The Journal of Neuroscience, May 15, 2001, 21(10):3409–3418 Loss of the Major GABAA Receptor Subtype in the Brain Is Not Lethal in Mice Cyrille Sur, Keith A. Wafford, David S. Reynolds, Karen L. Hadingham, Frances Bromidge, Alison Macaulay, Neil Collinson, Gillian O’Meara, Owain Howell, Richard Newman, Janice Myers, John R. Atack, Gerard R. Dawson, Ruth M. McKernan, Paul J. Whiting, and Thomas W. Rosahl Neuroscience Research Center, Merck Sharp and Dohme Research Laboratories, Harlow, Essex, CM20 2QR, United Kingdom ␣ ␤ ␥ ␣ Ϫ Ϫ The 1 2 2 is the most abundant subtype of the GABAA electrophysiological recordings from 1 / mice GABA cur- receptor and is localized in many regions of the brain. To gain rents in these neurons are dramatically reduced, and residual more insight into the role of this receptor subtype in the mod- currents have a benzodiazepine pharmacology characteristic of ulation of inhibitory neurotransmission, we generated mice ␣2- or ␣3-containing receptors. In contrast, the cerebellar Pur- lacking either the ␣1or␤2 subunit. In agreement with the kinje neurons from ␤2Ϫ/Ϫ mice have only a relatively small Ͼ ␤ Ϫ Ϫ reported abundance of this subtype, 50% of total GABAA reduction of GABA currents. In 2 / mice expression levels receptors are lost in both ␣1Ϫ/Ϫ and ␤2Ϫ/Ϫ mice. Surprisingly, of all six ␣ subunits are reduced by ϳ50%, suggesting that the homozygotes of both mouse lines are viable, fertile, and show ␤2 subunit can coassemble with ␣ subunits other than just ␣1. ␣ Ϫ Ϫ ␣ ␤ ␥ no spontaneous seizures. Initially half of the 1 / mice died Our data confirm that 1 2 2 is the major GABAA receptor prenatally or perinatally, but they exhibited a lower mortality subtype in the murine brain and demonstrate that, surprisingly, rate in subsequent generations, suggesting some phenotypic the loss of this receptor subtype is not lethal. drift and adaptive changes. Both adult ␣1Ϫ/Ϫ and ␤2Ϫ/Ϫ mice ␤ Ϫ Ϫ demonstrate normal performances on the rotarod, but 2 / Key words: GABAA receptor; mouse; cerebellum; radioli- mice displayed increased locomotor activity. Purkinje cells of gand; benzodiazepine; inhibitory current; locomotor activity; the cerebellum primarily express ␣1␤2␥2 receptors, and in rotarod The GABAergic system is the major contributor of the inhibitory heterozygotes have a normal life expectancy and demonstrate tone throughout the CNS. GABAA receptors are ligand-gated ion neophobia in a novel environment (Crestani et al., 1999). The channels that exist as a number of different subtypes. The lethality of the ␥2Ϫ/Ϫ mice can be rescued by transgenic overex- ␥ ␥ GABAA receptor is a pentameric structure, which is formed by pression of either the 2S or 2L subunit isoforms of the GABAA the coassembly of subunit polypeptides that exist in a large receptor indicating that both ␥2 subunit splice variants can sub- multigene family (McKernan and Whiting, 1996; Barnard et al., stitute for each other (Baer et al., 2000; Wick et al., 2000). Mice ␤ 1998). There are at least 16 different members of the GABAA lacking the 3 subunit of the GABAA receptor have cleft palate, receptor gene family, including 6␣,3␤,3␥, ␦, ⑀, ␪, and ␲ subunits epilepsy, and many behavioral characteristics of Angelman syn- (Whiting et al., 1999). The GABAA receptor genes are differen- drome (Culiat et al., 1995; Homanics et al., 1997; DeLorey et al., tially expressed both temporally and spatially throughout the 1998). Most of the ␤3Ϫ/Ϫ mice die as neonates, but the survivors, ␣ ␣ ␤ mammalian brain. For example the 2, 3, and 3 subunits are which are runted until weaning, can achieve normal body size by ␣ ␤ the major and subunits in the fetal brain, respectively, adulthood. In contrast, mice lacking the ␣6 subunit of the whereas the ␣1 and ␤2 subunits are mainly expressed after birth GABAA receptor, which is expressed exclusively in cerebellar (Zhang et al., 1991; Laurie et al., 1992b). Therefore, in the adult granule cells, have no major phenotypic abnormalities (Jones et ␣ ␤ ␥ brain the 1 2 2 subtype is the major subtype accounting for al., 1997). Expression of the ␦ subunit is inhibited in the ␣6Ϫ/Ϫ ϳ 43% of all GABAA receptors, whereas the remaining receptors mice, suggesting that both subunits form functional GABA ␣ ␣ A are made up mostly by 2- and 3-containing receptors together receptor subtypes in the cerebellar granule cells. Finally, mice with other combinations of quantitatively more minor GABAA deficient for the ␦ subunit are viable but show attenuated sensi- receptor subtypes (McKernan and Whiting, 1996). tivity to neuroactive steroids and epileptic seizures (Mihalek et Several mouse strains lacking individual GABAA receptor sub- al., 1999). units have been generated to study the physiological role of GABA receptors are the site of action of a number of clini- ␥ A GABAergic system in the living organism. Mice lacking the 2 cally