Sedation and anesthesia mediated by distinct GABA(A) receptor isoforms - PubMed (original) (raw)

. 2003 Sep 17;23(24):8608-17.

doi: 10.1523/JNEUROSCI.23-24-08608.2003.

Thomas W Rosahl, Jennifer Cirone, Gillian F O'Meara, Alison Haythornthwaite, Richard J Newman, Janice Myers, Cyrille Sur, Owain Howell, A Richard Rutter, John Atack, Alison J Macaulay, Karen L Hadingham, Peter H Hutson, Delia Belelli, Jeremy J Lambert, Gerard R Dawson, Ruth McKernan, Paul J Whiting, Keith A Wafford

Affiliations

Sedation and anesthesia mediated by distinct GABA(A) receptor isoforms

David S Reynolds et al. J Neurosci. 2003.

Abstract

The specific mechanisms underlying general anesthesia are primarily unknown. The intravenous general anesthetic etomidate acts by potentiating GABA(A) receptors, with selectivity for beta2 and beta3 subunit-containing receptors determined by a single asparagine residue. We generated a genetically modified mouse containing an etomidate-insensitive beta2 subunit (beta2 N265S) to determine the role of beta2 and beta3 subunits in etomidate-induced anesthesia. Loss of pedal withdrawal reflex and burst suppression in the electroencephalogram were still observed in the mutant mouse, indicating that loss of consciousness can be mediated purely through beta3-containing receptors. The sedation produced by subanesthetic doses of etomidate and during recovery from anesthesia was present only in wild-type mice, indicating that the beta2 subunit mediates the sedative properties of anesthetics. These findings show that anesthesia and sedation are mediated by distinct GABA(A) receptor subtypes.

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Figures

Figure 1.

Figure 1.

Generation of β2 N265S mutant mice. Targeting strategy for the GABAA receptor β2 N265S mice. Schematic representation of the wild-typeβ2 allele of the GABAA receptor (a) and targeting vector including the site-specificβ2N265S mutation indicated by an asterisk (b). _E, Eco_RI; K, _Kpn_I restriction sites; 6, 7, 8, exons 6, 7, and 8, respectively; black arrowhead, loxP site; neo, neomycin resistance gene; TK, thymidine kinase gene; P1–P6, oligonucleotides used for PCR (see Materials and Methods). Targeted β2 allele after homologous recombination before (c) and after (d) _cre_-mediated excision of the loxP flanked neomycin cassette.

Figure 2.

Figure 2.

In vitro validation ofβ2 N265S mice. Genotyping examples of tail biopsy samples from wild-type (WT), heterozygous (HE), and homozygous (HO) mice using primers P3 and P4 (see Material and Methods) and digestion of the resulting PCR product with the restriction endonuclease _Sca_I (A). The 800 bp PCR product is nearly fully cut into two 400 bp fragments for homozygous, less cut for heterozygous, and not cut at all for wild-type samples. M, Marker. Quantitative Western blots showing analysis of P2 membranes from wild-type (WT), heterozygous (HE), and homozygous (HO) mice using anti-β1 (B), anti-β2 (C), and anti-β3 (D) antibodies (green). Immunoreactive intensities were quantified and normalized against anti-actin controls (red). The comparison of β subunit expression across genotypes is represented graphically in E–G. OD, Optical density. Example autoradiographs of brain sections from wild-type (H, J) and β2 N265S (I, K) mice showing [35S]TBPS binding sites. In J and K, [35S]TBPS binding was performed in the presence of 5 μ

m

loreclezole to allosterically inhibit binding toβ2 andβ3 subunit-containing receptors. Scale bar, 2 mm.

Figure 3.

Figure 3.

Electrophysiological properties of cerebellar Purkinje neurons in wild-type andβ2 N265S mice. Representative recordings from dissociated cerebellar Purkinje neurons isolated from wild-type or β2 N265S mice in response to a predetermined EC20 concentration of GABA, in the absence and presence of 3 μ

m

loreclezole (a), 3 μ

m

etomidate (b), and 100 μ

m

pentobarbital (c). Associated histograms illustrate mean potentiation ± SEM from four or more cells in each case. d, Overlayed averaged mIPSCs recorded from Purkinje cells from wild-type (dark) and β2 N265S (light) mice. e, Representative traces illustrating the effect of 10 μ

m

etomidate on wild-type (i) and β2 N265S (ii) mIPSCs from Purkinje cells. f, Cumulative probability plots for _T_90 values in the presence and absence of 10μ

m

etomidate obtained from an exemplar neuron of wild-type (i) and β2 N265S (ii) mice show that etomidate preferentially prolonged mIPSC decay of wild-type mice [from 6.32 ± 0.47 to 61.9 ± 10.55 msec (n = 5) in wild-type mice, p < 0.01, paired Student's t test; from 5.56 ± 0.21 to 18.52 ± 0.62 msec (n = 5) inβ2 N265S mice, p < 0.001, paired Student's t test] Mean percentage increase of _T_90 values by 10 μ

m

etomidate from five neurons was 927 ± 220 and 225 ± 14 for wild-type andβ2 N265S mice, respectively (p < 0.01 by repeated-measures ANOVA).

