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_v3.1 knock-out mice to investigate the molecular mechanisms of hypnosis induced by the neurosteroid analogue (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH).

Results: Patch-clamp recordings showed that 3β-OH inhibited isolated T-currents but had no effect on phasic or tonic γ-aminobutyric acid A currents. Also in acute brain slices, 3β-OH inhibited the spike firing mode more profoundly in WT than in Ca_v3.1 knockout mice. Furthermore, 3β-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 3β-OH induced hypnosis in >60% of WT mice but failed to induce hypnosis in the majority of mutant mice. A subhypnotic dose of 3β-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 Ca_v3.1 knockout mice. In vivo recordings of LFPs showed that a hypnotic dose of 3β-OH increased δ, θ, α, and β oscillations in WT mice in comparison with Ca_v3.1 knock-out mice.

Conclusions: The Ca_v3.1 T-channel isoform is critical for diminished thalamocortical excitability and oscillations that underlie neurosteroid-induced hypnosis.

Keywords: calcium channel; electrophysiology; hypnosis; mechanisms of anaesthesia; neurosteroid; thalamus.

Figure 1

Inhibition of T-currents by 3β-OH in CeM neurones. (a) Original traces of T currents in a representative CeM neurone in control conditions recorded using a double-pulse protocol with 3.6 s long prepulses to variable voltages (from –115 to –80 mV in 5 mV increment; blue); traces from the same cell using the identical voltage protocol during an apparent steady-state inhibition of T-current by 3 μM 3β-OH (purple) recorded with TMA with ATP internal solution. (b) Average normalised steady-state inactivation (I/I_max) curves in control conditions and after application of 3β-OH in the same cells. 3β-OH induced a hyperpolarising shift in V₅₀ of 4.6 mV; the average V₅₀ value for steady-state inactivation was –94.9 (1.8) mV in control conditions (blue; six cells, three animals). After 3β-OH perfusion of the same cells, the V₅₀ value was –99.5 (2.1) mV (purple). In the inset of panel (b), the V₅₀ was significantly different after 3β-OH perfusion (two tailed paired t-test: t₍₅₎=4.08, P<0.01). (c) Average current density calculated from the steady-state inactivation protocol. Application of 3β-OH (purple line and data points; six cells, three animals) decreased current density when compared with control conditions (blue line and data points). Data were analysed with two-way RM analysis of variance (anova): interaction F_(12,60)=13.91, P<0.001; voltage F_(12.60)=46.66, P<0.001; 3β-OH F_(1,5)=15.81, P=0.011; Sidak's post hoc presented in figure). (d) Average normalised steady-state inactivation (I/I_max) curves in control conditions and after application of 3β-OH in the same cells recorded with Cs with ATP internal solution. 3β-OH induced a hyperpolarising shift in V₅₀ of 3.2 mV because the average V₅₀ value for steady-state inactivation was –80.9 (1.2) mV under control conditions (blue; seven cells, four animals), and after 3β-OH perfusion in the same cells, the V₅₀ value was –84.1 (1.3) mV (purple). In the inset of panel (d), the V₅₀ was significantly different after 3β-OH perfusion (two tailed paired t-test: t₍₆₎=3.92, P=0.008). (e) Averaged representative traces recorded under control conditions (blue) and after application of 3β-OH (purple) using a protocol Vt=−50 mV, Vh=−90 mV, 3β-OH decreased T-current amplitudes from 416.9 (17.5) pA to 291.9 (38.2) pA (two-tailed paired t-test: t₍₄₎=4.67, P<0.01; five cells, three animals) ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001. 3β-OH, (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile; ATP, adenosine triphosphate; CeM, central medial nucleus; RM, repeated measure; TMA, tetramethylammonium.

Figure 2

Potentiation of miniature inhibitory postsynaptic currents by alphaxalone but not by 3β-OH. (a) Representative traces from CeM neurones before and after perfusion with 3β-OH (top panel) or alphaxalone (bottom panel). (b) Decay tau was increased only after perfusion of alphaxalone but not 3β-OH; alphaxalone increased decay tau from 26.7 (3.4) to 62.9 (6.6) ms (two-tailed paired t-test: t₍₁₀₎=6.82, P<0.001). (c) Left panel shows that cumulative frequency distribution for decay tau confirms no change after application of 3β-OH (control 1479 events, 3β-OH 1497 events); right panel shows longer decay taus after alphaxalone perfusion (1523 events for alphaxalone, 2134 events for the control). (d) Representative traces showing tonic GABA current after 3 μM 3β-OH (purple trace) or 3 μM alphaxalone (green trace) perfusion. (b) Difference from baseline showed a minimal effect of 3β-OH on tonic current in comparison with alphaxalone (3β-OH five cells, alphaxalone six cells; unpaired two-tailed t-test: t₍₉₎=2.31, P=0.046). ∗P<0.05, ∗∗∗P<0.001. 3β-OH, (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile; CeM, central medial nucleus; GABA, γ-aminobutyric acid A.

