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

Abstract

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 Ca v 3.1 knockout mice to investigate the molecular mechanisms of hypnosis induced by the neurosteroid analogue (3b,5b,17b)-3hydroxyandrostane-17-carbonitrile (3b-OH). Results: Patch-clamp recordings showed that 3b-OH inhibited isolated T-currents but had no effect on phasic or tonic gaminobutyric acid A currents. Also in acute brain slices, 3b-OH inhibited the spike firing mode more profoundly in WT than in Ca v 3.1 knockout mice. Furthermore, 3b-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 À1 i.p. injections of 3b-OH induced hypnosis in >60% of WT mice but failed to induce hypnosis in the majority of mutant mice. A subhypnotic dose of 3b-OH (20 mg kg À1 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 Ca v 3.1 knockout mice. In vivo recordings of LFPs showed that a hypnotic dose of 3b-OH increased d, q, a, and b oscillations in WT mice in comparison with Ca v 3.1 knockout mice. Conclusions: The Ca v 3.1 T-channel isoform is critical for diminished thalamocortical excitability and oscillations that underlie neurosteroid-induced hypnosis.

Differential effects of the novel neurosteroid hypnotic (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile on electroencephalogram activity in male and female rats

“…36 These effects of 3b-OH and TTA-P2 are either absent or severely diminished in mice lacking the Ca V 3.1 Ca 2þ channel isoform that mediates T-currents. 34,36 In conclusion, we found that 3b-OH is a potent hypnotic in rats with a rapid onset of action and an EEG signature comparable with other neurosteroid hypnotics/anaesthetics (e.g. alphaxalone).…”

“…29 Alphaxalone, a neurosteroid analogue with prominent GABAergic properties 30 that also inhibits the Ca V 3.2 Ca 2þ channel isoform of T-currents in sensory neurones, 31 is another hypnotic with a typical pattern of EEG changes. 32,33 Although 3b-OH is devoid of direct GABAergic activity in both thalamic 34 and hippocampal brain slices, 4 it produces an apparently similar EEG signature in rats. We have shown previously that 3b-OH may suppress neuronal excitability by inhibiting T-channel-dependent rebound bursting 5 and by decreasing presynaptic glutamatergic transmission.…”

“… 4 Furthermore, 3β-OH may hyperpolarise thalamocortical neurones by inhibiting the baseline influx of Ca 2+ via T-type ‘window’ currents. 34 Although precise molecular mechanisms underlying sex-dependent differences of neurosteroid-induced hypnosis are not known, it is possible that 3β-OH, by decreasing neuronal excitability and hyperpolarising the neuronal membrane, may produce an EEG pattern resembling a typical GABAergic neurosteroid hypnotic, such as alphaxalone. Consistent with this hypothesis, a structurally unrelated T-channel selective antagonist (TTA-P2) also produces hyperpolarisation of thalamocortical neurones by inhibiting T-type window current and promotes generation of delta oscillations both in vitro 35 and in vivo.…”

“… 36 These effects of 3β-OH and TTA-P2 are either absent or severely diminished in mice lacking the Ca V 3.1 Ca 2+ channel isoform that mediates T-currents. 34 , 36 …”

Synthetic neuroactive steroids as new sedatives and anaesthetics: Back to the future

“…At higher concentrations above 10 μM, 3β‐OH blocks partially HVA currents and sodium currents 114 . Behavioural studies and mouse genetics of T‐channel isoforms, have shown that Ca V 3.1 and Ca V 3.2 deletion significantly decreases duration of hypnosis mediated by this neuroactive steroid when given intraperitoneally 45,46 . However, wild‐type and Ca V 3.1 and Ca V 3.2 KO still responded to toe and tail pinch, indicating that T‐channels play at least a partial role in the hypnotic effects of neuroactive steroids.…”