Deletion of the 1 or 2 Subunit of GABAA Receptors Reduces Actions of Alcohol and Other Drugs

Enhancement of the activation of GABAA receptors is a common feature of many sedative and hypnotic drugs, and it is probable that the GABAA receptor complex is a molecular target for these drugs in the mammalian central nervous system. We set out to elucidate the role of the two predominant ( 1 and 2) subunits of GABAA receptor in sedative drug action by studying mice lacking these two subunits. Both 1 ( / ) and 2 ( / ) null mutant mice showed markedly decreased sleep time induced by nonselective benzodiazepine, flurazepam, and GABAA agonist, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol. The sleep time induced by the -selective drug etomidate was decreased only in 2 ( / ) knockout mice. In contrast, 1 ( / ) mice were more resistant to the 1-selective drug zolpidem than 2 ( / ) or wild-type animals. Knockout mice of both strains were similar to wild-type mice in their responses to pentobarbital. The duration of loss of the righting reflex produced by ethanol was decreased in male mice for both null alleles compared with wild-type mice, but there were no differences in ethanol-induced sleep time in mutant females. Deletion of either the 1 or 2 subunits reduced the muscimolstimulated Cl influx in cortical microsacs suggesting that these mutant mice have reduced number of functional brain GABAA receptors. Our results show that removal of either 1 or 2 subunits of GABAA receptors produce strong and selective decreases in hypnotic effects of different drugs. Overall, these data confirm the crucial role of the GABAA receptor in mechanisms mediating sedative/hypnotic effects. GABA, acting via GABAA receptors, is the brain’s major inhibitory neurotransmitter system in the central nervous system. Because the ability to enhance the ion channel activation of GABAA receptors is a common feature of many sedative and hypnotic drugs, it is probable that the action on the GABAA receptor complex is a molecular target for these drugs in the mammalian central nervous system (Charney et al., 2001). Nevertheless, many of these drugs, including ethanol and pentobarbital, may have multiple sites of action and the importance of specific GABAA receptors in drug action is not clearly defined. The GABAA receptor has a pentameric structure, which is formed by the coassembly of subunit polypeptides that exist in a large multigene family (McKernan and Whiting, 1996; Barnard et al., 1998). There are at least 16 different members of the GABAA receptor gene family, including 6 , 3 , 3 , , , , and subunits (Whiting et al., 1999). The largest population of GABAA receptors in the rat brain has a subunit composition of 1 2 2, whereas together 2 3 2 and 3 2/ 3 constitute the next most prevalent subtypes (McKernan and Whiting, 1996). GABA affinity is mainly governed by subunits (Smith et al., 2001), but affinity and efficacy at the benzodiazepine site are influenced by both and subunits (McKernan et al., 1995; Buhr et al., 1996; Wingrove et al., 1997) but not subunits (Hadingham et al., 1993). The benzodiazepine site occurs at the interface of an and a subunit, with residues in both influencing modulation (Smith and Olsen, 1995). The presence of 2 confers the classical benzodiazepine pharmacology to GABAA receptors (Pritchett et al., 1989). In contrast, subunits control loreclezole and etomidate sensitivity (Stevenson et al., 1995). Mouse strains lacking individual GABAA receptor subunits provide insight regarding the role of GABA receptors. Mice lacking the 2 subunit die shortly after birth (Gunther et al., 1995), whereas mice deficient the 2L (long splice variant) subunit are viable and show small increases in sleep time responses to midazolam and zolpidem, but responses to nonbenzodiazepine agents such as ethanol, etomidate, and pentobarbital are unchanged (Quinlan et al., 2000). 3 null mice ( / ) did not show any changes in sleep times after administration of This work was supported by funds from the Texas Commission on Alcohol and Drug Abuse and National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism Grants AA06399 and AA13520. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.042960. ABBREVIATIONS: THIP, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol; ANOVA, analysis of variance. 