Bidirectional regulation of intravenous general anesthetic actions by α3-containing γ-aminobutyric acid A receptors

Abstract

Background: γ-aminobutyric acid A (GABAA) receptors mediate the actions of several intravenous general anesthetics. However, the contribution of α3-containing GABAA receptors to the action of these drugs is unknown.

Methods: The authors compared anesthetic endpoints (hypnosis, immobility, hypothermia) in response to various intravenous anesthetics in mice lacking the α3 subunit of the GABAA receptor (α3 knockout) and in wild-type mice. Furthermore, the authors generated and analyzed conditional mutant mice expressing the GABAA receptor α3 subunit exclusively in noradrenergic neurons.

Results: α3 knockout mice displayed decreased hypnotic and hypothermic responses to etomidate and midazolam, but an increased response to pentobarbital. The hypnotic response to ketamine was unaltered, whereas the hypothermic response was increased. In contrast, the hypnotic but not the hypothermic response to medetomidine was increased. The combination of ketamine/xylazine displayed increased hypnotic, immobilizing, and hypothermic effects in α3 knockout mice. Mice expressing the α3 subunit exclusively in noradrenergic neurons were generated to assess whether the lack of α3 subunits on noradrenergic neurons may be responsible for this effect. In these mice, the increases of the hypnotic and immobilizing actions induced by ketamine/xylazine were largely absent, whereas the increase in the hypothermic action was still present.

Conclusion: α3-containing GABAA receptors bidirectionally regulate essential anesthetic actions: they mediate anesthetic actions of etomidate and midazolam, known to selectively act at GABAA receptors, and they negatively constrain anesthetic actions of compounds with targets partly or exclusively distinct from GABAA receptors such as medetomidine, ketamine, and pentobarbital. Furthermore, our results indicate that α3-containing GABAA receptors on noradrenergic neurons may contribute to this constraint.

Figure 1. Effect of ketamine, pentobarbital, midazolam, and etomidate on duration of LORR (A) and core body temperature (B)

A. Duration of loss of righting reflex (LORR) was significantly increased in α3KO mice by 60 mg/kg pentobarbital (repeated measures ANOVA followed by t-tests with Bonferroni correction) and significantly decreased by 30 mg/kg etomidate (unpaired t-test). B. Repeated measures ANOVA showed a mean effect of genotype on core body temperature for 250mg/kg ketamine, 75mg/kg midazolam and 30 mg/kg etomidate. t-tests with Bonferroni correction revealed that the temperature decrease was more pronounced in α3KO mice compared to wild type mice 60 minutes and 120 minutes after administration of ketamine and pentobarbital, respectively and that the temperature decrease was less pronounced in α3KO mice compared to wild type mice 90 minutes after administration of midazolam or etomidate. Data represent the mean ± SEM; *, P<0.05, **, P<0.01 and ***, P<0.001, n=9 wild type and n=7 α3KO for ketamine; n=9 for pentobarbital; n=11 for etomidate and midazolam. WT, wild type; KO = α3KO.

Figure 2. Effects of medetomidine on locomotor activity (A), duration of LORR (B) and body temperature (C) in α3KO and wild type mice

A. Locomotor activity was comparable in both genotypes at all medetomidine doses over the whole 60 minute time period (insert) and looking at 5-minute time bins. B. Duration of loss of righting reflex (LORR) was significantly increased in α3KO mice by 250 μg/kg medetomidine compared to wild type mice (Sheffe post-hoc tests). C. Body temperature was comparable between α3KO mice and wild type mice. Data represent the mean ± SEM; n=9 per genotype.

Figure 3. Schematic of genetic rescue of the α3 knockout in noradrenergic neurons

A. α3KO and Rescue alleles. theα3KO allele (top) has integrated the insertion-type gene targeting vector for single reciprocal recombination in its entirety. It carries an artificial exon (light green, 5*) flanked by loxP sites (red), targeting vector-derived homologous genomic sequences (light brown) and the plasmid backbone of the targeting vector (blue). Genomic sequences that were not included in the targeting vector are shown as dark brown. The most relevant feature is that the α3KO allele contains the endogenous exon 5 (dark green, 5) and an artificial exon 5 (light green, 5*), which results messenger RNA (mRNA) degradation, which is the likely mechanism of the α3 knockout. In the Rescue allele, elimination of the artificial exon by Cre-loxP-mediated recombination results in α3 mRNA (orange) being made and thus the expression of the α3 subunit being restored. B. Exonic structure of predicted gene products from wild type (WT), α3KO and Rescue allele. Presence of the artificial exon 5, 5*, in the α3KO results in mRNA breakdown and thus a functional knockout. The exonic structure of wild type (WT) and Rescue alleles is identical.