important drugs, including benzodiazepines, barbiturates, subunit die shortly after birth (Gunther et al., 1995), whereas and anesthetics (Sieghart, 1995; Whiting et al., 1995). The ␣1␤2␥2 subtype is of particular interest in this context because it com- Received Dec. 19, 2000; revised Feb. 14, 2001; accepted Feb. 23, 2001. prises the major benzodiazepine binding site in the brain. We C.S., K.W., and D.R. contributed equally to different aspects of this work. addressed the question about the physiological role of this recep- Correspondence should be addressed to Dr. Thomas W. Rosahl, Merck Sharp and tor subtype by generating mice lacking either the ␣1or␤2 subunit Dohme Neuroscience Research Center, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, UK. E-mail: [email protected]. of the GABAA receptor which are thought to primarily coas- Copyright © 2001 Society for Neuroscience 0270-6474/01/213409-10$15.00/0 semble to form the ␣1␤2␥2 subtype. • 3410 J. Neurosci., May 15, 2001, 21(10):3409–3418 Sur et al. Mice Lacking the Major GABAA Receptor Subtype MATERIALS AND METHODS wild-type mice at postnatal days 11–17, and the vermal layer was isolated ␣ Ϫ Ϫ ␣ and placed into ice-cold oxygenated dissociation media containing (in Generation of 1 / mice. The GABAA receptor 1 gene-targeting mM): 82 Na SO ,30KSO , 5 MgCl , 10 HEPES buffer, and 10 glucose vector was constructed from the same genomic 129/SvEv ␭ fixII clone, 2 4 2 4 2 at pH 7.4. Tissue was then stirred for 7 min in 10 ml of dissociation media which has been used for the introduction of the ␣1H101R mutation containing 3 mg/ml of protease XXIII (Sigma) at 37°C. The tissue was (McKernan et al., 2000). However, for the complete gene knock-out, then washed in warmed oxygenated dissociation media containing 1 exon 4 was disrupted at the MscI restriction site by cloning the 1.2 kb mg/ml bovine serum albumin and 1 mg/ml trypsin inhibitor and main- BstBIϩMscI and the 7 kb EcoRVϩBamHI DNA fragment blunt-ended tained under oxygenating conditions at room temperature in Tyrode’s into the targeting vector. A phosphoglycerate kinase I (PGK) neo and a solution (in mM: 150 NaCl, 4 KCl, 2 CaCl , 2 MgCl , 10 HEPES, and 10 thymidine kinase (TK) cassette were also engineered blunt-ended into 2 2 glucose, pH 7.4). Tissue was withdrawn as needed and triturated with a the loxP site containing targeting vector. After linearization with NotI fire-polished Pasteur pipette to liberate individual cells. Cells were the targeting vector was introduced into AB2.2 embryonic stem (ES) plated onto a glass coverslip and left to settle for at least 30 min before cells (Lexicon Genetics) as described (Soriano et al., 1991; Rosahl et al., use. Purkinje cell bodies were identified by their characteristic size and 1993, 1995). Homologous recombinants were identified by PCR using the morphology. Cells could be used for up to 5 hr after preparation. primers 5Ј-ATTAATGGAGAGTGTGGTAATCTTT-3Ј and 5Ј-GGAT- Glass coverslips containing the dissociated cells were placed in a GCGGTGGGCTCTATGGCTTCTGA-3Ј and were further confirmed perspex recording chamber on the stage of a Nikon Diaphot inverted by genomic Southern blotting. Correctly targeted ES cell clones were microscope. Cells were perfused continuously with artificial CSF (aCSF) injected into C57BL6 blastocysts, and one of three clones gave rise to containing (in mM): 149 NaCl, 3.25 KCl, 2 CaCl , 2 MgCl , 10 HEPES, highly chimeric males, which transmitted the targeted allele into the germ 2 2 11 D-glucose, D(ϩ)-sucrose, pH 7.4, and observed with phase-contrast line. A colony of homozygous and wild-type control animals were estab- optics. Fire-polished patch pipettes were pulled on a WZ, DMZ- lished and used for the present study. Some ␣1Ϫ/Ϫ mice were crossed Universal puller (Zeitz-Instruments, Munich, Germany) using conven- with a cre-transgenic mouse (Schwenk et al., 1995) and further interbred tional 120TF-10 electrode glass. Pipette tip diameter was ϳ1.5–2.5 ␮M, to establish ␣1 homozygotes, which no longer contained the neomycin with resistances ϳ4M⍀. The intracellular solution contained (in mM): resistance gene marker. 130 CsCl, 10 HEPES, 10 BAPTA-Cs, 5 ATP-Mg, 0.1 leupeptin, and 1 Mice lacking the ␤2 subunit were generated in a similar way. A 17.5 kb MgCl , with 100 ␮M NaCO , pH-adjusted to 7.3 with CsOH and 320–340 genomic ␭/FixII clone containing exons 6, 7, and 8 of the ␤2 subunit was 2 3 mOsm. Cells were voltage-clamped at Ϫ60 mV using an Axopatch 200B subcloned into pBluescript via the NotI sites. An 8.5 kb SalI DNA amplifier (Axon Instruments, Foster City, CA). Drug solutions were fragment as a long arm and a 1.25 kb HpaI ϩ FspI DNA fragment as a applied to the cells via a multibarrel drug delivery system, which could short arm were cloned into the PGK neo and TK containing modified pivot the barrels into place using a stepping motor.