Etomidate Impairs Long-Term Potentiation In Vitro by Targeting α5-Subunit Containing GABAA Receptors on Nonpyramidal Cells

Abstract

Previous experiments using genetic and pharmacological manipulations have provided strong evidence that etomidate impairs synaptic plasticity and memory by modulating α5-subunit containing GABAA receptors (α5-GABAARs). Because α5-GABAARs mediate tonic inhibition (TI) in hippocampal CA1 pyramidal cells and etomidate enhances TI, etomidate enhancement of TI in pyramidal cells has been proposed as the underlying mechanism (Martin et al., 2009). Here we tested this hypothesis by selectively removing α5-GABAARs from pyramidal neurons (CA1-pyr-α5-KO) and comparing the ability of etomidate to enhance TI and block LTP in fl-α5 (WT), global-α5-KO (gl-α5-KO), and CA1-pyr-α5-KO mice. Etomidate suppressed LTP in slices from WT and CA1-pyr-α5-KO but not gl-α5-KO mice. There was a trend toward reduced TI in both gl-α5-KO and CA1-pyr-α5-KO mice, but etomidate enhanced TI to similar levels in all genotypes. The dissociation between effects of etomidate on TI and LTP in gl-α5-KO mice indicates that increased TI in pyramidal neurons is not the mechanism by which etomidate impairs LTP and memory. Rather, the ability of etomidate to block LTP in WT and CA1-pyr-α5-KO mice, but not in gl-α5-KO mice, points toward α5-GABAARs on nonpyramidal cells as the essential effectors controlling plasticity in this in vitro model of learning and memory.

Keywords: GABAA receptor; LTP; learning and memory; mechanisms of anesthesia.

Figure 1.

GABA_AR α5 subunit KO mice. A, Generation of the floxed and conditional KO alleles. P1–P3, PCR primers used to detect floxed and KO alleles (loxP sites and primers not drawn to scale). B, Immunohistochemical detection of the GABA_AR α5 subunit in the hippocampus of conditional and global α5 KO mice. Top row, fl–α5 mice (pseudo-WT) at 4, 8, and 15 weeks of age. Middle row, CA1–pyr–α5–KO mice at 4, 8, and 21 weeks of age. Bottom row, gl–α5–KO mice at 23 weeks of age. Note that CA1–pyr–α5–KO mice display apparently normal staining for the α5 subunit at 4 weeks of age, with progressive reductions at 8 and 21 weeks of age in the CA1 region driven by the expression of the CaMKIIα–Cre transgene starting in the third postnatal week (Tsien et al., 1996).

Figure 2.

LTP is suppressed by etomidate in fl–α5 and CA1-pyr-α5–KO mice but not gl–α5–KO mice. A, Etomidate (Etom) suppressed LTP in brain slices from fl–α5 mice. A TBS was delivered at time 0. Control (Ctrl) experiments were performed in drug-free conditions. Etomidate experiments were performed in the continuous presence of etomidate at the indicated concentrations, in brain slices that had been equilibrated with etomidate for at least 1 h before initiating the recording. Data points indicate mean ± SEM. Inset traces show representative fEPSPs recorded before TBS (thick lines) and 60 min after TBS (thin lines). B, Etomidate did not suppress LTP in brain slices from gl–α5–KO mice. C, Etomidate suppressed LTP in brain slices from CA1–pyr–α5–KO mice 8 weeks (gray circles) and 16-weeks-old (gray squares). D, Summary of LTP results. Bars show fEPSP slope (mean ± SEM) during the last 11 min (50–60 min) of recording. *p < 0.05, **p < 0.01.

Figure 3.

Etomidate potentiates tonic inhibition in fl–α5 and KO mice. A–C, Spontaneous inhibitory currents recorded in CA1 pyramidal neurons of brain slices prepared from fl–α5 (A), gl–α5–KO (B), and CA1–pyr–α5–KO (C) mice, in the absence (Ctrl) and presence of etomidate (Etom; 0.5 and 1 μm). Bars above traces represent application of the noncompetitive GABA_AR channel blocker PTX (100 μm). D, Summary bar plot of amplitudes (mean ± SEM) of tonic inhibition measured in the three genotypes under different conditions. **p < 0.005, ***p < 0.0005, ****p < 0.0001; n = 5–9 cells per group.

Figure 4.

Characterization of baseline responses in fl–α5 and KO mice. A, B, Input–output relationships for fEPSP slope (A) and PS amplitude (B), obtained over a range of stimulus intensities in brain slices prepared from fl–α5 (n = 7), gl–α5–KO (n = 6), and CA1–pyr–α5–KO (n = 8) mice. The slopes of fEPSPs recorded in the stratum radiatum and amplitudes of PSs recorded near the stratum pyramidale were normalized to the maximum values attained for each brain slice. The stimulus intensities applied to each brain slice varied; therefore, values were sorted and averaged in 50 μA bins. C, Input–output relationship between fEPSP slope and PS amplitude for each mouse strain. Data are shown as mean ± SEM.