Loss of the major GABA(A) receptor subtype in the brain is not lethal in mice

Abstract

The alpha1beta2gamma2 is the most abundant subtype of the GABA(A) receptor and is localized in many regions of the brain. To gain more insight into the role of this receptor subtype in the modulation of inhibitory neurotransmission, we generated mice lacking either the alpha1 or beta2 subunit. In agreement with the reported abundance of this subtype, >50% of total GABA(A) receptors are lost in both alpha1-/- and beta2-/- mice. Surprisingly, homozygotes of both mouse lines are viable, fertile, and show no spontaneous seizures. Initially half of the alpha1-/- mice died prenatally or perinatally, but they exhibited a lower mortality rate in subsequent generations, suggesting some phenotypic drift and adaptive changes. Both adult alpha1-/- and beta2-/- mice demonstrate normal performances on the rotarod, but beta2-/- mice displayed increased locomotor activity. Purkinje cells of the cerebellum primarily express alpha1beta2gamma2 receptors, and in electrophysiological recordings from alpha1-/- mice GABA currents in these neurons are dramatically reduced, and residual currents have a benzodiazepine pharmacology characteristic of alpha2- or alpha3-containing receptors. In contrast, the cerebellar Purkinje neurons from beta2-/- mice have only a relatively small reduction of GABA currents. In beta2-/- mice expression levels of all six alpha subunits are reduced by approximately 50%, suggesting that the beta2 subunit can coassemble with alpha subunits other than just alpha1. Our data confirm that alpha1beta2gamma2 is the major GABA(A) receptor subtype in the murine brain and demonstrate that, surprisingly, the loss of this receptor subtype is not lethal.

Fig. 1.

Generation and validation of α1 and β2 −/− mice. a, b, e,f, Schematic representation of the WT α1 (a, b) and β2 allele (e, f) of the GABA_A receptor and the corresponding targeting vectors.4, 6, 7, Exons 4, 6, and 7, respectively; 4Δ, partial deletion of exon 4;neo, neomycin resistance gene; TK,thymidine kinase gene; E, EcoRI restriction site;K, KpnI restriction site; P1, P2, P3, P4, PCR primers for detecting the mutant α1 and β2 allele, respectively. c,g, Targeted allele after homologous recombination for α1 and for β2 allele, and after cre-mediated removal of the neomycin resistance gene (d, h), respectively. i, Northern blot results using 45 mer oligonucleotides for the α1, α3, α6, β2, and β3 subunit genes of the GABA_A receptor and neomycin resistance (neo) and glyceraldehyde-3-phosphate dehydrogenase gene as probes; 5 μg of poly(A+) selected RNA was loaded per lane; α1+/+ and β2+/+: wild-type control samples; α1−/−[-neo] and β2−/−[-neo]: samples from α1 and β2 homozygotes lacking the neo gene in their genome; α1−/−[+neo] and β2−/−[+neo]: samples from α1 and β2 homozygotes with the neo gene in their genome. j, Western blot demonstrating the absence of α1 polypeptide in the brain of α1−/− mice.

Fig. 2.

Loss of GABA_A receptors in the forebrain and cerebellum of α1 −/− and β2 −/− mice. Color-coded autoradiograms for [³⁵S]TBPS (8 nm) binding to sections of mouse brain revealed a widespread reduction of GABA_A receptors. Major losses are observed in cortex (−66 and −46%), globus pallidus (−72 and −74%), thalamus (−65 and −63%), and cerebellum (−67 and −71%) for example of α1 −/− and β2 −/− mice, respectively. Scale bars, 1 mm.

Fig. 3.

Electrophysiological analysis of GABA currents recorded from cerebellar Purkinje neurons of wild-type and α1−/− and β2−/− mice. a, Example recordings from wild-type (WT), α1−/−, and β2−/− mice, of the response to 1 mm GABA, and potentiation of submaximal, EC₂₀ currents by 3 μm chlordiazepoxide or 100 nm zolpidem (this concentration would distinguish α1 from other subtypes). Amplitude is indicated by the scale bar in each case, and drugs were applied as shown by the bar above each response. B, Mean current amplitude in response to 1 mm GABA from isolated Purkinje neurons; nnumber is shown in parentheses above each column and includes all cells tested including unresponsive cells.c, Mean potentiation of GABA EC₂₀ by 3 μm chordiazepoxide; n number is shown inparentheses above each column. d, Mean potentiation of GABA EC₂₀ by 100 nm zolpidem;n number is shown in parentheses above each column.

Fig. 4.

Effects of loreclezole, etomidate, and pentobarbital on GABA receptors from WT and β2−/− Purkinje neurons.a, Recording from WT and β2−/− neurons showing a GABA response to 1 mm GABA followed by the potentiation of a GABA EC₂₀ response by 3 μm loreclezole, 30 μm etomidate, and 100 μm pentobarbital. Amplitude is indicated by the scale bar, and drugs were applied as shown by the bar above each response. b–d, Mean potentiation of the GABA EC₂₀ response by 3 μm loreclezole (b), 30 μmetomidate (c), and 100 μm pentobarbital (d); n number is shown inparentheses above each column.

Fig. 5.

Behavioral evaluation of α1 and β2 −/− mice.a, The duration a mouse remained walking on the rotarod revolving at different speeds (18–36 rpm) decreased as the speed was increased. α1 and β2 −/− mice did not differ from wild-type mice at any speed examined. Data are the means ± SEM;n = 6–9. The spontaneous locomotor activity of α1 −/− mice (b) did not differ from wild-type mice. In contrast, β2 −/− mice (c) showed a marked increase in activity (p < 0.005) compared with wild-type mice. Data are the mean number of cage crosses in 2 min time bins ± SEM; n = 10–12.

Fig. 6.

Generation-dependent upregulation of α2 and α3 subunits in cerebellum and forebrain of α1−/− mice.a, The loss of [³H]Ro15–1788 binding sites increased in cerebellum (left panel) and forebrain (right panel) of α1−/− mice from generation F2 to F5 to finally reach the proportion of [³H]Ro15–1788 sites immunoprecipitated by a selective α1 antibody from wild-type membrane (filled bars). ***p < 0.001; Student's t test. b, Evidence for an upregulation of α3 subunit in the cerebellum of α1−/− mice. Western blot showing an increase in α3 subunit expression in knock-out mice compared with wild-type (left panel). Quantitative immunoprecipitation with a α3 antibody demonstrated that α3 subunit-containing GABA_Areceptors account for 14 ± 3% (mean ± SEM;n = 3) and 92 ± 8% (mean ± SEM;n = 4) of total [³H]Ro15–1788 binding sites in the cerebellum of wild-type and α1−/− mice, respectively (right panel). c, Quantitative immunoprecipitation demonstrated a 42% increase in the expression of α2 and α3 subunit-sensitive [³H]Ro15–1788 binding sites in the forebrain of α1−/− compared with wild-type mice.