Molecular and Pharmacological Characterization of GABAA Receptor 1 Subunit Knockout Mice

GABAA receptors mediate fast inhibitory neurotransmission in the central nervous system (CNS), and approximately half of these receptors contain 1 subunits. GABAA receptor 1 subunits are important for receptor assembly and specific pharmacological responses to benzodiazepines. Plasticity in GABAA receptor 1 subunit expression is associated with changes in CNS excitability observed during normal brain development, in animal models of epilepsy, and upon withdrawal from alcohol and benzodiazepines. To examine the role of 1 subunitcontaining GABAA receptors in vivo, we characterized receptor subunit expression and pharmacological properties in cerebral cortex of knockout mice with a targeted deletion of the 1 subunit. The mice are viable but exhibit an intention tremor. Western blot analysis confirms the complete loss of 1 subunit peptide expression. Stable adaptations in the expression of several GABAA receptor subunits are observed in the fifth to seventh generations, including decreased expression of 2/3 and 2 subunits and increased expression of 2 and 3 subunits. There was no change in 4, 5, or subunit peptide levels in cerebral cortex. Knockout mice exhibit loss of over half of GABAA receptors measured by [ H]muscimol, [H]2-(3-carboxyl)3-amino-6-(4-methoxyphenyl)-pyridazinium bromide ([H]SR-95531), and t-butylbicyclophosphoro[S]thionate ([S]TBPS) binding. [H]Ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4Himidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([H]Ro154513) binding is reduced by variable amounts in different regions across brain. GABAA receptor 1 / mice lose all high-affinity [H]zolpidem binding and about half of [H]flunitrazepam binding in the cerebral cortex. The potency and maximal efficacy of muscimol-stimulated Cl uptake in cerebral cortical synaptoneurosomes are reduced in 1 / mice. Furthermore, knockout mice exhibit increased bicuculline-induced seizure susceptibility compared with wild-type mice. These data emphasize the significance of 1 subunit expression and its involvement in the regulation of CNS excitability. GABAA receptors are a family of ligand-gated ion channels that are the major target of the endogenous inhibitory neurotransmitter (GABA) and maintain the majority of fast inhibitory ion currents in the CNS. They are pentamers composed of subunits ( 1–6, 1–3, 1–3, , , , and ) that are encoded by a gene family with diverse expression patterns (Sieghart et al., 1999). GABAA receptors are the targets of several classes of drugs, including benzodiazepines (BZDs), barbiturates, alcohols, neurosteroids, and inhalation anesthetics (Sieghart, 1995). Additionally, GABAA receptors have been shown to be involved in epilepsy (DeLorey et al., 1998), various behavioral states such as depression and anxiety (Benson et al., 1998; Crestani et al., 1999), and learning and memory (Flood et al., 1992; DeLorey et al., 1998). Prevailing theory suggests that the subunit composition of an individual GABAA receptor confers a unique pharmacology that dictates the binding characteristics, functional capacity, and role of the receptor in maintaining the inhibitory tone of the CNS. The GABAA receptor 1 subunit is the most abundant subunit in adult brain, highly expressed throughout most brain regions, and is a component of 50% of GABAA receptors (Duggan and Stephenson, 1990; McKernan et al., 1991). Recombinant expression studies have indicated that 1 subunit expression confers specific pharmacological properties to the receptor, including GABA sensitivity (Levitan et al., 1988) and maximal efficacy of benzodiazepines (Puia et al., 1991). Furthermore, the expression of 1 versus 2, 3, and 5 in 2 receptors results in differential This study was supported by National Institutes of Health Grants AA09013 and AA11605 (to A.L.M.) and GM52035, GM47818, and AA10422 (to G.E.H.). The study was partially supported by the Academy of Finland (to E.R.K.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.036665. ABBREVIATIONS: CNS, central nervous system; BZD, benzodiazepine; mIPSC, miniature inhibitory postsynaptic potential; ANOVA, analysis of variance; TBPS, t-butylbicyclophosphorothionate; Ro15-4513, [H]ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate; SR-95531, 2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide; CL 218, 872, 3-methyl-6-(3-trifluoromethyl-phenyl)-triazolo[4,3-b] pyridazine. 