The general anaesthetic etomidate inhibits the excitability of mouse thalamocortical relay neurons by modulating multiple modes of GABAA receptor-mediated inhibition

Abstract

Modulation of thalamocortical (TC) relay neuron function has been implicated in the sedative and hypnotic effects of general anaesthetics. Inhibition of TC neurons is mediated predominantly by a combination of phasic and tonic inhibition, together with a recently described 'spillover' mode of inhibition, generated by the dynamic recruitment of extrasynaptic γ-aminobutyric acid (GABA)A receptors (GABAA Rs). Previous studies demonstrated that the intravenous anaesthetic etomidate enhances tonic and phasic inhibition in TC relay neurons, but it is not known how etomidate may influence spillover inhibition. Moreover, it is unclear how etomidate influences the excitability of TC neurons. Thus, to investigate the relative contribution of synaptic (α1β2γ2) and extrasynaptic (α4β2δ) GABAA Rs to the thalamic effects of etomidate, we performed whole-cell recordings from mouse TC neurons lacking synaptic (α1(0/0) ) or extrasynaptic (δ(0/0) ) GABAA Rs. Etomidate (3 μm) significantly inhibited action-potential discharge in a manner that was dependent on facilitation of both synaptic and extrasynaptic GABAA Rs, although enhanced tonic inhibition was dominant in this respect. Additionally, phasic inhibition evoked by stimulation of the nucleus reticularis exhibited a spillover component mediated by δ-GABAA Rs, which was significantly prolonged in the presence of etomidate. Thus, etomidate greatly enhanced the transient suppression of TC spike trains by evoked inhibitory postsynaptic potentials. Collectively, these results suggest that the deactivation of thalamus observed during etomidate-induced anaesthesia involves potentiation of tonic and phasic inhibition, and implicate amplification of spillover inhibition as a novel mechanism to regulate the gating of sensory information through the thalamus during anaesthetic states.

Keywords: nucleus reticularis; phasic inhibition; spill-over inhibition; thalamus; tonic inhibition.

Figure 1

Etomidate prolongs the duration of evoked phasic inhibition in VB neurons by potentiating synaptic and extrasynaptic GABA_A receptors. (A–C) Superimposed, representative, evoked IPSCs recorded from WT (A), α1^0/0 (B) and δ^0/0 (C) VB neurons under control conditions (black) and in the presence of 3 μm etomidate (grey). Currents were evoked by extracellular stimulation of the nRT (single shocks, 20 μs duration). (D–E) Bar graphs illustrating the effect of etomidate on eIPSC peak amplitude (D), τ_w (E) and charge transfer (F) in WT (black bars, n = 7), α1^0/0 (dark grey bars, n = 7) and δ^0/0 (light grey bars, n = 8) thalamic slices. **P < 0.01, ***P < 0.001 vs. control, paired t-test. ^†P < 0.05, ^††P < 0.01 vs. WT, mixed anova, Tukey's post-hoc test. Etom, etomidate.

Figure 2

Synaptic and extrasynaptic GABA_ARs influence phasic suppression of VB tonic firing. (A–C) Representative current-clamp recordings illustrating the suppression of tonic action potential discharge induced by evoked IPSPs in WT (A), α1^0/0 (B) and δ^0/0 (C) thalamic slices. The plots to the right of each trace depict the inter-spike interval (ISI) over the course of the illustrated recordings, and demonstrate the increase in ISI (i.e. spike suppression) during delivery of the eIPSP. Evoked IPSPs were generated by extracellular stimulation of the nRT. (D) Bar graph comparing the effect of eIPSPs on ISI during tonic spike trains recorded from WT (n = 13), α1^0/0 (n = 12) and δ^0/0 (n = 8) VB neurons. ***P < 0.001 vs. baseline ISI, paired t-test. ^†P < 0.05 vs. WT, mixed anova, Tukey's post hoc test.

Figure 3

Etomidate prolongs phasic suppression of VB tonic firing by potentiating synaptic and extrasynaptic GABA_ARs. (A–C) Whole-cell current-clamp recordings illustrating the suppression of VB tonic firing under control conditions (Ai–Ci), and in the presence of 3 μm etomidate (Aii–Cii), in WT (A), α1^0/0 (B) and δ^0/0 (C) thalamic slices. The plots to the right of each trace depict the inter-spike interval (ISI) during the illustrated tonic spike train, and demonstrate blockade of action potentials during the eIPSP. Note that the ISI during the eIPSP is increased in the presence of etomidate across all mouse strains. (D) Bar graph comparing the effect of eIPSPs on the ISI of tonic spike trains, before and after etomidate, in WT (n = 8), α1^0/0 (n = 9) and δ^0/0 (n = 6) VB neurons. The bar representations are depicted in the symbol key. ***P < 0.001 vs. baseline ISI, paired t-test. ^†††P < 0.001, ISI during eIPSP after etomidate application vs. control. The increased duration of the eIPSP-induced spike suppression observed in the presence of etomidate is not significantly influenced by mouse genotype (P = 0.58, mixed anova).