Figure 3

3β-OH influence on the excitability of CeM neurons. (a) Representative traces from a WT CeM neurone before application of 3β-OH (blue), after application of 3 μM 3β-OH (purple) and after wash (orange trace) with active membrane responses to a depolarising (125 pA) and hyperpolarising (–75 pA) current injection. (b) Top: averaged LTS amplitude was reduced by application of 3β-OH across all hyperpolarising current pulses from –50 to –225 pA in WT mice (eight cells, four animals; two-way RM analysis of variance [anova]: interaction F_(6,42)=0.41, P=0.865; current injection F_(6,42)=5.44, P<0.001; 3β-OH F_(1,7)=22.63, P=0.002). Bottom: bar graph shows 3β-OH increased the threshold for the occurrence of LTS (seven cells, four animals; paired two-tailed t-test t₍₆₎=4.07, P=0.007). (c) 3β-OH reduced tonic action potential firing frequency across all current pulses in WT mice (eight cells, four animals; from 50 to 225 pA, two-way RM anova: interaction F_(7,49)=1.66, P=0.140; current injection F_(7,49)=132.70, P<0.001; 3β-OH effect F_(1,7)=29.52, P=0.001). (d) 3β-OH decreased tonic frequency at 100 pA current injection in WT mice (eight cells, four animals; paired two-tailed t-test: t₍₇₎=4.80, P=0.002). (e) 3β-OH hyperpolarised RMP in WT mice (eight cells, four animals; paired two-tailed t-test: t₍₇₎=4.00, P=0.005). (f) 3β-OH did not affect IR measured with a hyperpolarising current injection of 100 pA in WT mice. (g) 3β-OH diminished tonic action potential firing frequency in Ca_v3.1 KO mice (eight cells, three animals; from 50 to 225 pA, two-way RM anova: interaction F_(7,49)=1.23, P=0.302; current injection F_(7,49)=63.29, P<0.001; 3β-OH effect F_(1,7)=8.26, P=0.024). (h) 3β-OH did not significantly affect tonic firing frequency at 100 pA current injection in Ca_v3.1 KO mice. (i) 3β-OH did not significantly affect RMP in Ca_v3.1 KO mice. (j) 3β-OH did not significantly affect IR measured with a hyperpolarising current injection of 100 pA in Ca_v3.1 KO mice. ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001. 3β-OH, (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile; CeM, central medial nucleus; GABA, γ-aminobutyric acid A; KO, knock-out; LTS, low threshold spikes; RM, definition; IR, input resistance; RMP, resting membrane potential; WT, wild-type.

Figure 4

Effect of 3β-OH on anaesthetic endpoints. (a) Dose–response curve for loss of righting reflex (LORR) in WT and Ca_v3.1 KO mice with ED₅₀ indicated on the top of the panel. (b) Left, percentage of animals that lost righting reflex under 80 mg kg⁻¹ i.p. 3β-OH in WT (17 animals) and Ca_v3.1 KO (20 animals) cohorts (80 mg kg⁻¹: χ² = 9.79, P=0.002). (b) Right: duration of LORR under 80 mg kg⁻¹ i.p. 3β-OH in WT (17 animals) and Ca_v3.1 KO (20 animals) mice (unpaired two-tailed t-test: t₍₃₅₎=2.94, P=0.006). (c) 20 mg kg⁻¹ 3β-OH lowered the percent of isoflurane needed for LORR in WT and Ca_v3.1 KO mice (10 mice in each group, two-way RM analysis of variance [anova]: interaction F_(1,18)=1.67, P=0.210; mutation effect F_(1,18)=1.35, P=0.260; 3β-OH effect F_(1,18)=42.19, P<0.001). (d) WT animals pretreated with 3β-OH needed significantly less time for induction with isoflurane compared with mutant animals (10 mice in each group, two-way RM anova: interaction F_(1,18)=0.71, P=0.410; mutation effect F_(1,18)=9.05, P=0.007; 3β-OH effect F_(1,18)=1.59, P=0.223). ∗∗P<0.01, ∗∗∗P<0.001. 3β-OH, (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile; KO, knock-out; WT, wild-type; ED₅₀, median effective dose; RM, definition.