0022-3565/02/3023-1037–1045$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 36665/1001748 JPET 302:1037–1045, 2002 Printed in U.S.A. 1037 at A PE T Jornals on July 3, 2017 jpet.asjournals.org D ow nladed from affinity for several benzodiazepine site ligands (Pritchett et al., 1989; Pritchett and Seeburg, 1990). Recently, several studies have begun to describe the in vivo role of the 1 subunit in GABAA receptor pharmacology, function, and related behavior. An association between heightened CNS excitability and reduced 1 subunit expression has been observed during ontogenic development, alcohol dependence and withdrawal, and in animal models of temporal lobe epilepsy (Aicardi and Chevrie, 1970; Mecarelli et al., 1988; Morrow et al., 1990; Devaud et al., 1997; BrooksKayal et al., 1998; Poulter et al., 1999). Previous studies have suggested various roles for subunit isoforms in specific BZD-related behaviors (Mohler et al., 1996; Rudolph et al., 1999; Crestani et al., 2000; Low et al., 2000). Recently, the production of two independent 1 knockout mouse lines has been described (Sur et al., 2001; Vicini et al., 2001). Global deletion of the 1 subunit gene results in viable mice that are surprisingly normal. Although initial electrophysiological studies revealed diminished mIPSCs and loss of zolpideminduced prolongation of mIPSC decay rates in cerebellar stellate cells (Vicini et al., 2001), we have now analyzed GABAA receptor subunit expression, ligand binding, and muscimol-stimulated Cl uptake in cerebral cortex as well as ligand binding autoradiography throughout brain. The results reveal interesting stable receptor adaptations that differ in some respects from adaptations observed in 1 / mice reported by Sur et al. (2001). The goals of the present studies were to identify in vivo relationships between GABAA receptor subunit expression, receptor adaptations, function, and seizure susceptibility. Materials and Methods Subjects. Male and female wild-type ( 1 / ), heterozygous GABAA receptor 1 subunit knockout ( 1 / ), and homozygous GABAA receptor 1 subunit knockout ( 1 / ) mice (Vicini et al., 2001) were derived from 1 / breeding pairs at the University of North Carolina (Chapel Hill, NC) or the University of Pittsburgh (Pittsburgh, PA). The wild-type allele consisted of a floxed allele in which the exon encoding nucleotides 1307 to 1509 of the 1 subunit was flanked by loxP sites that lacked a selectable marker gene. The knockout 1 allele consisted of the floxed allele after cre-mediated recombination. Briefly, the floxed allele was produced in Strain 129/Sv/SvJ embryonic stem cells. Chimeric offspring derived from these cells were mated to C57BL/6J mice and subsequently interbred for one generation. These mice were crossed with an actin-cre general deleter mouse line (FVB/N genetic background) to produce the recombined allele (Lewandoski et al., 1997). The cre transgene was subsequently eliminated from the pedigree. Mice that were heterozygous for the wild-type floxed allele and the recombined allele were interbred to produce the mice for experimental analysis. Thus, all mice were of the same mixed genetic background consisting of C57BL/6J ( 37.5%), 129/Sv/SvJ ( 37.5%), and FVB/N ( 25%). All animals were genotyped by Southern blot analysis, as described previously (Vicini et al., 2001). After weaning, mice were group housed with same sex littermates, given free access to standard rodent chow and water, and maintained on a 12-h alternating light/ dark schedule with lights on at 7:00 AM. All studies were conducted with mice derived from F5 to F7 generations and were 8 to 13 weeks of age. All studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by each institution’s Animal Care and Use committees. Western Blot Analysis. After decapitation, cerebral cortex was rapidly dissected over ice, frozen on dry ice, and stored at 80°C. P2 membrane fractions from cerebral cortex were prepared by homogenization in phosphate-buffered saline buffer (150 mM NaCl and 10 mM Na2HPO4/Na2H2PO4, pH 7.4). Aliquots of 25 g protein/lane were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions using an Xcell II minicell apparatus (Novex, San Diego, CA). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Blots were probed with GABAA receptor anti-peptide 1, 2, 3, 5, and 2 (Fritschy and Mohler, 1995), 4 (Kern and Sieghart, 1994), 2/3 (bd17; BMB, Indianapolis, IN), and (Quirk et al., 1995) antibodies. Antibodies were kind gifts from Drs. Jean-Marc Fritschy (University of Zurich, Zurich, Switzerland) and Werner Sieghart (University of Vienna, Vienna, Austria). Blots were then probed with horseradish peroxidase-conjugated anti-guinea pig ( 1, 2, 3, and 5 and 2), antirabbit ( 4 and ), or anti-mouse ( 2/3, actin) antibodies. Specific peptide labeling was detected by enhanced chemiluminescence (Pierce Chemical, Rockford, IL). Blots were apposed to X-ray film (Eastman Kodak, Rochester, NY) under nonsaturating conditions and analyzed