Figure 4.

Figure 4.

Etomidate-induced LORR is preserved but sedation is decreased in β2 N265S mice. The duration of LORR (a; mean ± SEM duration) induced by intraperitoneal etomidate was very similar for both wild type (WT) and β2 N265S at all three doses of etomidate examined (F(1,57) < 0.005; _p_ > 0.1). Likewise, 50 mg/kg intraperitoneal pentobarbital produced similar LORR durations in both wild-type andβ2 N265S mice (F(1,20) = 2.24; p > 0.1). n = 10–11 for etomidate and pentobarbital groups. Intravenous etomidate produced a dose-dependent increase in LORR duration, although this was significantly shorter for the β2 N265S mice at the higher doses (n = 6–8). Propofol, an intravenous anesthetic not effected by the point mutation, gave a similar LORR in both genotypes (n = 5). Intravenous etomidate produced a dose-dependent increase in LOPWR (b; mean ± SEM duration), although this was shorter in theβ2 N265S mice (n = 7–8). Low doses of etomidate (0.3–7.5 mg/kg, i.p.) dose-dependently decreased spontaneous cage crossings (c; exploration–locomotion) in wild-type mice. In contrast, these doses of etomidate did not reduce activity inβ2N265Smice. At higher doses of etomidate (10 and 12.5 mg/kg), both wild-type andβ2 N265S mice behaved similarly (n = 31 for vehicle; n = 12 for 0.3, 1, 3, and 5 mg/kg; n = 18 for 7.5 mg/kg; n = 6–7 for 10 and 12.5 mg/kg). In the rotarod test of motor coordination (d), wild-type mice showed a marked deficit compared with vehicle-treated mice at 5 and 7.5 mg/kg intraperitoneal etomidate, whereas the performance ofβ2 N265S mice was not significantly affected. Data shown are mean ± SEM time on rotarod; n = 5–6 for vehicle and n = 10–11 for etomidate; +p < 0.05 compared with vehicle-treated mice; *p < 0.05 compared with wild-type mice receiving the same dose of etomidate.

Figure 5.

Figure 5.

β2 N265S mice recover more quickly from etomidate-induced LORR. Mice were tested for recovery of motor coordination after administration of etomidate (5–15 mg/kg, i.v.) or propofol (30 mg/kg, i.v.). Each group of mice were repeatedly tested on the rotarod starting from the recovery of righting reflex. a–c,β2 N265S mice recovered maximal rotarod performance much more quickly than wild-type mice (WT) (genotype, F(1,38) = 63.1; p < 0.00005), and this effect was more pronounced at higher doses of etomidate (drug dose, _F_(2,38) = 4.52; _p_ < 0.02). _d_, Both genotypes recovered at a similar rate after propofol-induced LORR (_F_(1,8) = 1.35; _p_ > 0.1). n = 6–8 for all etomidate groups; n = 5 for propofol.

Figure 6.

Figure 6.

EEG analysis of anesthesia and recovery inβ2 N265S mice. a, Data from wild-type (WT) andβ2 N265S mice showing differences in EEG across different stages of vigilance. i, Wake EEG is characterized by a low-amplitude signal, containing higher frequencies. ii, During slow-wave sleep, signal amplitude is increased, with slower frequencies evident. iii, Etomidate anesthesia produces a very different type of EEG waveform that is defined as burst suppression. Large-amplitude, fast bursts are interspersed by periods of relatively isoelectric EEG. iv, Magnification of a portion of EEG trace shown in iii. Suppressed EEG was defined as a waveform of <5μV amplitude, of not <100 msec duration, and is defined by dotted window. b, Depth of anesthesia as defined by EEG burst suppression activity is similar in both wild-type and β2 N265S mice. i, Percentage of suppression in EEG per minute after etomidate anesthesia (12.5 mg/kg, i.v.) is not significantly different inβ2 N265S mice compared with wild type. ii, Percentage EEG suppression is dose dependent. Higher levels of suppression are evident at 12.5 mg/kg intravenously in both genotypes (wild type, filled squares;β2 N265S, open triangles) compared with 10 mg/kg intravenously (wild type, open squares;β2 N265S, filled triangles), although there is no effect of genotype at both doses. c, There is increased sedation in wild-type compared with β2N265S mice after recovery from etomidate anesthesia.i, After recovery from 10mg/kg intravenous etomidate, increased levels of slow-wave sleep (compared with baseline circadian matched control) are evident in the first 2 hr after recovery. Sleep in β2 N265S mice is not significantly different from baseline. ii, After recovery from 12.5 mg/kg intravenous etomidate, there is enhanced SWS in the wild-type mice in the following 3 hr. Amount of SWS is not significantly enhanced in theβ2 N265S mouse.

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