Figure 5

Oscillations in CeM neurones after injections of 3β-OH. (a) Total δ power after injections of the vehicle 2-hydroxypropyl-β-cyclodextrin (Day 1) and 3β-OH (Day 2) in WT and Ca_v3.1 KO mice. Mutant mice had less δ power on Day 1 (two-way RM analysis of variance [anova]: interaction F_(11,121)=0.27, P=0.990; time F_(11,121)=1.15, P=0.330; mutation effect F_(1,11)=5.93, P=0.033). 3β-OH enhanced δ power in WT animals (two-way RM anova: interaction F_(11,121)=1.51, P=0.137; time F_(11,121)=3.62, P<0.001; mutation effect F_(1,11)=11.99, P=0.005). (b) Total θ power after vehicle (Day 1) or 3β-OH (Day 2) in WT and Ca_v3.1 KO mice. 3β-OH enhanced θ power in WT animals (two-way RM anova: interaction F_(11,121)=4.07, P<0.001; time F_(11,121)=4.03, P<0.001; mutation effect F_(1,11)=26.60, P<0.001; Sidak's post hoc presented in figure). (c) Total α power for vehicle (Day 1) or 3β-OH (Day 2) in WT and Ca_v3.1 KO mice. 3β-OH enhanced α power in WT animals (two-way RM anova: interaction F_(11,121)=5.87, P<0.001; time F_(11,121)=5.10, P<0.001; mutation effect F_(1,11)=27.74, P<0.001; Sidak's post hoc presented in figure). (d) Total β power for vehicle (Day 1) or 3β-OH (Day 2) in WT and Ca_v3.1 KO mice. 3β-OH enhanced β power in WT animals (two-way RM anova: interaction F_(11,121)=1.16, P=0.324; time F_(11,121)=2.82, P=0.003; mutation effect F_(1,11)=8.62, P=0.013; Sidak's post hoc presented in figure). (e) Total γ power for vehicle (Day 1) or 3β-OH (Day 2) in WT and Ca_v3.1 KO mice. Mutant mice had less γ power on Day 1 (two-way RM anova: interaction F_(11,121)=0.53, P=0.879; time F_(11,121)=1.77, P=0.067; mutation effect F_(1,11)=6.73, P=0.025). 3β-OH did not statistically change γ power between WT and Ca_v3.1 KO animals. WT, seven animals, Ca_v3.1 KO, six animals; ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001. 3β-OH, (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile; CeM, central medial nucleus; KO, knock-out; LFP, local field potentials; WT, wild-type.

Figure 6

Oscillatory differences in CeM between WT and Ca_v3.1 KO mice after 3β-OH. (a) Representative spectrograms from the CeM at 30 min after i.p. injections of 3β-OH recorded from WT (upper image) and Ca_v3.1 KO mice (lower image). (b) Power density with electrode confirmation (inset in figure). Power density revealed decreased low frequency oscillations in Ca_v3.1 KO mice under 3β-OH. (c) Total power revealed less power in δ, θ, α, and β frequency range in Ca_v3.1 KO mice (two-way RM analysis of variance [anova]: interaction F_(4,44)=10.76, P<0.001; oscillations F_(4,44)=37.80, P<0.001; mutation effect F_(1,11)=32.50, P<0.001; Sidak's post hoc presented in figure). (d) Analysis of relative power after neurosteroid injection did not find significant differences between WT and Ca_v3.1 KO mice. (e) Change in relative power (relative power under 3β-OH minus relative power during baseline in CeM) revealed differences between WT and Ca_v3.1 KO mice in the θ frequency range (two-way RM anova: interaction F_(4,44)=3.30, P=0.019; oscillations F_(4,44)=8.08, P<0.001; mutation effect F_(1,11)=0.31, P=0.591; Sidak's post hoc presented in figure). WT, seven animals; Ca_v3.1 KO, six animals; ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001. 3β-OH, (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile; CeM, central medial nucleus; KO, knock-out; RM, repeated measure; WT, wild-type.