by densitometric measurements (NIH Image 1.47). All Western blots were conducted under conditions in which densitometric signals were linear with protein concentration as determined in preliminary experiments. Blots were reprobed with actin and normalized to verify equivalent protein loading. Radioligand Binding. After decapitation, brains were immediately removed and placed in ice-cold saline from which cerebral cortices were rapidly dissected over ice and either used immediately or frozen on dry ice and stored at 80°C. Membranes were prepared by homogenization of cerebral cortices from eight mice per genotype in 50 volumes of assay buffer (50 mM Tris-citrate, pH 7.4) with a Polytron homogenizer. Samples were centrifuged at 20,000g for 20 min and resuspended in wash buffer five times before freezing the pellets at 80°C overnight. Pellets were washed twice more to remove endogenous GABA and used at a final concentration of 1 mg/ml. High-affinity [H]muscimol (specific activity 30 Ci/mmol) (PerkinElmer Life Sciences, Boston, MA) binding was conducted over a concentration range of 0.5 to 100 nM in a final assay volume of 500 l and incubated for 90 min at 0–4°C. Nonspecific binding was determined using 100 M GABA. The reaction was terminated by rapid filtration under vacuum ( 25 in. Hg) using GF/B filter strips (Whatman, Maidstone, UK) pretreated with 0.03% polyethylenimine. Samples were washed twice with 3-ml aliquots of assay buffer at 0–4°C. Filters were dried, added to liquid scintillation cocktail, and counted in a liquid scintillation counter. Saturation binding curves were evaluated using Prism (GraphPad Software, San Diego, CA) to obtain the KD and Bmax values and compared between genotypes by one-way ANOVA. [H]SR-95531 (specific activity 59.1 Ci/mmol) binding (25 and 200 nM; PerkinElmer Life Sciences) was determined in fresh cerebral cortical membranes according to McCabe et al. (1988), with minor modifications. Tissue was prepared by homogenization of cerebral cortices from two mice per genotype in 40 volumes of 0.32 M sucrose assay buffer (50 mM Tris-citrate, pH 7.4) with a Teflon homogenizer. Samples were centrifuged at 1000g for 10 min at 4°C from which the supernatant was centrifuged at 40,000 rpm in a rotor 41Ti (Beckman Coulter, Inc., Fullerton, CA) for 45 min at 4°C. The pellet was resuspended in double distilled H2O and centrifuged at 40,000 rpm for 30 min at 4°C before freezing at 80°C overnight. The following day, the pellet was resuspended in wash buffer and washed twice by centrifugation at 40,000 rpm for 15 min and used at a final concentration of 1.5 mg/ml. The final assay volume was 1 ml, and incubation took 45 min at 0–4°C. Nonspecific binding was determined using 100 M GABA. The termination of the binding reaction, washing of the membrane filters, counting the radioactivity, and analyzing the data were carried out as described above for [H]muscimol. [H]Zolpidem (specific activity 48 Ci/mmol) (0.125–50 nM; PerkinElmer Life Sciences) and [H]flunitrazepam (specific activity 71 Ci/mmol) (0.125–25 nM; PerkinElmer Life Sciences) binding assays were conducted using membranes prepared by homogenization of cerebral cortex from two mice per genotype in 50 volumes of wash 1038 Kralic et al. at A PE T Jornals on July 3, 2017 jpet.asjournals.org D ow nladed from buffer (50 mM Tris-HCl, pH 7.4). Samples were centrifuged at 40,000g for 15 min and resuspended in wash buffer twice before freezing the pellets at 80°C overnight. Pellets were washed twice more before resuspension in assay buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl, and 5 mM KCl). The final tissue concentration was 1 mg/ml. Nonspecific binding for [H]zolpidem and [H]flunitrazepam studies was determined using 500 nM zolpidem and 1 M diazepam, respectively (BIOMOL Research Laboratories, Plymouth Meeting, PA). The final assay volume of 500 l was incubated for 45 min at 0–4°C. The termination of the binding reaction, washing of the membrane filters, counting the radioactivity, and analyzing the data were carried out as described above for [H]muscimol. Autoradiography. t-Butylbicyclophosphorothionate ([S]TBPS) and tritium-labeled ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4Himidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([H]Ro15-4513) were purchased from PerkinElmer Life Sciences. Flumazenil (Ro15-1788) was donated by F. Hoffmann-La Roche (Basel, Switzerland). Picrotoxin was purchased from Sigma-Aldrich (St. Louis, MO). For autoradiography, 14m horizontal or frontal serial sections were cut from three to five frozen mouse brains of each genotype using a cryostat (Microm), thaw-mounted onto gelatin-coated object glasses, and stored frozen under desiccant at 