The T-type calcium channel isoform Cav3.1 is a target for the hypnotic effect of the anaesthetic neurosteroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile.

BACKGROUND: The mechanisms underlying the role of T-type calcium channels (T-channels) in thalamocortical excitability and oscillations in vivo during neurosteroid-induced hypnosis are largely unknown. METHODS: We used patch-clamp electrophysiological recordings from acute brain slices ex vivo, recordings of local field potentials (LFPs) from the central medial thalamic nucleus in vivo, and wild-type (WT) and Cav3.1 knock-out mice to investigate the molecular mechanisms of hypnosis induced by the neurosteroid analogue (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH). RESULTS: Patch-clamp recordings showed that 3ß-OH inhibited isolated T-currents but had no effect on phasic or tonic γ-aminobutyric acid A currents. Also in acute brain slices, 3ß-OH inhibited the spike firing mode more profoundly in WT than in Cav3.1 knockout mice. Furthermore, 3ß-OH significantly hyperpolarised neurones, reduced the amplitudes of low threshold spikes, and diminished rebound burst firing only in WT mice. We found that 80 mg kg-1 i.p. injections of 3ß-OH induced hypnosis in >60% of WT mice but failed to induce hypnosis in the majority of mutant mice. A subhypnotic dose of 3ß-OH (20 mg kg-1 i.p.) accelerated induction of hypnosis by isoflurane only in WT mice, but had similar effects on the maintenance of isoflurane-induced hypnosis in both WT and Cav3.1 knockout mice. In vivo recordings of LFPs showed that a hypnotic dose of 3ß-OH increased δ, θ, α, and ß oscillations in WT mice in comparison with Cav3.1 knock-out mice. CONCLUSIONS: The Cav3.1 T-channel isoform is critical for diminished thalamocortical excitability and oscillations that underlie neurosteroid-induced hypnosis.