Figure 4. Localization and quantification of the α3 knockout in global rescue mice

A. Generation of global Rescue mice. An α3KO mouse (with artificial exon 5, the “STOP” signal) is bred with an EIIa-Cre mouse. Some of the offspring will carry both the α3KO allele and the EIIa-Cre transgene. This mouse is bred with a wild type mouse to breed out the EIIa-Cre transgene (depicted by two sequential horizontal arrows) to confirm that the artificial exon 5 (the “STOP” signal) has been removed from the germline, so that the α3 subunit is expected to be expressed in all cells in which it is naturally expressed. B. Immunoperoxidase staining of perfusion-fixed parasagittal brain sections: α3 subunit distribution pattern is equivalent in wild type and global rescue mice. Representative sections from 2 wild type and 2 global rescue mice are shown. C. Left panel: Representative Western Blot: α3 subunit expression level is decreased in global rescue mice compared to wild type mice. Right panel: Quantification of Western Blot signal: signals were normalized to the α3 subunit signal at 10 μg protein in wild type mice (100%). α3 subunit expression level in global rescue mice is 70±7% of wild type expression. Data represent the mean±SEM of five experiments.

Figure 5. Recue of α3 expression in defined neuronal cell types

A. An α3KO mouse is crossed with a dopamine β hydroxylase impoved Cre (DBH-iCre) mouse to obtain DBH-iCre rescue mice (in our studies hemizygous males) containing both the α3 knockout allele and the DBH-iCre transgene. The artificial exon 5 in the α3 knockout allele functions as a “STOP” signal resulting in messenger RNA degradation. In noradrenergic neurons of the rescue mouse, this “STOP” signal is removed by cre-loxP-mediated recombination. Thus, in the Rescue mice, the α3 subunit is expressed only in the noradrenergic neurons. Similar considerations apply to the generation of the dopamine transporter (DAT)-Rescue mice using the DAT-iCre transgene expressing iCre specifically in dopaminergic neurons. B. Neuron-specific rescue of α3 subunit expression in the locus coeruleus: Immunofluorescence double-labeling of the α3 subunit (red) and tyrosine hydroxylase (green) shows that the α3 subunit is highly expressed in the locus coeruleus (LC) in wild type mice (WT). In α3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue) α3 subunit expression is restricted to noradrenergic neurons. It is not detectable in the locus coeruleus of α3KO mice expressing the iCre recombinase exclusively in dopaminergic neurons (DAT-Rescue), nor in α3KO mice. Scale bar: 50μm. C. Neuron-specific rescue of α3 subunit expression in the substantia nigra pars compacta: Immunofluorescence double-labeling of the α3 subunit (red) and tyrosine hydroxylase (green) shows that α3 subunit expression is found in dopaminergic neurons of the substantia nigra pars compacta (SNpc) of wild type mice (WT) and in α3KO mice expressing the iCre recombinase selectively in dopaminergic neurons (DAT-Rescue). It is not detectable in the substantia nigra pars compacta of α3KO mice expressing the iCre recombinase selectively in noradrenergic neurons (DBH-Rescue), nor in α3KO mice. Scale bar: 50μm.

Figure 6. Effect of Ketamine (139 mg/kg)/Xylazine (21 mg/kg) on duration of LORR (A) and core body temperature (B)

A. Duration of loss of righting reflex (LORR) was significantly increased in α3KO mice compared to wild type mice, whereas mice expressing the α3 subunit exclusively in noradrenergic neurons [dopamine β hydroxylase (DBH)-Rescue] and mice carrying the improved cre (iCre) transgene (DBH-iCre) did not differ from wild type mice (1-way ANOVA followed by post hoc Dunnett's t-tests). B. Body temperature was significantly lower in α3KO mice compared to wild type mice 150 minutes and 180 minutes after injection. Body temperature in DBH-Rescue mice was significantly lower 180 minutes after injection compared to wild type mice (2-way repeated measures ANOVA followed by post hoc t-tests with Bonferroni correction). Data represent the mean ± SEM; ***, P<0.001 (α3KO compared to wild-type), *, P<0.05 (α3KO compared to wild-type), #, P<0.05 (DBH-Rescue compared to wild type); n=15 wild type and DBH-iCre, n=14 α3KO and DBH-Rescue. WT, wild type.