Figure 4

Etomidate delays the timing of post-inhibitory rebound burst firing in VB neurons. (A, B) Exemplar traces from a subset of current-clamp recordings obtained from WT (A) and δ^0/0 (B) mice in which rebound burst firing was observed at the offset of eIPSPs. The responses to three consecutive stimuli recorded before (black traces) and after (grey traces) application of 3 μm etomidate are superimposed, illustrating the delay to rebound burst firing in the presence of the anaesthetic. Action potentials are truncated for clarity. (C) Summary bar graph comparing the latency to rebound spiking before (black bars) and after etomidate (grey bars) application for WT (n = 4) and δ^0/0 (n = 5) VB neurons. **P < 0.01, ***P < 0.001, paired t-test.

Figure 5

Deletion of the α1 or δ subunit reduces the effect of etomidate on VB tonic inhibition. (A, C, E) Representative whole-cell recordings (left) and corresponding all points histograms (right), illustrating the effect of etomidate on the holding current of WT (A), α1^0/0 (C) and δ^0/0 (E) VB neurons. Evoked IPSCs were simultaneously monitored throughout the recordings, but have been truncated to emphasize changes in holding current (I_hold). Non-truncated eIPSCs denoted by i (control) and ii (etomidate) are shown in B, D and F for WT, α1^0/0 and δ^0/0, respectively, to confirm the effect of etomidate on eIPSCs. (G) Bar graph comparing the outward current induced by the bath application of etomidate (expressed as a change in I_hold) for WT (black bar, n = 8), α1^0/0 (dark grey bar, n = 9) and δ^0/0 (light grey bar, n = 7) VB neurons. *P < 0.05, **P < 0.01, ***P < 0.001, one-way anova, Tukey's post hoc test.

Figure 6

The excitability of VB neurons is not significantly altered in α1^0/0 or δ^0/0 mice. (A–C) Whole-cell current-clamp recordings from WT (A), α1^0/0 (B) and δ^0/0 (C) VB neurons, illustrating voltage responses to a family of hyperpolarizing and depolarizing current steps (−200 to +300 pA, 50 pA increments). For clarity, the responses to only a selection of tested current steps are shown, as indicated in the stimulation protocol (bottom). (D) Graph summarizing the number of spikes occurring within rebound bursts upon offset from hyperpolarizing current steps in recordings from WT (black squares, n = 23), α1^0/0 (dark grey circles, n = 12) and δ^0/0 (light grey triangles, n = 22) VB neurons. (E) Graph comparing the number of spikes generated in response to depolarizing current steps of increasing amplitude in VB neurons derived from WT, α1^0/0 and δ^0/0 mice. Symbols and n numbers are as indicated for D. The input–output relationship for each strain is fitted with a Boltzmann sigmoid curve.

Figure 7

The effect of etomidate on VB neuron excitability is attenuated by deletion of the α1 subunit and abolished by deletion of the δ subunit. (A) Representative whole-cell current-clamp recording from a WT VB neuron, illustrating superimposed voltage responses to the indicated current steps (middle) under control conditions (top) and in the presence of 3 μm etomidate (bottom). (B) Graph comparing the intra-burst spike number observed on rebound from a range of hyperpolarizing current steps (−200 to −50 pA), before (black symbols) and after (grey symbols) etomidate (n = 12). (C)Graph comparing the number of spikes generated in response to depolarizing current steps of increasing amplitude (50–300 pA) under control conditions (black symbols), and in the presence of etomidate (grey symbols). (D–F) Effect of etomidate on the excitability of α1^0/0 VB neurons (n = 11). Figure details are as described for A–C. (G–I) Effect of etomidate on the excitability of δ^0/0 VB neurons (n = 9). Figure details are as described for A–C. The input–output relationships determined for each condition are fitted with a Boltzmann curve. *P < 0.05, **P < 0.01, ***P < 0.001, paired t-test. Etom, etomidate.