Alpha5GABAA receptors mediate the amnestic but not sedative-hypnotic effects of the general anesthetic etomidate

Abstract

A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABAA receptors that contain the alpha5 subunit (alpha5GABAARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by alpha5GABAARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not alpha5 null mutant (alpha5-/-) mice. In addition, etomidate impaired memory performance of WT but not alpha5-/- mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The alpha5-/- mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and alpha5-/- mice. Deletion of the alpha5 subunit of the GABAARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABAAR subtype pharmacology.

Figure 1.

Etomidate selectively enhanced the tonic current recorded in cultured hippocampal pyramidal neurons from Swiss White mice. A, Current traces illustrate the effects of etomidate (0.1 and 1 μm) on the tonic current and mIPSCs. B, The increase in net charge transfer by etomidate was greater for the tonic currents than for time-averaged synaptic currents, as summarized in the bar chart. The low concentration of etomidate (0.1 μm) caused no increase in charge transfer associated with the mIPSCs.

Figure 2.

Low concentrations of etomidate increased the holding current in WT but not α5−/− neurons. A, Current traces illustrate the increase in an inward current at different concentrations of etomidate in WT neurons (top trace) and α5−/− neurons (bottom trace). Note the difference in amplitude of the scale bars. B, The bar chart shows that the amplitude of the holding current was greater in WT than in α5−/− neurons. Note that the scale of the y-axis was increased for 1.0 and 10 μm etomidate.

Figure 3.

Etomidate reduced the LTP of fEPSPs recorded in the CA1 region of hippocampal slices prepared from WT but not α5−/− mice. A, The superimposed traces show averaged fEPSPs recorded from WT mice in the presence of vehicle or etomidate (1 μm) at baseline (1) and 60 min after TBS (2). The time-dependent change in the slope of the fEPSPs recorded in the absence or presence of etomidate (1 μm) is summarized in the graphs. B, Current traces and time-dependent changes in the slope of the fEPSPs for recordings in hippocampal slices from α5−/− mice are shown. Etomidate (1 μm) significantly reduced LTP elicited by 1 s of TBS in pyramidal neurons from WT mice (n = 6) but not α5−/− mice (n = 6) at CA1-Schaffer collateral synapses.

Figure 4.

Etomidate impaired contextual fear conditioning in WT but not α5−/− mice. A, The bar graphs show the effects of vehicle (black) and etomidate (white; 4 mg/kg, i.p.) on the freezing scores (mean ± SEM). The scores were reduced in the WT but not the α5−/− mice, which indicates impairment of memory acquisition after the administration of etomidate. B, Compared with vehicle control (black), ketamine (white; 20 mg/kg, i.p., gray) caused a similar reduction in freezing scores, which indicates a similar impairment in fear conditioning for the same context. C, Spatial learning was impaired by etomidate (4 mg/kg, i.p.) in WT but not α5−/− mice. The Morris water maze probe trial showed that etomidate (black) reduced the amount of time the mice spent in the area where the platform had been located the previous day. Impaired memory retrieval was shown by WT but not α5−/− mice. D, In contrast, impairment by ketamine did not depend on the mouse genotype. E, No differences were detected in the visible platform trial (E) or swim speed (F) between the two genotypes (WT control, 0.24 ± 0.01 m/s, n = 16 vs WT etomidate, 0.24 ± 0.01 m/s, n = 15, p > 0.05; α5−/− control, 0.24 ± 0.02 m/s, n = 15 vs α5−/− etomidate, 0.24 ± 0.2 m/s, n = 15, p > 0.05).

Figure 5.

Etomidate impairment of motor coordination, spontaneous motor activity, and LORR was not increased in α5−/− mice. A, Etomidate (4 mg/kg) caused impairment of motor performance in the 12 rpm rotarod test. One preinjection trial was performed on the test day before the mice were treated with etomidate (WT, n = 16; α5−/−, n = 15). The results are expressed as the latency to fall off the rotarod (mean ± SEM). Both groups had impaired responses 5 min after injection (*p > 0.05, one-way ANOVA) but not at 30 or 60 min. B, Spontaneous locomotor activity (walking) was reduced by etomidate as shown for the open field test. No difference in baseline activity was observed between WT and α5−/− mice after injection of the vehicle control. Etomidate (4 mg/kg and 10 mg/kg, i.p.) caused a concentration-dependent reduction in locomotion that was similar in WT and α5−/− mice. C, Dose–response analysis for etomidate causing the LORR is shown. The data points represent the percentage of the WT (filled square) or α5−/− (open square) mouse populations that exhibited LORR at the dose indicated on the abscissa. Overlapping data points for the two genotypes are represented by a third symbol (filled circle). Mice were tested at doses of 5, 7.5, 10, 12.5, 15, and 20 mg/kg, and a total of 69 mice were studied. The fitted curves were generated using a sigmoidal equation that provided the following values: WT ED₅₀ = 9.6 mg/kg ± 1.1, Hill slope parameter h = 5.8 ± 1.8; α5−/− ED₅₀ = 9.2 ± 1.1, h = 5.7 ± 1.5, p < 0.05. D, The latency to LORR recorded from the time of etomidate injection. Error bars show the mean ± SEM. Each data point represents six to eight mice, and no differences were detected between WT and α5−/− mice. The time to LORR was significantly different at all doses of etomidate (ANOVA; p < 0.05) with the exception of 10 versus 12.5 mg/kg.