Extrasynaptic GABAA receptors of thalamocortical neurons: a molecular target for hypnotics

Abstract

Among hypnotic agents that enhance GABAA receptor function, etomidate is unusual because it is selective for beta2/beta3 compared with beta1 subunit-containing GABAA receptors. Mice incorporating an etomidate-insensitive beta2 subunit (beta(2N265S)) revealed that beta2 subunit-containing receptors mediate the enhancement of slow-wave activity (SWA) by etomidate, are required for the sedative, and contribute to the hypnotic actions of this anesthetic. Although the anatomical location of the beta2-containing receptors that mediate these actions is unknown, the thalamus is implicated. We have characterized GABAA receptor-mediated neurotransmission in thalamic nucleus reticularis (nRT) and ventrobasalis complex (VB) neurons of wild-type, beta2(-/-), and beta(2N265S) mice. VB but not nRT neurons exhibit a large GABA-mediated tonic conductance that contributes approximately 80% of the total GABAA receptor-mediated transmission. Consequently, although etomidate enhances inhibition in both neuronal types, the effect of this anesthetic on the tonic conductance of VB neurons is dominant. The GABA-enhancing actions of etomidate in VB but not nRT neurons are greatly suppressed by the beta(2N265S) mutation. The hypnotic THIP (Gaboxadol) induces SWA and at low, clinically relevant concentrations (30 nM to 3 microM) increases the tonic conductance of VB neurons, with no effect on VB or nRT miniature IPSCs (mIPSCs) or on the holding current of nRT neurons. Zolpidem, which has no effect on SWA, prolongs VB mIPSCs but is ineffective on the phasic and tonic conductance of nRT and VB neurons, respectively. Collectively, these findings suggest that enhancement of extrasynaptic inhibition in the thalamus may contribute to the distinct sleep EEG profiles of etomidate and THIP compared with zolpidem.

Figure 1.

The properties of synaptic and extrasynaptic GABA_A receptors of nRT and VB neurons. A, Overlayed averaged mIPSCs recorded from exemplar nRT and VB cells of WT, β_2N265S, and β₂^–/– mice. Note that the deletion of the β₂ subunit greatly decreases the amplitude of VB mIPSCs (of those neurons for which mIPSCs were detected), but with no effect on nRT mIPSCs. The β_2N265S mutation had no effect on VB or nRT mIPSCs. B, Bicuculline (30μm) has no effect on the holding current recorded from an exemplar nRT neuron but reveals a large tonic current in a VB neuron from a WT mouse. Note that the mIPSCs of both nRT and VB neurons are abolished by this antagonist. C, Bar graph showing the lack of a tonic current in nRT neurons and the influence of deleting (β₂^–/–) or mutating (β_2N265S) the β₂ subunit compared with wild type on the tonic current of VB neurons. Data are obtained from three to nine cells. Note that the deletion, but not the mutation, of the β₂ subunit significantly reduces the VB tonic current. *p < 0.05 versus wild type (unpaired Student's t test). Error bars indicate the SE of the arithmetic mean.

Figure 2.

The effect of etomidate on the mIPSCs and the tonic current of nRT and VB neurons. A, Etomidate (3 μm) has no effect on the holding current of the exemplar WT nRT neuron but prolongs the mIPSC decay (see expanded traces). B, C, Overlayed averaged mIPSCs recorded from exemplar VB neurons of WT and β_2N265S mice showing the effect of 3 μm etomidate (B) and 100 μm pentobarbital (C). Note that etomidate-induced but not pentobarbital-induced prolongation of VB mIPSCs is reduced by the β_2N265S mutation. D, Bar graph showing the percentage of increase in the decay time constant τ_w produced by etomidate (3 μm) and pentobarbital (100 μm) in VB neurons from WT (▪) and β_2N265S (□) mice. Data were obtained from six cells. *p < 0.05 versus control; **p < 0.001 versus control; ^†p < 0.01, wild type versus β_2N265S (two-way, repeated-measures ANOVA). Error bars indicate the SE of the arithmetic mean.

Figure 3.

The etomidate-induced but not the pentobarbital-induced enhancement of the VB tonic current is reduced by the β_2N265S mutation. A, The tonic current (calculated as the difference between the holding current (picoamperes) in the presence and absence of 30 μm bicuculline (see Materials and Methods) recorded from an exemplar VB neuron of a WT mouse is greatly enhanced by etomidate (3 μm). B, All-points histograms illustrating the amplitude of the holding current (normalized to the current in the presence of bicuculline) under control (Ctrl) conditions (gray), in the presence of etomidate (3μm; white), and after the application of bicuculline (30 μm; black) recorded from exemplar VB neurons of a WT (left) and β_2N265S (right) mouse. C, Bar graph showing the inward current induced by 3μm etomidate (left) and 100 μm pentobarbital (right) in VB neurons of WT, β_2N265S, and β₂^–/– mice. Data were obtained from five to six cells. **p < 0.001 versus wild type (unpaired Student's t test). Error bars indicate the SE of the arithmetic mean.

Figure 4.

THIP potently activates a tonic current in VB but not nRT neurons. A, The tonic current (calculated as the difference between the holding current (picoamperes) in the presence and absence of 30 μm bicuculline (see Materials and Methods) recorded from an exemplar VB neuron of a WT mouse is greatly increased by THIP (1μm). B, A corresponding all-points histogram illustrating the amplitude of the holding current (normalized to the current in the presence of bicuculline) under control (Ctrl) conditions (gray), in the presence of THIP (100 nm to 3 μm; white), and after the application of 30 μm bicuculline (black) recorded from an exemplar VB neuron of a WT mouse. C, Neither THIP (1μm) nor bicuculline (30μm) has any effect on the holding current recorded from an exemplar nRT neuron of a WT mouse. D, Bar graph showing the current induced by 30 μm bicuculline (i.e., tonic current), 3 μm etomidate (Etom), 1 μm zolpidem (Zolp), and 1–3μm THIP in VB and nRT neurons of WT mice. Data were obtained from four to nine cells. Error bars indicate the SE of the arithmetic mean.