0022-3565/03/3041-30–36$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 304, No. 1 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 42960/1028615 JPET 304:30–36, 2003 Printed in U.S.A. 30 at A PE T Jornals on M ay 3, 2017 jpet.asjournals.org D ow nladed from pentobarbital or ethanol, but they were more resistant to etomidate and midazolam (Quinlan et al., 1998). Mice lacking the 6 subunit of the GABAA receptor, which is expressed exclusively in cerebellar granule cells, have no major phenotypic abnormalities (Jones et al., 1997). Mice deficient the subunit are also viable but show attenuated sensitivity to neuroactive steroids and epileptic seizures (Mihalek et al., 1999). Thus, deletion of some of the less abundant GABAA receptor subunits reduces the action of some sedative drugs, but they do not provide evidence for a major role for GABA receptors in actions of many sedatives. It is important to note, however, that the only predominant GABAA subunit that has been deleted is the 2 and this proved lethal. Recently, mice lacking the most predominant GABAA receptor subunits, 1 and 2, were successfully generated (Sur et al., 2001; Kralic et al., 2002). Although the mice lack approximately 60% of the total number of brain GABAA receptors, adult 1 ( / ) and 2 ( / ) mice do not display major phenotypic abnormalities or spontaneous seizures. 1 ( / ) mice showed overexpression of the 2 and 3 subunit but lack approximately 40% of the GABAA receptors despite this apparent compensation. In contrast, 2 ( / ) mice displayed an equal reduction in all six subunits and a loss of GABAA receptors of about 50% (Sur et al., 2001). In this study, we asked how this substantial decrease of benzodiazepine receptors and reduction of expression of different GABA receptor subtypes might affect receptor function by measuring agonist stimulated Cl uptake in brain tissue from these mutant mice. For the behavioral studies, we used the loss of righting reflex and tested a GABAA agonist, 4,5,6,7tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP), a nonselective benzodiazepine, flurazepam, and 1-selective drug, zolpidem, a -selective drug, etomidate, and two drugs with activities on several channels in addition to GABAA, ethanol and pentobarbital. Materials and Methods Animals. Null 1 ( / ) and 2 ( / ) allele mice were created using homologous recombination and genotyped as previously described in Sur et al. (2001). Homozygotes of the F2 generation were interbred avoiding any brother-sister mating, and homozygous colonies of 1 ( / ), 2 ( / ) and wild-type ( / ) mice were established. In this study, the mice from the F6 to F7 generations of this interbreeding have been used. All mice were at least 14 to 18 weeks of age at the time of analysis; within each experiment, all mice were of similar age. Mice were grouped housed (3–5 per cage) under a 12-h light/dark cycle (lights on at 7:00 AM) and provided ad lib access to food and water. All experiments were conducted in an isolated behavioral testing room in the animal facility to avoid external distractions. All mice were allowed to recover for at least 1 week between each drug treatment. Each group of mice, however, was not used for more than for two different drugs, and drug testing was randomized. All experiments were approved by the Institutional Animal Care and Use Committee. Loss of Righting Reflex. Sensitivity to ethanol (Aaper Alcohol and Chemical Co., Shelbyville, KT; 3.4, 3.6, and 3.8 g/kg, i.p.), flurazepam (Sigma-Aldrich, St. Louis, MO; 225.0 mg/kg, i.p.), zolpidem tartrate (gift from Dr. J. S. Vedo; Pharmacia, Skokie, IL; 60.0 mg/kg, i.p.), pentobarbital (Sigma/RBI, Natick, MA; 50.0 mg/kg, i.p.), etomidate (40.0 mg/kg, i.p.), and THIP (Sigma/RBI; 55.0 mg/kg, s.c.) was determined using the standard sleep time assay (Kakihana et al., 1966). Ethanol was diluted in 0.9% saline (20.0% w/v) and administered in doses adjusted by injected volumes. Flurazepam, zolpidem, and etomidate were dissolved in 3 to 4 drops of Tween 80 (SigmaAldrich) before saline was added and injected at 0.01 ml/g b.wt. Pentobarbital was dissolved in 0.9% saline and injected at 0.01 ml/g b.wt. THIP was dissolved in 0.9% saline and injected at 0.005 ml/g