20°C. All experiments were carried out in parallel manner with respective genotype, eliminating any day-to-day variation in receptor assays between genotype. The autoradiographic procedures for regional localization of [H]Ro15-4513 and [S]TBPS binding were as described previously (Makela et al., 1997). Briefly, sections were preincubated in an ice-water bath for 15 min in 50 mM Tris-HCl, pH 7.4, supplemented with 120 mM. The final incubation in the same buffer was performed with 6 nM [S]TBPS at room temperature for 90 min and assays with 10 nM [H]Ro15-4513 at 0–4°C for 60 min. After incubation, sections in both assays were washed 3 15 s in ice-cold incubation buffer. Sections were then dipped into distilled water, airdried under a fan at room temperature, and exposed with plastic [H]methacrylate or [C]methacrylate standards to Biomax MR films (Eastman Kodak) for 1 to 8 weeks. Nonspecific binding was determined with 10 M Ro15-1788 and 100 M picrotoxin in [H]Ro15-4513 and [S]TBPS assays, respectively. Images from representative autoradiography films were scanned, processed with Adobe Photoshop (version 3.0; Adobe Systems, Mountain View, CA) and Corel Draw 5.0 programs, and printed for figures. The concentration of [H]Ro15-4513 (10 nM) was greater than or equal to the dissociation constants for a range of recombinant and native GABAA receptors (Pritchett et al., 1989; Luddens et al., 1990; Pritchett and Seeburg, 1990; Wisden et al., 1991). Therefore, the autoradiographic images should represent the density rather than affinity of binding sites. Autoradiography films were quantified using AIS image analysis system (Imaging Research, St. Catherines, Ontario, Canada) as described previously (Makela et al., 1997). Binding densities for each brain area were averaged from measurements of one to three sections per brain. The standards exposed simultaneously with brain sections were used as reference with the resulting binding values given as radioactivity levels estimated for gray matter areas (nCi/mg for H and nCi/g for C). Significance between the mouse lines in different brain regions was assessed by two-way ANOVA followed by Bonferroni’s post hoc test using Prism. Chloride Uptake Assay. After decapitation, brains were immediately removed and placed in ice-cold saline. Cerebral cortices of seven mice per genotype were pooled for each experiment. Synaptoneurosomes were prepared and Cl uptake was conducted as described previously (Morrow et al., 1988). The synaptoneurosomal pellet was resuspended in 6.6 volumes of ice-cold assay buffer (20 mM HEPES, 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, and 2.5 mM CaCl2, pH 7.4) for a final protein concentration of approximately 5 mg/ml. The homogenate was aliquoted 200 l/assay tube and preincubated at 30°C for 12 min. Muscimol-stimulated Cl uptake was initiated by addition of 0.2 Ci of Cl (PerkinElmer Life Sciences) in the presence of various concentrations of muscimol (1–200 M). The solution was vortexed and uptake terminated after 5 s by addition of 4 ml of ice cold assay buffer containing 100 M picrotoxin with rapid vacuum filtration over G6 filters (Fisher Scientific, Pittsburgh, PA) using a single manifold filter apparatus (Hoeffer, San Francisco, CA). After two more washes, filters were allowed to dry and radioactive counts determined by liquid scintillation spectroscopy. Basal chloride uptake was measured in the absence of muscimol and subtracted from all tubes to determine muscimol-stimulated chloride uptake. Concentration-response curves were evaluated using computerized nonlinear regression (Prism; GraphPad Software) to obtain the EC50 and Emax values and compared between genotype by one-way ANOVA. Bicuculline-Induced Seizure Threshold Test. Seizure thresholds were determined at the beginning of the light cycle as described previously (Devaud et al., 1995). Mice were restrained in a Plexiglas plunger-style mouse restraint (Braintree Scientific, Braintree, MA). Threshold determination was made by constant lateral tail vein infusion of bicuculline (Sigma-Aldrich) dissolved in 0.1 N HCl, and diluted with isotonic saline to a final concentration of 0.05 mg/ml, pH 7. The solution was infused at a constant rate of 0.5 ml/min; the endpoint was taken as the first myoclonic jerk of the head and neck. This time point precedes forepaw clonus and generalized tonic/clonic convulsions. Each animal was tested once. Seizure thresholds were determined by experienced observers who were blind to the experimental conditions. Seizure thresholds were calculated from the time of infusion dose of bicuculline per body weight and presented as milligrams per kilogram of bicuculline. Data were analyzed by oneway ANOVA using Prism.